Practical
Streptomyces
Genetics
Tobias Kieser, Mervyn J. Bibb, Mark J. Buttner,
Keith F. Chater, David A. Hopwood

John Innes Centre, Norwich Research Park,
Colney, Norwich NR4 7UH, England

2000
The John Innes Foundation
NorwichTHE JOHN INNES FOUNDATIONJohn Innes Centre
Norwich Research Park
ColneyNorwich
NR4 7UHEnglandISBN 0-7084-0623-8

Copyright© 2000 by
The John Innes Foundation

Printed by Crowes, Norwich, England

Contents
Preface1.

General introduction to actinomycete biology . . . . . . . . . . . . . . . . . 1

2.

Growth and preservation of Streptomyces . . . . . . . . . . . . . . . . . . . . 43

3.

Microscopical methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

4.

Specialised biochemical techniques . . . . . . . . . . . . . . . . . . . . . . . . . 75

5.

Mutagenesis of Streptomyces by irradiation or chemicals . . . . . . . 99

6.

Transposon mutagenesis in Streptomyces . . . . . . . . . . . . . . . . . . . . 109

7.

In vivo genetic analysis by conjugation and protoplast fusion . . 125

8.

Preparation and analysis of genomic and plasmid DNA . . . . . . . 161

9.

General considerations about gene cloning in Streptomyces . . . . 211

10.

Introduction of DNA into Streptomyces . . . . . . . . . . . . . . . . . . . . . 229

11.

Plasmids and their use for gene cloning . . . . . . . . . . . . . . . . . . . . . 253

12.

Streptomyces phages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271

13.

Cloning with phage vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289

14.

Gene disruption and gene replacement . . . . . . . . . . . . . . . . . . . . . 311

15.

Reporter systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339

16.

RNA methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353

17.

Production and secretion of proteins by Streptomyces . . . . . . . . . 385

18.

Analysing Streptomyces DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393

19.

Media, buffers and suppliers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 405

20.

Genome maps and genetically marked strains . . . . . . . . . . . . . . . 423

21.

Maps of DNA fragments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 439

22.

Maps of plasmids, transposons and phage genomes . . . . . . . . . . . 511

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 585

LLL

Preface“Genetic Manipulation of Streptomyces: a Laboratory Manual” was published by the John Innes
Foundation in 1985. Judging by the number of research papers that cited the manual as a primary sourceof materials and methods (1692 citations up to August 1999), and the number of copies distributed(nearly 3000), it has proved useful. However, for many years it has been badly out of date. We began
to prepare a new edition in 1994, hoping - naively as it turned out - that we might be able to publish iton the tenth anniversary of the first edition! However, we had grossly underestimated the size of the task.
The 1985 book was based primarily on the protocols used during the first EMBO practical course on
Streptomyces genetics held in Norwich in 1983 and so nearly all the procedures were home-grown andcould be easily checked. Now the picture has been transformed. Streptomyces genetics is widely
disseminated, and many laboratories have devised cutting-edge methodologies, or constructed state-ofthe-art vectors. It has therefore proved to be a major task to try to monitor all this progress, collectprotocols from colleagues, and put the book together: hence the delay. We hope that the final productwill be more useful as a consequence, even if the book arrives only with the new millennium. Because
in the end we found ourselves running as hard as we could to keep the book current, we were not able
to spend enough time to make all the chapters - written by five different authors - completely uniform
from an editorial point of view, but we hope that this will not detract too much from the ease of using
the book.
This new manual is so different from the first that we regard it as a new work rather than a newedition of the first. This time, instead of confining ourselves to a series of laboratory protocols, we haveincluded more information about the biology and genetics of Streptomyces and its near relatives. The
main growth areas in terms of methodology have been in DNA transfer into Streptomyces, genedisruption, gene expression including reporter systems, versatility of vectors, and physical analysis of
the chromosome (foreshadowing the complete DNA sequence for Streptomyces coelicolor A3(2) whichshould be available by late 2000). In order to make room for all this new material we have omitted
coverage of purely E. coli procedures and generalised in vitro DNA handling, because these are easily
available in other excellent manuals such as Sambrook et al. (1989) and Ausubel et al. (1994).
As indicated, the book is a manifestation of the combined efforts of the whole Streptomyces
community. Sadly, some of this community, who have made signal contributions to the field, have diedin the interval since our first manual appeared. We here mark the passing of Ed Katz, and the untimely
loss of Bengtåke Jaurin, Kathy Kendrick and Gilberto Hintermann.
The innumerable colleagues who have generously sent background material, protocols and detailsof vectors and host strains are generally cited at appropriate points in the book. However, we should liketo thank especially the following people who have provided sections of text, tables or figures: Joe
Altenbuchner (University of Stuttgart) for transposons; Janos Bérdy (Budapest) for the statistics ofantibiotic production; Celia Bruton (JIC) for phage methods; Eric Cundliffe (University of Leicester) forLY

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antibiotic targets and coupled transcription-translation; Arny Demain (Massachusetts Institute ofTechnology) for details of useful antibiotics; Paul Dyson (University of Wales, Swansea) for
transposons; Chuck Hershberger (Eli Lilly and Company) for plasmid maps; David Hodgson (Universityof Warwick) for the physiology of primary metabolism and the growth of Streptomyces for physiologicalstudies; Helen Kieser (JIC) for pulsed-field gel electrophoresis; Gabriella Kelemen (JIC) for RNA
methods; Karel Mikulik (Institute of Microbiology, Prague) for the preparation and germination of
spores; Mark Paget (JIC) for gene disruption/replacement and RNA methods; Julie Schwedock (Harvard
University) for immunological staining methods; Luis Servin-González (Autonomous University, Mexico
City) for plasmid maps; Colin Smith (UMIST) for gene disruption/replacement, RNA methods andexpression vectors; Bill Strohl (Ohio State University) for expression vectors and plasmid maps; Eriko
Takano (JIC) for RNA methods; Charles Thompson and Xin-Ming Lee (Biozentrum, Basel) for 2D
electrophoresis of proteins; Liz Wellington (University of Warwick) for the taxonomy, ecology andisolation of Streptomyces; Wolfgang Wohlleben (Eberhard-Karls University, Tübingen) for genedisruptions/replacement and plasmid maps;. Finally, we thank Claire Luness, Tim Chater and Julian
Chater for graphics, and a special thank you to Sue Redmond for her painstaking and skilful preparationof the book for publication.

Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman
JG, Smith JA, Struhl K (eds.) (1994) Current Protocols in
Molecular Biology. John Wiley and Sons, Inc.

Sambrook J, Fritsch EF, Maniatis T (1989) Molecular
Cloning: a Laboratory Manual, 2nd ed. Cold Spring Harbor,
Cold Spring Harbor, NY.

m01tk.wpd 20 April 2000 Tobias Kieser

Chapter 1

General introduction to actinomycete biology
CONTENTS

Taxonomy of Streptomyces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
The genera of actinomycetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
The genus Streptomyces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Ecology of Streptomyces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Streptomycetes as pathogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Some physiological features of primary metabolism in streptomycetes . . . . . . . . . . . . . . . . . . 7
Carbon sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Nitrogen sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Amino acid catabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9Some physiological novelties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Antibiotic production by Streptomyces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Streptomycetes as antibiotic producers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Antibiosis in soil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Physiology and regulation of antibiotic production . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Developmental biology of Streptomyces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
The Streptomyces chromosome and its genetic elements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
DNA base composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
The chromosome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Plasmids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Transposable elements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Phages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Restriction and modification of Streptomyces DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Genetic studies with streptomycetes and their near relatives . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Actinomycetes used for genetical studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Genetics and strain improvement for antibiotic and enzyme production . . . . . . . . . . 23
Safety guidelines for recombinant DNA experiments with Streptomyces . . . . . . . . . . 33
This chapter contains an introduction to actinomycete biology as a background to the molecularbiology and genetics. Books dealing in more detail with some of these areas include two edited
collections of chapters (Goodfellow et al., 1984, 1988). The proceedings of the triennial International



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Symposia on the Biology of Actinomycetes are also rich sources of information. The last four volumes
are: Szabó et al. (1986); Okami et al. (1988); Ensign et al. (1992); and Debabov et al. (1995). There isalso Volume 4 of Bergey’s Manual of Systematic Bacteriology (Williams et al., 1989) devoted to theactinomycetes.

Taxonomy of Streptomyces
The genera of actinomycetes
The Gram-positive bacteria include two major branches: the low G+C organisms, containing
genera such as Bacillus, Clostridium, Staphylococcus and Streptococcus; and the high G+C organisms
referred to as the actinomycetes. Many of the latter develop a mycelial habit - originally regarded as thehallmark of the actinomycetes - at least at some stage in their life cycle, but others do not.
A whole array of taxonomic tools has been used to define genera and suprageneric groups ofactinomycetes (Goodfellow, 1989; Embley and Stackebrandt, 1994), but partial sequence analysis of 16S
ribosomal RNA is the most significant. An abridged tree based on this analysis is shown in Fig. 1.1 togive a flavour of the diversity of the actinomycete families and the place of the genus Streptomyces in
the group.
The genus Streptomyces
The genus is defined by both chemotaxonomic and phenotypic (“phenetic”) characters. The
major emphasis is now on 16S rRNA homologies, in addition to cell wall analysis and fatty acid and lipid
patterns (Williams et al., 1989; Wellington et al., 1992). One of the quickest methods for preliminary
identification to genus level was the presence of the LL isomer of diaminopimelic acid (LL-DAP) as thediamino acid in the peptidoglycan. This feature, when combined with the characteristic substrate andaerial mycelium, was diagnostic for Streptomyces. However, it turned out that some strains, previouslyclassified as Kitasatosporia, may contain major amounts of DL-DAP in the vegetative mycelium only
(Wellington et al., 1992); species from this former genus were found by Wellington et al. (1992) to lie
within the 16S rRNA clade for Streptomyces, but this was recently contradicted when more strains werestudied (Zhang et al., 1997).
The genus Streptomyces has been subjected to numerous systematic studies during the past 30
years but it is still difficult to identify unknown isolates. Many type species have been described, butthere has been much over-speciation resulting from antibiotic patents and the consequent need to assign
a name to the producing organism. Since the International Streptomyces Project in 1964, an attempt wasmade to produce valid species descriptions with at least a minimal number of standard phenotypic
criteria. However, the criteria turned out to be too minimal and the proliferation of species continued,
without any real attempt to compare species thoroughly with each other. The first study to do this reliedon numerical phenetic techniques to define clusters of strains or species based on comparison of many
phenotypic traits (Williams et al., 1983). This established 23 major clusters and some 20 minor groups;
the clusters were equated with species, except for the largest, cluster 1, which was split into three species(Williams et al., 1989). Because the Williams et al. (1983) study included only one representative foreach species, very many species were reduced to synonyms. However their specific epithets are still in

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Fig. 1.1. Abridged phylogenetic tree of actinomycetes based on almost full 16S ribosomal RNA
sequences. The tree was constructed by using the neighbour-joining method with Bacillus subtilis as
the outgroup. There are now over 100 validly described actinomycete genera, but not all have been
assigned to families. About half of the genera omitted from this abridged tree belong to one of therecognised families. Kindly provided by M. Goodfellow.

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Fig. 1.2. Dendrogram obtained using phenotypic characters for selected major clusters derived using
simple matching coefficient (Ssm) and clustering by the unweighted pair group method based onarithmetic averages (UPGMA). Data were derived from Williams et al. (1983). Kindly provided by E.
M. Wellington. (Note that species of Chainia, Kitasatoa and Streptoverticillium, reduced to synonymy
with Streptomyces by Williams et al., 1989 and Witt and Stackebrandt, 1990, are included.)

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constant use; for example S. griseus is synonymous with S. anulatus, S. lividans with S. violaceoruber,and S. hygroscopicus with S. violaceoniger. The major species groups (Williams et al., 1983), are given
in Fig. 1.2, with some minor groups of interest also included. The names are those of the oldest extant
type species within each cluster.
A second numerical phenetic analysis was made by Kämpfer et al. (1991); it differed from theearlier study in including many more species, and more than one strain of each species when available.
Many of the clusters defined by Williams et al. (1983) were recognised; a striking example is thealbidoflavus/anulatus/griseus/halstedii group appearing as cluster 1 in both studies, in which 28 of theS. griseus strains were grouped. Despite the problems associated with phenotypic characterisation, mostof the strains sharing the same specific epithet grouped together, indicating previously reliable
identification, but there were some notable exceptions; for example S. hygroscopicus strains wererecovered in cluster 1 subcluster 6, 8, 9, 10, 13, 24, 25, 35, 53, 54, 55, 56, 57 and 85. This may indicate
either problems in identification of this group or considerable phenotypic variation.
Several studies have attempted to use sequence data from variable regions of 16S rRNA toestablish taxonomic structure within the genus, but the variation was regarded as too limited to help
resolve problems of species differentiation (Witt and Stackebrandt, 1990; Stackebrandt et al., 1991,
1992). For example, species with a phenotype characteristic of the streptoverticillia grouped as a clade,but were not distinct from other species in the genus, in contrast to the results of the phenotypic analysis(clusters 55, 56, 58 and 59). The close phenotypic relationship between S. lavendulae and thestreptoverticillia species (Fig. 1.2) was also confirmed by 16S rRNA sequence comparisons (Witt andStackebrandt, 1990). The type species of the genus, S. albus, retained a distinct position in thephylogenetic trees and had unique sequences in the variable and regions of the gene (Stackebrandt
et al., 1991).
Total DNA homology studies (Labeda, 1992) have indicated genetic heterogeneity within someof the large phenotypic species groups defined by numerical taxonomy. The S. cyaneus cluster 18 wasstudied in detail; strains showed DNA relatedness of 20-85% with the majority of values around 50%(Labeda and Lyons, 1991). Selected species within the cluster were reduced to synonymy with others
if the comparisons gave homology values >70%.

Ecology of Streptomyces
Streptomycetes are ubiquitous in nature. Their ability to colonise the soil is greatly facilitatedby growth as a vegetative hyphal mass which can differentiate into spores that assist in spread andpersistence. The spores are a semi-dormant stage in the life cycle that can survive in soil for long periods(Mayfield et al., 1972; Ensign, 1978): viable Streptomyces cultures were recovered from 70 year old soil
samples (Morita, 1985). The spores impart resistance to low nutrient and water availability, whereas the
mycelial stage is sensitive to drought (Karagouni et al., 1993). The relatively high numbers of
streptomycetes in soil exist largely as inactive spores for most of the time. When laboratory-grownspores were added to nonsterile soil, they exhibited very low germination efficiencies, probably becauseof competition with indigenous microorganisms, but pre-germinated spores grew for a short time and thenre-sporulated (Lloyd, 1969). Germination can be partially density-dependent, but the interaction did notcross species boundaries (Triger et al., 1991), suggesting special signalling factors between spores of thesame strain, causing inhibition of germination above a certain concentration. The advantage would be&KDSWHU*HQHUDOLQWURGXFWLRQWRDFWLQRP\FHWHELRORJ\

to limit the number of germinating propagules in accordance with available resources. Spore germination
requires exogenous nutrients, water and Ca2+ (Ensign, 1978), and nutrient status of the germination site
limits the extent of hyphal growth and the time to differentiation into aerial hyphae and eventually spores(Wellington et al., 1990).
Actinomycetes produce many extracellular enzymes in soil. By decomposing complex mixturesof polymers in dead plant, animal and fungal material (McCarthy, 1987; Crawford, 1988; Wang et al.,1989), they are important in soil biodegradation by the recycling of nutrients associated with recalcitrant
polymers (McCarthy and Williams, 1992). Addition of chitin to acidic soil led to chitinolytic activityof acidophilic streptomycetes, with subsequent ammonification of the soil and eventual colonisation by
neutrophiles (Williams and Robinson, 1981). The enzymes required to catabolise such a common
material as straw are very complex and recently much effort has been devoted to characterising thecellulases, xylanases, amylases, maltases, etc. involved.
In addition to their ability to colonise bulk soil, many streptomycetes successfully colonise therhizosphere (Watson and Williams, 1974). This ability may be partly due to the antagonistic
characteristics of streptomycetes in competition with other rhizosphere bacteria such as pseudomonads
and bacilli. An added advantage over Gram-negative soil bacteria is their ability to spread throughrelatively dry soil via hyphal growth; in wetter soil, motile bacteria such as Pseudomonas fluorescensshow more extensive colonisation of rhizospheres than streptomycetes (Elliott-Juhnke et al., 1987;
Karagouni et al., 1993; Milus and Rothrock, 1993).

Streptomycetes as pathogens
Streptomycetes have only a very limited medical importance: human diseases caused byactinomycetes are largely due to infections by Mycobacterium, Actinomadura, Nocardia and Actinomyces
species. However, S. somaliensis, an established human pathogen, is now known to be an authentic
member of the genus Streptomyces (M. Goodfellow, and M. A. M. Nasher, personal communications).
Very few plant diseases are caused by actinomycetes. The best known is potato scab. Althoughyield loss from severe infections can result if the watering regime is improperly managed, the common
complaint is surface damage of infected tubers (Lechevalier, 1988), while geosmin secreted by thecolonies in scab tissue may render the flesh inedible. "Common scab" is caused by S. scabies (which also
causes scab in other root crops such as red and sugar beet, carrot, parsnip, radish and turnip), while S.
acidiscabies causes "acid scab" in soils of low pH, which are not conducive to growth of S. scabies. S.
scabies is a facultative parasite capable of saprophytic growth in the soil.
An economically important disease in the U.S.A. is sweet potato soil pox (or rot) caused by S.
ipomoea (Kennedy and Alcorn, 1980). This causes yield loss and disfiguring lesions to tubers.
Streptomycetes have been found in xylem vessels of maples, causing early decay and dieback of tree
branches (Blanchette et al., 1981a,b). Several Streptomyces species have been isolated from these trees,
including S. parvulus and S. sparsogenes. A lignocellulose degrading S. flavovirens was found in
Douglas fir responsible for decomposition of intact phloem and parenchymatous walls (Sutherland et al.,1979).

&KDSWHU*HQHUDOLQWURGXFWLRQWRDFWLQRP\FHWHELRORJ\Some physiological features of primary metabolism in streptomycetes
Carbon sources
In most streptomycetes the Embden-Meyerhof-Parnas (glycolysis) pathway of glucose catabolism
operates (Cochrane, 1961), but S. antibioticus apparently uses only the hexose monophosphate shunt(Salas et al., 1984). S. clavuligerus cannot metabolise glucose as carbon and energy source but can use
starch (Garcia-Dominguez et al., 1989). Several streptomycetes switch from glycolysis to the hexose
monophosphate shunt during secondary metabolism (L. Dijkhuizen, I. S. Hunter and S. Oliver, personalcommunications). There is no evidence of any streptomycete using the Entner-Doudoroff pathway thatis common in pseudomonads. Sugars appear to be imported into the cell and then phosphorylated by
specific kinases (Sabater et al., 1972; Ikeda et al., 1984; Novotna and Hostalek, 1985), but evidence fora phosphoenolpyruvate:fructose phosphotransferase system (PTS) for the transport and phosphorylationof fructose has recently been found in S. coelicolor, S. lividans and S. griseofuscus (Titgemeyer et al.,1995).
The detailed study of glycerol and galactose catabolism has revealed interesting similarities toand differences from the systems seen in other bacteria. It appears that these "peripheral" carbon
catabolic pathways are all inducible and often subject to carbon catabolite repression. Carbon catabolite
repression (CCR) refers to the ability of an efficiently metabolised carbon source, such as glucose, toprevent utilisation of alternative carbon sources. CCR is widespread in bacteria. In streptomycetes, it
can also inhibit antibiotic production (e.g. Cortes et al., 1986); and the glucose-dependent phenotype ofmost bald mutants of S. coelicolor points to steps in differentiation that are subject to glucose repression(Champness, 1988).
In the Gram-negative and low G+C Gram-positive bacteria, the glucose-PTS plays a central rolein CCR, exerting its effect through inducer exclusion and by precluding the expression of genes that
require the cAMP-catabolite control protein complex for transcriptional activation. Streptomycetes lacka glucose-PTS, and cAMP is not implicated in CCR. The Streptomyces fructose-PTS (Titgemeyer et al.,1995) appears to lack the Ser-phosphorylation of Hpr that plays a regulatory role in the low G+C Grampositives, so whether it participates in CCR is unclear.
In contrast, the ATP-dependent glucose kinase (GlkA) of S. coelicolor plays a crucial role in
CCR. Although glucose transport is not affected in glkA mutants, they cannot utilise glucose, andglucose repression of a wide variety of genes involved in the utilisation of alternative carbon sources isrelieved (Angell et al., 1994, and references therein). The inability of alternative glucose kinases torestore glucose represssion in glkA mutants suggests a direct regulatory role for the GlkA protein inglucose repression, and implies that the relief of CCR is not simply due to loss of glycolytic flux (Angell
et al., 1994). Furthermore, the role of GlkA appears to extend beyond glucose repression: glkA mutants
are deficient in CCR even when the repressing carbon source is not metabolised via glucose kinase(Kwakman and Postma, 1994). GlkA exerts its effects at the transcriptional level (Angell et al., 1994,
and references therein), although it is unlikely to be a DNA-binding protein: it probably interacts withor modifies transcription factors that are directly responsible for regulating the expression of genes
subject to glkA-dependent CCR.
GlkA is not required for glucose repression of the xylose utilisation operon of S. rubiginosus(Wong et al., 1991) or the chi63 promoter from S. plicatus (Ingram and Westpheling, 1995) when they

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are expressed in S. coelicolor. Furthermore, mutants of S. kanamyceticus defective in glucose repressionof -amylase production have wild-type levels of glucose kinase activity (Flores et al., 1993). At least
one additional regulatory mechanism that is capable of exerting CCR must therefore exist in
streptomycetes. One potential contributor in S. coelicolor is ccrA1 (control of catabolite repression;
Ingram et al., 1995). In ccrA1 mutants, transcription from the glucose-repressible galP1, gyl and chi63
promoters, but not from the glucose-insensitive galP2 promoter, is increased under both repressing andnon-repressing conditions. While mutations in glkA tend to abolish CCR, transcription from the galP1,
gyl and chi63 promoters is still subject to glucose repression in a ccrA1 mutant.
Putative cis-acting regulatory sequences have been identified for several genes subject to glucose
repression in streptomycetes, including chi63 (Delií et al., 1992), gal (Mattern et al., 1993), aml (Virolle
and Gagnat, 1994), and gyl (Hindle and Smith, 1994), and direct and inverted repeats have been
implicated in mediating glucose repression. However, none of these elements share obvious sequencesimilarity. For chi63 and aml, mutations in direct and inverted repeats, respectively, markedly reduced
both glucose repression and substrate induction. This suggests either common or overlapping recognition
sites for distinct regulatory proteins involved in glucose represssion and substrate induction, or a single
regulatory protein for both functions. In this context, it is notable that mutations in gylR (Hindle andSmith, 1994) and malR (van Wezel et al., 1997) abolish both substrate induction and glucose repressionof operons under their control.
In addition to their obvious defects in morphological differentiation, bld mutants appear to bepleiotropically defective in glucose repression (Pope et al., 1996), suggesting that bld mutants cannot
sense carbon limitation, and that it is this defect that is responsible for their morphological phenotype.
The mechanisms that regulate the utilisation of alternative carbon sources may therefore also play a rolein differentiation.
Nitrogen sources
In all streptomycetes so far studied the main pathway of nitrogen assimilation is via glutamate
synthetase (GS) and glutamate: 2-oxoglutarate transaminase (GOGAT) (Fisher, 1988). Surprisingly, allstreptomycetes studied have two GSs; GSI resembles those found in most prokaryotes and GSII is moresimilar to the eukaryotic type (Behrmann et al., 1990). An additional surprise is that GSI is regulated
by adenylation, like the GSs of Gram-negative enteric bacteria, but unlike those of Gram-positive bacilli(Streicher and Tyler, 1981). Assimilation of nitrogen via GS is energy-expensive, but is possible at low
ammonia concentrations. In many bacteria a "cheaper" method of assimilation uses a glutamate
dehydrogenase (GDH):GOGAT couple, but this mechanism works only at high ammonia concentrations.
Many streptomycetes seem to have this couple but some, including S. clavuligerus and S. aureofaciens,
do not (Aharonowitz and Friedrich, 1980; Vancurova et al., 1988). This led to speculation that alanine
dehydrogenase and alanine:2-oxoglutarate transaminase could be used instead, but the latter enzyme
could not be detected in the species lacking GDH. One is forced to conclude that the “expensive”
GS:GOGAT couple is the sole means of nitrogen assimilation in some streptomycetes.

&KDSWHU*HQHUDOLQWURGXFWLRQWRDFWLQRP\FHWHELRORJ\Amino acid catabolism
Most amino acid catabolism in streptomycetes seems to be via pathways previously describedin other bacteria. An exception is arginine, which is catabolised via -guanidinobutyramide and guanidinobutyrate (Thaoi et al., 1966); the enzymes involved were induced more than 15-fold by
arginine. The histidine and proline catabolic pathways are induced by the cognate amino acid, but forhistidine the true inducer is an intermediate of the pathway (Kendrick and Wheelis, 1982; Smith et al.,1995). About half the pathways studied seem to be constitutive, but the levels of the enzymes are very
low and difficult to assay.
Global catabolite repression of amino acid catabolism has been reported in streptomycetes, but
carbon catabolite repression of amino acid catabolism, common in enteric bacteria, does not seem tooccur (Hood et al., 1992; Kendrick and Wheelis, 1982; Kroening and Kendrick, 1987). Ammonium
repression of amino acid catabolism has been reported, as has ammonium repression of secondary
metabolism. However, the two do not seem to be directly linked because mutations that abolish global
repression of primary metabolism do not affect secondary metabolism (Bascaran et al., 1989). Much
evidence has been presented that "ammonium repression" of some secondary metabolites is due toammonium inhibition of enzymes involved in amino acid catabolism that supply precursors to secondary
metabolism, e.g. valine dehydrogenase (®mura and Tanaka, 1986).
Biosynthesis
The pathways of amino acid biosynthesis have been examined in numerous streptomycetes. The
amino acids are apparently synthesised in the same way as they are in most bacteria, with a couple ofminor differences. Tyrosine is synthesised via arogenate as in pseudomonads, rather than via
hydroxyphenylpyruvate as in enteric bacteria (Keller et al., 1985). A more important difference is thepresence of a trans-sulphurase pathway in streptomycetes, which is characteristic of fungi (Kern andInamine, 1981); this means that sulphur can be transferred either way between methionine and cysteine
via cystathionine -lyase and cystathionine -synthase.
A major difference between amino acid biosynthesis in streptomycetes and the enteric bacteria
and bacilli is its apparent lack of regulation. In the unicellular bacteria, most regulation is at thetranscriptional level, with the amino acid product negatively controlling expression of the genes encoding
biosynthetic enzymes. The method of inhibition includes attenuation and amino acid binding to repressor
proteins. There is very little evidence of such feedback inhibition of gene expression in streptomycetes.
Most amino acid biosynthetic enzymes, like those for their catabolism, are expressed at very low
constitutive levels (so low it is often difficult to believe that the assays are meaningful) (Hood et al.,1992). There are reports that biosynthesis of arginine and the common aromatic amino acids may besubject to feedback repression. Recently an attenuator was found upstream of the trpEG gene, which
encodes the first enzyme of the tryptophan-specific pathway. Genes encoding the enzymes for the final
part of the pathway appeared to be regulated by growth phase (on in exponential phase, off in stationary
phase) and growth rate (the faster the growth the more enzyme) (Hu et al., 1999).
We may speculate that the dearth of tight biosynthetic regulation reflects the growth of thestreptomycetes under typically nutrient-limited conditions, compared with the enteric bacteria. In thegut a plentiful supply of nutrients washes through at regular intervals, so the enteric bacteria have



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developed the ability to react quickly to feast and famine. For the streptomycetes, starvation is the ruleand so they seem not to have evolved, or to have lost, the ability to regulate amino acid biosynthesis at
the gene level. How streptomycetes manage to avoid the apparent futile cycle of constitutive synthesisof an amino acid followed by its degradation by a constitutive catabolic enzyme is a mystery.
Some physiological novelties
The environment inside living cells is normally maintained in a highly reduced state so that -SHgroups, essential to catalysis, are not inappropriately oxidised. A major factor in this process is usually
the maintenance of the major low molecular weight thiol, glutathione, in reduced form. Unusually,
streptomycetes and their relatives use instead of glutathione a newly discovered alternative, mycothiol(Newton et al., 1996).
Streptomycetes are not generally noted for autotrophic capabilities but one species, S.
thermoautotrophicus, has recently been found to be an obligate hydrogen-oxidising chemolithotroph
capable of growing on carbon monoxide (Gadkari et al., 1990). It can fix nitrogen also (Gadkari et al.,1992)!

Antibiotic production by Streptomyces
Streptomycetes as antibiotic producers
It is well known that members of the genus Streptomyces produce a great many antibiotics andother classes of biologically active secondary metabolites, but it is useful to put this statement in context.
As seen from Table 1.1, actinomycetes do indeed make some two-thirds of the known antibiotics thatare produced by microorganisms (some definitions actually restrict the word "antibiotic" to microbial
products), and amongst them nearly 80% are made by members of the genus Streptomyces, with othergenera trailing numerically: Micromonospora is the runner-up with fewer than one-tenth as many as
Streptomyces. If we include secondary metabolites with biological activities other than anti-microbial,
actinomycetes are still out in front, with over 60% (again Streptomyces spp. account for 80% of these).
However, Table 1.1 also brings out the fact that it is not the overall chemical versatility of theactinomycetes that is unparalleled - fungi and higher eukaryotes beat them hands down - but the
proportion of their output that has a known biological effect. Table 1.2 describes a range of usefulactinomycete antibiotics.
Antibiosis in soil
Obtaining direct evidence of antibiotic production in soil has proved problematical because ofdifficulties in extracting and detecting specific antibiotics. Circumstantial evidence for production of
streptomycin in soil was obtained during microcosm experiments where a producing strain, Streptomyces
bikiniensis, was coinoculated with Salmonella dusseldorf in order to observe the fate of sludge-borne

&KDSWHU*HQHUDOLQWURGXFWLRQWRDFWLQRP\FHWHELRORJ\

Table 1.1 Approximate numbers of secondary metabolites produced by different groups of organisms,
as of 1994 Data kindly provided by J. Bérdy
Bioactive metabolites
Source"Inactive"
metabolites

Antibiotics

Other

Total

1400 (12%)

240 ( 9%)1640 (11%)

2000 - 5000

79001 (66%)
2600 (22%)

12201 (40%)
1540 (51%)

91201 (61%)
4140 (28%)

8000 - 10,000
15,000 - 25,000

Total
microorganisms

11,900 (100%)

3000 (100%)

14,900 (100%)

25,000 - 40,000

Lichens
Algae
Higher plants
Terrestrial animals
Marine animals

150
700
5000
500
1200

200 - 500
800 - 900
25,000 - 35,000
10,000 - 15,000
1500 - 2000

~ 10001000 - 2000
500,000 - 800,000
200,000 - 300,000
2000 - 3000

Total higherorganisms

7500

35,000 - 50,000> 1,000,000

Non-actinomycete
bacteria
Actinomycetes
Fungi

1

In each category, nearly 80% were found in Streptomyces and 20% in other actinomycetes

pathogens in the presence of soil streptomycetes. The Salmonella viable count rapidly declined incomparison to control microcosms where a non-streptomycin producing strain was coinoculated instead(Turpin et al., 1992). The presence and antagonistic action of streptomycin-producing streptomycetesin tropical soils was associated with the occurrence of streptomycin-resistant Gram-negative bacteria,such as Rhizobium and Bradyrhizobium species (Ramos et al., 1987; Roughley et al., 1992).
Recent evaluations of streptomycetes as potential antagonists of soil-borne plant pathogens has
focussed on their ability to produce natural antifungal metabolites. S. rochei and S. rimosus from thechickpea rhizosphere were strong antagonists of Fusarium oxysporum f. sp. ciceri (Bashar and Rai,
1994). S. hygroscopicus var. geldanus, grown in sterile soil, antagonised Rhizoctonia solani, the pea root
rot fungus, via geldanamycin production (Rothrock and Gottlieb, 1984). Antifungal polyene antibiotics
were detected in sterile peat by HPLC following inoculation and incubation of microcosms with S.
griseoviridis (Raatikainen et al., 1994). Antibiotics have not been detected in natural untreated soils, but
production has been detected in nonsterile amended (i.e. fortified with nutrients), sterile amended andunamended soil inoculated with producing strains of streptomycetes (Williams, 1982; Wellington et al.,
1993; Marsh and Wellington, 1994). Recently, S. scabies as a potato pathogen has been found to be
specifically controlled by an antibiotic produced by another streptomycete (Eckwall and Schottel, 1997).

&KDSWHU*HQHUDOLQWURGXFWLRQWRDFWLQRP\FHWHELRORJ\

Table 1.2 Useful actinomycete antibiotics
Antibiotic

Producer

Chemical class1

Target 2

Application

Actinomycin D

S. spp.

PeptideTranscription

Antitumour

Antimycin A

S. spp.

Macrolide

Cytochrome
systemTelocidal

Avermectin

S. avermitilis

Macrolide (PK)

Chloride ionchannels

Antiparasitic

Bambermycin

S. bambergiensis

Substituted amino- Peptidoglycan
glycosides (complex of at least 4
moenomycins)

Growth promotant

Bialaphos

S. hygroscopicus

PeptideGlutaminesynthetase

Herbicidal

Bleomycin

S. verticillus

Glycopeptide

DNA strand
breakage

Antitumour

Candicidin

S. griseus

Polyene macrolide(PK)

Membrane(pore former)

Antifungal

Cephamycin C

Nocardia
lactamdurans(and others)-lactam

Peptidoglycan

Antibacterial

Chloramphenicol

S. venezuelae

N-dichloroacyl
phenylpropanoid

R

Antibacterial

Chlortetracycline

S. aureofaciens

Tetracycline (PK)R

Antibacterial

Clavulanic acid

S. clavuligerus-lactam-lactamase
inhibitor

Combined witha -lactam as
antibacterial

Cycloserine

S. orchidaceus

Substituted cyclic
peptidePeptidoglycan

Antibacterial

Daptomycin

S. roseosporus

Lipopeptide

Lipoteichoic
acid?

Antibacterial

Daunorubicin(daunomycin)

S. peucetius

Anthracycline
(PK)

DNA
intercalation

Antitumour

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Antibiotic

Producer

Chemical class1

Target 2

Application

Desferrioxamine

S. pilosus

PeptideIron chelation

Iron purging iniron overload

Doxorubicin(adriamycin)

S. peucetius var.
caesius

Anthracycline
(PK)

DNA
intercalation

Antitumour

Erythromycin

Sac. erythraea

Macrolide (PK)R

Antibacterial

FK506(tacrolimus)

S. hygroscopicus

Macrolide (PK)

Binds to FK
protein

ImmunosuppressantFortimicin

Micromonospora
olivoasterospora

AminoglycosideR

Antibacterial

Fosfomycin

S. spp.

Phosphoric acid

Peptidoglycan

Antibacterial

Gentamicin

Micromonospora
spp.

AminoglycosideR

Antibacterial

Hygromycin B

S. hygroscopicus

Substituted
aminoglycosideR

Antihelminthic

Kanamycin

S. kanamyceticus

AminoglycosideR

Antibacterial

Lasalocid

S. lasaliensis

Polyether (PK)Membrane(ionophore)

Anticoccidial;
growth promotant

Lincomycin

S. lincolnensis

Sugar-amide

R

Antibacterial

Milbemycin

S. hygroscopicus

Macrolide (PK)

Chloride ionchannels

Antiparasitic

Mithramycin

S. argillaceus

Aureolic acid
(PK)

DNA
alkylation

Antitumour

Mitomycin C

S. caespitosus
S. verticillatus

Benzoquinone

DNA crosslinking

Antitumour

Monensin

S. cinnamonensis

Polyether (PK)Membrane(ionophore)

Anticoccidial;
growth promotant

Natamycin

S. nataensis

Tetraene polyene(PK)

Membrane(pore former)

Antifungal

&KDSWHU*HQHUDOLQWURGXFWLRQWRDFWLQRP\FHWHELRORJ\

Antibiotic

Producer

Chemical class1

Target 2

Application

NeomycinS. fradiae

AminoglycosideR

Antibacterial

Nikkomycin

S. tendae

Nucleoside

Chitinbiosynthesis

Antifungal;
insecticidal

Nocardicin

Nocardia
uniformis-lactam

Peptidoglycan

Antibacterial

Nosiheptide

S. actuosus

Thiopeptide

R

Growth promotant

NovobiocinS. niveus

Coumerin
glycoside

DNA gyrase( subunit)

Antibacterial

Nystatin

S. noursei

Polyene macrolide(PK)

Membrane(pore former)

Antifungal

Oleandomycin

S. antibioticus

Macrolide (PK)R

Antibacterial

Oxytetracycline

S. rimosus

Tetracycline (PK)R

Antibacterial

Paromomycin

S. rimosus forma
paromomycinus

AminoglycosideR

Antiamoebal

Phleomycin

S. verticillus

Glycopeptide

DNA strand
breakage

Antitumour

Polyoxins

S. cacaoi var.
asoensis

Nucleosidepeptide

Chitinbiosynthesis

Antifungal (plant
protection)

Pristinamycin

S. pristinaespiralis PeptidicR
macrolactone +polyunsaturated
macrolactone (PK)

Antibacterial

Puromycin

S. alboniger

Purine nucleosideR

Research

Rapamycin

S. hygroscopicus

Macrolide (PK)

Binds to FK
protein

ImmunosuppressantRifamycin

Amycolatopsis
mediterranei

Ansamycin (PK)RNA
polymerase

Antibacterial(tuberculosis andleprosy)

Ristocetin

Nocardia lurida

Glycopeptide

Peptidoglycan

Antibacterial

&KDSWHU*HQHUDOLQWURGXFWLRQWRDFWLQRP\FHWHELRORJ\

1
2



Antibiotic

Producer

Chemical class1

Target 2

Application

Salinomycin

S. albus

Polyether (PK)Membrane(ionophore)

Anticoccidial;
growth promotant

Spectinomycin

S. spectabilis

Aminocyclitol

R

Antibacterial

Spinosyns

Sac. spinosa

Tetracyclic
macrolide (PK)Unknown

Insecticidal

Spiramycin

S. ambofaciens

Macrolide (PK)R

Antibacterial

Streptogramins

S. graminofaciens

Macrocyclic
lactones

R

Antibacterial

Streptomycin

S. griseus

AminoglycosideR

Antibacterial

Streptothricin

S. lavendulae

N-glycosideR

Growth promotant;
plant protectant

Teichoplanin

Actinoplanes
teichomyceticus

Glycopeptide

Peptidoglycan

Antibacterial

Tetracycline

S. aureofaciens

Tetracycline (PK)R

Antibacterial

Thienamycin

S. cattleya-lactam

Peptidoglycan

Antibacterial

Thiostrepton

S. azureus

Thiopeptide

R

Growth promotant

Tobramycin

S. tenebrarius

AminoglycosideR

Antibacterial

TylosinS. fradiae

Macrolide (PK)R

Growth promotant

Validamycin

S. hygroscopicus

Aminoglycoside

R

Plant protectant

Vancomycin

Amycolatopsis
orientalis

Glycopeptide

Peptidoglycan

Antibacterial

Virginiamycin

S. virginiae

Macrocyclic
lactone (PK) +macrocyclic
peptidolactoneR

Growth promotant

PK = polyketide
R = binds to ribosomes and thus inhibits protein synthesis

&KDSWHU*HQHUDOLQWURGXFWLRQWRDFWLQRP\FHWHELRORJ\

Rigorous model experiments on agar (Wiener, 1996) have indicated that antibiotic production
by streptomycete colonies can play a significant role in preventing invasion by competing Bacillus
subtilis but it did not improve the ability of the antibiotic producer to invade an established populationof sensitive B. subtilis, nor help competition between established streptomycete and bacillus populations.
This fits strikingly the idea (Chater and Merrick, 1979) that the major role for antibiotics is to defend thecolony from competitors at the time of development of the aerial mycelium in the lysing vegetative
mycelial mat.
Physiology and regulation of antibiotic production
Antibiotic production in streptomycetes is generally growth phase-dependent. In liquid culture,
it begins as the culture enters stationary phase. In agar-grown cultures, it coincides with the onset ofmorphological differentiation, and the occurrence of mutants deficient in both antibiotic production andthe formation of aerial hyphae indicates at least some common elements of genetic control (Chater andBibb, 1997). Most antibiotics are the products of complex biosynthetic pathways, with a cluster of genes(generally 20-30) dedicated to the synthesis of any one compound. These gene clusters usually containpathway-specific regulatory genes which act as transcriptional activators, and which are themselves
subject to control by pleiotropic regulatory genes. The onset of antibiotic biosynthesis is determined andinfluenced by a variety of physiological and environmental factors. These include growth rate, diffusible-butyrolactone signalling molecules (Horinouchi and Beppu, 1994), imbalances in metabolism (e.g.
Hood et al., 1992) and various physiological stresses (Hobbs et al., 1992; Yang et al., 1995). In addition
to these positive effectors of antibiotic production, antibiotic synthesis may also be subject to metabolite
repression and/or inhibition by readily utilised sources of nitrogen (generally NH4+), phosphate and/or
glucose (Demain, 1992; Demain and Fang, 1995; Chater and Bibb, 1997). The various factors that
influence antibiotic production in streptomycetes are not readily accommodated in a simple unifying
model. However, it seems reasonable to propose an overall regulatory influence of growth rate, withsuperimposed pathway-specific regulatory effects influencing the production of individual antibiotics.
These effects may be elicited at the level of expression of the pleiotropic or pathway-specific regulatorsor the biosynthetic structural genes, and/or at the level of activity of the biosynthetic enzymes.
Most genetic studies of antibiotic regulation have used S. coelicolor A3(2), which produces atleast four different antibiotics (p. 93), permitting studies of pathway-specific and pleiotropic control ofantibiotic production to be carried out. Over 20 different pleiotropic genes influence antibiotic
production in S. coelicolor (Bibb, 1996; Chater and Bibb, 1997), several of which (the bld genes) are also
required for sporulation. While the roles of most of these genes are unknown, they include three twocomponent regulatory systems and one eukaryotic-like serine-threonine protein kinase, indicating animportant role for protein phosphorylation - and potentially phosphorylation cascades - in the regulationof antibiotic production. No satisfactory model exists to explain the roles of these genes, the interactions
between them, and the ways in which they activate expression of the pathway-specific regulatory genes.
In particular, little is understood of the signals that trigger the onset of antibiotic production, and that
presumably activate expression of some or all of these pleiotropic regulatory genes. These genes arelikely to form a complex regulatory web, consistent with the need of the organism to sense, integrate andrespond to a variety of environmental and physiological signals; their multiplicity might explain theconditional nature of many of the mutants that are pleiotropically defective in antibiotic production.

&KDSWHU*HQHUDOLQWURGXFWLRQWRDFWLQRP\FHWHELRORJ\Developmental biology of Streptomyces
The complex life cycle of the streptomycetes (Fig. 1.3) is one of the major reasons for studying
their genetics. During the growth of a colony from a spore, colonial complexity increases (Hodgson,
1992; Chater, 1993; Champness and Chater, 1994; Chater and Losick, 1996). After a suitable
germination trigger, the germ tube emerges from the unigenomic spore and its cell wall grows mainly at
the tip, at a rate that doubles with each replication of the chromosomes in the initially aseptate germ tube
(reviewed by Chater and Losick, 1996). The potentially catastrophically accelerating rate of tip
extension becomes stabilised after a few DNA replication cycles, because new tips arise through lateralbranching as further doublings in chromosome number take place. Many branches are close to septa.
Both branches and septa are most frequent in older parts of the mycelium, and are not found very close
to tips. The formation of septa at some distance behind tips produces an apical and a sub-apicalcompartment. Sub-apical compartments differ from apical compartments in various respects: they lacknew cell wall synthesis but DNA synthesis nevertheless takes place. If a branch forms in a sub-apicalcell, it regains some apical properties.
As colonies grow, the older mycelium becomes densely piled up, and further changes take place.
These changes may be responses to nutrient limitation or other physiological stresses (some perhaps
mediated via ppGpp: Ochi, 1986; Takano and Bibb, 1994; Chakraburtty et al., 1997), and/or to celldensity via extracellular signals (examples include A-factor in S. griseus (Horinouchi and Beppu, 1994)
and an apparent cascade of signals necessary for aerial growth in S. coelicolor A3(2) (Willey et al.,1993)). The changes are of at least five general kinds: increased production of some extracellular
proteins; onset of secondary metabolism; initiation of lysis of some compartments of the substrate
mycelium; onset of storage metabolism in substrate hyphae on the colony surface; and initiation of aerial
hyphal growth. Some of the bld genes appear to play a part in several of these attributes. For example,
bldA mutants in S. coelicolor A3(2) - depending on growth conditions - may lack aerial growth, antibiotic
production and glycogen (Plaskitt and Chater, 1995). Remarkably, bldA specifies the only tRNA able
efficiently to translate the leucine codon UUA, which (as TTA) is particularly rare in the high G+C DNA
of streptomycetes, but which is often present in pathway-specific regulatory genes for antibiotic
biosynthesis (Leskiw et al., 1991).
Several classes of bld mutants of S. coelicolor can take part in extracellular complementationinteractions: when pairs are grown near each other (at least on R2YE medium) one of the two may
develop a normal aerial mycelium. As many as five different extracellular molecules appear to be
involved in a signal cascade that culminates in production of a modified oligopeptide, SapB, which seems
itself to be a morphogen that facilitates aerial growth on this medium (Willey et al., 1993; Nodwell etal., 1996).
Once aerial hyphae begin to grow, they extend apparently mainly by tip growth (Miguelez et al.,1994), implying the need to supply nutrients by diffusion from the basal part of the colony. Under thecontrol of at least six specialised regulatory genes (whiA, B, G, H, I and J, one of which, whiG, encodes
an RNA polymerase sigma factor: Chater, 1993), the long, multigenomic and often curled aerial hyphaltips undergo multiple synchronous cell division to give cylindrical unigenomic prespore compartments
in which glycogen deposits form transiently. (This second phase of storage metabolism involves
glycogen biosynthetic isoenzymes genetically distinct from those involved in mycelium-associatedglycogen synthesis: Bruton et al., 1995.) At least partially under the control of another sporulationspecific sigma factor ( F) (Potúûková et al., 1995), the cylindrical compartments round up and acquire&KDSWHU*HQHUDOLQWURGXFWLRQWRDFWLQRP\FHWHELRORJ\

thickened spore walls pigmented with a grey, polyketide-derived aromatic compound. Glycogendisappears, probably having provided a local supply of precursors for the production of pigment, spore
wall and trehalose (an abundant component of spores that probably contributes significantly to theirresistance to desiccation: Braña et al., 1986).
From this brief description, it can be seen that Streptomyces colonies are multicellular,differentiated organisms exhibiting temporal and spatial control of gene expression, morphogenesis,
metabolism and the flux of metabolites: these are attributes most often associated with multicellular
eukaryotes, but here they are in the much more fully understood and more readily manipulatable context
of prokaryotic cellular organisation and molecular biology. An extensive knowledge of thedevelopmental biology of streptomycetes is not only of basic scientific interest, but also provides avaluable resource for the optimisation of their industrial exploitation, whether through the fuller
understanding of the genetic interconnections of development and secondary metabolism, and of
metabolic routing and re-routing, or in the preparation of inocula, or in controlling growth form infermenters.

The Streptomyces chromosome and its genetic elements
DNA base composition
As members of the high G+C branch of the Gram-positive bacteria, Streptomyces spp. have oneof the highest proportions of these residues in their chromosomal DNA, often quoted as 70-74 mole %,and sometimes with an even wider range. (Analysis of the coding regions of 64 different streptomycete
genes from 27 different species - not necessarily representative of the genome as a whole - covered therange 61-80% mole % G+C: Wright and Bibb, 1992.) Values for genera such as Saccharopolyspora,
Amycolatopsis and Micromonospora are in the same range. There is little or no evidence for extensive
blocks of chromosomal DNA with a composition significantly different from the average, which would
appear as a fraction of "satellite" DNA. However, short segments of lower G+C DNA have been foundby sequencing: for example in a non-coding segment of the actinorhodin gene cluster of S. coelicolor(Fernández-Moreno et al., 1992).
The base composition of plasmids native to these hosts has generally been found not to differ
significantly from the chromosomal DNA. For phages, not surprisingly, a much wider range isencountered. The best-studied temperate Streptomyces phage, C31, has 63% G+C; others are in therange of 60-73%, but some virulent phages have been reported with values as low as 49% (Cox and
Baltz, 1984).
The chromosome
Early attempts to estimate genome size in Streptomyces by renaturation kinetics gave a rather
wide range of values. The use of pulsed-field gel electrophoresis (PFGE) to analyse macrorestriction
fragments of the chromosome has yielded much more accurate values, which are very consistent: 7.8-8.0
Mb for S. coelicolor A3(2) (Kieser et al., 1992), S. lividans 66 (Leblond et al., 1993), three strains of S.
ambofaciens (Leblond et al., 1991) and S. griseus IFO03237 (Lezhava et al., 1995).

&KDSWHU*HQHUDOLQWURGXFWLRQWRDFWLQRP\FHWHELRORJ\Fig. 1.3. The life cycle of Streptomyces coelicolor A3(2). Under favourable conditions, one or two
germ tubes emerge from a spore and grow by tip extension and branch formation to give rise to a
substrate mycelium. After about 2-3 days, aerial hyphae grow up in a process that involves the actionof a large number of bld genes. The apical compartment of individual aerial hyphae forms a spiralsyncytium that contains many tens of genomes. When aerial growth stops, multiple septa subdivide
the apical compartment into unigenomic pre-spore compartments. These subsequently change in shape;
wall thickening occurs and grey spore pigment is deposited, to generate dessiccation-resistant spores.
Sporulation septation depends on six regulatory loci (whiA, B, G, H, I and J), which are also neededfor expression of at least one (sigF) of two late regulatory loci, whiD and sigF; these in turn play
important roles in the thickening of the spore wall and the deposition of the grey spore pigment (spore
pigment is specified by the whiE gene cluster). The terminal phenotypes of mutants blocked at
different stages, shown here by side-arrows before the relevant genes, are somewhat different fromintermediate stages of normal sporulation.

&KDSWHU*HQHUDOLQWURGXFWLRQWRDFWLQRP\FHWHELRORJ\

An exciting recent discovery is that the chromosome of many, probably most, streptomycetes isa linear structure (Lin et al., 1993; Leblond et al., 1993; Lezhava et al., 1995). The chromosomes carryproteins covalently bound to both free 5U ends; these probably act as primers for synthesis of the last
Okazaki fragment of the lagging strand when bidirectional DNA replication reaches the free ends, after
initiation at a typical oriC (Musialowski et al., 1994) located centrally in the chromosome (Chang and
Cohen, 1994; Chen, 1996; Redenbach et al., 1996). The ends of the chromosomes have long terminal
inverted repeats (TIRs), in the range 24-600 kb in different species.
Chromosome linearity is probably causally related to another special feature of Streptomyces
chromosomes, which has been known for many years, namely their tendency to suffer large deletions andamplifications, which can occur with a frequency as high as 0.1-1% spontaneously and even more
frequently after treatment with compounds that damage DNA or interfere with its replication, such as
acridines or ethidium bromide, or by cold shock or protoplasting (reviewed by Cullum et al., 1988;
Schrempf et al., 1989; Birch et al., 1990; Leblond et al., 1990). Recent work has shown that thesedeletions, which may together include nearly a quarter of the chromosome, occur near its ends. (Perhaps
the fourth strain of S. ambofaciens studied by Leblond et al. (1991), which had a genome size of only
6.5 Mb, was a spontaneous deletion variant.) Interestingly, terminal chromosomal deletions may span
both ends to yield a circular chromosome capable of replication (Lin et al., 1993; Redenbach et al.,1993), an event that can also be brought about artificially (Lin et al., 1993). In S. lividans 66 and S.
coelicolor A3(2), the most frequently deleted marker (at about 1% per spore) is a gene forchloramphenicol resistance (Altenbuchner and Cullum, 1984; Dittrich et al., 1991), located rather near
one end of the chromosome, and many of the chloramphenicol-sensitive variants had circularchromosomes (Redenbach et al., 1993). Amongst the chloramphenicol-sensitive strains, up to 25% thenlost a gene (argG) for argininosuccinate synthetase, causing arginine auxotrophy (Altenbuchner andCullum, 1984), leading to the concept of progressively extending deletions (Lin and Chen, 1997).
Plasmids
Most Streptomyces strains contain plasmids, nearly all of which are self-transmissible fertility
factors. Both linear and circular plasmids occur. The linear plasmids are between 10 and 600 kb in size
and, like the chromosome, have TIRs and proteins at the 5U ends. In one case studied, most of the 17 kb
linear plasmid pSLA2 from S. rochei was replicated from a centrally located replication origin, and only
synthesis of c. 280 bp at the ends was primed by the terminal proteins (Chang and Cohen, 1994). S.
coelicolor A3(2) has three fertility factors, SCP1, SCP2 and SLP1. SCP1 (350 kb) is linear, SCP2 (30kb) is circular and SLP1 is integrated in the chromosome (see below).
Some of the circular Streptomyces plasmids have phage -like int and xis genes for site-specific,
non-mutagenic integration into highly conserved tRNA genes. SLP1 (17 kb) of S. coelicolor and pSAM2(11 kb) of S. ambofaciens are examples (p. 264). These plasmids normally reside in the hostchromosome, but transfer independently by conjugation to strains lacking the plasmid, where they
become autonomously replicating CCC plasmids with a moderate copy number when either their int geneor att site is inactivated by mutation or is deleted during or following excision from the chromosome.
Non-integrating, "conventional" CCC plasmids are also common. The larger ones, e.g. SCP2,
have a low copy number and probably replicate by the “theta” mode, bidirectionally from an origin to
generate new double-stranded plasmid copies. The smaller ones tend to have higher copy numbers:

&KDSWHU*HQHUDOLQWURGXFWLRQWRDFWLQRP\FHWHELRORJ\pIJ101, 8.9 kb, has 300 copies per chromosome (p. 256). They replicate by a rolling circle mechanism(sigma mode) via a circular, single-stranded replication intermediate. Replication is initiated by acharacteristic, plasmid-encoded replication protein.
Most Streptomyces plasmids contain neither antibiotic resistance determinants nor antibiotic
biosynthesis genes (SCP1, with genes for the biosynthesis of and resistance to methylenomycin, is a rare
exception). It is also remarkable that even small plasmids, like pIJ101, contain an autonomous transfer
mechanism that does not depend on trans mobilisation by a larger plasmid. Most Streptomyces plasmids
conjugate with 100% efficiency (expressed per recipient). In a plasmid-free lawn, a plasmid startingfrom a single cell can spread over a diameter of up to 2 mm, resulting in "pock" formation; this is a very
sensitive, macroscopic means for detecting self-transmissible plasmids (pp. 153, 239).
Transfer of conjugative plasmids and mobilisation of a nonconjugative plasmid have been
demonstrated in sterile amended and unamended soil, and in nonsterile unamended soil (Rafii andCrawford, 1988; Wellington et al., 1988). Interspecific transfer of pIJ673 from S. violaceolatus to S.
lividans in nonsterile soil was also demonstrated (Wellington et al., 1988, 1990), and concurrent
microscopic observations showed that all gene transfer occurred during the brief period of mycelial
growth within two days of spore germination (Clewlow et al., 1990). In situ metabolic activity was
shown to be important in the frequency of gene transfer, because nutrient amendments of the soil resulted
in significant increases during peak mycelial growth. Peaks in mycelial growth could be accurately
monitored via quantitation of plasmid DNA by selective DNA extraction from the vegetative stage in soil
samples (Cresswell et al., 1992; Wellington et al., 1992).
Autonomously replicating and integrating plasmid vectors and the properties of their parental
plasmids are described in Chapter 11. Pocks are explained on p. 153. Methods for the analysis of CCC
plasmids are detailed in Chapter 8. Linear plasmids can be visualised by PFGE after removal of theterminal proteins by proteinase treatment (Chapter 8.). Plasmid vector maps are in Chapter 22.
Transposable elements
Several transposable elements have been discovered in streptomycetes but none contains anatural selectable marker. Five are known in S. coelicolor (Table 20.2); they are present in one, two or
three copies dispersed on the chromosome. IS117, the 2.6 kb "mini-circle" of S. coelicolor, integrates
at specific sites and has a circular transposition intermediate; derivatives with resistance markers are used
as integrating vectors (see pp. 265). Two randomly integrating elements, IS493 of S. lividans 66 andTn4556 of S. fradiae, have been modified for transposon mutagenesis. Also IS6100 from Mycobacterium
fortuitum has been adapted for random mutagenesis of Streptomyces. These mutagenic tools andmethods for their use are described in Chapter 6. Transposon maps are in Chapter 22.
PhagesMany phages active on Streptomyces spp. and their relatives have been described. Most havebeen isolated from soil, though some have been discovered after release by natural lysogens. Most
phages studied in detail show typical life cycles, and are most active on germ tubes and young mycelium.
Some, however, appear to be adapted to growth on old mycelium; this means that, in laboratory
conditions, plaques become visible only after prolonged incubation, or may continue to grow until a few
plaques cover the whole surface of a Petri dish (Haket et al., 1990).

&KDSWHU*HQHUDOLQWURGXFWLRQWRDFWLQRP\FHWHELRORJ\

Both lytic and temperate phages are abundant, and they may have narrow or wide host-ranges.
However, it seems to be rare to find Streptomyces phages that infect other genera of actinomycetes.
Among temperate phages, some (such as the particularly extensively studied C31), lysogenise byintegrating site-specifically into the host chromosome, while at least one example of a plasmid prophage
is known (Chung and Thompson, 1985). In some species (e.g. S. venezuelae: Stuttard, 1989) it has been
easy to find transducing phages, while in S. coelicolor A3(2) success has very recently been reported (J.
Westpheling, personal communication). We are not aware of any reports of Streptomyces phages that
contain RNA or single-stranded DNA genomes.
Details of C31 and other phages, vectors derived from them, and suggestions for their use arein Chapters 12 and 13. Phage vector maps are in Chapter 22.
Restriction and modification of Streptomyces DNA
Type II restriction systems (p. 276) are widespread in actinomycetes (many have yielded"workhorse" enzymes for genetic engineering, such as SalI, SstI, SstII, SphI, NcoI and NotI). In several
streptomycetes they have been implicated as barriers to interspecific gene transfer by the isolation of nonrestricting mutants; a tour de force is the work of Matsushima et al. (1987a) in which five different
restriction systems were eliminated sequentially from the tylosin-producing S. fradiae strain. There arealso indications of the occurrence of non-Type II restriction systems in some strains. In the "model"
S. coelicolor A3(2), a system that has resisted all attempts to eliminate it by mutation is likely to be ofthis type. It appears that at least a major part of the barrier to the transfer of cloned DNA from E. coli
to S. coelicolor is due to a restriction system that cuts methylated DNA and that can be circumvented by
the use of a totally non-methylating E. coli host as DNA donor. This finding arose from the discovery
of such a restriction system in S. avermitilis (MacNeil, 1988).
A different system of DNA modification was discovered in S. lividans 66. This is associatedwith the degradation of DNA in vitro rather than in vivo. This modification (which is not a basemethylation) renders the DNA susceptible to degradation by oxidative cleavage at preferred sites during
electrophoresis. A mutant, ZX1 and its derivative ZX7, lacking this property were isolated (Zhou et al.,1988), and buffer conditions can circumvent it (Ray et al., 1995; Evans and Dyson, 1993); these areparticularly useful for pulsed-field gel electrophoresis (p. 197) of DNA from S. lividans or from
S. avermitilis, which apparently has the same modification system (Evans et al., 1994). An S. lividans
gene cluster responsible for the degradation phenotype has recently been cloned and specificallyinactivated (strain ZX64: X. Zhou, personal communication).

Genetic studies with streptomycetes and their near relatives
Actinomycetes used for genetical studies
The predecessor of this Manual included primarily John Innes protocols suitable for S. coelicolorand S. lividans. Many of these have turned out to be readily applicable to other Streptomyces species,
or even to members of other genera of actinomycetes, but others have not. Moreover, many protocols
for novel procedures have been developed by other laboratories, starting with other species. In this book

&KDSWHU*HQHUDOLQWURGXFWLRQWRDFWLQRP\FHWHELRORJ\

we are attempting to include the most important of these, as well as modifications of earlier protocols
for use in other Streptomyces spp., at least by means of literature references.
Table 1.3 lists such strains and includes references to papers dealing with different aspects of
their genetics. (“Cluster” in the second column refers to taxonomic cluster according to Williams et al.,1983.) We have included antibiotic-producing strains that were earlier classified as streptomycetes - suchas Amycolatopsis mediterranei, Saccharopolyspora erythraea and Nocardia lactamdurans - as well as
Micromonospora spp. However, the extensive genetical studies on mycobacteria and corynebacteria are
not covered. Even though initiation of these studies owes a debt to Streptomyces genetics, they have now
diverged and developed to the point where they would need a separate treatment.
We have tried to make Table 1.3 as complete as possible, knowingly excluding only species thathave been used merely as donors of genes for study in other hosts, unless this has led to extensive genetic
knowledge about the donor (for example about an antibiotic gene cluster), but we are conscious of thefact that there may be serious omissions.
Genetics and strain improvement for antibiotic and enzyme production
For several decades, efficient chemical- or radiation-induced mutagenesis has been crucial inindustrial strain improvement programmes for antibiotic titre (Baltz, 1986). The use of natural matingor protoplast fusion to combine desirable genes from divergent lines of a mutation-screening pedigree
has also been successful on many occasions, but there is little or no satisfactory description of theseendeavours in the public domain, for reasons of commercial secrecy. This is frustrating for thoseinterested in developing and applying the required methodology, but is a fact of life! The use of genetic
engineering for titre improvement (Chater, 1990) is also little documented, but finally there is quotable
information on the enhanced expression of specific "rate-limiting" steps in a biosynthetic pathway, forcommercial antibiotic production, to enhance end-product titre (Baltz et al., 1997; Sezonov et al., 1997).
Attempts to engineer titre increases by the manipulation of pathway-specific or global regulatory genes,
which are now well known in many streptomycetes, may well have been successful in industry as they
have in academic studies of improved strains, but are not described in print.
A more visible use of genetic engineering is to produce novel, "hybrid" antibiotics. After the
first early reports (Hopwood et al., 1985; Epp et al., 1989), this topic is currently undergoing an
explosion of interest, especially in relation to polyketide antibiotics (McDaniel et al., 1995; Katz andDonadio, 1993; Hutchinson and Fujii, 1995; Tsoi and Khosla, 1995). No useful compounds have yet
been described, but it is early days! It would be very surprising if "combinatorial biosynthesis", alone
or in combination with chemical transformations, did not yield applications in the next few years.
Some of the hydrolytic enzymes secreted by streptomycetes are actual or potential industrial
products, and genetic engineering has potential for the construction of high yielding strains, as well as
strains expressing protein pharmaceuticals (Baltz, 1990; Binnie et al., 1997). Glucose isomerase, which
is typically an intracellular enzyme, has also been engineered successfully for increased thermal andalkaline stability for commercial production.

&KDSWHU*HQHUDOLQWURGXFWLRQWRDFWLQRP\FHWHELRORJ\

Table 1.3 Actinomycetes used for genetical studies
StrainCluster Types of studies

Selected references

S. achromogenes ssp.
rubradiris

19

DNA amplification

Hornemann et al. (1993)

S. acrimycini

1B2

Genetic mapping

Wright and Hopwood (1977)

S. alboniger
ATCC12461

1B

Puromycin biosynthesis(cloning of complete set ofgenes)

Lacalle et al. (1992)

S. albus G

16

Expression of foreign genes

Binnie et al. (1997);
Olano et al. (1995)

Genetics of SalI restriction
systemAlvarez et al. (1993)

Heat shock genes, including
gene disruption

Servant and Mazodier (1995)

Genetic mapping, genestructure and instability

Smokvina et al. (1988); Leblond etal. (1996); Dary et al. (1993);
Schneider et al. (1993)

Plasmid studies

Sezonov et al. (1995)

Spiramycin biosynthesis,
including transposon mutagenesis and hybrid antibiotic
production by cloning an S.
thermotolerans gene

Epp et al. (1989);
Richardson et al. (1990);
Tang et al. (1994);
Geistlich et al. (1992)

Genetic instability

Yu and Chen (1993)

Regulation of melanin
synthesis

Betancourt et al. (1992)

Stringent response

Kelly et al. (1991); Jones (1994)

S. ambofaciens

S. antibioticus
IMRU3720(actinomycin D
strain)23

31

S. antibioticus
ATCC11891(oleandomycin strain)

31

Oleandomycin biosynthesis
and resistance

Swan et al. (1994);
Olano et al. (1995)

S. aureofaciens
ATCC10762

14

Sigma factors, including
gene disruption

Kormanec et al. (1994)

Glycogen metabolism

Homerová et al. (1996)

&KDSWHU*HQHUDOLQWURGXFWLRQWRDFWLQRP\FHWHELRORJ\

StrainS. aureofaciens
ATCC10762

S. avermitilis

S. azureus
ATCC14921

Cluster Types of studies
14

191

18

Selected references

Bromoperoxidases

Pelletier et al. (1994)

Tetracycline biosynthesisBhal and Hunter (1995)

Genetic mapping

Ikeda et al. (1987)

DNA modification

MacNeil et al. (1993);
Evans et al. (1994)

Avermectin biosynthesis:
gene cloning, disruption,
mutation

MacNeil (1995);
Denoya et al. (1995);
Ikeda and ®mura (1995)

Mutation and lysogeny

Yamada et al. (1989, 1990)

Thiostrepton resistance

Strohl and Floss (1995)

S. bikiniensis var.
zorbonensis

1B

Genetic mapping

Coats and Roeser (1971)

S. cacaoi KCCSO352

16-lactamase expression

Urabe et al. (1990)

S. chartreusis

18

Host for cloned resistancegenes

Murakami et al. (1983)

S. chrysomallus
ATCC11523

13

Protoplast fusion

Keller et al. (1983)

S. cinnamonensis
A3823.5

612

Polyketide synthase genes;
gene disruption

Arrowsmith et al. (1992)

S. clavuligerus

71

Genetic mapping by
protoplast fusion

Illing et al. (1989)

Linear plasmid

Wu and Roy (1993)

Stringent response

Bascarán et al. (1991)

Cephamycin and clavulanic
acid biosynthesis; genedisruption

Yu et al. (1994);
Paradkar and Jensen (1995);
Ward and Hodgson (1993)

S. coelicolor A3(2)211

Numerous and diverse

This Manual; see index

S. coelicolor(Müller)

1A

Oxidative enzymes;H2O2resistant mutants

Lee et al. (1993)

Strain&KDSWHU*HQHUDOLQWURGXFWLRQWRDFWLQRP\FHWHELRORJ\

Cluster Types of studies

Selected references

S. curacoi
ATCC13385

18

Polyketide synthase genes

Bergh and Uhlén (1992)

S. espinosus?3

Protoplast transformation

Manis and Highlander (1982)

S. fradiae(tylosin strain)

68

Mutagenesis, protoplast
fusion and transformation,
restriction-modification,transduction, biosynthesis oftylosin, gene disruption,
attenuation

Baltz and Seno (1988);
Kreuzman et al. (1988);
Merson-Davies and Cundliffe(1994);
Tang et al. (1994);
Kelemen et al. (1994)

S. fradiae
ATCC10745(neomycin strain)

68

Neomycin resistance,translation

Jones et al. (1992)

S. fradiae Tü2717(urdamycin strain)

68

Urdamycin biosynthesis,
including gene disruption
and hybrid antibiotic
production by cloning
S. olivaceus gene

Decker and Haag (1995);
Decker et al. (1995)

S. fulvoviridis

3

Carbapenem biosynthesis

Nakata et al. (1989)

S. galilaeus

19

Biosynthesis of aclacinomycins, including hybrid
antibiotic production bycloning S. purpurascens
genes

Strohl and Connors (1992);
Tsukamoto et al. (1994);
Niemi and Mäntsälä (1995)

S. glaucescens
ETH22794

28

Genetic mapping

Baumann et al. (1974)

Genetic instability: deletions
and amplifications

Birch et al. (1991)

Fatty acid biosynthesis

Summers et al. (1995)

Biosynthesis of tetracenomycin, including
complete gene sequence andtranscription studies

Shen and Hutchinson (1993);
Summers et al. (1993)

Hybrid antibiotic synthesis
by cloning S. fradiae genes

Decker and Haag (1995)

Dihydrostreptomycin
biosynthesis

Piepersberg (1995)

&KDSWHU*HQHUDOLQWURGXFWLRQWRDFWLQRP\FHWHELRORJ\

StrainS. griseofuscus

S. griseus4(streptomycin strains,some formerly called
S. bikiniensis)

Cluster Types of studies
12

1B

Selected references

Cloning of heterologous
genes

Rao et al. (1987)

Transduction by FP43

Hahn et al. (1991)

Genome structure

Lezhava et al. (1995)

Gene expression

Wu et al. (1995)

Sporulation and septation,
including A-factor

Dharmatilake and Kendrick (1994);
Ueda et al. (1993); Ochi et al.
(1992); Horinouchi and Beppu(1994)

Biosynthesis of streptomycin Piepersberg (1995)
S. griseus
NRRL 3851(cephamycin strain)

1B

Genetic recombination

Liras and Martín (1981)

S. griseus IMET
JA3933(anthracycline strain)

1B

Daunorubicin biosynthesis,
including mutant
complementation

Krügel et al. (1993)

S. griseus K-63
(griseusin strain)

1B

Griseusin polyketide
synthase genes, including
gene disruption

Yu et al. (1994)

S. griseus ETH
A7796(nonactin strain)

1B

Nonactin biosynthesis andresistance

Plater and Robinson (1992)

S. halstedii
NRRL2381

1C

Spore pigment biosynthesis,
including gene disruption

Blanco et al. (1993)

Genetics of cellulases

Fernández-Abalos et al. (1992)

Two glutamine synthetase
genes

Kumada et al. (1990)

Bialaphos biosynthesis,
including gene disruption
and regulation

Anzai et al. (1988);
Holt et al. (1992);
Thompson and Seto (1995)

Generalised transduction

Süss and Klaus (1981)

S . hygroscopicus
ATCC 21705(bialaphos strain)

S. hygroscopicus(IMET strains)

32

32





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StrainCluster Types of studies

Selected references

S. hygroscopicus var
geldanus NRRL3602

32

Geldanamycin biosynthesis,
including gene disruption

Allen and Ritchie (1994)

S. hygroscopicus
NRRL5491(rapamycin strain)

32

Rapamycin biosynthesis(sequence of 26 genes)

Schwecke et al. (1995)

S. hygroscopicus
ssp. yingchengensis

32

Gene cloning, including
antibiotic biosynthesis

Hu et al. (1994);
Qin et al. (1994)

S. jumonjinensis

71

Protoplast transformation

Manome and Hoshino (1987)

S. kanamyceticus

42

Interspecific mating with
S. rimosus

Mazières et al. (1981)

S. kasugaensis?3

Protoplast transformation,
etc.

Okanishi et al. (1983);
Nabeshima et al. (1984)

S. lasaliensis NRRL
3382R

?3

Lasalocid biosynthesis,
including protoplast fusion

Donovan et al. (1989)

S. lavendulae(mitomycin C
strains)

61

Mitomycin C resistanceAugust et al. (1994)

S. lavendulae
ATCC8664

61

Genetic instability

Nakano and Ogawara (1980)

S. levoris A-9

1B2

Valinomycin biosynthesis,
including mutant
complementation

Perkins et al. (1990)

S. lincolnensis
NRRL293619

Lincomycin biosynthesis,
including transposon mutagenesis, gene amplification,and sequence of 30 genes

Chung and Crose (1990);
Peschke et al. (1995)

S. lividans 66

21

Genome mapping, structure
and instability

Hopwood et al. (1983); Lin et al.
(1993); Redenbach et al. (1994);
Eichenseer and Altenbuchner(1994); Rauland et al. (1995)

Plasmid studies

Kieser et al. (1982)

Cloning and expression offoreign genes

Ogawara (1989);
Brawner et al. (1991)

&KDSWHU*HQHUDOLQWURGXFWLRQWRDFWLQRP\FHWHELRORJ\

StrainCluster Types of studies

Selected references

S. narbonensis

5Interspecific protoplast
fusion with S. fradiae

Ikeda et al. (1984)

S. noursei

292

Plasmids

Kojima et al. (1985)

S. olivaceoviridis
ATCC11238

20

Gene encoding cellulosebinding protein

Schnellmann et al. (1994)

S. olivaceus Tü2353

1C

Elloramycin biosynthesis

Decker et al. (1995)

S. parvulus
ATCC12434

12

Lysogenisation

Shirai et al. (1991)

Expression of S. coelicolor
actinorhodin genes

Kim et al. (1995)

S. peucetiusssp. caesius
ATCC27925

422

Otten et al. (1995);
Daunorubicin biosynthesis,
Madduri and Hutchinson (1995);
including gene sequence,
Filippini et al. (1995)
transcriptional regulation,
mutant complementation andgene disruption

S. phaeochromo40
genes NRRLB3559(chloramphenicol strain)

Plasmid pJV1

Doull et al. (1983);
Bailey et al. (1986)

S. pilosus

37

Desferrioxamine
biosynthesis, including ironregulated gene expression

Günter et al. (1993)

S. platensis ssp.
malvinus

29

Platenomycin biosynthetic
mutants

Furumai and Suzuki (1975)

S. pristinaespiralis
ATCC25486

26

Pristinamycin biosynthesis,
including gene sequencing
and gene disruption

Blanc et al. (1995)

S. reticuli

1B2

Genetic instability

Schrempf (1982)

S. rimosus(oxytetracycline
strains)

42

Genetic mappingAlaûevií et al. (1973);
Rhodes et al. (1981)

Lysogenisation

Rausch et al. (1993)

Ribosomal RNA genes

Plohl and Gamulin (1991)

Electroporation

Pigac and Schrempf (1995)

Strain&KDSWHU*HQHUDOLQWURGXFWLRQWRDFWLQRP\FHWHELRORJ\

Cluster Types of studies

Selected references

S. rimosus(oxytetracycline
strains)42

Oxytetracycline biosynthesisBhal and Hunter (1995)

S. rimosus f.
paromomycinus

42Interspecific mating with S.
kanamyceticus

Mazières et al. (1981)

S. rochei

12Linear plasmids

Chang and Cohen (1994)

S. roseochromogenes KCC-SO264

1B2

Plasmids

Shindoh et al. (1984)

S. scabies

3

Zinc regulation of esterase
production

Babcock et al. (1992)

S. setonii ISP5395

1B

Lignin degradation,
including protoplast fusion

Pettey and Crawford (1984)

Carotenoid biosynthesis,
including transformation

Kato et al. (1995)

S. tanashiensis,
strain Kala

52

Kalafungin biosynthesis,
including mutant
complementation

Kakinuma et al. (1991)

S. tendae Tü901

12

Nikkomycin biosynthesis,
including 2-D protein
analysis, gene disruption andcloning of the whole
pathway in S. lividans

Möhrle et al. (1995);
Bormann et al. (1996)

S. tendae 4158

12

Tendamistat expression in
S. lividans

Fass and Engels (1996)

S. tenjimariensis?3

Protoplast fusion with
S. griseus

Yamashita et al. (1985)

S. thermotolerans

18

Carbomycin biosynthesis,
including mutant
complementation

Epp et al. (1989)

S. toyocaensis

29

Transformation, transduction Matsushima and Baltz (1996)
and conjugation from E. coli

&KDSWHU*HQHUDOLQWURGXFWLRQWRDFWLQRP\FHWHELRORJ\

StrainCluster Types of studies

S. venezuelae
ATCC10712

6

Selected references

Genetic mapping by transduction, conjugation, transformation and gene disruption, including chloramphenicol biosynthesis

Stuttard (1988);
Doull et al. (1986);
Paradkar et al. (1993)

Jadomycin biosynthesis,
including gene disruption

Han et al. (1994)

S. verticillus
ATCC15003?3

Bleomycin resistance genes

Calcutt and Schmidt (1994)

S. virginiae

61-butyrolactone
autoregulators

Okamoto et al. (1995)

S. viridochromogenes Tü494

27

Bialaphos resistance,
including transformation andintegration

Behrmann et al. (1990)

Bialaphos biosynthesis

Thompson and Seto (1995)

S. viridosporus

15

Lignin degradation,
including mutagenesis andprotoplast fusion

Crawford et al. (1984)

S. wadayamaensis?3

Protoplast transformation

Acebal et al. (1986)

Streptomyces sp.
Strain C5?3

Daunomycin biosynthesis,
including complementationand characterisation of
12 genes

Dickens et al. (1995)

Amycolatopsis
mediterranei(rifamycin producer)-

Genetic mapping

Schupp et al. (1975)

DNA amplification

Zhu et al. (1990)

Transformation andelectroporation

Madon and Hütter (1991);
Lal et al. (1991)

Rifamycin biosynthesis,
including protoplast fusion

Schupp et al. (1981)

A. methalonica-

Plasmid studies and cloning

Vrijbloed et al. (1995)

A. orientalis(vancomycin producer)-

Protoplast transformation

Matsushima et al. (1987b)





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StrainCluster Types of studies

Selected references

Micromonospora
echinospora
NRRL15839(calichiamicin producer)

Transcriptional regulation

Lin and Rothstein (1992)

M. olivasterospora(fortimicin producer)-

Protoplast transformationand isolation of fortimicin
biosynthetic genes

Hasegawa (1992)

M. purpurea-

Protoplast transformation

Kelemen et al. (1989)-

Protoplast transformation

Matsushima and Baltz (1988)-

Cephamycin biosynthesis:
cloning of the gene cluster

Coque et al. (1993)

Transformation

Kumar et al. (1996)

Transformation and gene
integration/disruption

Weber and Losick (1988);
Andersen and Hutchinson (1992);
Hsieh and Kolattukudy (1994)

Integrating plasmids

Brown et al. (1994)

Erythromycin biosynthesis

Donadio and Katz (1992);
Cortes et al. (1995)(gentamicin producer)
M. rosaria(rosariamicin producer)
Nocardia
lactamdurans

Saccharopolyspora
erythraea(erythromycin
producer)

1-

Sacc. hirsuta-

Polyketide synthase genes

Le Gouill et al. (1993)

Sacc. spinosa(spinosyn producer)-

Conjugal transfer of DNA
from E. coli;transformationof modified DNA

Matsushima et al. (1994);
Matsushima and Baltz (1994)

Identified by E. M. H. Wellington using the scheme of Williams et al. (1983).
Inferred from studies of Kämpfer et al. (1991); cluster according to Williams et al. (1983).
3
Unknown taxonomic status.
4
Several different streptomycin producing S. griseus strains have been used in genetic work; thePiepersberg group has mainly used a strain called N2-3-11 isolated by the Kaken Chemical Co., with the
Waksman type strain 18-16 (ATCC233345) in some work; the Beppu/Horinouchi laboratories have used
IFO13350; the Ochi group has used IFO13189 (ATCC11984); the Kendrick/Ensign laboratories haveused NRRLB-2682 (ATCC23345/23921 from Waksman’s 18-16); and the Baumberg group has used
ATCC12475.
2

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Safety guidelines for recombinant DNA experiments with Streptomyces
Small-scale recombinant DNA work with Streptomyces has become established in most countriesof the developed world over the last 15 years. As far as we are aware, no significant problems in
obtaining the necessary permissions have been encountered, and no difficult safety issues have cropped
up. For experiments involving the transfer of genetic material between members of the genus
Streptomyces, or of DNA from other non-pathogens into streptomycete hosts, no physical containment
beyond the lowest levels commensurate with ensuring sterility - in other words "good microbiologicalpractice" - seems to have been required. In the UK, it was agreed early on that interspecific transfer in
Streptomyces would count as "self-cloning" and so would actually fall outside the need for special
consideration. In some other countries, notably the USA and nations that followed the early NIH lead,
specific strains or host-vector combinations were named in the regulations, but there was no difficultyin obtaining permission to work with other strains. In the EU, Guideline 219, covering the contained useof recombinant organisms, deals in general principles, rather than with particular species or approved
host-vector systems. It is typically left to the national committees to classify experiments on a case-bycase basis.
For large-scale work, and particularly for the use of genetically engineered Streptomyces hosts
for commercial production, the situation is much too complex to deal with here. Suffice it to say that fulllicenses have indeed been obtained for some specific products.

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m02tkb.wpd 20 April 2000 Tobias Kieser

Chapter 2

Growth and preservation of Streptomyces
CONTENTS
Selective isolation of streptomycetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
Making a Streptomyces spore suspension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
Plating out a Streptomyces spore suspension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Streptomyces cultures on agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Growth of mycelium on cellophane overlays, nitrocellulose filters or agar . . . . . . . . . . . . . . . . 48
Growth of Streptomyces mycelium in liquid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Growth of Streptomyces for physiological studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Conditions for spore germination and reproducible exponential growth
and antibiotic production by Streptomyces coelicolor . . . . . . . . . . . . . . 50
Germination of Streptomyces spores . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Procedure 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Procedure 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
Preparation of aerial mycelium and spores separately . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
Aerial mycelium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Spores . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Sporulation of Streptomyces in submerged cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
Preparation of protoplasts from Streptomyces lividans 66 and S. coelicolor A3(2) . . . . . . . . . . 56
Preservation of Streptomyces strains and phages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
Preparation of lyophils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
Alternative I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
Alternative II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Growing a culture from the lyophil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

Selective isolation of streptomycetes
Most genetic studies are made on strains that are available from stock culture collections orcolleagues, but here we give an indication of methods that have been developed to isolate streptomycetes
and other actinomycetes from nature. Streptomycetes can be isolated from a wide variety of habitats but,because they are aerobic, they are less readily recovered from water-logged soils than facultative
anaerobes. Adding lime to the soil can enrich for streptomycetes (Wellington and Toth, 1994).
Most isolation techniques involve extraction from an environmental sample followed by biomass
dilution to allow culturing on solid media. Chemical disruption aids dispersal and subsequentdissociation of spores and mycelium from soil particles with which they are normally intimately

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associated. This can be achieved by gently shaking soil samples with the ion-exchange resin Chelex-100(Bio-Rad) and an eluent, resulting in exchange of Na+ for divalent cations such as Mg2+ and Ca2+, whichbind soil aggregates together (Herron and Wellington, 1990). Spores can then be separated from the bulkof mycelial material by differential centrifugation and filtration, which also serves to concentrate thebiomass. The final extract can be plated on selective media such as arginine glycerol salts agar (ElNakeeb and Lechevalier, 1963), HV agar (Hayakawa and Nonomura, 1987), colloidal chitin agar (Hsuand Lockwood, 1975), or reduced arginine starch salts agar (Herron and Wellington, 1990).
Inhibitors may be incorporated into isolation media to aid selection of streptomycetes(Wellington and Toth, 1994). Detergents like SDS, which kill vegetative cells, have been used to isolate
spores (Nonomura and Hayakawa, 1988). It is also useful to incorporate antifungal agents such as
cycloheximide and nystatin into solid isolation media to prevent overgrowth by fungi. Most
streptomycetes can then be isolated on the selective medium at 28o, whereas thermotolerant strains canbe isolated at 40o, and acidophiles on media adjusted to pH4.5 (Wellington and Toth, 1994). For
isolation of particular species groups, Wellington et al. (1987) chose selective media based on taxonomic
data, reflecting specific carbon or nitrogen source utilisation or resistance to various compounds.
Even on selective media, bacteria such as pseudomonads and bacilli may overgrow target
colonies. New methods are therefore being developed to allow specific recovery of desired propagules
using immunocapture. This facilitates initial separation of species from environmental samples, after
which enrichment on non-selective media and then on selective media provides sensitive recovery of atarget species. A monoclonal antibody to a major spore coat polypeptide exclusive to cluster 21
Streptomyces species, including S. lividans, was developed by Wipat et al. (1994). As a test, soil
samples were seeded with S. lividans spores, which were recovered by immunomagnetic capture using
magnetic beads coated with the antibody, and a magnet to concentrate the bead-spore complex.
Streptosporangium fragile was recovered from soil by indirect immunomagnetic capture using a rabbit
polyclonal primary antibody against the spores (Mullins et al., 1995); the antibody was added to soil
samples after treatment with blocking agents. Antibody-labelled spores were captured using magnetic
beads coated with sheep anti-rabbit second antibody, and concentrated on a magnet.

Making a Streptomyces spore suspension
Most streptomycetes produce copious, haploid, unigenomic spores under suitable culture
conditions. Although Streptomyces spores arise in chains in the aerial mycelium, individual spores canusually be readily separated by suspending and vortexing in water; for some strains, however, a wetting
agent such as 0.1% Tween 80 or 0.001% Triton X100 is needed. The resulting suspensions are used formany purposes, such as inoculating liquid medium to produce mycelium for isolating plasmid or
chromosomal DNA, RNA or enzymes, or for preparing protoplasts; for the isolation of mutants; and forthe analysis of recombination or plasmid transfer in crosses. Concentrated spore suspensions (c. 109
ml–1) are crucial for purposes like starting reproducible cultures for physiological or fermentation studies(p. 49), so it is worth experimenting with different media to find one that is good for a particular strain.
MS (mannitol soya flour; p 409) agar is particularly good for S. coelicolor and S. lividans.
Suspensions of spores in 20% glycerol, kept frozen at -20o, will usually remain viable for years,
even if they are repeatedly thawed and re-frozen for sampling purposes (but not if they start to&KDSWHU*URZWKDQGSUHVHUYDWLRQRI6WUHSWRP\FHV

germinate). Non-sporulating strains, such as whi and some bld strains, die rapidly on freezing in glycerol(though bldA strains retain viability). Spore suspensions frozen in water usually lose about 50% of their
viability on freezing and thawing. Suspensions can also be kept frozen in DMSO at -20o (useful for
glycerol-sensitive mutants).
Fresh plate culture (or slant) of the strain
Sterile water
20% glycerol in water (sterilised by autoclaving)
Rigid inoculating loop; pipettes; filter tubes containing non-absorbent cotton wool(see Fig. 2.1); centrifuge tubes; screw cap containers, e.g. 7.5 ml ("Bijou"
bottles) and 20 ml (wide-necked McCartney bottles or "Universal" containers)
Vortex mixer; bench centrifuge; compressed air supply
The following should be carried out in a laminar flow hood or other sterile environment.

1.

Add c. 9 ml of sterile water to the plate (or slant). It is convenient to keep a supply of
20 ml screw cap bottles (wide-necked McCartney bottles or "Universals") containing 9 ml
amounts of sterile water ready for making spore suspensions. Plate cultures usually yield more
spores than slants and are routinely used, provided there is access to a laminar flow hood for
inoculating them. Slants are less prone to contamination, if this is a special problem.

2.

Scrape the surface of the culture with an inoculating loop, first with gentle
pressure and then gradually more vigorously, so as to suspend the spores. Gentle
scraping is important for agarase-producing species such as S. coelicolor, otherwise lumps ofagar medium will clog the filter. Some use a small piece of cotton wool, held in sterilised
forceps, to rub the surface of the culture gently to suspend the spores. Others collect the spores
dry by rolling glass beads over the culture surface and then suspend the spores in water whenneeded.

3.

Pour the crude suspension back into the container that held the sterile water, or
use a sterile syringe or pipette, and agitate the liquid as violently as possible ona vortex mixer for about a minute to break up spore chains.

4.

Filter the suspension through non-absorbent cotton wool, using a filter tube (seeFig. 2.1). If a piece of cotton wool was used to suspend the spores the suspension may be freeenough of mycelial fragments and pieces of agar medium that filtration is superfluous.

5.

Pour the filtered suspension into a centrifuge tube and spin for 5-10 min at c.
1000 × g to pellet the spores. The centrifugation step is to remove compounds dissolved
from the growth medium. These may include growth factors that could interfere with theselective use of auxotrophic markers, or "staling" compounds that might reduce the longevity ofthe spores, or inhibit germination.

&KDSWHU *URZWKDQGSUHVHUYDWLRQRI6WUHSWRP\FHV

Compressed
air line
A
Glass
tube
Rubber
bung

B

C

F

Cotton woolplug
A
15 x 125 mm test
tube with hole
blown in bottom

D

Cotton wool
collarB

C

D

Small cottonwool padE
E

25 x 150 mm
test tube

Fig. 2.1. Filter tube for spore suspensions. Pour crude spore suspension into innertube. Apply air pressure by holding rubber bung of compressed air supply gently
over cotton wool stopper. Filtered spore suspension will pass through nonabsorbent cotton wool filter pad into the receiving outer tube. Remove inner tube
and cotton wool collar to recover spore suspension.

6.

As soon as the centrifuge stops, pour off the supernatant. If the spore pellet is left in
the tube, even for a few minutes, after the centrifuge has stopped, it will often become detachedfrom the wall of the tube.

&KDSWHU*URZWKDQGSUHVHUYDWLRQRI6WUHSWRP\FHV

7.



Agitate the tube on the vortex mixer for a few seconds to disperse the pellet in thedrop of water remaining in the tube. It is easier to disperse the spores in a minimum
volume of liquid than in the final volume.

8.

Add sterile 20% glycerol (usually 1-2 ml for the spores from a well-sporulating
slant or plate) and briefly agitate again. Transfer the suspension to a screw capbottle for freezing at -20E; 7.5 ml "Bijou" bottles are convenient for this. If thespores are just for immediate use, they can be suspended in water; if you then decide to keep
them, add a roughly equal volume of 40% glycerol and freeze.

Plating out a Streptomyces spore suspension
Streptomyces spores (and even mycelial fragments) are quite resistant to osmotic damage and sodilutions can be made directly into distilled water, even from 20% glycerol or concentrated sucrose
solutions. When using frozen spore suspensions, take care to keep them on ice on the bench if you plan
to re-freeze them, because they rapidly lose viability on re-freezing if spore germination has begun.
We usually transfer 0.5 ml or 1 ml of suspension using conventional glass pipettes to 4.5 ml or
9 ml of water, respectively, for each successive tenfold dilution step, but smaller volumes can of course
be handled accurately using automatic micro-pipettes (such as the Pipetman manufactured by Gilson).
A fresh pipette or tip must be used for each successive dilution step.
For plating, we usually spread 0.1 ml of spore suspension on each standard Petri plate using a
glass spreader. The same spreader can be used for a series of plates at the same dilution or when movingfrom more dilute to less dilute suspensions, because any carry-over of spores is then negligible. However,use a different spreader for selective plates containing different growth factors since the carry-over oftraces of these compounds can cause background growth. Note that, in plating undiluted spore stocks in
20% glycerol, significant amounts of this potential carbon source are added to the plate; the spores mayneed to be spun down and resuspended in water if this may be a problem.
When preparing dense lawns, for example when looking for pocks (pp. 153, 255) or for making"plate-crosses" (p. 152), we normally dry the plates in a laminar flow cabinet after spreading; drying them
before-hand, so that the suspension rapidly soaks in, may lead to patchy growth. When plating for isolatedcolonies, for example of recombinants from a cross, there is usually no need to dry the plates; just incubate
them with the agar surface facing upwards for the first day or so.

Streptomyces cultures on agar
To obtain confluent cultures on agar, the organisms have to be inoculated over the entire surfaceof the medium. This is because Streptomyces colonies, in contrast to those of most moulds, will spread
only over a very limited distance within a reasonable time and so point inoculation of Streptomyces will



&KDSWHU *URZWKDQGSUHVHUYDWLRQRI6WUHSWRP\FHV

not yield a confluent culture. It is best to use a suspension of inoculum in liquid as starting material. This
need not be a carefully prepared spore suspension, although this is very suitable.
It is undesirable to propagate cultures by successive rounds of mass culture. Instead they shouldbe plated, or streaked out, and a single colony taken to start the next culture. This precaution reduces theaccumulation of revertants and the gradual loss of unselected plasmids, which is otherwise an ever-present
possibility when the variant forms grow or sporulate better than the original genotype; as a visual
manifestation of this, mass subculture very often yields obvious morphological heterogeneity.
It is not uncommon to observe heterogeneity even in confluently grown lawns. Two features inparticular have been noted. One is the appearance of tiny plaque-like holes in the aerial mycelial mat,often reminiscent of plasmid-induced pocks (p. 153). It has usually proved difficult to establish the causeof such spontaneous "self-pocking", although there are reports of material resembling defective phages
being associated with some such pocks (Ogata et al., 1992). The other feature probably results from insitu germination of spores produced on a culture that has sporulated, so that white tufts may appear, or
the whole culture surface may even become white again (Dowding, 1973). Such germination is perhaps
stimulated by the gradual diffusion of nutrients up the aerial mycelium, causing a second developmental
cycle.

Growth of mycelium on cellophane overlays, nitrocellulose filters or agar
Streptomycetes, because they grow naturally on solid surfaces, or at water/air interphases in thesoil, are usually ill-adapted to undergo their full cycle of physiological and morphological development
in liquid culture, and so they often show particular aspects of their phenotype, such as antibiotic
production or sporulation, best on a solid surface. (Much of the early development work done on apromising new antibiotic-producer in an industrial laboratory is often aimed at finding a medium, andappropriate culture conditions, that allow the organism to make its antibiotic in submerged liquid culture.)
This explains why small-scale, academic work is sometimes done with mycelium scraped from cellophanelaid on the surface of agar plates. For antibiotic production, even agar-grown cultures can be used. The
agar can be chopped up and extracted with an organic solvent, as in the series of studies on "hybrid"
polyketides described by McDaniel et al. (1995 and references therein). Water-soluble metabolites caneasily be recovered from agar plates by freezing them whole at -20o and then allowing them to thaw,
whereupon the gel collapses and up to 60% of the total volume of the agar can be poured off as a crude
aqueous solution.
Cellophane discs need to be made of normal wettable cellulose acetate film, such as dialysis
membrane, not certain kinds of non-wettable plastic. The discs can be bought as pre-cut "jampot covers",
or can be cut from larger sheets (much cheaper than dialysis tubing). They are conveniently sterilised by
autoclaving in glass Petri dishes, interleaved with wet filter paper. (They can also be autoclaved dry but
must then be wetted before use.) After the mycelium has been grown it can be scraped easily from thecellophane and harvested in water or a suitable buffer. Streptomyces hyphae can grow throughnitrocellulose filters of 0.45 µm pore size into the agar (Wolf and Schoppmann, 1989); this was turned
to advantage by Kelemen et al. (1995) who peeled off the aerial mycelium on the filters and examined
ectopic sporulation in the remaining substrate mycelium.

&KDSWHU*URZWKDQGSUHVHUYDWLRQRI6WUHSWRP\FHVIt should be noted that, at least in our hands, and on some media, growth on cellophane discs
differs somewhat from that observed when cultures are grown directly on the same medium. The amountof biomass, particularly substrate mycelium, appears to be reduced, and the onset of sporulation isaccelerated.
When aiming to obtain a series of plates developing synchronously - for example so as to harvestgroups of plates at intervals to make a RNA time curve for developmental S1 mapping - it is very important
to dry the plates carrying the cellophane discs thoroughly in the laminar flow cabinet. Otherwise damp
patches develop on the surface and hinder the development of aerial mycelium and spores.

Growth of Streptomyces mycelium in liquid
For many purposes, such as for preparing protoplasts for fusion, transformation or transfection,or for isolating plasmid or chromosomal ("total") DNA or RNA or proteins, as well as for physiological,
biochemical or fermentation studies, streptomycetes are grown as mycelium in liquid cultures started froman inoculum of spores. Since streptomycetes are highly aerobic, the cultures need to be shaken during the
incubation, usually on an orbital incubator shaker, in vessels with sufficient space for good aeration; forexample 25-50 ml cultures are grown in 250 ml Erlenmeyer flasks, or 500 ml cultures in 2 l flasks. Manystrains, including S. coelicolor A3(2) and S. lividans 66, tend to grow as rather compact masses or pellets
of mycelium unless steps are taken to encourage dispersed growth. Dispersed growth is favoured onparticular media, for example those containing polyethylene glycol, or Junlon (Hobbs et al., 1989), or ahigh concentration of sucrose and/or by the use of flasks containing baffles. A simple baffle is obtainedby inserting a stainless steel spring into an ordinary Erlenmeyer flask. For each 250 ml flask we use a 30
cm length of spring, 1.3 cm diameter coil, 19 sw gauge, marine grade stainless steel wire (p. 419). The
new springs need to be washed very thoroughly with detergent before use to remove traces of inhibitorymaterial.
The growth of mycelium for the preparation of DNA and RNA is described in Chapters 8 and 16
as parts of the protocols for extracting nucleic acids. Here we deal with the growth of organisms forphysiological purposes and for preparing protoplasts for genetic work.

Growth of Streptomyces for physiological studies
Because bacteria are small the only way in which physiological and biochemical events can bemeasured is to work with populations. For the events that are measured in the population to be a true
reflection of what is happening in the individual cell, all or most of the cells must be in the same
physiological condition. For studies of primary metabolism, this is usually considered to be achieved
when culture growth is exponential. A problem with streptomycetes is that one of the aspects that makes
them such interesting bacteria to study physiologically, their multicellular lifestyle, makes that study very
difficult. Streptomycetes grow by elongation at the tips of the mycelium and by branching. This permits
the achievement of exponential growth by young mycelium. However, physiological homogeneity is notreadily sustained over many generations because older, central parts of the mycelium may become
nutrient-limited. This problem is exacerbated by the tendency of initially separate microcolonies to&KDSWHU *URZWKDQGSUHVHUYDWLRQRI6WUHSWRP\FHV

aggregate into larger clumps. Some species, such as S. rimosus, fragment in liquid medium to give fairly
homogeneous suspensions, but many, including the genetically most studied S. coelicolor and S. lividans,form large, dense mycelial clumps unless special media or growth conditions are used.
One way to carry out biochemical and physiological tests on a homogeneous cell population is
to work with spore germlings, but the disadvantage is that very large numbers of spores are needed toobtain enough biomass for most assays. This technique was used by Riesenberg and Bergter (1984) with
S. hygroscopicus. They were concerned to obtain balanced growth (i.e. growth in which each cellularcomponent increased by the same factor) and they found that growth remained balanced for up to five
doublings. Liquid medium was used, but spores can also be germinated on cellophane discs (p. 48)
overlaid on a solid medium and the germlings scraped from the cellophane.
To obtain large amounts of biomass that can be easily manipulated the ideal solution is to growthe organisms in a liquid medium. Several techniques have been developed to allow dispersed growth of
S. lividans and S. coelicolor A3(2). There is a rule of thumb that the more complex the medium, and so
the faster the growth rate, the less likely is "clumping" of the cells. Addition of 34% sucrose, which S.
lividans and S. coelicolor A3(2) cannot catabolise, to complex media such as YEME, and the use ofstainless steel springs as baffles, allows the generation of cultures that are relatively dispersed. The
problem comes when a fully defined salts medium is required, as in physiological studies where the inputs
and outputs in a culture need to be precisely measured. Addition of Casaminoacids enhances growth rateand hence aids dispersal, but amino acids can act as nitrogen, carbon and energy sources, as in NMMP
medium (p. 413).
Several materials have been used as dispersants in liquid minimal media. Table 2.1 lists theiradvantages and disadvantages. Some kind of baffle is always needed, in addition to the dispersant, or theculture has to be vigorously stirred. Stainless steel springs are convenient as baffles, but the disadvantage
is that antifoam (p. 413) must be used to avoid frothing (Hodgson, 1982). An alternative is to use 0.3 cm
diameter glass beads (c. 100 per 250 ml flask) (Doull and Vining, 1989).
Good dispersed growth depends on the state of the inoculum. The single most important point
is to use a large inoculum and to pre-treat spores by a procedure that allows germination but notnecessarily outgrowth. It is also important that the germinated spores are disaggregated before being used
as inoculum. They can either be vortexed vigorously or subjected to a short burst of ultrasound. Several
methods of pregermination have been used (see below). The recommended inoculum is that which gives
an initial OD450 of at least 0.03 ($107 spores ml–1). This inoculum size should allow at least six doublings
before stationary phase. When inoculum size was investigated in detail it was found that there was a clear
co-operative effect: the larger the inoculum the shorter the lag phase. It was speculated that carbon
dioxide might have been responsible for this cooperativity (Hodgson, 1980).

Conditions for spore germination and reproducible exponential growth andantibiotic production by Streptomyces coelicolor
These conditions were developed (by Strauch et al., 1991) for S. coelicolor M145 (prototrophic,
SCP1–, SCP2–), and may require modification for other strains. In SMM, which contains 0.2% (w/v)
Casaminoacids, M145 grows exponentially with a doubling time of about 2.2 h; in the absence of theCasaminoacids, the doubling time is about 4 h. The cultures enter stationary phase approximately 14 h

&KDSWHU*URZWKDQGSUHVHUYDWLRQRI6WUHSWRP\FHVTable 2.1 Dispersants used in liquid minimal medium
Dispersant

Sucrose(350 g 1– w/v, 1.1M)
Hopwood et al. (1985)

Polyethylene glycol
8000(50 g 1– w/v, 6 mM)
Hodgson (1982)

Junlon(2 g 1– w/v; p.419)
Hobbs et al. (1989)

Starch–1(50 g 1 w/v)
Doull and Vining (1989)

Agar–(1 g 1 w/v)
Hobbs et al. (1989);
Magnolo et al. (1991)

Carboxy-methylcellulose(10 g 1– w/v)
C. Potter (personalcommunication)

Advantages

Disadvantages

Fully soluble to give a clear solution
for OD measurements. Not
metabolised by most streptomycetes.
Can be removed by simple washing.
Unlikely to affect most enzymes.

Very high concentrations
needed; high osmotic potential.
Cannot be autoclaved at higher
concentration and autoclavingin the presence of other media
components releases glucose
and fructose.

Fully soluble to give a clear solution
for OD measurements. Can be
autoclaved or filter-sterilised as a
double strength solution. Removedby extensive washing of cells. Not
metabolised by streptomycetes.

Causes foaming and so
antifoam is necessary. Low
concentrations inhibit someenzymes and Folin-Ciocalteu’s
reagent. Autoclaved 10%
solution acquires growth
inhibitor on standing.

Low concentrations are effective.
Not metabolised by streptomycetes.

Insoluble colloidal gel makes
OD measurement difficult, but
not impossible. Cannot be
removed. Inhibits many
enzymes and interferes with
RNA isolation.

Unlikely to affect most enzymes.
Can be removed by amylase.

Relatively high concentrations
needed. Must be autoclavedwith medium. Colloid, making
OD measurement difficult.
Can be metabolised by moststreptomycetes.

Low concentrations are effective.
Unlikely to affect most enzymes. A
colloid, but low concentrations
unlikely to affect OD measurements.
Could be removed by agarase.

Catabolised by S. coelicolor
A3(2) as carbon, nitrogen andenergy source.

Gives a clear solution for OD
measurements. Can be removed bycellulase. Unlikely to affect mostenzymes.

Catabolised as carbon andenergy source by moststreptomycetes.

&KDSWHU *URZWKDQGSUHVHUYDWLRQRI6WUHSWRP\FHV

after inoculation at an OD450 of 1.5-2.0, yielding about 1 mg ml–1 dry weight after 7-8 cell doublings. The
high spore inoculum, coiled springs and PEG provide relatively dispersed growth. A higher
concentration of Casaminoacids will give a higher final OD450, but the increase in size of the mycelial
pellets may lead to greater physiological heterogeneity. Note that the cultures are nitrogen-, not carbonlimited.
A dense spore suspension (c. 1010 spores ml-1) of S. coelicolor
20 ml volumes of 2 × YT medium (p. 414)
SMM (Supplemented Minimal Medium) (p. 413)
250 ml siliconised flasks containing coiled springs. (We use 250 ml siliconised
PYREX flasks with stainless steel coiled springs (p. 419). To minimise differences inaeration and clumping between flasks, use the same length of spring (about 26 cm, with
4.5-5 turns per 2 cm length) in each, shake at 300 rpm at 30o and check frequently;
universal containers; pipettes
Bench centrifuge, vortex mixer; water bath; incubator shaker; sonicator.

1.

Inoculate 20 ml of 2 × YT in a 250 ml flask containing a coiled spring with 20µl of the dense spore suspension. We use 20 ml because this is convenient for spinning
down in a Universal, but 50 ml can also be used. If a smaller volume is required, use a Universal
with c. 10 ml of medium, containing a short coiled spring, instead of a flask.

2.

Incubate at 30o on the shaker at 300 rpm for 6-8 h, by which time emerging germ
tubes should be visible.

3.

Spin germinated spores down in a Universal at 1000 × g for 5 min, preferably ina swing-out rotor.

4.

Decant the supernatant, add 5 ml of SMM and vortex mix. Sonicate on ice for
5 sec only to disperse aggregated germlings. We use a MSE Soniprep 150 with finesonicator tip at full power and tuned to 50%. To sterilise the tip, sonicate in absolute ethanol fora few sec and store under ethanol; air dry immediately before use.

5.

Add a portion of the sonicated cells to the desired final volume of SMM to givean OD450 of c. 0.1 measured against water. It is important to mix the samples wellimmediately before measuring the OD. SMM has an OD450 of about 0.06 (mostly from theantifoam).

6.

Mix well. Dispense 50 ml samples into fresh 250 ml flasks and incubate as
above. This should be equivalent to a spore inoculum of 4 × 106 ml–1, but remember that someof the germlings will still be clumped, and that sonication may cause reduced viability.

&KDSWHU*URZWKDQGSUHVHUYDWLRQRI6WUHSWRP\FHVGermination of Streptomyces spores
Apart from the procedure just described, several others have been used for the germination of
spores. Here are two.
Procedure 1 (This method was included in Hopwood et al. (1985) and is probably still useful.)
A dense spore suspension (c. 1010 spores ml-1), either fresh or frozen
Sterile TES buffer (0.05M, pH8)
Double strength Germination Medium (Difco yeast extract, 1%; Difco
Casaminoacids, 1%; CaCl2, 0.01M (prepared and autoclaved separately as 5M solution;
add 2 µl per ml of medium)).
Screw cap bottles; centrifuge tubes; pipettes
Bench centrifuge; vortex mixer; 50o water bath; 37o orbital shaker;
spectrophotometer set at 450 µm; cuvettes; microscope.

1.

Pellet the spores on the bench centrifuge and re-suspend in e.g. 5 ml of TES
buffer. It is important to remove glycerol from the spore suspension, otherwise it may impair
spore viability during heat-shocking.

2.

Heat shock the spores (e.g. 50o for 10 min) and cool under cold tap. Various
combinations of time and temperature are suitable: e.g. 45o for 15 min or 50o for 10 min for S.
coelicolor; other strains may need different conditions.

3.

Add an equal volume of double strength Germination Medium. Incubate at 37owith shaking (300 rpm) for 2-3 h. Malt extract is not present; it hampers synchronous
germination. About 50% of spores should have microscopically visible germ tubes at the endof this stage.

4.

Pellet the germinated spores in the bench centrifuge and re-suspend in water or
TES buffer. Agitate on vortex mixer to disperse the clumps. Quite vigorous
agitation (or even a short burst of ultra-sound) is needed to disperse the clumps caused byadhesion of the germ tubes.

5.

Inoculate growth medium to OD450 of 0.03-0.05. A dense inoculum is needed: thespores from one or more slants (c. 2 × 107 spores) will be required for each 100 ml culture.
Spores treated in this way should establish exponential growth in c. 5 h or less in a chemicallydefined medium, depending on the carbon source.

&KDSWHU *URZWKDQGSUHVHUYDWLRQRI6WUHSWRP\FHV

Procedure 2 (From Mikulik et al., 1984)
Streptomyces spores
Chemically defined germination medium: 2.5mM of each of 20 amino acids; 0.01%
each of adenosine, guanosine, thymidine, uridine and cytidine; 0.02M potassium
phosphate, pH7.0. Complex germination medium: Casaminoacids, Difco, 0.2%; Yeast
extract, Difco, 1.0%; KH2PO4, 0.68%; (NH4)2SO4, 0.2%; MgSO4, 0.02%; CaCl2,
0.001%; pH7.0
Ultra-Turrax Turbomixer (p. 420) or High Speed Homogeniser System (p. 419) withspeed regulation from 0-20,000 rpm; reciprocating shaker

1.

Homogenise 1 g of spores in 2.5 ml of cold water at 20,000 rpm in the UltraTurrax Turbomixer at 0o (5 times 45 sec run followed by 30 sec cooling).
Homogenised spores prepared as above can be mixed with an equal volume of 40% (v/v)
glycerol and stored at -70o. The spores remain viable for several months of storage, andgerminate synchronously.

2.

Inoculate 0.5 ml of the homogenised spore suspension into 50 ml of germination
medium in a 500 ml flask and incubate on a shaker at 28o. After 3 h of cultivation,
outgrowth of germ tubes is microscopically visible. Synchronous germination takes placethrough a sequence of time-ordered events. RNA and protein synthesis start during the first 5
min and net DNA synthesis at 60-70 min of germination.

Preparation of aerial mycelium and spores separately
This procedure was supplied by K. Mikulik. It was developed for physiological studies ofsporulation and spore germination in S. granaticolor (St’astná, 1977; Mikulik et al. 1977, 1984; St’astná
and Janda, 1983).
Streptomyces mycelium
Sporulation agar (Yeast extract, 0.4%; malt extract, 1.0%; glucose 0.4% Oxoid No 4
Agar, 2.0%; pH7.0)
Cellophane discs (see p. 48)
Ultra-Turrax Turbomixer (p. 420) or High Speed Homogeniser System (p. 419) withspeed regulation 0-20,000 rpm; laminar flow hood; preparative centrifuge; vortex
mixer

1.

Pour a 7 mm layer of sporulation agar (45 ml in a 9 cm dish) in each Petri dishand dry in a laminar flow hood for 30-40 min.

&KDSWHU*URZWKDQGSUHVHUYDWLRQRI6WUHSWRP\FHV

2.

Lay a cellophane disc on the agar and remove air bubbles with a sterile spreader.

3.

Inoculate 3 ml of a culture of vegetative mycelium, spread the cells over thecellophane, and incubate at 28o. Then proceed to either 4a or 4b.

Aerial mycelium
4a.

After 48 h growth, strip the white layer of aerial mycelium and residues ofvegetative cells from the cellophane with a spatula.

5a.

Homogenise stripped cells in ice-cold water at 15,000 rpm for 30 sec in the
Ultra-Turrax Turbomixer.

6a.

Spin the mixture at 7,000 × g for 10 min at 4o. Vegetative hyphae sediment at
the bottom of the tube while hydrophobic aerial mycelium floats in the
supernatant.

7a.

Transfer the supernatant fraction to a centrifuge tube and remove water from thepellet by suction using a Pasteur pipette with its tip wrapped in glass wool.

8a.

Repeat steps 5a-7a.

9a.

Freeze the white aerial mycelium in liquid nitrogen and store at -70o.

Spores4b.

After 14 days growth, scrape the grey hydrophobic spores from the surface of thecellophane.

5b.

Transfer the harvested material to cold sterile distilled water and wash byvortexing for 1 min.

6b.

Spin the mixture at 7,000 × g for 10 min. Hydrophobic spores may float in the waterphase.

7b.

Collect the suspension of spores and remove water by suction as above.
Spores can be stored either frozen at -20o or mixed with an equal volume of 40% glycerol
and kept at -70o.

&KDSWHU *URZWKDQGSUHVHUYDWLRQRI6WUHSWRP\FHV

Sporulation of Streptomyces in submerged cultures
Although the typical reproductive cycle of Streptomyces takes place on solid surfaces, somestrains can sporulate in submerged liquid culture. This feature was first observed with S. griseus(Kendrick and Ensign, 1983) and has led to the use of certain strains of this species for studying
physiological events associated with sporulation (Ochi, 1987; McCue et al., 1992). Subsequently, otherspecies have been found to sporulate in liquid medium under suitable conditions: S. viridochromogenes(Koepsel and Ensign, 1984); S. roseosporus (Huber et al., 1987); S. acrimycini and S. albus (Daza et al.,1989); S. venezuelae (Glazebrook et al., 1990); S. albidoflavus (Lee and Rho, 1993). In most or all
respects, the spores from such cultures seem to resemble typical aerial spores of the same strain. In
contrast, only "spore-shaped bodies" lacking the normal resistance of spores to moist heat were produced
by S. coelicolor A3(2) and S. lividans 66 (Daza et al., 1989). When the S. coelicolor whiG gene,encoding a sporulation-specific sigma factor, was cloned at high copy, sporulation occurred in submergedliquid culture, but not to an extent usable for large-scale study (Chater et al., 1989). Recently, apparently
normal heat-resistant spores were found in submerged growth of S. coelicolor cultures provided they
were not shaken (E. Takano, personal communication).

Preparation of protoplasts from Streptomyces lividans 66 and S. coelicolor A3(2)Protoplasts are used both for fusion and for transformation and transfection. This protocol
describes their preparation up to the first wash (see the appropriate technique for their further handling:pp. 157, 232, 301 respectively). It was originally based on the work of Okanishi et al. (1974) and wasadapted and optimised for high transformation frequency by Bibb et al. (1978) and Thompson et al.
(1982). Many different Streptomyces species can be protoplasted and regenerated using this procedure.
Sometimes the age and/or physiological state of the mycelium is important. For example, thebest transformation or transfection frequencies in S. lividans are obtained with protoplasts prepared frommycelium grown to "late exponential" phase (36-40 h), by which time the culture develops a pink/orange
tinge, whereas for transfection of S. parvulus protoplasts needed to come from much younger mycelium(c. 18 h). The details may also depend on the particular genotype: many morphological mutants are
particularly idiosyncratic.
It is worth emphasising that a poor spore inoculum leads to a bad batch of mycelium, and thisin turn results in poor protoplasts and a low transformation frequency.
Streptomyces spores, fresh suspension (c. 109 spores ml–1) or frozen stock in 20%
glycerol (pp. 44-47)
YEME medium (containing 34% sucrose, 5 mM MgCl2, 0.5% glycine) (p. 412)
P buffer (p. 415)
10.3% sucrose
Lysozyme solution (1 mg ml-1 in P buffer, filter sterilised)
250 ml baffled Erlenmeyer flasks (see p. 419 for springs); 20 ml screw cap bottles;
pipettes (25 ml, 10 ml, 5 ml, 1 ml); filter tubes plugged with cotton wool (p. 46);
plastic tubes (disposable sterile tubes, e.g. 16 × 100 mm, round bottom, polycarbonate

&KDSWHU*URZWKDQGSUHVHUYDWLRQRI6WUHSWRP\FHVtest tubes, Cat. No. 142AS made by Sterilin, Teddington, Middlesex. These are muchpreferable to tubes which are washed and re-used because traces of detergent are thereby
avoided.); pro-pipettes; 30o orbital incubator; microscope; bench centrifuge;
30o water bath; refrigerator; -20E or -70E freezer.

1.

Add 25 ml YEME medium to a baffled flask. Add c. 0.1 ml spore suspension
and required growth factors. Incubate 36-40 h at 30o in an orbital incubator
shaker. Cultures of S. lividans and S. coelicolor are ready for harvesting when they start toproduce red pigment

2.

Pour culture broth into a 20 ml screw cap bottle and spin in the bench centrifuge(e.g. 1000 × g, 10 min). Before centrifugation, examine the culture for contamination byunicellular bacteria, usually indicated by turbidity: the Streptomyces mycelium will fall to thebottom in a few moments, while unicellular contaminants remain suspended. In case of doubt,
check in the microscope.

3.

Discard the supernatant.

4.

Re-suspend pellet in 15 ml 10.3% sucrose and spin in bench centrifuge as above.
Discard supernatant. Occasionally, mycelium will not pellet from the growth medium
containing 34% sucrose on centrifugation; in this case add 10.3% sucrose or water to reduce thedensity of the medium and so facilitate pelleting. Take care in pouring off the supernatant fromthe loose pellet.

5.

Repeat step 4. The mycelial pellet, without added liquid, can be frozen at -20o before
resuspension.

6.

Resuspend mycelium in 4 ml lysozyme solution; incubate at 30o, 15-60 min. For
most purposes, lysozyme is dissolved in P buffer; use of L buffer (p. 416) gives more efficienttransformation of S. lividans by plasmid DNA. Lysozyme treatment times vary from strain tostrain, e.g. 15 min is usually long enough for S. lividans whereas S. coelicolor requires 60 min;other strains may take longer. Protoplast formation can be monitored using the phase-contrast
microscope. For S. lividans the time (15-60 min) is not critical but protoplasts of other strains
may lyse (especially in L buffer) if left too long. If this happens, P buffer + lysozyme can besubstituted for L buffer and lysozyme treatments may be done at a lower temperature (e.g. room
temperature) or for a shorter time.

7.

Draw in and out of a 5 ml pipette three times and incubate for a further 15 min.

8.

Add 5 ml P buffer. Repeat step 7. This helps to free protoplasts from the mycelium sothat they will pass through the cotton wool filter used in step 9. At least with S. lividans, it is&KDSWHU *URZWKDQGSUHVHUYDWLRQRI6WUHSWRP\FHV

possible to obtain transformants with unfiltered material, but the washing (steps 9-10) is stillneeded to remove lysozyme.

9.

Filter protoplasts through cotton wool (using a filter tube: p. 46) and transfer toa plastic tube.

10.

Sediment protoplasts gently by spinning in a bench centrifuge (e.g. 1000×g,
7 min).

11.

Discard supernatant and suspend protoplasts in 1 ml P buffer. At this and any othersteps when pelleted protoplasts are to be re-suspended, re-suspend in the remaining drop of
liquid by tapping the side of the tube repeatedly with a finger until the protoplasts are dispersed
to form a creamy suspension, then add the suspending P buffer (otherwise the protoplast pellet
is difficult to disperse). Avoid vortexing, which induces foaming and consequent lysis.

12.

See one of the three techniques (protoplast fusion p. 157; transformation of
protoplasts (p. 232) or transfection of protoplasts, p. 301) for further
manipulations. To freeze the protoplasts for storage, place samples of the
protoplast suspension in small plastic tubes, close them and place them in ice ina plastic beaker. Place the beaker at -70o overnight. Free the frozen protoplasts
in their tubes from the ice and store at -70o. To thaw, shake the frozen tube
under running warm water (i.e. freeze slowly, thaw quickly). To assess the
proportion of non-protoplasted units in the suspension, samples can be diluted in parallel in P
buffer and in dilute detergent (e.g. 0.01% SDS) and plated on regeneration plates. Any colonies
arising after dilution in detergent are likely to have arisen from non-protoplasted units.

Preservation of Streptomyces strains and phages
Strains that are needed only for short-term use can be kept as cultures on agar slants or in sealed
Petri dishes at 4o, where they will usually remain viable for months, provided they sporulate before
refrigeration. Non-sporulating cultures need to be sub-cultured frequently.
A very convenient method for keeping strains for longer periods is to prepare spore suspension
in 20% glycerol (see "making a spore suspension", p. 44) and freeze them at -20o. Even with repeatedthawing and re-freezing for sampling purposes, viability remains high for years, provided the spores are
not allowed to remain at room temperature for extended periods before re-freezing: when you work withthem on the bench, keep them in an ice bucket. Another procedure, using mycelium instead of sporesand employed routinely at the Eli Lilly Company (E. T. Seno, personal communication), is to grow thestrains in a complex medium such as Tryptic Soy Broth (Difco) for two days, add an equal volume of
20% glycerol/10% lactose, and store in the vapour phase of liquid nitrogen. Again the cultures remain
viable for years.

&KDSWHU*URZWKDQGSUHVHUYDWLRQRI6WUHSWRP\FHVFor long-term storage, and as a back-up to frozen spore suspensions, lyophils (on filter paper
wicks) are prepared as described below.
Phages are usually kept in suspension in Difco Nutrient Broth (DNB; p. 412) at 4o in the dark.
Lyophils of phages may be prepared by dipping filter paper wicks into broth suspensions and proceedingin the same way as for spores.
Preparation of lyophils (From Hopwood and Ferguson, 1969)
Fresh slant cultures or spore suspensions in 20% glycerol
Skim milk (10% solution of Difco Bacto Skim Milk powder sterilised by autoclaving
at 121o, 5 min; this shortened time is to prevent caramelisation)
Glass ampoules or bottles previously sterilised by autoclaving; filter paper wicks(strips of Whatman 3MM paper, cut to fit the container); inoculating loop; long-jawed
forcepsFreeze dryer, either with manifold to accept ampoules or chamber to containbottles; gas flame, ampoule tester
Alternative I (using a freeze dryer with manifold)1.

Add 1 ml skim milk to a slant culture and scrape the surface to make a crude
spore suspension. If working from an existing spore suspension in glycerol, add
0.05 ml of dense spore suspension to 0.5 ml skim milk or, if the suspension is notvery dense, centrifuge it and re-suspend the spores in 0.5 ml skim milk.

2.

Using forceps, place several sterile filter paper wicks in the spore suspension andallow them to become saturated.

3.

Using long-jawed forceps (e.g. stainless steel angled aural forceps) place one
wick in each ampoule.

4.

Constrict the neck of the ampoule to about a quarter of its original diameter bydrawing it out in a fine flame, at a point c. 30 mm from the open end, and placeeach ampoule, after constriction, on the manifold of the freeze dryer.

5.

When the manifold is full, switch on the freeze dryer and leave it pumping until
a low reading (<10 mm mercury) is obtained on the vacuum gauge (this takes
several hours, so overnight pumping is convenient).

6.

Seal off each ampoule in turn and remove it from the freeze dryer.



7.

&KDSWHU *URZWKDQGSUHVHUYDWLRQRI6WUHSWRP\FHV

Each ampoule (or a sample of them) should be tested with a hand-held vacuum
tester to ensure that they contain a good vacuum.

Alternative II (using a freeze dryer with chamber and press for closing bottles: e.g. from Edwards High
Vacuum Ltd. p. 419)1.

Previously sterilise the bottles, each containing (say) 4 filter paper wicks, withthe rubber stopper loosely in place.

2.

Prepare a spore suspension in skimmed milk as in (I, step 1).

3.

Pipette 0.2 ml of spore suspension into each container so that the wicks soak itup.

4.

Replace the stopper loosely and place the container in the chamber of the freeze
dryer.

5.

Pump for several hours as in (I, step 5).

6.

Close the containers by screwing down the press, remove them from the chamberand crimp on a metal closure.

7.

Test the ampoules as in (I, step 7).

Growing a culture from the lyophil
To grow a culture from a lyophil, open the container, remove the wick (or one of them if several
were included in the same container, then re-evacuate and re-seal it) and place it on a plate of agar
medium (CM is better than MM and may be better than R2YE). When it has soaked up some moisture,
move it about over the agar surface to distribute the inoculum. Incubate.

Bibb MJ, Ward JM, Hopwood DA (1978) Transformationof plasmid DNA into Streptomyces at high frequency. Nature,274, 398-400.
Chater KF, Bruton CJ, Plaskitt KA, Buttner MJ, Méndez
C, Helmann J (1989) The developmental fate of S. coelicolor
hyphae depends crucially on a gene product homologous withthe motility sigma factor of B. subtilis. Cell, 59, 133-143.
Daza A, Martin JF, Dominguez A, Gil JA (1989)
Sporulation of several species of Streptomyces in submerged
culture after nutritional downshift. J.Gen.Microbiol., 135,2483-2491.
Doull JL, Vining LC (1989) Culture conditions promoting
dispersed growth and biphasic production of actinorhodin in
shaken cultures of Streptomyces coelicolor A3(2). FEMS

Microbiol.Lett., 65, 265-268.
Dowding JE (1973) Characterization of a bacteriophagevirulent for Streptomyces coelicolor A3(2). J.Gen.Microbiol.,76, 163-176.
El-Nakeeb MA, Lechevalier HA (1963) Selective isolationof aerobic actinomycetes. Appl. Microbiol., 11, 75-77.
Glazebrook MA, Doull JL, Stuttard C, Vining LC (1990)
Sporulation of Streptomyces venezuelae in submerged
cultures. J.Gen.Microbiol., 136, 581-588.
Hayakawa M, Nonomura H (1987) Humic acid-vitamin
agar, a new method for the selective isolation of soil
actinomycetes. J.Ferment.Technol., 65, 501-509.
Herron PR, Wellington EMH (1990) New method for the
extraction of streptomycete spores from soil and application

&KDSWHU*URZWKDQGSUHVHUYDWLRQRI6WUHSWRP\FHV
to the study of lysogeny in sterile amended and nonsterilesoil. Appl.Env.Microbiol., 56, 1406-1412.
Hobbs G, Frazer CM, Gardner DCJ, Cullum JA, Oliver
SG (1989) Dispersed growth of Streptomyces in liquid
culture. Appl.Microbiol.Biotechnol., 31, 272-277.
Hodgson DA (1980) Carbohydrate utilization in
Streptomyces coelicolor A3(2). Ph.D.Thesis, University of
East Anglia, Norwich.
Hodgson DA (1982) Glucose repression of carbon source
uptake in Streptomyces coelicolor A3(2) and its perturbationin mutants resistant to 2-deoxyglucose. J.Gen.Microbiol.,
128, 2417-2430.
Hopwood DA, Ferguson HM (1969) A rapid method forlyophilizing streptomyces cultures. J.Appl.Bact., 32, 434-436.
Hopwood DA, Bibb MJ, Chater KF, Kieser T, Bruton CJ,
Kieser HM, Lydiate DJ, Smith CP, Ward JM, Schrempf
H (1985) Genetic Manipulation of Streptomyces. A
Laboratory Manual. John Innes Foundation, Norwich.
Hsu SC, Lockwood JI (1975) Powdered chitin agar as aselective medium for enumeration of actinomycetes in waterand soil. Appl. Microbiol., 29, 422-426.
Huber FM, Piper RL, Mertz FP (1987) Sporulation ofStreptomyces roseosporus in submerged culture.
J.Ind.Microbiol., 2, 235-241.
Kelemen GH, Plaskitt KA, Lewis CG, Findlay KC,
Buttner MJ (1995) Deletion of DNA lying close to the glkA
locus induces ectopic sporulation in Streptomyces coelicolor
A3(2). Mol.Microbiol., 17, 221-230.
Kendrick KE, Ensign JC (1983) Sporulation ofStreptomyces griseus in submerged culture. J.Bacteriol., 155,357-366.
Koepsel R, Ensign JC (1984) Microcycle sporulation ofStreptomyces viridochromogenes. Arch.Microbiol., 140, 9-14.
Lee KJ, Rho YT (1993) Characteristics of spores formed bysurface and submerged cultures of Streptomyces albidoflavus
SMF301. J.Gen.Microbiol., 139, 3131-3137.
Magnolo SK, Leenutaphong DL, DeModena JA, Curtis
JE, Bailey JE, Galazzo JL, Hughes DE (1991)
Actinorhodin production by Streptomyces coelicolor andgrowth of Streptomyces lividans are improved by theexpression of a bacterial hemoglobin. Bio/Technology, 9,473-476.
McCue LA, Kwak J, Babcock MJ, Kendrick KE (1992)
Molecular analysis of sporulation in Streptomyces griseus.
Gene, 115, 173-179.
McDaniel R, Ebert-Khosla S, Hopwood DA, Khosla C(1995) Rational design of aromatic polyketide natural
products by recombinant assembly of enzymatic subunits.
Nature, 375, 549-554.
Mikulík K, Janda I, Masková H, St'astná J, Jiranová A(1977) Macromolecular synthesis accompanying thetransition from spores to vegetative forms of Streptomyces
granaticolor. Folia Microbiol., 22, 252-261.

Mikulik K, Janda I, Weiser J, St'astná J, Jiránova A(1984) RNA and ribosomal protein patterns during aerial
spore germination in Streptomyces granaticolor.
Eur.J.Biochem., 145, 381-388.
Mullins PH, Gurtler H, Wellington EMH (1995) Selective
recovery of Streptosporangium fragile from soil by indirect
immunomagnetic capture. Microbiol., 141, 2149-2156.
Nonomura H, Hayakawa M (1988) New methods for theselective isolation of soil actinomycetes. In Okami, Y.,
Beppu, T. and Ogawara, H. (eds.), Biology of Actinomycetes’88. Japan Scientific Societies Press, Tokyo, pp. 288-293.
Ochi K (1987) Changes in nucleotide pools during
sporulation of Streptomyces griseus in submerged culture.
J.Gen.Microbiol., 133, 2787-2795.
Ogata S, Matsubara H, Harada Y, Umeda A (1992)
Formation of spontaneously developing pocks and productionof phage taillike particles in thiostrepton-producing
Streptomyces laurentii ATCC 31255. Actinomycetol., 6, 2932.
Okanishi M, Suzuki K, Umezawa H (1974) Formation andreversion of streptomycete protoplasts: cultural conditions
and morphological study. J.Gen.Microbiol., 80, 389-400.
Riesenberg D, Bergter F (1984) Establishment of a system
for analysis of balanced and unbalanced growth ofStreptomyces hygroscopicus. J.Gen.Microbiol., 130, 25432548.
St'astná J (1977) A method of rapid wetting and synchronous
germination of streptomycete spores. Folia Microbiol., 22,
137-138.
St'astná J, Janda I (1983) Production, long termpreservation and synchronous germination of aerial spores ofStreptomyces. J.Microbiol.Meth., 1, 267-273.
Strauch E, Takano E, Baylis H, Bibb MJ (1991) The
stringent response in Streptomyces coelicolor A3(2).
Mol.Microbiol., 5, 289-298.
Thompson CJ, Kieser T, Ward JM, Hopwood DA (1982)
Physical analysis of antibiotic-resistant genes from
Streptomyces and their use in vector construction. Gene, 20,
51-62.
Wellington EMH, Toth IK (1994) Actinomycetes. Methodsof Soil Analysis, Part 2. Microbiological and Biochemical
Properties-SSSA Book Series, no. 5. Soil Science Society ofAmerica, Madison, pp. 269-290.
Wellington EMH, Al-Jawadi M, Bandoni R (1987)
Selective isolation of Streptomyces species-groups from soil.
Dev. Ind. Microbiol., 28, 99-104.
Wipat A, Wellington EMH, Saunders VA (1994)
Monoclonal antibodies for Streptomcyes lividans and theiruse for immunomagnetic capture of spores from soil.
Microbiol., 140, 2067-2076.
Wolf H, Schoppmann H (1989) Streptomycetes can growthrough small filter capillaries. FEMS Microbiol.Lett., 57,
259-264.

&KDSWHU *URZWKDQGSUHVHUYDWLRQRI6WUHSWRP\FHV

m03tkc.wpd 20 April 2000 Tobias Kieser

Chapter 3

Microscopical methods
CONTENTSLight microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Cellophane cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
Coverslip or slide cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
Impression preparations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
Staining for light microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
DAPI Staining of Streptomyces mycelium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Staining for respiratory activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
Immunofluorescent staining of Streptomyces mycelium . . . . . . . . . . . . . . . . . . 66
Electron microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
Electron microscopy of thin sections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
Sectioning of Streptomyces colonies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
Staining thin sections of Streptomyces colonies for glycogen . . . . . . . . . . . . . . . 72
Spatial visualisation of gene expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

A full description of light and electron microscopical procedures is outside the scope of theManual. However, since microscopical observations are crucial to some genetical studies, such as
establishing the phenotypic consequences of mutations in morphogenetic genes (Chater, 1972;
McCormick et al., 1994; Bruton et al., 1995; Keleman et al., 1995) or of cloning them at enhanced copy
number (Chater et al., 1989), a brief coverage of these techniques is included here.
Cytological and microscopical procedures for Streptomyces are not in general different fromthose used for other types of biological materials; the main challenge is to prepare Streptomyces
mycelium and spores for microscopy in such a way that good quality observations and pictorial records
can be obtained without disrupting the relationships between the various phases of development in acolony, or losing track of the origin of particular cells in, for example, electron micrographs of thinsections.

Light microscopy
The chief classes of preparations for either phase-contrast observations or the study of stainedorganisms have been cellophane, coverslip/slide, or impression preparations.

&KDSWHU0LFURVFRSLFDOPHWKRGVCellophane cultures
Small squares of cellophane, previously sterilised by autoclaving in a Petri dish in water (p. 48),are placed on the surface of agar and inoculated with a spore suspension. At intervals during
development, cellophane squares are removed and mounted between a slide and a coverslip for direct
phase-contrast examination (Hopwood, 1960) or for fixing and staining (Hopwood and Glauert, 1960).
Good optical conditions are difficult to achieve once growth becomes crowded, and are not perfect even
before that, so this method is in general suitable only for early stages of growth. However, a complete
developmental cycle may be achieved under starvation conditions (Hopwood and Glauert, 1960).
Coverslip or slide cultures
In order to try to overcome the problems of observing the aerial phase of Streptomyces colonies
at high resolution without disturbing the spatial organisation of the hyphae, Hopwood (1960) grewmicrocolonies of S. coelicolor in a thin agar layer on a coverslip in a moist chamber and madeobservations on them by inverting the coverslip over a slide, with lens tissue spacers, and filling the airspace with water to achieve reasonable phase-contrast conditions. Chater (1972) studied S. coelicolor
whi mutants by growing them in the acute angle between a coverslip inserted obliquely into an agar plate
and the surface of the medium. When the coverslip was carefully removed, adhering mycelium remained
attached to it. This method usually allows visualisation of aerial hyphae still attached to substrate
mycelium. Slides, rather than coverslips, can be used in a similar way (see below under“Immunofluorescent staining”, p. 66).
Impression preparations
To study the later stages of sporulation, by either phase-contrast microscopy (Hopwood, 1960;
Hopwood et al., 1970; Chater, 1972; Potúûková et al., 1995) or after staining (Hopwood and Glauert,1960), a simple procedure is to press a coverslip onto the surface of a colony, whereupon it picks upaerial hyphae, as well as spores if they are produced. When the coverslip is inverted and mounted in adrop of water or other suitable medium on a slide, good optical conditions are achieved. McCormick andLosick (1996) used 50% glycerol to mount impression preparation, with the advantages of very slow
drying out, long specimen life (they can be kept in the fridge or freezer), better wetting of thehydrophobic aerial mycelium and spores, and reduced Brownian movement.
Staining for light microscopy
Procedures under this heading include generalised staining methods designed to reveal thegeneral form of the mycelium (as an alternative to phase-contrast microscopy), and stains that are
specific, to a greater or lesser extent, for particular cellular components or functions. In the former
category are toluidine blue (G. Kelemen, personal communication), gentian violet and methylene blue
(Drouin, 1996). All that is required is a suitable fixative for the mycelium (e.g. 17.5% acetic acid),followed by staining in a dye solution for a short time (e.g. 30 sec), washing in water, and drying of the
preparation.

&KDSWHU0LFURVFRSLFDOPHWKRGVWe include two procedures for specific staining. The first is the use of DAPI as a stain for DNA
and thus for revealing the nucleoids. Such fluorochromes are more sensitive and/or more specific thanthe “classical” Feulgen and other chromosome stains (e.g. Hopwood and Glauert, 1960). The second
procedure is designed to detect respiratory activity as an index of metabolic competence.
DAPI Staining of Streptomyces mycelium (provided by K. Kendrick, based on the E. coli procedureof Hiraga et al.,1989)
Streptomyces mycelium
Methanol (100%)
Poly-L-lysine solution (5 µg ml–1 in water)
DAPI (40,6-diamidino-2-phenylindole) solution (5 µg ml–1 in 0.85% NaCl)
Slides; coverslips
Fluorescence microscope1.

Spread 5-10 µl of cell culture on an ethanol-cleaned slide, or make an impression
preparation by placing a cover-slip on a colony. A clean surface is important forsmooth spreading.

2.

Air dry the sample.

3.

Cover the slide surface with methanol and leave at room temperature until dry.

4.

Repeat step 3.

5.

Wash the slide very gently by submerging it in a beaker of distilled water. Air
dry the slide.

6.

Cover the sample area with 20-30 µl of poly-L-lysine solution. Air dry.

7.

Shortly (5-10 min) before observing the sample by fluorescence microscopy,
apply 5 µl of DAPI solution and cover with a coverslip.
Alternative to 6 and 7

6.

Place a coverslip carrying fixed cells on a slide coated with: 1% agarose; 0.85%
NaCl; 5µg ml–1 DAPI.

&KDSWHU0LFURVFRSLFDOPHWKRGV

Staining for respiratory activity
This procedure (from Pons et al., 1998) uses a solution of a tetrazolium salt, which can act as
an electron acceptor (as an alternative to molecular oxygen) to produce crystals of insoluble formazan;
these accumulate as visible granules inside the mycelium.
Streptomyces mycelium
Solution of INT [2(p-iodophenyl)-3-(p-nitro-phenyl)-5-phenyl-tetrazolium chloride],(0.2 g l–1 INT, 53.31 g l–1 KH2PO4, 105.8 g l–1 KH2PO4, 0.9 g l–1 glucose)
Methylene blue, 0.1%
Slides, coverslips
Phase-contrast microscope1.

Cover the specimen with the INT solution and incubate at 28o for 30 min.

2.

Wash in phosphate buffer.

3.

Counterstain with methylene blue for 1 min.

4.

Examine in the phase-contrast microscope with a green filter.

Immunofluorescent staining of Streptomyces mycelium
Immunofluorescence is a very sensitive method for visualising proteins at the subcellular level;
it is much simpler, as well as more sensitive, than electron microscopy of immunogold-labelled cells, butof course has a lower spatial resolution. This protocol was provided by Julie Schwedock, Harvard
University (Schwedock et al., 1997). It is based on a procedure developed for E. coli and B. subtilis(Harry et al., 1995; Pogliano et al., 1995, 1997). The procedure was optimised for visualising the FtsZ
protein of S. coelicolor, using a rabbit anti-FtsZ antibody and a goat anti-rabbit secondary antibody
coupled to a fluorescent dye, e.g. fluorescein isothiocyanate (FITC) or Cy3. Two or more antigens canbe visualised by using different such dyes. Counterstaining with propidium iodide (for nucleoids), and/or
fluorescent dye-coupled wheat germ agglutinin (WGA) (for oligomers of N-acetylglucosamine andmuramic acid released by lysozyme treatment of Gram-positive cell walls) can be included. This
protocol will need adjustment for other proteins and species; the chief variables are probably the age ofthe mycelium, the strength of the lysozyme treatment, the amount of glutaraldehyde in the fixative, andthe concentration of antibody. The non-specific background tends to vary from strain to strain; also, cells
that are completely lysed by lysozyme tend to have a higher background than more intact cells. Suitable
controls would include: omission of the primary antibody; replacement of the primary antibody by preimmune serum; and, if available, a null-mutant for the relevant protein. Two general references on
immunofluorescence are Larison (1992) and Pringle et al. (1991). See Haugland (1996) for information
about fluorescent reagents and specific details of the products of Molecular Probes Inc.

&KDSWHU0LFURVFRSLFDOPHWKRGVStreptomyces spore suspension or mycelial fragments
Agar medium (e.g. minimal medium with glucose replaced by mannitol),molten and held at c. 50o
PBS (10 mM sodium phosphate buffer, pH7.4; 150mM NaCl, 15mM KCl)
Glutaraldehyde fixative (per slide: 3.3 ml PBS; 0.7 ml 16% paraformaldehyde;
0.7 µl 25% glutaraldehyde) (TOXIC)
Lysozyme solution: 2 mg ml–1 in GTE (50mM glucose, 10mM EDTA, 20mM Tris,
pH7.5)
Bovine serum albumin solution, 2% in PBS
Primary antibody to detect protein of interest
Fluorescent-coupled secondary antibody or antibodies (from Jackson Immunoresearch Labs, p.418)
Counterstains, e.g.: propidium iodide (1 mg ml–1 aqueous stock); FITC-coupled
or AMCA-S-coupled WGA (wheat germ agglutinin) (10 mg ml–1 stock)
Slow Fade Kit (from Molecular Probes, p. 419)
Sterile microscope slides and plastic Petri dishes; forceps; long coverslips;
PAP hydrophobic marking pen (e.g. from Electron Microscopy Sciences,
p. 419)Fluorescence microscope (e.g. Olympus, with the following filter sets: UMWIBA for visualising FITC-conjugates; U-MWG for propidium iodide orCy3-conjugates; U-MNUA for AMCA-S conjugates)

1.

Pour molten agar medium into a Petri dish. Place a sterile microscope slide withsterilised forceps (both can be sterilised by flaming with ethanol) into the agar,
long side down, at a 45o angle with the two top corners just resting on the sidesof the dish. Allow the agar to solidify. Do not allow agar to contaminate the protruding
part of the slide: you want the mycelium to grow onto the glass surface itself. A second pair ofsterile forceps may help to position the slide.

2.

With a pipette, inoculate a spore suspension (about 50 µl of a suspension of c.
2 x 107 spores ml–1) or a suspension of broken up mycelial fragments (50-200µl depending on the concentration of fragments) into the acute angle between
slide and agar surface. Scrape the solidified meniscus of agar medium off theslide with the pipette during inoculation; again, this is to allow the growing
mycelium to reach the glass surface.

3.

Incubate at 30o (right-side up!). For FtsZ, 32-40 h was appropriate; other proteins mightneed different times.

&KDSWHU0LFURVFRSLFDOPHWKRGV

4.

Pull the slide from the medium, leaving as much agar behind as possible without
disturbing the mycelium attached to the glass. Wipe off the back of the slidewith a tissue. Place the slide in an empty Petri dish.

5.

Add glutaraldehyde fixative to the Petri dish and leave at room temperature for
15 min.

6.

Gently pipette off the fixative. Wash twice with PBS by gentle pipetting. Dry
the back of the slide and the Petri dish with paper towel and allow the surface ofthe slide to dry thoroughly in the air (15-20 min).

7.

Clean oval areas around the mycelial growth with cotton wool swabs and drythese areas with dry swabs. Draw oval outlines with a hydrophobic PAP
marking pen and allow the ink to dry for a few minutes.

Ink outline
Mycelium

Multiple areas allow different staining, or different antibodies, to be used on thesame slide.
8.

Gently add PBS to the areas within the hydrophobic outlines and leave 5 min to
rehydrate the mycelium. Use, say, 300 µl for a full-size oval, 150 for a half-size,and less for smaller ones. (In subsequent washing steps, these volumes can be
reduced by about one-third.)

9.

Gently remove the PBS (for example by aspiration with a Pasteur pipette).

10.

Add lysozyme solution and leave for 1 min. Remove the lysozyme solution andwash with PBS. 2 mg ml–1 lysozyme worked best for S. coelicolor grown on minimal medium
with mannitol. Depending on strain or medium the lysozyme concentration may need to be
reduced to as little as 0.1 mg ml–1; or alternatively the treatment time might need to be as long as
5 min.

&KDSWHU0LFURVFRSLFDOPHWKRGV



11.

Add bovine serum albumin solution and leave for 5 min for blocking to takeplace.

12.

Add an appropriate antibody in BSA/PBS and keep at 4o overnight covered. To
hinder evaporation, add a moist tissue to the edge of the Petri dish. The appropriate
antibody concentration must be determined empirically; it is usually 10 times higher than for aWestern blot.

13.

Remove the antibody solution and wash 10 times with PBS.
The remaining steps need to be carried out in low light.

14.

Add a fluorescent-coupled secondary antibody and other stain (e.g. propidium
iodide for nucleoids and/or fluorescent-tagged WGA for cell walls if
appropriate), as follows:
FITC-coupled secondary antibody (goat anti-rabbit): 1/100 dilution of 0.75 mg ml–1
stock;
Cy3-coupled secondary antibody (goat anti-rabbit): 1/200 dilution of 0.75 mg ml–1
stock;
Propidium iodide: 1/100 dilution of 1 mg ml–1 stock;
FITC-coupled WGA: 1/5000 dilution of 10 mg ml–1 stock;
AMCA-S-coupled WGA: 1/1000 dilution of 10 mg ml–1 stock.

Leave at room temperature for 1-3 h. For a primary antibody from a species other than
rabbit, appropriate secondary antibodies must be used. Cy3 and propidium iodide cannot be used
together because of spectral overlap. Similarly, only one FITC-conjugate may be used at a time.

15.

Wash eight times with PBS (include propidium iodide if using for staining), thentwice with Slow Fade equilibration buffer (component C). Leave the second
wash in contact with the mycelium for 5 min.

16.

Remove the buffer and add Slow Fade in PBS/glycerol. Remove a little of theliquid and add a long coverslip. Remove excess buffer from the edges of thecoverslip by strong aspiration. Expel any bubbles from under the coverslip bypressing with a clean pipette tip and aspirate further buffer to suck the coverslip
tightly to the slide. Do not allow the coverslip to move or PAP ink will spreadand spoil the specimen.

&KDSWHU0LFURVFRSLFDOPHWKRGV17.

Examine the specimen in the fluorescence microscope. A film such as Kodak
Ektachrome slide film, ASA400, is appropriate for taking micrographs. Blue hues (AMCA-S
fluorescence) can be difficult to capture on film. The slides can be stored at -20o. Allow themto warm up and then dry off condensation before viewing.

Electron microscopy
Transmission electron microscopy of whole cells, especially spores, sometimes negatively stained(e.g. Wildermuth, 1970a), has been used extensively in taxonomic studies, but reveals few cytologicaldetails (Hopwood and Glauert, 1961) because of collapse and distortion of the cells and because of theelectron opacity of the material. Scanning electron microscopy produces visually attractive pictures (e.g.
Miyadoh, 1997) and reveals valuable topological information (for example the lack of disturbance of thematerial and the depth of field can make it possible to follow a single spore chain for perhaps 100 µm).
Fine surface detail is hard to obtain without a cold stage and a high resolution instrument. Carbon replicas(Hopwood and Glauert, 1961) and freeze-etching (Wildermuth, 1970b) can yield much detail of thesurface layers and membranes. However, thin sectioning has been the most useful technique forobservations linked to genetic studies.
Electron microscopy of thin sections
In early work (Glauert and Hopwood, 1960; Wildermuth, 1970a, b; Hopwood et al., 1970), wholecolonies were cut from agar plates for fixing, embedding and sectioning; attempts were made to position
the colonies in a known orientation in the capsules of embedding medium so as to aid the recognition of
specific planes of sectioning. Wildermuth (1970a) found it useful to grow an auxotrophic mutant of S.
coelicolor under starvation conditions and so produce tiny, compact colonies suitable for anatomical
study.
In a later procedure (Plaskitt and Chater, 1995; Bruton et al., 1995), heavily seeded inocula weregrown on either cellophane or 0.45 µm Millipore filters laid on agar plates, with the advantage of allowing
easier orientation of areas of thin sections in the colony as a whole, and of somewhat greater synchronyof development. The following procedure is for thin sections of S. coelicolor.
Sectioning of Streptomyces colonies
Streptomyces colonies growing in agar or on cellophane
Sodium cacodylate buffer, 0.05M, pH7.2 (TOXIC)Glutaraldehyde, 2.5% in 0.05M sodium cacodylate buffer, pH7.2 (TOXIC)
Osmium tetroxide, 1% in 0.05M sodium cacodylate buffer, pH7.2 (TOXIC)Distilled water
Ethanol, 30%, 50%, 70%, 90%, 100%
LR White epoxy resin, pure and mixtures with ethanol 1:1, 2:1, 3:1
Gelatin capsules, size 00 e.g. from Electron Microscopy Sciences (p. 419)

&KDSWHU0LFURVFRSLFDOPHWKRGVCopper EM grids with carbon films
Uranyl acetate, 2% in water (TOXIC)Lead citrate, 1% in water (TOXIC)
Embedding oven (60o); ultramicrotome with glass knives; transmission electron
microscope1.

Fix individual colonies by cutting them from an agar plate with a scalpel, or by
cutting out small squares of membrane-grown mycelium, and transferring themto glutaraldehyde. Leave to fix overnight.

2.

Wash by draining the fixative from the colonies and replacing it with about 10 mlof 0.05M sodium cacodylate buffer. Leave for 5-15 min. Repeat the washing
twice more.

3.

Post-fix by replacing the washing buffer with 1% osmium tetroxide in 0.05M
sodium cacodylate buffer for 1 h at room temperature.

4.

Wash three times with c. 10 ml of distilled water, 15 min each.

5.

Dehydrate by successive transfer to c. 10 ml of 30%, 50%, 70%, 90% and 100%
ethanol, 15-60 min in each.

6.

Embed in LR White epoxy resin, by successive infiltration for 1 h each in about
5 ml of 1:1, 2:1, 3:1 resin: ethanol mixture, followed by about 5 ml of 100% resin,
and then by the same volume of fresh 100% resin overnight.

7.

Next morning, replace the liquid with about 5 ml fresh resin once more. In theafternoon, transfer each colony to a gelatin capsule, top up with fresh resin, andplace at 60o overnight to polymerise.

8.

Cut thin sections with a 45o glass knife. Collect sections from the water surface
on a carbon-coated copper grid and stain by floating upside down on uranyl
acetate and lead citrate. Uranyl acetate enhances the contrast of nucleic acid-containing
components, and lead citrate tends to stain the cell walls.

9.

Examine and photograph the sections in a transmission electron microscope.

&KDSWHU0LFURVFRSLFDOPHWKRGV

Staining thin sections of Streptomyces colonies for glycogen (Original procedure from Thiéry, 1967,
used to analyse Streptomyces by Braña et al. (1986) and Plaskitt and Chater, 1995)
Thin sections of Streptomyces colonies (p. 71, step 8)
Periodic acid, 1% (e.g. 4 µl of 50% periodic acid added to 196 µl of distilled water) as
drops on a wax sheet
Distilled water
TCH (thiocarbohydrazide; TOXIC) solution, 0.2% in 20% acetic acid; add 0.2 g of
TCH to a mixture of 20 ml glacial acetic acid and 80 ml distilled water and stir repeatedly
to dissolve
Acetic acid, 10%, 5% and 2.5%
Silver proteinate (BDH Chemicals), 1%: dissolve, say, 0.03 g in 3 ml distilled water.
Gold EM square mesh grids with carbon films
Transmission electron microscope1.

Collect thin sections on gold grids carrying carbon films.

2.

Invert the grids on drops of 1% periodic acid on a wax sheet and leave for 20 min
at room temperature in a covered container. Staining with uranyl acetate and lead citrate(p. 71, step 8) may be included before this step if it is desired to enhance the contrast of the cell
walls, for example.

3.

Wash the grids repeatedly in distilled water: three times for 10 sec each, followedby three times for at least 10 min each. The short washes are conveniently done bysquirting water from a wash bottle over the grids held in forceps

4.

Invert the grids on the surface of c. 1 ml of 0.2% TCH solution in a closed
Eppendorf tube and leave overnight. Thiocarbohydrazide takes some time to dissolve, so
prepare the solution in advance (e.g. while steps 2 and 3 are in progress). The solution can be
kept frozen in small volumes for at least a year.

5.

Next morning, carefully dispose of the TCH solution. Wash the grids repeatedly
with 10% acetic acid: three times for 10 sec each, then three times for 10 min
each.

6.

Wash the grids with 5% acetic acid for 10 sec; 2.5% acetic acid for 10 sec;
distilled water three times for 10 sec each, then three times for 10 min each (seenote 3).

7.

Invert the grids on drops of 1% silver proteinate for 30 min in the dark.

&KDSWHU0LFURVFRSLFDOPHWKRGV8.

Wash the grids repeatedly with distilled water: three times for 10 sec eachfollowed by three times for 10 min each.

9.

Dry the grids by placing them, right side up, on filter paper, and examine them inthe transmission electron microscope.

Spatial visualisation of gene expression
Use of methods to locate the expression of genes to regions of the developing Streptomycescolony, or to specific intra- or extracellular sites, is expected to increase over the next few years. As far
as we are aware, attempts at subcellular localisation have so far been limited to preliminary studies of
immunogold labelling of thin sections examined in the electron microscope, using antibodies to individual
proteins (an aromatic polyketide synthase, H. C. Gramajo, personal communication; and phenoxazinone
synthase, G. H. Jones, personal communication). Note that polyclonal antibodies against purified
Streptomyces proteins very often show weak, but perceptible, signals against a few other proteins in
Streptomyces cell extracts in Western blotting. This may contribute background in immunogold labelling
experiments. It has been suggested (but not rigorously tested) that the use of complete Freund’s adjuvant,which contains material from mycobacteria, may contribute to the development of "non-specific"
antibodies during immunisation, though even null rabbit sera often give weak signals in Western blots.
Hopefully, the use of monoclonal antibodies or phage antibody display libraries may give future
improvements in specificity.
On a grosser scale, the Vibrio harveyi luxAB genes (p. 346), combined with low power lightmicroscopy, have been used as transcriptional reporters to detect gene expression, either at different times
in development of the whole colony, or in different phases in development of the same colony - substrateor aerial mycelium - in vertical sections (Schauer et al., 1988). Exploitation of the fluorescence of Green
Fluorescent Protein in the analysis of spatial expression of Streptomyces genes, particularly when allied
to the use of laser confocal microscopy, is beginning to look promising (p. 347).

Braña AF, Méndez C, Díaz LA, Manzanal MB, Hardisson
C (1986) Glycogen and trehalose accumulation during colony
development in Streptomyces antibioticus. J.Gen.Microbiol.,
132, 1319-1326.
Bruton CJ, Plaskitt KA, Chater KF (1995) Tissue-specificglycogen branching isoenzymes in a multicellular prokaryote,
Streptomyces coelicolor A3(2). Mol.Microbiol., 18, 89-99.
Chater KF (1972) A morphological and genetic mapping
study of white colony mutants of Streptomyces coelicolor.
J.Gen.Microbiol., 72, 9-28.
Chater KF, Bruton CJ, Plaskitt KA, Buttner MJ, Méndez
C, Helmann J (1989) The developmental fate of S. coelicolor
hyphae depends crucially on a gene product homologous withthe motility sigma factor of B. subtilis. Cell, 59, 133-143.
Drouin J-F (1996) Caractérisation de la différenciation
cellulaire de Streptomyces ambofaciens en culture submergée.
Etude des relations avec la production de spiramycine paranalyse quantitative d'images. Institut National Polytechniquede Lorraine, Nancy.
Glauert AM, Hopwood DA (1960) The fine structure ofStreptomyces coelicolor I. The cytoplasmic membrane system.
J.Biophys.Biochem.Cytol., 7, 479-488.
Harry EJ, Pogliano K, Losick R (1995) Use of
immunofluorescence to visualize cell-specific gene expression
during sporulation in Bacillus subtilis. J.Bacteriol., 177, 33863393.
Haughland RP (1996) Handbook of Fluorescent Probes andResearch Chemicals. Molecular Probes Europe BV, Leiden,
The Netherlands.
Hiraga S, Niki H, Ogura T, Ichinose C, Mori H, Ezaki B,
Jaffé (1989) Chromosome partitioning in Escherichia coli:
novel mutants producing anucleate cells. J.Bacteriol., 171,1496-1505.
Hopwood DA (1960) Phase-contrast observations on
Streptomyces coelicolor. J.Gen.Microbiol., 22, 295-302.
Hopwood DA, Glauert AM (1960) The fine structure of&KDSWHU0LFURVFRSLFDOPHWKRGVStreptomyces coelicolor.
II. The nuclear material.
J.Biophys.Biochem.Cytol., 8, 267-278.
Hopwood DA, Glauert AM (1961) Electron microscopeobservations on the surface structures of Streptomyces
violaceoruber. J.Gen.Microbiol., 26, 325-330.
Hopwood DA, Wildermuth H, Palmer HM (1970) Mutantsof Streptomyces coelicolor defective in sporulation.
J.Gen.Microbiol., 61, 397-408.
Kelemen GH, Plaskitt KA, Lewis CG, Findlay KC, Buttner
MJ (1995) Deletion of DNA lying close to the glkA locus
induces ectopic sporulation in Streptomyces coelicolor A3(2).
Mol.Microbiol., 17, 221-230.
Larison KD (1992) Handbook of Fluorescent Probes andResearch Chemicals. Molecular Probes Inc., Eugene, Oregon.
McCormick JR, Losick R (1996) Cell division gene ftsQ isrequired for efficient sporulation but not growth and viability
in Streptomyces coelicolor A3(2). J.Bacteriol., 178, 52955301.
McCormick J, Su EP, Driks A, Losick R (1994) Growth andviability of Streptomyces coelicolor mutant for the cell
division gene ftsZ. Mol.Microbiol., 14, 243-254.
Miyadoh S (ed.) (1997) Atlas of Actinomycetes. Asakura
Publishing Co., Tokyo.
Plaskitt KA, Chater KF (1995) Influences of developmental
genes on localised glycogen deposition in colonies of amycelial prokaryote, Streptomyces coelicolor A3(2): a
possible interface between metabolism and morphogenesis.
Phil.Trans.R.Soc.B., 347, 105-121.
Pogliano K, Harry L, Losick R (1995) Visualization of thesubcellular location of sporulation proteins in Bacillus subtilis
using immunofluorescence microscopy. Mol.Microbiol., 18,459-470.
Pogliano K, Hofmeister AEM, Losick R (1997)
Disappearance of the sigma E transcription factor from theforespore and the SpoIIE phosphatase from the mother cell
contributes to the establishment of cell-specific gene
expression during sporulation in Bacillus subtilis. J.Bacteriol.,
179, 3331-3341.
Pons MN, Drouin JF, Louvel L, Vanhoutte B, Vivier H,
Germain P (1998) Physiological investigations by image
analysis. J. Biotechnol., 65, 3-14.
Potúûková L, Kelemen GH, Findlay KC, Lonetto MA,
Buttner MJ, Kormanec J (1995) A new RNA polymerase
sigma factor, F, is required for the late stages ofmorphological differentiation in Streptomyces spp.
Mol.Microbiol., 17, 37-48.
Pringle JR, Adams EEM, Drubin DG, Haarer BK (1991)
Immunofluorescence methods for yeast. Meth.in Enzymol.,
194, 565-602.
Schauer A, Ranes M, Santamaria R, Guijarro J, Lawlor E,
Méndez C, Chater K, Losick R (1988) Visualizing gene
expression in time and space in the morphologically complex
filamentous bacterium Streptomyces coelicolor. Science, 240,
768-772.
Schwedock J, McCormick JR, Angert ER, Nodwell JR,
Losick R (1997) Assembly of the cell division protein FtsZ
into ladder-like structures in the aerial hyphae of Streptomyces
coelicolor. Mol.Microbiol., 25, 847-858.
Thiery JP (1967) Mise en évidence de polysaccharides surcoupe fines en microscopie électronique. J.Microscopie(Paris), 6, 987-1018.
Wildermuth H (1970a) Development and organisation of theaerial mycelium in Streptomyces coelicolor. J.Gen.Microbiol.,60, 43-50.
Wildermuth H (1970b) Surface structure of streptomycete
spores as revealed by negative staining and freeze-etching.
J.Bacteriol., 101, 318-322.

m04tkd.wpd 20 April 2000 Tobias Kieser



Chapter 4

Specialised biochemical techniques
CONTENTS
Preparation of cell-free extracts from Streptomyces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
Cell-free extracts from mycelium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
Cell-free extracts from spores . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
Small-scale cell-free extract from spores . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
Large-scale cell-free extract from spores . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
Preparation of a coupled transcription-translation system from S. lividans . . . . . . . . . . . . . . . 78
Two-dimensional polyacrylamide gel electrophoresis and its application
to Streptomyces proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
In-vivo radioactive labelling of Streptomyces proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
Two-step liquid cultivation and radioactive labelling method for Streptomyces . . . . . 90
Growth and radioactive labelling of Streptomyces on zirconium-silica beads . . . . . . . 92
Detection and assay of S. coelicolor (and S. lividans) antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . 93
The S. coelicolor antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
Actinorhodin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
Undecylprodigiosin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
CDA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
Methylenomycin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
S. lividans as an antibiotic producer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
Secondary metabolite production in S. lividans transformants . . . . . . . . . . . . . . . . . . . 95
A complete coverage of biochemical techniques for Streptomyces would be inappropriate in amanual that deals primarily with genetic methodology. After some descriptions of cell-free extract
preparation we deal with two procedures that require special attention if their full potential is to berealised; these are coupled transcription-translation, and two-dimensional gel electrophoresis for the
separation and visualisation of the complete repertoire of cellular proteins or a subset of proteins
expressed under specific physiological conditions or in a particular genotype. DNA and RNA methods
are in Chapters 8 and 15.

Preparation of cell-free extracts from Streptomyces
Cell-free extracts from mycelium
Over the last few years research on Streptomyces enzymes involved in primary or secondary
metabolism has flourished and there are now innumerable studies that could be cited. Different

&KDSWHU6SHFLDOL]HGELRFKHPLFDOWHFKQLTXHV

laboratories will favour different procedures for the isolation of different types of enzymes, especiallybecause “methods by which cells are disintegrated have a profound effect on the quality and yield of theprotein isolated” (K. Mikulik, personal communication). Many of the precautions to be taken are wellknown to biochemists used to working with other biological systems. Thus, in general cells should be
washed with the extraction buffer several times before disintegration to remove residues of medium,
extracellular enzymes and soluble metabolites; this is especially important because streptomycetes
produce many hydrolytic enzymes, including nucleases, proteases and phosphatases, as well as
antibiotics, that could interfere with enzyme-catalysed reactions. Disintegration of cells should be gentle,
rapid and performed at low temperature. Vegetative mycelium of Streptomyces can be disintegrated bysonication (e.g. Crosby et al., 1995; Holmes et al., 1993); passage through a French press (White et al.,1992); lysis with lysozyme (Summers et al., 1995); grinding with glass beads (Lichá et al., 1995) oralumina; or by use of a bead beater (p. 418) The suspending medium should contain components toimprove the stability of proteins, such as buffer, salts, metal chelators, sulphhydryl reagents, and proteaseinhibitors. The protein extract should be concentrated, and stabilised with glycerol. Extracts frozen inliquid nitrogen can be stored at -70o.
Cell-free extracts from spores
Breaking spores presents a particular challenge. Several methods have been developed for thispurpose, including agitation with glass beads in a Vortex mixer (Quiros et al., 1986; McBride andEnsign, 1987) and grinding with glass beads in a precooled mortar (Weiser et al., 1989; Mikulik andZhulanova, 1995). Breakage of spores can be monitored by 400× phase-contrast microscopy.
Small-scale cell-free extract from spores (from K. Mikulik)
Spores, wet pellet in water frozen at -20E.
Extraction buffer, 0.1M Tris-HCl pH7.6, 10mM MgCl2, 6mM 2-mercaptoethanol(4.2 µl/10 ml), 1mM phenylmethylsulphonyl fluoride: TOXIC.
Glass beads, 0.1-0.2 mm in diameter. Prepare glass beads as follows: boil 100 g ofglass beads in 200-300 ml 6M HCl for 1 h. Remove the supernatant and wash the beads
with distilled water until the pH has reached that of the washing water. Dry the beads
on filter paper and then at 180o for 2 h.
Spin X microcentrifuge filter unit with 0.22 µm cellulose acetate sterile filter(p. 419); Sonicator with small tip; eye and ear protection.

1.

In a 1.5 ml Eppendorf tube, mix 0.3 g of wet spores (c. 8 plates) with 0.3 g ofglass beads and 0.5 ml of extraction buffer.

2.

Homogenise spores by sonication: ten 30 sec bursts at 80 W and 1 min
intermittent cooling in ice.

3.

Spin the mixture at 12,000 × g for 15 min at 4o.

&KDSWHU6SHFLDOLVHGELRFKHPLFDOWHFKQLTXHV

4.



Save the supernatant, and remove residues of membranes and nucleic acids by
ultrafiltration using the SpinX filter system. Total yield of protein from 0.3 g of wet
spores is about 0.5 mg. The filtrate containing spore proteins can be frozen in liquid nitrogenand stored at -70o; it is suitable for high resolution 2D electrophoresis and for enzyme assays.

Large-scale cell-free extract from spores (Mikulik and Zhoulanova, 1995)
Spores, wet pellet in water frozen at -20E.
Extraction buffer, 0.1M Tris-HCl pH7.6, 10mM MgCl2, 6mM 2-mercaptoethanol(4.2 µl/10 ml), 1mM phenylmethylsulphonyl fluoride: TOXIC.
Glass beads, 0.1-0.2 mm in diameter. Prepare glass beads as follows: boil 100 g ofglass beads in 30 ml 6M HCl for 1 h. Remove the supernatant and wash the beads with
distilled water until the pH has reached that of the washing water. Dry the beads onfilter paper and then at 180o for 2 h.
Liquid nitrogen; mortar and pestle; high speed centrifuge; ultracentrifuge; 400×
phase contrast microscope.

1.

Mix 10 g spores (obtainable from 20 square plastic dishes, 22 × 22 cm, NUNC,
p. 419) and 20 g of glass beads in a mortar precooled with liquid nitrogen.

2.

Fill the mortar partially with liquid nitrogen and homogenise the contents
25-30 min with the pestle until the spore suspension is viscous; add liquid
nitrogen as necessary to keep the homogenate frozen.

3.

Allow the liquid nitrogen to evaporate. Add 4 ml of ice-cold extraction buffer
and mix the homogenate.

4.

Remove glass beads by 5 min centrifugation at 3000 × g, 4o.

5.

Collect the supernatant solution and re-extract the sediment with 3 ml of
extraction buffer. Spin as above and combine the supernatants.

6.

Remove cell debris by centrifugation at 10,000 × g for 15 min at 4o.

7.

Spin the supernatant solution at 40,000 × g (e.g. 20,000 rpm in SS-34 rotor) for
30 min at 4E to sediment the membrane fraction. The supernatant can be further
fractionated by centrifugation at 150,000 × g (e.g. 30,000 rpm in SW 40 rotor) for 3 h at 4o. The
sediment is a source of crude ribosomes and the supernatant solution (S150 fraction) contains
soluble proteins.

&KDSWHU6SHFLDOL]HGELRFKHPLFDOWHFKQLTXHV

Preparation of a coupled transcription-translation system from S. lividans (Protocol
provided by S Fish and E Cundliffe, with slight modification by G H Jones; original reference: Thompson
et al., 1984)
The original coupled transcription-translation system for S. lividans has been used in manystudies. The same methodology has also been applied successfully to other Streptomyces spp., such as
S. azureus, S. coelicolor, S. fradiae, S. lincolnensis, S. pactum and S. virginiae (E Cundliffe, personalcommunication). The protocol below is in the form that would be relevant to experiments to identify theproteins encoded by particular open reading frames. Some references to more complex situations, suchas “fractionated systems” for the study of ribosome-determined antibiotic resistance, are: Calcutt andCundliffe (1989, 1990); Zalacain and Cundliffe (1991).

Materials (the procedures start on p. 81)
Precautions

Wear gloves for all steps in the procedure. Autoclave all solutions, buffers (except 2mercaptoethanol), water, glassware and the French press before use. Make all pH
adjustments at 25o. Use sterile Millipore Super Q water for all reagent solutions.

Spores harvested from plates of NEF agar
NEF agar
5g
1g
1g
0.5 g
20 g
1 litreGlucoseDifco yeast extract
Difco Casamino acids
Oxoid beef extract
Agar
1M KOH (to pH8.0)
Water

The pH is adjusted after the addition of agar andbefore autoclaving. Test pH by pipetting small
volumes onto narrow range pH paper.
Presumably media other than NEF would yieldsuitable spores, but NEF works especially well for
S. lividans.

YEME medium (p. 412) +PEG 6000 (5 g l–1)
CaCl2 (50mM)

GF/C discs, 2.5 cm diameter (Whatman)

EGTA-KOH, 25mM, pH7.0,

Scintillation fluid

Magnesium acetate, 0.1M

10% polyacrylamide, 0.1% SDS gel
0.27% methylene bisacrylamide

TCA, 10% and 5% w/v

&KDSWHU6SHFLDOLVHGELRFKHPLFDOWHFKQLTXHVGel loading buffer



375mM
Tris-HCl, pH6.8
4% (w/v)
SDS
35% (v/v)
glycerol
2.86 M 2-mercaptoethanol (200 µl ml–1 stock) or 20mM dithiothreitol (added
fresh from a 1M stock)

Equipment
2-litre conical flasks with,e.g. four side indentations (“baffles”)
Buchner funnel (12 cm diameter)
Vacuum flask and filter pump
Orbital shaker
Preparative centrifuge
French press and pressure cell

Spectrophotometer
Eye protection
Scintillation counterGel electrophoresis equipment
Boiling water bath

Template plasmid DNA
Plasmid purity is important. Qiagen column purification works well. Plasmid DNA
purified by routine use of caesium chloride is unreliable, unless followed by a further
purification: dissolve the ethanol-precipitated caesium-purified DNA in 200-250 µl of
TE and layer the solution over 5 ml of TE containing 1M NaCl in an appropriate
centrifuge tube, then spin for 7 h at 250,000 × g in a swinging bucket rotor (e.g. 45,000rpm in a SW 50.1 rotor). The DNA pellet is redissolved in TE, ethanol precipitatedagain and is then dissolved for use in the coupled system (step 5, p. 82)
Nuclease solution in buffer (used in step 4, p. 82)
The nuclease used (Boehringer 107921) has been variously described as“staphylococcal” or “micrococcal”, and most recently as S7. It is activated by Ca2+ andinactivated by chelation. Buy 15,000 units and dissolve in 200 µl of buffer (50mM
glycine-KOH pH9.2 containing 5mM CaCl2) to give a final activity of 75 units ml–1.
Unlabelled methionine, 440 µg ml–1 (3mM)[35S]-methionine
7 µl
3 µl
90 µl

[35S]-methionine >1000 Ci mol–1; 15 mCi ml–1 (377 TBq mol–1, 550 Mbq ml–1),
Amersham S.J. 204. (<15µM methionine, <1µM final concentration)
Unlabelled methionine (440 µg ml–1 = 3.3mM; 0.1mM final concentration)
2-mercaptoethanol (100mM = 7 µl ml–1)

This gives [35S]-methionine at a specific activity suitable for following incorporation into hot TCAprecipitable material directed by pUC18. For gel analysis of products use this mix without unlabeledmethionine.

&KDSWHU6SHFLDOL]HGELRFKHPLFDOWHFKQLTXHV

Washing buffer, S30 buffer, and S30 buffer+10% glycerol
For 1 litre buffer add
Stock solutions
Washing buffer

S30 buffer

S30 buffer+10% glycerol

1M HEPES-KOH pH7.5

10 ml

50 ml

50 ml

1M MgCl2

10 ml

10 ml

10 ml

4M NH4Cl

250 ml12.5 ml12.5 ml2-mercaptoethanol(14.3M, as purchased)

0.35 ml0.35 ml0.35 ml

Glycerol--

100 mlMillipore Super Q water

730 ml927 ml827 mlSynthesis mix The components must be mixed in the order listed
1.

200 µlHEPES/KOH, 2M, pH8.2

2.

25 µl

Dithiothreitol, 0.55M

3.

250 µlATP di-Na+ salt, 38mM, pH7.0, adjusted using KOH

4.

75 µl

CTP, GTP, UTP mixture, each at 88mM, pH7.0, adjusted using KOH
GTP (Na+)UTP (Na+)CTP (Na+)5.

500 µlPhosphoenolpyruvate, 0.42M pH7.0, adjusted using 1M Tris base,phosphoenolpyruvate tri-Na+ salt

6.

50 µl

19 amino acids, 55mM each, (minus methionine;
kit also contains cystine-omit)

7.

375 µlPEG 8000, 40% (w/v), in H2O

8.

100 µlFolinic acid, Ca2+ salt, 2.7 mg ml–1 in water9.

240 µlPyruvate kinase, 2000 U ml–1 in 20% glycerol

10.

66.5 µl

Ammonium acetate, 4.2M

11.

66.5 µl

Potassium acetate, 8.4M

&KDSWHU6SHFLDOLVHGELRFKHPLFDOWHFKQLTXHV

Protein size markers [14C]-methylated protein markers, Amersham CFA 626;
5 µl gives reasonable exposure after 24 h.
200 kDa
92.5 kDa + 100 kDa
69 kDaMyosin46 kDaOvalbumin

Phosphorylase b(100 kDa band is faint)

30 kDaCarbonic anhydrase

BSA

14.3 kDa

Lysozyme

Procedures
1.

Growth of cells. Inoculate flasks of YEME (containing MgCl2.6H2O, 1 g l–1 andPEG 6000, 5 g l–1) (1 l in 2 l flasks) with a spore suspension from a freshly
sporulating NEF plate (1 plate per litre of medium) and grow for 17-18 h withvigorous shaking. Add the appropriate antibiotic to NEF and YEME to maintain any plasmidpresent. A normal batch is 1.5-3 l YEME per preparation. The aim is to achieve as rapid growth
as possible: baffles in the flasks improve aeration and mycelial dispersion. The time ofincubation is chosen to give an appropriate yield of mycelium for step 3: it should be about 1.5 g
l–1 (more than 2 g l–1 is too much).

2.

Harvesting and washing mycelium. Harvest the mycelium by centrifuging at
7,000 rpm (e.g. Beckman JA10 or Sorvall GSA rotor) for 10 min at 4o. Wash
twice with washing buffer and once with S30 buffer (500 ml of each per litre ofculture).

3.

Preparation of S30. Resuspend washed mycelium in S30 buffer and filter
through Whatman no. 1 paper in a Buchner funnel until as dry as possible.
Weigh mycelium (to estimate growth yield - see note step 1) and resuspend at
about 1 g per 2.5 ml S30 buffer+10% glycerol, dispersing clumps with a sterile
spatula to give a smooth suspension. Rinse a chilled French pressure cell
thoroughly with sterile Super Q water and then with S30 buffer before use. Thisis very important: it must be perfectly clean. Press the mycelial suspension at
10-12 × 103 psi directly into a centrifuge tube. Centrifuge at 15,000 × g (e.g.
11,500 rpm in a Beckman JA21 rotor) for 30 min at 4o. Remove the supernatant(pool if more than one tube was used). This is termed the S30. Read A 260: it isusually about 300-350 units ml–1. Hold S30 at 0o while carrying out preliminary
nuclease treatment (step 4). If good cell breakage was not achieved by one passage throughthe French press, add a little more buffer to the 15,000 rpm pellet and press again. When you
assay the S30 in coupled transcription-translation reactions, you will find an unacceptably highbackground level of incorporation in the absence of added plasmid. This plasmid-independent&KDSWHU6SHFLDOL]HGELRFKHPLFDOWHFKQLTXHV

background is reduced by nuclease treatment. We sometimes carry out a preliminary nucleasetreatment to determine the optimal duration and then treat the bulk of the S30 likewise. With
experience, this preliminary nuclease treatment, which takes 15-20 min, may be unnecessary
because the optimal treatments vary little between preparations; usually we simply do a scaledup bulk nuclease treatment, for 20 minutes, at this stage (see step 6).

4.

Preliminary nuclease treatment (or final if scaled-up).
Mix in this order:
200 µlS30 (40-60 A260 units)
4.1 µl50mM CaCl2 to give a final CaCl2 concentration of 1mM.
2 µl
(150 units) nuclease. (Added after taking 30 µl untreated control.)Incubate at 30o. Remove 30 µl samples at 10, 20, 30 and 45 min, and inactivate
nuclease by adding 2.6 µl of 25mM EGTA-KOH pH7.0 to each sample to givea final EGTA concentration of 2mM. Assay samples, including untreatedcontrol, for coupled transcription-translation activity in the presence and absenceof plasmid (see step 5).

5.

Assaying the activity of the S30. Follow [35S]-methionine incorporation into
hot TCA-precipitable material in the presence and absence of plasmid (routinely
pUC18) by setting up the following assay.
Mix in this order (total volume=30 µl):
1. 10 µl S30 (2-3 A260 units)
2. 6 µl S30 buffer
3. 2 µl [35S]-methionine
4. 2 µl 0.1M magnesium acetate (12mM final)
5. 8 µl Synthesis mix
6. 2 µl Plasmid DNA, 2 µg, very pure or water blank
Incubate at 30o. Remove 5 µl samples into approximately 1 ml 10% (w/v) TCA
at 3, 6, 9 and 12 min. Incubate samples in boiling water bath for 10-20 min.
Make the volume up to 5 ml with 10% TCA. Filter through 2.5 cm GF/C discs(Whatman). Wash discs with c. 20 ml 5% TCA (3 washes of tube plus 1 direct
wash of filter, c. 5 ml each). Dry the filters and count in 5 ml scintillation fluid.
Typical incorporations in the presence of plasmid reach 15-30 × 103 cpm per 5 µl
sample after 12 min. From the above assay it is possible to determine the optimal durationof incubation with nuclease required to eliminate plasmid-independent synthesis (typically 15-20

&KDSWHU6SHFLDOLVHGELRFKHPLFDOWHFKQLTXHVmin, the time-point with the greatest difference between plasmid-dependent and plasmidindependent synthesis).

6.

Repeat nuclease treatment on remainder of S30 (if not already done).

7.

Gel analysis of protein products. Set up the assay exactly as in step 5, except
for the use of undiluted [35S] methionine (see note under preparation of [35S]
methionine, p. 79), using the now nuclease-treated S30. Incubate at 30o for 20
min, then add 2 µl unlabeled methionine and continue incubating at 30o for 10
min to allow completion of protein labelling. As a guide, 200,000 cpm per gel
track gives a clear, well-exposed fluorogram after 24 h. Take the appropriate
amount of sample, add one-third volume of gel loading buffer, and heat inboiling water bath for 10 min, then load on the gel. Samples may be kept at -70E
before running gels, but do not add loading buffer if samples are to be stored in this way. For
analysing the products, a 10% (w/v) polyacrylamide gel containing 0.1% (w/v) SDS gives sharpbanding patterns. Our gels are 12 cm wide, 14 cm long and 2 mm thick. If the bromophenol blue
dye is run to the bottom of the gel (about 5 h at 20 mA constant current at ambient temperature),
the 200 kDa myosin just runs into the separating gel and the 14.3 kDa lysozyme runs
approximately 0.5 cm from the bottom of the gel.

Two-dimensional polyacrylamide gel electrophoresis and its application to
Streptomyces proteins (Provided by Xin-Ming Li and Charles J. Thompson)
Depending on amino acid composition, most proteins are positively or negatively charged at agiven pH. Such zwitterionic molecules with a certain net charge will move towards the anode or thecathode in an electric field. This is the basic principle for all protein electrophoretic techniques(Westermeier, 1993). One application of this principle is isoelectric focussing (IEF), which separates
a protein mixture on a pH gradient. Each protein in the mixture will move in an electric field along thepH gradient until it reaches a point where the pH of the micro-environment titrates ionisable groups togenerate a protein with no net charge. The pH of this point defines the pI for this protein (Wrigley,1971). Proteins are denatured and negatively charged by binding to sodium dodecyl sulphate (SDS), an
anionic detergent. During electrophoresis, all proteins migrate towards the anode in a polyacrylamide
gel matrix, which serves as a molecular sieve to separate protein mixtures by their different masses (SDSPAGE; Laemmli, 1970).
Both IEF and SDS-PAGE have high resolving potentials for complex protein mixtures. When
combined, to separate proteins by IEF in the first dimension and then by SDS-PAGE in the second
dimension, the resolving power is greatly enhanced. This makes it possible to separate and visualise
individual proteins directly from a crude cell extract on a single gel (O’Farrell, 1975). This approach iscalled high resolution two-dimensional polyacrylamide gel electrophoresis (2D PAGE). Despite thesophisticated skills and the specialised equipment needed, the application of 2D PAGE has dramaticallyincreased since it was introduced in the mid-70s. Using automated systems, a set of 2D PAGE images

&KDSWHU6SHFLDOL]HGELRFKHPLFDOWHFKQLTXHV

can be recorded and digitised, and the resulting data can be statistically analysed. A 2D PAGE protein
database can be established, forming an important foundation for further biological study (Garrels, 1979).
A simple qualitative protein database can be merely a collection of cellular protein spots with an index
to the positions of individual proteins on a 2D gel; an advanced protein database should be interactivewith other related databases. This may include interactions to corresponding DNA sequence databases,or those describing biophysical, biochemical, or biological properties of the proteins.
Two-dimensional electrophoresis has been used successfully to study various aspects ofStreptomyces biology (e.g. Holt et al., 1992; Dary et al., 1993; Blanco et al., 1994; Möhrle et al., 1995;
Puglia et al., 1995). Currently, we are working on a 2D PAGE protein database which describes themorphological and physiological programme of S. coelicolor. For this purpose, we have developed
standardised techniques for cultivation and radio-isotope labelling of both liquid and solid phase culture.
For making 2D gels suitable for protein database construction, or gels comparable with an existing
database, reproducibility is the first requirement. Many factors may contribute to the final gel pattern,including the equipment, the sources of the chemicals, and the protocol. Several commercial 2D PAGE
systems are currently available, differing mainly in the type of ampholytes used in the IEF and the formatof the gel. Generally, the resolution of the IEF is determined by the quality of the pH gradient. There
are two types of gradient-forming techniques. One is based on free carrier ampholytes, which consistof mixtures of small zwitterionic molecules with high buffering capacity, forming a pH gradient during
the electrophoresis (Wrigley, 1971). The other is based on an immobilised pH gradient, which forms a
pH gradient fixed to a matrix. In this case the pH range is pre-determined by the buffering groupcovalently bound to the matrix (Görg et al., 1995). The format of the gel, both IEF and PAGE, strongly
affects its capacity and the resolution of the 2D gel. Chemicals from different sources may varyconsiderably. This is especially true for the ampholytes used in the IEF. Once satisfactory results areachieved from a specific batch, one should keep using the same batch, or at least material from the same
supplier. Another important factor, often overlooked, is the water.
The following 2D PAGE protocol has been routinely used in our laboratory for resolving crude
cellular proteins of S. coelicolor. A typical gel is shown in Fig. 4.1. We consider this gel system andthe protocol to be suitable for database construction, because: a large number of protein spots are
reproducibly found on each gel, representing most of the cellular proteins; most of the protein spots arehigh quality, dense, circular, and non-overlapping with others; proteins are evenly distributed along bothdimensions, resulting in protein spots scattered over the entire gel; and no obvious streaking is found ineither dimension. The protocol was adapted and improved from the technique first introduced by
O’Farrell (1975), and further developed by Garrels (1979, 1983). Of the systems available, we employ
the Investigator 2-D Electrophoresis SystemTM from Millipore. Two procedures for labelling
Streptomyces proteins in vivo are on pp. 90 and 92.

&KDSWHU6SHFLDOLVHGELRFKHPLFDOWHFKQLTXHV

Fig. 4.1 Autoradiograph of a typical 2D PAGE protein pattern generated by the described protocol. An
S. coelicolor J1501 agar-grown culture was labelled with 35S methionine during exponential growthphase. Total crude yeast extract (about 1,000,000 cpm) was applied, and the film was exposed for one
week.



&KDSWHU6SHFLDOL]HGELRFKHPLFDOWHFKQLTXHV

Materials (The procedures start on p. 87)
H2O. Deionised, distilled, and degassed water should be used throughout
2D sample buffer
3g
Urea
0.2 g
CHAPS180 mg
DTT
2.3 mlH2O
275 µlpH3-10 ampholytes/2D
25 µl
pH8-10.5 ampholytes

Mix and dissolve all components completely.
Dispense in 0.5 ml samples in Eppendorf tubes
and store frozen at -20o.
Ampholytes from Millipore

13-[(3-cholamidopropyl)dimethylammonio]-1propanesulphonate

Acrylamide 30% stock solution
30 gAcrylamide
0.8 g
N,N -methylene-bisacrylamide0

Dissolve in 60 ml H2O and bring to 100 ml.
Filter with 0.45 µm filter (Millipore).
Store at 4E

This stock is used only for IEF gel preparation.
Acrylamide is a potent neurotoxin and isabsorbed through the skin. Special care should
be taken when weighing the powder as well as
handling acrylamide solutions.

Duracryl® is a high-tensile strength acrylamide solution from Millipore used for large format second
dimension gels.
IEF gel stock solution (for 100 IEF gels)
22.8 g
Urea
0.8 g
CHAPS
15 ml
H2O
5.2 ml
30% acrylamide stock

Mix and dissolve completely.
The final volume of the solution is about 39 ml.
Filter the solution with a 0.45 µm filter.
Store at -20o in 6 ml lots.

Anode buffer, 100mM phosphoric acid. Dilute 2.6 ml of 85% phosphoric acid in 4 l H2O.
Cathode buffer, 100mM NaOH. Dissolve 3.2 g of NaOH in 800 ml H2O.
Sample overlay buffer. Dilute the 2D sample buffer 1:4 with H2O.

&KDSWHU6SHFLDOLVHGELRFKHPLFDOWHFKQLTXHV

Equilibration buffer
12.5 ml
1M Tris pH6.8
20 ml
Glycerol
30 ml
10% SDS
2.5 ml
0.5 mg ml–1 bromophenol blue

Add H2O to 100 ml.
Keep the solution at room temperature.
Immediately before each use, dissolve
0.23 g DTT in 30 ml of this solution.

Slab gel buffer
130.8 gTris base66.3 gTris HCl

Dissolve in H2O and bring up to 1 litre.
Final concentration is 1.5M, pH8.8.
Store the solution at 4o.



10% SDS solution (w/v). Keep at room temperature. SDS is irritating to the skin and respiratory
system and is dispersed easily.
10% APS (w/v), ammonium persulphate. Keep at 4o for up to 1 week.
Slab gel mixture (100 ml)
41.5 ml
Duracryl®
25 ml
Slab gel buffer
32.2 ml
H2O
1 ml
10% SDS
50 µl
TEMED
250 µl
10% APS

Slab gel running buffer (10 times
concentrated)
288.4 gGlycine60.6 gTris base
20 g
SDS
Dissolve in distilled water and bring up to 2 l.

Slab gel overlay solution. Mix equal volumes of isobutanol and H2O. Let stand to separate phases. Use
the upper isobutanol phase only.

Procedures
1.

Sample preparation for 2D PAGE. Mix 5 µl of the protein sample with 10 µlof 2D sample buffer at 25E (chilling the sample may precipitate the urea). The
protein loading capacity is up to 100 µg, but better resolution is achieved in the range of 10-50µg. For the 35S-methionine labeled protein samples, 106 cpm should be applied on each gel lane,in order to view the autoradiogram after a reasonable exposure time (1 week). Precipitate theprotein in cold acetone if the original sample is too dilute: Mix 2:1 (v/v) of cold acetone tooriginal sample; vortex and then put the mixture at -20o for 2 h or overnight. Centrifuge themixture at maximum speed (13,000 rpm) for 20 min in the bench-top Eppendorf centrifuge.
Remove as much supernatant as possible, then speed-vac the pellet for a few minutes.
Resuspend the pellet in 5 µl of H2O.



2.

&KDSWHU6SHFLDOL]HGELRFKHPLFDOWHFKQLTXHV

Preparation of IEF tube gels. Soak the tubes for 1 h in H2O. Drain the extra
water away and mark a length of 21 cm from one end of the tubes. Thaw onevial with 6 ml IEF gel stock solution completely, then add: 360 µl 3-10
ampholytes, and 40 µl 8.5-10 ampholytes, vortex and centrifuge at room
temperature for 5 min at about 1,500 × g. Polymerisation is initiated by adding
30 µl of 10% APS solution. At this stage one may use an IEF gel casting
apparatus (Garrels, 1983). Alternatively, pipette the whole mixture to the bottomof a glass or a transparent acrylic tube, 1.5 cm i.d., and 26 cm long with one end
closed by Parafilm. Insert up to 20 threaded tubes. Slowly add deionised wateron the surface of the gel mixture without disturbing it. Add more water toprovide hydrostatic pressure, thus pushing the gel solution up the threaded tube
to a height of 21 cm. Allow to polymerise for 3 h or overnight. We use Millipore
analytical IEF tubes routinely. The 200 µl tube is 1 mm inner diameter 26 cm long, with a 0.08
mm thread incorporated along its length. The thread serves as a flexible reinforcement
embedded into the polymerised IEF gel, making the gel stronger and easier to handle.

3.

IEF pre-running. Wash the ends of the fully polymerised IEF gel with H2O.
Add 30 µl of sample overlay buffer, which is a 1/5 dilution of the 2D sample
buffer and serves as a protection barrier between the protein sample and thecathode buffer. Put the gel tubes in place in the IEF running system.
Prepare a fresh solution of 100mM NaOH as the cathode buffer and
100mM phosphoric acid as the anode buffer. Pour the anode buffer into thelower tank and the cathode buffer into the upper. Be careful to remove all the
air bubbles in the tubes. Start the pre-run immediately. With a Millipore
Investigator system, the pre-run time is about 70 min until the voltage reaches
1,500.

4.

Sample-loading and the IEF running. After the pre-run, load the protein
sample prepared according to step 1 on top of the IEF gel with a Hamilton
syringe. Because of the high concentration of urea, the dense sample solution
will descend to the bottom, underneath the overlay buffer.
Run the isoelectric focussing at 1000 V for 15.5 h with the Millipore
Investigator system. High voltage (up to 2,500 V) is applied during the last
30 min for a total 16,000 volt-hours.

5.

Preparation of the slab gels for the second dimension. Use Duracryl® high-

&KDSWHU6SHFLDOLVHGELRFKHPLFDOWHFKQLTXHVtensile strength acrylamide for large-format gels. For S. coelicolor
proteins, 12.5% gels give good separation. All the chemicals and techniques
are essentially the same as SDS-PAGE (Laemmli, 1970) without
stacking gel.
It is a useful practice to put a piece of numbered paper at the lower-leftcorner of each gel, in order to have a permanent label.
Slowly fill the slab gel mixture till the level is 0.5 cm below the top edgesof the plates. Add 0.5 ml of the slab gel overlay solution to each gel. Fullpolymerisation will need about 1.5 h.
6.

IEF gel equilibration and second dimension loading. After IEF, put the tubes
on ice for 20-30 min, then extrude the IEF gel from the tube with compressed air.
Some water or equilibration buffer on top of the gel will ease extrusion andavoid air leakage between the gel and the wall of the glass tube. Leave the IEF
gel in the equilibration buffer for 2 min, then transfer the IEF gel to the surfaceof the second dimension slab gel. This is a critical step for the whole 2D PAGE
technique; therefore, one should be especially careful and practise first. Two small tricks mayhelp in this step: (1) remove all extra liquid remaining on top of the slab gel, which is normally
1-1.5 cm below the edge of the glass plates; (2) after aligning the IEF gel appropriately on theslot formed between the two glass plates using a small blunt spatula, slide the gel into the slot
from one end to the other, and finally smooth it on top of the slab gel, taking care not to trap any
air bubbles between the IEF gel and the slab gel.

7.

Second dimension running. Gel running buffer is the same as in SDS-PAGE.
Use double strength slab gel running buffer (dilute stock 5-fold) for large format
gels to cope with the prolonged running time. Keep the running temperature at
20o and stop the run when the bromophenol blue dye front is 1 cm from the endof the gel. For the Millipore Investigator system, this requires about 6 h at 14
W for each gel.

8.

Visualising the 2D PAGE result. For 35S-methionine-labelled protein samples,soak the gel with fixing solution (40% ethanol and 10% acetic acid in water); dry
the gel on a filter paper or transparent cellophane backing, and expose the geleither to a high-sensitive X-ray film or a phosphor-screen (Molecular Dynamics,
p. 419). The latter is 10-15 times faster than the former.
For all protein samples, the protein spots on the gel can be visualised by
Coomassie blue or silver staining (Kirkeby et al., 1993), depending on the&KDSWHU6SHFLDOL]HGELRFKHPLFDOWHFKQLTXHV

amount of protein in each spot. Both methods will fix the proteins irreversibly
to the gel matrix. If unfixed proteins are required for further processing (e.g.
Western blotting or protein sequencing) alternative reverse staining methods
should be applied, such as copper staining (Lee et al., 1987) or zinc staining(Fernándes-Patron et al., 1992).
9.

Computerised analysis and database construction. The 2D PAGE results,
including stained gels, developed films or exposed phosphor-screens, can bescanned, and the resulting data stored in a computer with appropriate software,e.g. QUEST, ELSIE/MELANIE, TYCHO, GELLAB, HERMES or LIPS. The
S. coelicolor protein database is in the PDQUEST® format (PDI, Inc.,
Huntington Station, NY, USA). After spot detection and quantitation, 2D PAGE
images can be edited and matched to form a matchset. Each image in thematchset can then be compared, at individual spot level, to the other images or
to a standard image which consists of all matchable spots from all images in amatchset. Further statistical analysis can be performed on one specific spot ora group of selected spots. A protein database is thus created which allows
efficient data input and output, data organisation and update, and convenient
access to the research community.

In-vivo radioactive labelling of Streptomyces proteinsTwo-step liquid cultivation and radioactive labelling method for Streptomyces (Provided by X-M
Li and C J Thompson)
J medium, rich medium for seed culture
100 g
Sucrose30 g
Tryptone soya broth (p. 412)
10 g
Yeast extract
10 g
MgCl2.6H2O

Dissolve the components in distilled water andbring the volume to 1 l.
No pH adjustment is required.
Autoclave at 121o for 30 minutes.

MG medium, defined minimal medium for labelling, based on the MG medium described by Doull andVining (1989). 800 ml MG Base and 200 ml MG carbon source are prepared separately and mixed after
autoclaving. The pH after autoclaving should be around 6.0.

&KDSWHU6SHFLDOLVHGELRFKHPLFDOWHFKQLTXHVMG Base (800 ml)
0.2 g
MgSO4.7H2O
9 mg
FeSO4.7H2O
1g
CaCl2.2H2O
1g
NaCl
4.5 mlTrace mineral solutiona
21 g
MOPS b Sigma M 8899
11.23 gMonosodium glutamate.1H2O
15 ml
Phosphate buffer, pH6.5, 1M caTrace mineral solution stock (1 litre)
39 mg
Cupric sulphate.5H2O
5.7 mgBoric acid
3.7 mgAmmoniummolybdate.4H2O
6.1 mgManganese sulphate.1H2O
880 mg
Zinc sulphate.7H2O
Autoclave 121o, 23-30 minb

MG carbon source (200 ml)
50 g
Maltose.1H2O

3-morpholinopropanesulphonic acidMix 34 ml 1M KH2PO4 and 16 ml 1M K2HPO4

c

Seed culture

1.

Inoculate 107-108 spores into 10 ml J medium or YEME in a test tube (20 cm
long, 2.5 cm inner diameter) containing a stainless steel spring (p. 49).

2.

Incubate the tube at a 45 degree angle for 48 hrs, with shaking at 200 rpm, 30E.

3.

The culture should be very dense with well-dispersed mycelium. Wash twicewith sterile water or MG medium and resuspend in 10 ml complete MG medium.
This is the starting material for the main culture.

Main culture

4.

Inoculate 500 ml of complete MG medium in a baffled 2 l flask with 10 ml
washed seed culture.

5.

Incubate on a rotary shaker at 200 rpm, 30o.

Labelling

6.

At any desired time-point during the cultivation, transfer 1 ml culture to a 50 ml
Falcon® conical tube, or 5 ml to a 25 ml flask.

7.

Add 100 µCi (3,700 KBq) 35S-methionine to 1 ml culture or 500 µCi(18,500 KBq) to 5 ml culture.

&KDSWHU6SHFLDOL]HGELRFKHPLFDOWHFKQLTXHV

8.

Incubate the tube at a 45 degree angle for 40 min, with shaking at 200 rpm, 30E.

9.

Spin the mycelium down and keep the pellet at -20E.

Growth and radioactive labelling of Streptomyces on zirconium-silica beads (Provided by X.-M. Li
and C. J. Thompson)
This procedure is an alternative to growth on cellophane (p. 48). Zirconium-silica beads mimic
the texture of soil. The culture is easier to handle and allows for growth and radioactive labelling of thecolonies without changing the environment of the growing mycelium (especially nutritional and secreted
substances). This improves the accuracy and reproducibility of the experiments. Another advantage ofthis method is that agar is not used in the medium, thus eliminating a source of impurities, especially forstrains such as S. coelicolor that utilise agar.
Zirconium-silica beads, 0.1 mm in diameter, Cat. No. 11079101Z from Cole Parmer
Instruments (p. 419)
35
S-methionine
Bead-beater (p. 418).

1.

Distribute 6 g of zirconium-silica beads, in flat-bottomed 25 ml flasks to forma smooth, shallow bead layer. Cover with a piece of aluminium foil. Autoclave
the flask at 121o for 30 min.

2.

Inoculate Streptomyces spores into a suitable liquid medium (MG, MS, R5
without agar, etc.) at c. 107 ml–1.

3.

Pipette 1 ml into each flask, spreading equally all over the surface of the beadlayer.

4.

Put the flasks in a closed box with wet paper towel to maintain humidity.
Incubate the box at 30o.

5.

For 35S-methionine labelling in MG medium, apply 150 µCi (555 KBq) to eachflask, and shake slowly (25 rpm) for 2 h.

6.

Add 20 ml 0E water, shake a few times and withdraw the water from beneath thebeads. Repeat once.

7.

Store the washed culture at -20o. The mycelium can be lysed by using a Beadbeater.

&KDSWHU6SHFLDOLVHGELRFKHPLFDOWHFKQLTXHV

Detection and assay of S. coelicolor (and S. lividans) antibiotics
The S. coelicolor antibiotics
S. coelicolor A3(2), like many other streptomycetes, makes several chemically diverse antibiotics(Fig. 4.2). Historically the most notable is actinorhodin, a red-blue acid-base indicator pigment firstdescribed as “der rote Farbstoff” (“the red pigment”) in the Göttingen strain of S. coelicolor (Brockmann
and Hieronimus, 1955) and first studied genetically in S. coelicolor A3(2) by the isolation of blocked
mutants (Wright and Hopwood, 1976a; Rudd and Hopwood, 1979). Undecylprodigiosin, a red pigment
first described in other strains, was identified as an A3(2) product by Rudd and Hopwood (1980).
Methylenomycin, first described in S. violaceoruber (a close relative of the A3(2) strain) by Haneishi
et al. (1974), was identified in A3(2) derivatives carrying the SCP1 plasmid either autonomously (SCP1+)or chromosomally integrated (the NF state) (Wright and Hopwood, 1976b) and was shown to be specified
by the plasmid. The calcium-dependent lipopeptide antibiotic (CDA) was first described in S. coelicolor
A3(2) (Lakey et al., 1983) and was chemically characterised by Kempter et al. (1997).
Actinorhodin
It has been known for many years that S. coelicolor A3(2) colonies that become red-purple
because of actinorhodin production will rapidly turn blue on fuming the colonies with ammonia,
consistent with the known pH indicator properties of the compound (Rudd and Hopwood, 1979).
However, depending on the particular A3(2) derivative and on the growth conditions, cultures will often
produce copious extracellular blue pigment without ammonia fuming, and this also was assumed to be
actinorhodin. Recently the situation has been clarified with the demonstration that the spontaneous blue
colour is actually due to the lactone form of the antibiotic, -actinorhodin (Christiansen, 1970), and thatup to three genes in the act gene cluster are involved in the conversion of actinorhodin to -actinorhodin
and its export (Bystrykh et al., 1996). Conditions for the assay of the two compounds were described.
To analyse extracellular -actinorhodin, culture supernatants were acidified with 1N HCl to
pH2-3, and ½ vol of methanol: chloroform (1:1) was added. The sample was shaken vigorously andcentrifuged at 3000 × g for 5 min. The chloroform phase was collected and the A542 in chloroform wasmeasured ( 542= 18600 for the pure compound, Christiansen, 1970).
To analyse intracellular actinorhodin, mycelium grown under acidifying conditions (e.g.
ammonium instead of organic nitrogen source or nitrate) was harvested by centrifugation (15,000 × g for
10 min), washed twice with 0.1N HCl (to reduce impurities interfering with further extraction),
resuspended in 1N KOH, thoroughly mixed, and centrifuged (3000 × g for 5 min). The supernatant wascollected and the A640 in 1N KOH was measured ( 640 = 25320 for the pure compound). Alternatively,
actinorhodin was quantified in dioxane solution using 523 = 4200 (Brockmann and Hieronimus, 1955).
For this, KOH extracts were acidified to pH2-3 with 4N HCl, allowed to stand on ice for 15 min, thencentrifuged. Precipitated pigment was redissolved in dioxane.
To determine the total of actinorhodin and -actinorhodin combined, cultures were treated with
KOH (1N final concentration), centrifuged (3000 × g for 5 min), and the A640 of the supernatants wasdetermined.

&KDSWHU6SHFLDOL]HGELRFKHPLFDOWHFKQLTXHV

Fig. 4.2 The S. coelicolor antibiotics.

&KDSWHU6SHFLDOLVHGELRFKHPLFDOWHFKQLTXHV

Undecylprodigiosin
Undecylprodigiosin is often referred to as the “red pigment” of S. coelicolor A3(2), or “RED”(even though, as we saw above, this was the original name for actinorhodin). It is actually a mixture ofat least four prodiginines, with undecylprodigiosin and butylcycloheptylprodiginine predominating (Tsaoet al., 1985: Fig. 4.2). The red pigment is normally recovered after vacuum drying of mycelium byextraction into methanol, acidification with HCl (to 0.5M) and spectrophotometric assay ( 530 = 100,500).
CDA
CDA is a cyclic lipopeptide consisting of 11 amino acid residues with a 6-carbon hydrocarbon
chain at its N-terminus (Kempter et al., 1997). CDA production is assayed biologically by growing thestreptomycete on Oxoid Nutrient Agar and then overlaying with Soft Nutrient Agar containing Bacillus
mycoides and Ca(NO3)2 at a concentration to give 12mM throughout the plate. A zone of inhibition,
which is absent when Ca(NO3)2 is omitted, is diagnostic for CDA.
Methylenomycin
This SCP1-specified antibiotic is normally detected by its inhibitory effect on the growth anddevelopment of an SCP1– S. coelicolor culture when a producing strain is spotted onto a lawn of sporesof the indicator strain; controls are provided by using an SCP1+ strain as a producer and as the lawn
(which is then resistant to inhibition). It can be quantified by HPLC (Hobbs et al., 1992).

S. lividans as an antibiotic producer
S. lividans 66 also produces actinorhodin and undecylprodigiosin, but usually not so abundantly
as does S. coelicolor A3(2). Indeed, under many physiological conditions, very little pigment is
produced, a circumstance that allowed Horinouchi et al. (1983), and later others (e.g. Fernández-Moreno
et al., 1992; Romero et al., 1992), to clone regulatory genes from S. coelicolor and other strains by theirability to cause copious pigment production in S. lividans. Methylenomycin is not produced naturally
by S. lividans, but it acquires methylenomycin biosynthesis and resistance when SCP1 is transferred into
it by conjugation. Originally it was noted that S. lividans was resistant to CDA - most other species weresensitive - though it appeared not to produce CDA. Recently this finding has been contradicted by thediscovery that S. lividans is a conditional producer of CDA (Podmore, 1995).
Secondary metabolite production in S. lividans transformants: a note of caution
The innate, but “semi-cryptic”, capacity of S. lividans 66 and its derivatives to produce
antibiotics is of some significance because of the use of S. lividans as a surrogate host for the cloning ofantibiotic biosynthetic genes from other actinomycetes. Experimenters have certainly been misled on
occasion into concluding that a compound made by an S. lividans clone was the product of the cloned
genes, when its biosynthesis was actually directed by host genes that had become activated, in partprobably as a “shock” response.

&KDSWHU6SHFLDOL]HGELRFKHPLFDOWHFKQLTXHV

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Bystrykh LV, Fernández-Moreno MA, Herrema JK,
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&KDSWHU6SHFLDOL]HGELRFKHPLFDOWHFKQLTXHV

m05tkb.wpd 13 March 2000 Tobias Kieser

Chapter 5

Mutagenesis of Streptomyces by irradiation or
chemicals
CONTENTS

General remarks about mutagenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
The optimal amount of mutagenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
Expression of mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
Spores or mycelium? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
Choice of mutagen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
Precautions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
Mutagenesis of Streptomyces spores by ultraviolet light (UV) . . . . . . . . . . . . . . . . . . . . . . . . . . 102
Mutagenesis of Streptomyces spores by N-methyl-N0-nitro-N-nitrosoguanidine (NTG) . . . . . 103
Isolation of specific classes of mutant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
Resistant mutants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
Mutants that have to be isolated by screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
Auxotrophs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
Other classes of mutants screened by replica plating . . . . . . . . . . . . . . . . . . . 106
Mutants recognised by visual examination . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106

General remarks about mutagenesis
The optimal amount of mutagenesis
It is not surprising that all mutagens kill a proportion of the treated cells. This in part reflects
the induction of “lethal” mutations (a relative term, since a mutation may be lethal under someconditions, but not others), but this is certainly not the whole story. Otherwise it would be difficult toaccount for the enormously different proportions of a given mutant type at the same level of survival after
treatment with different mutagens. Thus some agents cause much non-mutagenic killing and others
relatively little. N-nitroso-N0-nitro-N-nitrosoguanidine (NTG) is an example of a mutagen in the second
category and it is therefore useful when high absolute numbers of mutants are needed. For any mutagen,the absolute number of viable mutants in the population will increase with dose up to a certain level, andthen fall as killing overtakes the induction of new mutants. However, in most situations, it is not theabsolute number but the proportion of mutants amongst the survivors that is the important thing, since&KDSWHU0XWDJHQHVLVRI6WUHSWRP\FHVE\LUUDGLDWLRQRUFKHPLFDOV

only the survivors have to be screened. This proportion in general also increases with dose, sometimes
in an approximately linear fashion, as would be expected on simple first principles, though often
proportionally to a higher power of the dose, up to a dose well above the optimum for absolute numbers.
Eventually, with increasing dose, the proportion of mutants often begins to fall. This may be due to thestarting population having been heterogeneous (for genetic or physiological reasons) in respect ofsensitivity to the killing and mutagenic effects of the mutagen so that, at high doses, the more resistantcomponent comes to make up an increasing proportion of the survivors. This is seen in a plot of thelogarithm of the total survivors against dose, when the expected straight line for one-hit killing gradually
tails off at high doses (Hopwood, 1970). Thus for each mutagen, and each organism, and even each classof mutants, there is usually an optimum “dose” (a combination of mutagen concentration, time andconditions of treatment) which leaves the highest proportion of a particular mutant class amongst thesurvivors.
However, we may not necessarily want to aim for the highest proportion of mutants obtainable,
because this may lead to “over-mutagenesis”. For example, NTG is such a potent mutagen that
auxotrophic mutants may constitute up to, say, 20% of the survivors when the maximum level isachieved. Assuming that mutations to auxotrophy occur at random in the population, if 20% of thesurvivors are auxotrophic mutants, one-fifth of them will harbour more than one auxotrophic mutation,and other classes of non-lethal mutation will also be carried by the auxotrophs. Thus a high proportionof the isolated mutants will be troublesome to characterise phenotypically, and their genetic analysis will
also be complex. Probably mutagenesis should be moderated, to yield a smaller proportion of multiple
auxotrophs.
As explained in detail by Baltz (1986), there is a particular reason for avoiding over-mutagenesis
when searching for mutants with enhanced antibiotic productivity. This is because productivity is
influenced by a very large number of genes, and most mutations in them will have a negative effect.
Thus, at high dose rates, any yield-enhancing mutation is likely to end up in the same survivor as at least
one yield-reducing mutation and may well be unable to exert its effect. The optimum dose is therefore
expected to be one which results in an average of just a single productivity-affecting mutation (positiveor negative) per survivor. Based on the Poisson model of random mutagenesis, an average of one
mutation per survivor means that 37% of the survivors, would carry none and so be unchanged in
antibiotic productivity (Baltz, 1986). This value can be determined experimentally: in a large-scale
screening operation, in which the productivity of many survivors of the mutagenic treatment is measured
and recorded, it will emerge from the data and so mutagenesis can be monitored and adjusted if
necessary.
Expression of mutations
In order for a mutagenic treatment to lead to the isolation of a desired mutant strain, mutations
have to be expressed. This term covers several different processes (Hopwood, 1970). First the DNA
damage caused by the mutagen may have to undergo error-prone repair in order to cause a heritable
mutation. Next, segregation may be needed to convert a mutation established as a base change on only
one strand of the DNA to the double-stranded condition. When a cell or hyphal segment carrying amutation contains more than one copy of the gene, the mutant form must also be segregated into a
different cell from any wild-type alleles. Finally, it may take time, and further segregational events,

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before the mutant phenotype becomes established because there may still be expression of the phenotype
conferred by the wild-type allele. Such “phenotypic lag” is illustrated by streptomycin-resistance, whichis expressed only when pre-existing streptomycin sensitive ribosomes are diluted out by ribosomes thatare resistant because of mutation in a ribosomal protein gene.
Taking account of segregational delay and phenotypic lag is not so straightforward with amycelial organism like Streptomyces as with a unicellular bacterium or a yeast. The easiest solution is
to grow a mass culture from the mutagen-treated spores on a non-selective agar medium and make a spore
suspension from the resulting culture. Mutants are then sought amongst these “expressed” spores.
Disadvantages are that mutants may tend to be lost by competition with wild-type individuals during theexpression period, and that clonal copies of the same mutation may arise, so in general only one mutant
of a given phenotype should be saved from each spore population.
Spores or mycelium?
We always treat spore suspensions, rather than mycelial fragments, with mutagens because they
provide the easiest access to a large population of unigenomic, haploid cells, which are needed if mutants
are to be found with a minimum of segregational delay. The Lilly group, on the other hand, has madeextensive use of mycelial fragments; this provides a clear advantage for poorly-sporulating strains.
Details are in Baltz (1986).
Choice of mutagen
We have used only two agents for routine mutagenesis in Streptomyces: short wavelength
ultraviolet light (UV) and NTG. Use of other mutagens (hydroxylamine; 4-nitroquinoline-1-oxide; ethyl
methanesulphonate; methyl methanesulphonate) is described by Baltz (1986); none of these has foundsuch wide published application as NTG. This is also true of long wavelength (near) ultraviolet light in
the presence of 8-methoxypsoralen (NUV + 8-MOP), which was described in the predecessor of thisbook.
UV is a very convenient, comparatively safe mutagen, but it may fail to be effective in somespecies, either because a strain lacks the error-prone repair system needed to convert premutagenic
lesions (pyrimidine dimers) into heritable mutations, or for unknown reasons, as in S. fradiae (Baltz,
1986). NTG is a very effective mutagen when used under appropriate conditions (which are rather
different from those employed in E. coli: Deliì et al., 1970, p. 103); it yields the highest mutant
frequencies of any mutagen at low lethality (for example up to 20% auxotrophs) when used under
optimal conditions of time, concentration and pH. In some other organisms NTG has been found to cause
clustered lesions in DNA and very often to induce additional unwanted mutations. The latter effect is
not well documented in Streptomyces, but the phenomenon of clustered mutagenesis was reported in S.
coelicolor (Randazzo et al., 1976). NTG may well be the mutagen of first choice, and should certainly
be considered seriously when a desired class of mutation is not found after UV mutagenesis.
Precautions
Except with ionizing radiations such as X-rays, there is little likelihood of the experimenter
accidentally inducing mutations in his or her own gonads. However probably all mutagens are&KDSWHU0XWDJHQHVLVRI6WUHSWRP\FHVE\LUUDGLDWLRQRUFKHPLFDOV

carcinogenic under some conditions, as well as being potentially harmful in other ways, so that great careshould always be taken in their handling and disposal after use.

Mutagenesis of Streptomyces spores by ultraviolet light (UV)
Spore suspension of the Streptomyces strains, freshly prepared or frozen in 20%
glycerol (p. 44)
Dilution tubes containing sterile water (or 20% glycerol) for survival curves
Plates of suitable agar medium (for direct plating) or slants or plates of non-selective
medium (for inoculation for mutant “expression”)
Glass Petri dish for irradiation; glass spreaders; pipettes (10 ml, 1 ml)“Germicidal” (low pressure mercury vapour) UV lamp (emitting most of its energy
at 254 nm), in a yellow-lit photographic darkroom (Wratten OB filter or sodium
vapour lamp (589 nm) of the kind used in street lighting), or dimly lit laboratory, if thestrain has a photoreactivation system; 30o incubator.

1.

Make sure that the UV lamp has been switched on for at least 15 min before use
to allow it to reach maximum emission.

2.

Place the spore suspension (e.g. 10 ml) in a glass Petri dish. (Remove a sample
if an unirradiated control is needed.) The spores must be suspended in a medium that
does not absorb at 254 nm: water or 20% glycerol are suitable, but Tris-containing buffers arenot. A glass Petri dish is recommended because the surface is easily wetted by the spore
suspension.

3.

Irradiate the suspension under the UV lamp with constant agitation (protect yourskin and eyes from irradiation) for an appropriate time (timing is conveniently
done by removing the lid of the dish for the correct interval). Agitation is needed
to avoid “shading” by the spores in a dense suspension and because UV penetrates poorly even
“UV transparent” solutions; for short periods this can be done by hand, or a flatbed shaker could
be used. The most appropriate time of irradiation is best determined by first plotting a dosesurvival curve (plate samples of the suspension after different periods of irradiation and countthe resulting colonies) and then using the time that gives a high kill (e.g. 99 or 99.9%) but notso high as to cause the survival curve to “tail” at high dose. Some strains (e.g. S. lividans 66)
have a photoreactivation (PR) system, while others (e.g. S. coelicolor A3(2)) lack one. PR (bylight of wavelength shorter than c. 525 nm) repairs a proportion of potentially mutagenic UVinduced lesions in DNA (pyrimidine dimers), resulting in the need for a higher UV dose toachieve the desired result, and, in practice, poorly reproducible results. If the strain has a PR
system, carry out the irradiation and later platings under a suitable yellow safelight.

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4.



Inoculate slants or plates of non-selective medium with the dense irradiatedspore suspension (if “expressing” the mutations) or plate out appropriate
dilutions on a suitable medium if not.

Mutagenesis of Streptomyces spores by N-methyl-N0-nitro-N-nitrosoguanidine(NTG) (Deliì et al., 1970)
Spore suspension of the Streptomyces strain, freshly prepared or frozen in 20%
glycerol (p. 44)
N-methyl-N0-nitro-N-nitrosoguanidine (NTG or MNNG). HAZARDOUS
TM buffer: make up a solution containing 0.05M Tris base and 0.05M maleic acid andadjust to pH8 or 9 (see note 3)
Dilution tubes containing sterile water (or 20% glycerol)
Plates of suitable agar medium (for direct plating) or slants or plates of non-selective
medium (for inoculation for mutant “expression”)
1N NaOH
Pipettes (10 ml and 1 ml); glass spreaders
Balance; fume cupboard; bench-top centrifuge and sterile tubes; 30o incubator

1.

Weigh out a small quantity of NTG crystals (e.g. 3-12 mg: see note 3) into a preweighed sterile centrifuge tube. NTG is believed to be most dangerous (as a potential
carcinogen) in the dry state when small crystals might be inhaled. Weighing might therefore bedone in a fume cupboard, taking precautions not to spill any of the mutagen on the balance or
its surroundings. Do not try to weigh out the precise amount; note the weight and achieve thecorrect final concentration of NTG by adjusting the volume of spore suspension added in step
3.

2.

Pellet the spores in the bench-top centrifuge and re-suspend in TM buffer ofsuitable pH (see note to step 3).

3.

In a fumehood, add a suitable measured volume of the spore suspension in TM
buffer to the NTG crystals to give the correct final NTG concentration and
immediately agitate on a vortex mixer until the crystals are completely dissolved.
The concentration of NTG should be 1-3 mg ml–1 for most Streptomyces strains. We usually use
1 mg ml–1 at pH8 for 1 h for routine mutagenesis. 3 mg ml–1 is the highest concentration that canreadily be obtained. At pH9 NTG decomposes very rapidly to the mutagenic diazomethane,
resulting in a very intense burst of mutagenesis (Deliì et al., 1970). Therefore the most intense
convenient treatment is 3 mg ml–1 at pH9 for 2 h.

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

4.

Incubate at 30o for 1-2 h in a fume cupboard.

5.

Pellet the spores in the bench-top centrifuge and re-suspend in water (forimmediate use) or 20% glycerol (for freezing). NTG solutions and contaminatedglassware can be made safe by immersion in a container of alkali (e.g. 1N NaOH) in a fume
cupboard for a few hours.

6.

Inoculate slants or plates of non-selective medium with the mutagenised spore
suspension (if “expressing” the mutations) or plate out appropriate dilutions ona suitable medium if not.

Isolation of specific classes of mutant
Resistant mutants
A convenient feature of mutants that are resistant to an agent to which the wild-type is sensitive whether this is an antibiotic, a synthetic chemical, a metal salt or a bacteriophage - is that the mutant canbe directly selected by plating on a medium containing the inhibitory agent. For this class of mutation,
one can sometimes dispense with mutagenesis and select for rare spontaneous mutants pre-existing in
the population (choosing only one from the descendants of any culture derived from each independentcolony to avoid picking members of clones). If mutagenesis is used, it is crucial to allow time for the“expression” of induced mutations (p. 100).
Mutants that have to be isolated by screening
Auxotrophs
No effective procedure for a penicillin-type enrichment for auxotrophs has been properly
documented for Streptomyces, so screening by replica plating is normally used. In our experience inisolating auxotrophic mutants, replication to a set of plates each containing a pool of potential growthrequirements such that each compound is present in two different pools (Holliday, 1956) is notstraightforward in Streptomyces. In theory, a typical simple auxotroph is identified by its growth on two
pools of the set, but many ambiguous reactions are usually encountered, probably caused by effects ofthe various combinations of metabolites on the physiology of the organisms. When the object is simply
to seek a sample of auxotrophs for use as genetic markers, rather than to isolate a specific class of mutant,
we have usually proceeded as follows:1.

Plate the “expressed” spore suspension ( p. 101) on complete medium (CM) toyield c. 100-150 colonies per plate.

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

2.

Replicate the resulting colonies to minimal medium (MM).

3.

Compare the replicas and the originals to identify potential auxotrophs (manyStreptomyces auxotrophs are slightly leaky under these conditions, and in any
case well-sporulating colonies leave a visible patch of spores on the replica
plate, even if they do not grow; thus auxotrophs are usually best recognised by
scrutinising the replica plate very carefully under appropriate illumination to
identify the “ghost” colonies, as well as comparing the pattern of colonies on
replicas made on MM and CM).

4.

Pick possible auxotrophs to a grid of 20 squares (p. 132) on CM.

5.

Replicate the resulting patches of growth to MM.

6.

Identify potential auxotrophs; many of the original candidates fail at this step,
perhaps because they were mixed, unstable or leaky.

7.

Make a new master plate of the candidates on CM.

8.

Replicate the resulting patches to a series of plates containing, in nine groups,
the 25 metabolites likely to be required by useful auxotrophs (see below). Someof the groups are chosen rationally to allow for expected double (or multiple)
requirements - e.g. isoleucine + valine; adenine + thiamine; arginine + uracil;
methionine + threonine; phenylalanine + tyrosine + tryptophan - while others arearbitrary and contain compounds unlikely to interfere with each other. Ourpreferred set of nine combinations is as follows (see p. 415 for concentrations):
A.
B.
C.
D.
E.

9.

arginine, uracil, thymine
phenylalanine, tyrosine, tryptophan
methionine, cystine, threonine
isoleucine, valine, leucine
serine, glycine

F.
G.
H.
I.

histidine, glutamic acidproline, lysine
adenine, guanine, thiamine
nicotinamide, p-aminobenzoic acid,pyridoxine, riboflavine

Confirm and further characterise the identified auxotrophs by plating on
individual compounds, or pairs of compounds, as indicated by the plate on which
the mutant grew at step 8.

&KDSWHU0XWDJHQHVLVRI6WUHSWRP\FHVE\LUUDGLDWLRQRUFKHPLFDOVOther classes of mutants screened by replica plating
Replica plating has been used to isolate several other classes of mutants besides auxotrophs.
Temperature-sensitive mutants are identified using pairs of replicas incubated at 30o and 38o (themaximum growth temperature for S. coelicolor: Hopwood, 1966); and ultraviolet sensitive mutants wererecognised by using pairs of replicas, one of them exposed to UV radiation (Harold and Hopwood, 1970).
Phage-sensitive mutants - such as the pgl (phage growth limitation) mutants for the phage C31 forexample (p. 276) - are seen by replicating to plates spread with a phage suspension (Lomovskaya et al.,1972); and mutants that cannot use a sugar or nitrogen source by replication to a medium containing theappropriate compound. For this, the ability of S. coelicolor A3(2) to utilise agar as carbon source poseda practical problem, but this can be suppressed by adding homoserine at. 0.5% (w/v) (Hodgson, 1980).
Mutants recognised by visual examination
Several kinds of mutants can be isolated by visual inspection of colonies growing directly on themedium used for plating out the “expressed” spore population. These include differentiation mutants,
either bld (bald) mutants that fail to produce aerial mycelium (Merrick, 1976) or whi mutants that
produce aerial mycelium but no spores (strictly speaking, no spore pigment) (Chater, 1972). Obviously
a medium that promotes good sporulation of the wild-type is a pre-requisite to avoid too many false
negatives; but choice of medium may also be crucial because some bld mutants are conditional,
depending on the carbon source. Some classes of bld mutants have been found to sporulate in response
to extracellular stimulation by adjacent wild-type colonies (Willey et al., 1991) and these can be missedon crowded plates. Other mutants can be recognised by colour reactions, for example for an enzyme suchas urease (Hopwood, 1972).
Sometimes - when the antibiotic is pigmented - antibiotic non-producing mutants canconveniently be recognised by visual selection of mutants lacking or altered in pigmentation (Rudd andHopwood, 1979, 1980). Usually, however, screening for antibiotic non-producers is more laborious. It
is rarely possible to pick them reliably by overlaying the primary plates with a sensitive indicator
microorganism because antibiotic production by producer colonies usually interferes with the detectionof non-producers. Normal practice is to pick the colonies individually for testing, for example in smallliquid cultures, used in many high throughput screens; on isolated agar blocks (Ichikawa et al., 1971);
or in segmented dishes (Hopwood and Wright, 1983).

Baltz RH (1986) Mutagenesis in Streptomyces spp. In
Demain, A.L. and Solomon, N.A. (eds.), Manual of Industrial
Microbiology and Biotechnology. American Society for
Microbiology, Washington DC, pp. 184-190.
Chater KF (1972) A morphological and genetic mapping
study of white colony mutants of Streptomyces coelicolor.
J.Gen.Microbiol., 72, 9-28.
Deliì V, Hopwood DA, Friend EJ (1970) Mutagenesis by
N-methyl-N’-nitro-N-nitrosoguanidine (NTG) in Streptomyces
coelicolor. Mut.Res., 9, 167-182.
Harold RJ, Hopwood DA (1970) Ultraviolet-sensitive
mutants of Streptomyces coelicolor I. phenotypiccharacterisation. Mut.Res., 10, 427-438.
Hodgson DA (1980) Carbohydrate utilization in

Streptomyces coelicolor A3(2). Ph.D.Thesis, University of
East Anglia, Norwich.
Holliday R (1956) A new method for the identification ofbiochemical mutants of micro-organisms. Nature, 178, 987.
Hopwood DA (1966) Nonrandom location of temperaturesensitive mutants on the linkage map of Streptomyces
coelicolor. Genetics, 54, 1169-1176.
Hopwood DA (1970) The isolation of mutants. In Norris, J.R.
and Ribbons, D.W. (eds.), Methods in Microbiology, Vol. 3A.
Academic Press, London, pp. 363-433.
Hopwood DA (1972) Genetic analysis in microorganisms. In
Norris, J.R. and Ribbons, D.W. (eds.), Methods in
Microbiology, Vol 7B. Academic Press, London, pp. 29-158.
Hopwood DA, Wright HM (1983) CDA is a new

&KDSWHU0XWDJHQHVLVRI6WUHSWRP\FHVE\LUUDGLDWLRQRUFKHPLFDOV
chromosomally-determined antibiotic from Streptomyces
coelicolor A3(2). J.Gen.Microbiol., 129, 3575-3579.
Ichikawa T, Date M, Ishikura T, Ozaki A (1971)
Improvement of kasugamycin-producing strain by the agar
piece method and the prototroph method. Fol.Microbiol., 16,
218-224.
Lomovskaya ND, Mkrtumian NM, Gostimskaya NL,
Danilenko VN (1972) Characterisation of temperate
actinophage C31 isolated from Streptomyces coelicolor
A3(2). J.Virol., 9, 258-262.
Merrick MJ (1976) A morphological and genetic mapping
study of bald colony mutants of Streptomyces coelicolor.
J.Gen.Microbiol., 96, 299-315.
Randazzo R, Sciandrello G, Carere A, Bignami M, VelcichA, Sermonti G (1976) Localized mutagenesis in
Streptomyces coelicolor A3(2). Mut.Res., 36, 291-302.
Rudd BAM, Hopwood DA (1979) Genetics of actinorhodinbiosynthesis by Streptomyces coelicolor A3(2).
J.Gen.Microbiol., 114, 35-43.
Rudd BAM, Hopwood DA (1980) A pigmented mycelial
antibiotic in Streptomyces coelicolor : control by achromosomal gene cluster. J.Gen.Microbiol., 119, 333-340.
Willey J, Santamaría R, Guijarro J, Geistlich M, Losick R(1991) Extracellular complementation of a developmental
mutation implicates a small sporulation protein in aerial
mycelium formation by Streptomyces coelicolor. Cell, 65,
641-650.

&KDSWHU0XWDJHQHVLVRI6WUHSWRP\FHVE\LUUDGLDWLRQRUFKHPLFDOV

m6tkb.wpd 20 April 2000 Tobias Kieser



Chapter 6

Transposon mutagenesis in Streptomyces
CONTENTS

General points about transposon mutagenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
Discovery of Streptomyces transposable elements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
Streptomyces transposons suitable for mutagenesis of Streptomyces . . . . . . . . . . . . . 110
Use of heterologous transposons for mutagenising Streptomyces genes . . . . . . . . . . . 110
Transposon delivery vectors for Streptomyces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
Isolation of independent mutant strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
Cloning transposon-tagged Streptomyces DNA in E. coli . . . . . . . . . . . . . . . . . . . . . . . 111
Tn4556 from S. fradiae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
A. Transposition of Tn4560 from pUC1169 to a chromosomal location . . . . . . . . . . 112
B. Protocol for inserting Tn4560 into S. coelicolor NF strains . . . . . . . . . . . . . . . . . . 113
C. Tn4560 mutagenesis of SCP2* plasmids in S. lividans . . . . . . . . . . . . . . . . . . . . . . 114
D. Tn4560 mutagenesis of pock-forming SCP2* in S. coelicolor . . . . . . . . . . . . . . . . . 115
IS493 from S. lividans 66 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
E. Sectoring method for delivering IS493 derivatives using temperature-sensitive
plasmids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
IS6100 from Mycobacterium fortuitum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
F. Transposon mutagenesis of S. lividans using pSIT151 . . . . . . . . . . . . . . . . . . . . . . 119
Tn5493 derived from Tn5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
G. Mutagenising S. lividans using pJOE2577 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
H. Alternative method for isolating S. lividans with random insertions of Tn5493 . 122

General points about transposon mutagenesis
Transposable elements that insert in vivo, fairly randomly and stably, into DNA can be very
useful for generalised mutagenesis and gene tagging. Transposon insertions usually inactivate genes
completely and unconditionally, and often exert polar effects on downstream genes in operons.
Sometimes they also induce DNA rearrangements in their vicinity. Some transposable elements containpromoters that activate silent genes, or resolution sites that stabilise the inheritance of some plasmids.
Reviews describing non-Streptomyces transposons and their applications include: Berg and Howe (1989);
Berg et al. (1989); Calos and Miller (1980); De Lorenzo and Timmis (1994); Grindley and Reed (1985);
Kleckner (1981, 1990); Kleckner et al. (1977); Miller (1992); Reznikoff (1993).

&KDSWHU7UDQVSRVRQPXWDJHQHVLVDiscovery of Streptomyces transposable elements
Several transposable elements have been discovered in streptomycetes (see review by Kieser andHopwood, 1991). Surprisingly, none of them naturally contains a selectable marker. Initially, they were
found inserted into under-packaged phages, deleterious cloned genes, or unstable plasmids. Solenberg
and Burgett (1989) developed an SCP2*-derived vector, pCZA126, specifically to trap transposons.
More transposons are emerging from sequence analysis.
Streptomyces transposons suitable for mutagenesis of Streptomyces
Tn4556 from S. fradiae UC8592, and IS493 from S. lividans 66 have been developed as tools
for random mutagenesis of Streptomyces. Derivatives of IS1373 from S. lividans (flanking AUD2) havealso been shown to transpose efficiently in S. lividans strains that lack this sequence (Volff and
Altenbuchner, 1997). (IS117 from S. coelicolor A3(2) integrates into preferred sites in S. lividans andhas been developed as an integrating vector; p. 267.) Different protocols have been developed for thedelivery of each of these transposons; they are described as specific examples, with notes facilitating
adaptation for different transposons and strains. Transposon maps are in Chapter 22.
Use of heterologous transposons for mutagenising Streptomyces genes
Smith and Dyson (1995) constructed Tn1792, a derivative of IS6100 from Mycobacterium
fortuitum, for transposon mutagenesis in S. lividans. Volff and Altenbuchner (1997) constructed a Tn5
derivative, Tn5493, that transposes at high frequency in S. lividans. Tn5 and Tn10 readily transpose intohigh G+C Streptomyces DNA cloned in E. coli, and selection for the Tn5 neo marker has been used to
introduce mutated genes into Streptomyces by gene replacement (Occi et al., 1993; Richardson et al.,1990).
Note that several resistance genes from transposons of Gram-negative bacteria are useful for
selection in Streptomyces: neo of Tn5, km of Tn903, cat of Tn9, aacC1 from Tn1696, and aadA from
Tn7. See Chapter 21 for maps.
Transposon delivery vectors for Streptomyces
The transposon delivery vector is very important. Ideally, such a vector can be introduced
efficiently into the target strain, so that many independent transformants and transposon insertions canbe isolated. After transposition, unless a cointegrate is sought (see IS6100), the delivery vector must be
eliminated ("cured") so that the rare progeny with transposon insertions in the genome can be selectedusing the transposon marker.
Wide host-range, multicopy plasmid vectors derived from the naturally temperature sensitive
pSG5, and temperature-sensitive mutant derivatives of pIJ702, pMT660 and pKU112 ( Ikeda et al., 1993)
have been used for transposon delivery to the chromosome and to plasmids. Note that pIJ702 itself, as
well as the temperature-sensitive derivative pMT660, are unstable in NF strains of S. coelicolor.

&KDSWHU

7UDQVSRVRQPXWDJHQHVLVFor the mutagenesis of plasmids, it is most convenient to start with strains containing thetransposon integrated into the chromosome. Plasmids containing transposon insertions can then be
selected after transformation or conjugal transfer to a new host.
Isolation of independent mutant strains
For mutagenesis, independent clones containing transposon insertions must be isolated. The
number of independent clones needed to saturate a genome cannot be estimated mathematically, because
most transposons have some sequence preference. Random transposition of Streptomyces should givec. 2% auxotrophs with many different nutritional requirements. Determination of spontaneoustransposition frequencies requires a fluctuation test (Luria and Delbrück, 1943).
Unfortunately, none of the Streptomyces transposons has useful restriction sites in the terminal
inverted repeats. This means that for the precise restriction mapping of the insertion sites, the orientationof each transposon insert must be determined. Transposons with rare AseI, AflII, DraI, PacI, SpeI, SspI,
etc. sites can facilitate PFGE macro-restriction mapping in Streptomyces (p. 197).
Cloning transposon-tagged Streptomyces DNA in E. coli
Non-restricting E. coli strains should be used: EcoK (hsdR+) restricts unmethylated DNA; mcr
and mrr systems of E. coli K restrict methylated DNA. Non dam-methylated bifunctional plasmids
excised from the Streptomyces genome and religated transform dam+ E. coli strains at low efficiency (PDyson, personal communication).

Tn4556 from S. fradiae
Tn4556 is a 6625 bp 68% G+C transposon from S. fradiae UC8592, discovered at the Upjohn
Company by Chung (1987), and sequenced by Siemieniak et al. (1990). Tn4556 resembles Tn3 in the
38 bp terminal inverted repeats (70% identical), the 97 kd transposase (61% identical), the putative 16 kd
resolvase (45% identical), and a conserved cointegrate resolution site. The functions of two other ORFs
are not known: one resembles mercuric reductases (but does not confer mercury resistance) and anothermight be an O-methyl-transferase. Insertion creates 5 bp duplications of the target sequence. (SeeSherratt, 1989, for a detailed review about Tn3.)
Several Tn4556 derivatives with vph and neo resistance, and lux or xylE indicator genes, havebeen constructed and are described in Chapter 22.
Tn4560, containing the viomycin phosphotransferase gene vph, was first to be used fortransposon mutagenesis. It transposes efficiently (0.1-1% of progeny) from the temperature-sensitive,
multicopy pUC1169 to the chromosome of several Streptomyces strains. Tn4560 also transposes, withsimilar frequency, from pUC1169, or from chromosomal locations, to SCP2* and autonomously
replicating SLP1 derivatives. (Surprisingly, no Tn4560 insertions could be obtained in the naturally
temperature-sensitive pSG5 derivatives, wild-type pIJ101 or the linear plasmid SLP2.) Tn4560 stabilises
unstable ( Stability) SCP2* derivatives (p. 263) and the plasmid prophage SF1, possibly because it
provides a resolution function (Brolle et al., 1993).

&KDSWHU7UDQVSRVRQPXWDJHQHVLVTransposition of Tn4560 is not completely random. Ikeda et al. (1993) observed a wider target
range in S. avermitilis if transposition occurred at 39E rather than at 30E.
As for Tn3, only one copy of Tn4560 seems to be tolerated per replicon: transformation ofstrains containing a Tn4560 insert with a differently marked Tn4556 derivative results in efficienttransposon exchange, presumably by site-specific recombination at the transposon ends. These elements
can therefore also be used as integrative vectors (Chung and Crose, 1990).
For practical reasons, the protocols that have been used for Tn4560 mutagenesis produce poolsof mutant colonies, some of which contain identical, clonally related transposon insertions. If phenotypic
screening is difficult, the number of pools should be increased and their sizes reduced. Viomycin
resistance, conferred by the Tn4560 vph gene, is best selected on MM (low salt concentration isimportant). The protocols can be simplified for other resistance markers as indicated in the notes after
the procedure steps. Protocol A is a generally applicable protocol producing plasmid-free transposoncontaining Streptomyces strains. Protocol B is specific for use with NF S. coelicolor strains. Protocols
C and D are for transposon mutagenesis of Streptomyces plasmids.
A. Transposition of Tn4560 from pUC1169 to a chromosomal location (Chung, 1987)
pUC1169 (=pMT660::Tn4560) DNA fortransformation
Streptomyces protoplasts for transformation
R2YE and MM, supplemented for the markersof the target strain and for the desired
resistances: thiostrepton (50 µg ml–1); viomycin(concentration depends on medium; 30-50 µg
ml–1 for S. coelicolor or S. lividans on MM)

1.

vph
Tn4560pMT660tsr

Introduce pUC1169 by transformation into
Streptomyces protoplasts. Spread the transformed protoplasts onto several
plates, regenerate non-selectively, and replica-plate to select viomycin resistant(transposon marker) transformants at 39E. Incubation at 39E increased the randomness
and also the frequency of transposition in S. avermitilis (Ikeda et al., 1993).
With markers other than vph, transformants can be selected by overlaying theregeneration plate. Selection for the tsr marker on the vector, pMT660, would also work, but
it may yield fewer independent transposon-induced mutations. Using a large pool oftransformants increases the chance of obtaining many independent transposition events.

2.

Harvest the cells separately from the different transformation plates, andpropagate at 39E without antibiotic selection. Harvesting the spores or mycelium andre-plating at high density is better than replica plating, which may transfer too few spores. Nonselective growth at 39E improves the frequency of plasmid curing. Loss of pUC1169 isinfrequent if its high copy number is maintained by selection for the vector or the transposon
markers.

&KDSWHU

7UDQVSRVRQPXWDJHQHVLVAdditional rounds of non-selective growth may be necessary to achieve sufficient
plasmid curing. Alternatively, consider destabilizing pUC1169 by introducing incompatible
SCP1 or a Sti+ pIJ101 derivative(p. 256); these plasmids may interfere with later experiments.
Some pUC1169 may persist integrated in the chromosome as cointegrate transpositionintermediates; 100% curing of the tsr marker may be impossible.
Use of conjugation to separate the pUC1169 (and similar plasmids) from chromosomes
containing transposon insertions is described in Protocol B.

3.

Harvest spores and plate at 39E for single viomycin resistant colonies. Screen
for the desired phenotype(s) and confirm absence of the tsr plasmid marker. In
principle, only one VioR ThioS colony of each desired mutant phenotype should be picked,
because other phenotypically identical colonies might be of clonal origin. However, Tn4560
transposes very efficiently, and therefore colonies from the same pool often contain independent
insertions; these can be checked by Southern hybridisation, using labelled Tn4560 as probe.

B. Protocol for inserting Tn4560 into S. coelicolor NF strains (Schauer et al., 1991)
S. coelicolor NF strains contain most of the 350 kb linear plasmid SCP1 integrated in the
9 o'clock region of the chromosome. In NF × SCP1– crosses, the NF chromosome is transferred to SCP1–
strains with a bidirectional marker gradient and all the recipients inherit the NF fertility properties (Fig.
7.1, p. 129). When inserted into the chromosome, Tn4560 is transferred like any other marker, but
pUC1169 is not mobilised along with the chromosome. Additionally, pUC1169 is destabilised by SCP1.
If the NF donor strain is counterselected using a distal marker (e.g. cysD), most of the VioR progeny haveTn4560 inserted into the chromosome and lack pUC1169 (ThioS). Note that SCP2* should be absent
from both the NF and SCP– strains because it is a favoured target for Tn4560 and transfers with 100%efficiency.
The marker gradient makes the recovery of transposon insertions near the SCP1 insertion site
more likely than at distal sites, but for about half the chromosome, centred on the NF integration site,
NF × SCP1– crosses give the highest chromosome mobilising ability. All the markers are inherited atsimilar frequencies in NF × NF crosses (fertility - 1%; p. 126) which would be more suitable for theisolation of transposon insertions nearer the chromosome ends.
NF/pUC1169
vph
Conjugation

cys-

tsr

NF

and recombination

cys+

NF

pUC1169

pUC1169 (or a similar plasmid)
Spores of S. coelicolor NF strain containing "distal" counterselectable marker
Spores of S. coelicolor SCP1– strain

&KDSWHU7UDQVSRVRQPXWDJHQHVLVMedium for crosses (R2YE or HT agar and selection (MM), supplemented forrequirements of the strains and for the desired resistances; thiostrepton (50 µg ml–1);
viomycin (concentration depends on medium; 30-50 µg ml–1 for S. coelicolor or
S. lividans on MM)

1.

Prepare an S. coelicolor NF strain containing pUC1169. This can be achieved either
by protoplast transformation or by crossing an SCP1– SCP2–/pUC1169 strain with an SCP2– NF
strain; most ThioR VioR recipient-type progeny will be NF, recognisable by methylenomycin
production. It is best to work with several independent strains to obtain clonally independenttransposon insertions. Propagation of the strain at 39E may increase the frequency andrandomness of transposition (Ikeda et al., 1993)

2.

Cross spores of S. coelicolor NF/pUC1169 and the SCP1– strain and plate on
supplemented R2YE. Incubate at 30E until sporulation.

3.

Harvest the spores and plate for VioR, recipient-type colonies using counterselection against a distal marker of the NF donor strain. Screen for desired
phenotypes. Most of the selected colonies have Tn4560 insertions in the chromosome andlack pUC1169 (ThioR).

C. Tn4560 mutagenesis of SCP2* plasmids in S. lividans (Brolle et al., 1993)
Tn4560 does not seem to transpose into wild-type pIJ101, pSG5 or SLP2.
S. lividans TK503, prototrophic, or similar strain containing a copy of Tn4560 in
the chromosome
pIJ903 (tsr) or other transfer-proficient SCP2* derivative not containing vph(transposon marker)
R2YE and MM plates
YEME+34% sucrose+0.5% glycine
Viomycinvph
Antibiotic for selection of SCP2*
derivative

1.

Introduce pIJ903 into S. lividans TK503 bytransformation or conjugation. Working witha large number of independent transformants or exconjugants increases the chance of obtaining many
independent transposition events.

pIJ903tsr

&KDSWHU

2.



7UDQVSRVRQPXWDJHQHVLVHarvest spores separately from several pools of TK503/pIJ903 clones andpropagate in liquid medium, maintaining selection for the tsr plasmid marker.
Incubation at 37-39E may increase the frequency and randomness of transposition (Ikeda et al.,1993). Multiple rounds of propagation may increase the yield of Tn4560-containing plasmids,
but may also increase clonal populations.

3.

Isolate plasmid DNA from the liquid cultures and use to transform a plasmid-free
S. lividans strain. Aim for about 150 transformants per plate by using
sufficiently small amounts of DNA. Regenerate the transformed protoplasts nonselectively so that pocks (p. 153) can form. For target plasmids that do not form pocks
it is better to select transformants by overlaying the regeneration plates. Viomycin is not suited
for selection by overlay; use the plasmid marker instead.

4.

Replica-plate the well-sporulated plates to MM with 30-50 µg ml–1 viomycin.
Score the plates after 2 days incubation at 30E. Sporulation is usually delayed in thepock areas, so premature replication may not transfer plasmid-containing mycelium. Eachgrowth area represents a plasmid containing Tn4560. If the growth area is very small, Tn4560
has probably inserted into a SCP2* transfer gene. Large, pock-sized areas of VioR growth
represent transfer-proficient plasmids, possibly with Tn4560 insert in a cloned gene. Early
scoring of the plates is essential for recognizing pock-forming plasmids.
For analysis of the Tn4560-containing plasmids, total DNA preparations and Southernblots, using a probe for the region of interest, may be more efficient and easier to interpret than
CCC plasmid preparations (include total DNA from a plasmid-free host and pIJ903 DNA as
controls). Plasmids from the same pool may be clonal, but because of the high Tn4560
transposition frequency, about 50% of the isolates from most pools are different; 5-10% of theisolates may be rearranged or deleted.

D. Tn4560 mutagenesis of pock-forming SCP2* in S. coelicolor(modified from Davis and Chater, 1990)
This procedure takes full advantage of the conjugative
ability (100% transfer) of many SCP2* derivatives, and the fact that
pUC1169/pMT660::Tn4560, tsr vph is not mobilised in trans by
SCP2*. This procedure is also suitable, and would be more
efficient, for transposon delivery from the chromosome, rather thanfrom pUC1169. It is not very suitable for SCP2* derivatives withlarge inserts of host chromosomal DNA because such artificial
SCP2* primes give up to 10% chromosomal recombinants by"donor crossing over", making isolation of pUC1169-free SCP2*
recipient strains difficult. Also, the SCP2* derivative and pUC1169
should not share sequences (e.g. tsr) because this would result invery efficient mobilisation by cointegrate formation (p. 262).

p
vph
tsr

SCP2*
tra

pUC1169

Conjugation
J1501

SCP2*
tra



&KDSWHU7UDQVSRVRQPXWDJHQHVLVThe previous procedure, requiring a strain with a chromosomally integrated Tn4560, ispreferable, except if a poorly transformable host strain needs to be used.
SCP2* derivative, preferably without tsr or other identity with pUC1169
S. coelicolor M124 (pro arg cys) or similar multiply auxotrophic strain containing
the Tn4560 delivery plasmid pUC1169 (tsr, vph)
S. coelicolor J1501 (his ura str) or a similar plasmid-free strain
R2YE and MM plates with growth supplements
YEME+34% sucrose+0.5% glycine
Viomycin
Antibiotic for selection of SCP2* derivative

1.

Introduce SCP2* derivative into M124/pUC1169. Conjugation or transformation canbe used. Working with a large number of independent ex-conjugants or transformants increases
the chance of obtaining many independent transposition events.

2.

Harvest spores separately from several pools of M124/pUC1169/SCP2* clones
and propagate in liquid or on solid medium, maintaining selection for both
plasmid markers. Incubation at 39E may increase the frequency and randomness oftransposition (Ikeda et al., 1993). Multiple rounds of propagation may increase the yield of
Tn4560-containing SCP2* plasmids, but may also increase clonal populations.

3.

Cross several independent pools of M124/pUC1169/SCP2* with strain J1501 on
supplemented R2YE. Incubate at 30E c. 5 days until sporulation occurs. Platecrosses (p. 152) work well because SCP2* transfers with 100% efficiency.

4.

Harvest spores separately fromdifferent pools, dilute to obtainsingle colonies, and select Pro+
Arg+ Cys+ StrR (J1501-type) VioR
progeny containing SCP2*
derivatives with Tn4560 insertions(on MM + his + ura + str + vio).

his

+

+

+

J1501

arg

pro

M124
vph
tsr

+

pUC1169

SCP2*
tra

cys

The vast majority of the selected colonies
ura+
should be ThioS and therefore lack+
pUC1169. If this is not the case,str
propagation at 37E on medium without
thiostrepton and viomycin may cure mostof pUC1169, and another cross with asuitably marked strain (repeat steps 2 and 3) may give the desired result. This has worked even

&KDSWHU

7UDQVSRVRQPXWDJHQHVLVwhen there were sequence homologies between the SCP2* derivative and the S. coelicolor
chromosome and pUC1169.
It may be possible to use, for the last stage, a recipient strain that allows phenotypic
detection of the inactivation of the cloned target gene. The plasmids need to be analysed as
described for the previous procedure.

IS493 from S. lividans 66
IS493 is a 1641 bp, 66% G+C insertion sequence with two probably translated ORFs and 59 bp
imperfect terminal inverted repeats. It was discovered and sequenced at the Eli Lilly company by
Solenberg and Burgett (1989). ORFB encodes the transposase; the function of ORFA is unknown andit has been deleted from some IS493 derivatives (Baltz et al., 1992). IS493 resembles slightly IS1373
which flanks AUD2 of S. lividans. The consensus target site is gnCanTgnny; insertion duplicates thethree underlined bases (Solenberg and Baltz, 1994).
There are three copies of IS493 in S. lividans 66, so IS493 derivatives cannot be used in thisstrain. Sequences hybridizing with IS493 have not been observed in other Streptomyces species including
S. coelicolor A3(2), S. ambofaciens BES2281, and S. griseofuscus C581.
Several derivatives of IS493 with resistance markers, suitable for transposon mutagenesis ofStreptomyces, are described in Chapter 22. They transpose at a frequency of c. 10–6, much less frequently
than Tn4560. Hyper-transposing derivatives, Tn5099-10 and Tn5100-4, transpose up to 1000-fold more
frequently (depending on the host strain) into the same consensus target sequence; they are mutated in
one of the terminal repeats (Solenberg and Baltz, 1994). Some of the transposons contain PL, allowing
resistance selection in E. coli to be made as well as in Streptomyces.
IS493 is usually delivered to the chromosome by temperature-sensitive, Streptomyces multicopy
plasmids like pSG5 (p. 258), which can be introduced into Streptomyces by protoplast transformation
or by RP4-mediated conjugation from E. coli (p. 249). A pMT660 derivative, pRHB106, containing thehft fragment for FP43-mediated transduction (p. 300), has also been used to deliver an IS493 derivative(McHenney and Baltz, 1991). An elegant sectoring method is used for isolating chromosomal insertions(procedure E). Delivery from non-replicating E. coli vectors is also possible but inefficient.
E. Sectoring method for delivering IS493 derivatives using temperature-sensitive plasmids(Solenberg and Baltz, 1991)
ApraR ThioR

For simplicity, the procedure is described
specifically for the delivery of Tn5096 (aac(3)IV,
apramycin resistance) from the pSG5 derivative
pCZA186 (temperature-sensitive replication; tsr,
thiostrepton resistance) to the chromosome of S.
griseofuscus, the strain for which the procedure was
first developed.

aac(3)IV
tsr

Tn5096ori pSG5

39 o
ApraS ThioS

ApraR ThioS&KDSWHU7UDQVSRVRQPXWDJHQHVLV

pCZA186, or similar transposon delivery plasmid with temperature-sensitive
replication
S. griseofuscus C581 or other target strain able to grow at $35E
TSB (p. 412)
Bennett’s agar (for 1 litre: 1 g yeast extract, 1 g beef extract, 2 g N-Z amine or casitone,
10 g glucose, 15 g agar, adjust to pH7.3 with NaOH); thick, 30 ml plates are needed instep 3
Apramycin and thiostrepton to select for transposon and vector.
29E and 39E incubators

1.

Introduce pCZA186 into S. griseofuscus by transformation or by RP4-mediatedconjugation from E. coli. Select ThioR (vector marker) strains.

2.

Grow several independent S. griseofuscus/pCZA186 isolates separately at 29E
in 25 ml TSB containing 25 µg ml–1 thiostrepton. C. 2 days incubation produces a midlog-phase culture. It is important to use several independent cultures: some may haveexperienced transposition early and yield many identical clones, while others may contain aspontaneously arisen mutant derivative of pCZA186 unable to deliver Tn5096. For the hypertransposing IS493 derivatives in some strains, it may be possible to avoid this step and plate
spores from step 1 directly onto apramycin agar as described in step 3. Very low transposition
frequencies in some strains may necessitate further rounds of cultivation.

3.

Homogenise the mycelium using a Potter homogeniser (p. 419), dilute, andspread out to obtain not more than 100 colonies per plate (30 ml) of Bennett’s
agar containing 25 µg ml–1 apramycin (transposon marker). Incubate 2-3 days
at 29E. Thick plates, rich medium and low colony density are important for sector growth instep 4. The colonies should reach about 1-2 mm in diameter; different strains may requiredifferent incubation times. Slightly longer incubation may be necessary if spores rather thanmycelial fragments are plated. Homogenised mycelium can be stored in 15% glycerol at -70E.

4.

Transfer the plates to 39E and incubate for 10-14 days. The
plates may need to be kept sealed to prevent desiccation.
Two to three apramycin resistant, thiostrepton sensitive
sectors should grow from each colony.

5.

Purify spores/mycelium from sectors by streaking on
medium containing apramycin and incubate again at 39E.
Before analysing a large number of sectors from a pool,make sure that the isolates are mostly ThioS and containdifferent insertions.

ApraR
sectors

Apra S

&KDSWHU

7UDQVSRVRQPXWDJHQHVLVIS6100 from Mycobacterium fortuitum
IS6100 is an 800 bp, 60% G+C insertion sequence with one transposase ORF (tnp) and 14 bp
terminal inverted repeats. It originates from the direct terminal repeats of the composite Tn610, present
in four copies in Mycobacterium fortuitum FC1 (Martin et al., 1990). IS6100 seems to have a wide hostrange: identical copies have been found on a Flavobacterium plasmid and close homologues exist in
pseudomonads (Kato et al., 1994). IS6100 belongs to the IS6 family which has representatives in Grampositive and Gram-negative bacteria; they all transpose replicatively, producing cointegrates.
Two IS6100-containing, multifunctional plasmids, pUCS30 and pSIT151, have been constructed
and used for mutagenesis of S. lividans, producing c. 0.2% auxotrophic mutations (Smith and Dyson,
1995; Herron et al., 1999).
Both plasmids contain the pSG5 temperature-sensitive, multicopy Streptomyces origin of
replication (p. 258). Increasing the incubation temperature above 34E, while maintaining selection forthe plasmid markers, results in clones containing the whole plasmid integrated into the host genome. The
transposable part of the plasmids becomes directly repeated, as is usual for replicative transposition.
These cointegrates are fairly stable, but on prolonged culture, the plasmid may excise (resolution of
cointegrate by homologous recombination between the direct IS6100 repeats), leaving behind one copyof the IS, and secondary transposition events may occur (Fig 6.1)
Unusually for transposable elements, IS6100 can be induced up to 100-fold by transcription fromexternal promoters; for this purpose, pUCS30 and pSIT151 contain the thiostrepton inducible tipAp
promoter upstream of the IS6100 tnp gene. Note that tipAp must be induced at 28-30E, and that
activation requires an active tipA gene from the host. S. lividans 1326, the original wild-type strain, andS. lividans ZX1 are competent for tipAp induction, but strains TK64 and JT46 are not. Induction
competent revertants of TK64 can, however be isolated (Weaden and Dyson, 1998; P. Dyson personalcommunication).
F. Transposon mutagenesis of S. lividans using pSIT151 (Herron et al., 1999)
This method is not suitable for pUCS30 (Smith and Dyson, 1995) because S. lividans/pUCS30
sporulates poorly at 40E; use the sectoring method (procedure E) instead. The media are usually
supplemented for the desired mutations: gentamicin, kanamycin, thiostrepton.
pSIT151 (IS6100 derivative containing Tn1792 on pSG5 derivative)
S. lividans 1326 or other strain, preferably supporting tipAp induction (p. 386)
R2YE
MS agar (p. 409)

1.

Introduce pSIT151 by transformation into S. lividans protoplasts. Selectgentamicin (30 µg ml–1) and kanamycin (50 µg ml–1) resistant colonies byoverlaying c.12 h after plating the transformed protoplasts. Grow at 30Euntil&KDSWHU7UDQVSRVRQPXWDJHQHVLV

ori
pSG5

neo

tsr

pSIT151
11.4 kb

PtipA
tnp
Transposase

Gentamicin

aacC1
EcoRI

PstI

Tn1792Thiostrepton induced
transposition into
chromosome
10 -5
SalI

EcoRI

PstI

aacC1

SalI
Tn1792

Tn1792
neo

ori
pSG5

tsr

tnp

aacC1

Resolution byhomologous
recombination

Tn1792Chromosome

aacC1

Fig 6.1. Integration of pSIT151 into the genome of S. lividans. pSIT151 contains the transposition
functions of IS6100: the transposase, tnp, is expressed from the thiostrepton inducible tipAp promoter.
The terminal inverted repeats (<=) flank the aacC1 gentamicin resistance gene; together they constituteTn1792 which is duplicated on transposition. The origin of replication (ori) is from the temperaturesensitive pSG5. tsr, thiostrepton resistance gene; neo, kanamycin resistance gene from Tn5. The
positions of the SalI sites in the chromosome are unknown; there are additional SalI sites in pSIT151
between EcoRI and PstI, but not in Tn1792. (Partial sequence is in AJ006518.)

sporulation occurs. It is important to avoid thiostrepton selection because this would induce
premature transposition.

2.

Harvest spores and plate on MS agar containing sub-inhibitory amountsof thiostrepton (20 ng-2 µg ml–1) . Incubate at 30E for 24 h, then transfer to 40E
and incubate until sporulation occurs. The thiostrepton concentration must be subinhibitory, otherwise plasmid curing is inefficient.

&KDSWHU

3.



7UDQVSRVRQPXWDJHQHVLVHarvest spores and plate 106-108 spores onto R5 + 30 µg ml–1 gentamicin +50 µg ml–1 kanamycin. Individual colonies should grow. Incubate at 40E until
sporulation occurs. Analyse clones for phenotypes. For Southern analysis of genomic
DNA use a Tn1792-specific probe (e.g. the PstI-EcoRI fragment of pSIT151), which should give
two bands for the expected cointegrates when using BamHI-digested total genomic DNA. For
the cloning of both sides of the tagged DNA, directional cloning of EcoRI-SalI, PstI-Sal or SphISalI fragments, e.g. in pUC19, and selection for gentamicin resistance in E. coli seems more
efficient than direct rescue of the plasmid. (SphI, PstI, and EcoRI sites are relatively rare in
Streptomyces DNA; SalI sites occur about every 3 kb).

Tn5493 derived from Tn5
Tn5493 (Volff and Altenbuchner, 1997) is a 1.1 kb mini-transposon specifically designed for
Streptomyces. It consists of the tsr gene flanked by the inner and outer inverted repeats of IS50 from thecomposite transposon Tn5. Transposition from the vector pJOE2577 is induced by a high-frequency
transposition mutant form of the Tn5 transposase tnpA. The tnpA gene is expressed from the S. lividansmer (mercury resistance operon) promoter and a Streptomyces ribosome binding site. The transposondelivery plasmid, pJOE2577, is a derivative of the temperature-sensitive plasmid pSG5 (p. 258).
Transposition is very efficient in S. lividans: 1-6% of the progeny contained Tn5493 transposed into thechromosome and, as expected for a Tn5 derivative, there seems to be no problem with cointegrate
formation. The sectoring method described for IS493 (procedure E) does not seem to work for Tn5493,
possibly because the transposition frequency is too high (J. Altenbuchner, personal communication).
G. Mutagenising S. lividans using pJOE2577 (Volff and Altenbuchner, 1977)
pJOE2577 DNA for transformation
S. lividans protoplasts for transformation
HT (Hickey-Tresner) agar (p. 407)
Thiostrepton
Kanamycin

1.

ori
pSG5

tsr
neo
tnpA

tsr

Introduce pJOE2577 by transformation into
S. lividans. Select transformants by overlaying with thiostrepton. It is best to startwith many independent transformants to obtain many independent transposition events.

2.

Harvest spores from pools of transformants and spread onto HT agar containing
50 µg ml–1 thiostrepton to obtain confluent growth. Incubate at 30E.



&KDSWHU7UDQVSRVRQPXWDJHQHVLV

3.

Harvest spores and plate dilutions on HT agar with and without thiostrepton.
Incubate the plates at 39E. 1-6% of the colonies are expected to be thiostrepton resistant.

4.

Isolate ThioR kanamycin sensitive (15 µg ml–1 kanamycin in HT agar) strains.
These contain the Tn5493 and have lost pJOE2577.

H. Alternative method for isolating S. lividans with random
insertions of Tn5493 (J Altenbuchner personal communication)pJOE2577 DNA for transformation
S. lividans protoplasts for transformation
HT (Hickey-Tresner) agar (p. 407)
Thiostrepton
Kanamycin

1.

1

30

Thio

2

39

Thio

3

39

No Thio

430

No Thio

530

No ThioTransform S. lividans with pJOE2577 and selectthiostrepton-resistant transformants by overlay; incubate at 30E. Each transformant
is expected to produce at least one (independent) transposition event.

2.

After sporulation, replicate onto HT agar containing thiostrepton and incubate
at 39E.

3.

After sporulation, replicate onto HT agar without thiostrepton and incubate at
39E.

4.

Replicate on HT agar without thiostrepton and incubate at 30E. This gives better
sporulation than 39E.

5.

Harvest spores and plate dilutions at 30E for single colonies on HT containing
thiostrepton.

6.

Replicate to confirm that most colonies are sensitive to 15 µg ml–1 kanamycin in
HT agar.

&KDSWHU

7UDQVSRVRQPXWDJHQHVLVBaltz RH, Hahn DR, McHenney MA, Solenberg PJ (1992)
Transposition of Tn5096 and related transposons in
Streptomyces species. Gene, 115, 61-65.
Berg DE (1989) Transposon Tn5. In Berg, D.E. and Howe,
M.M. (eds.), Mobile DNA. American Society for
Microbiology, Washington, D.C., pp. 185-210.
Berg DE, Howe MM (eds.) (1989) Mobile DNA. American
Society for Microbiology, Washington, D.C.
Brolle D-F, Pape H, Hopwood DA, Kieser T (1993)
Analysis of the transfer region of the Streptomyces plasmid
SCP2*. Mol.Microbiol., 10, 157-170.
Calos MP, Miller JH (1980) Transposable elements. Cell,
20, 579-95.
Chung ST (1987) Tn4556, of a 6.8 kb transposable elementof Streptomyces fradiae. J.Bacteriol., 169, 4436-4441.
Chung ST, Crose LL (1990) Transposon Tn4556-mediatedDNA insertion and site-directed mutagenesis. In Heslot, H.,
Davies, J., Florent, J., Bobichon, L., Durand, G. and Penasse,
L. (eds.), 6th International Symposium on Genetics ofIndustrial Microorganisms, GIM 90, Proceedings. SocietéFrancaise de Microbiologie, Strasbourg, pp. 207-218.
Davis NK, Chater KF (1990) Spore colour in Streptomyces
coelicolor A3(2) involves the developmentally regulatedsynthesis of a compound biosynthetically related topolyketide antibiotics. Mol.Microbiol., 4, 1679-1691.
De Lorenzo V, Timmis KN (1994) Analysis and constructionof stable phenotypes in gram-negative bacteria with Tn5- andTn10-derived minitransposons. Meth.in Enzymol., 235, 386405.
Grindley ND, Reed RR (1985) Transpositional
recombination in prokaryotes. Ann. Rev. Biochem., 54, 86396.
Herron PR, Evans MC, Dyson PJ (1999) Low target site
specificity of an IS6100-based mini-transposon, Tn1792,
developed for transposon mutagenesis of antibiotic-producing
Streptomyces. FEMS Microbiol.Lett., 171, 215-221.
Ikeda H, Takada Y, Chang-Hong P, Tanaka H, ®mura S(1993) Transposon mutagenesis by Tn4560 and applications
with avermectin-producing Streptomyces avermitilis.
J.Bacteriol., 175, 2077-2082.
Kato K, Ohtsuki K, Mitsuda H, Yomo T, Negoro S, Urabe
I (1994) Insertion sequence IS6100 on plasmid pOAD2,
which degrades nylon oligomers. J.Bacteriol., 176, 1197-200.
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Meth.in Enzymol., 204, 430-458.
Kleckner N (1977) Translocatable elements in procaryotes.
Cell, 11, 11-23.
Kleckner N (1981) Transposable elements in prokaryotes.
Ann.Rev.Genet., 15, 341-404.
Kleckner N (1990) Regulation of transposition in bacteria.
Ann. Rev. Cell Biol., 6, 297-327.

Luria S, Delbrück M (1943) Mutations of bacteria fromvirus sensitivity to virus resistance. Genetics, 28, 491-511.
Martin C, Timm J, Rauzier J, Gomez-Lus R, Davies J,
Gicquel B (345) Transposition of an antibiotic resistanceelement in mycobacteria. Nature, 345, 739-43.
McHenney MA, Baltz RH (1991) Transposition of Tn5096
from a temperature-sensitive transducible plasmid in
Streptomyces spp. J Bacteriol, 173, 5578-81.
Miller JH (1992) A short course in bacterial genetics. Cold
Spring Harbor Press, Cold Spring Harbor, N.Y.
Occi JL, Gibbons PH, Wong E, MacNeil DJ (1993)
Insertion of transposon Tn5seq1 into G+C-rich DNA of
Streptomyces avermitilis: generation of 8-, 9-, and 10-bp
duplications. Plasmid, 30, 167-9.
Reznikoff WS (1993) The Tn5 transposon.
Ann.Rev.Microbiol., 47, 945-63.
Richardson MA, Kuhstoss S, Huber MLB, Ford L,
Godfrey O, Turner JR, Rao RN (1990) Cloning ofspiramycin biosynthetic genes and their use in constructing
Streptomyces ambofaciens mutants defective in spiramycin
biosynthesis. J.Bacteriol., 172, 3790-3798.
Schauer AT, Nelson AD, Daniel JB (1991) Tn4563
transposition in Streptomyces coelicolor and its application toisolation of new morphological mutants. J.Bacteriol., 173,
5060-5067.
Sherratt D (1989) Tn3 and related transposable elements:
site-specific recombination and transposition. In Berg, D.E.
and Howe, M.M. (eds.), Mobile DNA. American Society for
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Siemieniak DR, Slightom JL, Chung ST (1990) Nucleotide
sequence of Streptomyces fradiae transposon Tn4556: a class
II transposon related to Tn3. Gene, 86, 1-9.
Smith B, Dyson P (1995) Inducible transposition in
Streptomyces lividans of insertion sequence IS6100 from
Mycobacterium fortuitum. Mol Microbiol, 18, 933-41.
Solenberg PJ, Baltz RH (1991) Transposition of Tn5096 andother IS493 derivatives in Streptomyces griseofuscus.
J.Bacteriol., 173, 1096-1104.
Solenberg PJ, Baltz RH (1994) Hypertransposing
derivatives of the streptomycete insertion sequence IS493.
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Solenberg PJ, Burgett SG (1989) Method for selection oftransposable DNA and characterization of a new insertion
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171, 4807-4813.
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Weaden J, Dyson P (1998) Transposon mutagenesis with
IS6100 in the avermectin-producer Streptomyces avermitilis.
Microbiol., 144, 1963-70.



&KDSWHU7UDQVSRVRQPXWDJHQHVLV

m07tkb.wpd 13 March 2000 Tobias Kieser

Chapter 7

In vivo genetic analysis by conjugation andprotoplast fusion
CONTENTS
The modes of gene exchange in Streptomyces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
Mating . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
Protoplast fusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
Transduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
Chromosomal recombination by transformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
Electroporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
The practicalities of making crosses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
Quantitative analysis of crosses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
Recombination frequency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
Frequency of plasmid transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
Linkage mapping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
Establishing a genetic map by the "four-on-four" procedure . . . . . . . . . . . . . . . . . . . . . . . . . 141
The "classical" way of analysing the results of a "four-on-four" cross . . . . . . . . . . . 144
Analysing the results of a "four-on-four" cross by minimizing multiple crossovers . 147
Mapping a new marker by a single selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
Plate-crosses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
Detection of conjugative plasmids by "pock" formation by transconjugants . . . . . . . . . . . . . 153
Protoplast fusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156
Fusion of protoplasts of S. coelicolor or S. lividans . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
Genetic analysis by protoplast fusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158

The modes of gene exchange in Streptomyces
MatingNatural gene exchange is very widespread in Streptomyces and in related genera like
Saccharopolyspora and Amycolatopsis. In the laboratory it requires the mixed culture of pairs of strains
on agar ("matings"). Conjugation occurs non-synchronously during several hours of mixed growth,
involving successive rounds of recombination, as can be deduced from the progeny of triparental matings(Bibb and Hopwood, 1981). No mating has been detected in liquid cultures, even though clumping ofyoung mycelium arising from different spores often occurs in liquid. Quantitative analysis ofrecombinant progeny from matings has been the main means of genetic mapping in Streptomyces.

&KDSWHU,QYLYRJHQHWLFDQDO\VLVE\FRQMXJDWLRQDQGSURWRSODVWIXVLRQTable 7.1 Recombination induced by some commonly used plasmids Data from Bibb and Hopwood(1981); Hopwood et al. (1983); Bailey et al. (1986)
Plasmid status1
c.m.a.2

Host
S. coelicolor

S. lividans

Comments

Parent 1

Parent 2

-

-

10–8

The c.m.a. may be from SLP1INT

SCP1
SCP1

SCP1
-

10–5
10–6

No entry disadvantage; SCP1 in bothparents actually increases c.m.a.

SCP2
SCP2

SCP2
-

10–7
10–6

A small entry disadvantage reduces
c.m.a. for SCP2

SCP2*
SCP2*

SCP2*
-

10–3
10–3

No entry disadvantage; SCP2* is a goodsex factor; random marker exchange

SCP2*

SCP2

10–5

SCP2* partially overcomes the entry
disadvantage of SCP2SCP1NF

SCP1NF1%

Random marker exchange

SCP1NF-

100%

Bidirectional marker gradient (Fig. 7.1);
all progeny are SCP1NF

-

-

<10–8

Strains lacking the natural SLP2 seem tobe self-sterile

SLP2
SLP2

SLP2
-

10–6
10–4

Entry disadvantage strongly reduces
c.m.a. for SLP2

SLP1
SLP1

SLP1
-

10–4
10–4

No entry disadvantage for SLP1

pIJ101
pIJ101

pIJ101
-

$10–3
$10–3

No entry disadvantage; pIJ101is an
excellent sex factor

SCP2*
SCP2*

SCP2*
-

10–4
10–4

No entry disadvantage for SCP2*

pJV1
pJV1

pJV1

#10–7
5 × 10–6

Entry disadvantage reduces c.m.a.;pJV1is a poor sex factor

For footnotes, see opposite page.

-

&KDSWHU,QYLYRJHQHWLFDQDO\VLVE\FRQMXJDWLRQDQGSURWRSODVWIXVLRQRecombination always seems to depend on the activity of plasmid sex factors. Since manystrains naturally harbour such plasmids, genetically marked strains derived from the same starting culture
usually generate recombinants when they are mated together. Often the recombination frequency is quite
high (10–6-10–3), as in S. coelicolor A3(2) (Hopwood, 1959), S. glaucescens (Baumann et al., 1974),
Amycolatopsis mediterranei (Schupp et al., 1975) and S. avermitilis (Ikeda et al., 1987), but sometimes
it is below 10–6. Where analysed, this low frequency has been explained by plasmid "entry
disadvantage", which causes recombinants to be very infrequent when the same plasmid is present in bothmating partners, but much higher when only one partner carries it. This occurred with S. lividans 66,
where good recombination frequencies were obtained only when the SLP2 plasmid was eliminated frommarked derivatives of strain 66 so that SLP2+ × SLP2– matings could be made (Hopwood et al., 1983).
Similar findings were reported for S. rimosus, in which SRP1 showed entry disadvantage (Friend et al.,1978), and in S. venezuelae, in which three plasmids, SVP1, SVP2 and SVP3, all showed this property
to varying degrees (Doull et al., 1986); in Sacc. erythraea, SEP1 showed a small entry disadvantage(DeWitt, 1985). Some species that lack a convenient sex plasmid have been rendered fertile by the
introduction into them of a broad host-range sex plasmid (Kieser et al., 1982; Smokvina et al., 1988).
Table 7.1 gives examples of recombination frequencies in S. coelicolor and S. lividans induced byvarious plasmids.
Chromosomal markers usually appear to be mobilised randomly by autonomous plasmids, and
it is not possible to distinguish between donor and recipient strains, even if the plasmid was initiallypresent in only one of the parents; presumably rapid transfer of the plasmid occurs, followed by rare
chromosomal mobilisation in each direction. Chromosomal mobilisation ability (c.m.a.) by integrated
plasmids has been studied only in S. coelicolor A3(2). In this strain the SCP1 plasmid integrates into thechromosome to form various donor strains which yield very high recombinant frequencies in matings
with SCP1– strains (Hopwood et al., 1984b): NF was the first example of such a donor state, with SCP1
being integrated in the "9 o’clock" region of the chromosome. All NF strains in the culture collection
derive from this one original integration event. Use of NF × SCP1– matings allowed efficient mapping
for specific regions of the chromosome, following from the donation of chromosomal segments carrying
the 9 o’clock region of the NF chromosome (Fig. 7.1) to the SCP1– recipient (e.g. Schauer et al., 1991).
However, crosses in which the SCP2* plasmid, in one or both parents, provides the c.m.a. are often themost convenient for preliminary mapping of new mutations because they usually show fairly randomtransfer of different regions of the chromosome at a reasonable frequency.

Footnotes for Table 7.1.
1
S. coelicolor A3(2) is naturally SCP1+ SCP2+; SCP1NF strains (originally called just NF) are descended
from a strain in which most of SCP1 became integrated at 9 o’clock in the chromosome (Kieser et al.,1992) (SCP2* is present in many NF strains; others are SCP2+). SCP1– strains were originally called UF(for “ultra-fertility” when crossed with NF strains) but most, at least, would have been SCP2+, so thedesignation UF is no longer used; similarly, SCP1+ strains (again SCP2+) were at first called IF (for“initial fertility”). SCP2* is a highly fertile variant of SCP2. S. lividans 66 is naturally SLP2+ SLP3+;
SLP3 is conjugative but has no c.m.a. SLP1 is the autonomous form of SLP1INT, which is found stably
integrated into the S. coelicolor chromosome; SLP1 cannot replicate autonomously in S. coelicolor.
pIJ101 is from S. lividans ISP5434. pJV1 is from S. phaeochromogenes NRRLB3559.
2
c.m.a., chromosome mobilising ability (see p. 137 for calculation).



&KDSWHU,QYLYRJHQHWLFDQDO\VLVE\FRQMXJDWLRQDQGSURWRSODVWIXVLRQInterspecific Streptomyces matings have occasionally generated novel combinations of presumed
chromosomal genes (e.g. Mazières et al., 1981), but usually very few recombinants arise. The reasons
have not been analysed, but could include: sequence divergence; genome rearrangements; restriction;
inhibition of partners by antibiotics or bacteriocins; or even zygotic induction of prophages.
Efficient plasmid transfer can occur in interspecific crosses, and can be useful in the discoveryof plasmids. For example, crossing strains of interest with a well-characterised recipient, such asplasmid-free S. lividans 66 derivatives, can lead to the discovery of new pock-forming plasmids (p. 153).
Mating between E. coli and Streptomyces (Mazodier et al., 1989), mediated by such plasmids as RP4
with oriT on the mobilised plasmid, has become one of the standard procedures for introducing plasmids
into Streptomyces. This is covered in Chapter 10, p. 249.
Protoplast fusion
Genetic recombination occurs after protoplast fusion, which is induced very effectively bypolyethylene glycol (PEG) treatment. This recombination does not depend on plasmids. Protoplast
fusion can be used for mapping, especially for closely linked markers (p. 159). However, it isparticularly adapted to strain construction, since the very high frequencies of recombinants produced
allow complex combinations of non-selectable chromosomal markers to be built up: recombination canbe so frequent that recombinants can be isolated without counterselecting the parents. Protoplast fusion
can also lead to the transfer of plasmids from strain to strain.
Transduction
Natural generalised transduction of chromosomal markers has not been generally available in
Streptomyces, but has been developed using the phage SV1 in S. venezuelae (Stuttard, 1989). Veryrecently, generalised transduction has been described in S. coelicolor and S. lividans (J. Westpheling,
personal communication). The artificial introduction of genes into Streptomyces on vectors derived fromthe phage C31 (others have been used to a small extent), often using infection by engineered phages,
is dealt with in Chapter 13; use of such vectors is complementary to that of plasmids for gene cloning.
Transduction of special plasmids by phage FP43 is also a useful tool (p. 300).
Chromosomal recombination by transformation
There is no well documented natural competence for DNA uptake in Streptomyces, or in any true
actinomycete (the competent Thermoactinomyces vulgaris (Hopwood and Wright, 1972), with low G+C
DNA, is now recognised not to be a close relative of the actinomycetes). Reports of the production ofrecombinants via fusion of protoplasts with liposomes containing chromosomal DNA (Makins and Holt,
1981), or of PEG-induced transformation by chromosomal DNA (Isogai et al., 1981; Ochi. 1982), do notappear to have been followed up, but recently PEG-induced transformation by denatured DNA was foundto occur at low but reproducible frequencies (Oh and Chater, 1997). Artificial polyethylene glycolinduced transformation of protoplasts by plasmids or transfection by phage DNA are the standard
methods of introducing naked DNA into Streptomyces. These methods are described in detail in
Chapters 10 and 13.

&KDSWHU,QYLYRJHQHWLFDQDO\VLVE\FRQMXJDWLRQDQGSURWRSODVWIXVLRQ

C

960

E 690

I 290 L140
H115

G480
M

110

C1650

G



F610

280

J200
E
820

B

S. coelicolor A3(2)1770

8000 kb

H

380

Q
10

D900

A

A

2100

1500

D820
OK
16

190

N
45

F530
P11

B 1300250 kb

Fig. 7.1 The S. coelicolor A3(2) chromosome, showing the frequencies with which various genes are
inherited by recombinant progeny in NF × SCP1– crosses (Hopwood et al., 1984b). The restriction map
for the two rare-cutting enzymes AseI and DraI is shown in the inner arcs (fragment sizes are in kb). The
outer arc shows the linear chromosome, with its terminal proteins (). The scale represents the lengthsof 10 average-sized cosmid inserts from the minimal ordered set, allowing for overlaps (Redenbach et al.,1996). (The total average insert size in each cosmid is 37.5 kb, of which 12.5 kb overlaps adjacentcosmids on either side; each cosmid therefore contributes 25 kb of sequence to the map.) There are
currently three short gaps in the cosmid set, as shown. Eight of the markers (including the integrated
SCP1 plasmid) are located precisely on the cosmid set, while the positions of the other five markers(indicated by asterisks), which have been mapped only genetically, have been interpolated using theirinheritance frequencies, which fall exponentially in both directions from the SCP1 integration point.

&KDSWHU,QYLYRJHQHWLFDQDO\VLVE\FRQMXJDWLRQDQGSURWRSODVWIXVLRQElectroporation
Electroporation, in which bacteria are exposed to a pulse of direct current in a suitable buffer
containing DNA, results in DNA uptake. This technique has become the standard means of introducing
DNA into several bacteria, including mycobacteria (Snapper et al., 1988) but, in spite of considerable
effort, is so far of only limited use in Streptomyces. Success was first achieved with S. rimosus (Pigac
and Schrempf, 1995). A significant factor may be the tendency of S. rimosus to become fragmented into
small units during liquid growth. Recently, electroporation of several species, including S. lividans, wasachieved using germinated spores (Tyurin et al., 1995). Electroporation is described in Chapter 10,
p. 237.

The practicalities of making crosses
Historically, crosses were made on CM but now we normally use R2YE because better
sporulation usually occurs on it. Crosses are normally incubated until sporulation occurs (4-10 days),
but crosses that are not producing spores can also yield recombinants (this is important when using bld
mutants: p. 17). The spores from a cross are typically plated out, at various dilutions, on one or more
selective media containing antibiotics or lacking growth factors, so as to allow the growth of the desiredclass(es) of progeny and to prevent growth of parental types. Thus, by counting the colonies on different
media, the frequencies of various recombinant classes, or of plasmid transfer events, can be calculated.
Colonies will often be picked from the primary selection plates, or replica plated, for purification andphenotypic classification. Very occasionally, recombinant frequencies are so high that the spores froma cross can be plated on a non-selective medium and the resulting colonies classified as being of parental
or any recombinant genotype (e.g. Hopwood et al., 1969).
Parental spores: fresh or frozen spore suspensions of the parental strains
R2YE supplemented with any growth factors required by the parental strains, as
Petri plates or as slants (17 ml in 25 mm × 150 mm tubes) plugged with cotton wool
Sterile water; 20% glycerol
Inoculating loop; pipettes; filter tubes; centrifuge tubes
28-30o incubator; vortex mixer; bench centrifuge

1.

Making the cross. Crossing is extremely simple: just mix spores of the twostrains together on the surface of the agar with circular movements of a loop, orwith a glass spreader. Incubate the culture. If the titres of the parental spore
suspensions are known, about 106 spores of each should be used. Plates are now generally usedinstead of slants for crossing, but are more susceptible to contamination; one contaminant spore
can ruin the cross!

2.

Harvesting the cross. Follow the procedure for "making a Streptomyces spore
suspension" (p. 44). You now have a suspension of progeny spores in 20%

&KDSWHU,QYLYRJHQHWLFDQDO\VLVE\FRQMXJDWLRQDQGSURWRSODVWIXVLRQglycerol. This can be plated out immediately or it (or the remainder after
immediate plating) can be kept frozen at -20o for long periods. Filtration andvortexing are especially important to remove mycelial fragments and to break up spore chains,so that pure colonies arise from single spores. Note that many developmental mutants are
sensitive to freezing and so must be plated immediately.

3.

Plating out the cross (see Box 7.1 for choice of media). See "plating out aStreptomyces spore suspension" (p. 47) for notes on procedure. Plate the spore
suspension from the cross on different selective media to recover various classes
of progeny. Ideally the numbers should be in the range from a few tens to acouple of hundred to obtain accurate counts and reliable estimates ofrecombination frequencies. Especially when working with unfamiliar markers,
comparable numbers of pure parental spores should be plated on selective media
to control for spontaneous reversion of auxotrophic markers or mutation to drug
resistance. The number of spores produced per cross varies with the strains andmedium; for S. coelicolor a typical slant yields c. 108 spores on CM or c. 109 on
R2YE. With most of our strains, countable plates carrying unselected progeny,
or progeny with the genotype of one or other parent, will therefore arise at 10–4,
10–5 or 10–6 dilutions (regarding the original suspension as 100 and plating 0.1
ml: e.g. 120 colonies at 10–5 = 1.2 × 108 spores ml–1 original suspension);experience soon teaches how to guess appropriate plating dilutions to bereasonably sure of obtaining the right density of colonies without wasting too
many plates (and of course the frozen suspension is there for re-plating later ifnecessary). The right dilutions for selective platings will depend on the expected
frequency of recombination or plasmid transfer: sometimes even 0.1 ml of the
100 suspension will yield only a few recombinants.
Selection may not be clean: some auxotrophs are "leaky", resulting inbackground growth. Growing colonies may stimulate satellite growth bysecreting growth factors; this is usually more obvious on dense plates.
Background growth tends to increase with time, so it is useful to view the plates
periodically and choose the best time for counting or for picking colonies. (Ifsome media give suitable growth before the others the plates can be stored in thefridge for a day or two until the colonies on all the media are ready.) Here is aquick way to obtain approximate tenfold dilutions, suitable for the generation of isolated
recombinant colonies for picking; it is not suitable for quantifying recombination. Place 0.1 ml&KDSWHU,QYLYRJHQHWLFDQDO\VLVE\FRQMXJDWLRQDQGSURWRSODVWIXVLRQFig. 7.2 Grids for preparing master plates.

&KDSWHU,QYLYRJHQHWLFDQDO\VLVE\FRQMXJDWLRQDQGSURWRSODVWIXVLRQ

of a concentrated spore suspension and then, using the same spreader, spread the drops of water on
successive plates. The method works because a typical glass spreader carries about 0.01 ml of liquid with
it after spreading.

4.

Making master plates. When classifying recombinants in respect of nonselected markers, it is usually best to pick off a sample of colonies to a "master
plate" of medium with the same composition as the original selective plate andreplicate the ordered array of patches to a series of "diagnostic media". Even
when there is no visible background growth there may be many viable parental
spores on the selection plates so it is important to maintain the initial selection
when propagating the colonies on master plates.
We routinely use two types of master plates, with 20 or 50 colonies;
usually the latter are satisfactory, provided all the markers are easy to score.
They are made by placing a fresh plate of medium over a grid (Fig. 7.2) andinoculating a colony into each square. Streptomyces colonies do not spread
rapidly like mould colonies so it is best to inoculate much of the surface area of
the agar in the appropriate square rather than just stabbing the centre of thesquare. (Colonies can be picked even when they are not sporulating by digging
out part of the mycelium with a flattened needle or toothpick.) It is obviously
crucial to analyse a random sample of the selected population, so all colonies
should be picked from a pre-designated area of the plate: do not pick and choose!
The recently introduced Easi-Grid™, manufactured by Jencons, Cherrycourt Way Industrial
Estate, Stanbridge Road, Leighton Buzzard, Bedfordshire, LU7 8UA, UK, is a device to hold thePetri plate over a suitable grid: those supplied are for 25, 50, 70, 100, 150 and 200 squares, aswell as 6 and 12 sectors.

5.

Replica plating of master plates to diagnostic media (see Box 7.1, for choiceof media). Master plates can usually be replica plated, using velvet, after 4-7
days’ incubation, even if they are not sporulating copiously, and especially if thestrain has a tendency to give "friable" growth (that is soft, as opposed to toughand leathery). Usually, the same velvet can be used for making replicas on 3-5
diagnostic plates in succession (but do not try to re-charge it with spores bygoing back to the master plate, since it is virtually impossible to align it
accurately enough). If one of the markers leads to a requirement for only trace
amounts of its supplement (like vitamin auxotrophs), or is "leaky" (such as thoserequiring cysteine or methionine), make sure that the corresponding replica(lacking the vitamin, etc.) is printed as the first plate with a fresh velvet tominimise carry-over of traces of the nutrient from replica to replica.

&KDSWHU,QYLYRJHQHWLFDQDO\VLVE\FRQMXJDWLRQDQGSURWRSODVWIXVLRQBox 7.1 CHOICE OF PLATING AND DIAGNOSTIC MEDIA
The choice of suitable media for plating the spores from the cross is illustrated in the first tablebelow. The example deals with a five-factor cross. The parents have the genotypes (1) his leu phe str
and (2) arg. (Note that his, leu, phe and arg represent the mutant alleles for requirement of histidine,
leucine, phenylalanine or arginine, respectively, while str represents the mutant allele for streptomycin
resistance.) The new marker to be mapped is leu. Two "recombinant" media are chosen in thisexample; each selects one marker from each parent and leaves the other three markers non-selected,
allowing various classes of recombinants to be recovered.
Medium type

Selected markers

Medium contains

Parental (1)leu his phe str

Parental (2)arg

Non-selected markers

Recombinant (1)his+ strarg leu phe str

arg leu phe

Recombinant (2)arg+ phe+leu hisleu his str

We need to classify a sample of the recombinants for their genotypes in respect of the three
non-selected markers on each medium. There are eight possible combinations of three markers, butthere is no need to replicate to eight media! After picking recombinants to master plates of the same
selective medium as that on which they were growing, we replicate the resulting patches to just four
diagnostic media, one to classify each non-selective marker and a fourth, identical to the plating
medium, as a control, as shown in the table below.
(1) his+ str recombinants

1(2) arg+ phe+ recombinants

Designation

Supplements

Designation

Supplements-arg

leu phe str1

-his

leu

-leu

arg phe str1

-leu

his

-phe

arg leu str1

+str

leu his str

Controlarg leu phe str1

Controlleu hisStreptomycin is included to maintain the original selection

After incubation of the replicas, each recombinant is classified for growth or no growth oneach replica plate and its genotype is thus determined: the absence of growth on a medium lacking a
growth factor (or its presence on an antibiotic-containing plate) tells us the recombinant carries themutant allele for that locus.

&KDSWHU,QYLYRJHQHWLFDQDO\VLVE\FRQMXJDWLRQDQGSURWRSODVWIXVLRQPatch onmaster plate

leu arg str leu arg str leu arg
leu arg
leu arg
strstrleu arg str leu arg str
phestrarg
strstrphearg
leu arg str leu arg str leu arg str leu arg str leu arg str
leu arg
arg strphestrleu arg str
phe
arg
str
leu arg str leu arg str leu arg str leu arg str leu arg str leu arg str leu arg str
phe arg str
pheargarg
strarg str leu arg str leu arg str leu arg str
leu arg str leu arg str leu
str leu
phe arg str
leu arg str leu arg
str leupheargargstrstr leu arg str leu arg str leu arg str leu arg str
leu arg str leu arg str leu arg str leuphearg strleustrarg str leu arg str leu arg strargphe
leu arg str leu arg str leu arg str leu arg str leu arg str leu arg str leu arg
strarg str
arg
leu arg str leu arg str
leuar
arg str leuphearg strleustrarg str leu arg str leu arg str
phe
arg
str
leu arg str leu arg str leu arg str leu phearg strleu strarg str leu arg str leu arg str
arg
leu arg str leu arg str leu arg str leu arg str leu arg str leu arg str leu arg str
pheargargstrstr
leu arg str leu arg str leu arg str leu arg str leu arg str leu arg
str leu
leu arg str leu arg str leu arg
leu arg
leu arg
phestrarg
str str leu arg str
phestrarg
strstr leu arg
pheargargstrstr
leu arg str leu arg str leu arg str leu arg str leu arg str leu arg
str leu
phe arg str
leu arg str leu arg str leu arg str leu arg str leu arg str leu arg str leu arg str
phe arg str
leu arg str leu arg str leu arg str leu arg str leu arg str leu arg str leu arg str
pheleu arg str leu arg str
leu arg
arg strstr
leu arg str leu arg str leu arg str leu arg str

leu arg str leu arg str leu arg str leu arg str leu arg str leu arg str leu arg str
leu arg str leu arg str leu arg str leu arg str leu arg str leu arg str leu arg str
leu arg str leu arg str leu arg str leu arg str leu arg str leu arg str leu arg str
leu arg str leu arg str leu arg str leu arg str leu arg str leu arg str leu arg str
leu arg str leu arg str leu arg str leu arg str leu arg str leu arg str leu arg str
leu arg str leu arg str leu arg str leu arg str leu arg str leu arg str leu arg str
leu arg str leu arg str leu arg str leu arg str leu arg str leu arg str leu arg str
leu arg str leu arg str leu arg str leu arg str leu arg str leu arg str leu arg str
leu arg str leu arg str leu arg str leu arg str leu arg str leu arg str leu arg str
leu arg str leu arg str leu arg str leu arg str leu arg str leu arg str leu arg str
leu arg str leu arg str leu arg str leu arg str leu arg str leu arg str leu arg str
leu arg str leu arg str leu arg str leu arg str leu arg str leu arg str leu arg str
leu arg str leu arg str leu arg str leu arg str leu arg str leu arg str leu arg str
leu arg str leu arg str leu arg str leu arg str leu arg str leu arg str leu arg str
leu arg str leu arg str leu arg str leu arg str leu arg str leu arg str leu arg str
leu arg str leu arg str leu arg str leu arg str leu arg str leu arg str leu arg str

- phe



Patterns ofgrowth on
diagnostic
platesphe arg str phe arg str
phe arg str phe arg str
phe arg str
phe arg str
phe arg str
phe arg str
phe arg str
phe arg str
phe arg str
phe ararg strphe arg str

phe arg str
phe arg str
phe arg str
phe arg str

phe arg str
phe arg str
phe arg str- leu

Fig. 7.3 A patch of growth on a master plate containing two different recombinant genotypes and thepatterns of growth after replication of the master plate to two different diagnostic media.

Cotton velvet without too much dressing and with a short dense pile isbetter than velvet made with man-made fibres (p. 420). We use squares (c. 16
cm for standard 9 cm Petri dishes) of velvet. Good quality squares can be
washed and re-used hundreds of times. It is best to put the squares through thewashing cycle several times before starting to use them so as to remove any
dressing. After use, the velvets are washed and squeezed out but not dried.
They are wrapped in packets of aluminium foil (say 20 per packet) andautoclaved thoroughly (e.g. 30-40 min at 121o). The ends of the packets are thencarefully opened to allow moisture to escape and dried in an oven at 80o before
being re-closed. In use, they are placed over a cylindrical block (80 mm
diameter) which fits comfortably inside a standard Petri dish and are held inplace by a metal or plastic ring (85 mm internal diameter).



6.

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Scoring of replica plates. The scoring is usually done after 2 days’ incubation.
It is convenient to make the primary scoring on an enlarged copy of the master
plate grid, before tabulation. This facilitates the re-checking of particulargenotypes and/or their isolation for subsequent use or further characterisation.
In classifying the patches of growth on the replica plates, care must be
taken to detect mixtures of genotypes, since these will distort the results if notrecognised and excluded from the analysis. There will always be at least a few
percent of mixed patches arising from the close association of two recombinantplating units, or perhaps from a mycelial fragment containing more than one
recombinant genotype. Misclassification of mixed patches can be minimised bycomparing the shape of the patches of growth on the various replica plates. For
example, a patch containing a mixture of the two genotypes phe arg str and leu
arg str will give rise to growth on the -leu and -phe plates as well as the control(see Box 7.1) and so might be mis-classified as arg str; but different patterns
may be seen on the -leu plate (caused by the phe arg str component of the
mixture) compared with the -phe plate (the leu arg str component) and thecontrol plate (where both components grow) (Fig. 7.3).
Special kinds of "mixed" colonies may also appear. One class is the"heteroclones", which are frequent only when selection is for recombination
between two close markers (pp. 140 and 141). The other class, "heterokaryons",
have been described in crosses of some species, such as S. rimosus (Friend andHopwood, 1971). They are prototrophic colonies that grow on the selective
medium by complementation between two complete parental genomes and canbe propagated as mycelium on the same medium, but give the two parental types
at sporulation. They can not therefore be transferred by replica plating to thesame selective medium as that on which they arose. Heterokaryons are notexpected on a selective medium containing streptomycin, because resistance isrecessive (e.g. Wright and Hopwood, 1977). Heteroclones and heterokaryons areusually ignored in analysing crosses.

Quantitative analysis of crosses
Recombination frequency
As described above, we normally plate dilutions of the spore suspension derived from a crosson the two media that each select for one of the parental genotypes, and on one or more media that selectfor recombinants, and count the colonies that grow. The precise recombination frequency would be the

&KDSWHU,QYLYRJHQHWLFDQDO\VLVE\FRQMXJDWLRQDQGSURWRSODVWIXVLRQsum of all recombinant types divided by the sum of the parental type progeny. Estimating this value isusually impractical because not all genotypes can be recovered by selection. In practice, recombination
frequencies (c.m.a.) are approximated (underestimated) as the average colony count on the media
selecting recombinants, divided by the sum of the counts on the two "parental" media. Parental-typeprogeny should be recovered in similar numbers (a "balanced" cross) for the values to be reproducible.
This may be helped if equal numbers of spores of the two parents are used to make the cross, but equal
input does not guarantee equal output of the two parents.
Frequency of plasmid transfer
Plasmid transfer frequency in Streptomyces is usually expressed as the fraction of the recipient
genotype that acquires the plasmid. To calculate it one needs to measure the total frequency of therecipient genotype, by plating on a medium that recovers it, and also the frequency of transconjugants(that is recipients carrying the plasmid). If the plasmid carries a selectable marker, the transconjugant
frequency can be measured by plating on a medium which selects the recipient genotype plus the plasmid
marker. If plasmid transfer is greater than c. 10%, a more accurate estimate is made by simply platingon the medium that selects the total recipient genotype, replica plating the resulting colonies to a medium
selective for the plasmid marker, and making direct counts of colonies that grow on the replica and of
those that do not. If the plasmid has no selectable marker, but forms pocks (p. 153), the frequency oftransconjugants can be estimated from the number of pock-forming units by plating a sample of spores
from the cross on a medium selective for the recipient genotype and on another plate with an added
excess of spores of the same plasmid-free recipient, to reveal pocks. These are counted and related to
the number of recipient colonies on the plate without the added spores. Alternatively, one can replica
plate recipient colonies to a lawn of plasmid-free spores to detect those that show "tramlines" (p. 154).
A further possibility is to detect a plasmid that has the c.m.a. property by “plate-crossing” (p. 152).
Linkage mapping
Since crosses in Streptomyces consist of growing cultures of two (or occasionally more) strainson the surface of agar, there is no equivalent of the familiar interrupted mating of E. coli genetics, andmeaningful kinetic analysis of the mating process in Streptomyces has not proved possible. Instead, one
analyses the end results of a series of events taking place over a period of days, during which manyrounds of conjugation and recombination may well have taken place.
The classical problem with genetic mapping in bacteria arises from the fact that progeny
produced by gene exchange between individuals (the equivalent of sexual reproduction in eukaryotes
with a sexual stage in the life-cycle) are almost always very rare compared with asexually produced
progeny, and so recombinants have to be recovered by the use of selectable genetic markers, such as
prototrophy or antibiotic resistance, that prevent the parental genotypes from growing. It follows that
only certain classes of recombinants can be recovered, and their frequencies depend not only on linkage
between the non-selected markers but also between them and the selected markers. This makes thestarting of a genetic map from scratch a tricky business. It was to overcome this problem that the "fouron-four" procedure (p. 141) was devised; it is so called because it involves the plating of a four-factor
cross (i.e. one containing four selectable or counterselectable markers) on four media, each selecting one&KDSWHU,QYLYRJHQHWLFDQDO\VLVE\FRQMXJDWLRQDQGSURWRSODVWIXVLRQof the four possible combinations of markers that counterselect the parental genotypes; four genotypes
are recovered on each selection. The resulting data set gives an unambiguous order of the four markers,
and approximate indications of the distances between them. This procedure, originally developed for
S. coelicolor A3(2) (Hopwood, 1959, 1972), was found to be easily applicable to several other strains,such as S. glaucescens (Baumann et al., 1974), S. rimosus (Friend and Hopwood, 1971), S. lividans(Hopwood et al., 1983), S. acrimycini (Wright and Hopwood, 1977), Amycolatopsis mediterranei(Schupp et al., 1975), S. venezuelae (Doull et al., 1986), S. avermitilis (Ikeda et al., 1987) and S.
ambofaciens (Smokvina et al., 1988). Once some genes have been located on the map, new markers canbe mapped by making just one or two selections and considering the segregation of the new marker in
relation to that of non-selected markers of known map locations (p. 147).

Box 7.2 RECOMBINATION AND GENOME TOPOLOGY IN STREPTOMYCES
A single circular linkage group
Early studies of genetic linkage, using conjugal crosses, revealed two separate linkage groups
in S. coelicolor A3(2); by 1964, they carried 19 and 20 loci respectively (Hopwood, 1965a). The
following year, the two groups of genes were shown to be parts of a single linkage group, and thisappeared to be circular (Hopwood, 1965b). Such a conclusion was not unprecedented, because
Escherichia coli K-12 and Salmonella typhimurium LT2 were known by then to have circularchromosomes. However, it did not follow that the chromosome of S. coelicolor was a closed circular
structure. This is because mating gave rise to incomplete zygotes, or merozygotes (again as in the E.
coli paradigm). Each zygote contained a complete chromosome from one parent and a chromosome
fragment - on average representing about one-fifth of the chromosome - from the other (Hopwood,1967). The fragments appeared to have more or less random end points and could come from eitherof the two parents in a mating, with the other parent contributing the complete chromosome.
Assuming that the chromosome is actually a linear structure with fixed ends, some representative
members of the merozygote population could be shown as follows:

Continued .....

&KDSWHU,QYLYRJHQHWLFDQDO\VLVE\FRQMXJDWLRQDQGSURWRSODVWIXVLRQWhen recombination occurred in such merozygotes, uneven numbers of crossovers, forexample a single crossover, would give rise to two incomplete chromosomes, and these could notyield viable progeny: see diagram below (a). On the other hand, even numbers of crossovers, forexample two crossovers, would produce complete, recombinant, chromosomes which could be
inherited by viable progeny: see diagram (b). Only in the - presumably rare - case when the parental
fragment included a chromosome end would a single crossover yield a viable recombinant: see
diagram (c)(a)

1 crossover(b)

2 crossovers(c)

1 crossoverContinued .....



&KDSWHU,QYLYRJHQHWLFDQDO\VLVE\FRQMXJDWLRQDQGSURWRSODVWIXVLRQThus there would be automatic selection for the results of even numbers of crossovers, meaning that(nearly) all viable recombinants would inherit both ends of their chromosomes from the same parent.
The ends would therefore show complete linkage; in other words the extremities of the linkage map
would be joined to make a circle (Stahl, 1967).

Genome topology
Because of these considerations, and in the absence of the kind of microscopic evidence
available for E. coli, a choice could not be made between circularity and linearity for the S. coelicolor
chromosome. However, notwithstanding this ambiguity, it appeared to be possible at least to deducethat the chromosome was either circular or circularly permuted. This conclusion arose from theresults of an experiment involving heteroclones (Hopwood, 1966). Heteroclones are colonies thatarise when the progeny of a cross are plated out on a medium selecting the dominant marker at eachof two closely linked loci; they originate from partially heterozygous plating units and give rise to
haploid recombinant progeny when they sporulate. Samples of the spores can be analysed by platingthem out, and the resulting genotypes reveal the extent of the heterozygous segment in a particular
heteroclone. On the simplest hypothesis that the heterozygous segment thus deduced represented asingle continuous region of the chromosome, it was found that this could extend, in different classesof heteroclones, over every interval between adjacent loci on the linkage map: there was no intervalthat was not bridged in this way, suggesting that the chromosome lacks constant ends.
No further information on the topology of the S. coelicolor chromosome (or that of any otheractinomycete) was obtained for the next 25 years. Then, the use of pulsed-field gel electrophoresisof macro-restriction fragments led to the construction of a physical map for the chromosome. Thisat first appeared to be circular (Kieser et al., 1992) but a re-examination of the data after the S.
lividans chromosome had unexpectedly been found to be a linear structure with fixed ends showedthat the S. coelicolor chromosome is also linear with constant ends (Lin et al., 1993). This seems to
be characteristic of streptomycetes in general (Lin et al., 1993; Lezhava et al., 1995; Leblond et al.,1996).
It is still not clear how to rationalise these findings with the results of the heteroclone
experiment, which seemed to exclude constant chromosomal ends. The finding that the S. lividans
chromosome, and by analogy that of S. coelicolor, can be circularised, either spontaneously or
artificially (Lin et al., 1993), might have provided an answer, but physical analysis of the two strainsused in the heteroclone experiment has suggested that neither is a variant with a circularchromosome. Perhaps the cultures might harbour a substantial minority of variants with circularchromosomes, which could perhaps have provided the chromosomal fragments spanning thechromosome ends, but the classes of heteroclones containing them were probably too numerous forthis to provide a satisfactory explanation. Another possibility is a non-covalent association between
the chromosomal ends, perhaps mediated by proteins bound to the series of DNA repeats in the end
regions of the chromosome (Chen et al., 1994). In this case, the non-covalent association between
the ends might have to be preserved during the conjugation process.
Even more important to understand is the way in which the DNA is transferred during mating,
and whether the pieces transferred represent random, and so usually internal, fragments of the wholechromosome, thus justifying the argument above about even-numbered crossovers being required togenerate viable recombinants. Alternatively, if the end regions were preferentially involved intransfer, all, or a majority, of the merozygotes might be able to generate viable recombinants by oddnumbered crossovers. Perhaps both possibilities arise, depending on the conjugal plasmid responsible
for the c.m.a. (C. W. Chen, personal communication). Hopefully some of these questions will soon
be addressed experimentally.

&KDSWHU,QYLYRJHQHWLFDQDO\VLVE\FRQMXJDWLRQDQGSURWRSODVWIXVLRQIn S. coelicolor A3(2) (Hopwood, 1967) and a few other strains, such as S. rimosus (Alaúeviì,1976), heteroclones have been used in mapping. Heteroclones are colonies arising from partiallyheterozygous plating units which each give rise to spores representing a population of recombinant andparental genotypes. They provide a rare opportunity to make a non-selective linkage analysis. However,
the analysis of heteroclones is laborious and specialised and so they are largely of historical interest (e.g.
in the development of ideas about genome topology: Hopwood, 1966, 1967, and Box 7.2); we do notconsider them further here as a mapping tool.
The results of genetic mapping experiments in S. coelicolor, as well as in other species that havebeen studied less intensively, generate a circular linkage map even though the chromosome itself is alinear molecule with fixed ends (see Box 7.2). Indeed, it is still valid and convenient to use the logic of
circular chromosomes to consider the results and to represent the outcome of most purely genetic
mapping experiments. Recent physical analysis of the S. coelicolor chromosome has shown that theearlier in vivo genetic analysis had been quite accurate, and has also re-kindled interest in it. Physicalmapping of many of the gene loci earlier mapped genetically revealed no clear example of an incorrect
map order (Kieser et al., 1992; Redenbach et al., 1996). In general, the approximate relative spacing of
the genes was also found to be remarkably accurate, except for a general underestimation of the lengthof the "3 o’clock silent region" (now known to include DNA within about 1 Mb of each end of thechromosome). With the recent construction of a set of ordered cosmids covering the chromosome of theS. coelicolor A3(2) derivative M145, and the finding that individual cosmids (and even pools of cosmids)
can be introduced into the chromosomes of S. coelicolor mutants by direct transformation using cosmid
DNA isolated from E. coli, resulting in complementation of the mutants (Redenbach et al., 1996; H. M.
Kieser, personal communication), map location has become valuable in attempts to clone genes. It
enables transformation to be limited to just a handful of candidate cosmids.
Details of genetic maps and strains of S. coelicolor and S. lividans are in Chapter 20.

Establishing a genetic map by the "four-on-four" procedure
From a starting strain, two strains are derived that differ by four characters, typically by virtueof two auxotrophic mutations in each strain, or one antibiotic resistance and two auxotrophic markers
in one strain and one auxotrophic marker in the other. The strains are crossed, the crosses are allowed
to sporulate, and a spore suspension is prepared. Recombinants are then selected from equal volumesof the spore suspension by plating it on four selective media, each with a different non-parental
combination of two growth factors. Accurate counts are made of the numbers of colonies on eachmedium. (Samples may also be plated on the two media selecting parental genotypes, so as to estimate
total recombination frequency, but these counts are not used in the linkage analysis.) All, or a random
sample, of the colonies from each of the four media are then classified in respect of the two non-selectedmarkers on that medium, by picking the colonies to master plates and characterising them by replica
plating. There are four possible genotypes on each medium. The numbers found give the figures inparentheses in Table 7.2. These figures are then scaled, using the total colony count on that medium,to give an estimate of the frequency of each genotype per unit volume of the spore suspension; these are
the numbers outside of the parentheses in Table 7.2.

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&KDSWHU,QYLYRJHQHWLFDQDO\VLVE\FRQMXJDWLRQDQGSURWRSODVWIXVLRQTable 7.2 Analysis of a four-factor cross (ade his × arg lys): Friend and Hopwood (1971)

Genotypes of progeny(in complementary
pairs, 1-8)

ade +Number of each genotype per unit volume(actual counts in parentheses) on
selective media containing
Adenine andarginine

AdenineandlysineArginineandhistidine

Averagefrequency
for eachpair ofgenotypes

Histidineandlysinehis +1Parental - do not grow on selective media
+

arg +

lys

+

+

+

+

(14)

24

(45) 36(14) 33(6) 26230
ade arg his lys
ade +

+

+

(17)

*

*

*

*

29

(30) 24

*

*

3

27
+

arg his lys

+

arg +

+

(42)

*

*

*

*

72

*

(48) 112

*

4

92
ade +

his lys

*

*

*

*

+

his +

*

*

(86) 202(92) 393+

5298
ade arg + lys

*

*

*

*

+

*

(51) 41*(3) 13

+

+

lys

6

27
ade arg his +ade arg +

+

*

*

*

*

(76) 130

*

*

*

7

100
+

+

his lysade + +

*

lys

*

(0)

*

*

(16) 70

0

*

*

8

3
+

arg his +Heterokaryons1

(1)

*

*

(2)

5

*

2

(24) 19(0)0(33) 142

Total(150) 257
(150) 120(150) 352
(150) 644
Recombinant classes that do not grow because the selective medium does not contain all their growthrequirements.
1"Heterokaryons" are prototrophic colonies that generate spores of the two parental genotypes only
(p. 136); they are ignored in the analysis.
*

&KDSWHU,QYLYRJHQHWLFDQDO\VLVE\FRQMXJDWLRQDQGSURWRSODVWIXVLRQTable 7.3 The four 2 × 2 tabulations of segregation of pairs of unselected markers, using the absolutecounts in parentheses from Table 7.2
Selected alleles
his+/lys+
arg+

arg–

lys+

lys–

ade+
ade–

14
17

42
76

45
30

51
0

( 2)

0.30 (0.96)(independent)

Unselected markers

Probability of independence1

arg+/his+<0.001 (27)(dependent)

Selected alleles
ade+/lys+
arg+

arg–

lys+

lys–

his+
his–

14
86

48
2

6
92

3
16

( 2)

<0.001 (92)(dependent)

Unselected markers

Probability of independence1

arg+/ade+

0.15 (2.1)(independent)

1

Determined using the 2 test. A simple way to calculate this for a set of values
a, b, c, d is shown below:

2

a

b

(a + b)cd(c + d)

(a+c)

(b+d)

(a+b+c+d)= (ad - bc)2 (a+b+c+d) ÷ (a + b) (c + d) (a + c) (b + d)

The probability of independence can be determined using a 2 distribution table for one
degree of freedom; the smaller the value of 2, the higher the probability of
independence. A key value is 2 = 3.8 for a probability of independence of 0.05; this is
a commonly used confidence limit.
A four-factor cross yields 16 possible genotypes of progeny, in eight complementary pairs (acomplementary pair is one in which each locus is represented by the mutant allele in one member andthe wild-type allele in the other). The genotypes are arranged in complementary pairs in Table 7.2. Pair
number 1 represents the parental genotypes. Most of these parental types are produced by asexual
reproduction, and so their frequencies do not tell us anything about the linkage relationships of themarkers. The other seven pairs are recombinants. It is a special feature of the four-on-four procedurethat at least one member of every complementary pair of recombinants can be selected, on one or moreof the media. Crucially, both members of two of the pairs (numbers 7 and 8) can be recovered, each ona different selective medium. This gives us a chance to check whether their frequencies are&KDSWHU,QYLYRJHQHWLFDQDO\VLVE\FRQMXJDWLRQDQGSURWRSODVWIXVLRQ

his
+
his

+

4

-

ade
ade

+ade

3

-

his

1

-

+

arg
arg

lys

-

2

+
his

4

arg
+

+

-

ade
ade

+ade

-

+

+

3

+

+
lys

1

-

arg
arg

lys

+
lys

-

arg
+

+

2

+

Fig. 7.4 Arrangement of four markers deduced from the analysis of a four-on-four cross in Table 7.3.
On the left is the his+/lys+ selection and on the right the ade+/lys+ selection (triangles indicate the selected
alleles).
approximately the same, as expected if the parents are contributing equally to the progeny (that is, there
are approximately equal numbers of zygotes with the complete chromosome coming from either parent).
When this is borne out, we assume that the same would be true for the pairs where we have only one
member, and so we can use its frequency to stand for both. We can now calculate the notional averagefrequency of each pair of recombinant genotypes, using all the data; these values are in the right-handcolumn of Table 7.2. Note that the estimates of the frequency of the same genotype on different media
give us a chance to check for possible distortions that are sometimes caused by differential survival ofdifferent genotypes, which is an ever-present possibility in all genetic analysis. In Table 7.2 they varyby about as much, overall, as the frequencies of complementary genotypes, reinforcing the view that thelatter may be regarded as having equal frequencies.

The "classical" way of analysing the results of a "four-on-four" crossHaving completed Table 7.2, we can conduct the linkage analysis in more than one way. The"classical" method is to begin by asking whether the two pairs of unselected alleles on each medium
behave independently of each other. For example, on the medium containing arginine and adenine, allprogeny must be his+ and lys+ in order to grow (these are the selected alleles), but they may be arg+ or
arg–, and ade+ or ade– (the unselected alleles). Similar reasoning applies to the other media. We cantherefore draw up a 2 × 2 table containing the number of each of the four possible combinations of
unselected alleles for each of the four media (Table 7.3), and then ask whether the ratio of, for example,
arg+ to arg– is the same among ade+ as among ade– colonies. A simple statistical test ( 2) may be applied
here to estimate the probability that this is not true: this test requires that the actual numbers scored in

&KDSWHU,QYLYRJHQHWLFDQDO\VLVE\FRQMXJDWLRQDQGSURWRSODVWIXVLRQTable 7.4 Crossovers required to generate various genotypes of recombinants(a) Selection of recombinants on arginine plus
adenine (his+ and lys+ selected)(b) Selection of recombinants on arginine plus
histidine (ade+ and lys+ selected)

Intervals in Fig. 7.4 in which crossing over is required to form detectable recombinants(In parentheses: numbers of recombinants found, from Table 7.2)

ade+
ade–

arg+

arg–

1,3 (14)
1,4 (17)2,3 (42)
2,4 (76)

his+
his–

arg+

arg–

1,3 (14)3,4 (86)

2,3 (48)
1,2,3,4 ( 2)the samples be used (the numbers in parentheses in Table 7.2), rather than the calculated numbers of eachgenotype per unit volume. We see in this example that the ratios are the same: that is 14:42 is
statistically equal to 17:76, as indicated by the low 2 value of 0.96. The arg and ade genes are therefore
said to be segregating independently. The same is true of the lys and his genes ( 2 = 2.1). In contrast,
arg and his do not segregate independently (the 2 value is high: 27) . The same is true of the lys andade genes ( 2 = 92).
By combining the information from all four 2 × 2 tables, the order of the four loci on the circular
linkage map is immediately apparent (it is the one shown in Fig. 7.4), because only one of the threepossible arrangements of four markers on a circle fits the results, for the following reason. Independent
segregation of two non-selected markers results when one is located in each of the two intervals that
separate the two selected markers on the circular linkage map (they are said to be "non-adjacent"), while
dependent segregation is diagnostic of the situation in which both non-selected markers are in the sameinterval (they are "adjacent"). Why is this? The key to the answer is that an even number of crossovers(two or four) is required to generate a viable haploid recombinant chromosome by recombination
between a fragment of the donor chromosome and a whole circular chromosome of the recipient (p. 139).
(With a linear chromosome, exceptions to this situation would correspond to cases when the fragment
included a chromosome end.) The numbers (1, 2, 3, 4) in Table 7.4 identify the intervals in which suchcrossovers must occur to generate each class of recombinants.
In (a) of Table 7.4, all four recombinant classes can be generated by two crossovers, one in eachof the two arcs separating the selected markers; that is in interval 1 or 2 and 3 or 4. Therefore the pairsof alleles should segregate independently because the ratio of probabilities 1,3 : 2,3 should equal the ratioof 1,4 : 2,4. In (b) the situation is different, because one class of recombinants (arg– his–) require fourcrossovers to produce them instead of only two; clearly the ratio of the probabilities 1,3 : 2,3 is not equal
to that of 3,4 : 1,2,3,4 and so the pairs of alleles will not segregate independently. The segregation oflys and his follows the same pattern as in (a), while that of ade and lys resembles that in (b).
Only one of the three possible arrangements of four markers is compatible with the segregation
patterns observed in a particular cross, so the result is unambiguous. (Note, however, that it is notinevitable to find two independent and two dependent segregation patterns as seen in this example; theoutcome will reflect the order of the selected and non-selected markers and how they are distributed

&KDSWHU,QYLYRJHQHWLFDQDO\VLVE\FRQMXJDWLRQDQGSURWRSODVWIXVLRQTable 7.5 Relative recombination frequencies in each interval
ade/arg

ade/hisade/lysarg/hisarg/lyshis/lys

30

27

30

30

92

30

298

298

92

27

27

27

27

100

298

27

100

92

100

3

3

3

3

100

455

428

423

87

222

249

between the two parents. The other possible situation is that all four 2 × 2 tabulations can show
dependent segregation; this occurs when, in each parent, the selected and non-selected alleles alternate
around the circle instead of, as in Fig. 7.4, where they are in adjacent pairs.)
Although it is useful to calculate 2 values as an aid to recognising independent or dependent
segregation, these values should not be interpreted too slavishly. Often, viability disadvantages of certain
genotypes, or a tendency for the cross to be polarised (with the complete chromosome coming
preferentially from one parent), cause a segregation to depart from independence even when the nonselected markers are not adjacent. One should be prepared to make allowances for such imperfectionsin the data; experience with S. coelicolor, in which map locations deduced genetically have been
extensively confirmed physically (p. 141), shows that this approach works!
The final step is to use the average frequencies of pairs of complementary genotypes (from therightmost column in Table 7.2) to give the relative recombination frequencies between pairs of markers(Table 7.5), and so to assign approximate lengths to the four map intervals. For example, recombination
between ade and arg is required to form any recombinant in which arg+ is combined with ade+ or arg–
with ade– - i.e. the pairs 2, 5, 6 and 7 in Table 7.2 - and so their frequencies are summed to give the valueof 455 (Table 7.5). Similar reasoning applies to the other pairs.
From Table 7.5 we see that the recombination frequency between arg and his is smaller than theothers, so interval arg/his is clearly the shortest. The fact that the intervals arg/lys and his/lys are shorter
than the remaining three intervals, which all involve ade, suggests that his, arg and lys form a group of
linked loci in one region of the chromosome, with ade further away.
Note that the new double mutant genotypes (pairs 7 and 8 in Table 7.2) that arise in the cross
may be used in two further crosses with each other to strengthen the conclusions on linkage derived fromthe first cross; a good example of the analysis of a complete set of three four-on-four crosses is in a paperon S. glaucescens (Baumann et al., 1974).
The above method of deducing linkage relationships from the four-on-four cross was the one
originally devised and, as far as we know, it has been followed in all published examples (see references
p. 138). It may seem laborious as described here, but once the theory is assimilated the analysis andpresentation of the data are very straightforward.

&KDSWHU,QYLYRJHQHWLFDQDO\VLVE\FRQMXJDWLRQDQGSURWRSODVWIXVLRQ



ade

his
arg

Fig. 7.5 Arrangement of three markers on a circle.

Analysing the results of a "four-on-four" cross by minimizing multiple crossovers
An alternative way of analysing the data has been used by T. Kieser (unpublished). The data arecollected and treated in exactly the same way up to the stage of completing Table 7.2, but are thenconsidered differently. As in the "classical" method, only the genotypes that were found and theiraverage frequencies are needed for the analysis. These are copied from Table 7.2 to Table 7.6. For eachgenotype, the alleles from one of the parents are shaded.
There is only one arrangement of three markers (say ade, arg, his) on a circle representing thechromosome (Fig 7.5). We can now deduce where the fourth marker (lys) fits best in the sequence forade arg his. The lys allele is placed in each of the three intervals and (to the right in Table 7.6) the
number of crossovers that would be needed to generate each genotype is noted. With the parent-specificshading, this is simple: crossing over must occur where the shading changes. (Note that for each positionof lys there is only one complementary pair of genotypes that requires four crossovers.) The interval
requiring the smallest number of four crossovers is the likely map position: lys maps in interval 1,
between ade and arg.

Mapping a new marker by a single selection
Once the relative positions of at least four loci are known, further markers can be mapped byanalysing progeny from a cross on a single selective medium (Hopwood, 1967). Selection is best madefor two markers that lie approximately opposite each other on the circular map. The resulting
recombinants are classified in respect of the non-selected markers, including the new gene, and theresults are used to deduce the position of the new marker. The method is demonstrated here using aneight-marker cross of the two genotypes his phe hom str and pro arg cys ura. In this cross, hom needs
to be mapped with respect to the seven known markers. Table 7.7 lists the genotypes obtained withselection for his+ and str, in an arbitrary order of frequency. A total of 356 progeny were analysed,
representing 22 of the 64 (26) possible genotypes.

&KDSWHU,QYLYRJHQHWLFDQDO\VLVE\FRQMXJDWLRQDQGSURWRSODVWIXVLRQTable 7.6 Analysis of the data of Table 7.2 by minimising the multiple crossover frequency

Average Number of crossovers
frequency
for each interval

Genotypes of progeny
1

2

3

1

2

3

Parental

0

0

0

+

30

2

2

4

+

+

27

2

2

2

arg

+

+

92

2

2

2

+

+

his

+

298

2

2

2

6

+

+

+

lys

27

2

2

2

7

ade
+

arg
+

+
his

+
lys

100

2

4

2

8

ade
+

+
arg

+
his

lys
+

3

4

2

2

3

100

30

1

ade
+

+
arg

his
+

+
lys

2

+

+

+

3

ade

+

4

+

5

Number of four crossovertypes if lys is in each of
the three intervals:

arg

lys
1

+

+

2

his

ade

+

3

+

30

The first stage of the analysis consists of calculating, from the numbers in Table 7.7, the overallfrequencies of the two alleles at each locus (the "allele ratio") among the progeny. These ratios areshown in Fig. 7.6. At the selected loci, indicated by ù, only one allele is of course represented, so theratios are 356 : 0 and 0 : 356. The allele ratios at the non-selected loci take intermediate values, fallingon a continuous gradient in each of the two arcs. It follows that any new locus can be assigned to justtwo possible positions, one in each arc, by virtue of its allele ratio alone. In this example, the allele ratio
for hom shows that it lies either between str and cys or between str and phe (Fig. 7.6).

&KDSWHU,QYLYRJHQHWLFDQDO\VLVE\FRQMXJDWLRQDQGSURWRSODVWIXVLRQTable 7.7 Genotypes of recombinants from the cross his phe hom str × pro arg cys ura (Fig. 7.6)
Genotype+ura

Number

Genotype

Number

pro

arg

cys

+

70

phe ura

pro

arg

cys

hom

6

phe +

pro

arg

+

hom

68

phe ura

pro

arg

cys

+

6

+

pro

arg

+

hom

49

+

ura

pro

arg

+

+

4

phe +

+

+

+

hom

34

phe ura

pro

+

+

hom

3

phe +

+

arg

+

hom

28

phe +

+

arg

cys

hom

3

phe +

pro

+

+

hom

16

phe +

+

arg

cys

+

3

phe +

pro

arg

cys

hom

14

+

+

pro

arg

cys

+

2

phe +

pro

arg

cys

+

14

+

ura

+

+

+

hom

1

+

ura

pro

+

+

hom

13

+

+

+

arg

+

hom

1

+

ura

pro

arg

cys

hom

12

phe +

pro

+

cys

+

1

phe ura

pro

arg

+

hom

7

+

+

+

cys

+

1

ura

ura

The next step is to make a choice between these two alternative map positions. This is done byconsidering the segregation of hom in relation to the alleles at the non-selected loci that would be
adjacent to it on the two alternative positions: these loci are cys and phe respectively. The segregations
are analysed using 2 × 2 tables, constructed from the table of genotypes; these are at the bottom of Fig.
7.6. We see from these tables that the segregations of hom and cys are very far from independent of one
another. We can describe this as reflecting a "deficiency of the hom+ cys+ class". In contrast, thesegregations of hom and phe are much closer to independence. We therefore conclude that hom and cys
are on the same side of the selected marker, str, whereas hom and phe are on opposite sides of str. In
other words, the correct position for hom is between str and cys. The explanation for the "deficiency ofthe hom+ cys+ class" referred to above is now obvious: this class needs two crossovers between str andcys to produce it, whereas the other three combinations of the hom and cys alleles need either nocrossover (hom cys+) or just one (hom+ cys and hom cys). (The reason that the segregations of hom andphe are not truly independent, even though the two loci are separated from each other by the selectedmarker, str, is because the zygotes are incomplete. This means that any zygote containing str on an
incomplete fragment can give recombinants inheriting str only if there is a crossover on both sides of str,
within the limits of the fragment, and so these crossovers tend to be correlated with each other rather thanbeing independent. The result is a relative excess of the double crossover phe+ hom+ genotype: see
Hopwood (1967) for further explanation.)
An alternative way of analysing the same data is to consider the pattern of crossovers required togenerate the set of recombinant genotypes, with the unknown marker, hom, placed in each of the sevenintervals between the already mapped loci, and to choose the position that results in the minimum numberof crossovers. The first step is to tabulate the genotype frequencies, which would usually have beencollected initially in an arbitrary order (Table 7.7), in a new table (Table 7.8) in which the known markers

&KDSWHU,QYLYRJHQHWLFDQDO\VLVE\FRQMXJDWLRQDQGSURWRSODVWIXVLRQ0
his 71
pro+

5

4
his +
356

pro 285arg 287

112
hom +

3

ura +
184

69
arg+

2

172
ura 153phe+hom 244

hom -

hom +

phe+

76

77

phe -

168

35

132
cys -

0
str+
7

1
phe 203

6

cys +
224

str
356
hom -

hom +

cys -

24

108

cys +

220

4

Fig. 7.6 Mapping the hom marker with reference to seven other markers of known
map location by a single selection for his+/str (indicated by triangles). In the first
method of mapping described in the text, the allele ratio at the hom locus is used toposition hom in interval 1 or interval 7 between the known markers, and the 2 × 2
analysis serves to choose interval 7 because of the highly dependent segregation ofhom–/hom+ in relation to that of cys–/cys+, with a marked shortage of the hom+ cys+
recombinant class. In the second method of mapping, the boxed numbers of genotypes
requiring “extra” crossovers (from Table 7.8) are relied upon: interval 7 is againchosen for the position of hom because of the minimum number (4) of such genotypes.
are listed in their correct order, starting at an arbitrary point on the circular map. The selected markers,
str and his, are included. The unknown marker, hom, is written to the right. The map intervals arenumbered (1-7). For each genotype, the alleles of one of the parents are shaded.
The number of crossovers required to generate each genotype, disregarding the unknown hom
marker, is now considered and inserted on the right-hand part of the table. Note that, with the parentspecific shading, this is simple: crossing-over must occur where the shading changes, and at least two

&KDSWHU,QYLYRJHQHWLFDQDO\VLVE\FRQMXJDWLRQDQGSURWRSODVWIXVLRQTable 7.8 Analysis of the data of Table 7.7 by minimising multiple crossover classes

Parental genotypesA

str phe

+

+

his

+

+

hom

B

+

ura pro+arg cys++Number of colonies

StrR His+ recombinants
Intervals:

1

2

3

4

5

6

7

1

Number of crossovers if
hom is in interval
2
3
4
5
6

7

1

str

+

ura pro

+

+

+

hom

13

2

2+2 2+2 2+2

2

2

2

2

str

+

ura pro

+

arg

+

hom

49

2

2+2 2+2 2+2 2+2

2

2

3

str

+

ura pro+arg cys

hom

12

2

2+2 2+2 2+2 2+2 2+2

2

4

str

+

ura pro+arg cys

+

70

2

2

2

2

2

2

2

5

str

+

ura pro

+

arg

+

+

4

2

2

2

2

2

2

2+2

6

str phe ura pro

+

+

+

hom

3

2

2

2+2 2+2

2

2

2

7

str phe ura pro

+

arg

+

hom

7

2

2

2+2 2+2 2+2

2

2

8

str phe ura pro+

arg cys

hom

6

2

2

2+2 2+2 2+2 2+2

29

str phe ura pro+

arg cys

+

6

2+2

2

2

2

2

2

2

10

str phe

+

pro

+

+

+

hom

16

2

2

2

2+2

2

2

2

11

str phe

+

pro

+

arg

+

hom

68

2

2

2

2+2 2+22

2

12

str phe+pro+arg cys

hom

14

2

2

2

2+2 2+2 2+22

13

str phe+pro+arg cys+14

2+2 2+2

2

2

2

2

2

14

str phe

+

pro

+

+

cys

+

1

4+2 4+2

4

4

4

4

4

15

str phe

+

+

+

+

+

hom

34

2

2

2

2

2

2

2

16

str phe

+

+

+

arg

+

hom

28

2

2

2

2

2+2

2

2

17

str phe+++arg cys

hom

3

2

2

2

2

2+2 2+2

2

18

str phe+++arg cys+3

2+2 2+2 2+2

2

2

2

2

19

str

+

+

pro

+

arg cys

+

2

4

4

4

4

4

4

4

20

str

+

ura

+

+

+

+

hom

1

6

6+2

6

6

6

6

6

21

str

+

ura

+

+

+

cys

+

1

6

6

6

6

6

6

6

22

str

+

+

+

+

arg

+

hom

1

4

4

4

4

4+2

4

4

24

94

94 189 188 35

4

Number of types with extra crossovers
if hom is in each of the seven intervals:

&KDSWHU,QYLYRJHQHWLFDQDO\VLVE\FRQMXJDWLRQDQGSURWRSODVWIXVLRQ

crossovers are needed to generate a recombinant phenotype. For each genotype, the unknown hom allele
is placed into each of the map intervals and, to the right in Table 7.8, it is noted in the appropriate column
when insertion of hom into a particular map interval would require two additional crossovers. Then, forhom in each map interval, the numbers of colonies requiring two additional crossovers are summed. Theinterval requiring the smallest number of additional crossovers - interval 7, between str and cys - ischosen as the map position for hom (see the numbers in "boxes" in Fig. 7.6).
As with the four-on-four procedure, the two ways of looking at the data will of course indicate thesame map position, with the same degree of confidence, because the underlying genetic logic is the sameand the same data have been used.
Often it is a good idea to confirm a map location by analysing recombinants obtained by selecting
for a second pair of markers in the same cross. In the present example, selection for arg+/phe+recombinants would be suitable since these markers are again nearly opposite each other on the linkage
map, and the allele ratios for non-selected loci will tend to be reversed compared with the first selection,
so that the effect of any gross disadvantage of one allele over another will be seen.
Further mapping will be dictated by the needs of the experimenter. Sometimes an approximate map
location - such as deduced here - will suffice. If not, the next step would be to make a selection forrecombination between two loci that flank the new marker; these should be as close as possible but with
one or more other non-selected markers in the same map interval also free to segregate. In this way amore precise map location for the new marker can be obtained. Examples are in Chater (1972) and Rudd
and Hopwood (1979).

Plate-crosses
The "plate-crossing" technique is useful when there are many strains to be crossed with a common
parent in order to assay the frequency of chromosomal recombination or of plasmid transfer. It is usuallya two-stage procedure. First, a "master plate" carrying the set of strains to be tested is prepared and, after
sporulation, this is replicated to a lawn of spores of the common parent on non-selective medium (CMor R2YE) to form the crosses. When the crossing plate has sporulated well, it is replicated to one ormore selective media to recover recombinants or transconjugants. Often only about 20 individual strains
can be tested on a single master plate, because a fairly large area of culture may be needed to obtainenough recombinants to assess the chromosomal recombination frequency if this is rather low (e.g. about
20 recombinants will usually arise from a well sporulating 1.5 cm2 patch with a c.m.a. of 10–3). If a highfrequency is expected, 50 can be handled on one master plate, or even individual colonies on a plate
carrying 100 or more can be accurately analysed (Hopwood et al., 1969). The procedure may be
extended through further rounds of replication, since Streptomyces replication gives very accurate"prints". This opens the possibility of rapid mapping by plate-crossing, such that the recombinants
initially selected may be roughly assessed for the frequency among them of a second marker. This
procedure is particularly effective when the second marker is very closely linked to one of the initiallyselected markers, as demonstrated by Chater (1974) and Seno et al. (1984).

&KDSWHU,QYLYRJHQHWLFDQDO\VLVE\FRQMXJDWLRQDQGSURWRSODVWIXVLRQDetection of conjugative plasmids by "pock" formation by transconjugants
The transient inhibition of growth of a plasmid-free Streptomyces culture when a conjugativeplasmid is transferred into it was first reported by Bibb et al. (1977) and called "lethal zygosis" (Ltz) byanalogy with the situation involving the F plasmid of E. coli, even though the latter probably has a very
different molecular basis. The Ltz reaction can be seen around patches of plasmid-containing cultures
replicated onto a lawn of a strain lacking the same or a related plasmid as narrow, sharply defined parallellines of inhibition of aerial mycelium formation ("tramlines": Fig. 7.7). These zones accurately follow
the outline of the donor culture and so the appearance of "tramlines" is quite different from the effect ofa diffusible inhibitory agent such as an antibiotic or bacteriophage; diffusible agents cause zones ofinhibition that are broader where two colonies are close together (Fig. 7.8). The Ltz reaction is also seen
as small circular zones surrounding individual spores (or regenerating protoplasts) of a donor strain in
a confluent lawn of a plasmid-free recipient (Bibb et al., 1978); these are called "pocks" (Fig. 7.7).
The Ltz reaction is believed to be a direct consequence of conjugation. Mycelial development in
the pock or tramline areas is delayed, but the recipient cells are not necessarily killed. Most spores fromthis area inherit the plasmid, which may travel up to 2 mm; this is much further than the distance reached
by donor hyphae in a confluent lawn. The Ltz reaction is shown by many, perhaps all, conjugative
Streptomyces plasmids and has played a significant part both in the discovery of Streptomyces plasmids,
including those originating by excision of integrated elements (Bibb et al., 1981), and in analysis of thegenetic control of conjugation. For example, the discovery of small-pock variants of pIJ101 gave riseto the concept of plasmid "spreading", as a distinct phase in the colonisation of a recipient culture by a
newly acquired plasmid: plasmid copies are proposed to spread by an intramycelial process, which iscontrolled by genes - called spd for spread - distinct from the one or more tra genes needed forintermycelial mating itself (Kieser et al., 1982). This hypothesis is now accepted, though not proven.
It, and other aspects of the biology of conjugative Streptomyces plasmids, are discussed in Chapter 11.
S. lividans 66 is a particularly good indicator of pocks because the pigmented antibiotics
actinorhodin and undecylprodigiosin are often produced precociously in the pocks, perhaps as a "stress"
response; pocks therefore become visible, not only on the surface of the culture as areas of reduced
sporulation, but also on the reverse as pinkish-purple rings. There are two further advantages: (1) S.
lividans 66 behaves as a largely non-restricting strain (increasing the likelihood that plasmid DNA fromother strains can be successfully transferred); (2) self-sterile derivatives of S. lividans 66 have been
prepared (using the Ltz reaction) which lack the two endogenous plasmids, SLP2 and SLP3, and show
no physical or genetic evidence of the presence of other plasmids (Hopwood et al., 1983), so SLP2–SLP3– derivatives are unlikely to contain any plasmid incompatible with an incoming plasmid or toconfer resistance to lethal zygosis elicited by it.
For the detection of pocks after conjugation with other cultures, the two SLP2– SLP3– S. lividans
strains TK23 and TK24 (Hopwood et al., 1984a) are valuable because each contains an antibiotic
resistance mutation (for spectinomycin or streptomycin, respectively). Most streptomycetes are sensitive
to one or both of these antibiotics, which can therefore be used to counterselect them, after crossing with
S. lividans. The cross between S. lividans TK23 or TK24 and the strain under study is made in the usualway (p. 130). Sometimes, one partner will strongly inhibit the other and so a more balanced outcome
may be achieved if the sensitive partner is inoculated before the other, say 24 h earlier. Harvest the cross&KDSWHU,QYLYRJHQHWLFDQDO\VLVE\FRQMXJDWLRQDQGSURWRSODVWIXVLRQFig. 7.7 “Tramlines” and “pocks” caused by the spreading of conjugative plasmids within a lawn ofa plasmid-free strain. Top: “Tramlines”. Left: Replicated patches of an SCP2* S. coelicolor strain(M142) on a lawn of an SCP2– strain (M132); note the tramlines surrounding the SCP2* culture andtheir absence around the SCP2+ strain (M143) (from Bibb et al., 1977). Magnification × 6. Right:
Replicated patches of an SLP2+ SLP3+ culture of S. lividans (top) and of an SLP2– SLP3+ culture(bottom) on a lawn of an SLP2– SLP3– culture; note the “tramline” caused by SLP2 around the top patch
and the much narrower tramline (really a line of isolated pocks) caused by SLP3 around both patches(From Hopwood et al., 1983). Magnification × 1.3. Bottom: “Pocks”. Note the varying appearanceof pocks caused by SLP2 in lawns of S. coelicolor (left), S. lividans (middle) and S. parvulus (right).
(From Hopwood et al., 1983). Magnification × 3.5.

&KDSWHU,QYLYRJHQHWLFDQDO\VLVE\FRQMXJDWLRQDQGSURWRSODVWIXVLRQRetarded development

+
Ltz
+
+
Ltz Ltz
+
Ltz

+
Ltz
+
+
Ltz Ltz
+
Ltz

Antib
Antib Antib
Antib

Antib
Antib Antib
Antib

Fig. 7.8 Different kinds of zones of inhibition caused by(non-diffusible) plasmid spreading around Ltz+ patches(above) and by a diffusible antibiotic (below).
and prepare a spore suspension (p. 44). Plate serial dilutions of the spores on a medium (usually R2YE)
containing spectinomycin (for TK23) or streptomycin (for TK24). Before spreading the spores, add adrop of spores of TK23 or TK24 to each plate; this is to make sure that there are enough spores of theplasmid-free recipient strain on each plate to reveal pocks. Usually, 100, 10–1 and 10–2 dilutions areplated; exceptionally, it may be necessary to go down to 10–3 or even 10–4 to reveal well separated pocks.
Tramlines and pocks are not always easy to detect. Optimal light conditions and timing can help,
but microscopic observation does not. The composition of the medium is important for good pockformation. We usually use R2YE, but sometimes R2 is preferred, and other media might be better forsome strains and plasmids. At intervals during the incubation of the cultures, examine them for thepresence of pocks: sometimes pocks become distinct on one day and are harder to see the next. Genuine
pocks should appear in proportion to the number of spores of the cross plated, whereas occasional
circular zones of unknown origin, present in many cultures including S. lividans, appear sporadically.
To purify transconjugants containing putative plasmids, touch a very fine, cooled wire to the centre
of a pock and streak out on a new plate. Replicate the resulting colonies to plates of R2YE previously
spread with spores of TK23 or TK24 and allowed to dry. After incubation, plasmid-carrying colonies
give rise to replicas surrounded by "tramlines". If the sampling of the pock was done accurately, and theplasmid is stable, it is easy to obtain a pure culture by this simple procedure; otherwise it may benecessary to go through further rounds of streaking and replica plating.
The purified transconjugant culture (as well as the plasmid donor strain and the recipient) can thenbe examined for the presence of CCC DNA. We have encountered the following situations. (1) Bothdonor and transconjugant yield similar CCC DNA, suggesting that a plasmid that can be isolated has beentransferred, e.g. SCP2* from S. coelicolor (Bibb et al., 1977). (2) Neither donor nor transconjugant yields
CCC DNA, suggesting that a plasmid that is difficult or impossible to isolate by usual techniques has
been transferred, e.g. SCP1 from S. coelicolor (Hopwood and Wright, 1973), and SLP2 from S. lividans(Hopwood et al., 1983). At this point attempts can be made to isolate the plasmid by pulsed-field gel

&KDSWHU,QYLYRJHQHWLFDQDO\VLVE\FRQMXJDWLRQDQGSURWRSODVWIXVLRQelectrophoresis (PFGE, p. 197), in case it is linear, as turned out to be true for both SCP1 (Kinashi et al.,1987) and SLP2 (Chen et al., 1993). (3) The transconjugant yields CCC DNA not found in the donor(nor in the unmated recipient), suggesting that an autonomous plasmid has been produced by excisionof chromosomal sequences in the donor and transferred to the transconjugant, e.g. SLP1 from S.
coelicolor (Bibb et al., 1981; Omer and Cohen, 1984), pIJ110 from S. parvulus; pIJ408, from S.
glaucescens (Hopwood et al., 1984a); or pSAM2 from S. ambofaciens (Pernodet et al., 1984). (4) The
donor shows CCC DNA but the transconjugant does not, suggesting that the plasmid identified in thedonor has not been transferred, but another (unseen) plasmid has been. Again, it may be possible toidentify this as a linear plasmid by PFGE.

Protoplast fusion
By far the most effective and convenient agent for bringing about the fusion of protoplasts of
microorganisms (or plant protoplasts or animal cells) is polyethylene glycol (PEG). The most importantparameters bearing on the use of PEG for inducing recombination were elucidated in early work withplant protoplasts and subsequent studies with animal cells. There is much evidence that they also apply
for Streptomyces protoplast fusion (Hopwood, 1981).
PEG concentration is critical. The optimal (w/v) concentration is essentially independent of themolecular weight of the PEG used: it is the concentration of the ethylene glycol monomers that is
important, irrespective of their degree of polymerisation. However, the choice of polymer is not
irrelevant because, for a given concentration, the osmotic pressure rises with a decrease of molecularweight, while the viscosity rises with an increase of molecular weight. In practice, molecular weights
in the range 1000-6000 are normally used. A concentration of about 50% is optimal, with a slight
reduction in fusion frequency (as measured by recombinant formation) at 40% or 60%. Below 40%, thefrequency of fusion falls dramatically. Few studies have used concentrations above 60% because evensolutions of PEG 1000 at 70% concentration are really too viscous to work with. In our own work wehave seen no reason to use the very viscous PEG 6000, although some authors have recommended it; wealways use PEG 1000. In our early work we used 15% DMSO and a PEG concentration of 40%,
following the report that this was effective for animal cell fusion. It certainly worked well in S.
coelicolor. However, there seems to be no advantage in using 40% PEG with DMSO rather than 50%
PEG without it (Hopwood and Wright, 1979). (Note that the optimal PEG concentration fortransformation of protoplasts by naked DNA is lower than for protoplast fusion: p. 231.)
High concentrations of calcium ions are necessary for the fusion reaction and these are provided(at 0.2M) in P buffer. As for the time of treatment, it is generally accepted that initial effective contacts
between the membranes of the protoplasts are established very rapidly during treatment with theconcentrated PEG solution, and that these are converted to fusion events during subsequent incubation
after dilution of the PEG. Thus, a brief period of treatment (a few minutes) with the fusing solution iseffective, provided that the protoplasts are handled gently during subsequent dilution and plating.
Different batches of PEG can give different results. There are reports that some batches are actually
toxic for the protoplasts, and that even non-toxic batches vary in their biological effect, at least on
transformation frequency (Baltz and Matsushima, 1983). It is therefore a good idea to keep a stock ofa batch of PEG that has been shown to be effective.

&KDSWHU,QYLYRJHQHWLFDQDO\VLVE\FRQMXJDWLRQDQGSURWRSODVWIXVLRQRecombination frequencies are reported to depend on conditions during the preparation of the
protoplasts and also during protoplast regeneration. Some such effects apparently operate via
regeneration efficiency, but others appear to be independent of it (Baltz and Matsushima, 1981). We
have not investigated the effects of such variables on our strains of S. coelicolor and S. lividans.
A way to increase the frequency of recombinants recovered from a protoplast fusion by a factor ofc. 10 was described by Hopwood and Wright (1979). This involved irradiating the parental protoplasts
with UV light, to a survival of c. 1%, immediately before fusing them. The effect was explained by the
introduction of lethal hits into the parental chromosomes, which could be recombined out to yield viable
recombinant progeny; the recovery of higher proportions of multiple crossover progeny than in control
fusions supported this interpretation.
Fusion of protoplasts of S. coelicolor or S. lividans
Protoplasts of parental strains(freshly prepared or frozen at -70E, p. 58)
P Buffer
PEG 1000 50% in P buffer. Add 1 ml sterile P buffer to autoclaved 1 g samples of PEG inscrew cap bottles; heat to dissolve and cool to 25o
R2YE plates (with growth factors for parental strains)
Pipettes (10 ml, 5 ml, 1 ml); Pasteur pipettes; plastic tubes; glass spreaders (optional)
Bench-top centrifuge; 30o incubator
Proceed as follows, after step 11 of "preparation of protoplasts" (p. 58). Most of the labour isconcerned with preparing the parental protoplasts; if protoplasts are already available the fusion
procedure takes less than 30 min.

1. Adjust the parental protoplast suspensions to have approximately the same
turbidity, as judged by eye, and mix equal volumes of each.
2. Sediment the protoplasts gently on the bench centrifuge (e.g. 1000 × g, 7 min) anddiscard the supernatant.
3. Re-suspend the pellet, by tapping, in the drop of liquid left in the tube, then add 5
ml P buffer and repeat step 2, draining the pellet thoroughly by shaking out any
drops of liquid. The pellet is usually quite coherent and "sticky". The object is to remove any
liquid which could reduce the effective concentration of PEG significantly below 50% in step 4.
4.

Re-suspend the pellet by tapping and add 0.8 ml of 50% PEG in P buffer and
immediately suspend the protoplasts by pipetting rapidly in and out of a Pasteur
pipette once. Vortex mixing should probably be avoided since it might damage the protoplasts.
The object is to coat the protoplasts with the PEG solution as rapidly as possible and then to leavethem undisturbed to establish effective fusion contacts.

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5. Leave for 2 min at room temperature.
6. Plate samples of the protoplasts on fresh plates of R2YE (supplemented withrequired growth factors) by spreading with a loop or glass spreader without
applying pressure to avoid risk of separating the fusing protoplasts, and incubate
at 28-30o. Plate 0.2 ml of the undiluted suspension as a "100" sample and transfer successive 0.1
ml samples to 0.9 ml of P buffer to prepare 10–1 and 10–2 dilutions (great accuracy is not needed).
Normally, one or more of the three plates (100, 10–1, 10–2) will produce a dense, even lawn of growthfrom which spore suspensions can be prepared for subsequent plating. The regeneration plates
should carry at least several thousand colonies to ensure a statistical sampling of possible
genotypes, but with some strains very dense plates may show evidence of inhibition of theregenerating protoplasts by precociously developing colonies arising from non-protoplasted units(mycelial fragments or even spores: Hopwood et al., 1977; Baltz, 1978). Higher dilutions (10–310–6) may be useful if individual fusion colonies are needed (e.g. Hopwood and Wright, 1978).
R2YE and 28-30o are suitable for S. coelicolor, S. lividans and some other strains, but various
authors have described other media and temperatures for particular strains. Some recommend
plating in soft agar overlays rather than spreading on the surface of agar plates. Some strains needa different osmotic stabiliser than the 0.3M sucrose in P buffer and R2YE regeneration medium.

7. When good sporulation occurs, harvest the spores from confluent plates and plateappropriate dilutions to obtain single colonies on selective or non-selective media
as for conjugative crosses (p. 130).
Genetic analysis by protoplast fusion
In principle, samples of the mixture of fused protoplasts could be plated out to regenerate on one
or more media selecting for recombinants: for example those inheriting a prototrophic marker from eachparent (drug resistance is not in general suitable because many resistances are recessive and so the
diploid fusion bodies resulting from protoplast fusion would be killed). Samples would be plated inparallel on the media selecting parental genotypes, so that frequencies of recombination could beobtained, as in a cross (p. 136). However, the problem is that the frequency of "recombinants" obtained
is not very meaningful; it can give only an approximate idea of the frequency of fusion events leading
to the emergence of particular classes of recombinants, but not a true recombination frequency(Hopwood, 1981). Also, the resulting colonies will almost certainly be genetically heterogeneous,
because of the diploid nature of the fusion bodies in which recombination takes place (Hopwood andWright, 1978). It is therefore far better for most purposes to plate the fused protoplasts on a nonselective regeneration medium at step 6 in the above procedure to give a confluent lawn of growth andthen harvest the resulting haploid spores and plate them on various selective media for analysis.
The frequency of recombination after protoplast fusion can be so high - 10-15% was routinely
observed in S. coelicolor (Hopwood and Wright, 1978) - that it is feasible to plate the spore suspension
at this stage on a non-selective medium and analyse a random sample of all progeny. For example, in

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possible genotypes (Hopwood and Wright, 1978). However, this is labour-intensive and it is usuallymore efficient to plate spores on one or more media selective for recombinants and proceed as for amating. An example is Hopwood et al. (1983), where the results of a protoplast fusion in S. lividans arecompared with those of a cross, in which mating was promoted by a derivative of the sex factor pIJ101.
These data nicely illustrate a striking feature of protoplast fusion, namely that crossing over per unit map
length is much greater than in matings (Hopwood et al., 1977; Hopwood and Wright, 1978). This result
was attributed both to the diploid (rather than merodiploid) nature of the fusion bodies and to theopportunities for several rounds of recombination during and immediately after protoplast regeneration.
This feature reduces the reliability of protoplast fusion for mapping distant markers - they may appear
essentially unlinked if mapping is attempted simply by minimising crossovers (p. 147). On the other
hand, close markers are easier to resolve by protoplast fusion than by crosses (Hopwood et al., 1983;
Hranueli et al., 1983), and fusion is also very useful for obtaining rare multiple crossover recombinants
for future use. Recombinants inheriting markers from up to four parents in a fusion of protoplasts fromfour different strains can even be obtained, but at low frequencies, necessitating their selective isolation(Hopwood and Wright, 1978).

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m08tk33b.wpd 20 April 2000 Tobias Kieser

Chapter 8

Preparation and analysis of genomic andplasmid DNA
CONTENTS
Isolation of genomic DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162
Discussion of individual steps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162
A. Kirby mix procedure for the isolation of genomic DNA . . . . . . . . . . . . . . . . . . . . . 168
B. Salting out procedure for the isolation of genomic DNA . . . . . . . . . . . . . . . . . . . . 169
C. CTAB procedure for the isolation of genomic DNA . . . . . . . . . . . . . . . . . . . . . . . . 170
Isolation of CCC plasmid DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
Discussion of individual steps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172
A. Plasmid isolation by neutral lysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
B. Standard CsCl-ethidium bromide gradient centrifugation . . . . . . . . . . . . . . . . . . 178
C. CsCl density gradient for removing polysaccharides from DNA . . . . . . . . . . . . . . 182
D. Plasmid isolation by alkaline lysis and potassium acetate precipitation . . . . . . . . 184
E. Plasmid isolation by alkaline lysis and phenol precipitation . . . . . . . . . . . . . . . . . 187
F. Plasmid purification using QIAGEN anion exchange column chromatography . 189
G. Plasmid isolation by the boiling method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190
H. Alkaline denaturation of partially purified DNA samples . . . . . . . . . . . . . . . . . . . 191
I. Purification of samples using ethidium bromide . . . . . . . . . . . . . . . . . . . . . . . . . . . 192
J. Acid phenol (pH4) extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193
K. “Jurassic preps” plasmid purification using guanidine thiocyanate . . . . . . . . . . . 194
Standard agarose gel electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195
Pulsed-field gel electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198
General considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198
Standard procedure for preparing Streptomyces chromosomal DNA for PFGE . . . . 199
Phenol wash procedure for preparation of actinomycete DNA for PFGE . . . . . . . . . 201
Digestion of DNA with restriction endonucleases in agarose blocks for PFGE . . . . . 202
Loading gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203
Running conditions for PFGE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204
Removal of small DNA fragments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
Ammonium acetate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
EDTA pH8 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206
Lithium chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206
Sodium chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206

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Sodium hydroxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
Potassium acetate and sodium acetate, 3M . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
Tris-HCl buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208

Isolation of genomic DNA
Streptomyces DNA can be isolated using methods developed for other bacteria. The Kirby mix
procedure (A; p. 168) is most commonly used by us and gives DNA fragments of about 40 kb. The
salting out procedure (B; p. 169) is simpler and avoids phenol. The CTAB procedure (C; p. 170)
removes polysaccharides. Lysozyme treatment, most effective on mycelium grown in the presence ofglycine, is necessary for lysis of most strains. DNase activity may be abundant and some streptomycetes
produce antibiotics that bind strongly to DNA (e.g. undecylprodigiosin, actinomycin).
The concentration of the mycelium, optimal timing of the lysis step, and the care and skill
exerted in the solvent extraction, phase separation and precipitation steps may be more important thanthe choice of method. DNA fragments should be at least four times as long as the fragments to be used
after digestion with restriction endonucleases. Preparing DNA fragments of more than 200 kb (70 µm
long!) for cosmid cloning can be quite a challenge; DNase action and mechanical shearing (shaking, rapid
pipetting through small-bore tips) must be minimised.
Removal of RNA from DNA preparations, a relic from the times when DNA was used forrenaturation studies (cot curves), is usually not necessary. It may be more convenient to add RNase(DNase-free, 5 µg ml–1; p. 166) together with the restriction enzyme or to the gel loading buffer. A
disadvantage is that with RNA present, DNA concentrations cannot be determined
spectrophotometrically (p. 167).
It is important to guard against contamination of genomic DNA preparations with vector plasmid
to avoid unwanted transformants in cloning experiments.

Discussion of individual steps(Complete procedures are on pp. 168-171.)
Growing mycelium. Media giving vigorous dispersed growth are best. Glycine (usually 0.5%)
improves the action of lysozyme, but may cause premature lysis and consequent nucleolytic degradationof DNA in some strains. About 0.5-1 mg of DNA can be expected from a 25 ml culture nearing
stationary phase. If difficulties are expected, grow sufficient mycelium for several experiments. For
S. coelicolor and S. lividans, we use YEME with 34% sucrose + 0.5% glycine. On solid media, cultures
are best grown on cellophane membranes (p. 48).
Harvesting mycelium. Streptomycetes can be harvested using vacuum filtration (two sheetsof Whatman No. 1 paper on top of each other; change filters as soon as they start clogging) or by low
speed ($500 × g, e.g. 2000 rpm in SS-34 rotor) centrifugation. If the density of the medium is high (e.g.
YEME+34% sucrose, density= 1.15 g ml–1), centrifugation produces loose pellets. Dilution of the culture

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with water helps. Washing of the mycelium with 10mM EDTA pH8 in water or in 10% sucrose forfragile strains (MacNeil and Gibbons, 1986; p. 190) removes extracellular nucleases, proteinases (could
inactivate lysozyme), polysaccharides (difficult to separate from DNA; pp. 164, 173), and phosphate(precipitated by alcohol). Turbid filtrates or supernatants signal contamination with unicellular bacteria
or yeast, recognisable under the microscope.
Storing mycelium. Mycelium may be stored frozen at -20E without added liquid to facilitate
thawing. Freezing and thawing assists lysis. Wrapping the mycelium in aluminium foil makes for
efficient storage, but beware of cross contamination with plasmid-containing material.
Resuspension. It is usual to resuspend the cells in about 20% of the original volume (#0.2 g ml–1
filtered wet weight). Complete resuspension at a suitable cell density is important. Too high a density
will result in unmanageably viscous samples. Mycelial clumps may be disrupted gently using a teflon
homogeniser (BDH 406/0313/0315) fitted to an electric drill. Fresh wooden toothpicks contain
nucleases and must be autoclaved. Make sure that the tubes are resistant to chloroform and suitably large
for mixing the samples after all the additions. Mycelium clings to the walls of tubes; reserve some buffer
for rinsing when transfer is necessary.
Lysozyme treatment. Lysozyme treatment is essential for most streptomycetes (1-5 mg ml–1,
e.g. Sigma L6876; filter sterilise; lysozyme is very stable in water at slightly acid pH and remains active
above 90E). Digestion is at pH8, usually for 10-60 min at 37E; higher temperatures cause DNA
degradation. Triturate with a pipette, before the lysis step. If cell wall-bound DNases (Mg2+-dependent,
most active at low ionic strength; Aparicio et al., 1991) are a problem, mycelium should be partiallydigested with lysozyme and then washed again to remove the released proteins. Lyse the cells as soon
as possible. Nuclease activity may vary with growth phase and medium. Drops of cell suspensions canbe tested by mixing with 10% SDS on a glass microscope slide. A highly refractile, clear and viscousproduct indicates complete lysis; partially lysed samples may become viscous, but they remain turbid.
Mycelium that does not lyse after lysozyme treatment may be ground in liquid nitrogen (Rao et al., 1987).
Mutanolysin (5 µg ml–1; Sigma M9901; Assaf and Dick, 1993) has been used to assist cell lysis.
Lysis. Several alternative methods are used.
1% SDS ± proteinase K (0.1-1 mg ml–1) at 55-70E. We use "specially pure" sodium laurylsulphate (BDH 44244). Avoid inhaling SDS dust. Autoclave 10% stock solution, keep at 20E and warm
before use if a white precipitate appears.
Proteinase K (Sigma P2308, Boehringer etc.) is rather expensive, but free of DNase activity (nopredigestion necessary for DNA purification), and works best at 50-55E in SDS at pH6.5-9.5. 5mM Ca2+
stimulates the activity but is not added because it prevents (partial) self destruction of proteinase K andstabilises the mycelium. Proteinase K degrades most but not all proteins including RNase, DNases andlysozyme, and eventually it will destroy itself (solutions must be made fresh or kept frozen as single use
aliquots).
2 × Kirby mix (TPNS (1% sodium tri-isopropylnaphthalene sulphonate: Kodak P 3513) + 3%
phenol/chloroform isoamyl alcohol + 6% sodium 4-amino-salicylate (BDH)) was designed for efficient
cell lysis and inactivation of RNase and DNase activity (Kirby et al., 1967). The detergent TPNS is more
lipophilic than SDS and toxic. 4-amino-salicylate is a chelating agent (procedure A, p. 168).
1% Sarkosyl (sodium lauroyl sarcosinate, NaOCO-CH2-N(CH3)-CO-(CH2)10-CH3; BDH 44275,
Sigma L9150), unlike SDS, is soluble in CsCl; lysates can be used directly for CsCl gradientcentrifugation (Sambrook et al., 1989).

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Solvent extractions. Phenol, chloroform or a mixture of the two denature and precipitate protein
which ends up in the interphase after centrifugation (RNA and polysaccharides remain with the DNA).
There must be a separate organic phase for the extractions to work. If necessary, add more solvent. The
shaking time required depends critically on how well the sample is emulsified (increasing surface area).
If interphases are bulky, they are best removed with a spatula before transferring the very viscous DNAcontaining upper phase, and the extraction should be repeated till no more interphase appears. DNA loss
can be reduced by re-extracting the phenol interphase with buffer. For large sample volumes it is safer
to do several extractions with 1/5 volume instead of the usual equal volume of solvent; upper aqueous
phases can be separated cleanly by decanting. In practice, usually one or two solvent extractions are
performed for genomic or plasmid DNA isolations.
Phenol (BDH 10188, colourless crystals, density=1.076 g ml-1) is used without further
purification. 0.1% 8-hydroxyquinoline (BDH 10131) is added as antioxidant (m-cresol and-mercaptoethanol additions are not used in our lab). Phenol is an acid (pKa=10, pH of water phase =4.5-6) and must be neutralised (to 500 g crystalline phenol add 120 ml water, 7 ml 1M Tris-HCl pH8 and
15 ml 1N NaOH) and saturated with aqueous buffer containing the correct amount of salt. The aqueous
phase, tested with indicator paper, should be pH8.0. DNA is lost at low pH (acid phenol is used forplasmid isolation procedures E (p. 187), H (p. 191) and J (p. 193)). Phenol mixtures should be stored
at 4E, airtight in the dark. Phenol is more stable when saturated with water than in pure form.
After centrifugation, the yellow (8-hydroxyquinoline) phenol phase is usually at the bottom of
the tube, but with dense sucrose or salt solutions inversion is possible. If the phases cannot be separated,
add chloroform. At high salt concentration, single-stranded but not double-stranded DNA goes to thephenol phase (c. 25% of the salt from the aqueous phase is extracted into the phenol phase). Phenol andphenol/chloroform very effectively remove ethidium bromide from DNA solutions (p. 182).
Phenol is poisonous by ingestion and by absorption through the skin; $1 g per person can belethal; small amounts phenol dissolved in water are not dangerous. Phenol burns are often not recognised
immediately because phenol is a local anaesthetic. Use eye protection and disposable gloves. Wash
contaminated skin immediately with water; in severe cases (large exposure, nausea) seek medical
attention.
Chloroform (density=1.484) denatures and precipitates proteins less effectively than phenol. To
reduce foaming, 1/24 v/v isoamyl alcohol is usually added. Phase separation is easier than with the lessdense phenol. Chloroform can also be used to remove traces of phenol from aqueous solutions.
Chloroform is not flammable, but mixing with strong bases or acetone can cause explosions.
Polycarbonate and some other plastic tubes dissolve rapidly in chloroform. Chloroform is hepatotoxic
and carcinogenic, and must be disposed of correctly.
Polysaccharides can be removed by chloroform/isoamyl alcohol extraction of DNA solutions
containing 1% CTAB (cetyltrimethylammonium bromide, hexadecyltrimethylammonium bromide,
CH3(CH2)15N(CH3)3Br; Sigma H5882, BDH 27665) and NaCl >0.7M at >15E. Note that CTAB
precipitates DNA if the salt concentration is below 0.5M. More CTAB must be added if the procedure
is repeated (procedure C, p. 170).
Phenol:chloroform:isoamyl alcohol 25:24:1 pH8 is commonly used to deproteinate DNA. It
gives better phase separation than phenol alone. Make sure the phenol is at the correct pH. DNA from
agarose gels should first be extracted with phenol alone before phenol/chloroform is used. Store as
phenol mixtures and consult safety and disposal rules for chloroform and phenol.

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

Dialysis. Dialysis (with several changes of buffer) is a very gentle (no mechanical shearing) but
slow method for removing low molecular weight compounds including SDS and phenol from DNA
solutions. Leave sufficient space when the samples contain high salt concentrations (CsCl); otherwise
tubes may burst. The volume of the sample does not increase if, for the first dialysis, 20% PEG 8000
(Sigma P5413) or PEG 20,000 (BDH 29864; Longmire et al., 1987) is used. Organic molecules are
removed more efficiently if activated charcoal (e.g. Fluka 05105 or BDH 33203) is added to the dialysis
buffer. Some streptomycetes produce cellulases (probably inactivated by phenol or proteinase K) thatcan damage dialysis membranes and also Sephadex columns.
Dialysis tubing is prepared by boiling for 10 min in 2% sodium hydrogencarbonate (bicarbonate;
BDH 10247) and 1mM EDTA, then 10 min in distilled water. Store in 50% ethanol. Boil tubes for 2
min in water before use.
Microdialysis with Millipore VMWP filters (pore size = 50 nm, much larger than normal dialysis
membranes) is fast, efficient, but expensive: sterilise filters in 70% ethanol, rinse in dialysis buffer, floaton buffer in Petri dish, shiny side up, add up to 100 µl DNA sample (less if sample contains high salt
concentration), wait 30 min, remove sample with pipette; >95% recovery of DNA can be expected.
Single-stranded DNA and DNA with bound protein may bind to the filters (Marusyk and Sergeant, 1980).
Precipitation of DNA. DNA precipitation removes salts, SDS and phenol. Protein,polysaccharides, much RNA and inorganic phosphate precipitate with DNA. Salts including phenolate
may also precipitate, especially at low temperatures.
Ethanol, 2.2 vol The salt concentration of the DNA solution is important. 2.5M LiCl, 1-2M
NaCl or CsCl (p. 181), 0.3M potassium or sodium acetate (p. 207), or 2.5M ammonium acetate (p. 205;
keep airtight) are used for low DNA concentrations; less salt or ethanol can be used for concentrated
DNA samples. tRNA or glycogen (BCL) (20 µg ml–1) is added as carrier for precipitating #1 µg ml–1 ofpure DNA. Potassium acetate is more soluble in ethanol than sodium acetate. Ammonium acetate
evaporates under vacuum (ammonium ions inhibit polynucleotide kinase).
Ice cold ethanol is usually used. Mixing is important. There is no need to cool the samples to-20E. For low DNA concentrations the centrifugation time and speed are important, mainly to make thepellets adhere to the bottom of the tubes. The purification achieved by precipitations critically depends
on how well the supernatant is removed (decant, spin and remove remaining liquid with a pipette).
Genomic DNA samples should produce clearly visible, stringy precipitates which are bestspooled onto a sealed Pasteur pipette (the remaining turbidity is probably RNA and possibly plasmidDNA) and can then be washed effectively in 70% ethanol to remove remaining salt. It is best not toprecipitate these samples by centrifugation because large compacted pellets may be impossible todissolve. Never dry samples completely because this makes them insoluble. DNA dissolves best in low
ionic strength buffer pH $8 (do not store DNA in water). Heating to 55E in TE buffer and gentle
agitation accelerate solution; pipetting shears DNA. In angle rotors, much of the DNA sticks to thewalls; mark its position.
Isopropanol precipitation at 0-20E (salt may precipitate at lower temperature). This is similar
in its salt requirement to ethanol precipitation; 0.54 volumes isopropanol are sufficient for precipitating
DNA; 0.6-1.0 vol isopropanol is usually used. Small RNA molecules precipitate less efficiently than

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Table 8.1. PEG 6000 precipitation of genomic and plasmid DNA
Final concentrationµl PEG stockfor 1 ml DNA solution

PEG w/v

NaCl

30% PEG1

50% PEG2

Genomic DNA>20 kb

(4%)
5%
6%
7%

0.7M
"
"
"

154
200
250
304

87
111
136
163

CCC plasmid
DNA

8%
10%
12%

0.7M
"
"

364
500
667

190
250
316

PurposeIncubation
time at
0-4E

Recoveryof DNA$5 minSpool

4-16 h10,000 × g3
20 min1

For 1 litre use 300g PEG 6000, 38.1 g NaCl, 692 ml water. 2For 1 litre use 500 g PEG 6000, 38.1 g NaCl,
535 ml water. 310,000 g = 10,000 rpm in SS-34 or 8,000 rpm in GSA rotor. For samples containing much
chromosomal DNA centrifuge 2600 × g (4,600 rpm in SS-34 or 4,000 rpm in GSA rotor).
DNA if the isopropanol concentration is kept low. The pellets tend to be "glassy" and more difficult to
see than pellets after ethanol precipitation. Large pellets tend to slide and may be lost if the tubes areleft inverted. Isopropanol precipitates more salt and is less volatile than ethanol. Wash precipitates with
70% ethanol before air drying the samples.
PEG 6000 precipitation (Table 8.1). This is suitable for large volumes (polyethylene glycol,
Fisons P/3681/53, BDH 44271, or PEG 8000, Sigma P5413; 30% or 50% w/v stock solution in wateror in 0.7M NaCl, autoclave and store at 4E). The DNA solution (>10 µg ml–1) after the addition of PEG
must contain $0.5M NaCl. Genomic DNA precipitates instantaneously and can be spooled. The PEG
concentration needs to be increased for small DNA and RNA fragments. Plasmid DNA precipitatesslowly and must be harvested by centrifugation (Table 8.1); samples contaminated with much
chromosomal DNA (p. 177) become difficult to dissolve if spun too hard (Humphreys et al., 1975; Lis,
1980; Sambrook et al., 1989). Remove all PEG, redissolve samples in TE and remove traces of PEG byethanol precipitation (p. 165). If in doubt, add more PEG 6000 to the supernatant, incubate 24 h at 0E
and centrifuge again.
Spermine (0.5-5mM). This precipitates DNA from low salt buffers (e.g. 10mM Tris, 1mM
EDTA pH8) and discriminates against very small DNA fragments (p. 174).
RNase treatment. 40 µg ml–1 DNAse-free pancreatic RNase A (Sigma R4875; stock solution:
4 mg ml–1 (400 units) stock in 10mM sodium acetate pH5.2, 15 min 100E, add Tris-HCl pH7.4 to
100mM, cool slowly to 20E, store in plastic container at -20E; RNase A precipitates when boiled atneutral pH; binding to glass may cause significant loss). RNase A cleaves double-stranded RNA and
RNA-DNA hybrids at salt concentrations below 0.3M; it is specific for single-stranded RNA at higher
salt concentration. Complete inactivation of RNase A requires proteinase K digestion (p. 163) andseveral phenol/chloroform extractions. Avoid contaminating equipment and solutions used for RNA
techniques. Large RNA molecules and polysomes can be removed by precipitation with 2.5M LiCl(p. 173); small RNA molecules are not precipitated by PEG 6000 or 0.54 vol isopropanol.

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For many purposes, RNase may be added together with restriction enzymes. If RNA is notremoved, spectrophotometric determination of the DNA concentration is not possible, but DNA
concentrations can be estimated from ethidium bromide-stained agarose gels (p. 175).
CsCl gradient centrifugation. For the purification of genomic DNA, ethidium bromide (700 µg–1ml ) is usually added to reduce the density of the DNA, allowing faster rotor speeds, and as a means ofvisualizing the DNA band. A 10 ml gradient may take about 2 mg of DNA and is prepared and run as
for plasmid DNA isolation (p. 178), except that gradients are not relaxed so that CCC remains close to
the linear DNA. Use a 18-gauge needle for harvesting and dialyse (p. 165) the DNA rather than
precipitating to remove CsCl.
DNA fragments can be separated according to their G+C content using the AT-specific dye
Hoechst 33258 (p. 175).
Storage of DNA. DNA can be stored at c. 0.5 mg ml–1 in TE buffer, either at 4E with a drop ofchloroform to maintain sterility (recommended for genomic DNA), or frozen at -20E. DNA is also stablein 4.8M CsCl solution (direct from the gradient after removal of ethidium bromide).
Quantitation of genomic DNA. 1 g of wet mycelium yields 0.5-1 mg DNA. The viscous, highmolecular weight DNA is difficult to sample because it tends to slide out of the pipette. 50 µg ml–1 pureand completely dissolved DNA gives OD260=1.0 (phenol and phenolate absorb maximally at 272 and
290 nm, respectively). To achieve equal loadings on gels (e.g. for Southern blots) it is often best to
estimate the DNA concentration visually on ethidium bromide-stained agarose gels (595 nm emission)
after digestion with a restriction enzyme. See also quantitation of plasmid DNA (p. 175).
Hoechst 33258 (Sigma B22883) fluoresces much more with DNA than RNA (345 nm excitation,
460 nm emission, photograph using yellow filter e.g. Wratten No. 3; sensitivity 0.2-1 µg 50% G+C DNA)and can be used for spot tests to determine DNA concentrations on agarose plates (0.1M NaCl, 10mM
EDTA, 10mM Tris-HCl pH7, 2.5 µg ml–1 Hoechst 33258, 1% agarose; incubate $1 h before reading theplates; Rieber and Rieber, 1990).
For Southern blots containing digests of plasmid clones and genomic DNA, make sure thatspecific sequences are equally represented. The amount of plasmid DNA required per gel slot depends
on how much genomic DNA is used per lane, and the size of the plasmid in relation to the genome:µg (plasmid DNA) = µg (genomic DNA) × size (plasmid, kb) ÷ size (genome = 8,000 kb).

Checking the quality of the DNA. The average size of the DNA molecules can be determinedby pulsed-field gel electrophoresis (p. 198). OD260÷OD280 < 2.0 indicates protein or phenol
contamination.

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Common problems
Cells do not lyse: increase glycine concentration in growth medium; Kirby mix gives best lysis;try other enzymes (p. 163).
DNA does not dissolve: remove all alcohol, PEG 6000 or spermine; remove salt by washing with
70% ethanol; increase volume; incubate at 55E; increase pH; spool instead of centrifugation; dialyse
instead of precipitation (p. 165).
DNA fragments too small (solution not very viscous): nuclease activity can be reduced by quick
lysis (p. 163) using Kirby mix (below) or proteinase K (p. 163). Reduce mechanical shearing, longexposure of DNA to acidic phenol or to cell extract at low pH. Also prolonged incubation of the cells
at high temperature (50-90E) results in degradation of the DNA.
DNA cannot be cleaved with restriction endonucleases: remove all phenol, SDS and salt (p. 165);
increase the reaction volume; add bovine serum albumin. There may not be sufficient enzyme present(some enzymes die off quickly) or Streptomyces DNA may have fewer restriction sites than expected (p.
212).
Polysaccharides: CTAB extraction (pp. 170, 173); CsCl gradient centrifugation without
ethidium bromide (pp. 174,179, 182); QIAGEN purification (p. 189).

A. Kirby mix procedure for the isolation of genomic DNA
Slightly modified from Hopwood et al., 1985. This procedure depends on lysis with phenolic
detergent mix and phenol/chloroform extraction.
Mycelium from 25 ml culture
TE25S buffer: 25mM Tris-HCl, pH8, 25mM EDTA pH8, 0.3M sucrose
Lysozyme: 60 mg ml–1 in water
Phenol: 500 g phenol (AnalaR), 0.5 g 8-hydroxyquinoline (AnalaR) saturated with
50mM Tris-HCl pH8 (p. 164)
Phenol/chloroform/isoamyl alcohol: 50 ml equilibrated phenol, 50 ml chloroform,
1 ml isoamylalcohol
2 × Kirby mix: 2 g TPNS (sodium tri-isopropylnaphthalene sulphate, Kodak P3513;
SDS can be used instead), 12 g sodium 4-aminosalicilate (BDH), 5 ml 2M Tris-HCl
pH8, 6 ml equilibrated phenol pH8.0, make up to 100 ml with water
3M unbuffered sodium acetate (p. 207); (sodium acetate pH4.8 is not suitable)
Pre-boiled RNase A: 4 mg ml–1 (p. 166)
Isopropanol, 70% ethanol, TE buffer
20 ml phenol/chloroform-resistant screwcap low speed centrifuge bottles; 10 ml
pipettes and propipettes; sealed Pasteur pipette; eye protection and disposable
gloves
37E water bath; low speed centrifuge

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

1.

Resuspend mycelium in 3 ml TE25S buffer, add 100 µl lysozyme solution,
incubate 10 min at 37E. Final lysozyme concentration is 2 mg ml–1.

2.

Add 4 ml 2 × Kirby mix and agitate for 1 min on a vortex mixer. Use gentler
agitation for longer to obtain DNA fragments >40 kb.

3.

Add 8 ml phenol/chloroform/isoamyl alcohol and agitate for 15 sec as above

4.

Centrifuge 10 min, $1500 × g (e.g. 3500 rpm in SS-34 rotor).

5.

Transfer upper (aqueous) phase to a fresh tube containing
3 ml phenol/chloroform/isoamyl alcohol and 600 µl 3M unbuffered sodium
acetate, agitate as in step 2 and centrifuge as in step 4.

6.

Add 0.6 vol isopropanol, mix, spool DNA onto a sealed Pasteur pipette, wash
DNA in 5 ml 70% ethanol.

7.

Optional: redissolve pellet in 5 ml TE, add 50 µl RNase A and incubate 30 min
at 37E. Extract with phenol/chloroform/isoamyl alcohol as in step 5, precipitate,spool and wash as in step 6. Final RNase concentration is 40 µg ml–1.

8.

Air dry pellet and redissolve in 0.5-1 ml TE at 55E.

B. Salting out procedure for the isolation of genomic DNA
Pospiech and Neumann (1995). Uses proteinase K and chloroform, but no phenol or RNase.
Mycelium from 30 ml culture
SET buffer: 75mM NaCl, 25mM EDTA pH8, 20mM Tris-HCl pH7.5
Lysozyme: 50 mg ml–1 in water
Proteinase K: 20 mg ml–1 in water
SDS, 10% (may be preheated to 55E)
NaCl, 5M; chloroform; isopropanol; 70% ethanol;TE buffer
Sealed Pasteur pipette; two 20 ml screw cap bottles (chloroform-resistant); two
Eppendorf tubes; 10 ml pipettes; propipette; yellow tips and 200 µl pipette
37E and 55E water baths or incubators; centrifuge

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1.

Resuspend mycelium in 5 ml SET buffer, add 100 µl lysozyme solution, incubate
30-60 min at 37E. Lysozyme concentration is 1 mg ml–1.

2.

Add 140 µl proteinase K solution, mix, add 600 µl 10% SDS, mix by inversion,
incubate 2 h at 55E, invert occasionally. Final proteinase K concentration is 0.5 mg ml–1,
SDS concentration is 1%.

3.

Add 2 ml 5M NaCl, mix thoroughly by inversion, let cool to c. 37E. Final NaCl
concentration is 1.25M.

4.

Add 5 ml chloroform, mix by inversion for 30 min at 20E.

5.

Centrifuge 15 min, 4500 × g at 20E (e.g. 6000 rpm in SS-34 rotor).

6.

Transfer supernatant to a fresh tube, add 0.6 vol isopropanol and mix byinversion. After c. 3 min spool DNA onto a sealed Pasteur pipette, rinse DNA
in c. 5 ml 70% ethanol, air dry, and dissolve in 1-2 ml TE at 55E.

C. CTAB procedure for the isolation of genomic DNA
Adapted from Murray and Thompson,1980; Ausubel et al., 1994; Rogers and Bendich, 1988.
Uuses proteinase K, CTAB (removes polysaccharides) and chloroform, but no phenol or RNase; timec. 4 h.
Mycelium from 30 ml culture
TE25S buffer: 25mM Tris-HCl pH8, 25mM EDTA pH8, 0.3M sucrose
Lysozyme: 100 mg ml–1 in water
Proteinase K: 20 mg ml–1 in water
SDS: 10% (may be preheated to 55E)
CTAB/NaCl: 10% CTAB (cetyltrimethylammonium bromide, Sigma H5882) in 0.7M
NaCl (add CTAB slowly to 65E NaCl solution, autoclave)
NaCl 5M; isopropanol; 70% ethanol; TE buffer
Chloroform/isoamyl alcohol 24:1
Sealed Pasteur pipette; 20 ml chloroform-resistant screw cap bottles; two
Eppendorf tubes; 10 ml pipettes; propipette; yellow tips and 200 µl pipette
37E and 55E water baths or incubators; centrifuge

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

1.

Resuspend mycelium in 5 ml TE25S buffer, add 100 µl lysozyme solution,
incubate at 30-60 min at 37E. Final lysozyme concentration is 2 mg ml–1.

2.

Add 50 µl proteinase K solution, mix, add 300 µl 10% SDS, mix by inversion,
incubate 1 h at 55E, invert occasionally. Proteinase K concentration is 0.18 mg ml–1,
SDS concentration is 0.5%.

3.

Add 1 ml 5M NaCl, mix thoroughly by inversion. Final NaCl concentration is 0.8M.

4.

Add 0.65 ml CTAB/NaCl, mix thoroughly and incubate 10 min at 55E.

5.

Cool to c. 37E, add 5 ml chloroform/isoamyl alcohol, and mix 30 min by
inversion.

6.

Centrifuge 15 min, 13,500 × g at 20E (e.g. 10,000 rpm in SS-34 rotor). If a second CTAB
extraction is required (steps 4-6), transfer supernatant, again add 1/10 vol CTAB/NaCl, incubate
at 55E, and extract with chloroform/isoamyl alcohol.

7.

Transfer supernatant to a fresh tube, add 0.6 vol isopropanol and mix byinversion. After c. 3 min spool DNA onto a sealed Pasteur pipette, rinse DNA
in 5 ml 70% ethanol, air dry, and dissolve in 0.5-1 ml TE at 55E.

Isolation of CCC plasmid DNA
For Streptomyces plasmids, an adaptation of the alkaline lysis procedure of Birnboim and Doly
(1979) is most commonly used (procedure D, p. 184). The hot alkaline lysis-acid phenol method(procedure E, p. 187; Kieser, 1984) produces CCC Streptomyces and E. coli plasmids for screening
undigested on agarose gels within a very short time. These samples become suitable for restriction
digests after removal of phenol and SDS. Alkaline lysis may not be suitable for all plasmids (seeGonzález and Carlton, 1980, for non-Streptomyces examples). A rapid boiling method has also beendeveloped (procedure G, p. 190; MacNeil and Gibbons, 1986). Neutral SDS lysis (procedure A, p. 177;
Bibb et al., 1977) is more laborious and the timing of lysis is more critical than for alkaline lysis
procedures, but the lysates are better suited for CsCl-ethidium bromide gradient centrifugation (procedureB, p. 178). Further purification of plasmids of all sizes can be achieved with QIAGEN columns(procedure F, p. 189) or CsCl-ethidium bromide gradient centrifugation (p. 174,). Procedures H-J (pp.
191-193) are for rescuing plasmid preparations containing too much chromosomal or open circular (OC)DNA and they also remove protein and single-stranded DNA. Procedure K (“Jurassic preps”; p. 194)



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uses a cheap “glass milk” alternative for the isolation of plasmid DNA after alkaline lysis and can also
be used for the purification of DNA fragments from agarose gels. Polysaccharides can be removed using
CTAB (p. 170, 164), CsCl density gradient centrifugation without ethidium bromide (procedure C, p.
182) or the QIAGEN procedure (procedure F, p. 189). Beware of cross-contamination between plasmid
preparations.

Discussion of individual steps
Complete procedures are in the next sections; plasmid structure and classical methods forplasmid isolation are reviewed by Clowes (1972); more specific information about Streptomyces plasmid
isolation is in Kieser (1984).
Growing mycelium, harvesting, storage, and lysozyme treatment. These are as for theisolation of genomic DNA (p. 162). Lower antibiotic concentrations than in agar media are used for the
maintenance of unstable plasmids in liquid culture. Fast growing, dense cultures (use large inoculum of
spores or starter culture) give more and cleaner CCC DNA than slow growing cultures. The copy numberof pIJ101 increases late in growth (Wrigley-Jones et al., 1993), but CCC forms of SLP1.2-derived
plasmids are more abundant in young than in old cultures. Little is known about the influence of the hostgenotype, which is important for E. coli plasmid preparations (the endA strains DH5 , JM105, JM109
and SURE (Stratagene) are better than e.g. HB101, JM101, NM522 or TG-1; Taylor et al., 1993).
Plasmid amplification by chloramphenicol (10 µg ml–1; Frenkel and Bremer, 1986) or spectinomycin(50 µg ml–1), which works for the ColE1-type plasmids pBR322 and p15A, has not been observed in
Streptomyces.
Alkaline lysis. This reliably disintegrates the lysozyme-treated mycelium and releases all DNA.
Immediate mixing of the samples is essential because locally high concentrations of alkali (pH>12.7)
may result in irreversible denaturation of plasmid DNA. Gentle mixing is essential when chromosomal
DNA is precipitated with potassium acetate; vigorous mixing is best before phenol precipitation(procedure E, p. 187). Sodium hydroxide solutions should be kept airtight in plastic containers. See p.
163) for comments about SDS and alternative detergents).
Neutralisation of alkaline samples is achieved by potassium acetate pH4.8 (better soluble inalcohol than sodium acetate) or by acid phenol/chloroform. Both methods precipitate much but not allof the single-stranded chromosomal DNA and protein, but CCC DNA, RNA and polysaccharides remainin the aqueous phase. Note that cell lysates at pH4.8 gradually nick DNA. Nicking can be prevented byadding small amounts of phenol/chloroform.
More single-stranded DNA can be removed by adsorption to nitrocellulose (filter through0.45 µm membrane, optimal binding at pH5-7 and high salt, 0.5M Na+, 3mM Mg2+; capacity c. 20 µg
cm–2, filters clog easily), or by extraction with phenol/chloroform after the volume of the sample has been
reduced. S1 nuclease digestion (100-100 units S1 nuclease for 20 µg ssDNA, 30 min in 50 NaCl, 30mM
NaOAc pH4.6, 5% glycerol, 1mM ZnSO4; Waldeck et al., 1976) is used only for diagnostic purposes
because CCC DNA may have single-stranded regions due to partial unwinding and may become
linearised; excess S1 nuclease may digest dsDNA. Single-stranded DNA fragments can spoil CsClethidium bromide gradients (see below) and contaminate QIAGEN-purified preparations.

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Neutral lysis (SDS, pH8). This has the advantage that no single-stranded DNA is produced, but
cleared Streptomyces lysates contain much more chromosomal DNA than E. coli lysates. Note that theboiling method (G, p. 190) denatures the chromosomal DNA.
Removal of polysaccharides. Polysaccharides can be extracted with chloroform/isoamyl
alcohol from pH8 solutions containing $0.7M NaCl and 1% CTAB (pp. 164,170; 0.5% SDS does notinterfere). CsCl gradient centrifugation without ethidium bromide (procedure C, p. 178) and the
QIAGEN procedure (H, p. 189) also remove polysaccharides.
Precipitation of cleared lysates. Plasmid DNA, unlike chromosomal DNA, cannot be recoveredby spooling. Isopropanol (0.54-1.0 vol) is preferred to ethanol (2.2 vol) because of the smaller volume(see genomic DNA isolation, precipitation of DNA (p. 165). Sometimes it is useful to add salt and thencentrifuge the samples (10 min, 11,000 × g, 4E; discard pellet) before adding alcohol. Use isopropanol
at 20E to prevent precipitation of salt, and mix thoroughly before centrifugation. Lysates from amplified
E. coli plasmids may give stringy precipitates similar to genomic DNA. For low DNA concentrations
centrifuge hard and long. Angle rotors leave much of the sample at the side of the centrifuge tube;
isopropanol pellets tend to be invisible (mark position) and may slide out if the tubes are left inverted.
Always spin briefly after decanting the supernatant and remove the rest of the liquid using a pipette.
Always wash isopropanol precipitates with ice-cold 70% ethanol to remove salt and accelerate air drying.
Even bulky precipitates dissolve readily as long as they have not been vacuum-dried.
PEG 6000 precipitation (8% final concentration, 0.7M NaCl). This gives excellent results, but
good recovery of plasmid DNA requires several hours incubation at 4E (pp. 166, 177).
Removal of RNA. This is important for end-labelling with polynucleotide kinase; it also
obscures small fragments on agarose gels. RNase A (pre-boiled; p. 166) is included as an optional
ingredient in several plasmid isolation procedures. Sometimes it is practicable to add RNase A torestriction digests.
Lithium chloride (2.5M final concentration, no alcohol; p. 206) precipitates large RNA but isuseless after RNase treatment or heating alkaline samples: redissolve sample in 300 µl TE, add 300 µl
5M LiCl (21.2 g 100 ml–1) or 36 µl 8M LiCl (33.9 g 100 ml–1). Incubate 10 min on ice, centrifuge 10 min$10,000 × g at 4E, discard the bulky pellet, precipitate DNA with 2.0 vol ethanol and wash with 70%
ethanol (there is no need to add extra salt). Li+ inhibits reverse transcriptase.
Small RNA molecules are removed by the QIAGEN procedure, and also by PEG and isopropanol
precipitation if exactly 0.54 vol isopropanol is used (p. 165). CsCl-ethidium bromide gradientcentrifugation precipitates most RNA species except tRNA which bands with CCC plasmid DNA.
Removal of remaining phenol, SDS and salt. For restriction digests and transformation, alltraces of phenol and SDS must be removed. A single alcohol precipitation does not reliably eliminate
these contaminants.
Additional isopropanol precipitations (0.54-1 vol; use above 0E to prevent precipitation of salt)
or ethanol precipitations (2.2 vol, 0E or below) can be used (p. 165). A single precipitation is not
sufficient, especially when the pellets are large. Precipitate the sample from a large volume to achieve
good purification. Ammonium acetate (2.5M), unlike potassium and sodium acetate (0.3M), evaporates
under vacuum and leaves oligonucleotides #30 nt mostly in solution. Ammonium ions inhibit
polynucleotide kinase. Carefully wash pellets in 70% ethanol (it is easy to lose whole pellets at thisstage). Remove all liquid using a pipette but do not vacuum-dry excessively. Carrier tRNA or glycogen(20 µg ml–1) is not required unless DNA is pure and the concentration <1 µg ml–1.

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Discard precipitates that form in the salt solution before adding the alcohol. Samples with very
high ionic strength (e.g. CsCl) and sucrose gradients need to be diluted by adding 3 vol TE before alcohol
precipitation is possible.
Spermine (0.5mM; H2N(CH2)3NH(CH2)4NH(CH2)3NH2@(HCl)4; FW=348.19; Sigma S1148).
This precipitates DNA $200 bp (and RNA and polysaccharides) rapidly from low salt buffers (pH8; seeprocedure E, p. 187). Spermine, like spermidine, at low concentrations increases the activity and thespecificity (less star activity) of restriction endonucleases (Hoopes and McClure, 1981; Pingoud, 1985;
Kieser, 1984). Large pellets from too concentrated DNA solutions are difficult to dissolve.
Dialysis (p. 165) and gel filtration chromatography using Sephacryl S-1000 (Pharmacia;
cation exchanger, functional group: -CH2-SO32–; Perbal, 1988; Gomez-Marquez et al., 1987), Sepharose
6B-Cl, or Sephadex (Pharmacia). These also remove small molecules, including nucleotides andoligonucleotides, from plasmid preparations.
CsCl density gradient centrifugation. This is still the method of choice for preparing large
amounts of pure CCC DNA (procedure B, p. 178). Some polysaccharides, tRNA, irreversibly denatured
CCC DNA and single-stranded DNA (produced by alkaline lysis and natural single-stranded
intermediates from rolling circle replication) accumulate in the same region as CCC DNA.
Polysaccharides can be separated from DNA in caesium chloride gradients without ethidium bromide(procedure C, p. 178). tRNA (inhibitor of EndA activity p. 172; good substrate for polynucleotide
kinase) should be degraded by RNase A at <300mM Na+ before centrifugation.
SDS precipitates in CsCl and must be removed before ultracentrifugation e.g. by precipitation
with potassium acetate; sarkosyl is soluble in CsCl and can be used instead of SDS (p. 163) if small-scale
samples are to be loaded directly onto a gradient (consider the density increment from the DNA solution).
Single-stranded DNA is only partially removed by high salt phenol extraction. Large amounts
form a diffuse fluorescent background across the gradient and may completely obscure faint plasmid
bands, causing loss of the sample.
Excessive amounts of double-stranded chromosomal DNA may also obscure plasmid bands, butharvesting from the lower part of the well-defined double-stranded DNA band enriches for plasmid DNA.
Overloaded, viscous gradients take a long time to equilibrate. Step gradients allow shorter
centrifugation times (Sambrook et al., 1989). Samples harvested from several gradients can be pooled
for re-centrifugation which usually reveals the CCC plasmid band(s). Ethidium bromide may need tobe replenished if the sample appears pale. Low ethidium bromide concentration lowers the bands in thegradient and reduces the separation between CCC and linear DNA.
Streptomyces has c. 72% G+C and is therefore 22 µg ml–1 denser than E. coli DNA with 50%
G+C. Plasmids with less than 72% G+C (e.g. Streptomyces-E. coli hybrid replicons) migrate closer tothe linear Streptomyces DNA; for 50% G+C the distance would be halved. The absolute distances
depend on the rotor speed (higher speed positions the bands closer to each other), rotor tube angle(vertical gradients give the best separation, swingout rotors are worst), and on the variable supercoil
density. The higher the (negative) supercoil density of the plasmid (in the absence of ethidium bromide),
the smaller the distance between the plasmid and linear DNA. Non-supercoiled (relaxed) CCC DNA
bands twice as far as from linear DNA as fully supercoiled CCC DNA. These facts are summarised inFig. 8.1.
Some polysaccharides (bind weakly to ethidium bromide) band near the plasmid DNA in CsClethidium bromide gradients but float far above the DNA in the absence of ethidium bromide. Banding

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

the DNA in a gradient with 0.7 µg ml–1 ethidium bromide (minimal amount, for staining purposes only),
followed by a (normal) gradient with 700 µg ml–1 ethidium bromide (procedure B, p. 178), produces
plasmid DNA free of polysaccharides. CTAB/chloroform extraction (pp. 164,170) and the QIAGEN
procedure (p. 189) also remove polysaccharides.
Substitution of propidium iodide (300 µg ml–1; Sigma P5264; Grossman et al., 1974) for ethidium
bromide doubles the distance between CCC and linear DNA. The procedure is similar in all aspects,
including CsCl density and extraction of dye, to CsCl-ethidium bromide gradient centrifugation, but
normal plasmids will be near the bottom of the centrifuge tube; inspect the gradient tube before relaxing
the rotor speed. Propidium iodide has not been used widely for plasmid DNA isolation and not at all for
Streptomyces plasmids.
The relatively inexpensive AT-specific dye Hoechst 33258 (bisbenzimide; 300 µg ml–1; Sigma
B2883) was used to separate plasmids with 8% less G+C than the host chromosome (Sorvall TV 865
vertical rotor, 34,000 rpm, >15 h; density of CsCl=1.7046; Fennewald et al., 1978). Hoechst 33258
bound to DNA fluoresces yellow under 365 nm UV irradiation (p. 167).
Miscellaneous methods. Lysis with non-ionic detergents (Brij 58, sodium deoxycholate or
Triton X-100 at 20E; Clewell and Helinski, 1970), which is useful for isolating relaxation complexes, has
not been reported for Streptomyces. (The boiling method denatures the chromosomal DNA.)Mono Q or Q Sepharose fast flow anion exchange column chromatography (functionalgroup: -CH2-N+(CH3)3; Pharmacia). This gives excellent purification of milligram amounts of plasmids
smaller than 15 kb; larger plasmids elute as unacceptably wide peaks in the salt gradient (T. Kieser,
unpublished).
Binding to glass (“Jurassic preps”, procedure K, p. 194) has not been used for very large
plasmids because nicking of CCC DNA has been reported (Marko et al., 1982).
Sucrose gradients. These have a relatively small capacity, and produce wide peaks.
Overloading produces unpredictable results. Preparative agarose gel electrophoresis (e.g. Sambrook etal., 1989) may be a more useful alternative.
Quantitation of plasmid DNA. OD260 measurements (50 µg ml–1 DNA gives OD=1.0) are notvalid if the sample contains RNA or phenol (peaks at 270 and 290 nm; p. 167). Hoechst 33258
fluorescence (p. 167) can be used with a standard curve. Comparison of band intensities on ethidium
bromide-stained agarose gels is reliable and convenient, but not very accurate. It is best to use digested
plasmid samples because CCC DNA binds less ethidium bromide and fluoresces c. 30% less than OC
or linear DNA. Plasmids may also give multiple bands (p. 195) that are difficult to integrate. Singlestranded DNA does not fluoresce strongly, except when it is partially annealed, as is common for highG+C sequences.
Common problems
Loss of plasmid DNA during precipitation steps: common if copy number is low. Monitor
different steps of purification to detect losses. Monitoring is simplest with Method E (alkaline lysis andphenol precipitation). Improve recovery by longer and harder centrifugation; if possible start with moreconcentrated mycelium suspension.
Low plasmid yield: Plasmid may be unstable; maintain antibiotic selection. Optimise growth
conditions (p. 172).

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1.40

65,000 rpm

45,000 rpm

50%

50%

72%

72%

1.50

1.45
1.50

1.55

Linear + OC

1.55
1.60

1.60

1.65
1.70

CCC supercoiled
CCC relaxed

1.65

1.75
1.80

1.70

Fig. 8.1 Positions of 50% G+C (E. coli) and 72% G+C (Streptomyces) DNA in a CsCl-ethidium
bromide gradient of an average density of 1.59 g ml–1 after centrifugation to equilibrium in the Beckman
VTi 65 vertical tube rotor. Not shown are protein which floats on top, RNA which precipitates andsticks to one side of the tube (bottom in angle and swingout rotors). Single-stranded DNA forms a
diffuse background below the band of linear DNA. Irreversibly denatured CCC DNA, tRNA and somepolysaccharides band near the native supercoiled CCC DNA. ), 0 and  mark the average CsCl
densities recommended for E. coli plasmids by Sambrook et al. (1989), or by Ausubel et al. (1994), andfor Streptomyces plasmids, respectively.

Background smear on agarose gels: remove single-stranded DNA (p. 172).
Plasmid DNA cannot be digested: see section on genomic DNA (p. 168). The samples may
contain too much salt or low pH buffering capacity. Polysaccharides may be inhibitory. Consider
possibility that plasmid may have no recognition site for restriction endonuclease. Faint linear bands
could be the result of non-specific nicking (check with double digest). Note that denatured CCC DNA
and single-stranded DNA are not cleaved by most restriction endonucleases. DNA may be modified(many streptomycetes have restriction-modification systems such as SacI, SalI).
Polysaccharides: remove using QIAGEN column (procedure F, p. 189), CTAB extraction(procedure C for genomic DNA, pp. 164,170), or CsCl centrifugation (plasmid isolation procedure C,
p. 182).
Plasmid samples contain too much linear or OC DNA: try second alkaline denaturation (H, I,
pp. 191, 192) or phenol (pH4) extraction (J, p. 193). Nicked plasmid DNA causes artifacts in sequencing
and exonuclease III deletion formation. endA E. coli strains give better quality plasmid preparations(p. 172).
Low transformation frequencies: phenol and SDS are more inhibiting for E. coli (CaCl2-heatshock method) than for Streptomyces protoplast transformation. Salt may increase Streptomycestransformation frequencies; ethanol, isopropanol, spermine, PEG are not critical (Kieser, 1984). The salt
concentration must be very low for transformation by electroporation.
Multiple bands on gel: all bands may be different forms of the same plasmid: CCC withdifferent numbers of supercoil turns, OC, multimers, single-stranded and denatured forms (p. 195).

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Precipitates difficult to dissolve: see section on genomic DNA (pp. 165, 168). The causes areusually polysaccharides, remaining protein and chromosomal DNA, and precipitation from too small a
volume. Pure plasmid DNA dissolves readily. Consider using methods H or I below.

A. Plasmid isolation by neutral lysis
Slightly modified from Bibb et al. (1977). This procedure uses SDS lysis, salt and PEG
precipitation and, CsCl-ethidium bromide gradient centrifugation; time 2 days, intermittent.
Mycelium from 500 ml culture
Sucrose, 34% (autoclaved; 100 ml per prep., 50 ml at 0E)
EDTA, 0.25M, pH8 (40 ml per prep., 30 ml at 0E; p. 206)
Lysozyme, 50 mg ml–1 (5 ml per prep.)
SDS, 10% (22 ml per prep., at 50E)
NaCl, 5M (44 ml per prep.; p. 206)
PEG 6000, 30% in water (p. 166)
TE: 1mM EDTA pH8, 10mM Tris-HCl pH8
One 1000 ml flask; 250 ml measuring cylinder; six 50 ml centrifuge tubes; two
250 ml centrifuge bottles; 25 and 10 ml pipettes
Centrifuge; ice bath; material and equipment for CsCl-ethidium bromide
centrifugation (procedure B, p. 178)

1.

Resuspend mycelium from 500 ml culture in 50 ml 34% sucrose in 1 l flask, add
1 ml 1M Tris pH8, 10 ml 0.25M EDTA pH8, and 5 ml 50 mg ml–1 lysozyme,
mix. Incubate at 30E for 10-45 min until the viscosity of the suspension
increases. The mycelium appears "glassy" just before the suspension turns viscous.

2.

Transfer to ice bath, add 50 ml ice-cold 34% sucrose and 30 ml 0.25M EDTA
and mix. Composition: 150 ml, 23% sucrose, 67mM EDTA, 7mM Tris-HCl pH8. This could
be done at 55E with proteinase K, but too expensive. Adding RNase might be dangerous becauseof activation of EndA activity. Phenol/chloroform could be added.

3.

To six 50 ml centrifuge tubes, each containing 3.6 ml 50E 10% SDS, add 25 mlof the above cell suspension and mix each tube immediately by inverting several
times. Add 7.2 ml 5M NaCl, mix by inversion. Leave for at least 2 h toovernight at 4E. Composition: 1% SDS, 1M NaCl.

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4.

Centrifuge, 30 min, 32,000 × g (16,000 rpm in SS-34 rotor) at 4E. Pool
supernatants in 250 ml measuring cylinder.

5.

Add 0.5 vol cold 30% PEG 6000, mix by inversion, incubate 4-24 h at 4E.
Centrifuge, 20 min, 2,600 × g (4000 rpm in GSA rotor) at 4E. Final concentrations:
10% PEG 6000, 0.7M NaCl. 8% PEG 6000 may be sufficient and harder centrifugation may benecessary; for samples with low contamination of chromosomal DNA use 10,000 × g for 20 min;overcentrifugation makes samples difficult to dissolve.

6.

Redissolve pellets in a total of 12 ml TE. Proteinase K might be added together withsarcosine. Try CTAB extraction, LiCl precipitation, EB treatment, alkaline denaturation. The
samples usually contain substantial amounts of double-stranded chromosomal DNA. Proceedwith CsCl-ethidium bromide gradient centrifugation using two 14 ml tubes for the first run. Pool
plasmid DNA in a single tube and repeat CsCl-ethidium bromide centrifugation.

B. Standard CsCl-ethidium bromide gradient centrifugation
For more detailed instructions consult e.g. Sambrook et al., 1989. Ultracentrifuges and rotors
must be used as specified in the manufacturers’ manuals.
DNA solution, #0.2 mg ml–1 in TE buffer (no SDS)
CsCl, dry solid (Fisons C/0680/60 or BDH 44157)
Ethidium bromide, 10 mg ml–1 (25mM; BDH 44159; mutagen, keep in dark)
CsCl/ethidium bromide solution density = 1.591 g ml–1, refractive index = 1.3893:
1.13 mg CsCl, 1 g H2O, 100 µl 10 mg ml–1 ethidium bromide
Light paraffin oil (BDH 29436)
Precision balance (±10 mg); refractometer (optional); centrifuge and tubes for low
speed centrifugation; ultracentrifuge; angle or vertical rotor with matching tubes
and caps (including fitting tools)1.

Add 1.13 g CsCl per g DNA solution (1 ml=1 g) and dissolve at 30E. Density =1.636 g ml–1, refractive index = 1.394. See Table 8.2 for preparing specific volumes of CsCl
solution. For E. coli DNA use 1.100 g CsCl per g DNA solution (Density = 1.623 g ml–1,
refractive index = 1.392).

1.0

0.1 ml 9
1.705

1.709
1.400

1.400
9.624

9.684

9.981

7.319

7.894

8.062

CsCl

6.747

7.405

7.324

7.319

7.176

7.135

H2O

0.675

7 µl

7 µl

0.732

0.718

0.714

EB

For 10 ml final volume
after addition of EB2

12.99213.07413.4759.880

10.657

10.884

CsCl

9.109

9.997

9.887

9.880

9.689

9.632

H2O

0.911

10 µl

10 µl

0.988

0.969

0.963

EB

For 13.5 ml final volume
after addition of EB

CsCl

H2O

EB

For . . . ml final volume
after addition of EB10

RI = refractive index. 2H20 = aqueous DNA solution, 1 g = 1 ml. 3EB = 10 mg ml–1 ethidium bromide, 4700 µg ml–1 final concentration.
Amounts recommended by Ausubel et al. (1994). 6Amounts recommended by Sambrook et al. (1989). 7Final concentration = 0.7 µg ml–1 ethidium bromide.
Check maximal permissible rotor speed (Table 8.3). 8Conditions used by Fennewald et al. (1978) for Pseudomonas aeruginosa (67% G+C, 1.726 g ml–1). 9The
AT-specific dye Hoechst 33258, 5 mg ml–1, is used instead of ethidium bromide (pp. 167,175). 10Space for recording amounts for a different tube size.

5

1

1.426

Hoechst3325881.0

0.1 µl 7

1.309

1.402

50% G+C
Genomic

1.731

0.1 µl 7

1.364

72% G+C
Genomic

1.0

1.348

1.388

1.579

1.537

1.0

1.000

0.1 ml 4

1.392

1.623

0.1 ml 4

"

"

-

1.389

1.591

1.388

1.0

1.100

0.1 ml 4

1.394

1.636

1.579

"

"

-

EB3

RI1

-

1.0

H2O2

1.130

CsCl

Densityg ml–1
20E

50% G+C
CCC6Standard

50% G+C
CCC5Standard

72% G+C
CCC
Standard

PurposeStarting with 1 ml aqueous
DNA solution

Table 8.2. Preparation of CsCl solutions of specific density (All quantities in g unless specified otherwise; warm to 37E to dissolve CsCl)&KDSWHU 3UHSDUDWLRQDQGDQDO\VLVRIJHQRPLFDQGSODVPLG'1$

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
10

10

9

9

8

8

7

7

6

6

5

5

4

4

3

3

2

2

1

1

0

0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

g CsCl solution
Fig. 8.2. Volume of CsCl solutions. When balancing centrifuge tubes, use thebalance to determine the weight in g that needs to be added or removed, then read thecorresponding volume from the graph. Beware of cross contamination.

2.

Add 100 µl 10 mg ml–1 ethidium bromide and mix immediately by inversion.
Centrifuge 10 min, 8000 × g (8000 rpm in SS-34 rotor) at 20E. Discard floatingpellet. Store samples in dark. Density = 1.591 g ml–1, refractive index = 1.389. For E. coli
DNA density = 1.579 g ml–1, refractive index = 1.388.

3.

Fill centrifuge tubes and balance to ±10 mg using CsCl/ethidium bromide
solution (Fig. 8.2). Fill tube completely with light paraffin oil. Seal tubes andsqueeze carefully to make sure that the seal is tight.

4.

Centrifuge at 20-25E. Maximal allowable speed and centrifugation times depend on typeof rotor (Table 8.3); consult manual. Equilibrium is reached faster and higher initial rotor speeds
are possible without danger of CsCl precipitating if the tubes are only partially filled with CsCl
solution and topped up with light paraffin oil. Decelerate as slowly as possible in the last stage
before the rotor stops to facilitate reorientation of the bands.
Angle rotors are often used at 55,000 rpm (280,000 × g) for 16 h for quick band formation andthen at 35,000 rpm (113,000 × g) for $8 h to obtain a shallower CsCl gradient and thus betterband separation. Very viscous, overloaded gradients need longer to produce sharp DNA bands.

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

Table 8.3. Examples of maximal speeds for CsCl solutions in different rotors

Manufacturer(Rotortype)

Beckman(vertical)

Sorvall(vertical)

Beckman(angle)Beckman(swingout)

Rotor

Tube
volume(ml)

NumberofplacesMaximal speed (rpm÷1000) using fulltubes and 20-25E1 CsCl solutions(k factor2)
Density=1.60 g ml–1

Density=1.73 g
ml–1

VTi 65

5.1

8

65

(10)

53

(15)

VTi 50

39

8

50

(36)

38

(65)

TV-865

6

8

56

(14)

51

(17)

TV-865B

18.5

8

55

(30)

39

(60)

Ti 70.1

13.5

12

45

(90)

32

(170)

Ti 70

33

8

39

(130)

28

(290)

Ti 50

13.5

12

40

(122)

29

(240)

SW 50

5

6

36

(115)

28

(184)

1

Beckman gives information for 20E, Sorvall for 25E.
The k factor gives an indication of the required centrifugation time (large values indicate that longcentrifugation times are required). It is not strictly relevant for gradient centrifugation because theslope of the gradient is not taken into account.

2

Vertical rotors, e.g. VTi65, can be run at higher speeds than angle rotors and the short pathaccelerates band formation. The density gradient is shallower in vertical rotors than in angle
rotors at the same g-force, resulting in better separation of CCC from linear DNA.
Note that the nominal speed of the rotor is not a reliable indication of the permissible
speed, which is in most cases limited by the danger of CsCl precipitation (density of solid CsCl
= 4 g ml–1). Note that often significantly faster speeds can be used if the tubes are partially filledwith CsCl solution and topped up with light mineral oil. Consult specific rotor manuals.
Centrifuge at 20-25E. For faster band formation, centrifuge first at maximal speed and then atc. 70% of maximum to obtain better separation of the bands.

5.

Harvesting DNA from CsCl-ethidium bromide density gradients. Wear UV eye
protection and gloves. Fix gradient tubes near 365 nm UV light above waste container indarkroom. If the gradient is overloaded and the bands are not sharp, longer centrifugation may
help. Remove grease/oil from tube with ethanol and stick Sellotape where the needle is to enteror use vacuum grease; this reduces the danger of leaks. Insert venting needle in top of tube.
Harvest bands using 21-gauge (green) needle for plasmids or 18-gauge needle (pink) for genomic

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DNA, and a 2 ml syringe. It may be convenient first to remove the top band containing the linear
DNA. Identical plasmid samples can be pooled and centrifuged again without adjusting thedensity of the CsCl solution. If samples appear pale, replenish ethidium bromide.

6.

7.

Removal of ethidium bromide. Here are three alternative methods:
1.

Extraction with 3-4 changes of equal volumes of isoamyl alcohol whichmust be saturated with 5M NaCl in TE buffer.

2.

Filtration through AG50W-X8 cation exchange resin (BioRad 143-1441).
Prepare resin by resuspension first in five volumes 5M NaCl (p. 206),
then 1 N HCl (8.5 ml conc/100 ml), then two batches of water and finallyTE buffer. Store in 1M NaCl, 10mM EDTA, 0.2% sodium azide, 100mM
Tris-HCl pH8. For up to 2 ml CsCl/ethidium bromide solution, pour 1 ml
column and equilibrate with TE. Start collecting sample immediately
after loading column; rinse with 1.2 vol TE.

3.

Phenol extraction: CsCl/DNA solution forms the lower phase; phenol
must be removed by dialysis or by two ethanol precipitations and 70%
ethanol wash (see below).

Removal of CsCl by dialysis or ethanol precipitation. Dialyse four times >2 h against>50 vol of cold stirred TE buffer. Leave sufficient room for volume to expand. Using
20% PEG 20,000 in TE for the first stage reduces the swelling of the tubes and also accelerates
the dialysis (p. 165). (CsCl solution is 4.8M.) Ethanol precipitation: to 1 ml CsCl solution add
2 ml TE to reduce the CsCl concentration to 1.6M. Then add 6 ml of ethanol, mix andcentrifuge. Wash precipitate with 70% ethanol.

C. CsCl density gradient for removing polysaccharides from DNA (Halford and Johnson, 1981)
Some polysaccharides have a density of about 1.60 in CsCl, about the same as CCC DNA in
standard CsCl-ethidium bromide gradients. Centrifugation in 1.73 g ml–1 CsCl without ethidium bromide
separates Streptomyces DNA (72% G+C, density = 1.731, no separation of CCC from other DNA) frompolysaccharides, which form a diffuse non-fluorescent band in the upper part of the gradient). (Most
RNA pellets and proteins float on top.)
See Table 8.2 (p. 179) for the amount of CsCl to use. 0.7 µg ml–1 ethidium bromide is tovisualise the DNA. Use the highest allowable speed for the density of the CsCl solution (Table 8.3); thelimiting factor is precipitation of CsCl.

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2 4 6 8 10

14

18

GSA
230 ml

50

60
50

40

40

30

30
SS-34
40 ml

20
10
0



20
10

GSA 180 ml

0
2 4 6 8 10
14
Krpm

18

Sorvall rotor features:
Rotor

Radius (cm)Type

maximal rpm

maximal g

H600A

16-26

swingout

5000

4500-7300

GSA

3.8-14.6

fixed angle

13,0007200-27600

SS-34

3.3-10.7

fixed angle

20,00014,800-48,000

Fig. 8.3. Centrifugation conditions equivalent to 10 min at 12,500 rpm in a
microfuge

CCC DNA can be separated from linear and OC DNA after increasing the ethidium bromide
concentration to 700 µg ml–1 and reducing the density of the DNA solution to 1.591 g ml–1:
to 1 g CsCl solution add 124 µl TE and 14 µl 10 mg ml–1 ethidium bromide.
orto 1 ml CsCl solution add 215 µl TE and 24 µl 10 mg ml–1 ethidium bromide.
Fill the centrifuge tube to the desired weight with 1.591 g ml–1 CsCl-ethidium bromide solution(procedure B) and centrifuge as for standard CsCl-ethidium bromide gradients. Do not pool samples atthis stage.

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D. Plasmid isolation by alkaline lysis and potassium acetate precipitation (modified from Birnboim
and Doly, 1979)
MyceliumTEGLR: 25mM Tris-HCl pH8, 10mM EDTA pH8, 50mM glucose (18 µl 50% stock
solution for 1 ml), 1 mg ml–1 lysozyme, optional: 5 µg ml–1 pre-boiled RNase A
NaOH/SDS: 0.2 N NaOH, 1% SDS (p. 207)
3M potassium acetate pH4.8
Acid phenol/chloroform (see procedure E, p. 187), possibly phenol pH8 (p. 164)
Isopropanol
TE buffer
Unbuffered 3M potassium acetate
5M ammonium acetate (p. 205)
100% and 70% ethanol, possibly 100mM spermine@HCl (0.348 g ml–1, autoclave andstore at room temperature; p. 174)
Ice; 37E water bath; eye protection and gloves
Centrifuge and chloroform-resistant high speed centrifuge tubes with lids (e.g.
Sarstedt No. 55.518);
The volumes of reagents and the centrifugation conditions given in the procedure below
are for 10 ml (bold in Table 8.4); the table gives reagent volumes and centrifugation conditions forlarger and smaller preparations. Fig. 8.3 shows the relationship between centrifuge speed and required
centrifugation time.

1.

Resuspend mycelium from not more than 50 ml culture in 1.4 ml TEGLR (total
volume #1.7 ml). Incubate c. 30 min at 37E.

2.

Add 2.8 ml NaOH/SDS; mix immediately by several inversions.
Incubate 20 min at 20E. Final conc: 133mM NaOH, 23mM SDS, 16mM glucose, 8mM
Tris, 3mM EDTA.

3.

Add 2 ml 3M potassium acetate pH4.8 and 0.2 ml acid phenol/chloroform andmix immediately by inversion. Incubate 5 min in ice. Centrifuge
15 min,12,000 × g at 4E (e.g. 10,000 rpm in SS-34 rotor). Phenol/chloroform
reduces nicking of DNA; no separate phase is formed (Kieser, 1984). Final conc: 1M KOAc,
90mM NaOH, 16mM SDS.

4.

Optional: extract with phenol pH8, cool in ice, centrifuge as above. This step
removes ssDNA and remaining protein (contributed by J. Ryding).

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

Table 8.4. Reagent volumes (ml) for different size plasmid preparations by alkaline lysis andpotassium acetate precipitation, procedure D ( p. 184)

Max. volume (ml)1.4

Rotor1

Microfuge

Tubes2

1.5 mlOpen

Open

SealedOpen

Sealed3

Speed (rpm)

12,5008,000512,00012,00010,00010,000Time4 (min)

10

22

10

20

10

30

1

TEGLR

0.200

1.4

4.0

5.7

25

30

2

NaOH/SDS

0.400

2.8

8.0

11.4

50

60

3

3M KOAc pH8

0.300

2.0

6.0

8.6

38

45

Acid Phenol/CHCl3

0.030

0.20

0.60

0.86

3.8

4.5

Isopropanol 0.6 vol

0.540

3.6

10.8

15.4

68

81

10

30

40

Sorvall SS-34

180

230

Sorvall GSA

Step

5

Steps 6-8 remove ssDNA, optional
6

TE

0.070

0.50

1.50

2.0

9.0

11.0

7

Unbuffered 3M
KOAc

0.014

0.10

0.30

0.40

1.80

2.20

Acid Phenol/CHCl3

0.014

0.10

0.30

0.40

1.80

2.20

Unbuffered 3M
KOAc

0.056

0.40

1.20

1.60

7.20

8.80

TE

0.630

4.50

13.5

18

81

100

Isopropanol 0.6 vol

0.460

3.30

10.0

13

59

74

70% ethanol

1.2

9

27

35

160

200

TE

0.150

1.00

3.0

4.0

20

25

5M NH4OAc

0.150

1.00

3.0

4.0

20

25

Ethanol

0.660

4.40

13.2

17.6

88

110

70% ethanol

1

7

21

25

130

160

Dissolve: TE

0.050

0.35

1.0

1.4

6

8

TE

0.950

7.0

20

27

120

160

Spermine

0.050

0.35

1.0

1.4

6.0

8.0

Unbuffered
3M KOAc

0.030

0.20

0.60

0.90

4.0

5.0

TE

0.300

2.0

6.0

9.0

40

50

8

9a

9b

Ethanol
0.726
14.5
22
97
121
4.8
2
Rotor radius: microfuge, 3.1-6.3 cm; SS-34, 3.3-10.7 cm; GSA, 3.8-14.6 cm. The useful volume
depends on whether or not the tubes can be sealed for centrifugation. 3Rotor speeds are below the
maximal speed, to reduce tube failures. 4Note that longer centrifugation times are needed with full tubes.
Fig. 8.3(p.182) gives centrifugation times. 5The rotor speed needs to be reduced because of the adaptors.
1

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5.

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Transfer aqueous phase (or supernatant) (c. 6 ml) to a new centrifuge tube
containing 3.6 ml 20E isopropanol (0.6 vol). Mix by inversion and centrifuge as
in step 3. Decant supernatant, spin for a few seconds and remove all liquid.
Steps 6-8 remove more ssDNA. They are optional, but important for CsCl-ethidium
bromide gradients. Steps 9a and 9b are optional alternative ways to remove traces of SDS,
phenol and salt, which can be applied after step 5 if necessary.

6.

Dissolve precipitate in 0.5 ml TE. It is important to keep the volume small (p. 172).
Samples are suitable for agarose gel electrophoresis but not for enzyme reactions.

7.

Transfer solution to 1.5 ml tube containing 100 µl unbuffered 3M potassium
acetate and 100 µl acid phenol/chloroform. Emulsify by vortexing. Cool sample
in ice and centrifuge 5 min, 11,000 × g in cold.

8.

Transfer upper aqueous phase (c. 0.55 ml) to a new 10 ml tube containing 0.4 ml
unbuffered 3M potassium acetate and 4.5 ml TE. Add 3.3 ml isopropanol(0.6 vol), mix and centrifuge as in step 3 and remove all liquid. Wash pellet(much of it is on the side wall of the tube) with 5 ml 0E 70% ethanol, remove all
liquid.

9.

May be done after step 5. Remaining traces of SDS, phenol and salt are removed bya further precipitation step after dissolving the sample in a relatively large volume.
Alternatively, the samples may be dissolved in c. 350 µl TE for CsCl-ethidium bromide
gradient centrifugation. Steps 9a and 9b are alternatives.

9a.

Ethanol-ammonium acetate precipitation: dissolve DNA in 1 ml TE, add 1 ml
5M ammonium acetate, mix, centrifuge, transfer supernatant to a new tube
(discard pellet), add 4.4 ml ethanol, mix and centrifuge, 10 min,12,000 × g. Remove all liquid, wash with 7 ml 70% ethanol, remove all liquidand dry in vacuum (to remove traces of ammonium acetate). Dissolve in 350 µl
TE.

9b.

Spermine precipitation: dissolve DNA in 7 ml TE (no salt to be added), add
350 µl 100mM spermine, mix and incubate on ice for 5 min. Centrifuge 10 min,12,000 × g and remove all liquid. 5mM spermine is c. 5-fold more than necessary.
Add 200 µl unbuffered 3M potassium acetate, 2.0 ml TE and 4.8 ml
ethanol, mix and incubate $5 min at 20E (the time required depends on the
thickness of the pellet). Centrifuge 10 min, 12,000 × g. Remove all liquid, wash

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with 10 ml 70% ethanol, air dry and dissolve in 350 µl TE. Note that spermine isextracted from the undissolved pellet.

E. Plasmid isolation by alkaline lysis and phenol precipitation
Kieser, (1984). This procedure is suitable for rapid screening and for enzyme reactions andtransformations; time c. 1 h to step 3, 3-4 h for completion.
MyceliumTESLR: 25mM Tris-HCl pH8, 25mM EDTA pH8, 0.3M sucrose, 0.02% bromocresol
green (BDH 20013, acts as alkali-stable loading dye), 2 mg ml–1 lysozyme, 5 µg ml–1
pre-boiled RNase A (optional)
NaOH.3/SDS: 0.3 N NaOH, 2% SDS
Acid phenol/chloroform: 5 g phenol, 5 ml chloroform, 1 ml H2O, 5 mg 8-hydroxyquinoline (density = 1.26 g ml–1; do not add buffer; p. 164)
Isopropanol
TE buffer
Unbuffered 3M potassium acetate (p. 207)
5M ammonium acetate (p. 205) or 100mM spermine@HCL (348 mg/10 ml; p. 174 )100% and 70% ethanol
Chloroform-resistant centrifuge tubes with lids; eye protection and gloves
37E and 70E (or 55E) water baths; vortex mixer; microcentrifuge
The volumes below are for 1.5 ml microcentrifuge tubes; all centrifugations #20E, 11,000 × g.
Reduce all volumes 5-fold for screening individual colonies. For scaling up, the maximum volume thecentrifuge tube nees to hold is 2.4 × the volume of the cell suspension in step 1; see Fig. 8.3 (p. 183) for
centrifugation conditions.

1.

Resuspend mycelium from #5 ml culture in a total volume of 500 ±25 µl
TESLR. Incubate 30 min at 37E (on ice for E. coli). Eppendorf tubes can be labelledpermanently by engraving with a hot needle in an area stained with a black permanent marker.

2.

Add 250 µl NaOH.3/SDS and mix immediately by vortexing (larger volumes are
best mixed using a disposable syringe). Open tubes and incubate 15 min at 70E(or 30 min at 55E for plasmids $20 kb). >40 kb CCC plasmids survive the vortexing insteps 2 and 3. Suitable also for E. coli plasmids. RNA is partially hydrolyzed. Final conc:
100mM NaOH, 12mM SDS, 17mM Tris, 17mM EDTA.

3.

Cool tubes to #37E. Add 80 µl acid phenol/chloroform. Emulsify by vortexing.
Centrifuge 10 min. If mixing is difficult, vortex briefly, then wait and vortex again. If no&KDSWHU 3UHSDUDWLRQDQGDQDO\VLVRIJHQRPLFDQGSODVPLG'1$

phenol/chloroform phase appears, add more, vortex and centrifuge again. The CCC plasmid
samples are stable at pH10 and ready for agarose gel electrophoresis.

4.

Transfer supernatant (c. 700 µl) to a new centrifuge tube containing 50 µl
unbuffered sodium acetate and add 450 µl isopropanol. Mix and centrifuge 10
min; remove all liquid using a pipette p. 173. Pellets may slide if left inverted.

Steps 5-7 remove more ssDNA (p. 172). This is optional, but important for CsCl-ethidium
bromide gradients. These steps are identical to steps 6-8 of the preceding procedure except thatthe volumes are for 1.5 ml tubes.

5.

Dissolve precipitate in 70 µl TE.

6.

Add 14 µl unbuffered 3M potassium acetate and 14 µl acid phenol/chloroform.
Emulsify by vortexing. Cool sample in ice and centrifuge for 5 min in cold . Try
CTAB extraction etc. at this stage.

7.

Transfer upper aqueous phase (c. 77 µl) to a new tube containing 56 µl
unbuffered 3M potassium acetate and 630 µl TE. Add 460 µl isopropanol(0.6 vol), mix, centrifuge 5 min and remove all liquid. Wash pellet with 1.2 ml
0E 70% ethanol, remove all liquid.

8.

May be done after step 4. Remaining traces of SDS, phenol and salt are removed bya further precipitation step after dissolving the sample in a relatively large volume.
Alternatively, the samples may be dissolved in 50 µl TE for CsCl-ethidium bromide
gradient centrifugation (procedure B) or microdialysis (p. 165). Steps 8a and 8b are
alternatives.

8a.

Ethanol-ammonium acetate precipitation. Dissolve DNA in 150 µl TE, add
150 µl 5M ammonium acetate, mix, centrifuge, transfer supernatant to a newtube (discard pellet), add 660 µl ethanol, mix and centrifuge, 10 min. Remove
all liquid, wash with 1 ml 70% ethanol, remove all liquid and dry in vacuum (toremove traces of ammonium acetate). Dissolve in 50 µl TE.

8b.

Spermine precipitation. Dissolve DNA in 950 µl TE (no extra salt), add 50 µl
100mM spermine, mix and incubate on ice for 5 min. Centrifuge 10 min andremove all liquid. Add 30 µl unbuffered 3M potassium acetate, 300 µl TE and726 µl ethanol, mix and incubate $5 min at 20E (the time required depends on
the thickness of the pellet). Centrifuge 10 min, 11,000 × g. Remove all liquid,

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is extracted from the undissolved pellets.

F. Plasmid purification using QIAGEN anion exchange column chromatography (slight
modification of standard procedure of Quiagen Inc.: see Quiagen Plasmid Handbook, 1992; Brolle et al.,1997)
Frozen Mycelium
QIAGEN Tip-100
P1+lysozyme: 50mM Tris-HCl pH8, 10mM EDTA pH8, 100 µg ml–1 pre-boiled
RNase A (optional), 4 mg ml–1 lysozyme
P2: 0.2M NaOH, 1% SDS (207; make sure there is no SDS precipitation)
P3: 3M potassium acetate pH5.5 (294.4 g KOAc, 16.5 ml glacial acetic acid in1000 ml)
QBT (Equilibration buffer): 0.75M NaCl, 50mM MOPS pH7.0 with NaOH
(3-N-morpholinopropane sulfonic acid; MW=209; Sigma M5162), 15% ethanol
0.15% Triton X-100 (use 10% autoclaved stock solution; Sigma X-100) (43.83 g NaCl,
10.46 g MOPS (acid), adjust to pH7.0 with NaOH, 150 ml ethanol, 15 ml 10%
Triton X-100 in 1000 ml)
QC (Wash buffer): 1M NaCl, 50mM MOPS pH7.0, 15% ethanol (58.44 g NaCl,
10.46 g MOPS (acid), adjust to pH7.0 with NaOH, 150 ml ethanol in 1000 ml)
QF (Elution buffer): 1.25M NaCl, 50mM Tris-HCl pH8.5 (p. 208), 15% ethanol(73.05 g NaCl, 6.055 g Tris base, 150 ml ethanol, adjust pH8.5 at 20E with HCl, in
1000 ml)
Ice
High speed 30 ml centrifuge bottles; 10 ml pipettes; 37E water bath
Note. The QIAGEN procedure removes proteins, polysaccharides and small RNA, but lesseffectively large RNA and ssDNA. Chromosomal DNA and SDS are removed in step 4. The QIAGEN
Tip-100 has a maximal capacity of 100 µg dsDNA, less for larger plasmids. The procedure below
increases yield by reusing the column once (see step 9).

1.

Resuspend mycelium from #100 ml culture in 8 ml P1+lysozyme. Incubate
30 min at 37E.

2.

Add 8 ml P2 (NaOH/SDS), mix immediately by 4-6 inversions and incubate
5 min on ice. Final conc: 100mM NaOH, 17mM SDS.

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3.

Add 8 ml P3 (3M KOAc), mix by inverting 6-8 times and incubate 10 min on
ice.

4.

Centrifuge 30 min >30,000 × g (e.g. >16,000 rpm in SS-34 rotor). Transfer
supernatant to new tube and centrifuge 15 min as above to obtain a clear
supernatant. Try filtration through paper or a second centrifugation if the supernatant cannot
be cleared.

5.

Equilibrate QIAGEN Tip-100 with 4 ml QBT (1.5 min gravity flow).

6.

Immediately after centrifugation, apply 12 ml (½) supernatant from step 4 to
QIAGEN column (5 min gravity flow).
Alternatively use #100 µg DNA dissolved in 750mM NaCl, 50mM MOPS pH7.0 (QBT without
Triton X-100 and ethanol; add 5 µg ml–1 pre-boiled RNase A).

7.

Wash column with 2 × 10 ml QC (8 min gravity flow).

8.

Elute DNA with 5 ml QF, collect in 30 ml tube.

9.

Reuse of the QIAGEN column: repeat steps 5-8 using the same column and theremaining 12 ml of supernatant from step 4 (15 min gravity flow).

10.

Add 6 ml (0.6 vol) 20E isopropanol, mix and centrifuge 30 min at 4E as above.
Remove all liquid, wash with 10 ml 70% ethanol, air dry and dissolve DNA in
100 µl TE.

G. Plasmid isolation by the boiling method (MacNeil and Gibbons, 1986; adapted from Holmes andQuigley, 1981)
Mycelium grown in YEME+34% sucrose + 5mM MgCl2 + 0.5% glycine
EDTA/sucrose: 10mM EDTA pH8, 10% (0.3M) sucrose
STET+lysozyme: 0.1M NaCl, 10mM Tris-HCl pH8, 1mM EDTA pH8
5% Triton X-100 (BDH 30632; use 10% autoclaved stock solution), 1 mg ml–1 lysozyme
Phenol/chloroform pH 8 (p. 164)
Unbuffered 3M sodium acetate (p. 207)
Isopropanol
70% ethanol

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Ultracentrifuge tubes for large-scale preparations
37E and 100E water baths (add 10% ethylene glycol (car antifreeze) to prevent
boiling

1.

Wash mycelium from 5 ml culture with 5 ml EDTA/sucrose. Resuspend in
400 µl STET+lysozyme, incubate 15 min at 37E.

2.

Incubate at 100E for 90 sec and centrifuge immediately, 12 min, 12,000 × g, 4E.
Scaled-up version for mycelium from 500 ml culture: distribute the 40 ml cell suspension into
four 13 ml polyallomer ultracentrifuge tubes; balance precisely; heat 2 min 100E; centrifuge
30 min 92,000 × g at 4E (e.g 35,000 rpm in SW-41 rotor).

3.

Decant supernatant into tube containing 200 µl phenol/chloroform. Emulsify byvortexing, centrifuge 5 min as above.

4.

Take 200 µl aqueous supernatant, add 15 µl unbuffered sodium acetate and
129 µl (0.6 vol) isopropanol. Centrifuge 5 min as above. Remove all liquid.
Wash with 70% ethanol and remove all liquid. Precipitate again by one of themethods described (p. 173). Dissolve in 50 µl TE. For larger volumes, PEG
precipitation (8% PEG 6000, 0.7M NaCl; p. 166) may be more convenient; samples are suitablefor CsCl-ethidium bromide gradient centrifugation (procedure B, p. 178).

H. Alkaline denaturation of partially purified DNA samples
Double-stranded linear and OC DNA can be removed from CCC DNA preparations by alkaline
denaturation. Less NaOH is used than for alkaline lysis of whole cells. Glucose buffers at pH12.5(Marko et al., 1982). Keep the volume small (#1/5 of the volume of the cleared lysate) to precipitate asmuch single-stranded DNA as possible; time 40 min.
TEGR: 25mM Tris-HCl pH8, 10mM EDTA pH8, 50mM glucose (use 18 µl 50%
glucose stock solution for 1 ml), 5 µg ml–1 pre-boiled RNase A (optional; p. 166).
Low NaOH/SDS: 0.1N NaOH, 1% SDS
3M potassium acetate pH4.8 (p. 207) or acid phenol/chloroform (p. 187) andunbuffered 3M sodium acetate (p. 207)
Isopropanol

1.

Dissolve DNA in 200 µl TEGR (maximum volume for 1.5 ml tube).

2.

Add 400 µl Low NaOH/SDS, mix by inversion, incubate 2 min at 20E.

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Steps 3a and 3b are alternatives:3a.

Add 300 µl 3M potassium acetate pH4.8, mix, incubate 5 min in ice andcentrifuge 15 min, 12,000 × g at 4E. Transfer supernatant (c. 900 µl) to a tube
with 540 µl (0.6 vol) 20E isopropanol, centrifuge, remove all liquid andprecipitate once more with ethanol and ammonium acetate or spermine as inplasmid isolation procedure E, step 8a or 8b (p. 188).

3b.

Add 100 µl acid phenol/chloroform and 60 µl unbuffered 3M sodium acetate,
vortex and centrifuge 15 min, 12,000 × g at 4E. Transfer upper phase (c. 600 µl)
to a tube containing 360 µl (0.6 vol) isopropanol. Mix, centrifuge 10 min as
above and remove all liquid. Continue with step 5 (second phenol/chloroform
extraction) or step 8 (second precipitation) of the plasmid isolation procedure E.

I. Purification of samples using ethidium bromide
Ethidium bromide precipitates impurities from high salt DNA solutions. This procedure isadapted from Zhang and Kerr (1993). Keep volume small (#1/5 of the volume of the volume of thecleared lysate) to precipitate as much single-stranded DNA as possible; time 1 h.
TEGR: 25mM Tris-HCl pH8, 10mM EDTA pH8, 50mM glucose (use 18 µl 50%
glucose stock solution for 1 ml), 5 µg ml–1 pre-boiled RNase A (optional; p. 166)
0.1N NaOH (no SDS), made from 10 N stock solution (p. 207)
Ethidium bromide, 10 mg ml–1 (25mM)
3M potassium acetate pH4.8 (p. 207)
Phenol/chloroform/isoamyl alcohol: 5 ml phenol pH8, 5 ml chloroform, 0.1 ml
isoamyl alcohol (p. 164,)
Isopropanol

1.

Dissolve DNA in 100 µl TEGR.

2.

Add 200 µl 0.1N NaOH, mix by inversion and incubate 2 min at 20E.

3.

Add 150 µl 3M potassium acetate pH4.8, mix, add 40 µl 10 mg ml–1 ethidium
bromide, mix, incubate 5 min in ice and centrifuge 15 min, 12,000 × g at 4E.
Final conc: 1.2M NaOAc, 1 mg ml–1 ethidium bromide.

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4.



Extract twice with 500 µl phenol/chloroform/isoamyl alcohol. Ethidium bromide
goes to the organic phase.

5.

Transfer aqueous supernatant (c. 350 µl) to a new tube, add 210 µl (0.6 vol) 20E
isopropanol, centrifuge and remove all liquid.

6.

Precipitate once more with ethanol and ammonium acetate or spermine as inplasmid isolation procedure E, step 8a or 8b (p. 188).

J. Acid phenol (pH4) extraction
Phenol at pH4 denatures and dissolves linear and OC DNA (Azad et al., 1992, and references
therein). CCC DNA remains in the aqueous phase. Keep the sample cold and the time short to reduce
depurination (exonuclease III has apurinic site endonuclease activity; Rogers and Weiss, 1980). Plasmids
up to 75 kb have been purified by acid phenol extraction and (double-stranded) plasmid DNA from theendA+ strain TG-1 was made suitable for sequencing (Weikert and Chambliss, 1989). Time 1 h.
0.5M sodium acetate pH4.0: 0.5M sodium acetate + 3.155M acetic acid. For 100 ml:
6.804 g CH3COONa@3H2O (or 16.7 ml unbuffered 3M solution); adjust to pH4.0 with
8.67 ml (9.10 g) glacial acetic acid
Phenol pH4.0: equilibrate 3 × with 0.5 vol 50mM sodium acetate pH 4.0
100mM magnesium chloride (for 100 ml: 2.03 g MgCl2@6H2O; BDH 10149)
Chloroform:isoamyl alcohol 24:1
3M unbuffered sodium acetate (40,82 g 100 ml–1, p. 207)

1.

Dissolve DNA in 300 µl TE, cool in ice.

2.

Add 30 µl 0.5M sodium acetate pH4.0 and 5 µl 100mM magnesium chloride,mix, add 360 µl phenol pH4, vortex 1 min and centrifuge 10 min 11,000 × g at
4E. Final conc: 50 µM sodium acetate and 1.5mM magnesium chloride.

3.

Transfer upper aqueous phase (300 µl) to tube with 30 µl unbuffered 3M sodium
acetate, mix, add 400 µl chloroform/isoamyl alcohol, vortex 10 sec andcentrifuge as above.

4.

Transfer upper phase (300 µl) and precipitate with 180 µl (0.6 vol) isopropanol;
wash pellet with 70% ethanol.

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K. “Jurassic preps” plasmid purification using guanidine thiocyanate(modified from Carter and Milton (1993); contributed by W.R. Strohl)
4 M Guanidine thiocyanate solution: 44.28 g guanidine thiocyanate (Sigma G 6639), 0.61 g
Trizma base (50mM), 0.75 g Na2 EDTA·H2O (20mM), water to 100 ml. Adjust pH to 7.0, heatto 60-70Eto dissolve components and then filter sterilise.
Amber Resin (10 mg ml-1): Suspend 1 g of diatomaceous earth (Sigma D 5384) in 10 ml waterand let settle for 3 h in a graduated cylinder. Remove supernatant containing the fines.
Resuspend sediment in 100 ml 4M guanidine thiocyanate solution.
Lysozyme solution:3 mg ml-1 lysozyme in 25mM Tris 10mM EDTA pH8
Lysis solution: 0.2M NaOH 1% SDS
Neutralizing solution: 3M potassium acetate pH4.8
Column wash solution: 200mM NaCl, 20mM Tris pH 7.5, 5mM EDTA
Wizard mini columns (Promega syringe filter column A 7211)
37Eand 65E water baths, 1 ml syringes, Eppendorf tubes

1.

Resuspend mycelium in 200 µl lysozyme solution in an Eppendorf tube, mix andincubate c. 1 h at 37E. This procdeure can also be used to purify DNA fragments from gelsdissolved in 3 volumes of 6M sodium iodide.

2.

Add 200 µl neutralizing solution, mix by inversion and centrifuge 5 min.

3.

Transfer c. 500 µl supernatant to a fresh tube containing 1 ml of amber resin.
Mix 2 min by inversion. Resuspend amber resin before dispensing.

4.

Attach a Wizard mini column to a 1 ml disposable syringe barrel (plunger
removed) and attach the setup to a vacuum manifold.

5.

Pipette the resin/DNA mixture into the column and draw the resin into thecolumn by vacuum. DNA fragments from gels dissolved in 3 volumes of 6M sodium iodide
can also be purified on these columns.

6.

Rinse column 3 times with 1 ml 4M guanidine thiocyanate solution. Discard thesyringe.

7.

Transfer the Wizard mini column to a centrifuge tube and spin for 30 sec toremove any residual wash solution from the column.

8.

Transfer the Wizard mini column to a fresh centrifuge tube and 50-100 µl 65E
TE buffer. Spin 30 sec to elute DNA.

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9.



Centrifuge the DNA solution for 1 min to precipitate insoluble particulate matter.
Transfer supernatant to a new tube for storage.

Standard agarose gel electrophoresis
We routinely use commercial standards for estimating the sizes of linear Streptomyces DNA
fragments: the G+C content does not influence the migration of the DNA on normal agarose gels. For
pulsed-field gels electrophoresis (PFGE), however, use of phage
concatemers results in anunderestimation of the Streptomyces DNA fragments by c. 30% for a 60 kb fragment and 7.5%
overestimation for a 500 kb fragment (Gravius et al., 1994). The effect of G+C content on the migrationof DNA fragments may depend critically on the running conditions. It may be useful to include as
standards AseI digests of S. coelicolor M145 DNA whose complete sequence will soon be known.
Streptomyces CCC plasmids often occur in multiple forms differing in the number of supercoil
turns. In addition, single-stranded forms (rolling circle replication intermediates or denatured OC forms),
irreversibly denatured CCC DNAs, plasmid multimers, OC and linear forms may be present. These
plasmid forms are not unique for Streptomyces, but they are much more prominent than in E. coli plasmid
preparations. Fig. 8.4 shows alkaline lysis preparations (procedure E, p. 187) of 3.3-45 kb plasmids.
The identities of the multiple bands are explained in Fig. 8.5. The most intense band in each lane is the
fully supercoiled CCC form. The incompletely supercoiled plasmids form a distinct “ladder” for thesmaller plasmids up to 8.9 kb, and a prominent smear for the larger plasmids.
The non-supercoiled CCC forms presumably migrate slowest, in the same positions as therespective OC forms. Nicking of the multiple CCC forms of a plasmid by ethidium bromide staining and
254 nm UV irradiation, followed by second-dimension electrophoresis, produces OC forms that migrate
together (Kieser et al., 1982; Hinterman et al., 1981). This technique is also useful for analysing
mixtures of multiple plasmids (e.g. Kieser et al., 1982). Note that the CCC forms of the plasmid dimers
always migrate very close to the OC forms of the respective monomers. The OC forms can be converted
to ss DNA (one linear molecule and one circle when they carry a single nick) which renatures slowly.
The samples in lanes (a) in Fig. 8.4 were stored at room temperature for several days before
electrophoresis and the samples in the lanes (b) were renatured immediately before electrophoresis. OC
forms are reduced in lanes (b), with a corresponding increase in the fast-migrating ss DNAs (linear andcircular forms co-migrate).
The usually faint bands between the fully supercoiled CCC forms and the ss DNAs are(irreversibly) denatured CCC forms. They form when CCC DNA is exposed to pH12.5, e.g. whencomplete mixing of the samples is delayed after the addition of alkaline SDS (Fig. 8.6).
The ss DNAs and irreversibly denatured CCC forms can be removed by digestion with S1
nuclease (which is active in the presence of SDS), but they are not affected by most restriction
endonucleases.
Depending on the plasmid size and the gel conditions, the (irreversibly) denatured CCC forms
may migrate very close to, or even behind, the fully supercoiled CCC forms. This can give theimpression of incomplete restriction endonuclease digests. The denatured CCC forms renature readily
only after nicking to produce regular OC forms. Denatured CCC forms probably transform Streptomyces



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Fig. 8.4 Plasmid preparations run on a 0.7% agarose gel in Tris-acetate buffer for 14 h
at 1 V cm–1. Alternate lanes show (a) alkaline lysates after extraction with acidphenol/chloroform (step 3 in procedure E) and (b) the same preparations after a second
alkaline denaturation with alkaline SDS and extraction with acid phenol/chloroform. (Thesamples were precipitated with isopropanol as in step 4 of the procedure, redissolved in
500 µl lysozyme solution, and denatured with 250 µl alkaline SDS for 15 min at 70E.)
The second denaturation step converted the OC forms and linear DNA to ssDNA. A small
proportion of the CCC DNA became irreversibly denatured. The strains in 1-6 containmulticopy plasmids (pIJ101 and derivatives), 7 is an SLP1.2 derivative, 8 is an SCP2*
derivative and 9 is an E. coli cosmid (there is some contamination from lane 6b, marked
with an asterisk). = HindIII-cleaved phage DNA. The origin of the bands is further
explained in the schematic diagram in Fig. 8.5.
protoplasts efficiently, and denaturation of (non-replicating) plasmids stimulates recombination (p. 235;
Oh and Chater, 1997).
Linear forms sometimes appear in CCC plasmid preparations. Their positions relative to the
CCC forms are highly variable, depending on the gel conditions. Increasing the agarose concentration(e.g. from 0.8 to 1.1%), increasing the voltage (e.g. from 2 to 8 v cm–1), and substituting Tris-borate
buffer for Tris-acetate, which has a higher conductivity (and needs to be recycled for longer runs), retards
CCC and OC DNAs relative to the linear DNA.

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

Fig. 8.5 Schematic diagram explaining the bands in Fig 8.4. The bands containing CCC DNA
are labelled with the size of the molecules and are connected to the corresponding OC bandby a vertical line. Bands of partially supercoiled CCC molecules (“stepladder”) fall into theregion of the vertical line. They are not included in the diagram. Only the bands that are
visible on the original print of Fig 8.4 are drawn. The identity of the individual bands wasdetermined with the help of two-dimensional gel electrophoresis by the method of Hintermann
et al. (1981), which identifies CCC, OC, and linear DNA molecules. Restriction digests wereused to confirm the presence of dimers. The additional alkaline denaturation step which
generated single-stranded (ss) DNA and denatured CCC DNA helped to identify these bands(Fig. 8.4 lanes labelled “b”, and Fig. 8.6). The rightmost lane gives the sizes and positions of
the linear phage DNA fragments generated by digestion with HindIII. The 28 kb fragment
consists of the 4.3 and 23.5 kb fragments held together by the phage cohesive ends.

When isolating DNA fragments from gels for cloning purposes, it is best when the desired
fragment migrates ahead of the CCC DNAs as this reduces the chance of contamination.
Samples of Streptomyces plasmid and chromosomal DNA often produce smears on agarose gels(particularly when the DNA has been isolated from poorly growing cultures that produce large mycelial
pellets). Running long agarose gels dilutes the smear while the specific bands remain sharp. S. lividans
66, S. avermitilis, and probably some other Streptomyces strains produce modified DNA that is cleaved
during electrophoresis by Tris peracid generated at the anode (Ray et al., 1995). Such cleavage is always
evident in PFGE, but only occasionally in normal agarose gels. Addition of 50µM thiourea to Tris-borate
electrophoresis buffer or replacing Tris with HEPES buffer prevents degradation.

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Fig. 8.6 Denatured CCC DNA generated by treating the samples
with excess alkaline SDS at high temperature. The samples in thelanes labelled “b” are the same as in Fig. 8.4. The samples in thelanes labelled “c” were treated with 400 µl alkaline SDS andincubated for 5 min at 90E. Most of the CCC DNA in these samples
has been converted to denatured forms. The gel conditions were thesame as in Fig. 8.4. See Fig. 8.5 for the identification of the bands.

Pulsed-field gel electrophoresis
General considerations
Following the pioneering discovery of methods for separating very large DNA fragments on
agarose gels by pulsed-field gel electrophoresis (PFGE) (Schwartz and Cantor, 1984; Carle and Olson,
1984, 1985; Smith et al., 1987), developments of the technique have been used to characterise and map
the chromosomes and large linear plasmids of several Streptomyces spp. (Kinashi and Shimaji, 1987;
Leblond et al., 1990; Kieser et al., 1992; Redenbach et al., 1993; Beyazova and Lechevalier, 1993;
Lezhava et al., 1995).
PFGE does not normally use DNA that has been isolated in the conventional way; instead, thecells are embedded in small blocks of low melting-point (LMP) agarose and subsequent steps of cell lysis

&KDSWHU 3UHSDUDWLRQDQGDQDO\VLVRIJHQRPLFDQGSODVPLG'1$and endonuclease treatment are carried out on these blocks. This avoids mechanical shearing of theDNA. The blocks are then inserted into agarose slab gels for electrophoresis.
Two alternative procedures for cell lysis in agarose blocks are described. The first (or minor
variations of it) is used in many laboratories; the second, incorporating a phenol extraction of the cells
before lysis, has been developed for strains that gave unsatisfactory results with the first procedure.
Normally, a protease treatment (proteinase K, pronase E or subtilisin) is used after lysis of themycelium with lysozyme and detergent (sarcosine or SDS) to help release DNA from the cell debris.
However, the linear chromosomes and plasmids of Streptomyces spp. have proteins covalently bound to
the 50 ends of the DNA, and these proteins are removed by any protease treatment. If it is desired to leavethem intact, for example to identify end fragments, the protease treatment is omitted.
The most difficult variable to control is the optimal concentration of mycelium in the agarose
blocks. OD600 measurement of the mycelial suspension gives only a rough guide and so it may benecessary to prepare a few blocks with different concentrations of mycelium to discover which is best
before preparing large numbers of blocks.
For Streptomyces DNA, with its high G+C content, the most suitable restriction enzymes forgross mapping are those that recognise hexameric sites containing all A and T residues, namely AseI(AT0TAAT), DraI (TTT0AAA) and SspI (AAT0ATT). They cleave the Streptomyces chromosome intoc. 8-30 fragments. BfrI (C0TTAAG) is another useful enzyme that cuts the chromosome of S.
ambofaciens 30 times (Berger et al., 1996).
To achieve good separation of DNA fragments over a range of sizes from a few tens of base pairs
up to one or more megabases, it is usually necessary to use more than one combination of pulse time andvoltage for different gels and/or to vary these parameters during the course of a single electrophoretic
separation.
For the DNA of strains like S. lividans and S. avermitilis, which degrades during electrophoresis
because of the Tris in the buffer (Kieser et al., 1992; Evans et al., 1994), 100µM thiourea should be
added to the gel and the running buffer.

Standard procedure for preparing Streptomyces chromosomal DNA for PFGE (from Kieser et al.,1992)
Mycelium from a 25 ml culture grown in YEME (with 34% sucrose, 5mM MgCl2
and 0.5% glycine). For S. coelicolor and S. lividans c. 40 h cultures are optimal. The
mycelium can be stored as a frozen pellet at -20E
TE25-sucrose (25mM Tris-HCl pH8, 25mM EDTA pH8, 0.3M sucrose)
NDS (0.5M EDTA pH8, 10mM Tris-HCl pH8, 1% SDS, or 1% lauryl sarcosine)
Lysozyme solution (2 mg ml–1 in TE25-sucrose)
Proteinase K solution (1 mg ml–1 proteinase K, Sigma P6556, in NDS pre-incubated
at 50E for 30 min. Solutions should be fresh; alternatively solutions frozen immediately
after preparation can be used)&KDSWHU 3UHSDUDWLRQDQGDQDO\VLVRIJHQRPLFDQGSODVPLG'1$

PMSF (phenylmethanesulphonyl fluoride, serine proteinase inhibitor, Sigma P7626.
100mM stock solution in isopropanol: dissolve at 50E for 5 min and store at 20E.
Caution: this is a lethal toxin if inhaled or swallowed)
LMP Agarose (1.5% molten solution of low melting point agarose e.g. FMC insert
LMP, p. 418, cooled to 50E)
TE (10mM Tris-HCl pH8, 1mM EDTA pH8)
Plug mould (100 µl holes e.g. Pharmacia), clean or disposable
10 or 50 ml tubes with screw caps; caps with holes (e.g. Biorad) to facilitate draining
37E and 50E water baths

1.

Resuspend mycelium in 25 ml TE25-sucrose and centrifuge 5 min at 1000 × g.
Remove supernatant.

2.

Resuspend mycelium in TE25-sucrose to give OD600=1.9-2.0. The cell density canbe increased up to 5-fold to give higher DNA concentrations; this is useful for visualizing verylarge fragments which are sometimes present in lower amounts, and small DNA fragments, andalso for preparative purposes. Mycelium can be stored at -20E in TE25-sucrose.

3.

Add 500 µl of mycelial suspension to 500 µl of 1.5% 50E LMP agarose. Mix
immediately using a 1 ml pipette tip with the end cut off, and fill holes in aprecooled plug mould. Avoid the formation of air bubbles which cause
electrophoresis artifacts. Leave agarose to set for 10 min at 4E. Plug moulds canbe re-used after washing with detergent at 70E, soaking in 0.25N HCl for 1 h at 70E, and rinsing
4 times with sterile distilled water.

4.

Remove blocks from mould (a sealed bent Pasteur pipette end is useful for this)and incubate at 37E for up to 2 h in 5 ml lysozyme solution in a 10 ml tube. Mix
occasionally by inversion. S. clavuligerus must only be incubated 5 min in lysozyme.

5.

Remove lysozyme solution and add 5 ml proteinase K solution (pre-digested at
50E for 30-60 min). Incubate for 12-24 h at 50E. Mix occasionally by inversion.
Berger et al. (1996) used 5 mg ml–1 Pronase E (Sigma P6911) instead of 1 mg ml–1 proteinase
K, and omitted PMSF in Step 6.

6.

Remove proteinase K solution and wash blocks once for 1 h in 10 ml TE+0.1mM
PMSF, then three times with TE for at least 1 h each. Mix frequently byinversion. Remove all liquid after each wash either with a pipette or bydecanting from a tube with a perforated lid. Plugs containing $ 5 mg ml–1 DNA should
be clear if the protease digestion in step 5 was complete.

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7.



The agarose blocks are now ready for treatment with restriction endonucleases,
but can be stored for up to 1 month at 4E in TE; for longer periods store in 0.5M
EDTA pH8.

If telomeric proteins are to be preserved the amount of mycelium in the agarose blocks (instep 3) is increased by a factor of three to compensate for the lower yield of DNA.
In step 5 the protease treatment is omitted and the SDS concentration in the NDS is doubled to2%; the blocks are incubated at 50E for 40-60 h.
In step 6, wash blocks at least eight times with TE for 40 min at 50E. Mix frequently byinversion. Remove all liquid after each wash. The agarose plugs will remain opaque. It is very importantto remove all SDS as the presence of this will allow fragments of DNA with telomeric proteins to enter
the running gel.

Phenol wash procedure for preparation of actinomycete DNA for PFGE (from Beyazova andLechevalier, 1993)
Mycelium from 2-3 day old cultures grown at 28E, 250 rpm, in TSB (p. 412).
Younger cells are better; cells can be frozen in 10% glycerol at -80E
0.5M and 10mM EDTA pH8
Phenol, water-saturated
Chloroform-isoamyl alcohol 24:1
2% SDS + 5mM EDTA pH8
HE buffer (10mM HEPES, Sigma, 1mM EDTA pH8)
TE buffer
5mM EDTA pH8 + BSA fraction V, 1mg ml–1 (Sigma)
LMP agarose (Beckman) (2% molten solution, cooled to 50E)
Lysozyme (1 mg ml–1 in 6mM Tris-HCl pH7.6, 1M NaCl, 100mM EDTA, 0.5% Brij-58,
0.2% deoxycholate, 0.5% sarkosyl, 20 µg ml–1 RNase)
Subtilisin (protease similar to proteinase K), 1 mg ml–1 in HE buffer
Tissue grinders
Phenol and chloroform-resistant centrifuge tubes
Spectrophotometer and cuvettes
Ice
50E and 56E incubators

1.

Wash mycelium twice with 0.5M EDTA pH8.

2.

Disperse pellets by using a tissue grinder.



3.

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Extract mycelium for 15 min in an equal volume of 65E water-saturated phenol.
Spin down the cells. The phenol probably removes or inactivates cell wall-associatedDNases.

4.

Wash the cells three times with chloroform-isoamyl alcohol at room temperature.
The mycelium floats on top of the chloroform.

5.

Incubate cells for 30 min at 65E in 2% SDS-5mM EDTA.

6.

Wash cells three times in 5mM EDTA pH8 + 1mg ml–1 BSA.

7.

Wash cells three times with 10mM EDTA pH8.

8.

Resuspend cells in TE buffer and adjust concentration to OD600=1.8.

9.

Mix cell suspension with an equal volume of 1% 50E LMP agarose and fill plug
mould as in step 3 of the previous procedure.

10.

Treat the agarose blocks for at least 2 h at 37E with the lysozyme solution. The
size of the blocks is important for the incubation times.

11.

Treat the agarose blocks for at least 3 h at 56E with 1 mg ml–1 subtilisin.

12.

Wash agarose blocks twice for at least 2 h at 56E with TE buffer. The plugs areready for digestion with restriction endonucleases, or can be stored in HE buffer for at least 18
months.

Digestion of DNA with restriction endonucleases in agarose blocks for PFGE
For enzyme reactions it is important to use DNA embedded in LMP (hydroxyethylated) agarose.
The speed at which the enzymes diffuse into the blocks depends on their size and on the agarose
concentration.
Agarose blocks containing DNA
Concentrated restriction endonucleases, preferably certified for PFGE
Enzyme buffers
TE
0.5M EDTA (pH8)
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

Clean glass cover slips
Eppendorf tubes
37E incubator

1.

If the agarose blocks have been stored in 0.5M EDTA wash with 50 vol of TE
for 1 h. Place the blocks (or parts of them cut with a glass coverslip, not a metal
knife) in 1.5ml Eppendorf tubes containing 1 ml of 1 × restriction enzyme buffer
and incubate at room temperature for 15 min. Mix occasionally by inversion.
Sometimes it is advantageous to remove small fragments of degraded chromosomal DNA andplasmids from the agarose blocks electrophoretically before digestion with restriction
endonucleases. This greatly improves the clarity of gels (Whatling and Thomas, 1993). See p.
205 for the procedure.

2.

Remove buffer by aspiration and add just enough fresh 1 × buffer to cover theblocks. Add 1 µl of bovine serum albumin and 30-50 U of (high concentration)
restriction endonuclease. Mix very gently and incubate at 37E for at least 6 h.
After c. 2 h add a second sample of AseI (10-12 U), to ensure complete
digestion. Omit this second addition for DraI, because digestion for longer than
6 h causes serious non-specific degradation.

3.

Remove the liquid, and add 1 ml TE. Mix gently. The blocks can be inserted
directly into the agarose slab gels for PFGE without washing. If they are to be
stored for longer than c. 24 h, remove all TE and store blocks at 4E to prevent fragments smaller
than c. 50 kb from diffusing out of the blocks.

Loading gels
We normally use 1% agarose in 0.5 x TBE buffer. With the gel comb in position the agarose
block (or pieces) are placed directly on the front surface of the teeth. Excess buffer is removed and after
10 min the agarose (at 50E) is poured around the comb and samples. When the agarose is set and the
comb removed the sample will remain in the gel. The wells behind the samples will be filled with bufferwhen the gel is submerged and any air bubbles should be removed.
Alternatively, the gel is cast, with a suitably sized comb, and allowed to set before the comb isremoved. The agarose blocks (or pieces) are then inserted into the empty wells. Using sealed Pasteur
pipettes and coverslips to handle them the blocks are pressed firmly against the front walls of the wells.
Excess liquid is removed from the wells and the wells are filled with 1% LMP agarose.

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Table 8.5 Examples of PFGE running conditionsa
Fragment sizes

Pulse times(linear ramping)

Duration ofelectrophoresis

Suitable size standardsb

Uncut chromosome (8 mb)

30-120 min

3 daysS. pombe chromosomes

150-1500 kb

70-130 sec

24 h

S. cerevisiae
chromosomes

50-1000 kb

50-90 sec

24 hladder10-200 kb

10-50 sec

24 h

5 kb laddera

For uncut chromosomes, it is useful to reduce the field angle from the normal 120E to 95E; also to
reduce the voltage to 1.5-2 V cm–1 instead of the usual 6 V cm–1.
bCommonly used standards are yeast chromosomes and phage (48.5-50 kb) multimers (Biorad orother). The 5 kb ladder contains concatemers of pBR328 (Biorad). Gravius et al. (1994) found thathigh G+C phage DNA runs differently under PFGE conditions from the low G+C DNA (see p.
195).
Running conditions for PFGE
For Streptomyces genomes digested with restriction endonucleases, we usually first make a
PFGE run using 1% agarose in 0.5 × TBE buffer (with the addition of 100 µM thiourea for S. lividans
and S. avermitilis) at 6 V cm–1 and 13E, with an initial switch time of 70 sec and a final switch time of
130 sec for 24 h. This can be ramped (continuous increase of pulse time) or in “steps” of e.g. 70 sec for
9 h, 100 sec for 9 h, 130 sec for 6 h. Depending on the resulting banding pattern, compressed regions
can be expanded using different switch and run times. Sample digests can be run on the outside tracksof the gel. These pieces of gel can be cut off after the first run, and stained with ethidium bromide; theswitch and run times can then be altered for the remainder of the gel. (After staining, intercalation ofethidium bromide prevents fragments >300 kb from migrating in PFGE.) Some examples of running
conditions are in Table 8.5. For further information, consult the PFG apparatus manufacturer’s
handbook.

Distinguishing plasmid from chromosomal DNA
All linear Streptomyces plasmids have 50 terminal, covalently bound proteins (telomeric proteins)which virtually prevent the DNA from entering the gel during electrophoresis and block attack byexonucleases if they are left intact (Lin et al., 1993). See p. 201 for the preparation of DNA for PFGE
preserving these proteins.
Relatively little is known about the migration of circular DNA molecules during PFGE. It seems
that they migrate almost the same as during normal electrophoresis: CCC forms are more strongly
retarded by higher agarose concentrations than linear molecules; they accelerate relative to linear DNA
when the voltage is reduced. For example SCP2 (31 kb CCC) might migrate in the same position as ac. 200 kb linear DNA at one voltage, but in the position corresponding to a smaller linear DNA at a lower

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voltage. There is a size limit above which CCC molecules are no longer resolved (Hightower and Santi,1989).

Removal of small DNA fragments
Place the gel comb - with slots large enough to accept the agarose blocks without compressing
them - in position c. 2 mm from the surface of the mould. Pour the gel to c. 6 mm thick and allow to set.
Carefully remove the comb. Place the agarose blocks containing DNA in the slots, using a sealed Pasteur
pipette and glass coverslip for handling. Make sure that the agarose blocks are flush against the front
wall of the slots. Remove any liquid from the slots and allow the gel to stand for 10 min. Submerge thegel very carefully in electrophoresis buffer in the gel tank and run at, for example, 13E, 45 sec, 6 V cm–1
for 6 h. This will eliminate DNA fragments smaller than c. 500 kb. Remove the gel carefully from theapparatus. Remove the agarose blocks from the gel slots into 1.5 ml Eppendorf tubes and proceed withrestriction enzyme digestion (p. 202).

Solutions
Ammonium acetate
NH4OAc, CH3COONH4, FW=77.08 (BDH 10013; Fisons A/3440/53). Do not autoclave, store
airtight, best at -20E. pH=7 without adjustment; loss of NH3 makes solution acidic.

Stocksolution

Amount for
1 litreWater to make1 l solution

Add to make 2.5M
solution

5M

385 g

675 ml

1 vol

7.5M

578 g

503 ml

0.5 vol

8M

617 g

469 ml0.45 vol

10M

770 g

331 ml0.33 vol

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EDTA pH8
Na2EDTA@H2O, ethylenediamine tetraacetate disodium salt@2H2O, diaminoethanetetra-acetic aciddisodium salt, FW=372.24 (BDH 10093; Fisons D/0700/53) is insoluble in pure water; it dissolves
slowly when NaOH is added (heating accelerates dissolution). To prepare 100 ml 0.5M EDTA pH8.0,
mix either:
2.00 g (20 pellets)

NaOH

88 ml
18.6 gor

5.00 ml (5.45 g)

10N NaOH solution

water

84.6 mlwater

Na2EDTA@2H2O

18.6 gNa2EDTA@2H2O

Measure the pH when the Na2EDTA is completely dissolved (the electrode takes about 1 h toequilibrate and may not be accurate with 0.5M solutions). If necessary, adjust to pH8.0. Adding 100 mg
NaOH (1 pellet) or 250 µl 10N NaOH increases the pH by c. 0.25; 500 mg solid Na2EDTA@H2O
decreases the pH by c. 0.1.

Lithium chloride LiCl, FW=42.39 (BDH 10374; p. 173)

Stock solution

Amount per
1 litreWater to make1 l solution

Add to make
2.5M solution

5M

212 g

898 ml

1 vol

7.5M

318 g

834 ml

0.5 vol

8M

339 g

833 ml0.45 volSodium chloride NaCl, FW=54.44

Stock solution

Amount per litre

Water to make1 l solution

5M

292 g

897 ml1M

54.4 g984 ml

0.7M

38.1 g989 ml

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

Sodium hydroxide
NaOH FW=40. NaOH is hygroscopic and reacts with CO2 from the air and with glass. Keepsolutions airtight in plastic containers. Pellets containing white NaCO3 should be washed briefly anddried with paper towel. Protect eyes: solutions overheat!
Stock solution

NaOH for 100 ml

Water for 100 ml

10M

40 g

92.8 ml

0.3M

1.2 g

100 ml

0.2M

0.8 g

100 ml

0.1M

0.4 g

100 mlPotassium acetate and sodium acetate, 3M
KOAc, CH3COOK, FW=98.14 (BDH 10350)
NaOAc, CH3COONa@3H2O, FW=136.08 (BDH 10235)
pH adjustment, with glacial acetic acid MW=60.05, 99.6%, 17.4M, density=1.05 g ml–1,
pKa=4.78.
Amounts for 1 litre 3M solution
Solid potassium or
sodium acetate294.4 g

KOAc

or
408 g

NaOAc@3H2O

pH

Aceticacid

H2O

Unbuffered

6

830 ml4.76

90.5 ml(95.0 g)

740 ml4.8

82.5 ml(86.6 g)

747 ml5.0

57.1 ml(54.7 g)

778 ml5.5

16.5 ml(17.3 g)813 ml6.0

5.21 ml(5.47 g)825 ml

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Tris-HCl bufferTris(hydroxymethyl)aminomethane pKa=8.3. The pH is temperature-dependent ( pKA/E=-0.031: 25E pH8, 5E pH8.6, 55E pH7.1) and not all electrodes are suitable for Tris buffers. Hydrochloric
acid (MW=36.5; conc=36% w/w, 11.64M, density=1.18) or Tris hydrochloride can be used together with
Tris base to make buffers. Tris base MW=121.1, hygroscopic (Sigma T8524 or T1503); Tris
hydrochloride FW=157.6, very hygroscopic (Sigma T7149)

For 1 litre 1M Tris-HClbuffer
Using solid Tris base and HCl
pH at
25E

Using solid Tris baseand Tris-HCl

Using 1M stock
solutions

Tris
base

Conc HCl

H2O

Tris
base

TrisHCl

H2O

Tris
base

TrisHCl

g

ml

(g)

ml

g

g

ml

ml

ml

7.2

121

76.1

(89.8)

817

13.4

140

875

111

886

7.5

121

69.1

(81.5)

824

23.6

127

878

195

804s

8.0

121

48.3

(57.0)

845

53.0

88.8

886

438

562

8.5

121

23.9

(28.2)

869

87.2

44.2

897

720

280

9.0

121

8.25

(9.73)

885

109

15.2

904

903

96

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&KDSWHU 3UHSDUDWLRQDQGDQDO\VLVRIJHQRPLFDQGSODVPLG'1$
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m09tkb.wpd 20 April 2000 Tobias Kieser



Chapter 9General considerations about gene cloningin Streptomyces
CONTENTS
Features of Streptomyces genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212
Restriction-modification and other host factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
Preparation of vector and target DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214
How many clones are required for making a representative gene library? . . . . . . . . . . . . . . 214
Choice and use of restriction endonucleases for making gene libraries . . . . . . . . . . . . . . . . . . 214
Choice of cloning vector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
Vector host-range . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
Size of the target DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216
Integrating vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216
Low copy number plasmid vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217
High copy number plasmid vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217
Plasmid versus phage vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217
Positive selection vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218
E. coli vectors containing Streptomyces selection markers . . . . . . . . . . . . . . . . . . . . . . 218
Using bifunctional vectors that replicate in E. coli and Streptomyces . . . . . . . . . . . . . 219
Selective markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219
Antibiotic resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219
Counterselectable markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220
Ligation conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220
Transformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221
Finding the desired clone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221
Sib-Selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222
Antibiotic biosynthetic genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224
Confirmation of clones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224
Does the phenotype depend on the cloned DNA? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224
Is the cloned DNA rearranged? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
Is the promoter for a cloned gene present on the cloned DNA? . . . . . . . . . . . . . . . . . . 225
What if the desired gene cannot be cloned? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226
Assessing the quality of Streptomyces gene libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226
Storing gene libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226

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Features of Streptomyces genes
Streptomycetes, like other bacteria, contain relatively small, uninterrupted genes which aredensely packed on the DNA of chromosomes, phages and plasmids. On average, a gene, based on thedeveloping complete genome sequence (p. 424), occupies c. 1.13 kb. Streptomyces genes are oftentranscribed from multiple promoters which can be as far as 300 bp upstream of the start of the ORF(Strohl, 1992). Some genes (e.g. ermE and aphI) have no leader mRNA upstream of the start codon;
these ORFs have no upstream ribosome binding sequence (RBS) (Janssen et al., 1989; Wu and Janssen,
1996; Wu et al., 1992; see p. 386). Inverted repeats downstream of genes have been shown to act astranscription terminators in a few cases. The presence of such a sequence in cloned DNA may contributeto the stability of the construct (Kieser and Melton, 1988).
Operons are common in streptomycetes, and genes in operons often overlap (most often by 1 or
4 bp), suggestive of translational coupling (Schümperli et al., 1982). Antibiotic biosynthesis genes occur
in clusters containing several operons and single genes, including all the genes necessary to synthesise
the antibiotic, and often also regulatory and antibiotic resistance genes.
Streptomycetes are good at expressing genes (promoters, RBSs, CDSs, and terminators) fromlow G+C organisms, but Streptomyces genes are usually more difficult to express in E. coli because mostpromoters do not function, and translation may be inefficient unless the initial amino acid codons arechanged to lower G+C alternatives (p. 390).
Fig. 9.1 summarises general features of Streptomyces gene structure, and indicates the uses ofdifferent types of cloning vectors.
Streptomycete DNA has a fairly uniformly high G+C content of c. 73%. Promoter regions, andtransposable DNA probably acquired from other bacteria, often have a lower G+C content. Some
Streptomyces phages also have lower G+C content; e.g. C31 contains 63.6% G+C. The G+C content
affects the frequency of restriction sites. AT-rich recognition sequences are rare (e.g. AseI AT0TAAT,
DraI TTT0AAA) and GC-rich recognition sequences are frequent (e.g. SmaI CCC0GGG). The precise
frequency of restriction sites cannot, however, be predicted statistically: SalI (G0TCGAC) cuts
Streptomyces DNA more frequently than BamHI (G0GATCC) even though the two recognition sequences
are permutations of the same bases. The distribution of restriction sites in Streptomyces plasmids
generally follows that in the chromosome, but Streptomyces phages often have unexpected restriction
site frequencies, probably because their lifestyle leads them frequently to encounter the Type II
restriction systems that abound in streptomycetes (e.g. SalI, SacI, SacII, SphI, etc.).
The high G+C content increases the melting temperature of Streptomyces DNA by c. 10E
compared to that of 50% G+C DNA. This does not usually need to be taken into account in hybridisation
experiments, but PCR reactions, especially for the production of long PCR-amplified fragments, more
often give problems than with low G+C DNA, though this does not inhibit most uses of PCR.
No major G+C effects have been reported for the migration of high G+C DNA in normal agarose
gels. A slight effect has been found in pulsed-field gels (Gravius et al., 1994). (PAGE is not suitable forsize determination even with 50% G+C DNA because sequence-dependent DNA bends affect migration.)
The increased density of high G+C DNA is noticeable in CsCl gradients (Fig. 8.1, p. 176).

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-35

DNA

Clone internal fragments
into ∆ attP phages
etc. for mutational
cloning
ATG
GTG
? SD

-10

PromoterSD

Activator Repressor
bindingbinding0-300 bp

ORF

300 bp - 18,000 bp
100 aa - 6000 aa
10 kd - 600 kd



TGA

ATG

ORF without promoter? translational coupling

Terminator

mRNA

Regulatory region- clone onto multicopy vectors totitrate activators or repressors- clone into promoter-probe vectors

Clone ORFs into expression vectors

1-10 kb, whole genes, clone into phage and plasmid vectors
5-40 kb gene cluster, clone into SCP2* or cosmid vectors

Fig. 9.1 Features of Streptomyces genes in relation to issues discussed in this chapter.

Restriction-modification and other host factors

S. lividans 66 and, to a lesser extent, S. griseofuscus C581 (ATCC23916) are commonly usedhost strains that have no or weak restriction against heterologous DNA. S. coelicolor A3(2) and S.
avermitilis have strong methyl-specific restriction that prevents transformation or transfection with Dam(GAMeTC), Dcm (CMeCA/TGG), or Hsd (A AMeCN6GTGC and GCAMeCN6GTT)-methylated DNA fromwild-type E. coli K-12 (MacNeil, 1988). Several other streptomycetes probably also have methylspecific restriction systems. RP4-mediated conjugation from wild-type E. coli K-12 to S. coelicolor isinefficient unless a non-methylating mutant strain such as ET12567 (p. 230) is used as plasmid donor.
S. lividans 66 can be transformed quite efficiently with DNA from E. coli, but somewhat more efficiently
with non-methylated DNA (Orekhov et al., 1989). S. coelicolor can be transformed equally well with
its own DNA, DNA propagated in S. lividans, and DNA propagated in E. coli ET12567. PCR
amplification also generates unmethylated DNA suitable for transformation of S. coelicolor.
Many Streptomyces strains contain Type II restriction-modification systems (SalI, SacI, etc) that
restrict DNA from (non-methylated) heterologous sources. It is not known whether DNA from thesestrains is restricted by the methyl-specific system(s) of S. coelicolor.
Streptomyces phages are subject to classical restriction by endonucleases and also to other, lesswell understood, phage defence systems. They also often have mechanisms (generally not understood)
to combat restriction (p. 275). The pgl system of S. coelicolor prevents plaque formation, but not
lysogeny, by C31. Pgl& S. coelicolor strains arise spontaneously at c. 0.1% frequency. Lysogeny
prevents plaque formation by the same phages (superinfection immunity), but it is possible to make C31
double lysogens by sequential infection (pp. 290 and 304).
Plasmids with the same replication functions are incompatible, but note that e.g. SCP2*containing strains can be transformed efficiently with SCP2*-derived vectors. No pocks will develop
and the unselected plasmid will disappear. Maintaining incompatible plasmids in the same cells byselection results in cointegrate formation. Incompatible (same replicon) multicopy plasmids segregate
slowly when selection is relieved. There is, however, “strong incompatibility” that prevents co-existenceof Sti+ and Sti& derivatives of many multicopy plasmids; without selection, the Sti+ plasmid prevails

&KDSWHU*HQHUDOFRQVLGHUDWLRQVRQ6WUHSWRP\FHVFORQLQJ(Deng et al., 1988; p. 256). Plasmids such as SLP1 and pIJ408 that integrate into (different) tRNA genes
are often incompatible (Hopwood et al., 1984). Otherwise, the different plasmid replicons described in
Chapters 11 and 22 seem to be fully compatible.

Preparation of vector and target DNA
Methods for DNA purification are described in Chapter 8. DNA preparation is not usually done
under sterile conditions other than those imposed by the bactericidal effects of phenol and chloroform
extractions and alcohol precipitation. For cloning purposes, including PCR amplification, it is importantto guard against cross-contamination of genomic and vector DNA samples with plasmids that persist on
recycled pipettes, and centrifuge rotors. For the cloning of large DNA fragments, e.g. in cosmid vectors,
it is important that the chromosomal DNA is sheared as little as possible before digestion with restriction
endonucleases (routine DNA handling shears genomic DNA preparations down to about 50kb). The sizeof large DNA fragments can be checked using PFGE (p. 197).

How many clones are required for making a representative gene library?
The number of clones required for generating a representative library can be calculated
statistically (Seed et al., 1982). A useful guide is that the collection of independent clones should contain
about five genome equivalents of cloned DNA. For a typical Streptomyces genome of c. 8000 kb andan average insert size of 5 kb, this requires 8000 independent clones.
In addition to the single linear chromosome, many streptomycetes contain autonomously
replicating linear and CCC plasmids. Some of these plasmids occur at high copy numbers of up to 300
per chromosome, resulting in the over-representation of plasmid genes in gene libraries. Streptomycetes
sometimes also contain highly amplified chromosomal DNA sequences (pp. 20 and 427).

Choice and use of restriction endonucleases for making gene libraries
Partial or complete digestion using the six-cutters BamHI (G0GATCC), BglII(A0GATCT), BclI (T 0GATCA), KpnI (GGTAC0C), PstI (CTGCA0G), SacI (GAGCT0C), SphI
(GCATG0C), or XhoI (C0TCGAG) has been used frequently to clone specific genes. However, formaking representative gene libraries, partial digestion with MboI (0GATC) has been most commonly usedbecause the fragments can be cloned into BamHI, BclI and BglII sites or, after partial infill, into SalI and
XhoI sites (Zabarovsky and Allikmets, 1986; see scheme opposite). Other useful combinations that could
be explored are TaqI (T0CGA, cuts Streptomyces DNA less frequently than MboI) and MspI(=HpaII,C0CGG, cleaves Streptomyces DNA very frequently), both of which can be ligated to ClaI(AT0CGAT, some sites are protected by dam methylation in E. coli.), BstBI (TT0CGAA), and other sites.
Determining the correct enzyme concentration and digestion time for obtaining the desired DNA
fragment size range by partial digestion usually requires a pilot experiment. It is difficult to dispense the

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MboI
-- G t c G A T C --- C A G C T a g -SalI

very viscous high molecular DNA accurately, and the restriction endonucleases may diffuse only slowly
into the viscous samples. This makes it difficult to reproduce conditions precisely. We find it best to
digest c. 10 DNA samples with two-fold dilutions of the restriction endonuclease. Note that on ethidium
bromide-stained agarose gels the average size of DNA fragments is usually overestimated. Seed etal.(1982) showed that the centre of the highest fluorescence intensity corresponds to twice the average
size of randomly cut DNA fragments. Samples which contain DNA fragments of the desired sizes canthen be pooled for further processing by electrophoresis or sucrose gradient centrifugation. The poolingprocess increases the representativeness of the resulting library because the fragments in a particular size
range have different length biases when isolated from under-digested or over-digested samples. Sizefractionated DNA gives much better libraries than unfractionated partial digests.
Shearing of DNA by sonication is particularly useful for producing small, truly random
fragments for DNA sequencing, and is being used in the S. coelicolor genome project (p. 424).

Choice of cloning vector
The uses of plasmid and phage cloning vectors are described in Chapters 11 and 13 respectively.
Here we discuss only general considerations which are important when planning cloning experiments.
Vector host-range
The replication host-range of Streptomyces phages and plasmids is generally restricted to
streptomycetes (but pIJ101, for example, has been used in several other actinomycete genera); however,within the genus host range is not usually very narrow (but note that many strains carry integrated
plasmids, that prevent replication of SLP1 and related plasmids, p. 264). The host range for integration
is often wider: thus IS117, Tn4560 and pSAM2 can integrate into the Mycobacterium smegmatis
genome, and C31 lysogenises the rapamycin producing S. hygroscopicus, Pgl+ S. coelicolor and C31
lysogens, even though no phage plaques are formed on these strains. In cases where plasmid
incompatibility or other plasmid-plasmid interactions, phage-phage interactions or host restriction
mediated by restriction endonucleases prevent replication of vectors, permissive mutations cansometimes be isolated (e.g. Qin et al., 1993).

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Size of the target DNA
C31-derived vectors have upper and sometimes lower size limits for clonable fragments (thehighest capacity C31 vectors can usefully accommodate up to c. 8 kb: p. 293). Plasmid vectors haveno strict size limits for inserts, but in practice ligation does not readily generate circles that are muchlarger than four times the size of the vector alone. Although the size of circular DNA significantly
affects the efficiency of transformation of competent E. coli using CaCl2 treatment, transformation ofStreptomyces protoplasts appears to be little affected by plasmid size.
Large DNA fragments are cloned most efficiently into cosmid vectors. Cosmid vectors with twocos sites (e.g. Supercos 1) are particularly convenient for preparing equal amounts of the left and rightcosmid arms, and the kanamycin resistance gene present in SuperCos 1 makes it possible to select fortransformation of Streptomyces. Transformation depends on integrational rescue of the cosmid, mediatedby homologous recombination of the insert with the chromosome (Redenbach et al., 1996). There arebifunctional cosmid vectors which contain a Streptomyces replicon or integration function, permitting
the cosmids to be introduced into streptomycetes that do not have high homology to the insert. For all
cosmids, the combined size of vector and insert must be between 36 and c. 53 kb to allow packaging into
heads. This necessitates the use of starting DNA preparations with fragments at least 100 kb long.
Multicopy clones, especially when containing large inserts, are often structurally unstable,
producing deleted plasmids. Structural instability of pIJ101-derived vectors is greatly reduced in S.
lividans hosts (e.g. JT46, ZX7, ZX64: p. 425) containing the rec-46 mutation (Tsai and Chen, 1987;
Kieser et al., 1989). Use of pIJ699, in which terminators flank the cloning site, may also minimise somecauses of instability.
Integrating vectors
Integrating vectors, which can be lysogenic phages, integrating plasmids or transposons, provide
unit copy number and optimal segregational stability. Phage vectors that integrate via the natural attPand attB sites or, in the case of attP-deleted vectors, by homologous recombination between cloned
fragments and the host genome, can easily be recovered as phage particles because of low-level natural
phage release. Also, plasmids integrated into the host chromosome via a single insert-directed crossoverexcise spontaneously and can usually be recovered by transformation of E. coli provided they have asuitable replication function and a selectable marker. The longer the region of sequence identity, themore frequent is excision. IS117-derived vectors also form non-replicating circles spontaneously (hence
the name “mini-circle”). Other transposable elements, and C31-derived and pSAM2-derived attcontaining integrating vectors, do not excise spontaneously, at least in principle, though restriction
fragments containing selective markers can be re-cloned, and fragments that contain a selective marker
and an E. coli replicon can be circularised by ligation at low DNA concentration and used to transformE. coli. However, it has become apparent that such vectors often integrate as tandemly duplicatedmolecules, and when this happens free circular monomers can be generated spontaneously byhomologous recombination and recovered by transformation of E. coli.
Integrated vectors are well tolerated by the host whereas high copy number autonomously
replicating plasmid vectors have been reported to reduce antibiotic biosynthesis (e.g. Solenberg et al.,1996).

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Low copy number plasmid vectors
The only available Streptomyces low copy number vectors are derived from SCP2*. (Note that
SCP2*-derived vectors have copy numbers of 1-2, 10 or 1000, depending on the sequences from thereplication region that are present: Larson and Hershberger, 1986.) The low copy number SCP2*-derived
vectors are large and more difficult to isolate than multicopy plasmids or phages. Despite this they havea particularly good record for cloning genes by the complementation of mutants. For the DNA analysisof clones, instead of plasmid preparations it may be more efficient to use total DNA preparations andSouthern blots. Many, but not all, SCP2* vectors are conjugative, and this has permitted the very
convenient use of arrayed libraries for the complementation of many different mutants, by replication
to lawns of the mutants to permit the introduction of the library by mating (Davis and Chater, 1990, 1992;
Ryding et al., 1998). Transfer of conjugative plasmids works well between mutant derivatives of thesame species, except that plasmid transfer cannot be completely separated from chromosome
mobilisation. SCP2* clones containing large (>10 kb) inserts of homologous DNA mobilised nearby
parts of the S. coelicolor chromosome with up to 10% efficiency (p. 262). This can potentially give theappearance of complementation. For this reason, putative complementation obtained in primaryconjugation-based screening should be confirmed by transformation of the indicator strain with isolated
plasmid DNA. Conjugative plasmid transfer between different species occurs at low frequencies or not
at all, and the plasmid in the new species may be rearranged. SCP2* clones are also suitable formutagenesis using Tn4560 (pp. 114-115).
Low copy number plasmids must have a specific partition function to ensure their stable
inheritance, and several SCP2* vectors lack it (pp. 261-262). They can, however, be maintained using
selective pressure. Spontaneous plasmid loss is often useful for making sure that a phenotype is indeed
conferred by the plasmid and not by a secondary mutation.
High copy number plasmid vectors
High copy number plasmids are easy to isolate and analyse physically, but if a plasmid is
structurally unstable, it may not be the predominant DNA species observed on agarose gels that confers
the observed phenotype. High copy number vectors are particularly useful for cloning (but not studying)regulatory genes or sequences which enhance or decrease gene expression or antibiotic biosynthesis
when present in multiple copies. Regulatory genes have been discovered by cloning host or heterologous
DNA onto multicopy plasmids (e.g. Fernández-Moreno et al., 1992), and a high copy number of cisacting regulatory sequences has been found to titrate a host sigma factor, as in the production of aphenotype similar to WhiG deficiency (Tan and Chater, 1993).
Surprisingly, plasmids with a very high copy number are often segregationally unstable, eventhough this contradicts statistical expectations based on random plasmid partitioning.
Plasmid versus phage vectors
Plasmids are often preferred because they are conceptually simpler than phages, and because
there are no strict constraints on the sizes of the clonable fragments. Phage cloning does, however, haveimportant benefits. Phage plaques can be obtained overnight while plasmid transformants can take up
to a week to sporulate. Plaque-lifts for the detection of clones by hybridisation are easier than

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Streptomyces colony hybridisation. Phage clones can be moved between different strains by simple
replication of phage plaques, often after killing the host strain using chloroform vapour (take care that
exposure is not so long that the plastic of the Petri dish begins to dissolve!). Transferring plasmids toother strains by transformation is seldom so convenient. Transformation efficiencies vary greatly andin some strains protoplasting and regeneration is highly mutagenic, probably causing mainly deletionsof the chromosome end(s). Phage infection is not known to cause collateral mutations.
Phages have been avoided by many industrial laboratories for fear of phage infection of large
production cultures. It is doubtful whether this fear is justified; over many years we have notexperienced any problems with phage infections in our laboratory cultures.
Positive selection vectors
Some vectors provide a positive selection for the presence of a cloned insert. C31 requires atleast c. 35 kb between the cos sites for packaging. If the left and right arms of the phage add up to less,then a fragment must be cloned to give phage plaques (e.g. KC505, KC518; Table 13.2, p. 293). pIJ699
provides positive selection because of the “inviability” of plasmids with long ($ 50 bp) palindromes inmany situations in bacteria (Leach, 1994). pIJ699 is a derivative of pIJ101 that can be prepared in E.
coli, but the E. coli replicon is replaced on cloning. An SCP2* derivative, pIJ698, constructed on thesame principle, turned out not to exhibit significant palindrome inviability, but nevertheless has proved
to be the most convenient SCP2*-based vector for cloning by mutant complementation (e.g. Ryding etal., 1998). pIJ4642 is an E. coli positive selection vector derived from pACYC184. When using these
vectors it is important to avoid restriction endonuclease “star” activity (induced by low salt, organic
solvents such as glycerol and ethanol, and excess enzyme), which could remove the palindromic
sequence and disable positive selection for inserts.
For the construction of stable engineered strains (e.g. for large-scale fermentations), transposons(IS117, Tn4593) have also been suggested for use as (integrating) cloning vectors (Smokvina et al., 1994;
Motamedi et al., 1995; Baltz et al., 1997; Chung and Crose, 1990).
E. coli vectors containing Streptomyces selection markers
E. coli plasmids do not replicate autonomously in Streptomyces, but if they contain suitableselective markers they can be used as suicide vectors for gene disruption and replacement. This requires
recombination between the insert and a homologous sequence in the recipient genome, which is not very
efficient, so optimal transformation conditions are required. The denaturation procedure of Oh andChater (1997, p. 235) is often helpful here. A special case is that represented by ordered cosmid libraries(e.g. Redenbach et al., 1996). In particular, the S. coelicolor library has been much used for both mapbased and more random cloning (e.g. Aínsa et al., 1999; Kim, 1999). Transformants are selected byusing the kanamycin resistance conferred by the neo gene of the SuperCos 1 vector. It is necessary to
passage cosmids through E. coli ET12567 to obtain unmethylated DNA (at least one other nonmethylating host has been found less effective; J. Nodwell, personal communication) before transforming
S. coelicolor strains. The large inserts in cosmids considerably increase recombinational rescue in
Streptomyces, so the use of denaturing transformation conditions may not be necessary: indeed,transformation with pools of cosmids has been successful (Redenbach et al., 1996). Complementing
cosmids can often be recovered from complemented strains by direct retransformation of E. coli (usually

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by electroporation, to improve the transformation frequency for such large DNA molecules: Oh andChater, 1997).
RP4-mediated conjugation of oriT-containing plasmids from E. coli is used wherever possible
because it is usually more efficient than transformation (p. 249).
Using bifunctional vectors that replicate in E. coli and Streptomyces
Bifunctional vectors that replicate in E. coli and Streptomyces (p. 267) are very attractive because
it is still easier to manipulate DNA in E. coli than in streptomycetes. For making hybridisation probes
and PCR templates it may also be advantageous to avoid residual Streptomyces chromosomal DNA.
Probably the most useful bifunctional vectors are those containing oriT for RP4-mediatedmobilisation from E. coli to Streptomyces. Many Streptomyces strains that are difficult to transform are
amenable to inter-generic conjugation. Conjugative plasmid transfer is subject to host restriction, but
the high conjugation frequencies may permit plasmids to escape restriction occasionally. Methyl-specificrestriction can be avoided by using a non-methylating E. coli donor strain such as ET12567/pUZ8002(p. 249).
Bifunctional constructs are sometimes structurally unstable in Streptomyces. This has been aserious problem, particularly with pIJ101 derivatives. The reasons for the instability are unknown.
(Inserting transcriptional terminators between the Streptomyces and E. coli segments of the plasmid has
improved stability on at least one occasion, suggesting that readthrough from very active E. coli
promoters into the Streptomyces part may cause instability.) Bifunctional pIJ101 derivatives suffer fewer
deletions and appear stable in strains like S. lividans JT46 that contain the rec-46 mutation (p. 425).

Selective markers
Antibiotic resistance
Only a limited number of antibiotic resistance markers are currently used for selection in
Streptomyces (p. 241 and Chapter 21). Many Streptomyces strains are naturally resistant to several
antibiotics (e.g. S. coelicolor and S. lividans contain chloramphenicol and tetracycline resistance and S.
lividans has an inducible erythromycin resistance also; and -lactam antibiotics are generally ineffective
as selective agents). Therefore, it is important to test the resistance profile of new strains. Note thatsome antibiotics are much more expensive than others. The potency of some antibiotics depends
critically on the medium. In particular, bacteria are most sensitive to aminoglycosides (e.g. hygromycin,
kanamycin and neomycin) and viomycin on media containing low salt concentrations.
The antibiotic concentration required to inhibit large numbers of cells, and the concentration thatstill allows the development of a single resistant cell into a colony, should be determined by plating
different cell dilutions onto antibiotic plates. Valid, semi-quantitative results can be obtained by spotting
2 µl of each dilution onto agar media: up to c. 48 spots can be placed on a 9 cm plate. Take note of theinhibitory concentrations for each cell density, and the possible appearance of resistant clones whichsometimes arise with high frequency (maybe the strain contains an inducible antibiotic resistance thatcan easily become constitutive).

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Antibiotic resistance can arise from spontaneous mutation, and if transformation frequencies are
low, the proportion of mutants naturally increases. Having a second (selective or non-selective) marker
on the vector can be very useful for distinguishing rare transformants from spontaneous mutants. When
working with multiple resistance genes, note that there is sometimes cross-resistance, especially withaminoglycosides (see above). Note that certain spontaneous streptomycin-resistance mutations enhance
antibiotic production (Hosoya et al., 1998).
Counterselectable markers
Suicide plasmids, or temperature-sensitive plasmids, such as the pSG5-derived vectors, or attdeleted or C31-derived vectors that integrate via cloned DNA, are useful for obtaining integration via
a single crossover, but do not help elimination of the vector via a second crossover. The addition of acounterselective marker (glkA, rpsL: pp. 320-323) can facilitate the search for progeny that no longer
contain the vector (e.g. after a second crossover). If the use of these is considered, it is important to lookahead, because both selections depend on the employment of particular hosts (glkA-deleted or
streptomycin-resistant strA mutants respectively), which may have physiological abnormalities: glkA
mutants are resistant to glucose repression (Hodgson, 1982), and strA mutants may be partially depressed
for antibiotic production (Hosoya et al., 1998). This may impose additional steps on experiments. For
example, if a gene has been disrupted by a resistance gene with the help of one of thesecounterselections, the disruption may need to be transferred from the glkA or rpsL mutant backgroundinto a standard one (such as M145) by transformation with linear DNA, crossing or, if suitable systems
develop, by transduction (Chapters 7 and 10).

Ligation conditions
The following parameters are important for the ligation reaction: (1) the total concentration ofcompatible, ligatable DNA ends in the ligation mix; (2) the vector:insert ratio; (3) the nature of the DNA
ends, including the presence or absence of 50 phosphates; (4) the sizes of the DNA fragments; and (5) the
ligation buffer and ligase concentration.
Higher concentrations of ends favour inter-molecular over intra-molecular ligation: the speedof DNA circle formation is concentration-independent, but only at low DNA concentrations aremonomeric circles the main ligation product. The DNA concentration that gives 50% inter-molecular
ligations and 50% intra-molecular ligations is c. 20 µg ml–1 for 10 kb fragments and c. 10 µg ml–1 for 30
kb fragments.
For generating inserts in circular vectors, total DNA concentrations of 2-10 µg ml–1 are used.
(Note that DNA concentrations are often lower than those used for restriction digests. For phage cloning
experiments involving three fragments, c. 200 µg ml–1 is used.)
The vector:insert ratio should be 1:1 at the time of ligation. Small insert fragments that selfcircularise rapidly are often added in 2-5 fold molar excess. (Note that fragments #200 bp do not
circularise and therefore have a high chance of ligating to other fragments.)
Optimizing ligation conditions is difficult for vector and target DNAs that differ greatly in theirsizes (Bercovich et al., 1992; Graf, 1979; Ferretti and Sgaramella, 1981). Whenever possible ligate

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vector and insert molecules cleaved with two different restriction enzymes creating non-compatible ends,
or use partial infilling (p. 215).
Dephosphorylation is useful for preventing the recovery of self-ligated vector molecules in E.
coli and Streptomyces. Alternatively, dephosphorylation of the target DNA can be used to prevent
formation of multiple inserts. It is counterproductive to add the dephosphorylated vector fragments inexcess because useless linear molecules are generated with the target DNA ligated between two
dephosphorylated vector molecules.
Testing the success of ligation reactions by gel electrophoresis is recommended. It is important
to compare equal amounts of samples before and after ligation. Reduction of the intensity of bands
indicates ligation. For making circles it is best if several distinct new bands appear, which may represent
linear and circular products. If the main ligation products are large concatemers, the yield of clones will
be low and ligation should be done at a lower total DNA concentration.

Transformation
Protoplast transformation gives the highest frequencies for most streptomycetes; for others
electroporation may be better (p. 237). The transformation frequencies obtained with Streptomyces
protoplasts are proportional to the amount of DNA used over a wide range, indicating that all the
protoplasts are equally competent for DNA uptake. Excessive DNA concentrations can result in
cotransformation. If this happens, probably the smaller plasmid will become established and the larger
plasmid will be lost. The amount of DNA that can be used for introducing single plasmids into theprotoplasts depends on the transformation frequency and on the number of protoplasts used.
Phenol/chloroform extraction of the ligation mix probably increases the number of transformants,
but the solvents kill protoplasts and must be removed completely. (If the absolute transformation
frequency decreases with increasing DNA concentration, the DNA may contain traces of phenol,
chloroform or SDS.) High salt concentration (NaCl, sodium acetate, CsCl) in the DNA samples increases
rather than decreases the number of transformants.
After transformation, resistance genes must be expressed before antibiotic selection can beapplied. For this, the protoplasts must be spread on regeneration agar; incubation in P buffer to allow
expression is ineffective. It is useful to leave at least one plate without antibiotic overlay to observe
regeneration of the Streptomyces lawns, which should be confluent. Appearance of well separated
colonies indicates inefficient protoplast regeneration and probably low transformation frequencies.
The processes of protoplasting and regeneration seem to be highly mutagenic for some strains.
Phage infection or RP4-mediated mobilisation of oriT-containing plasmids may provide usefulalternatives in these cases, and they are often preferred even when transformation is unproblematic.
Competent E. coli cells give normal, high transformation frequencies with DNA from
Streptomyces. ColE1-type plasmids propagated in Dam+ E. coli strains give c. 100-fold reduced
transformation frequencies when introduced into Dam– E. coli because the first round of replication
produces hemi-methylated plasmids that start another round of replication inefficiently (Russell andZinder, 1987; Smith et al., 1985).

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Finding the desired clone
Complementation of recessive mutations (and its opposite, mutational cloning), hybridisation,
PCR screening, antibody tests and enzyme assays can be used to find the desired clone.
Complementation works best with complete genes, but homologous recombination between incomplete
clones and chromosomal sequences occurs readily and can restore gene function. This situation can berecognised by the emergence of a minority of individual colonies which have the mutant phenotype,while full trans complementation restores the wild-type phenotype in nearly all transformed cells (aminority of colonies may have the mutant phenotype because of gene conversion/homogenotisation inwhich both the chromosomal gene and the cloned gene become mutant through recombinational
interactions: wild-type +/+ homogenotes may also arise: see Kieser and Hopwood, 1991, and Chapter
14 for further explanation). The occurrence of homozygous -/- colonies can be exploited: for example,
if it occurs, it provides evidence that the cloned DNA corresponds to the locus being complemented, as
opposed to some kind of suppressor effect. In mutational cloning (Chapter 13) insert-directed insertionof the vector disrupts a gene or transcription unit, to create a mutant phenotype that can be recognised.
Although mutational cloning has been done mainly with C31-derived attP-deleted vectors, it can also
be done with E. coli plasmid vectors containing a marker selectable in Streptomyces. Examples of suchvectors include pDH5 and pSET151 (the latter being conjugally mobilisable from suitable E. coli hosts
into Streptomyces). The use of phage vectors for mutational cloning makes it particularly easy to recover
the clone through the occasional release of plaque-forming phages, and then to recreate the mutant
phenotype either for confirmation or in a different genetic background. Mutational cloning has beensuccessful with polycistronic transcription units, and is particularly appropriate for antibiotic production
gene clusters. However, its use for single genes is constrained by the low efficiency of insert-directed
integration mediated by small DNA fragments (c. 500 bp or less). We note in passing that it may also
be possible to use the ability to integrate by homologous recombination as a means of screening both forthe presence of DNA inserts and, more specifically, for inserts of DNA, such as a plasmid, that is present
in one strain but absent from a control strain (Chater and Bruton, 1983).
Colony hybridisation is possible with Streptomyces, but lysis of the colonies is more difficult,and the background activities are high compared to E. coli. A quite effective method has been widelyused (pp. 242-244). Although it works best with multicopy clones, it has also been used successfully on
single copy number DNA. Streptomyces plaque lifts are unproblematical (p. 303).
PCR screening can often be done on mycelium taken with a toothpick from colonies that release
traces of DNA. No lysozyme treatment is necessary to release the DNA; lysozyme inhibits Taq
polymerase and should be avoided. Many laboratories have devised their own variations on PCRscreening. An example was published by Soliveri and Chater (1999).
Antibody screens (Willey et al., 1991) and colour tests for enzyme activity are usually easily
performed for secreted proteins. However, for the detection of new or increased enzyme activities orantigenic proteins which are intracellular, one can consider a sib-selection for finding the desired clone(s)as is explained in the following example. The maximum number of transformants that can be used in
the first pools depends on the sensitivity of the assay.

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Sib-Selection
This is a hypothetical example of sib-selection; see Jones and Hopwood (1984) for a real example.

1.

Spread transformed protoplasts on 10 regeneration plates and overlay with antibiotic
to select for transformants. Assume that 1000 separate colonies grow on each plate. 10,000
transformants with inserts average >4 kb give a good representation of the Streptomyces genome.

2.

Make two or three replicas of each plate on selective medium and make sure that the
replica plates can later be lined up with the original plate. Making several replicas may
greatly accelerate the sib-selection (steps 6 and 7). If agar contamination must be completely
avoided for the assay, or if the strain produces extracellular agarase that softens the gel (e.g. S. coelicolor
except NF strains), the agar surface can be covered with a sterile cellophane membrane before replication(p. 48). Note that the presence of glucose in the medium causes significant repression of agarase.

3.

Store the original plates at 4E and incubate the replica plates as is best for the later
assay.

4.

Harvest the cells from each replica plate producing 10 pools. Gentle scraping of the dry
surface (do not add water) with a sharp, rounded scalpel blade is very effective for harvesting substrate
mycelium, aerial mycelium and spores. The assay may be possible using the scraped-off mycelium, or
further incubation in liquid culture may be necessary. Liquid cultures grow very well when inoculated
directly with scraped-off vegetative mycelium: spores are not necessary.

5.

Test the 10 pools as appropriate for enzyme activity, PCR product etc. The pool withthe highest activity probably contains one or several of the desired clones. Take thesecond replica of the corresponding transformation plate (from step 2) and subdivide
it into 10 numbered sectors, each containing c. 100 colonies. Carefully harvest the cells
from each sector as in step 4, and perform another test. Rectangular sectors are easier toharvest than radial segments.

6.

Depending on the sensitivity of the test, the most active sectors can be further
subdivided into numbered pools of c. 10 colonies using the third replica plate.

7.

At this stage, further sub-division of the replica plates may not be possible. Locate theregion of the desired activity on the original plate, and harvest spores from this area.
It may be most convenient to cut out the agar with a scalpel. The agar block can then be vortexed inwater with added glass balls to free the spores. Mycelial fragments and most agar are removed byfiltration through cotton wool (p. 46).

8.

Plate out spores on selective medium to produce 10 plates, each containing c. 100
colonies.

9.

Make three replicas as in step 2. Test each of the 10 plates for activity using replica 1.
All pools may give activity. Subdivide the pool with the highest activity into 10 pools
containing 10 colonies each. Test, and then find individual active clones. There should
be several active clones which are siblings. If the plasmids recovered from these are not identical,
structural instability should be suspected.

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Antibiotic biosynthetic genes
Antibiotic biosynthesis genes are of special interest in Streptomyces. The very fact that pathwayspecific genes for production, resistance and regulation are virtually always clustered makes it possible
to take a variety of approaches.
1.

2.
3.

4.

5.

6.

Complementation of antibiotic non-producing mutants. For this, low copy number plasmids are
preferred. If it is difficult to introduce DNA into the relevant mutant, it may be worth trying tocomplement a mutation of a related antibiotic biosynthesis cluster, hoping for the synthesis ofa “hybrid” compound which may be easy to detect if it is pigmented or has antibiotic activity.
Cloning of resistance genes which are often part of the biosynthetic cluster. (Note that
streptomycetes do not need or contain resistance genes against most antifungal antibiotics.)
Hybridisation using heterologous probing or PCR amplification using “guessomer” primers.
Polyketide synthases, peptide synthetases, and deoxysugar biosynthesis genes have been very
useful for heterologous probing (Malpartida et al., 1987; Stachelhaus and Marahiel, 1995;
Stockmann and Piepersberg, 1992).
Mutational cloning. The use of C31-derived vectors (p. 291) has the benefit that protoplasttransformation and regeneration do not have to be developed for the producing organism(provided that it is C31-sensitive).
Cloning of an entire gene cluster in a heterologous host that does not produce the antibiotic inquestion. This requires making libraries with very large ($ 30 kb) inserts. Nevertheless, successhas been reported for the puromycin cluster (Lacalle et al., 1992)
Cloning of specific activator genes which may give antibiotic overproduction when overexpressed. Most antibiotic biosynthesis gene clusters contain activator genes, though repressors
may also be found (Chater and Bibb, 1997). Such activator genes may also be active and easily
detectable in a surrogate host that normally makes a similar antibiotic. Thus S. lividans, which
in most conditions makes very low amounts of actinorhodin, can be stimulated to over-produce
this blue compound by cloned homologues of its pathway-specific activator gene actII-ORF4(but various pleiotropically-acting regulatory genes have also been cloned in this test system:
reviewed by Chater and Bibb, 1997).

Confirmation of clones
Does the phenotype depend on the cloned DNA?
Putative clones identified in a gene library by complementation need to be confirmed to rule out
possible reversion of the host mutation or correction of the genotype by extragenic suppression. It isreassuring when independently generated clones containing the same or similar overlapping DNA
fragments give the same phenotype, but this still leaves open the possibility of a suppressor effect.
Loss of the vector should restore the original phenotype of the host (unless homogenotisation has
generated a +/+ homozygote: see above). Most plasmid vectors, including multicopy plasmids, are lost
spontaneously at $1% after one or two rounds of non-selective growth. Derivatives of the naturally
temperature-sensitive pSG5 are routinely cured by propagation at 39E. Also curing of the pIJ101

&KDSWHU*HQHUDOFRQVLGHUDWLRQVRQ6WUHSWRP\FHVFORQLQJderivative pIJ303 is increased at higher growth temperature. Plasmids can often be eliminated byintroducing a differently selectable derivative of the same replicon, exploiting plasmid incompatibility,
Re-transformation is the most commonly used method for confirming plasmid clones. Phage
clones integrated into the host attB site or integrated by homologous recombination can be confirmed
easily by infecting fresh host cells using the rare phages that are spontaneously released from the lysogens.
IS117 clones can be rescued easily by transformation because CCC forms occur spontaneously
in S. lividans. Non-replicating plasmids integrated by homologous recombination into the Streptomyces
genome loop out of the chromosome by a reversal of the integration process and can be rescued bytransformation of E. coli. Note that the E. coli cells need to be highly competent for transformation if theplasmids are large (e.g. cosmid clones) or if the inserts are small and therefore undergoing very little
recombinational looping-out from the Streptomyces chromosome.
Unexpectedly, some means of rescue of clones based on integrating plasmid vectors using C31
or pSAM2 attP sites is often possible because tandem integration has proved to be the rule rather thanthe exception (see above). For simple single integration events, though, the clones may need to be
excised using restriction endonucleases and recircularised before transformation of E. coli.
Is the cloned DNA rearranged?
DNA inserts sometimes differ from the chromosomal segment they are supposed to represent.
Multiple, unrelated DNA fragments may have been joined together, or the original recombinant plasmidor phage clone may be structurally unstable because of over-expression of a deleterious gene, phage
packaging constraints, etc. Obtaining several identical independent clones suggests that no DNA
rearrangements have taken place. Southern hybridisation using the cloned DNA as a labelled probe canprove colinearity of the genomic sequence and the clone. Note that these tests may be more difficult tointerpret when partially digested or randomly sheared DNA was cloned.
Is the promoter for a cloned gene present on the cloned DNA?
Streptomyces promoters may sometimes be several hundred bp upstream of the start of the gene,and so are readily separated from their gene during cloning. The promoter is probably present when thesame phenotype is obtained with the cloned DNA inserted into the vector in both orientations. Promoter
activity from the vector into cloned genes, to give potential anti-sense transcripts, does not seem to affectgene expression in a dramatic way (thus anti-sense transcripts cannot be used as a reliable means toprevent gene expression). Vectors in which the cloning site is flanked by transcriptional terminators
usually require that a promoter is cloned together with the gene. Note that many Streptomyces promoter
regions are complex, and may include tandem sites for recognition by different sigma factors. Promoter
activity in DNA fragments can be detected using S1 protection assays (Chapter 16) or cloning into
specific promoter-probe plasmids (Chapter 15).

What if the desired gene cannot be cloned?
Possible reasons for failure to find a specific clone in a gene library include: (1) too few insertcontaining clones in the library; (2) the insert sizes may be too small; (3) the gene may be cleaved by the&KDSWHU*HQHUDOFRQVLGHUDWLRQVRQ6WUHSWRP\FHVFORQLQJ

restriction endonuclease used in complete digests; (4) the gene may not be tolerated on a particular vector;or (5) the mutation being used for complementation is dominant.
Problems (1) to (3) are dealt with by using partially digested, size-fractionated target DNA andensuring that the experiments are done on a reasonable scale (start with tens of µg of DNA!). Use of low
copy number phage or plasmid vectors minimises problem (4). Problem (5) can be dealt with in several
ways, which depend on the kind of phenotype being analysed. It may be appropriate to clone from DNAof the mutant (e.g. Guthrie et al., 1998). If a cosmid library is used, whether made with mutant or wildtype DNA, the frequency of homogenotisation may be high enough to generate homozygotes with twocopies of the recessive wild-type allele; alternatively, recovery of the wild-type and mutant allele isexpected to be approximately equal when cosmids are recovered in E. coli (Oh and Chater, 1997), so that
shuttling back into the wild-type and mutant Streptomyces with pooled recovered cosmids may also
facilitate clone identification.

Assessing the quality of Streptomyces gene libraries
The most reliable test for the quality of a gene library is probably the analysis of a sample ofplasmids to characterise the sizes of the inserts. For large, low copy number plasmid vectors, it may be
best to prepare total genomic DNA and analyse the sizes of the undigested plasmids by probing Southerntransfers with labelled vector as probe. Analysis of plasmids can be tedious for libraries containing many
vectors without inserts. Alternatively, one can add a small amount of a resistance gene fragment or acolour indicator such as the tyrosinase gene to the target DNA. The frequency with which these extra
fragments are recovered in clones will be proportional to the frequency with which target fragments ofsimilar sizes are represented in the library. Even simpler, and particularly useful when making
Streptomyces gene libraries for the first time, is to use a promoter-probe vector such as pIJ486, whichcontains the promoterless Tn5 neo gene. Constitutive promoters strong enough to activate the neo geneand give kanamycin/neomycin resistance are quite frequent in Streptomyces DNA. A high ratio of
kanamycin-resistant to kanamycin-sensitive signals a high frequency of insert-containing clones (inpractice, 30% resistance to 20 µg ml–1 kanamycin indicates that most plasmids have inserts of c. 5 kb;
smaller inserts will give fewer resistant clones).

Storing gene libraries
Phage libraries can be stored as phage stocks (p. 276) or as lysogens. Ligation mixtures seem todeteriorate when frozen, so storage at 4E, with a drop of chloroform to prevent fungal growth, ispreferable. Storing gene libraries as collections of identifiable, individual clones requires a lot of effort
and freezer space, and it is often inefficient to use such libraries. A good compromise is to store genelibraries as several independent pools of spores frozen in 20% glycerol, or as phages stored at 4E.
Note that Dam– E. coli strains have an increased spontaneous mutation rate and therefore should
not be used to store DNA that should remain functional (Barras and Marinus, 1989).

&KDSWHU*HQHUDOFRQVLGHUDWLRQVRQ6WUHSWRP\FHVFORQLQJ
Ainsa JA, Parry HD, Chater KF (1999) A response
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m10tkb.wpd 20 April 2000 Tobias Kieser

Chapter 10

Introduction of DNA into Streptomyces
CONTENTS

Methods available . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230
Restriction barriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230
Use of single-stranded DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230
Transformation and transfection in Streptomyces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
Polyethylene glycol (PEG) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
Transformation and transfection frequencies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232
PEG-assisted transformation of Streptomyces protoplasts with plasmid DNA . . . . . . . . . . . . 232
Standard procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232
Rapid small-scale procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234
Use of denatured DNA for protoplast transformation . . . . . . . . . . . . . . . . . . . . . . . . . 235
Spot-transformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236
Cosmid transformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236
Electroporation of mycelium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237
Recognition, selection and screening of Streptomyces transformants . . . . . . . . . . . . . . . . . . . . 239
Lethal zygosis reaction (pocks) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239
Fertility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239
Resistance markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239
Selection of antibiotic-resistant transformants by overlaying or flooding . . . . . . . . . 240
Soft agar overlays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240
Flooding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240
Detection of melanin-producing colonies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240
Screening for plasmid DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242
Colony hybridisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242
Using nitrocellulose filters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242
Using Whatman 541 paper . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243
Preparation of colony replicas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244
Hybridisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245
50 end labelling of oligonucleotide probes . . . . . . . . . . . . . . . . . . . . . . . 246
Using Whatman 540 paper . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247
Use of the Polymerase Chain Reaction (PCR) to identify transformants . . . . . . . . . . 248
Conjugation from E. coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249



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Methods available
There are several ways in which DNA can be introduced into streptomycetes. These include
transformation with plasmid, cosmid and chromosomal DNA, transfection with DNA containing a phage
replicon, phage transduction of chromosomal and plasmid DNA, conjugation between streptomycetes
and intergeneric conjugation from E. coli. Until recently, transformation and transfection haveexclusively involved the use of protoplasts, but recently electroporation has been developed to introduce
plasmid DNA into mycelial fragments of some species (Pigac and Schrempf, 1995; Tyurin et al., 1995;
Mazy-Servais et al., 1997), and could presumably be used for phage-based recombinant molecules also.
This procedure by-passes the need to develop protocols for protoplast formation and regeneration, but
may require specific conditions for different strains. Thus, its general utility has yet to be demonstrated.
Transduction has not found widespread application, reflecting in part the lack of a wide host-range
generalised transducing phage (but see p. 274). Intergeneric conjugation of mobilisable plasmids from
E. coli into streptomycetes (Gormley and Davies, 1991; Mazodier et al., 1989; Bierman et al., 1992) isincreasingly used, both for gene disruption/replacement and for genetic complementation, partly because
the required constructs can be made relatively easily in E. coli, and partly because of the simplicity of
the conjugation protocol.

Restriction barriers
Many streptomycetes possess restriction systems that can drastically reduce transformation
frequencies, depending on the origin of the DNA, e.g. S. coelicolor A3(2) has a potent methyl-specificrestriction system that can be circumvented by isolating DNA from a methylation-deficient E. coli host(e.g. the E. coli strain ET12567 dam13::Tn9 dcm6 hsdM hsdR recF143 zjj201::Tn10 galK2 galT22
ara14 lacY1 xyl5 leuB6 thi1 tonA31 rpsL136 hisG4 tsx78 mtli glnV44 F–; MacNeil et al., 1992), or by
first passaging it through S. lividans (MacNeil, 1988). (Note that PCR-generated DNA would also be
unmethylated, and has been used to transform highly restricting strains of corynebacteria; Ankri et al.,1996.) Alternative procedures which may be used to reduce the restriction barrier involve heat treatmentof protoplasts prior to transformation (Bailey and Winstanley, 1986; Engel, 1987), which presumably
serves to reduce the activity of restriction enzymes, and in vitro modification prior to transformation(Matsushima and Baltz, 1994). Another approach involves the isolation of restriction-deficient mutants(Matsushima et al., 1987a; Muchova et al., 1991; Qin et al., 1994).

Use of single-stranded DNA
Single-stranded (ss) DNA has been used to promote recombinational interactions of introduced
DNA with its chromosomal homologue, mainly to permit gene disruption and replacement (Hillemannet al., 1991). In the latter experiments, ss DNA was derived from a vector (pDH5, p. 570) with a f1
origin for ss DNA synthesis, but ss DNA derived by denaturation in vitro prior to transformation also
results in higher frequencies of incorporation (p. 235); this seems to reflect an enhanced ability to

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

recombine with the chromosome rather than reduced levels of restriction (Oh and Chater, 1997). The
high frequency of integration obtained by conjugation from E.coli may also reflect enhanced
recombinational properties, in addition to possible avoidance of host restriction by ss DNA arising during
plasmid transfer.

Transformation and transfection in Streptomyces
Although there have been reports of natural transformation and transfection of streptomycetes,
there is no such system generally applicable to most species. A system for competence induced by cold
shock and calcium treatment, which was crucial to the development of transformation and hence ofplasmid-mediated gene cloning in E. coli, has not been developed for Streptomyces. Instead, the initial
work by Okanishi et al. (1974) on the formation, regeneration and transfection of protoplasts led to thediscovery that protoplasts were transformed by plasmid DNA at very high frequency in the presence ofpolyethylene glycol (Bibb et al., 1978). This procedure is still widely used in transformation andtransfection experiments in Streptomyces, though minor modifications have been built into the original
procedures. Among other actinomycetes, PEG 3350 treatment of protoplasts is used to transform
Saccharopolyspora erythraea (M. Weber, personal communication), while PEG 1000 and CsCl treatment
has proved effective for transformation of mycelium of Nocardia lactamdurans (Kumar et al., 1994).
In Amycolatopsis mediterranei, direct transformation of mycelium with plasmid DNA is possible using
PEG and alkaline cations (Madon and Hütter, 1991). In Amycolatopsis methanolica, whole cells can betransformed at high frequency (106 transformants per µg) with both circular and linearised plasmid DNA,
provided that the pMEA300-encoded stf gene is present on the vector (Vrijbloed et al., 1995). Other
pertinent references are Acebal et al. (1986); Daza et al. (1990); Garcia-Dominguez et al. (1987); GayerHerkert et al. (1989); Hooley and Wellington (1985); Hussain and Ritchie (1991); Illing et al. (1989);
Kojic et al. (1991); Lampel and Strohl (1986); Lyutzkanova et al. (1993); MacNeil and Klapko (1987);
Matsushima et al. (1987b, 1989); Matsushima and Baltz (1985, 1988); Ogata et al. (1984); Schupp andDivers (1986).
Protoplast transformation has recently been applied to chromosomal DNA fragments in S.
coelicolor and, particularly after denaturation of the DNA, the frequencies are sufficient to permit theready movement of selectable DNA (such as antibiotic resistance-marked disrupted genes) between
different strains (Oh and Chater, 1997; p. 235).
Polyethylene glycol (PEG)
Batches of PEG from different sources are not necessarily equivalent, and there may even bebatchwise differences in ostensibly identical material from a single source. We use PEG 1000 supplied
by NBS Biologicals (catalogue number 14805-B, p. 419) at a final concentration of about 25% (w/v).
Very little protoplast fusion is detectable by recombination at this concentration (Hopwood and Wright,
1979). It thus seems likely that fusion is not a prerequisite for DNA uptake.

&KDSWHU,QWURGXFWLRQRI'1$LQWR6WUHSWRP\FHVTransformation and transfection frequencies
Plasmid monomers are taken up effectively by plasmid-free and also by plasmid-containing
protoplasts. Some 106-107 S. lividans or S. coelicolor transformants can be expected per microgram of
CCC DNA of plasmids up to at least 60 kb in size. Open circular and linearised plasmid molecules withcomplementary "sticky" ends transform with a 10-100 fold lesser efficiency (Bibb et al., 1980).
Linearised plasmids with complementary dephosphorylated ends transform with a reduced frequency,
while those with non-complementary or blunt ends give few or no transformants (T. Kieser, unpublishedresults). Integrative transformation with cosmid DNA isolated from the methylation-deficient host
ET12567 (p. 230) yields 102-104 transformants per µg when the DNA has been denatured (the frequency
is somewhat dependent on the recipient genotype; in our experience, J1501 gives 10-100 fold moretransformants than M145). Frequencies with undenatured cosmid DNA are about 100-fold lower.
Many authors have investigated the parameters influencing protoplast regeneration andtransformation frequency, and attempted to optimise the conditions for individual species (e.g. Thompson
et al., 1982; Okanishi et al., 1983; Baltz and Matsushima, 1981; Mirdamadi-Tehrani et al., 1986). Of
special importance are the growth phase of the mycelium at the time of protoplasting, the temperature
at which the mycelium and the regenerating protoplasts are incubated, the number of protoplasts used
per transformation, the dryness of the regeneration plates, and of course the composition of the medium.
Obviously, a reasonable regeneration frequency of protoplasts is necessary for good transformation, butconditions for maximum regeneration may not always exactly coincide with those for the besttransformation frequency.
It seems important to wash the protoplasts immediately before adding the DNA and then toproceed with the addition of the polyethylene glycol immediately after mixing of DNA and protoplasts,
probably because nucleases liberated from the protoplasts can destroy plasmid DNA (Baltz andMatsushima, 1981; Kieser, 1984). RNase, which is often added to plasmid preparations, reduces thefrequency of protoplast regeneration and transformation (Kieser, 1984). Generally, higher transformation
frequencies are obtained with ligation mixes that have been treated with phenol:chloroform and thenpreciptated using ethanol or isopropanol to remove any traces of phenol.
Parameters affecting transfection frequency are dealt with on p. 301.

PEG-assisted transformation of Streptomyces protoplasts with plasmid DNA
The "standard procedure" was developed by Thompson et al. (1982). The second procedure,
called "rapid small-scale procedure", incorporates all the important features of the "standard procedure"
but does not require any centrifugation steps after the addition of the PEG solution (T buffer). Bothprocedures give similar transformation frequencies.
Standard procedure (Bibb et al., 1978; Thompson et al., 1982)
Protoplasts: c. 4 × 109 (equivalent to about 2 ml of a YEME + 34% sucrose culture;p. 58) per transformation in a detergent-free plastic tube
DNA in up to 20 µl TE buffer

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

10 R2YE plates per transformation (with supplements added for auxotrophic markers)
dried in laminar flow cabinet for 2-4 h to c. 85% of their original weight
P buffer; T buffer or 25% PEG in P buffer
Pipettes (l ml, 10 ml); Gilson Pipetman (P20 or equivalent) and tips; spreaders;
plastic centrifuge tubes (10 ml)
30o incubator; bench-top centrifuge; phase-contrast microscope (c. 450 ×) andhaemocytometer

After step 10 of "Preparation of protoplasts" (p. 58).
1.

Count the protoplasts in a haemocytometer and distribute samples of c. 4 × 10 9
protoplasts (amount used for one transformation) into Sterilin tubes for
immediate use (for freezing and storage, see p. 58). Protoplast suspensions generally
need to be diluted 10-fold with P buffer for counting. O.D. measurements may be very
unreliable if the protoplasts are pigmented. Haemocytometer grid:

Depth of the chamber = 0.02 mm. Therefore protoplasts per ml =(number of protoplasts in the smallest size of square) × 2 × 107.

2.

Spin down one sample of protoplasts in a bench-top centrifuge (c. 3000 rpm,
700 × g) for 7 min at room temperature). This centrifugation also serves as a wash. If
the required volume of protoplast suspension is very small, dilute with P buffer to c. 5 ml.

3.

Resuspend the protoplasts in the drop (about 50 µl) of buffer left after pouring
off the supernatant, by tapping the tube (not by vortex mixing).

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

4.

Add DNA in up to 20 µl TE buffer. Larger volumes of DNA in TE buffer may lyse theprotoplasts.

5.

Immediately add 0.5 ml T buffer (which contains PEG) or 25% PEG made in P
buffer and mix by pipetting up and down once, using the same pipette. If DNA
is left incubating with the protoplasts it may be degraded.

6.

As soon as possible (no longer than 3 min after adding the PEG) add 5 ml P
buffer and spin in the bench-top centrifuge.

7.

Pour off supernatant, resuspend as in step 3 above, and dilute in l ml P buffer.

8.

Plate out 0.1 ml on each of 10 R2YE plates and incubate at 30o.

Rapid small-scale procedure
This procedure facilitates the introduction of larger numbers of DNA preparations into
Streptomyces protoplasts. The main labour is in preparing and labelling the R2YE plates; transformation
itself takes very little time.
Protoplast suspension (c. 1010 ml–1) freshly washed by centrifugation
DNA in up to 5 µl TE buffer
Dried and appropriately supplemented R2YE plates (usually 2 per transformation)
T buffer or 25% PEG in P buffer (best prepared fresh since prone to contamination)
Adjustable micropipettes (20 µl, 200 µl) with tips; spreaders (l ml pipettes can beused instead); Eppendorf or similar tubes (will not be used for centrifugation)
30o incubator; bench-top centrifuge

1.

Dispense 50 µl samples of protoplasts (c. 1010 ml–1) into as many tubes as there
are transformations. We usually spin the protoplasts down immediately before thetransformation experiment. This eliminates substances that may have leaked out of the
protoplasts during storage and the contents of protoplasts which have lysed spontaneously(which may include nucleases).

2.

Complete steps 2a-c for each transformation individually.

2a.

Add up to 5 µl DNA solution to protoplasts and mix immediately by tapping
tube.

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2b.



Add 200 µl T buffer or 25% PEG 1000 in P buffer and mix by pipetting up anddown four times (be careful not to contaminate the barrel of the pipette). 25%
PEG in P buffer is c. 50% as effective as T buffer.

2c.

Spread protoplast suspension (100-200 µl) on two dried R2YE plates. Use P
buffer to make dilutions if required. l ml pipettes can be used instead of spreaders. The
solution will spread to some extent by itself if the plates are left on a horizontal surface.

3.

Incubate plates at 30o. After 14-20 h, overlay for selection ( p. 240) or leave tosporulate. Score for resistant colonies or pocks after 3 d.

Use of denatured DNA for protoplast transformation
In studies done with S. coelicolor derivatives, the number of transformants obtained with nonreplicating plasmids that integrate by homologous recombination into the recipient chromosome is greatly
stimulated (102-103 fold) by simple denaturation of the CCC donor DNA (Oh and Chater, 1997). This
procedure is very effective for cosmids with large inserts, where the transformation frequency is
increased from 10-100 transformants µg–1 unmethylated DNA to c. 103-104 µg–1, as well as with smaller
plasmid constructs, facilitating cloning and subcloning experiments as well as gene replacement anddisruption. DNA denaturation also enhances, at least ten-fold, the low transformation frequency
observed with linear segments of chromosomal DNA (Oh and Chater, 1997). The procedure is notsuitable for plasmids that replicate or integrate site-specifically in Streptomyces. Either of the following
procedures can be used to denature DNA, although alkali treatment is generally preferred for its
simplicity.
Glycol/heat treatment (not suitable for cosmids)

1.

Thoroughly mix DNA (in a volume of 8 µl TE) with 5 µl plasmid
denaturing agent (10mM Tris-HCl pH7.5, 1mM EDTA, 50% glycerol,
50% ethylene glycol) in a microcentrifuge tube.

2.

Incubate at 90-100o for 5 min, then chill rapidly on ice.

Alkali treatment

1.

Thoroughly mix DNA (in a volume of 9 µl TE) with 2 µl 1M NaOH in a
microcentrifuge tube.

2.

Incubate at 37o for 10 min then chill rapidly on ice.

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

3.

Add 2 µl 1M HCl (use the same micropipette for both NaOH and HCl toensure addition of equal volumes and neutralisation).

The DNA samples are then used immediately to transform protoplasts using either of theprevious procedures.
Spot-transformation
The following procedure was developed by Muth et al. (1988) to allow multiple S. lividans
transformations to be carried out on a single agar regeneration plate, and is particularly useful whentransforming with a large number of different plasmid constructions.

1.

Prepare protoplasts in the usual manner.

2.

Spread 50 µl of protoplast solution containing 106-107 protoplasts on a pre-dried
9 cm R5 plate.

3.

Spot 5 µl samples of DNA in TE at marked positions on the agar (24 different
DNA samples can be applied readily to one plate).

4.

Add 10 µl of 25% PEG 1000 in T buffer to each spot.

5.

Dry the plate under a laminar air flow for 30 min and incubate at 30o for 16-20 h.

6.

Overlay the plate with antibiotic to select transformants (p. 240).

Cosmid transformation
The availability of an ordered cosmid library of the S. coelicolor genome (Redenbach et al.,1996) makes it possible to clone and map genes by direct cosmid transformation of mutants. The libraryconsists of 320 cosmids containing S. coelicolor M145 DNA cloned in the SuperCos-1 vector. The latter
carries the neo gene from transposon Tn5, which confers kanamycin resistance, not only on E. coli, butalso on Streptomyces. The vector lacks a replication origin for streptomycetes. Consequently,
introduction of the cosmids into S. coelicolor and selection for kanamycin resistance results in insertdirected recombination with the chromosome. The single crossover event that occurs results in the
duplication of the cloned segment in the chromosome. Assuming a complemented phenotype that canbe readily assessed on plates, it is then possible to isolate segregants in which the mutant allele has been
replaced by the wild-type gene present on the cosmid by screening for kanamycin-sensitivity ( p. 241).
Since S. coelicolor possesses a methyl-specific restriction system, it is necessary to isolate cosmid DNA
from the non-methylating E. coli host ET12567 (p. 230) prior to transformation of S. coelicolor
protoplasts. It is usual to use alkali-denatured cosmid DNA to increase transformation frequency

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(p. 235), though in some circumstances the lower frequency obtained without denaturation may be
preferred (for example, if a few well-separated colonies are more useful for screening than a large
number in close contact).
Approximately 1 µg of an individual cosmid DNA preparation, isolated from E. coli ET12567,
is used per transformation by the "standard procedure" (p. 232); when using pooled cosmid DNA(derived from a set of 20 cosmids), approximately 1 µg is again used. DNA samples are denatured usingalkali (p. 235). After plating out the treated protoplasts on R2YE plates, kanamycin-resistanttransformants are selected by overlaying or flooding with kanamycin sulphate (Km) after 16-20 h to givea final concentration in the plates of 200 µg ml–1 (i.e. 4 mg Km is added per plate). The transformants
are then transferred to minimal medium (MM) containing 20 µg ml–1 Km.
Once stable cosmid integrants have been obtained and the desired phenotype confirmed, it ispossible to recover the cosmid by carrying out a standard plasmid isolation procedure, transforming E.
coli and selecting for Km-resistance (50 µg ml–1 on L agar) (MacNeil et al., 1993). Since recovery
depends on a low level of excision of the non-replicating cosmid from the chromosome by homologous
recombination, the level of free cosmid DNA is low, and 500 ml cultures are recommended ( p. 176).

Electroporation of mycelium
Electroporation involves the application of a brief, high voltage pulse to a suspension of cells
and DNA, resulting in the formation of transient membrane pores and subsequent uptake of DNA. It has
been used to transform mycelium of several Streptomyces species (Pigac and Schrempf, 1995; Tyurin
et al., 1995; Mazy-Servais et al., 1997). Its main advantage is that it by-passes the need to develop
conditions for protoplast formation and regeneration. On the other hand, conditions for electroporation
may also be somewhat strain-specific. The procedure reported by Pigac and Schrempf (1995) apparently
works better on strains that fragment during growth. An alternative procedure using freshly germinatedspores (Tyurin et al., 1995) may thus prove more generally useful. Limited non-protoplasting lysozyme
treatment is used to weaken the cell wall and improve accessibility for DNA uptake. Electroporation
requires that the cell-DNA mixtures are in a low-conductivity (low salt) buffer.
The following procedure was used for S. rimosus by Pigac and Schrempf (1995).

1.

Grow mycelium in 100 ml CRM (g l–1: glucose, 10; sucrose, 103; MgCl2.6H20,
10.12; tryptic soy broth (either Difco Tryptic Soy Broth or Oxoid TSB: p. 412),
15; yeast extract, 5) at 30o for 24 h and harvest by centrifugation at 4o and 10,000
rpm.

2.

Resuspend the cells in 100 ml ice-cold 10% sucrose and recentrifuge; resuspend
in 50 ml of 15% ice-cold glycerol, and recentrifuge.

3.

Resuspend the mycelium in 10 ml 15% glycerol containing 100 µg ml–1
lysozyme, and incubate at 37o for 30 min; wash the mycelium twice with 10 ml
ice-cold 15% glycerol.

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4.

Resuspend the pellet in 1-5 ml of 30% PEG 1000, 10% glycerol, 6.5% sucrose.
Dispense 50 µl samples in Eppendorf tubes, freeze rapidly on dry ice/methanoland store at -80o.

5.

For electroporation, thaw a 50 µl sample at room temperature, place on ice, andadd 0.01-1 µg of plasmid DNA in 1-2 µl water and mix by pipetting; avoid
introducing air bubbles.

6.

Transfer the suspension to an ice-cold 2 mm-gapped electrocuvette (Bio-Rad
Laboratories, Richmond, California or similar). Tap the cuvette to ensure thatthe cells are at the bottom. Subject to a 2 kV electric pulse (10 kV cm–1) froma Gene Pulser (Bio-Rad), connected to a Pulse Controller (25 µF capacitor;
parallel resistance 400 Ohm).

7.

Immediately dilute pulsed mycelium with 0.75 ml ice-cold CRM (see step 1 forcomposition) and incubate with shaking for 3 h at 30o.

8.

Add 200 µl CRM and plate dilutions on TSB (p. 412) containing an appropriate
concentration of the required antibiotic (e.g. 30 µg ml–1 thiostrepton). For S.
rimosus, 105-106 transformants per µg of pIJ486 CCC plasmid DNA were obtained.
The following alternative procedure was devised by Tyurin et al. (1995) for electroporation of
freshly germinated mycelial fragments of several Streptomyces spp.

1.

Inoculate 109-1010 freshly harvested spores into 25-30 ml TSB containing 1%
glucose, 0.5 % beef extract, 25 mg ml–1 MgCl2 and incubate at 28o for 90-120
min until germ tubes appear. Assess germination microscopically.

2.

Harvest the cells by centrifugation, and wash twice with an equal volume of 0.10.2M sucrose.

3.

Resuspend the cells in 1.5-2 pellet volumes of 7mM HEPES pH7.4, 75mM
sucrose, 1mM MgCl2, and store on ice.

4.

For each electroporation, use 50 µl of cells and 10 µl DNA (in water) and pulse
as described above in step 6 of the preceding procedure.

5.

Dilute the treated cells immediately 10-fold with cold TSB, leave on ice for 10
min and incubate at 28o for 1.5 h.

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6.



Plate dilutions on selective agar plates and incubate at 28o.

Recognition, selection and screening of Streptomyces transformants
Lethal zygosis reaction (pocks)
SCP2*, SLP1.2, pIJ101 and many other naturally occurring, self-transmissible Streptomyces
plasmids produce pocks. (Many cloning vectors, such as pIJ702, pIJ486, pIJ916 and pIJ943, have lost
this function.) Pock formation reflects the ability of a plasmid to spread by conjugation into a lawn ofa plasmid-free recipient. A single pock-forming spore or transformed protoplast can be detected visually
by its ability to form a pock in a lawn of 109 plasmid-free spores or protoplasts on a single 9 cm agar
plate. Pocks are visible in confluent lawns only on some media (e.g. R2, R2YE but generally not onMM). Some strain/plasmid combinations give very obvious pocks; the recognition of pocks with othercombinations may require experience and frequent examination of the cultures under good illumination.
Microscopic observation has never been helpful for recognizing pocks. S. lividans pocks often give acolour reaction (red rings), visible from the reverse side, before aerial mycelium develops. Note that S.
lividans and its derivatives frequently show features which look similar to pocks but have nothing to dowith (known) plasmids. They can normally be distinguished from the real pocks by comparing a control
lawn which does not include any plasmid-containing cells. (For references and further information on
pocks, see pp. 48 and 153.)
Fertility
The ability of many plasmids to mobilise chromosomal markers can be used potentially to detect
plasmid-containing transformants. This method has not been used routinely since all known fertility
plasmids produce pocks.
Resistance markers
After transformation the cultures can be left to sporulate and then replicated to selective media.
This is generally done with pock-forming plasmids which give small, confluent patches of resistant
growth corresponding to the area of pocks. More usually, the regeneration plates are overlaid (orflooded) with antibiotic after allowing time for the expression of the resistance markers. The optimal
time for expression is somewhat variable, depending on the resistance mechanism, the strain, the plasmid
and the dryness of the regeneration plates. Flooding with antibiotic can be advantageous, especially for
non-pock forming (non-transmissible) plasmids because plasmid-containing cells can be overgrown by
the plasmid-free mycelium or unstable plasmids can be lost when there is no antibiotic selection.

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Selection of antibiotic-resistant transformants by overlaying or flooding
Soft Nutrient Agar (SNA; 2.5 ml plate–1) must be completely molten (90o water bath or
micro-wave oven) and then cooled to 45-50o (bottles crack when cooled too quickly). For antibiotic stock
solutions, see Table 10.1.
Soft agar overlays

1.

Mix Soft Nutrient Agar (45-50o) with antibiotic in a prewarmed 20 ml screw capbottle.

2.

Overlay each plate with 2.5 ml SNA containing the antibiotic and leave to set for
at least 10 min.

3.

Incubate at 30o; colonies should be visible after c. 2 d.

Flooding

1.

Add stock antibiotic solution to sterile water in a 20 ml screw cap bottle to give
an appropriate final concentration in the regeneration plate. Thiostrepton will forma milky solution on dilution with water. This does not interfere with its use as a selective agent.

2.

Add 1-2 ml of diluted antibiotic solution to each plate and swirl the plate
vigourously to cover the entire surface.

3.

Dry the plates in a laminar flow hood and incubate at 30o; colonies should be
visible after c. 2 d.

Detection of melanin-producing colonies
Melanin production, encoded by melC (carried, for example, by pIJ702; p. 536), can be used toscreen for transformants. Melanin results from oxidation of tyrosine by the enzyme tyrosinase.
Tyrosinase contains copper in the active site and needs O2 for the reaction. For optimal melanin
production by S. lividans derivatives, MM is supplemented with 10 ml tyrosine (suspension at 7.5 g l–1),
4 ml 30% Casamino acids, 1.5 ml Tiger Milk (p. 410) and 4 ml 50% glucose per 200 ml, to give MMT.
For soft agar overlays on R2YE plates it is sufficient to add 100 µl tyrosine suspension per ml SNA(tyrosine in R2YE is said to inhibit protoplast regeneration).

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protoplast regeneration plates
Antibiotic(concentration ofstock solution in mg
ml–1)

Final concentration in
25 ml agar plate in µg
ml–1
Overlay
Flooding

Amount of stock solution(µl / µg per plate)
Overlay

Flooding

Apramycin (50)50

25 / 1250

20 / 1000

Erythromycin(100) in 50%
ethanol

20

5 / 500

Gentamicinsulphate (50)100

5 / 2500

Hygromycin B(50) (light sensitive)

50 (S. coelicolor) 40
200 (S. lividans) 80

25 / 1250
100 / 5000

20 / 1000
40 / 2000

Selection is more effective in
media with low salt

Kanamycin sulphate
(50)

200

100 / 5000

80 / 4000

Selection is more effective in
media with low salt. Used forselection of Tn5 neo of
integrated SuperCos 1

Lincomycin
(100)

20

Neomycin sulphate(100)

60 (Tn5 neo)
10 (aphI)

Spectinomycin
(200)

400

50 / 10000

aadA gene

Streptomycin
(50)

30

15 / 750

aadA gene; probably< 100% recovery oftransformants

Thiostrepton(50) in DMSO

12

8

6 / 300

4 / 200

Viomycin sulphate
(50)

100

320

50 / 2500

160 / 8000

40

160

Lincomycin is better for
S. lividans (and perhaps
S. coelicolor) sinceerythromycin induces MLS
resistance5 / 50060
10

15 / 1500
2.5 / 250

Comments

Better than erythromycin for
S. lividans (and perhaps
S. coelicolor); does not induce
endogenous MLS resistance
15 / 1500
2.5 / 250

Selection is more effective in
media with low salt

Selection is more effective in
media with low salt

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Screening for plasmid DNA
Detection of transformants by screening random colonies for plasmid DNA may be feasible whena high proportion of the protoplasts have been transformed (this may well occur when excess DNA isused for transformation). (See "Plasmid isolation by alkaline lysis", p. 187.)
Colony hybridisation
Colony hybridisation is used to identify directly clones containing a particular DNA sequenceand is extremely useful if the cloned fragment fails to elicit a readily observed phenotype. This
procedure is a modification of that employed to screen E. coli colonies. Colonies are lysed in situ on thefilter and the DNA released is denatured with alkali and fixed to the filter by baking. The fixed DNA
can then be hybridised with the appropriate probe and the desired clones recognised. References:
Hanahan and Meselson (1980); Kuczek and Mordarski (1989); Schrempf (1982).
Using nitrocellulose filters
R2YE plates
TE buffer
1% SDS in 1M NaOH
1M Tris-HCl pH7.5
5M NaCl
Lysozyme solution (4 mg ml–1 in TE buffer)
90% ethanol
Whatman 3MM paper
Nitrocellulose filter (BAS5 Schleicher and Schüll or Millipore pore size 0.45 µm).

1.

If pre-cut filters of the appropriate size are not available, cut the nitrocellulose
to produce circles that just fit inside a Petri dish. Saturate the filters with water,
sandwich them between Whatman 3MM paper, wrap them in aluminium foil,
and autoclave for 30 min at 15 lb psi (10,546 kg m-2, non-liquid cycle). Store atroom temperature in a sealed plastic bag.

2.

Place a filter on a R2YE plate and press with a glass spreader on the surface ofthe filter to remove any air bubbles.

3.

Replica plate colonies (50-10,000) of a Streptomyces strain on to the platecontaining the nitrocellulose filter (press firmly).

4.

Incubate the plates 20-30 h at 30o (only young mycelium should be formed).

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

5.

Carefully remove the filter and mark it and the original plate at two asymmetricpositions, using a pencil for the filter.

6.

Transfer the filter (colony side up) on to three sheets of Whatman 3MM paperthat have been placed in a tray and soaked with the lysozyme solution; incubate
for 30-45 min to allow formation of protoplasts.

7.

Transfer the filter (colony side up) on to three sheets of Whatman 3MM paper
soaked with 1% SDS and 1M NaOH.; leave the filter for 20 min on the paper.

8.

Transfer the filter to a dry sheet of Whatman 3MM paper to remove excess
liquid.

9.

Place the filter (colony side up) on three sheets of Whatman 3MM paper soakedin neutralising solution (7 parts 1M Tris-HCl pH7.5 and 3 parts 5M NaCl) for
5 min; repeat the procedure three times.

10.

Place the filter briefly on three sheets of Whatman 3MM paper soaked with 90%
ethanol and dry it between two sheets of Whatman 3MM paper at room
temperature.

11.

Bake the filter in a vacuum oven at 80o for 1.5-2 h. If the filter is not to be used
immediately, store at room temperature in a plastic bag.

12.

Hybridisation as for Southern blots.

This, and the procedure using Whatman 540 paper, have also been used successfully to probe
streptomycete colonies.
Using Whatman 541 paper (N. Davis and D. Henderson, personal communication)
This procedure describes the preparation of Streptomyces colony replicas on paper filters readyfor DNA hybridisation. Stronger signals are obtained by hybridisation to these replicas than to those
prepared on much more expensive and fragile nitrocellulose filters. The method has been used for S.
coelicolor and S. lividans, and should be applicable to other Streptomyces strains. The procedure is acombination (with modifications) of the use of Whatman 541 paper (Gergen et al., 1979), and the method
described above for the preparation of nitrocellulose replicas of Streptomyces colonies. A similar
procedure has been published by Landers (1990), and is also described below.

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In general, oligonucleotide probes have been used to probe colony replicas on Whatman 541
paper, but nick-translated probes may also be used. The main advantage of using Whatman 541 paper,
rather than nitrocellulose or nylon membranes, is that it can be boiled during the denaturation step, giving
greater cell lysis and DNA denaturation, which result in more DNA being bound (Maas, 1983). The
filters are tough as well as cheap and spores are transferred more evenly by replication to Whatman 541
paper than to nitrocellulose. In addition, the procedure is quick, because baking of the filters andprehybridisation are not required. Indeed, adding dry filters directly to the hybridisation solution allows
full and even penetration of the probe to all parts of the filters, which is particularly important whenhybridising to many filters in a stack (as many as 24 filters have been probed successfully with anoligonucleotide in one Petri dish). Only enough hybridisation solution to wet every filter completely isneeded. The removal of unincorporated label from oligonucleotide probes is important in reducing
background, but Denhardt’s solution and SDS appear to give no improvement. For nick-translated
probes, the main reduction in background is achieved by removing unincorporated label, and Denhardt’s
solution and SDS are not required (Gergen et al., 1979). Use of sheared DNA should reduce non-specifichybridisation of nick-translated probes, whereas tRNA is more effective, though not always necessary,for oligonucleotide probes (Meinkoth and Wahl, 1984).
Preparation of colony replicas
Streptomyces colonies, cultures or spores
4 mg ml–1 lysozyme in TE (10mM Tris-HCl pH8, lmM EDTA)
0.5M NaOH, 1% SDS
0.5M Tris-HCl pH7.5
90% ethanol
Whatman 541 filter papers labelled with pencil and autoclaved
Crystallizing dish or large glass beaker

Proceed with either (i) or (ii).
1.

(i) Replicate sporulated Streptomyces cultures to Whatman 541 filters by placing
the filter on the culture plate, allowing the filter to become wet with moisturefrom the agar plate by carefully pressing down with a spreader. Lift off the filter
and transfer it to a fresh plate, "spore-side" downwards.
(ii) After transformation or plating out spores, incubate overnight to allow
regeneration and colony attachment, then overlay with sterile Whatman 541
paper soaked in water or antibiotic solution at the correct concentration.

2.

Incubate approximately 40 h. The colonies grow into the paper and are retained during
subsequent treatment.

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

3.

Peel off filters and retain plates at 4o.

4.

Soak filters together in 4 mg ml–1 lysozyme in TE at 37o for 10 min (use 5 ml ofsolution per filter, adding filters one at a time in a crystallizing dish). Do not overincubate.

5.

Immerse in boiling 0.5M NaOH, 1% SDS for 5 min. Use 20 ml per filter. Thisis hazardous because NaOH solutions tend to superheat: take great care and use
a boiling water bath rather than a microwave oven if possible.

6.

Immerse in 0.5M Tris-HCl pH7.5 for 5 min (room temperature). Use 20 ml per
filter.

7.

Repeat step 6 with fresh Tris-HCl.

8.

Immerse in 90% ethanol (c. 5 ml per filter) and then air dry on Whatman 3MM
paper. No baking is required.

Hybridisation
Whatman 541 replica filters
Labelled probe
Hybridisation solution: 6 × SSC, 0.5 g l–1 sodium pyrophosphate
Crystallizing dish or large glass beaker
Shaking water bath1.

Add the heat-denatured labelled probe to 1-2 ml hybridisation solution per filter
in a Petri dish or beaker. No prehybridisation is required.

2.

Add the dry filters one by one, allowing the solution to wet each one completely
before adding the next.

3.

Hybridise overnight at 70o.

4.

Wash twice for 30 min in hybridisation solution at 70o with agitation.

6.

Wash further in lower concentrations of SSC, depending on the stringency
required.

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7.

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Dry filters between sheets of Whatman 3MM paper before autoradiography.
Autoradiography time will vary depending on colony size, plasmid copy number and the specificactivity of the probe but often requires only a few hours without intensifying screens.

50 end labelling of oligonucleotide probes
T4 polynucleotide kinase
Oligonucleotide
10 × Kinase buffer (0.5M Tris-HCl pH7.6, 1mM EDTA, 0.1mM spermidine, 0.1mM
MgCl2, pH7.6)
100mM DTT (dithiothreitol)–32P ATP (5000 Ci mmol–1)
Whatman DE52 resin (washed in TE/1M NaCl then equilibrated in TE)
TE/0.2M NaCl
TE/0.5M NaCl
20 × SSC
5% sodium pyrophosphate, pH8
Pasteur pipette plugged with siliconised glass wool or a glass bead; Petri dish or
beaker with lid
Geiger counter; 37o water bath

1.

Label 10 ng oligonucleotide (approximately 2 pmol of a 17-mer) in a total
volume of 50 µl kinase buffer, 4mM DTT with 10 µCi -32P ATP (2 pmol) and
8-12 U T4 DNA kinase for 1 h at 37o. To label larger amounts of oligonucleotide, thevolume should be increased proportionately.

2.

Load the reaction onto a 0.5 ml bed volume column of Whatman DE52 resin ina Pasteur pipette. Collect fractions in microfuge tubes.

3.

Remove unincorporated label with 4-5 1 ml washes of TE/0.2M NaCl. The final
wash should register <50 cpm on the Geiger counter.

4.

Elute oligonucleotide with 4-5 0.5 ml washes of TE/0.5M NaCl and poolfractions containing oligonucleotide. The first and/or last washes may containvery little oligonucleotide. Use fractions registering >100 cpm. Over 50% of thelabel should be incorporated into the oligonucleotide fraction.

5.

Make up hybridisation solution (1-2 ml per filter) consisting of 6 × SSC, 0.05%
sodium pyrophosphate and labelled probe (1-2 ng ml–1) in a Petri dish or beaker.

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6.



The sodium concentration in the pooled probe fractions should be taken into
account when making up the hybridisation solution by reducing the amount of
20 × SSC used (1 × SSC = 0.15M Na+). Denhardt’s solution and SDS are not usedbecause they do not reduce background on Whatman 541 filters (Gergen et al., 1979). tRNA
(100 µg ml–1) and NP-40 (0.5%) may reduce non-specific hybridisation (Meinkoth and Wahl,
1984).

Using Whatman 540 paper
3 mg ml–1 lysozyme in 25mM Tris-HCl pH8, 25mM EDTA, 10% sucrose
10 µg ml–1 thiostrepton
0.5M NaOH
0.5M Tris-HCl pH7.5, 10mM EDTA
50mM Tris-HCl pH7.5, 1mM EDTA
Phenol:chloroform:isoamyl alcohol (50:49:1)
95% ethanol
Whatman 540 water-hardened filter paper
Petri dish1.

Discs of Whatman 540 water-hardened filter paper (82 mm diameter for standard
9 cm plates) are cut, marked asymmetrically by clipping three notches in theedge, and sterilised by autoclaving.

2.

To apply antibiotic selection (the example given is for thiostrepton) toregenerated protoplasts after the expression period, prepare a sterile 100 µg ml–1
solution of thiostrepton (about 1 ml is needed for each filter). Pour the required
volume (up to about 50 ml) into a sterile Petri dish. Using two pairs of sterile
forceps, wet filters thoroughly in the thiostrepton solution, then apply to thesurface of the plates containing the bacteria. With practice, the filters can be put
down without trapping bubbles. If a bubble is caught, it should be worked out
to the edge gently. The filters should be left in place until colonies (2-3 mmdiameter) can be seen when looking through the plate at a light, or until they canjust be seen on the surface of the paper. Mark the underside of the plate tocorrespond to the notches in the filter for later alignment. Remove the filter toa sheet of plastic wrap and mark for identification with a soft pencil. At this point,
filters can be wrapped in aluminium foil or cling film and stored at -20o for later hybridisation.

3.

Filters are prepared for hybridisation by a modification of the method of Tauband Thompson (1982). The following manipulations are done at room

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temperature in a glass or plastic dish with 3-5 ml of solution per filter. They
seem to work equally well with or without shaking. Between each step, filters
are blotted briefly on paper towels.
4.

Filters are first incubated for 5 min in lysozyme solution (3 mg ml–1 lysozyme in
25mM Tris-HCl pH8, 25mM Na EDTA, 10% sucrose, freshly made each day).

5.

Cells are lysed by incubation for 5 min in 0.5M NaOH. When large colonies are
lysed, the excess chromosomal DNA should be washed or wiped off with a tissue.

6.

The filters are neutralised by incubating for 5 min, first in 0.5M Tris-HCl pH7.5,
10mM EDTA and then in 50mM Tris-HCl pH7.5, 1mM Na EDTA.

7.

Protein and lipids are removed by extracting the filters with phenol:chloroform:
isoamyl alcohol (50:49:1) for 15 min with shaking. After pouring off the organic
phase, rinse the filters thoroughly under running water until all visible
phenol:chloroform is gone.

8.

Rinse the filters three times for 2 min each in 95% ethanol, then allow to air dry.
From this point on, the filters can be treated like nitrocellulose (steps 11 and 12) for baking,blocking and hybridisation.

Use of the Polymerase Chain Reaction (PCR) to identify transformants
Provided appropriate sequence information is available, PCR can be used to screen fortransformants, including those giving rise to gene disruption or replacement. Reproducible results havebeen obtained using protoplasts derived by scaling down the standard procedure for protoplast formation(p. 56). After washing with 10.5% sucrose and harvesting by centrifugation, the protoplast pellet isresuspended in water. Standard PCR is conducted using 5-10 µl of "lysed protoplast solution" in a total
volume of 100 µl.
It has also proved possible to apply PCR directly to DNA isolated from single colonies (S-H Oh,
personal communication). DNA is isolated from a single colony grown on agar medium using the
Dynabeads DNA DIRECTTM kit (p. 418). The DNA is eluted in a final volume of 30 µl, and up 5 µl canbe used directly in the PCR reaction without the Dynabeads adversely affecting the reaction. PCR
reactions contain 10mM Tris-HCl pH8.3, 50mM KCl, 1.5mM MgCl2, 0.001% gelatin, 0.2mM eachdNTP, 0.4µM of each primer, and 2.5 U of AmpliTaq Gold (Perkin Elmer, p. 418). AmpliTaq Goldpermits the use of hot start PCR, which may assist in preventing priming at non-homologous sites.
This DNA has also been used for sequencing. The PCR product is first purified using aTM
Wizard PCR Prep System and the DNA eluted in 50 µl TE buffer (10mM Tris-HCl pH8, 1mM EDTA)
or water. 25-50 ng of the PCR product is then subjected to cycle sequencing (p. 394).

&KDSWHU,QWURGXFWLRQRI'1$LQWR6WUHSWRP\FHVConjugation from E. coli
Intergeneric transfer of plasmids from E. coli to Streptomyces strains was demonstrated after
modifying the mobilisation system established by Simon et al. (1983) for Gram-negative bacteria. It
works well also for non-replicative vectors that integrate into the chromosome either site-specifically at
the C31 (e.g. Bierman et al., 1992) or pSAM2 (e.g. Smokvina et al., 1990) attachment sites, or via
insert-directed homologous recombination. The vectors contain oriT from the IncP-group plasmid RP4(also designated RP1/RK2), and require transfer functions to be supplied in trans by the E. coli donor
strain. The latter is generally either the methylation-proficient S17-1 (Simon et al., 1983), which carries
an integrated derivative of RP4, or the methylation-deficient ET12567 (MacNeil et al., 1992; see p. 230
for genotype) carrying the self-transmissible pUB307 (Bennett et al., 1977; Flett et al., 1997) or the nontransmissible pUZ8002 (M.S.B. Paget, J.W. Wilson and D.H. Figurski, personal communication).
There are several advantages to using conjugation from E. coli as a means of introducing DNA
into streptomycetes and other actinomycetes: (1) it is simple and does not rely on the development ofprocedures for protoplast formation and regeneration; (2) restriction barriers may be by-passed orseverely reduced by the transfer of single-stranded concatemers of plasmid DNA (Matsushima et al.,1994); (3) a variety of versatile oriT vectors are available that permit site-specific or insert-directed
chromosomal integration; (4) since these vectors replicate in E. coli, the production of required
constructs is considerably facilitated.
Several published protocols exist for conjugation of mobilisable plasmids and cosmids from E.
coli into streptomycetes (Mazodier et al., 1989; Smokvina et al., 1990; Bierman et al., 1992; Matsushima
et al., 1994; Wohlleben and Muth, 1993; Flett et al., 1997). Optimal conditions for different strains mayvary, and although conjugation with freshly germinated spores may prove convenient, mycelial fragments
may give higher numbers of recombinants (see Matsushima et al., 1994 for studies on cosmid
conjugation into Saccharopolyspora spinosa). The procedure given below is a modification (Flett et al.,1997) of the method of Mazodier et al. (1989) that reproducibly gives large numbers of exconjugants of
S. coelicolor using both insert-directed and site-specific recombination. It is generally used for
conjugation from the non-methylating E. coli donor ET12567 (pp. 230 and 232).
Chloramphenicol, 25 mg ml-1 in ethanol
Kanamycin, 25 mg ml-1 in water
Antibiotic to select for oriT plasmid
Nalidixic acid, 25 mg ml-1 in 0.15M NaOH
MS agar (p. 409); 2×YT broth; LB (Lennox broth)37E shaking incubator, 50E water bath, 30E incubator
Spreaders1.

Prepare competent cells of E. coli ET12567/pUZ8002 grown in the presence ofKm (25 µg ml–1) and Cm (25 µg ml–1) to maintain selection for pUZ8002 and thedam mutation, respectively. High competence may be required. The non-methylating
E. coli donor must be used because S. coelicolor A3(2) restricts methylated DNA. ET12567 is&KDSWHU,QWURGXFWLRQRI'1$LQWR6WUHSWRP\FHV

also tetracycline resistant. With pUB307, but not with pUZ8002, triparental matings are also
possible (ET12567/pUB307 × ET12567/oriT plasmid × Streptomyces).

2.

Transform competent cells with the oriT-containing vector, and select for theincoming plasmid only.

3.

Inoculate a colony into 10 ml LB containing Cm, Km and the antibiotic used to
select for the oriT-containing plasmid and grow overnight. CmS or KmS segregants
arise frequently among transformants, so set up more than one culture.

4.

Dilute the overnight culture 1:100 in fresh LB plus antibiotic selection and growat 37o to an OD600 of 0.4-0.6.

5.

Wash the cells twice with an equal volume of LB (to remove antibiotics thatmight inhibit Streptomyces) and resuspend in 0.1 volume of LB.

6.

While washing the E. coli cells, for each conjugation add approximately 108
Streptomyces spores to 500 µl 2 × YT broth, heat shock at 50 o for 10 min, thenallow to cool. If using mycelial fragments for conjugation, harvest from a 3-4
d old culture growing on MS agar (p. 409) using 2-3 ml of 20% glycerol. Vortex
thoroughly and use 0.5 ml of the mycelial fragments for each conjugation,
omitting the heat shock. The number of spores and the amount of mycelial fragments used
can be varied depending on the efficiency of the conjugation.

7.

Add 500 µl of E. coli cells to 0.5 ml heat-shocked spores or mycelial fragments.
Mix and spin briefly. Pour off most of the supernatant, then resuspend the pellet
in the residual liquid.

8.

Plate out on MS agar + 10mM MgCl2 and incubate at 30o for 16-20 h. Drying the
MS plate for 1 h in a laminar flow hood before plating greatly assists in absorbing the 1 ml ofsolution added in step 9.

9.

Overlay the plate with 1 ml water containing 0.5 mg nalidixic acid and theappropriate plasmid selection (e.g. 1 mg apramycin for those vectors conferring
apramycin resistance), using a spreader to distribute the antibiotic solution
evenly. Continue incubation at 30o.

10.

Pick off potential exconjugants to selective media containing nalidixic acid (25µg ml-1.

&KDSWHU,QWURGXFWLRQRI'1$LQWR6WUHSWRP\FHV
Acebal C, Rubio V, Marquez G (1986) A method totransform the -lactam antibiotic producer Streptomyces
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Bailey CR, Winstanley DJ (1986 ) Inhibition of restriction
in Streptomyces clavuligerus by heat treatment.
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Bennett PM, Grinsted J, Richmond MH (1977)
Transposition of TnA does not generate deletions.
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

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

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m11tkb.wpd 13 March 2000 Tobias Kieser



Chapter 11

Plasmids and their use for gene cloning
CONTENTS
General properties of Streptomyces plasmids and their use for gene cloning . . . . . . . . . . . . . 253
Wild-type plasmids that have been used extensively to construct cloning vectors . . . . . . . . . 256
pIJ101 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256
pJV1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258
pSG5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259
SCP2* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260
Higher copy number derivatives of SCP2* . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263
SCP2* vectors as delivery systems for gene disruptions . . . . . . . . . . . . . . . . . 264
SLP1 and pSAM2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265
Other integrating vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265
List of special purpose vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267
Bifunctional E. coli-Streptomyces plasmids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267
oriT (RK2) vectors for conjugation between E. coli and Streptomyces . . . . . . . . . . . . 267
cosmid vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267
Expression vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268
Vectors with promoterless reporter genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268
Positive selection vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268
Integrating vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268
Unstable and temperature-sensitive plasmids useful for gene replacement
and transposon delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268
Vectors without the tsr gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268
Vectors with resistance markers other than the common ones . . . . . . . . . . . . . . . . . . 268
Vectors with blue/white selection (lacZ ) in E. coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269

General properties of Streptomyces plasmids and their use for gene cloning
Chapter 9 explained general considerations for choosing between different types of phage andplasmid vectors. Here, we describe some of the properties of individual plasmid replicons that have beenused to construct series of vectors. Many plasmid vector maps are in Chapter 22.
With the sole exception of the extremely wide host-range plasmid RSF1010, no plasmid from
Gram-negative bacteria, the low G+C Gram-positive bacteria, or even the high G+C corynebacteria andmycobacteria has been reported to replicate in Streptomyces. This made it necessary to construct cloning

&KDSWHU3ODVPLGVDQGWKHLUXVHIRUJHQHFORQLQJ

stiessential

ori

korB

pIJ101

clt 9 kd
6598 NotI

ORF56

kilB

8830 bp

korA

15 kd

25 kd

ORF66

kilA

spdB
31 kd

tra

spdA
66 kd

10 kd

Fig. 11.1 Restriction and functional map of pIJ101 (Kendall and Cohen, 1988; Accession number
M211778). The shaded arcs indicate the essential replication region and the sequence present in pIJ702,
respectively. ori, ds origin for anticlockwise rolling circle replication (RCR); rep, replication protein;sti, locus conferring “strong incompatibility”; tra, transfer protein, essential for plasmid transfer, c.m.a.
and pock formation; korA, repressor for tra and itself; clt, cis-acting transfer functions, probably oriT;
kilA, promoter and amino-terminal region of tra, causing lethality in the absence of repression by korA;
spdAB, “spread” genes; kilB, lethal gene when expressed in absence of korB; korB, regulator for kilB;
ORF56, unknown function. Dotted arcs with arrowheads indicate regulatory interactions. There are nosites for AflII, AseI, ClaI, DraI, EcoRI, EcoRV, HindIII, HpaI, NdeI, NsiI, ScaI, SphI, SspI, StuI, XbaI;and several sites for AatII, ApaI, NaeI, NarI, NcoI, PvuI, SalI, SmaI. Note that many of the cloning
vectors derived from pIJ101 lack the unique BamHI site in rep.
vectors using Streptomyces replicons, the host-range of which is restricted to streptomycetes and someof their closest relatives. Moreover, because Streptomyces plasmids, including those used for

&KDSWHU3ODVPLGVDQGWKHLUXVHIRUJHQHFORQLQJconstructing cloning vectors, lack antibiotic or heavy metal resistance genes (and other recognisedfunctions except transfer genes), selectable markers had invariably to be added to the plasmids to makethem into usable vectors.
The different Streptomyces replicons that have been developed into cloning vectors differ in theirsises, mode of replication, copy number, and reported host-ranges. pSG5 and pJV1 probably replicate
in the widest selection of actinomycetes. Integrating plasmids may have an even wider host range: oneof them, pSAM2, has been found to integrate site-specifically into the Mycobacterium smegmatis
genome.
Nearly all Streptomyces plasmids - circular, linear or integrating - contain transfer functions thatallow conjugative plasmid transfer and provide different levels of chromosome mobilising ability (c.m.a:
pp. 126-127) The Streptomyces transfer functions are surprisingly simple, consisting of a single transfer
gene and its repressor as the only essential functions. This is consistent with the finding that
streptomycetes do not produce sex pili (or any other pili), which require complex genetic determinationin Gram-negative bacteria. Inactivation of the repressor results in over-expression of the plasmid transfer
gene, and this in turn causes cell death. For this reason, the repressor genes are called kill override (kor)genes, and the transfer genes may be referred to as kil (kill) genes.
Most conjugative Streptomyces plasmids transfer very efficiently between strains. A single
plasmid-containing spore, mycelial fragment or protoplast in a dense lawn of plasmid-free cells (c. 107
cells evenly spread on the surface of a 9 cm Petri dish containing R2YE agar) may transfer the plasmid
to all the surrounding growth within a radius of 0.5-2 mm. The area of plasmid spread is often
macroscopically visible as a zone of retarded development of aerial mycelium which is called a "pock"(p. 153). Spreading is controlled by so-called spread genes (spd) the inactivation of which reduces thepock size (small pock mutants), but plasmid transfer in balanced crosses remains 100%, and c.m.aremains unchanged.
Some of the smaller plasmids, including pIJ101, pJV1, pSG5, pSN22 and pSAM2, multiply byrolling circle replication (RCR; reviewed by Khan, 1997). Presumably, the plasmid-encoded replication
protein (Rep) nicks one plasmid strand at the double-strand origin (ds ori), binds to the exposed 50 end andsynthesises new DNA using the exposed 30 end as a primer. The old strand peels off as it is replaced by
the newly synthesised DNA. When the entire plasmid has been copied, the pealed-off strand is recircularised by the Rep protein which is then released from the DNA in an inactive form. The Rep protein
is rate-limiting for replication, and inhibition of protein synthesis does not result in plasmid amplification
as is known for the ColE1 and p15A replicons in E. coli (Frenkel and Bremer, 1986). The single-stranded
circle is then made double-stranded by second-strand DNA synthesis starting at the single-strand origin(ss ori), which may be outside the minimal replication region consisting of the ds ori and rep. Rolling
circle replication produces plasmid monomers even when starting with a dimeric plasmid (ServinGonzález, 1993). This is different from the rolling circle replication of phage , which produces longconcatemers suitable for packaging. Plasmid preparations of SCP2*, which presumably replicates in thetheta ( ) mode, contain a higher proportion of CCC dimers than preparations of RCR plasmids like pIJ101.
Many of the currently used plasmid vectors lack the ss ori. They can still replicate, presumably
because of the presence of low-activity, secondary single-strand origins, or because second-strand
synthesis may also be primed by (fragments of) plasmid-encoded mRNA read in the appropriate
direction. These plasmids do, however, accumulate substantial amounts of strand-specific, singlestranded, circular plasmid DNA (see pp.194-195 for the appearance of ss plasmid DNA on agarose gels).

&KDSWHU3ODVPLGVDQGWKHLUXVHIRUJHQHFORQLQJ

The lack of the ss ori and/or the accumulation of ss plasmid DNA has been suspected of causing plasmid
instability (Pigac et al., 1988). This is, however, not always the case because pIJ680, for example, isstructurally and segregationally stable in S. lividans. Removal or inversion of the ss ori of pIJ101changes
the “strong incompatibility” (Sti) phenotype of pIJ101 (see below).

Wild-type plasmids that have been used extensively to construct cloning vectors
pIJ101, pJV1, pSG5 and SCP2* seem to be compatible for replication in the same cell. SLP1
and pSAM2 could possibly show incompatibility with each other but are probably compatible with all
the other vector replicons.
pIJ101
pIJ101 (Fig. 11.1) from S. lividans ISP5434 occurs at c. 300 copies per chromosome or 30% of
the total DNA of the strain. pIJ101 makes large pocks in S. lividans and S. coelicolor, and is a good
fertility factor. All the currently used pIJ101-derived cloning vectors are non-transmissible because theylack tra. Most of them also lack sti (strong incompatibility) and therefore accumulate single-stranded
plasmid DNA. Sti– plasmids have a lower copy number of c. 50 per chromosome. They cannot co-exist
with Sti+ plasmids. Very low transformation frequencies are observed with Sti– plasmids and host strainsthat contain a Sti+ plasmid; the few resulting transformants lack the Sti+ plasmid and probably result fromthe transformation of protoplasts that have spontaneously lost the Sti+ plasmid. When a Sti+ plasmid is
introduced by transformation or conjugation into a strain containing a resident Sti– plasmid, the Sti–
plasmid is invariably lost (Deng et al., 1988). Different Sti– pIJ101 derivatives can be introduced into
the same cells by transformation and, consistent with their high copy number, they co-exist for some time
without forming cointegrates. The same applies to two differently marked Sti+ plasmids.
If sti is inverted, the plasmid becomes phenotypically Sti– (single-stranded DNA accumulation,compatible with Sti– plasmids), but when introduced into a strain containing a Sti+ plasmid, stablecointegrates form in which the orientation of the two ds ori sequences is inverted. These cointegrates
may have an advantage over the original Sti+ plasmid because they have two ds origins and thus a betterchance in the competition for the rate-limiting Rep protein (Deng et al., 1988).
pIJ101 has two kil/kor functions. Inactivation of korA, e.g. by insertion into the unique BglII site,
results in over-expression of tra and cell death. Deletion of part of korA, together with the aminoterminal part of tra, produces viable plasmids. korB and kilB interact in a similar way (Stein et al., 1989;
Stein and Cohen, 1990).
The region between sti and rep may contain a function that is necessary for plasmid stability.
Deletion of this region has produced very useful vectors for gene replacement (pHZ1351 and pHZ1358).
Still the most popular general purpose pIJ101-derived cloning vectors are pIJ702 and thepromoter-probe vector pIJ487. Note that pIJ487, for example, is lost spontaneously from c. 1% of
S. lividans spores after one round of non-selective growth while pIJ860, for example, is very difficult tocure. Crameri et al. (1986) used ethidium bromide to cure pIJ101 derivatives, and Wrigley-Jones et al.
(1983) found that pIJ101-derivative pIJ303 is lost rapidly during fermentation at 37E. pMT660 is atemperature-sensitive mutant derivative of pIJ702 (Birch and Cullum, 1985). Many derivatives of pIJ101

&KDSWHU3ODVPLGVDQGWKHLUXVHIRUJHQHFORQLQJ


EcoRI
MscI
BspHI
XhoI
Bsu361
Bpu1102I
ApalI
SnaBI
PflMI

A

rep
traR
ori

rep

1
18
146
155
164
197
238
290
473

SacII

592

MluI

612

NarI
Eco57I
SfiI
AscI
NruI
KpnI
BclI
AlwNI
BbsI

933
970
1070
1087
1095
1319
1421
1434
1467

PstI

1527

SacII

1799

FspI

1982

PmlI

2233

XmnI

2414

pSG5

9.45 BglII

12.2 kb

traB
ds
ori

PstI

2442

BspEI
PvuI
SphI

2483
2604
2704

SrfI

2884

AvrII

3151

PvuII

3276

Fig. 11.2 Restriction and functional map of pSG5 (Maas et al., 1998; Accession number X80774). rep,
the A-Fragment required for low level transfer; ds ori, origin of rolling circle replication (the ss ori is
outside the minimal replication region); traB, main transfer gene (two spread genes are located between
traB and traR); traR, repressor of traB. The region present in vector plasmids is enlarged (the filled
triangles indicate an inverted repeat).
containing E. coli replication functions are structurally unstable. Tsai and Chen (1987) isolated
S. lividans JT46 which maintains these plasmids much more stably as a result of reduced intra-plasmid
recombination.

&KDSWHU3ODVPLGVDQGWKHLUXVHIRUJHQHFORQLQJ

Replication
Stability

SCP2*
31.4 kb

Transfer

DeletedinpSCP103

Homology
to SCP1

Fig. 11.3 Restriction and functional map of SCP2*. 9 kb duplications have been observed after
conjugative transfer from S. coelicolor to S. parvulus; an inverted repeat structure has been observed inthis region (Pel’ts et al., 1983), as well as an ORF encoding a transposase-like protein (T. Kieser and
R. Melton, unpublished). Integration into the linear plasmid SCP1 resulted in deletion of c. 4 kb
between the arrows labelled SCP1 (Kinashi, 1989).
pJV1
pJV1 (EMBL Accession number U23762) is a 11143 bp multicopy plasmid from S.
phaeochromogenes (a producer of SphI). The replication region is very similar to that of pIJ101, but thetransfer region is much more similar to that of pSN22, a multicopy plasmid of S. nigrifaciens (Kataoka
et al., 1994; Servin-González et al., 1995). In contrast, pIJ101 and pSN22 show little similarity. pJV1
seems to have a different, probably wider, host-range than pIJ101, which includes Saccharopolyspora
erythraea (Vara et al., 1989). Several cloning vectors have been developed from pJV1, including thepB series of plasmids and the cosmid vector pFD666.

&KDSWHU3ODVPLGVDQGWKHLUXVHIRUJHQHFORQLQJ



1
26
103
240

BclI
KpnI
BclI

FspI

239

KpnI
FspI

345
355

SphI

375

Psp1046I

644

SacII

708

SacI
MluI
BclI
Eco57I

1
79
159

ORF 1
BsiWI

897

BstXI

1131

ORF 2

ApaI

956

PvuII

978

ApaLI

1334

BclI

1449

Eco57I 1667
SrfI

1529

SphI

1690

1713
BssHII
1835
Esp3I
Eam1105I 1860

tra A
Mr=46494

SphI

2056

PmlI
BspEI
XmnI
ScaI
AgeI

2132
2158
2324
2326
2388

BbsI
PvuI
SacII

2547
2578
2632

DraIII

2871

EarI

2973

Tth111I
PstI

3184
3262

ORF 3

ORF 4

PflMI

1921

ApaLI

2466

BspHI 2651

ORF 5
(Kor homologue)

SacII

2821

KpnI
PvuI
ApaI

2968
3004
3079

SfiI

3115

ApaI

3153

SnaBI

3189

PvuI

3229

EcoNI 3268
BclI

3556

rep SCP2*
Fig. 11.4 The tra and rep regions of SCP2* from Accession number X72857
and C.L. Hershberger, personal communication, respectively.
pSG5
pSG5 (Fig. 11.2) is a naturally temperature-sensitive 12208 bp plasmid from S. ghanaensis 5/1B
(DSM 2932) (Muth, et al., 1998, 1989, 1995). It replicates by a RCR mechanism and has a wide hostrange in Streptomyces. pSG5 occurs at a copy number of 20-50. Replication stops above 34E, resulting
in gradual plasmid loss. pSG5 derivatives are fairly stable in chemostat cultures under nitrogen
limitation, range in Streptomyces. pSG5 occurs at a copy number of 20-50. Replication stops above 34E,
resulting in gradual plasmid loss. pSG5 derivatives are fairly stable in chemostat cultures under nitrogen
limitation, but less stable under carbon limitation (Roth et al., 1994). pSG5 transfers at a relatively low
frequency of 10–1 between S. lividans strains and does not form pocks, probably because its spread genes
are not transcribed. Inactivation of TraR, the repressor of the main transfer gene, reduces the transfer

&KDSWHU3ODVPLGVDQGWKHLUXVHIRUJHQHFORQLQJ

pSCP10324.5 kb
neo vph p15A ter

tsr hyg
pIJ698

pIJ698tsr

pBR327

pIJ903

pIJ903

vph
pIJ920pIJ920tsr
pIJ922

pIJ922tsr hygpIJ940pIJ940tsr hygpIJ941

pIJ941hyg attM

vph
pIJ4231

pIJ4231

tsr pIJ2923 ter
pKU306

pKU306PL

tsr
pVE1043

pVE1043
Transfer

Replication

Stability

Fig. 11.5 SCP2* vectors derived from pSCP103 carrying stability and transfer functions.
frequency 10-fold and retards sporulation of the S. lividans host, but it does not result in cell deathbecause traB does not have a kill function.
The cloning vectors derived from pSG5 feature only the rep gene and the ds ori. Surprisingly,
some of these small plasmids are still self-transmissible at a low frequency of 10 -4 between plasmid-free
S. lividans strains. The presumably non-coding DNA (A-fragment) downstream of rep is necessary forthis low-level plasmid transfer (Wohlleben et al., 1994; Maas, et al., 1998).
pSG5 vectors have proved extremely useful for transposon delivery (pp. 119-122) and for making
gene disruptions and gene replacements (p. 318). When containing homologous DNA fragments, pSG5
vectors integrate efficiently into the host chromosome and remain integrated as long as the nonpermissive growth temperature and antibiotic selection for the plasmid are maintained. Integration often
occurs in tandem. There is, however, selective pressure for the excision of the integrated plasmids (bya second crossover) and this is very useful for making gene replacements. If the selection allows, > 50%of the antibiotic resistant transformants contain gene replacements rather than single crossover
integrations. Using analogous E. coli suicide vectors, only 1-3% are gene replacements and 97% aresingle crossover integrations (Wohlleben and Muth, 1993).

SCP2*
SCP2 (Fig. 11.3) is a 31kb plasmid of S. coelicolor A3(2) (Schrempf et al., 1975; Bibb et al.,1977). A high-fertility variant, SCP2*, was identified by Bibb et al. (1977) and studied by Bibb andHopwood (1981) and Lydiate et al. (1985). SCP2* has now been almost completely sequenced (J.
Altenbuchner, personal communication). It is not known to carry any functions other than those

&KDSWHU3ODVPLGVDQGWKHLUXVHIRUJHQHFORQLQJpSCP10324.5 kbtsr

mel

pIJ943

aphI
pIJ80

hph cos
pJAR4

pIJ943

tra

pIJ80

stab

pJAR4

stab

pKC505stab

pMT603stabaac(3)IV cos
pKC505mel tsrpMT603tsr pBR327
pRM5 tra stabpRM5
Transfer

Replication

Stability

tsr pIJ2923 ter
pIJ943

pIJ943Fig. 11.6 SCP2* vectors derived from pSCP103: all lack either the stability or transfer functions or both
(symbols in right hand column refer to the deleted functions).
associated with being a plasmid and at least one insertion sequence (IS118) which also occurs in the S.
coelicolor chromosome. Restriction maps of the replication origin and of part of the transfer region are
in Fig. 11.4. The regions of SCP2* present in different vectors, all of which are derived from pSCP103(Bibb et al., 1980), are summarised in Fig. 11.5-11.7.
SCP2* is of prime importance because it is the progenitor of many very low copy number, stable,
vectors. The predecessor of this manual gave details of pIJ916 (tsr), pIJ922 (tsr), pIJ940 (tsr, hyg),
pIJ941 (tsr, hyg) and pIJ943 (tsr, mel), which are all rather basic vectors. Among them, pIJ916 andpIJ943 are transfer-defective. pIJ922 has found the most use as a vector (e.g. Malpartida and Hopwood,
1984; Davis and Chater, 1990). However, a more recent derivative of SCP2*, pIJ698 (Kieser and Melton,
1988), has shown great utility. This bifunctional plasmid can be propagated in E. coli (including the nonmethylating strain ET12567 (p. 230) if the DNA is to be used to transform S. coelicolor). Its E. coli
replicon is located between mirror-image arrangements of restriction sites (BamHI, HindIII and XbaI),and is prepared for ligation by cleavage with one of these enzymes, followed by agarose gelelectrophoresis (0.9% LMP agarose, TAE buffer) to separate the larger, Streptomyces-specific part fromthe E. coli replicon. The isolated fragment is then ligated with suitably digested target DNA (often
Sau3AI partial digests fractionated by sucrose gradient centrifugation to enrich for fragments in the 5-20
kb range). If Sau3AI-generated inserts are ligated between the BamHI sites of pIJ698, they can be cut
out intact by XbaI or HindIII for subcloning. Although SCP2* vectors are rather large (usually 20-25 kb),
they transform S. coelicolor efficiently (and the frequency is not greatly reduced by the presence of aresident normal SCP2* plasmid).
The construction of such libraries in transmissible SCP2*-derived vectors allows a very effective
strategy for complementation of mutants: colonies resulting from transformation with a ligation mixture
are picked to master-plate arrays (which can be kept at 4° for several months) and then replicated (p. 152)
to the recipient indicator strain (this has been particularly successful with morphological mutants: Davisand Chater, 1990; Ryding et al., 1998, 1999). By using an appropriately marked primary host (thehistidine and uracil auxotroph J1501 has often been used), it is straightforward to select exconjugants

&KDSWHU3ODVPLGVDQGWKHLUXVHIRUJHQHFORQLQJ

pSCP10324.5 kb

pHJL197

1

pBR322

aphI* (neomycin) tsr

pHJL21010pBR322

aphI* (neomycin) tsr

pKC53110

cos4

aac(3)IV (apramycin) cos

pKC575

10

pLambdaneo cos

pHJL40110

pUC19

tsr lacZ

pKC121810pUC19aac(3)IV (apramycin) lacZ oriT

pHJL302

1000

pUC19

lacZ tsr

Replication

Fig. 11.7 SCP2* vectors derived from pSCP103 lacking both stability and replication
factors. The columns to the right of the plasmid name include (from left to right) the
approximate copy number, the E. coli replicon, and markers present in the plasmid.
(e.g. by omitting histidine and uracil from the selective medium and incorporating thiostrepton tocounterselect the indicator strain). The appearance of complementation can be misleading, because
SCP2* derivatives containing parts of the chromosome can mobilise nearby chromosomal DNA withquite high efficiency (Hopwood et al., 1985; p. 217). For this reason, cases of complementation should
be confirmed by isolation of the plasmid from a subculture originating from the primary master plate andits use to transform the indicator strain (it is safer to avoid isolating plasmid from the complemented
indicator strain, because it may have picked up the mutant allele of the gene of interest byrecombination).
Conjugative SCP2* derivatives readily form cointegrates by homologous recombination with coresident pIJ101 derivatives. Only short ($100 bp) stretches of sequence identity are required. The
cointegrates have a high copy number, similar to that of the pIJ101 derivative, and they seem structurally
stable. Resolution into separate plasmids is very rare (Xiao et al., 1994).
More recently, the SCP2* derivatives (and other plasmids) constructed by Bierman et al. (1992)
have been used with great success. They contain an E.coli replication origin and oriT from RP4 forconjugation from E. coli (p. 249), and cos sites for in vitro packaging into phage heads.
The bifunctional vector pRM5 was designed and is used specifically for the cloning andexpression of polyketide synthase gene clusters, including the synthesis of a wide range of “unnatural
natural products” by combinatorial biosynthesis (McDaniel et al., 1995, 1999).

&KDSWHU3ODVPLGVDQGWKHLUXVHIRUJHQHFORQLQJ



aphI
Replication

attPTransfer∆pCAO200

pCAO106km

21.1 kb∆ SLP1.2
and pIJ61

bla

tsr

ori
p15A

pCAO170
pCAO200
pIJ61

Fig. 11.8 Restriction and functional map of SLP1 derivatives. pCAO106 contains thecomplete SLP1 sequence cloned into pACYC177. pCAO106 and pCAO170 integrate
into the S. lividans chromosome (via crossing-over at attP), are transfer proficient andform pocks. pCAO200 integrates but is transfer-deficient (no pocks). SLP1.2 andpIJ61 are non-integrating, self-transmissible, pock-forming CCC plasmids (Omer et al.,
1988; Thompson et al., 1982) aphI, bla, km, tsr, resistance to neomycin, -lactams,
kanamycin or thiostrepton.
Higher copy number derivatives of SCP2*When just the region around the replication origin is used as the basis of vectors, the copy
number may be increased to about 10 (as in pHJL401, Fig. 11.7 a vector that has proved useful for
subcloning) or many hundreds (as in pHJL302, which has not been used very much, and is very unstable).

&KDSWHU3ODVPLGVDQGWKHLUXVHIRUJHQHFORQLQJ

+

rep

spdD
spdC
spdB
spdA

xis
int

tra

pSAM2
10.92 kb

pra

attP

+

ss
ori ds
ori

ORF
131

kor

Fig. 11.9 Physical and functional map of pSAM2 (from Sezonov et al., 1995). kor, pra
and ORF 131 are regulatory genes; rep, xis and int form an operon involved in plasmid
replication, and integration into the host chromosome via crossing-over at attP; the traand spd genes are involved in plasmid transfer and spreading (pock formation).
SCP2* vectors as delivery systems for gene disruptions
The delivery of disrupted genes or simple mutations to the chromosome has been done effectively
with SCP2* derivatives (p. 319), because exchange between homologous sequences on the chromosome
and a plasmid is quite frequent (given homology of more than c. 1 kb on either side of the disruption or
mutation). The SCP2* derivatives without the stability region (Figs 11.6 and 11.7) are lost from $ 50%of the culture after one round of sporulation. More stable SCP2* plasmids which are lost with afrequency $1% after one round of sporulation can be displaced by a differently marked unstable SCP2*
derivative.

&KDSWHU3ODVPLGVDQGWKHLUXVHIRUJHQHFORQLQJSLP1 and pSAM2
SLP1 and pSAM2 are examples of Streptomyces plasmids that normally reside integrated intoa specific, highly conserved, chromosomal tRNA sequence. Eight such plasmids were reviewed by
Kieser and Hopwood (1991). These plasmids contain as their attachment sequence a precise c. 40 bp
copy of the 30 end of the respective tRNA gene. Therefore integration, mediated by a plasmid-encoded
int (integrase) gene, does not destroy the essential tRNA gene of the host.
SLP1, a 17.2 kb plasmid from S. coelicolor, was discovered because it readily transfers by
conjugation to S. lividans (p. 153; Bibb et al., 1981). In 10% of the S. lividans transconjugants SLP1 reintegrates into the appropriate tRNATyr gene. The remaining 90% suffer deletions, including the attP site,
and therefore cannot integrate into the chromosome. They form autonomously replicating, conjugative,
pock-forming CCC plasmids with a copy number of 4-10 per chromosome. The SLP1 derivative pIJ61
and bifunctional plasmids derived from pCAO106 (Fig. 11.8) have been used extensively for cloning in
S. lividans. They are, however, not suitable for S. coelicolor because the integrated SLP1 prevents
autonomous replication. Rare S. coelicolor transformants contain tandem integrations of the introduced
vector and the wild-type SLP1.
pSAM2 (11.2 kb) was discovered as a CCC plasmid in S. ambofaciens JI3212, which also
contains pSAM2 integrated into a tRNAPro gene (Pernodet et al., 1984; Simonet et al., 1987; Boccard etal., 1989a,b). The restriction and functional map of pSAM2 is shown in Fig. 11.9. Note that the ds origin
for rolling circle replication and the rep gene are at opposite sides of the circle (Hagège et al., 1993,
1994). The attP, int, xis, and rep operon (Sezonov et al., 1998) has been used for the construction of
pSAM2 integrating vectors which have a wide host-range including Mycobacterium smegmatis(Smokvina et al., 1990; Kuhstoss et al., 1989, 1991; Martin et al., 1991). pSAM2 integration has beenachieved in E. coli by expressing the pSAM2 integration functions (Raynal et al., 1998). pSAM2 has also
been observed to integrate into non-tRNA sites (Seoane et al., 1997).
Other integrating vectors
Integrating plasmid vectors carrying the integration functions of C31 (Bierman et al., 1992;
p. 300) transform much more efficiently than the integrating vectors derived from pSAM2. Anotherphage-derived integrating vector integrates (at relatively low efficiency) into a tRNA Pro gene (Van
Mellaert et al., 1998). Also transposons derived from Tn4556 and IS493 have been used for integrating
genes into specific sites in the chromosome. E. coli plasmids such as pDH5 and pSET151with added
homologous sequences from the host can, of course, also be used for (reversible) integration as is
described under gene disruption in Chapter 14.
IS117 (2527 bp) exists in two copies (Fig. 11.10) integrated at different sites in the S. coelicolor
chromosome (Lydiate et al., 1986, 1989; Henderson et al., 1989). The CCC form, the so-called “minicircle”, is a non-replicating transposition intermediate and occurs at one copy per 10-50 chromosomes.
Cloning of the CCC IS117 in pBR327 and addition of selectable markers produced integrating vectors
which insert into a preferred attachment site (attB) in S. lividans. About 103-105 transformants are
obtained per µg of CCC DNA. Cotransformation of two molecules seems to be necessary to givetransformants (Smokvina et al., 1994b). Insertion into attB is normally in tandem. Transformation ofan S. lividans strain lacking attB results in a c. 10-fold reduced frequency, and insertion into a few,



&KDSWHU3ODVPLGVDQGWKHLUXVHIRUJHQHFORQLQJ

BglI

SacII

ApaI*

HindIII

MluI

ORF1

ORF2

Transposase

ORF3

IS117 2527 bp

hyg
ori

attB
IS117

SCP2*

Stability
SCP2*

pIJ423128.3 kb

Transfer
SCP2*

vph

Fig. 11.10 Physical and functional map of IS117 and pIJ4231 (Henderson et al., 1990), which carries
the S. lividans IS117 preferred attachment site, attB. The SCP2* functions are explained in Fig. 11.3.
hyg, vph, resistance to hygromycin or viomycin.

discrete secondary IS117 attachment sites (Smokvina and Hopwood, 1993). The host-range of IS117 is
probably very wide because it can also be used in M. smegmatis (Bhatt and Kieser, 1999). Several
vectors have been constructed using IS117 (Henderson et al., 1989; Smokvina et al., 1994; Motamedi et

&KDSWHU3ODVPLGVDQGWKHLUXVHIRUJHQHFORQLQJ

al., 1995). They have the advantage over pSAM2 and C31int vectors (but see p. 216) that they can berecovered easily by transformation of E. coli because of the low-abundance free form that always exists
in Streptomyces (but not in M. smegmatis).
The S. lividans IS117 attB site has been cloned in the SCP2* plasmid pIJ4231(Henderson et al.,
1990; Fig. 11.10). The plasmid attB site is preferred over the chromosomal site and single integrations
rather than tandems are observed. IS117 also transposes readily from the plasmid to the chromosome andfrom the chromosome to the plasmid. It is therefore possible to clone DNA first into a small IS117 vectorin E. coli and then transfer the clones to pIJ4231 which has a low copy number in Streptomyces and isconjugative and pock-forming. If necessary, the pIJ4231 derivative can be cured and Streptomyces
containing a chromosomally integrated clone can be isolated. These clones, in contrast to the situation
after direct transformation, contain single inserts of IS117 in the chromosome. Surprisingly, a reduced
level of thiostrepton resistance has been observed with such IS117 derivatives (Smokvina et al., 1994).

List of special purpose vectors (Boldface indicates location of a restriction map.)
Bifunctional E. coli-Streptomyces plasmids
Integrating vectors: pIJ5971 p. 498; pIJ8600 p. 389, 531; pIJ8630 p. 531; pIJ8660 p. 531; pKTO2 p. 390, 532; pMT3226
p. 390, 532; pOJ436 p. 530; pOJ444 p. 530; pPM927 p. 390, 563; pSET152 p. 530; pHM11A Motamedi et al.
(1995); pIJ4201 p. 573; pIJ4210 p. 573; pIJ4696 p. 574; pIJ4698 p. 574; pIJ4700 p. 574.
pIJ101 derivatives: pANT1200 p. 538; pANT1201 p. 538; pANT1202 p. 538; pHZ1351 p. 542; pHZ1358 p. 542; pHZ1271
p. 540; pHZ1272 p. 540; pIJ699 p. 216, 218, 541; pSKO2 p. 543; pUWL series p. 544; pWHM3 p. 319, 544.
pSG5 derivatives: pCZA185 p. 551; pGM160 p. 318, 320, 550; pKC1139 p. 551; pSIT151 p. 119, 120; pUCS30 p. 119, 552.
Other multicopy vectors: pRES18/19 p. 545; pFD666 p. 546.
SCP2* derivatives: pCJR29 p. 558; pHJL302 p. 553; pHJL401 p. 262, 350, 553; pIJ4680 p. 319, 559; pIJ698 p. 218, 260,
557; pIJ903 p. 260, 554; pJAR4 p. 261, 560; pKC1064 Kuhstoss and Rao (1991); pKC1218 p. 268, 561; pOJ446
p. 562; pRM5 p. 261, 557.
SLP1 derivatives: pCAO106 p. 263; pCAO170 p. 263.
Plasmids containing transposable elements: pHM11A (Motamedi et al., 1995); pIJ4201 p. 573; pIJ4210 p. 573; pIJ4696
p. 574; pIJ4698 p. 574; pIJ4700 p. 574; pCZA163 p. 577; pCZA258 p. 577; pCZA276 p. 577; pJOE2577 p. 121,
581.

oriT (RK2) vectors for conjugation between E. coli and Streptomyces
Integrating vectors: pIJ5971 p. 498; pIJ8600 p. 389, 531; pIJ8630 p. 531; pIJ8660 p. 531; pMT3226 p. 390, 532; pOJ436
p. 530; pOJ444 p. 530; pSET152 p. 530
Autonomously replicating plasmids: pHZ1358 p. 542; pCZA185 p. 551; pHZ132 p. 550; pKC1139 p. 551; pSIT151 p. 119,
120; pUCS30 p. 119, 552; pKC1064 Kuhstoss and Rao (1991); pKC1218 p. 268, 561; pOJ446 p. 562; pPM927
p. 390, 563.
E. coli plasmids without Streptomyces replicon or integration function: pKC1250 p. 566; p. ; pHM10A p. 390, Motamedi
et al. (1995); pIJ8668 p. 348, 571; pIJ8671 p. 567; pIJ6650 p. 566; pKC1132 p. 566; pKC1138 p. 566; pOJ260 p.
566; pPM2208 p. 317, 571; pSET151 p. 570
Vectors containing transposable elements: pCZA258 p. 577; pCZA276 p. 577.

cosmid vectors
pOJ436 p. 530; pHZ1358 p. 542; p. ; pFD666 p. 546; pJAR4 p. 261, 560; pKC505 p. 261, 560; pOJ446 p. 562;
pSuperCos 1 p. 216, 218, 236, 571; pVE616 p. 571.



&KDSWHU3ODVPLGVDQGWKHLUXVHIRUJHQHFORQLQJ

Expression vectors
pIJ8600 p. 389, 531; pMT3226 p. 390, 532; pANT1200 p. 538; pANT1201 p. 538; pANT1202 p. 538; pANT849 p. 389, 537;
pANT850 p. 389, 537; pANT851 p. 389, 537; pHZ1271 p. 540; pHZ1272 p. 540; pIJ4090 p. 389, 535; pIJ4123 p. 539;
pIJ459 p. 534; pIJ6021 p. 389, 539; pMT3206 p. 389, 535; pCZA185 p. 551; pCJR29 p. 558; pKC1064 Kuhstoss and Rao(1991); pRM5 p. 261, 557; pPM927 p. 390, 563; pCJR24 p. 567

Vectors with promoterless reporter genes (see also Chapter15, p. 339)
aacC1:
EGFP :galK:
lac:
lux AB:
mdh:
melC:
neo:
redD:
xylE:

pGM11 p. 548
pIJ8630 p. 531; pIJ8660 p. 531; pIJ8668 p. 348, 571
pSKO2 p. 543
pDS5000 p. 350
pRS1105 p. 346, 543; pIJ5971 p. 498 Tn5351 p. 580; Tn5353 p. 580
pTMA1 p. 348, 561
pMT3010 p. 342, 536, pMT3020 p. 342
pIJ486, pIJ487 p. 256, 342, 533; KC310, KC850 p. 342
pIJ2527, pIJ2585 p. 350
KC858 p. 293, 297; KC859 p. 297, 523; KC860 p. 293, 297,308, 527; KC861 p. 293, 528; KC862
p. 293, 528; pMT3226 p. 390, 532; pIJ4083 p. 341; pXE4 p. 341

Positive selection vectors
KC401 (pp 290-294), KC505 (pp. 292-294, 526) and KC518 (pp.292-294, 526) are phage replacement vectors where the arms
are smaller than the minimal packaging size. pIJ699 p. 216, 218, 541; pJOE875 p. 541; pIJ698 p. 218, 260, 557; pIJ4680 p.
319, 559; pIJ666 p. 565; pIJ4642 p. 218, 565.

Integrating vectors
pIJ8600 p. 389, 531; pIJ8630 p. 531; pIJ8660 p. 531; pKTO2 p. 390, 532; pMT3226 p. 390, 532; pOJ436 p. 530; pOJ444
p. 530; pPM927 p. 390, 563; pSET152 p. 530; pHM11A Motamedi et al. (1995); pIJ4201 p. 575; pIJ4210 p. 575; pIJ4696
p. 576; pIJ4698 p. 576; pIJ4700 p. 576. Also C31 c+ att+ derivatives (p. 292) can be used as integrating vectors.

Unstable and temperature-sensitive plasmids useful for gene replacement and transposon delivery
KC573 p. 524; pHZ1351 p. 542; pHZ1358 p. 542; pMT660 p. 536; pCZA185 p. 551; pGM11 p. 548; pGM16 p. 549;
pGM160 p. 318, 320, 550; pGM17 p. 549; pGM9 p. 548; pHZ132 p. 550; pKC1139 p. 551; pSIT151 p. 119, 120; pUCS30
p. 119, 552; pIJ80 p. 261, 559; pIJ4680 p. 319, 559; pIJ2581 p. 321, 571.

Vectors without the tsr gene
KC211 p. 293; KC212 p. 292; KC401 p. 293; KC466 p. 524; KC517 p. 293; pIJ8600 p. 389, 531; pIJ8630 p. 531; pIJ8660
p. 531; pMT3226 p. 390, 532; pOJ436 p. 530; pOJ444 p. 530; pSET152 p. 530; pANT1201 p. 538; pANT1202 p. 538;
pANT850 p. 389, 537; pANT851 p. 389, 537; pJOE829 p. 541; pB45 p. 547; pB48 p. 547 ; pGM11 p. 548; pGM17 p. 549;
pKC1139 p. 551; pSIT151 p. 119, 120; pIJ4231 p. 266; pIJ80 p. 261, 559; pIJ920 p. 260, 555; pIJ921 p. p. 555; pJAR4 p.
p. 261, 560; pKC1218 p. 268, 561; pKC505 p. 261, 560; pOJ446 p. 562; pCAO106 p. 263; pIJ4696 p. 574; pIJ4700 p. 574

Vectors with resistance markers other than the common ones
Several vectors contain markers other than the following: aac(3)IV (apramycin), aphI (neomycin), hyg (hygromycin),
km (kanamycin), neo (kanamycin, neomycin, Tn5), tsr (thiostrepton), vph (viomycin).
pGM17 (ble catSa) p. 549; pGM9 (ble) p. 548; pSIT151 (aacC1) p. 119, 120; pUCS30 (ble aadA) p. 119, 552; pIJ4680(aadA) 319, 559; pPM927 (aadA) p. 390, 563; pPM2208 (aadA) 317, 571; pIJ4698 (aadA) p. 574; pIJ4700 (aadA) p. 574.

&KDSWHU3ODVPLGVDQGWKHLUXVHIRUJHQHFORQLQJVectors with blue/white selection (lacZ ) in E. colipSET152 p. 530; pUWL series p. 544; pWHM3 p. 319, 544; pRES18/19 p. 545; pKC1139 p. 551; pKC1218 p. 268, 561;
pKC1250 p. 567; pDH5 p. 230, 570; pKC1138 p. 566; pOJ260 p. 566; pSET151 p. 570; pUC19 p. 568.

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Streptomyces and their use in vector construction. Gene, 20,
51-62.
Tsai JF-Y, Chen CW (1987) Isolation and characterizationof Streptomyces lividans mutants deficient in intraplasmid
recombinatioin. Mol.Gen.Genet., 208, 211-218.
Van Mellaert L, Mei L, Lammertyn E, Schacht S, Anné J(1998) Site-specific integration of bacteriophage VWB
genome into Streptomyces venezuelae and construction of a
VWB-based integrative vector. Microbiology, 144, 3351-8.
Vara J, Lewandowska-Skarbek M, Wang YG, Donadio S,
Hutchinson CR (1989) Cloning of genes governing thedeoxysugar portion of the erythromycin biosynthesis pathway
in Saccharopolyspora erythraea (Streptomyces erythreus). J
Bacteriol, 171, 5872-81.
Wohlleben W, Muth G (1993) Streptomyces plasmid vectors.
In Hardy, K.G. (ed.) Plasmids: A practical Approach. Oxford
University Press, Oxford, pp. 147-175.
Wohlleben W, Hartmann V, Hillemann D, Krey K, Muth
G, Nussbaumer B, Pelzer S (1994) Transfer andestablishment of DNA in Streptomyces (a brief review). Acta
Microbiol.Immunol.Hung, 41, 381-9.
Wrigley-Jones CA, Richards H, Thonmas CR, Ward JM(1993) Stability of plasmid vector pIJ303 in Streptomyces
lividans TK24 during laboratory scale fermentations.
Biotechnol.Bioeng., 41, 148-155.
Xiao J, Melton RE, Kieser T (1994) High-frequency
homologous plasmid-plasmid recombination coupled withconjugation of plasmid SCP2* in Streptomyces.
Mol.Microbiol., 14, 547-55.



m12tk1b.wpd 20 April 2000 Tobias Kieser

Chapter 12

Streptomyces phages
CONTENTS
The relevance of phages to Streptomyces genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271
Occurrence, isolation and storage of Streptomyces phages . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272
Lytic and temperate phages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272
Phages from soil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272
Phages from lysogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272
Phages as industrial contaminants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273
Streptomyces phage genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273
In vivo physiological and genetic studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273
Deletion mutants and DNA packaging limitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273
Phage DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273
Uses of wild-type phages in the study of their hosts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274
Transduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274
Localised mutagenesis using generalised transduction . . . . . . . . . . . . . . . . . . . . . . . . . 274
Restriction-modification systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275
Storage of Streptomyces phages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276
Plaque assay of Streptomyces phages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277
Single-plaque isolation of Streptomyces phages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278
Preparation of high-titre Streptomyces phage stocks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278
Isolation of new Streptomyces phages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279
Isolation procedure I (direct method) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280
Isolation procedure II (specific enrichment) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281
Selection of potential transducing phages by pyrophosphate resistance . . . . . . . . . . . . . . . . . 281
Generalised transduction of S. venezuelae using SV1 phage . . . . . . . . . . . . . . . . . . . . . . . . . . 282
Large-scale preparation of Streptomyces phage DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282
Small-scale preparation of Streptomyces phage DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285

The relevance of phages to Streptomyces genetics
Streptomyces phages are interesting subjects for detailed study, especially when considered (asin the case of the most studied example, C31) in the context of well-studied host genetics. The interest
comes both from their own biology and diversity and because they can provide useful tools for studies
or exploitation of their hosts. They are also valuable teaching aids, allowing reliable, quick, economical
and informative class experiments. In this chapter we describe methods for isolating, propagating and

&KDSWHU6WUHSWRP\FHVSKDJHV

characterising Streptomyces phages, and procedures in which phages are used to study host genetics. In
Chapter 13 we cover the exploitation of phages in cloning and manipulating Streptomyces DNA.

Occurrence, isolation and storage of Streptomyces phages
Lytic and temperate phages
Streptomyces phages are recognised by plaque formation (usually overnight) in soft agar. The
distinction of lytic from temperate phages by plaque turbidity may be ambiguous. This is because, by
the time the background of resistant lysogens causes temperate phage plaques to be turbid, lytic phageplaques may also show some turbidity, because of the development of phenotypic resistance by older
mycelium (a widespread observation with Streptomyces phages, though one Saccharopolyspora phage,
JHJ-1, continues to propagate even when host growth has almost ceased: Haket et al., 1990). Ultimately,
the definition of a new phage as temperate requires the demonstration that lysogenic host cultures canbe obtained. This is usually done by restreaking the surviving growth from infected areas on plates andshowing that individual colonies not only are immune to superinfection by the same phage, but also
release free phages (pp. 303-306).
Phages from soil
The natural habitat of most streptomycetes is soil, and their phages are readily isolated from soil,
provided the pH is above c. 5.0 (S. T. Williams, personal communication). Sometimes phages can beisolated directly from aqueous extracts of soil, but the most successful procedure is to enrich for phages
by mixing together soil, nutrient broth and spores of a chosen indicator and incubating overnight before
plating for plaques (pp. 279-281). Nearly all soil samples readily yield phages that give plaques on S.
lividans 66, which is a generally permissive host for Streptomyces phages that have a wide host-range
within the genus (another such host is S. griseofuscus: Cox and Baltz, 1984). Most phages isolated fromsoil have a wide host-range, as expected because the laboratory indicator strains used are unlikely to bevery closely related to the streptomycetes that were most abundant in any particular soil sample. An
exception concerns phages with a high specificity for S. venezuelae, which are present in many soil
samples; this may reflect the ubiquity and abundance of S. venezuelae strains (Stuttard, 1989).
Phages from lysogens
Streptomycetes themselves provide a second source of phages, since they can harbour prophages.
It is not always easy to obtain phages in this way, because the endogenous phages often have a narrow
host-range, and a certain amount of luck is therefore needed in the choice of a suitable indicator strain(of course, the original lysogen is expected to be immune to plaque formation by the phage it carries).
Moreover, few Streptomyces prophages are UV-inducible, though at least two (SAt-1 and R4) are
induced by mitomycin C (Ogata et al., 1984; H. Takahashi, personal communication). Most often,
occasional spontaneous induction events during growth on solid or liquid media provide the free phagesfor detection by plaque formation on a suitable indicator strain.

&KDSWHU6WUHSWRP\FHVSKDJHVPhages as industrial contaminants
A few phages have been collected in circumstances where they were neither sought nor desired i.e., following the lysis of industrial fermenter cultures after contamination by an unusually virulent
phage. Such rare events have given phages an unnecessarily bad name in industrial laboratories. We do
not believe there to be any good reason for such laboratories to avoid the use of well-understood
Streptomyces phages. We have never observed unexpected, potentially phage-induced lysis in laboratory
batch cultures of S. lividans (a good host for phages), even though we frequently employ vectors derived
from C31, a phage which was originally isolated on S. lividans.

Streptomyces phage genetics
In vivo physiological and genetic studies
Traditional methods for phage study can be applied to Streptomyces phages (Lomovskaya et al.,
1980; Chater, 1986); they include adsorption studies, one-step growth curves and single burst
experiments. Genetic analysis by complementation and mapping has also been done with mutations in
C31, the most-studied Streptomyces phage (Lomovskaya et al., 1980). Mutants can be isolated either
spontaneously or after mutagenic treatments. Types of mutant phenotype used in Streptomyces include:
clear plaques (often, but not always, due to mutations affecting the regulatory gene(s) for establishingand maintaining lysogeny, but not usually affecting integrase genes or att sites); other plaque morphology
changes; altered host-range (potentially due to changes in adsorption or to altered responses to hostspecific restriction-modification systems); and temperature-sensitive plaque formation. Genetic mappingand complementation tests done by co-inoculating pairs of mutants into liquid cultures or solid cultures(in soft agar overlays) usually yield high frequencies of recombination. Since no attempts to obtain
nonsense-suppressing Streptomyces hosts have been successful, suppressor-sensitive mutants ofStreptomyces phages equivalent to amber mutants of E. coli phages have not been used in this genus.
Deletion mutants and DNA packaging limitation
For some phages that package their DNA by site-specific cleavage at cos sites, deletion mutants
can be isolated by a procedure borrowed from lambda genetics: such mutants have increased resistanceto chelating agents (Chater et al., 1981). (It may be necessary to test different chelating agents withdifferent phages.) The use of deletion mutants has been critical for the development of phage cloning
vectors, since the insertion of additional DNA into a wild-type phage genome may make the genome toolong to be packaged into a stable particle. For C31, the maximum and minimum lengths for packaging
are about 43 kb and 35 kb, respectively, compared with a normal genome size of about 41.6 kb. This
property has been exploited in the use of obligatory replacement vectors (Table 13.2, p. 293).
Phage DNADNA can readily be isolated from most Streptomyces phages by standard procedures (pp. 282287). To obtain sufficient phage particles, the simplest method is to inoculate standard agar overlays withenough phages to give just confluent lysis. Inoculation with too many phages often gives a lower yield.

&KDSWHU6WUHSWRP\FHVSKDJHVWhen a particular phage is to be studied in detail, it may be useful to develop liquid culture procedures
for large-scale propagation.
Once isolated, phage DNA is usually kept at 4o, as large linear molecules may be damaged byfreezing and thawing. However, where vector DNA preparations have been stored at -20o there have notbeen obvious problems. Preparations may become viscous because end-to-end concatemers form throughthe joining of sticky cos ends (see below). Pipetting should therefore be gentle, to minimise shearing.
Restriction mapping of phage DNA often reveals a distribution of sites quite different from thatof the host or its plasmids (Chater, 1986). This may reflect a difference in overall base composition, orbe the result of natural selection, since wide host-range phages may have encountered many hosts withdifferent restriction systems in their evolutionary history, although these explanations do not account for
all the observed departures from expected frequency. Because many phages have DNA with fixed,
cohesive (cos) ends, the end fragments in restriction digests can join together. As a result, the end
fragments are often apparently under-represented after electrophoresis, and perceptible amounts of ajunction fragment may be observed. A brief heat treatment (e.g. 70o, 10 min), followed by rapid cooling,
immediately before electrophoresis greatly reduces the amount of such junction fragments.

Uses of wild-type phages in the study of their hosts
Transduction
The classical use of temperate phages is for generalised transduction of host genes. For many
bacteria this is especially useful for transferring transposon-induced mutations from one genetic
background to another, whether for strain construction or to demonstrate 100% linkage of the transposon
with the mutant phenotype. Similar tests can be done for mutations in which a resistance marker has
been used to disrupt a gene - a very frequent situation. It has therefore been particularly frustrating thatunambiguous reports of transduction have until very recently been confined to chloramphenicolproducing strains of S. venezuelae and S. phaeochromogenes, and the neomycin-producing strain of S.
fradiae, despite many attempts to find transducing phages for S. coelicolor A3(2) or S. lividans (Stuttard,1989). (Note, though, that direct transformation of protoplasts with genomic DNA preparations (p. 235)
provides a valuable, albeit less efficient, substitute for transduction.) However, as this volume was being
prepared, J. Burke and J. Westpheling (personal communication) reported an effective generalised
transduction system for S. coelicolor A3(2). Their success in discovering this came from a simple trick
previously used by D.A. Hodgson (personal communication) in isolating transducing phages for Listeria
monocytogenes: this was to propagate isolate phages at a temperature closer to that prevailing in thenatural environment (i.e. 20o instead of the usual 30o ).
Generalised transduction requires phages that package DNA by the headful. Because suchphages are often relatively resistant to chelating agents, pre-enrichment for such resistance (p. 281) has
been recommended before direct tests of the transducing ability of newly isolated phages (Stuttard,1989).
Localised mutagenesis using generalised transduction
Since transduction usually involves the introduction of a segment of DNA extending
considerably beyond the selected marker, nearby donor genes (i.e. within the 40 kb genome length of the

&KDSWHU6WUHSWRP\FHVSKDJHVTable 12.1. An example of “classical” restriction and modification involving phage SH5 (from Klaus
et al., 1981)
Previous hosts (most recent given last)
lev = S. levoris
hyg = S. hygroscopicus

Relative efficiency of plaque formation (EOP)on S. hygroscopicus 0477 (compared withpermissive host S. levoris)

lev

10–4

lev, hyg

1lev, hyg, lev

10–4

S. venezuelae phage SV1) may simultaneously be transduced (Stuttard, 1983). It follows thattransduction of a particular marker by mutagenised donor phage preparations may lead to the cotransduction of newly induced mutations in closely linked genes, providing a means of localised
mutagenesis. This concept was first exploited in Salmonella typhimurium LT2 by Hong and Ames
(1971). Stuttard (1983) used the same procedure to produce lysA-linked mutations of S. venezuelae. We
do not give the details here, but note that anti-SV1 antiserum is useful in order to eliminate free SV1
from transductants on subculturing. No doubt it will be possible to use other generalised transducing
systems, including that newly discovered in S. coelicolor A3(2) (see above), in the same way.
Restriction-modification systems
Phages have also often been used to detect and study host-specific restriction-modification (RM)
systems (Chater, 1986). In the simplest case, a comparatively low efficiency of plaque formation (EOP)
is observed when a phage is first assayed on a new host, but phages isolated from such rare plaques havea high EOP when re-assayed on the same host. Further confirmation that this is due to modification
rather than to a phage mutation comes from reintroducing the phage into its original host, then reassaying the progeny on the restricting host, when the original low EOP should reappear (see Table 12.1
for an example).
Several such situations have been described for streptomycetes, and it has been possible toexploit this in the isolation of restriction-deficient (R–) mutants (e.g. Chater and Wilde, 1980), which may
then be better recipients in transformation experiments (another approach to isolating such mutants was
described by Matsushima et al., 1987). The basic method is to replicate colonies (after mutagenesis) toa plate spread with unmodified phages, at a concentration slightly too low to kill the restricting host’s
growth. In order to maximise the killing of any R– mutants the phages should be either lytic, or clear
plaque mutants of a temperate phage. R– mutants are expected to show obvious phage-sensitivity aftera brief (c. 24 h) incubation (the difference between R– and R+ colonies may disappear on longer
incubation).
Phage-host interactions are sometimes more complex, and the first plating on a new host may
also select phage mutants with improved plaque-forming ability: thus, after subsequent passage througha non-modifying host, the EOP on the restricting host may be higher than in the first test (Chater, 1986;
see Table 12.2). Such g mutants were described for C31 and R4, two phages that have been developed



&KDSWHU6WUHSWRP\FHVSKDJHV

Table 12.2. A more complicated example of restriction and modification, in which R4 phage genes
interact with the host-controlled RM system of S. albus G (from Chater and Carter, 1979)
Previous hosts1(most recent given last)

Relative EOP on S. albus G (R+M+) compared with permissive host
(J1074 S. albus G R–M– mutant)<10–9(1) R–M–(2) R–M+
10–6
–
+
+ +
(3) R M , R M
5×10–1(4) R–M+, R+M+, R–M–
10–5
1
R, restriction phenotype; M, modification phenotype (of the SalI RM system).
(1) Absence of plaques because of a combination of (a) restriction of unmodified DNA and (b) inhibitionof modification by g gene, preventing initiation of plaques even when restriction is bypassed.
(2) The g function has prevented modification except in the case of rare g mutants, which are responsible
for plaque formation on the R+M+ strain.
(3) The whole population now consists of g mutants hence giving a high level of modification; occasional
failure to modify every SalI site causes the EOP to be slightly less than 1.
(4) The unmodified phage is heavily restricted; in this case the rare plaques reflect a bypass of therestriction barrier, perhaps because of occasional R–M+mutants in the host population.
as vectors. The mutants give plaques (albeit at an initial low frequency) on some hosts on which theparental phages give none, so they may sometimes provide a useful extension to host-range.
Apart from conventional RM systems, there are other kinds of host resistance mechanisms, anotable one being the Pgl system of S. coelicolor A3(2), which specifically confers resistance to C31
and closely related phages. Phages unrelated to C31 are not affected by the Pgl system. C31sensitive Pgl– mutants of S. coelicolor can readily be isolated by the replication method used for isolating
R– mutants. The mutants occur (and often revert) spontaneously at a high frequency (c. 10–3)(Lomovskaya et al., 1980; Chater, 1986; Laity et al., 1993). Defined, stable Pgl– null mutants have been
constructed (Bedford et al., 1995).

Storage of Streptomyces phages
For most purposes, phages are stored at 4o in the dark as suspensions obtained as soak-outs fromsingle plaques or confluent plates. Filtration (0.45 µm nitrocellulose filter, Swinnex unit) is necessary
to minimise loss of phage titre by adsorption to host debris. (It is inadvisable to use CHCl3 to sterilise
suspensions without first testing the phage for CHCl3 resistance - many Streptomyces phages areinactivated rapidly by 1% CHCl3.) C31 suspensions often show a c. 10-fold drop in titre over the firstfew days, and then decline much more slowly. For long-term preservation, it is advisable to store phages
as DNA (usually at 4o; recover the phages by transfection) and/or as lyophils (using filter-paper strips
dipped into a high-titre suspension). To recover phages from lyophils, simply place the filter paper strip
on a fresh conventional overlay plate (p. 277), containing spores of the host strain in the top layer, andincubate overnight.
If phages have been pelleted by centrifugation, they are resuspended gently in buffers such as
SM buffer for storage (p. 416).

&KDSWHU6WUHSWRP\FHVSKDJHVPlaque assay of Streptomyces phages (after Dowding, 1973)
Streptomyces phages, like those of other bacteria, are routinely assayed as plaque-forming units(pfu) by counting plaques on the traditional type of double-layer plate: a basal layer of nutrient agar witha thin top layer of dilute (soft) agar containing a dense inoculum of host bacteria.
Phage suspension (e.g. in DNB)
Spores of Streptomyces indicator strain
Freshly poured (same day, not over-dried) 5 cm Petri plates of Difco Nutrient Agar
containing 0.5% glucose and appropriate divalent cations (Table 12.3). Oxoid
Nutrient Agar and Broth are unsuitable
Difco Nutrient Broth (DNB)
Molten Soft Nutrient Agar (SNA) top layers kept at 45-50o
Dilution tubes; pipettes
30o incubator; 45o-50o water bath

1.

Dilute the phage suspension (e.g. in a tenfold series) using DNB. Make the dilution
series immediately before use; 100-10–8 will cover most phage concentrations. For C31, single
plaques may contain c. 107 pfu and just confluent plates c.1010-1011 pfu.

2.

Pipette 0.1 ml samples of the dilutions and the undiluted phage onto 5 cm Petriplates.

3.

Add 0.8 ml SNA top layer (containing spores of the indicator stain) to each plate;swirl to cover the whole plate. Cool SNA to c. 45o before adding spores to give a justperceptibly turbid suspension (c. 107 ml–1), and dispense as quickly as possible. A 0.6 ml SNA
top layer will just cover the 5 cm plate, but up to 1 ml may be used. If the top layer is alreadysetting during swirling, plaques may be difficult to detect.

4.

Leave to set undisturbed for 5-10 min.

5.

Invert plates and incubate overnight (c. 18 h) at 30o. Alternative temperatures may be
used as appropriate within the range tolerated by the host. Very often, the maximum temperature
for plaque formation is lower than the maximum for the host.

6.

Score plaques next morning. Where plaques are easily countable (i.e. fewer than 100) the
titre can be calculated: e.g. if 47 plaques were counted after plating 0.1 ml on a 10–5 plate, thetitre would be 4.7 × 107 ml–1.

Table 12.3 shows examples of optimised concentrations of Ca2+ and Mg2+ salts for various Streptomyces
phages. When working with a new phage, try 4mM Ca(NO3)2 in the first instance (Dowding, 1973).

&KDSWHU6WUHSWRP\FHVSKDJHV

Table 12.3. Divalent cations for phage propagation (A.T. Carter, unpublished)
Phage

Final concentrations (ml stock solution1 per 200 ml Difco Nutrient Agar)MgSO4

Ca(NO3)2

10mM (2 ml)

8mM (2 ml)

R4

10mM (2 ml)

25mM (6.2 ml)

VP5

3mM (0.6 ml)

4mM (1 ml)

VP7

6mM (1.2 ml)

6mM (1.5 ml)-

8mM (2 ml)

C31

448

S14, Pal64mM (1 ml)
Stock solutions: 1M MgSO4; 0.8M Ca(NO3)2. The sterile solutions (together with glucose to a final
concentration of 0.5%) are added to the molten agar immediately before pouring plates. There is no needto add divalent cations to the soft agar overlays.

1

Single-plaque isolation of Streptomyces phages
In any genetic work with phages it is very important to purify each stock rigorously by several
rounds of single plaque isolation. A well isolated plaque on a freshly grown top layer (phages will havediffused significantly beyond the plaque margins in older plates) is sucked into the tip of a sterile Pasteur
pipette, and transferred into 1 ml DNB. After 2 h at room temperature a dilution series is prepared fromthe “soak-out” and used for a second round of single
plaque isolation using the plaque assay systems
described in the previous section. Single plaque soakouts to be retained should be filter-sterilised.
More economical purification, especiallysuitable when dealing with large numbers of phagesamples, is achieved by using a fine platinum wire tostab a plaque, then streaking to a fresh Difco Nutrient
Agar plate (heating the wire between each reorientationof the plate as in single colony purification of bacteria)
and finally overlaying with Soft Nutrient Agar
Add soft agar so that it spreads from thecontaining indicator spores (see previous section).
most dilute area towards the main area of
inoculum. Do not swirl the top layer.

Preparation of high-titre Streptomyces phage stocks
Single-plaque-purified phage stock
Spores of appropriate Streptomyces indicator strain
Fresh (same day, not overdried) 5cm Petri plates of Difco Nutrient Agar containing
0.5% glucose and appropriate divalent cations (Table 12.3)
Difco Nutrient Broth (DNB); molten Soft Nutrient Agar (SNA) kept at 45-50 o

&KDSWHU6WUHSWRP\FHVSKDJHVSterile 5 ml screw cap bottles; dilution tubes; tube rack; 25 mm Swinnex filter
units containing cellulose nitrate filters of pore size 0.45 µm; 10 ml syringes
30o incubator (or other appropriate temperature); 45o-50o water bath

1.

Dilute 0.1 ml of the phage suspension in a tenfold series down to 10–5 using
DNB.

2.

Pipette 0.1 m samples of the dilutions and the undiluted phage onto 5 cm Petriplates in duplicate.

3.

Add 0.8 ml SNA top layer (containing spores of the indicator strain) to eachplate and swirl to cover the whole plate. Cool SNA to c. 45o before adding spores togive a slightly turbid suspension, and dispense as quickly as possible.

4.

Leave to set undisturbed for 5-10 min.

5.

Incubate the plates inverted overnight at 30o (or other appropriate temperature).

6.

Next morning flood the two almost confluent plates each with 2.5 ml DNB.
Almost confluent plates give the highest possible phage titre.

7.

Stand at room temperature for c. 2 h.

8.

Pipette off the lysate from the two plates; pool and filter through a sterile 0.45
µm cellulose nitrate filter, using a 10 ml syringe, into a 5 ml screw cap bottle.

9.

Assay (see separate procedure, p. 277). The titre should normally be at least 109-1010 pfu
ml–1, but mutant phages may give a lower titre.

10.

Store at 4o. Phages may lose viability more rapidly at room temperature and in bright light.

Isolation of new Streptomyces phages (after Dowding, 1973 and C. Stuttard, personalcommunication)
Most soils yield Streptomyces phages, which can be isolated either by the direct plating of soil
extracts on an indicator strain using conventional double layer plates or by enriching for phages capableof growth on that strain by growth in liquid medium before plating. With direct plating, each plaque on
the primary plates represents a separate phage particle in the soil sample. Specific enrichment may allow
detection of plaques in soil samples that are not productive in direct platings.

&KDSWHU6WUHSWRP\FHVSKDJHV

Soil samples (c. 25 g)
Spores of Streptomyces indicator strain(s)Fresh (same day, not over-dried) 5 cm Petri plates of Difco Nutrient Agar
containing 0.5% glucose and 4mM Ca(NO3)2
Difco Nutrient Broth (DNB)
Molten Soft Nutrient Agar (SNA) top layers kept at 45o-50o
50% glucose
0.8M Ca(NO3)2
Pipettes; 20 ml screw cap bottles; 250 ml Erlenmeyer flasks; dilution tubes; 25 mm
filter units containing cellulose nitrate filters of pore size 0.45 µm; 10 ml syringes
Incubator (usually 30o); orbital incubator shaker (usually 30o); bench centrifuge;
45o-50o water bath; tube rack
Isolation procedure I (direct method)1.

Mix a small quantity (c. 1-5 g) of soil with 3 times its own volume of DNB in a
20 ml screw cap bottle.

2.

Stand at room temperature for 2-3 h with occasional agitation.

3.

Remove particulate matter by centrifuging on a bench centrifuge (c. 3000 rpm)
for 10 min.

4.

Carefully transfer supernatant to a fresh 20 ml screw cap bottle and filter througha sterile 0.45 µm cellulose nitrate filter, using a 10 ml syringe, into a 10 ml screw
cap bottle.

5.

Dilute 0.1 ml of the filtrate 10–1 and 10–2 in DNB.

6.

Plate 0.1 ml samples of the undiluted and diluted filtrate onto 5 cm Petri plates,
overlay with 0.8 ml SNA containing spores of the indicator strain, swirl
immediately and briefly, leave 5 min to allow agar to solidify, and incubate
overnight (usually at 30o, but lower temperatures such as 20o may yield different phages, more
often including transducing phages). Cool SNA to c. 45o before adding spores to give a slightly
turbid suspension and dispense as quickly as possible.

7.

Plaques are then purified by single plaque isolation (see separate procedure,p. 277) before being plated as almost confluent lawns for high-titre stocks (seeseparate procedure, p. 278).

&KDSWHU6WUHSWRP\FHVSKDJHVIsolation procedure II (specific enrichment)1.

Mix 10-20 g soil in a 250 ml Erlenmeyer flask with 20 ml DNB containing 0.5%
glucose and 4mM Ca(NO3)2 and add spores of the required Streptomyces hoststrain to give a final concentration of about 107 cfu ml–1 (c. 107 cfu ml–1 can beobtained by adding spores to give a slightly turbid suspension). Other rich media
can be used, such as RGMC (Anné et al., 1984).

2.

Incubate overnight with vigorous shaking in an orbital incubator (usually at 30o, but
lower temperatures such as 20o may yield different phages, especially transducing phages).

3-7.

As Procedure 1.

Selection of potential transducing phages by pyrophosphate resistance (C. Stuttard,
1989 and personal communication)
As mentioned previously (p. 274), generalised transducing phages usually package DNA nonspecifically in “headfuls” and are often comparatively resistant to chelating agents. In order to identify
potential transducing phages (and to identify some minor sub-populations of different phages after the
initial crude enrichment), treatment with sodium pyrophosphate of the potentially mixed phages isolated
from soil has been used.
Overlay plates containing fresh plaques from soil enrichment
Spores of indicator strain
Fresh (same day, not over-dried) 5 cm Petri plates of Difco Nutrient Agar
containing 0.5% glucose and 4mM Ca(NO3)2
Sterile phage buffer (3 ml per 5 cm plate)
Sterile APB (10mM Tris-HCl pH7.5, 4mM CaCl2, 0.005% gelatin: Vats et al., 1987)
Sterile 25mM Na4P2O7
Pipettes; 25 ml screw cap bottles; dilution tubes; 25 mm filter units containing
cellulose nitrate filters of pore size 0.45 µm
30o incubator; 30o water bath; 45o-50o water bath; tube rack1.

Flood each 5 cm plate containing plaques of newly isolated phages with 3 ml
APB, leave 2 h at room temperature, and withdraw the fluid.

2.

Filter; dilute filtrate 10–2 into 25mM Na4P2O7 and into distilled water (control).

3.

Incubate at 30o for 30 min.


4.

&KDSWHU6WUHSWRP\FHVSKDJHV

Dilute treated and control suspensions a further 10–2 and 10–4 in distilled waterand assay for pfu. The control counts will usually be higher because most phages are
sensitive to chelating agents5.

Purify potentially pyrophosphate-resistant phages by three serial single plaqueisolations.

6.

Test for transducing ability (see next section). Note that the strain used to propagate
a transducing lysate and the recipient strain for transduction must be derived from the same wildtype strain.

Generalised transduction of S. venezuelae using SV1 phage (Stuttard, 1979)
The procedures for handing SV1 are essentially the same as for C31. The basic transduction
methodology could not be simpler: recipient spores (107-108 per plate) and transducing phages (usuallyin excess: c. 109 per plate) are mixed either directly on a selective minimal medium (when selecting for
prototrophic transductants) or on a non-selective medium later to be flooded with selective antibiotic(when transduction of a resistance marker is sought). Transductants (typically 10-100 colonies) appear
after a few days of incubation. Frequencies may be enhanced up to 10-fold (i.e., c. 100-1000
transductants per plate) by pre-irradiating the transducing phage preparation with UV (256 nm) to 0.5%
survival, to minimise killing of the host by viable phages; but this could introduce new mutations, so
caution is advised if transductants are to be used for further study. Supplementation of MM with proline(56 µg ml–1) was reported to enhance the recovery of prototrophic transductants, and a low level oftryptophan was necessary for transduction of a tryptophan auxotroph (trp-1). The most useful negative
control is an experiment using, as the donor, phage SV1 propagated on the recipient strain.

Large-scale preparation of Streptomyces phage DNA
The procedure extends over three days (but the work is not continuous). It consists of theharvesting of phages from large areas of just confluently lysed overlays, partial purification andconcentration by differential centrifugation, further phage purification on caesium chloride gradients, andDNA extraction.
Freshly assayed, single-plaque-purified phage in Difco Nutrient Broth (DNB), atleast 106 pfu ml–1
Spores of Streptomyces indicator strain
Two containers each of approximately 0.15 m2 surface area of Difco Nutrient Agar
supplemented with 0.5% glucose and appropriate divalent cations (or equivalentarea in Petri plates) (prepare on day of use)
Difco Nutrient Broth (DNB)

&KDSWHU6WUHSWRP\FHVSKDJHVMolten Soft Nutrient Agar (SNA) at 45o
SM buffer
TE buffer (sterile and non-sterile)
CsCl
20×SSC
5M NaCl
Liquid paraffin
Phenol solution (p. 164)
Ether
Absolute ethanol
70% ethanol
Pipettes (10 ml, 5 ml, 1 ml); Pasteur pipettes (sealed and unsealed); Parafilm;
centrifuge bottles and tubes (500 ml, 50 ml, 12 ml, 11 ml glass conical-bottomed);
glass spreaders; rubber teats; propipettes; screw cap bottles (5 ml and 20 ml);
500 ml Erlenmeyer flask or beaker; eye protection and disposable gloves; 2 ml
disposable syringes; gauge 21 disposable needles; 1.5 ml Eppendorf centrifuge
tubes
Bench, high-speed and ultra-centrifuges; 30o incubator; -20o freezer; refrigerator(or cold room); 45-50o and 60o water baths; tube racks

1.

Inoculate 2 × 70 ml SNA at 45E with c. 7 × 108 Streptomyces spores each andphages (1.5 × 105 pfu for C31). Overlay 70 ml top layers onto each 0.15 m2
agar surface or 2.5 ml onto each of 50 Petri dishes. Incubate overnight.
Adjustment may be necessary for phage-host combinations giving very small or very large
plaques. Add enough spores to SNA to give a slightly turbid suspension and dispense as quickly
as possible.

2.

Using a spreader, scrape off top layers into 2 × 100 ml DNB in 250 ml centrifuge
bottles; mix by shaking and stand at room temperature for 2 h.

3.

Centrifuge at 10, 000 rpm (16, 000 × g) for 10 min at 4o to remove agar.

4.

Pour supernatants into 500 ml flask or beaker (store at 4o). Re-extract agar with
2 × 50 ml DNB; pool and stand at room temperature for 1 h.

5.

Repeat step 3 with second extracts. Pool supernatants and dispense into
8 × 50 ml screw cap centrifuge tubes.

6.

Centrifuge at 17, 000 rpm (34, 000 × g) overnight or 25, 000 rpm (54, 500 × g)
for 75 min at 4o to sediment phage gently.

&KDSWHU6WUHSWRP\FHVSKDJHV

7.

Discard supernatant and add 0.5 ml SM buffer to each tube; stand tubes (tilted
so that the pellet is immersed in the buffer) at 4o for about 3 h, or agitate on arotary shaker at 37o for 15-30 min.

8.

Pool phage suspensions and make up to a final volume of 7 ml with SM. Add
5.95 g CsCl (0.85 g ml–1).

9.

Transfer to 12 ml centrifuge tube, overlay with liquid paraffin, cap the tube andcentrifuge at 36, 000 rpm (85, 000 × g) for 18 h at 20o.

10.

Open the tube and remove the bluish phage band by piercing the side of the tube
with a 21-gauge syringe needle; collect phage in a 2 ml syringe. Phages may be
stored in the CsCl at this stage at 4o.

11.

Dialyse for 4 h at room temperature against 2 × 100 ml SM buffer. Transfer toan 11 ml conical-bottomed centrifuge tube.

12.

Add one-twentieth volume 20 × SSC, cover with parafilm, heat to 60o for 10
min, and cool to room temperature.

13.

Add 5M NaCl to 0.25M final concentration.

14.

Protect your eyes and wear gloves. Add an equal volume of phenol solution,
cover with parafilm, and invert 50 times.

15.

Centrifuge on bench centrifuge (c. 3, 000 rpm) for 10 min.

16.

Carefully remove upper (aqueous) phase with broken-off Pasteur pipette, andtransfer to fresh conical tube.

17.

Re-extract upper (aqueous) phase twice with phenol as in steps 14-16.

18.

Re-extract phenol phases with an equal volume of TE buffer containing 0.25M
NaCl, as in steps 14-16.

19.

Pool aqueous phases and transfer to 20 ml screw cap bottle; extract phenol byadding an equal volume of ether and shaking gently; centrifuge for 2 min on

&KDSWHU6WUHSWRP\FHVSKDJHVbench centrifuge (c. 3,000 rpm). Beware of open flames. Aqueous phases must betransferred to a screw cap bottle before ether extraction because ether dissolves parafilm.

20.

Remove upper (ether) phase and discard into waste solvent container.

21.

Re-extract aqueous phase twice more with ether as in step 19.

22.

Add 2.5 volume of absolute ethanol and swirl tube until DNA precipitates as afibrous clot. If the DNA concentration is very low (i.e. the volume is large) it may benecessary to precipitate the DNA at -20o for 2 h; then spin on bench centrifuge (c. 3,000 rpm)
for 10 min. Pour off ethanol, dissolve DNA in 1 ml TE buffer, transfer to 5 ml screw cap bottle,
add 50 µl 5M NaCl and 2.5 ml absolute ethanol and swirl; remove all the ethanol with a Pasteur
pipette and proceed to step 24.

23.

Spool DNA on a sealed Pasteur pipette and transfer to an Eppendorf tube.

24.

Wash DNA precipitate with 1 ml 70% ethanol.

25.

Remove ethanol with a Pasteur pipette and then dissolve the DNA in 0.5-1 mlsterile TE buffer. Store at 4o.

Small-scale preparation of Streptomyces phage DNA (based on procedures of J.E. Suárez
and H. Ikeda)
Twelve phage preparations can be processed at the same time. Phages isolated from overlays
are partially purified and concentrated by differential centrifugation, treated with RNase, then disrupted
with buffered SDS-EDTA solution. The addition of concentrated potassium acetate precipitates thedetergent and protein, and the DNA is subjected to further purification steps. The whole procedureincluding phage propagation takes about 48 h.
Freshly assayed, single plaque purified phage in Difco Nutrient Broth, at least 5 ×
105 pfu ml–1
Spores of Streptomyces indicator strain
Six (9 cm) Petri plates (per phage preparation) of Difco Nutrient Agar containing
0.5% glucose and appropriate divalent cations (prepared on day of use)
Difco Nutrient Broth (DNB)
Molten Soft Nutrient Agar (SNA) at 45o
SM buffer containing 40 µg ml–1 RNase. (Dilute RNase from frozen heat-treated stock
solution of 10 µg ml–1 in SM buffer; heat treatment of 5 min at 95o eliminates DNase)&KDSWHU6WUHSWRP\FHVSKDJHVSDS mix (mix shortly before use: 1 vol 2M Tris-HCl pH9.6, 2 vol 0.5M sodiumEDTA pH7.4, and 1 vol 10% SDS)
8M potassium acetate (unbuffered, filtered through Whatman No.1 paper)
TE buffer (sterile)
3M sodium acetate
Absolute ethanol
Ice
Pipettes (10 ml, 5 ml, 1 ml, 200 µl); Pasteur pipettes; centrifuge tubes (12 ml, 30 ml
and 50 ml); Eppendorf tubes; glass spreaders; rubber teats; propipettes
30o incubator; 37o orbital incubator; microcentrifuges (one at 4o, one at room
temperature); high-speed and ultracentrifuges; 45-50o and 70o water baths; tube
racks

1.

Grow phage overnight in 2.5 ml top layers (containing host spores) on six plates
each inoculated with 0.1 ml DNB containing 2 × 104 pfu per plate. It is usuallypossible to assume that a fresh overnight plaque will yield 107 pfu. From this, 2 × 104 pfu should
give just confluent lysis on 9 cm Petri plates for C31. Adjustments may be needed for phages
giving very small or very large plaques. Cool SNA to c. 45o before adding spores to give aslightly turbid suspension, and dispense as quickly as possible.

2.

Using a spreader, scrape off top layers and pool them in 25 ml DNB in a 50 ml
centrifuge tube. Suck the mixture into and out of a 10 ml pipette twice, andleave it at room temperature for 2 h. Take care to avoid cross-contamination of phages
by aerosols formed when blowing out the pipette. The top layer can be left to soak out for up
to 6 h but no more.

3.

Centrifuge at 2, 000 × g (10, 000 rpm, Sorval SS-34 rotor) for 10 min at 4o to
remove agar.

4.

Gently pour the supernatant into a 30 ml centrifuge tube, and centrifuge at 25,
000 rpm (55, 000 × g) for 75 min at 4o to sediment phage.

5.

Discard supernatant and resuspend phage pellet in 2 ml RNase solution; incubate
at 37o on orbital incubator for 20 min.

6.

Transfer to 12 ml centrifuge tube and add 400 µl SDS mix; incubate at 70o for
30 min in water bath.

7.

Immediately add 500 µl 8M potassium acetate and leave on ice for 15 min.

&KDSWHU6WUHSWRP\FHVSKDJHV



8.

Centrifuge at 2o for 30 min at 30, 000 × g (17, 000 rpm, Type 30 rotor).

9.

Decant supernatant into 50 ml centrifuge tube.

10.

Add 7 ml TE.

11.

Add 25 ml absolute ethanol and leave at -20o overnight to precipitate.

12.

Centrifuge at 4o for 15 min at 7, 500 × g (8, 000 rpm, Sorval SS-34).

13.

Pour off supernatant and resuspend the pellet in 1 ml TE.

14.

Add 0.33 ml 3M NaOAc to a final concentration of 0.3M.

15.

Add 3 ml absolute ethanol, and swirl tube until DNA precipitates as a fibrous
clot.

16.

Spool DNA on a sealed Pasteur pipette and transfer to a 1.5 ml Eppendorf tube.

17.

Resuspend in 300 µl TE, and add 33 µl 3M NaOAc (final concentration 0.3M).

18.

Add 1 ml absolute ethanol and precipitate DNA at room temperature.

19.

Remove ethanol with a Pasteur pipette.

20.

Wash precipitated DNA with 250 µl 70% ethanol.

21.

Remove all ethanol with pipette tip.

22.

Dissolve in 25-100 µl TE buffer and store at 4o. 3 µl DNA should be enough for each
10 µl digest. Problems have been encountered with digestions using SphI and EcoRI
endonucleases (excess of enzyme is often necessary for digestion).



&KDSWHU6WUHSWRP\FHVSKDJHVAnné J, Wohlleben W, Burkardt HJ, Springer R, PühlerA (1984) Morphological and molecular characterization ofseveral actinophages isolated from soil which lyse
Streptomyces cattleya or S. venezuelae. J.Gen.Microbiol.,
130, 2639-2649.
Bedford DJ, Laity C, Buttner MJ (1995) Two genes
involved in the phase-variable C31-resistance mechanism ofStreptomyces coelicolor A3(2). J.Bacteriol., 177, 4681-4689.
Chater KF (1986) Streptomyces phages and their application
to Streptomyces genetics. In Queener, S.W. and Day, L.E.
(eds.), The Bacteria. A treatise on structure and function,
vol. 9, Antibiotic-producing Streptomyces. Academic Press,
Orlando, Florida, pp. 119-158.
Chater KF, Carter AT (1979) A new wide host-range,
temperate bacteriophage (R4) of Streptomyces and its
interactions with some restriction-modification systems.
J.Gen.Microbiol., 115, 431-442.
Chater KF, Wilde LC (1980) Streptomyces albus G mutants
defective in the SalGI restriction-modification system.
J.Gen.Microbiol., 116, 323-334.
Chater KF, Bruton CJ, Springer W, Suarez JE (1981)
Dispensable sequences and packaging constraints of DNA
from the Streptomyces temperate phage C31. Gene, 15,249-256.
Cox KL, Baltz RH (1984) Restriction of bacteriophageplaque formation in Streptomyces spp. J.Bacteriol., 159, 499504.
Dowding JE (1973) Characterization of a bacteriophagevirulent for Streptomyces coelicolor A3(2). J.Gen.Microbiol.,76, 163-176.
Haket J, Jr., Desmarais D, Mehindate K, Déry CV (1990)
Saccharopolyspora hirsuta strain 367 releases JHJ-1, abacteriophage capable of propagation on old mycelium.
J.Gen.Microbiol., 136, 573-579.
Hong J-S, Ames BN (1971) Localized mutagenesis of any

specific small region of the bacterial chromosome.
Proc.Natl.Acad.Sci.USA, 68, 3158-3162.
Klaus S, Hartmann M, Krügel H, Roth M, Walter F,
Rautenstein YI, Solovyeva N (1981) Restriction ofStreptomyces phage SH5 by endonuclease ShyI from
Streptomyces hygroscopicus 0477. Mol.Gen.Genet., 184, 286288.
Laity C, Chater KF, Lewis CG, Buttner MJ (1993) Genetic
analysis of the C31-specific phage growth limitation (Pgl)system of Streptomyces coelicolor A3(2). Mol.Microbiol., 7,329-336.
Lomovskaya ND, Chater KF, Mkrtumian NM (1980)
Genetics and molecular biology of Streptomyces
bacteriophages. Microbiol.Rev., 44, 206-229.
Matsushima P, Cox KL, Baltz RH (1987) Highly
transformable mutants of Streptomyces fradiae defective inseveral restriction systems. Mol.Gen.Genet., 206, 393-400.
Ogata S, Suenaga H, Koyama-Miyoshi Y, Hayashida S(1984) Cloning of the his gene of Streptomyces azureus intemperate phage SAt1. J.Gen.Appl.Microbiol., 30, 405-409.
Stuttard C (1979) Transduction of auxotrophic markers in achloramphenicol-producing strain of Streptomyces.
J.Gen.Microbiol., 110, 479-482.
Stuttard C (1983) Localized hydroxylamine mutagenesis,
and cotransduction of threonine and lysine genes, in
Streptomyces venezuelae. J.Bacteriol., 155, 1219-1223.
Stuttard C (1989) Generalized transduction in Streptomyces
species. In Hershberger, C.L., Queener, S.W. and Hegeman,
G. (eds.), Genetics and Molecular Biology of Industrial
Microorganisms. American Society for Microbiology,
Washington,DC, pp. 157-162.
Vats S, Stuttard C, Vining LC (1987) Transductional
analysis of chloramphenicol biosynthesis genes in
Streptomyces venezuelae. J.Bacteriol., 169, 3809-3813.

m13tkb.wpd 20 April 2000 Tobias Kieser



Chapter 13

Cloning with phage vectors
CONTENTS
General features of C31 and its vector derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290
Shotgun cloning with C31 vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290
Choice of C31 vectors for mutational cloning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291
Choice of C31 vectors for screening by complementation of mutantsor acquisition of new capabilities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292
Ligation conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 294
Maximising and estimating insert frequency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 294
Construction and stability of lysogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 294
Homogenotisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295
Application of C31 to gene fusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 296
General features of the C31::xylE vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 296
KC862: a xylE-containing C31 derivative that gives yellow plaques
only when carrying inserts with active promoters . . . . . . . . . . . . . . . . . . . . . . . . . . . . 296
A single copy number promoter-probe vector, KC859 . . . . . . . . . . . . . . . . . . . . . . . . . 298
Vectors for in situ fusions of xylE to chromosomally located transcription units . . . 298
Other phage-based cloning systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299
Prophage transformation with phage SAt-1 of S. azureus . . . . . . . . . . . . . . . . . . . . . . 299
Phage-mediated transduction of plasmids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299
Vectors based on other Streptomyces phages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300
Use of integration functions of Streptomyces phages . . . . . . . . . . . . . . . . . . . . . . . . . . . 300
Transfection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301
Transfer of Streptomyces phage DNA onto nitrocellulose filters for“plaque hybridisation” . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303
Preparation of C31 lysogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303
Procedure A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 304
Procedure B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305“Low-tech” method for detecting C31 derivatives containing resistance genes . . . . . . . . . 306
Use of glkA counterselection to select deletions from, or loss of, C31 prophages . . . . . . . . 307



&KDSWHU&ORQLQJZLWKSKDJHYHFWRUVGeneral features of

C31 and its vector derivatives

The temperate phage C31 is reminiscent of coliphage lambda in morphology and generalgenetics (Lomovskaya et al., 1980). It forms plaques on and/or lysogenises about two-thirds ofstreptomycetes. The prophage integrates into the chromosome via specific phage and chromosomal att
sites, and is maintained by a repressor specified by the phage’s c gene. Each round of vegetative phage
infection takes about 50 min, with burst sizes of 10-100. C31 DNA isolated from virions is 41.4 kb inlength, contains 63% G + C, and has cohesive (cos) ends. Restriction maps of C31 DNA are on pp.
520-522. The sequence of C31 has been fully determined (Sinclair and Bibb, 1988; Kuhstoss and Rao,
1991; Rausch and Lehmann, 1991; Hartley et al., 1994; Smith et al., 1999), and an overview diagram ofits genetic organisation is in Fig. 13.1.
Vectors were initially made from C31 by a series of manipulations using in vivo deletions(selected by exposure of the phage to chelating agents), insertion of pBR322 at an EcoRI site, further invivo deletions and recombination, and insertion (into the pBR322 region) of Streptomyces antibiotic
resistance genes. Relevant restriction map information on some C31 vectors is on pp. 520-529, anda general scheme for a typical C31-mediated cloning experiment is as follows:
C31 vector DNA digest + donor DNA digest
9
ligate9transfect S. lividans protoplasts
9
replicate plaques to lawn of indicator strain
9
allow sporulation
9
replicate to plate containing appropriate antibiotic
9score relevant phenotype

Shotgun cloning with

C31 vectors

In most primary cloning experiments using C31 vectors the phenotypic expression of cloned
DNA is examined after inserting the vector into the chromosome, using the resistance genes present in
the vector to select lysogens. Integration can be through att-specific recombination (e.g. in Table 13.1),
but most shotgun cloning experiments have used att-deleted vectors (because they have more space for
DNA insertion). These can integrate by homologous recombination, either into a pre-existing prophage
to form a double lysogen (in which case a vector with a deleted c gene can be used; e.g. KC401, KC505,
KC518 in Table 13.2) or at the chromosomal site homologous with a DNA fragment cloned into thevector (in which case the vector needs an intact c+ gene to form a stable prophage: useful examples suchas KC515 are listed in Table 13.2). For the cloning space of vectors, see Tables 13.1 and 13.2.
The use of att-site-mediated integration (including the double prophage route) allows clone
recognition and analysis by conventional mutant complementation (e.g. Piret and Chater, 1985).

&KDSWHU&ORQLQJZLWKSKDJHYHFWRUVFig. 13.1 Genetic organisation and basic restriction map of C31
Complementing DNA cloned by this route usually contains a fully functional gene. Integration directedby the cloned DNA may also be used to complement mutations, but in this case the complementing DNA
may sometimes lack one end of the functional gene. An alternative approach is offered if the cloned
DNA is entirely internal to a functional gene (or transcription unit), since a mutation is generated whenthe vector is integrated via the cloned fragment (p. 312). Such “mutational cloning” (e.g. Chater andBruton, 1983; Seno and Chater, 1984) can allow effective cloning to be done without recourse to mutantsor protoplasting technology in the strain of interest.
Note that S. lividans is usually the primary host for transfection, and that the final host for clone
recognition does not have to be converted into protoplasts. It may be beneficial to expose the S. lividans
overlays containing plaques to CHCl3 vapour, if the indicator strain proves subject to inhibition by S.
lividans carried over during replication. For example, this has been observed with S. albus G. Exposureusually involves inversion over a glass Petri dish containing a few drops of CHCl3. It must be done ina fume hood. Exposure for more than 10 min is unnecessary and will result in gradual disintegration ofplastic Petri dishes.
Choice of

C31 vectors for mutational cloning

Where there is reason to expect that transcription units of several kilobases are involved, as is
likely for antibiotic production genes for example, mutational cloning should be considered. This should
be done with inserts of 1-2 kb: i.e. large enough to allow integration to take place readily, but smallenough to fall within many polycistronic transcription units. The most frequently used vectors for thispurpose are KC515, KC516 and PM8 (Table 13.2 and pp. 525, 529), which allow the insertion of up to
4 kb or 3.3 kb, respectively, at any of several restriction sites. However, the use of KC857 or other xylEcontaining vectors (Table 13.2, Figs. 13.2 and pp. 523, 527-528) offers the possibility of simultaneously

&KDSWHU&ORQLQJZLWKSKDJHYHFWRUV

Table 13.1

C31-based att+cloning vectors For maps, see Chapter 22

Phage

Resistance genes andother features

KC210
KC212

Sites for cloning

Comments

tsr, fd terminatoraac(3)IV

BglII, PstI
BglII, BamHI,
PstI, StuI

Up to 4 kb insert
Up to 4 kb insert

KC304

vph, tsr

BamHI
SstI
PstI
PvuII

Up to 5 kb insert; tsr selection
Up to 3.5 kb insert; tsr selection
Up to 3 kb insert; vph or tsr selection
Up to 3 kb insert; vph selection

KC859

tsr (xylE promoterprobe, contains fd
terminator)

BglII, BamHI

Up to 2.9 kb insert

constructing transcriptional fusions to the xylE reporter gene (which have proven helpful in studiesof antibiotic production: Guthrie and Chater, 1990; Bruton et al., 1991; Brian et al., 1996: Fig 13.3 andp. 343). Moreover, some of the xylE vectors contain relatively large numbers of restriction sites in thecloning polylinker, and in one of them (KC857) the presence of a transcriptional terminator upstream ofthe polylinker greatly reduces the likely influence of transcription from the vector into the chromosomeof lysogens obtained by insert-directed integration (this feature is also present in PM8). If sizefractionated MboI-generated fragments are inserted into any of these vectors, the fragments can be reexcised (with little additional DNA) via nearby restriction sites.
Choice of C31 vectors for screening by complementation of mutants or acquisition of newcapabilities
The C31 c+ att+ derivative KC304 (Table 13.1 and p. 523) has been successfully used forshotgun cloning of DNA complementing several morphological mutants (e.g. Elliot et al., 1998). Newer
KC304-like vectors (KC210 and KC212: C.J. Bruton, personal communication) offer slightly more spaceor alternative selections (Tables 13.1 and 22.1, p. 513). The very efficient lysogenisation via the att site
makes it easy to obtain transductants, even on some strains that do not support plaque production.
However, for inserts of more than 4 kb, att-deleted vectors offer more space. The indicator strain may
then either be a C31 lysogen (in which case the indicator does not need to contain DNA homologous
to the cloned insert and the vector can be a high-capacity c-deletion derivative such as KC401, KC505
or KC518; Table 13.2 and p. 526), or a non-lysogen (in which case the indicator must contain DNA
homologous with the insert, and the vector must contain a functional c gene: suitable vectors are listed
in Table 13.2).

&KDSWHU&ORQLQJZLWKSKDJHYHFWRUV

Table 13.2

C31-based att– cloning vectors For maps, see Chapter 22

Phage

Resistance genes
and other features

Sites for cloning

Comments

KC211
KC401

aac(3)IV
vph, c-deleted

BamHI, BglII, PstI
PstI

Up to 5 kb insert; apramycin selection
2-8 kb replacement positively
selected; vph selection
Up to 4 kb insert; vph selection

PvuII
KC505

vph, tsr, c-deleted

PstI
BamHI

a

2-8 kb replacement positively
selected; vph selection
1.5-7.5 kb replacement vector; noselection (also sites for PvuII and SstI)KC515,
KC516

vph, tsr

PstI,BamHI,BglII, XhoI
SstI
PvuII

Up to 4 kb insert; vph or tsr selection
Up to 4.5 kb insert; tsr selection
Up to 4 kb insert; vph selection

KC517

vph

PstI,BglII,XhoI
SstI

Up to 6 kb insert; vph selection
Up to 6.5 kb insert; no selection

KC518

vph, tsr, c-deleted

PstI
BamHI
PvuII
SstI

Up to 6 kb insert; vph and tsr selection
2-8 kb replacement positively
selected; vph selection
Up to 6 kb insert; vph selection
Up to 6.5 kb insert; tsr selection

KC857

vph, tsr, xylE,
fdter

BamHIa (XhoI, ScaI,
DraI)bUp to 2.2 kb inserts. Useful for in situ
fusions to xylE

KC858

vph, tsr, xylE

BamHI, BglII (XhoI,
ScaI)bUp to 2.5 kb insert. Useful for in situ
fusions to xylE

KC860

vph, tsr, xylE

BamHI (BglII, XhoI,
ScaI, DraI)b

Up to 2.8 kb inserts. Useful for in situ
fusions to xylE

KC861

tsr, xylE

SstI, BamHI (BglII,
XhoI, ScaI, DraI)b

Up to 4 kb inserts. Useful for in situ
fusions to xylE

KC862

tsr, xylE oriented
right to left

BamHI, SstI (BglII,
XhoI, DraI)b

Up to 3.4 kb inserts. Used for in situ
xylE fusions and detecting promotersactive during C31 lytic cycle

PM8hyg, tsr, ter(fd)

PstIa, BamHIa, BglIIa,
XhoI
PvuII
SstI

Up to 3.3 kb insert; hyg or tsr
selection
Up to 3.3 kb; hyg selection
Up to 3.3 kb; tsr selection

Sites located next to fdter, preventing readthrough transcription
Sites not upstream of xylE reporter gene

b

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&KDSWHU&ORQLQJZLWKSKDJHYHFWRUV

Ligation conditions
Because of the large size of C31 compared with the inserts, much higher DNA concentrations(usually 100-200 µg ml–1) are used in ligation mixtures than is typical with plasmids (for a relevant
discussion, see Williams and Blattner, 1980). The molar ratio of vector:target DNA is usually about 1:2.
Alkaline phosphatase treatment of the cleaved vector has been used to prevent the formation of multimers(Ikeda et al., 1984), but does not seem to be very important. Reconstruction experiments suggest thatlow DNA concentrations (5 µg ml–1 vector, 5 µg ml–1 donor fragments of about 5 kb) in the ligation
mixture may be effective when the cohesive ends of the vector have been ligated together before cleavage
by a restriction enzyme and treatment with alkaline phosphatase (H. Ikeda, personal communication).
This should be useful if donor DNA is available in very small amounts (as may happen with the subcloning of gel-purified fragments), but it is probably applicable only to relatively large donor fragments(several kilobases).
Maximising and estimating insert frequency
In some vectors there is a positive selection for inserts because of packaging constraints(“replacement vectors”: Table 13.2), so that the fraction of transfectant plaques with inserts should
usually be very high, provided that the vector was efficiently digested before ligation, and that the“stuffer” fragment to be replaced has not been re-inserted in too high a fraction of plaques. The
efficiency of vector digestion is checked by electrophoresis and ideally also by loss of transfection
efficiency. Re-insertion of the stuffer fragment is reduced by cleaving (if possible) with a second enzymethat cuts within the stuffer fragment but not elsewhere in the vector, and by adding a few-fold molar
excess of donor fragments. Measurement of insert frequency into PstI-cut KC401 or KC505 and BamHIcut KC518 may be done by replica-plating plaques to a lawn of J1501/ C31 mixed 1:1 with nonlysogenic J1501 on R2YE medium, followed 4 days later by replication to thiostrepton-containing
medium (replacement of the tsr-containing vector fragment results in loss of ability to generatethiostrepton-resistant transductants).
With insertional vectors, such as KC515 or KC516, there is no positive selection for inserts, but
recognising and quantifying inserts is conveniently done by replication to a lawn of a strain containing
DNA homologous with that being cloned. For example, an S. coelicolor DNA library in KC515 plaques
can be replicated to S. coelicolor J1501 on R2YE, followed after 4 days by further replication to medium
containing thiostrepton or viomycin. Plaques containing inserts (of more than 1 kb) will give a few
resistant transductants, which may subsequently be screened for particular phenotypic changes.
If a defined piece of DNA is being inserted, the quickest way to detect plaques containing theinsert is plaque hybridisation (p. 303 )Construction and stability of lysogens
Most of the available experience has been with S. coelicolor, and its applicability to other species
may need investigation.
It is sometimes necessary to make lysogens of strains of a specific genotype, for use as indicatorstrains with attP-deleted vectors. The c+ attP+ aacC(3)IV-, vph- or tsr-containing phages KC212, KC300

&KDSWHU&ORQLQJZLWKSKDJHYHFWRUV

and KC301 are very useful for this purpose (Tables 13.1 and 22.1, p. 513) and are apparently very stable
in S. coelicolor and some other species. (However, S. lividans lysogens constructed with att+ C31
vectors seem less stable, and we have usually avoided their use. This is particularly marked with phages
carrying the ¶W12 deletion, which ends very close to attP (e.g. KC212, KC300, KC301, KC304, KC859:
Table 13.1 and p. 523); these give rise predominantly to unstable (possible pseudo) lysogens in S.
lividans, although reasonably stable lysogens can usually be obtained eventually.) The selectable
markers of KC212, KC300 and KC301 are particularly helpful with strains which can be lysogenised by
C31 but do not support plaque formation, a phenomenon observed with the wild-type Pg1+ S. coelicolor(but possibly of wider occurrence) (p. 276).
Double lysogens made by superinfection of a lysogen with an attP-deleted vector are moderately
stable on agar medium (usually about 50-85% of spores contain both prophages after a round of nonselective growth and sporulation). Lysogens made by insert-directed integration are more stable thantandem double lysogens (usually at least 99% of spores retain the prophage after a round of non-selective
growth on agar medium), but there is circumstantial evidence of more frequent prophage loss in liquid
cultures (Ikeda et al., 1984), or when attP-deleted phages are targeted into transmissible plasmids (Chaterand Bruton, 1983).
Homogenotisation
Recombination between cloned sequences and their homologues in the chromosome maycomplicate the interpretation of data obtained with cloned DNA. There seems to be a rather dynamic
recombinational interrelationship between sequences cloned in plasmids and their chromosomal
counterparts, which interferes with true complementation and dominance tests (e.g. Seno and Chater,
1984). Prophages integrated at the chromosomal attB site, either via integrase-mediated recombination
or by recombination with a preexisting prophage, are less subject to such problems, though during several
rounds of sub-culture homogenotes may eventually become predominant. Thus, provided that relevant
analysis is done on freshly made heterogenotes of this kind, they provide a useful route for functional
tests.
When a prophage is formed by insert-directed recombination into a recipient carrying a different
allele of the cloned DNA, homogenotisation is quite frequent (often more than 10% of primary lysogens)(pp. 328-332). This is probably associated with the recombination event by which the prophage wasinserted (for an interpretation of similar events in Myxococcus, see Shimkets et al., 1983). It has beenused as a convenient route for the insertion of a particular allele into a vector, to allow further functional
and physical analysis. However, caution may be necessary: if a mutant contains more than one difference
from the wild-type gene, homogenotisation may not occur at both/all base differences. Moreover, at least
for work with S. coelicolor, the convention of propagating the phages on S. lividans, which has c. 99%
DNA sequence identity to S. coelicolor A3(2) in many genes, allows for possible recombination between
the insert and the chromosome of the host used for propagation. In some circumstances, pgl– mutants of
S. coelicolor could be used instead of S. lividans, but it is important to remember that in that case theligating DNA molecules must be free of methylation, to avoid severe restriction (p. 230).


Application of&KDSWHU&ORQLQJZLWKSKDJHYHFWRUV

C31 to gene fusions

Attempts have been made to exploit C31 in the study of gene expression, by incorporating
reporter genes next to cloning sites (see also Chapter 15). Constructs involving lacZ (King and Chater,1986) and vph (Rodicio et al., 1985) proved of little practical utility. Most success has been withtranscriptional fusion vectors containing the xylE reporter gene, encoding catechol 2,3-dioxygenase,which gives a yellow product when catechol is present (the Ylo+ phenotype). Three kinds of vector areavailable: those in which expression of cloned promoters can be detected in plaques during phage
multiplication; attP+ vectors in which the activity of cloned promoters can be assessed at single copy
when integrated at the host’s C31 attB site; and attP-deleted vectors in which insert-directed phage
integration places the reporter gene inside a transcription unit at its correct chromosomal location.

General features of the

C31::xylE vectors

The structures and properties of six xylE-containing C31 derivatives are summarised in Fig.
13.2, Table 13.1 and Table 13.2. All these vectors contain a functional repressor gene (c), needed toestablish and maintain lysogeny, and the tsr (thiostrepton resistance) gene for selection of lysogens.
Several also contain the alternative selective gene vph which confers viomycin resistance. The xylE open
reading frame is preceded by a strong ribosome binding site, upstream of which is a polylinker containing
a unique BamHI site flanked on one side by an XbaI site and on the other by several restriction sites (inKC857, KC858 and KC859 including another XbaI site). This will usually allow the ready recovery of
MboI-generated fragments inserted at the BamHI site. In the 72 bp of vector DNA between the BamHI
site and the xylE translational start codon, there are translation stop codons in each possible reading
frame, so that transcriptional, not translational, fusions to xylE are ensured (M. Zukowski, personalcommunication).
KC862: a xylE-containing
with active promoters

C31 derivative that gives yellow plaques only when carrying inserts

The natural transcription of C31 during lytic infections (Ingham et al., 1993) leads tosignificant left-to-right readthrough transcription into the “cloning region” of C31 vectors. The fd
major terminator has little effect on this readthrough (King and Chater, 1986; Bruton et al., 1991), eventhough this terminator is effective in other situations in Streptomyces (Ward et al., 1986). It is tentatively
assumed that an antiterminator is active during C31 lytic infections. In most of the xylE-containing
phages, xylE is inserted with a left-to-right orientation, and readthrough from vector promoters gives riseto the Ylo+ phenotype in plaques (for details of the detection of xylE expression in plaques, see p. 344).
To circumvent this, KC862 was constructed with xylE orientated right-to-left (Fig. 13.2). With thisphage, the Ylo+ plaque phenotype is obtained only when a promoter-active insert is placed upstream of
xylE. Although this provides a method of cloning and analysing some promoters, it is important to beaware that many host promoters are shut down during C31 lytic infections (Rodriguez et al., 1986;
Clayton and Bibb, 1990), so it is likely that this will limit the range of promoters to which KC862 canbe applied in this mode.

&KDSWHU&ORQLQJZLWKSKDJHYHFWRUV

Fig.13.2 Six xylE-containing C31 vectors (after Bruton et al., 1991). For the availability of MCS sites
for cloning and for detailed restriction maps, see pp. 523 and 527-528.

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Fig. 13.3 Insert-directed fusion of xylE into a chromosomally located transcription unit, using an attP–,
xylE-containing C31 vector
A single copy number promoter-probe vector, KC859
KC859 (Fig.13.2 and Table 13.1) contains the C31 attP site and can establish lysogeny very
efficiently. Colonies of the lysogens have a Ylo– phenotype unless a suitably orientated promoter iscloned in the vector. This allows the use of KC859 to assess promoter activity in cloned DNA at a single
copy number and separated from its normal context. Because lysogens made by attP-mediatedintegration are highly stable, selection for the vector is not necessary even during prolonged culture once
the lysogen has been constructed. For details of catechol 2, 3-dioxygenase detection and assay, see
pp. 343-345.
Vectors for in situ fusions of xylE to chromosomally located transcription units
KC857, KC858, KC860, KC861 and KC862 (Fig 13.2, Table 13.2 and pp. 523, 527-528) allow
insertion of xylE into chromosomal transcription units (Fig.13.3). For this purpose, it is not necessary
for the cloned DNA fragment to contain a promoter, nor for the vector to contain a terminator upstreamof the cloning sites (insert-directed integration separates the fused transcription unit from any possible
interference from upstream vector sequences). Nevertheless, the presence of the fd terminator upstreamof the polylinker in KC857 may be helpful when the vector is used analytically in operon disruption by
mutational cloning (p. 334) since, being more than 95% effective in terminating transcription in
Streptomyces plasmids, the terminator is expected to reduce greatly any potential problems caused byreadthrough transcription from the vector prophage into the adjacent chromosome. Any supposed
antiterminator activity encoded by C31 (p. 296)) is irrelevant in lysogens, since the phage’s lytic
functions are repressed.

&KDSWHU&ORQLQJZLWKSKDJHYHFWRUV

It is worth noting that promoters cloned in att– xylE C31 vectors can alternatively be integrated
at the C31 attB site, if desired, by superinfecting a strain that already contains a C31 prophage in the
attB site. The >30 kb of sequence homology shared by the incoming phage and the prophage allows
efficient recombination to give moderately stable double lysogens (see “Construction and stability oflysogens”, p. 294).

Other phage-based cloning systems
Prophage transformation with phage SAt-1 of S. azureus
Ogata et al. (1984) used a narrow host-range temperate phage, SAt-1, for primary cloning andgene replacement in S. azureus. The strategy used for shotgun cloning with SAt-1 is different from thoseused with C31, so we summarise it here.
The method, first used in Bacillus subtilis by Kawamura et al. (1979), was used to clone a his
gene of S. azureus (Ogata et al., 1984). Chromosomal DNA fragments were ligated with partiallydigested phage DNA so that the ligation mixture was expected to contain the desired his+ fragment
flanked on both sides by fragments of phage DNA, some of them in the same configuration as in the SAt1 genome. When the ligation mixture was used to transform a his– SAt-1 lysogen of S. azureus, such
constructs could generate his+ transformants by integrating into the prophage by a double crossover.
Although many such recombinant prophages would have lost the ability to form viable, plaque-forming
phages, some did not (presumably essential phage genes had not been disrupted or deleted in these cases),so they could release viable his+-transducing phages on induction (SAt-1 is inducible by mitomycin C
treatment).
Phage-mediated transduction of plasmids
Phages package their own DNA either by cleavage at specific cos sites, in the case of phages
such as or C31, or, in phages that use headful packaging (including typical generalised transducing
phages), by sequential cleavage of headful lengths (just over one genome length) starting from a definedpac site. It follows that cos or pac sequences in “non-phage” DNA potentially render such DNA
packageable by cognate phages. Thus, phage preparations grown on hosts that contain cognate cos or
pac sequences may contain a certain, usually minor, proportion of particles in which non-phage DNA
has been packaged. The natural adsorption and injection mechanisms of the particles may then allow theeasy introduction of this DNA into the next host encountered.
Several attempts have been made to capitalise on the possibility of such in vivo packaging as aconvenient means of moving plasmids from one Streptomyces host to another. In one case the cohesiveend site (cos) of the wide host-range temperate phage R4 was incorporated into pIJ365 (a pIJ101
derivative) to give pR4C1 (6.9 kb), which could easily be packaged as a linear concatemer during R4
infection of S. lividans/pR4C1 to permit its transduction to a recipient strain. Up to 3 × 10–3
transductants were obtained per infectious phage (Morino et al., 1985, 1986). Kobler et al. (1991)
reported less efficient, but potentially useful, transduction of a C31 cos-containing plasmid by C31.
Both the R4 and the C31 in vivo cosmid systems appear to have potential utility, but neither has beendeveloped into a widely adopted system.

&KDSWHU&ORQLQJZLWKSKDJHYHFWRUVMore use has been made of a system based on FP43, a wide host-range temperate Streptomyces
phage that uses headful packaging (Hahn et al., 1991). Starting from a defined pac site, FP43 DNA ispackaged from the concatemeric replicated form as c. 56 kb headfuls. McHenney and Baltz (1988) foundthat pIJ702 containing pac in a 7.8 kb fragment of FP43 (the plasmid was called pRHB101) was
efficiently transduced by FP43 between S. griseofuscus strains. The transducing phages containedpRHB101 DNA in the form of linear concatemers. The transducing lysates could introduce pRHB101
into many other Streptomyces species.
Successful transduction was also obtained with
Saccharopolyspora spp. but not with representatives of nine other genera of actinomycetes. Further tests
showed that transduction frequencies could be increased by strain-specific optimisation, the main
parameters being the recipient’s growth phase and cultivation temperature (Matsushima et al., 1989;
McHenney and Baltz, 1989). Use of the FP43/pRHB101 transduction system may have potential to
bypass transformation problems with “difficult” strains. However, its main use has been in the deliveryof IS493 derivatives such as Tn5096 (p. 117), using a temperature-sensitive version (pRHB106) ofpRHB101. FP43 is available as a lysogen of S. griseofuscus (NRRL B-18184), and pRHB106 is carried
by S. griseofuscus NRRL B-18183.
Vectors based on other Streptomyces phages
Developments along the same lines as those for C31-based vectors have been reported for atleast three other wide host-range temperate phages, though only in the case of the S. cattleya phage TG1
was the system used for cloning purposes. TG1 resembles C31 in its overall genome organisation, andits host-range is somewhat complementary to that of C31 (Foor et al., 1985). Shuttle TG1 plasmid
vectors were constructed containing the E. coli plasmid pACYC177 (Foor and Morin, 1990). A TG1
derivative was used to clone the argG gene of S. cattleya (Streicher, 1983). Another phage, HAU3,
was used to construct pIJ8301, a phasmid (phage-plasmid chimaera) containing an E. coli cosmid (Zhouet al., 1994). In addition, a derivative of phage R4 termed L4, containing a single BamHI site for cloning,
and with space for up to 4 kb, was constructed by Ishihara et al. (1983).
Use of integration functions of Streptomyces phages
The att-int region of C31 has been used as the foundation of several integrating vectors (pp.
216 and 218), which appear to have the advantages of higher integration frequency and simpler
integration patterns than equivalent vectors based on the pSAM2 att-int region (Kuhstoss et al., 1991).
A DNA segment extending from ClaI site “5.49" to KpnI site “9.36" (c. 4 kb) (p. 522) was used in
pKC796 (a simple integrating plasmid) and pKC731 (an integrating cosmid). Subsequent analysis
showed that only 2.1 kb of this 4 kb fragment was needed for integration (Kuhstoss and Rao, 1991). The
2.1 kb segment extends rightwards from a position about 300 bp left of EcoRI site 19 (Fig. 13.1). Morerecently, att-int containing plasmids that can be transferred by conjugation from E. coli to Streptomyces
spp. have found increasing use (p. 249). Matsuura et al. (1996) described pAT98, an E. coli plasmid that
contains tsr and the att-int regions of phage R4 and is capable of efficient and stable transformation of
S. parvulus to thiostrepton resistance. However, S. coelicolor and S. lividans appear to lack attB sites
for R4.

&KDSWHU&ORQLQJZLWKSKDJHYHFWRUVTransfection
Phage DNA is introduced into Streptomyces by PEG-assisted transfection of protoplasts, using
conditions much like those for transformation with plasmids. Parameters affecting transfection frequency
have been studied using DNA from various Streptomyces phages (Suarez and Chater, 1980; Isogai et al.,1980). One particularly useful tip is to avoid using too many protoplasts (see instruction 2 of theprocedure below). The age of cultures used for protoplasting may have some effect, although there havebeen contradictory results with S. parvulus: Suarez and Chater (1980) found it necessary to use young
cultures, whereas Isogai et al. (1980) found older cultures very satisfactory. Generally, conditions similar
to those used for transformation give about 105 plaques per microgram of native C31 or R4 DNA,
irrespective of whether or not the DNA has been circularised in vitro by ligation of the cohesive ends.
For different phages, the effect of increasing DNA amount on transfection frequency may differ.
However, for C31 in S. lividans or R4 in S. parvulus there is a direct proportionality up to at least 100
ng in the transfection mixture, with some evidence of a slight fall in frequency (though a continued
increase in absolute numbers) with 1 µg of DNA.
In early experiments with C31 and S. lividans protoplasts, an improvement in transfection was
obtained by the addition of positively charged DNA-free liposomes to the mixture together with anincreased concentration of PEG (Rodicio and Chater, 1982). New batches or sources of lipids later
proved ineffective, and we no longer use liposomes. Meanwhile, frequencies have in any case improved
as the transfection technique has been refined. 105-107 plaques per microgram are routinely obtainedwith uncut linear phage DNA, with a reduction of about 103-fold when ligation mixtures are used.
Although there is a significant decrease in transfection frequency with high amounts (100-1000 ng) of
native C31 DNA, we have not usually observed similar reductions with ligation mixtures. Phosphatasetreatment of C31 DNA does not reduce transfection frequencies (H. Ikeda, personal communication).
The procedure described here was optimised for S. lividans 66 but it also works well with some otherstrains. Almost certainly some further optimisation could help for particular strains. In outline, DNA,
protoplasts and PEG are briefly mixed and then plated in half-strength agar regeneration medium (“softR2 agar”) containing S. lividans spores as an overlay on R2YE base plates. This medium supports C31
infection, and transfected protoplasts give rise to plaques after overnight incubation.
Freshly prepared S. lividans protoplasts from fresh or frozen mycelium (p. 58)
Spores of S. lividans
DNA in TE buffer or ligation buffer (up to 20 µl per transfection)
9 cm or 5 cm Petri plates of R2YE
Soft R2 agar (equal volumes of R2YE and P buffer, freshly mixed together andmaintained at c. 70o until just before use; the soft agar can be kept at temperatures as low
as 45o, but you may need nimble fingers to pour the overlays before the agar sets)
25% PEG solution (1 g sterile molten PEG 1000 added to 3 ml P buffer)
P buffer
Pipettes; sterile plastic tubes
Bench centrifuge (e.g. c. 5cm radius to tube centres); 30o incubator ; 45-50o and 70owater baths



1.

&KDSWHU&ORQLQJZLWKSKDJHYHFWRUV

Sediment protoplasts gently in the bench-top centrifuge (e.g. 3,000 rpm, 7 min)and resuspend immediately in 10 ml P buffer. We do not recommend frozen protoplasts
for transfection since the number of transfectants obtained is at least tenfold lower than whenusing fresh protoplasts. However, with protoplasts freshly prepared from frozen mycelium there
is no difference in the transfection frequency compared with protoplasts from fresh mycelium.
Protoplasts can be stored for up to 24 h at 4o.

2.

Divide protoplast suspension into the required number of aliquots in plastic tubes
and add P buffer to a final volume of 10 ml. Centrifuge in bench-top centrifuge(c. 3,000 rpm) for 7 min. While the centrifuge is running, prepare for steps 4 and5. For step 5 it is useful to pre-load a Pasteur pipette with 0.5 ml of 25% PEG
solution. About 12-20 transfections can be made with the protoplasts from a 25 ml culture.
The pellet after centrifuging each aliquot of protoplasts should be only just visible. The
transfection frequency is reduced if much larger amounts of protoplasts are used.

3.

Discard supernatant, and resuspend the protoplast pellet in the drop of P buffer
left after pouring off the supernatant.

4.

Immediately add DNA (up to 20 µl) to the protoplast suspension. Usually, 0.3-1µg of DNA is used per transfection with ligation mixtures, to give c. 102-103 plaques. It is
usually wise to keep at least half of a ligation mixture at 4 o in case the transfection fails, and tocarry out a control transfection with 10 ng uncut C31 DNA.

5.

Immediately add 0.5 ml 25% PEG solution from a Pasteur pipette and pipette up
and down in the same pipette three times to mix the components.

6.

Plate 0.1 ml of undiluted transfection mixture, and of tenfold serial dilutions in
P buffer, on R2YE plates. Do not use a spreader, but immediately overlay with
soft R2 containing S. lividans spores (c. 107 ml–1), and briefly mix by rapidly
sliding the plate backwards and forwards a few times. Do not overdo this - youmust stop before the soft agar begins to set. Use 5 cm plates when subsequent
replication will not be required, and 9 cm plates if they are to be replicated. Cool soft R2 to c.
45o before adding spores to give a slightly more turbid suspension (soft R2 is itself turbid), anddispense as quickly as possible. Use 3-4 ml soft R2 on 9 cm plates and 1.2 ml soft R2 on 5 cm
plates. Storage of residual transfection mixture overnight at 4o for further plating is notrecommended.

7.

Allow at least 10 min for the agar to set before incubation overnight at 30o.

&KDSWHU&ORQLQJZLWKSKDJHYHFWRUVTransfer of Streptomyces phage DNA onto nitrocellulose filters for “plaquehybridisation”
The procedure is based on that of Benton and Davies (1977). The filters can be probed withprobes labelled radioactively or by the DIG system of Boehringer Mannheim.
Fresh plates with phage plaques (usually up to 104 per plate)
Denaturation solution (0.5M NaOH, 1.5M NaCl)
Neutralizing buffer (1M Tris-HCl, pH7.5 containing 1.5M NaCl)
2 × SSC (0.3M NaCl, 0.03M Na citrate)
Whatman 3MM paper
Nitrocellulose filters (BA85 Schleicher and Schuell or Millipore) to fit Petri dishes
Tray; forceps; vacuum oven1.

Keep the plates at 4o for two hours to harden the top agar.

2.

Place a dry nitrocellulose filter over the plaques for 10 min (avoid air bubbles
between the agar and the filter).

3.

Mark the filter (with a pencil) and the plate at two positions (asymmetrically).

4.

Remove the filter and place it (plaque side up) on two sheets of Whatman 3MM
paper soaked in denaturation solution (on a tray).

5.

Remove the filter after 10 min and transfer it for 10 min onto two sheets ofWhatman filter paper soaked in neutralizing buffer.

6.

Transfer the filters again onto Whatman paper soaked in 2 × SSC for 10 min.

7.

Dry the filters between Whatman filter paper at room temperature for 30 min.

8.

Bake the filter for 1½-2 h in a vacuum oven at 80o.

9.

Store filters at room temperature in a plastic bag until needed for hybridisation.

Preparation of

C31 lysogens

Cloning with vectors derived from C31 or other kinds of phages in Streptomyces usually
involves phenotypic analysis of strains carrying integrated insert-containing prophages. To generate
suitable lysogens requires a functional repressor gene (c) to repress the vector’s lytic functions. Physicalintegration into the genome may be achieved very efficiently by site-specific recombination with achromosomal attB site, or, in the case of vectors lacking the full set of integration functions, by the muchless efficient process of insert-directed integration using the host’s recombination machinery. In some

&KDSWHU&ORQLQJZLWKSKDJHYHFWRUV

situations vectors lacking a functional c gene are used. These can integrate into the host genome onlyin the presence of a separately provided c gene, typically that present on a wild-type C31 prophagealready present in a pre-constructed lysogenic host. For the latter purpose lysogenic indicator strains
must be prepared. For strains which allow C31 plaque formation the lysogens may be obtained fromthe surviving growth in turbid plaques produced by the wild-type phage (Procedure A). However, forstrains which do not allow plaque formation but which can be lysogenised - for example, Pgl+ strains
such as wild-type S. coelicolor A3(2) - it is necessary to use a c+ phage carrying a resistance marker(aac(3)IV, vph and tsr are available in C31 KC212, KC300 and KC301 respectively); lysogens may
then be detected as antibiotic-resistant colonies found only in areas of a plate onto which phage has been
introduced (Procedure B).
Single-plaque-purified phage stock
Concentrated spore suspensions of S. lividans and other relevant Streptomyces
strains
9 cm Petri plates of R2YE
Difco Nutrient agar containing 0.5% glucose, 8mM Ca(NO3)2 and 10mM MgCl2(poured on day of use)
MM containing appropriate supplements (or for procedure B, R2YE with
supplements and antibiotics)
25mM tetrasodium pyrophosphate (filter-sterilised)
Sterile distilled water
Pipetting equipment for 10 ml, 5 ml, 1 ml, 0.1 ml, 20 µl volumes; sterile glass
spreaders; sterile Pasteur pipettes; teats, sterile 5 ml and 25 ml screw cap bottles;
sterile Pasteur pipettes containing cotton wool plugs at the neck of the drawn-out
tip for small-scale filtration; sterile 25 mm filter units (disassemblable, e.g.
Swinnex) containing membrane filters of pore size 0.45 µm; sterile 5 ml syringes;
sterile dilution tubes; tube rack; replicating block and velvets
30o incubator; vortex mixer
Procedure A (using wild-type C31)

1.

Spread R2YE with c. 0.1 ml concentrated spore suspension and allow to dry
thoroughly.

2.

In an area marked on the base of the plate, spot c. 20 µl of phage suspension andallow to dry.

3.

Incubate at 30o until the area within the spot is sporulating (4-6 days).

4.

Harvest the spores within the marked area by using a sterile bacteriological loop
to suspend them in 1 ml of 25mM tetrasodium pyrophosphate, then sucking theminto a Pasteur pipette. Transfer into a 5 ml sterile screw cap bottle and leave at

&KDSWHU&ORQLQJZLWKSKDJHYHFWRUVroom temperature for 30 min. Sodium pyrophosphate, like other chelating agents,
inactivates free C31 particles fairly rapidly (unless they are extensively underpackaged deletionmutants).

5.

Vortex-mix for 30 seconds and filter through a cotton wool plug in a Pasteur
pipette.

6.

Using a 5 ml syringe and a filtration unit, collect the spores on a 0.45 µm
membrane filter.

7.

Wash the filter by passing 2 × 5 ml 25mM tetrasodium pyrophosphate throughthe filter unit.

8.

Aseptically transfer the membrane filter to a 5 ml screw cap bottle, add 1 ml
sterile distilled water and vortex-mix thoroughly.

9.

Dilute 0.1 ml of the spore suspension in sterile distilled water in a tenfold series
down to 10–4.

10.

Spread 0.1 ml samples of the dilutions and the undiluted spore suspensions on
R2YE plates and incubate at 30o until sporulated (about 4 days).

11.

Replicate sporulating plates containing about 100 colonies gently onto freshlyprepared, refrigerated Difco Nutrient Agar plates with SNA overlays containing
S. lividans spores. Prior refrigeration at 4Emakes the overlay less liable to disintegration
during subsequent replication.

12.

Incubate overnight and identify lysogens as colonies which have released phage.

Procedure B (using c+ att+ phages with cloned resistance genes, such as KC212, KC301, KC304 etc;
Table 13.1)

1-3.

As Procedure A.

4.

Replicate to plates of appropriately supplemented MM (preferred, if suitable forthe strain) or other medium, containing suitable antibiotic, such as apramycin (50µg ml–1), viomycin (20-30 µg ml–1) or thiostrepton (50 µg ml–1). The use of MM isadvantageous in giving a lower background of spontaneous resistant colonies.



5.

&KDSWHU&ORQLQJZLWKSKDJHYHFWRUV

Incubate for about 4 days to obtain lysogens as sporulating resistant growthcorresponding to phage-infected areas on original plate. Growth on thiostreptoncontaining plates is usually slower and may sporulate poorly.

6.

Purify lysogens by single-colony isolation, again preferably on MM. MM ispreferred because free phages tend to be lost relatively easily during culture on this medium.

“Low-tech” method for detecting

C31 derivatives containing resistance genes(after Lomovskaya et al., 1996)
Derivatives of C31 vectors can be used for chromosomal gene replacement (p. 318). The mosttypical strategy first of all involves construction in E. coli of a plasmid in which the gene of interest has
been interrupted by a resistance gene suitable for selection in Streptomyces. Secondly, the disrupted geneis ligated into an attP-deleted C31 derivative (such as KC515: Table 13.2), to give a construct which
can be used in a third step to replace the chromosomal gene with its disrupted version. To facilitate thesecond step, Lomovskaya et al. (1996) described a convenient plate-based method (i.e. without the needfor hybridisation) for detecting plaques that contain the disrupting resistance gene. The method isapplicable for most situations in which a selectable resistance gene is cloned into C31 derivatives. It
takes advantage of a simple trick (Chater et al., 1982) in which phages to be tested are used to infect a
mixture of lysogenic and non-lysogenic hosts spread on a suitable agar surface: the non-lysogens permit
multiplication of the phage to a high titre, and the lysogens provide a target for integration of theresistance gene-containing phages by recombination, using the extensive homology of the prophage andincoming phage genomes. Lysogens containing the resistance gene are identified by overlaying the plateswith the relevant antibiotic after an initial overnight incubation, followed by a further overnightincubation.
9 cm plates with overlays containing plaques arising from transfection with asuitable ligation mix
Spore suspensions of S. lividans and its C31 lysogen
Difco Nutrient Broth dispensed in 0.2 ml amounts
R2YE plates (but lacking sucrose)
R2YE soft agar (sucrose-free R2YE made up with half the usual agar concentration
kept molten at 45o-50o)
Stock solution of relevant antibiotic
Pipetting equipment for 10 µl volumes; sterile Pasteur pipettes or platinum wire;teats; sterile Eppendorf tubes
30o incubator; water bath at 45o-50o

1.

Transfer single plaques to 0.2 ml of DNB using Pasteur pipettes or platinum
wire.

&KDSWHU&ORQLQJZLWKSKDJHYHFWRUV



2.

Pour overlays of R2YE soft agar freshly inoculated with equal mixture of sporesof S. lividans lysogen and non-lysogen (3 ml overlay per R2YE base-plate; c. 107
spores ml–1).

3.

Pipette 10 µl of each sample from step 1 as spots on overlay plates from step 2.
Allow to dry (e.g. in laminar flow hood) and incubate at 30o for c. 17 h. Keep
phage suspensions at 4o.

4.

Overlay with 3 ml soft R2YE containing the selective agent (antibiotic) to givea final concentration calculated on the basis of the total volume of agar in theplate. Incubate overnight at 30o. Recombinant phages carrying the additional
resistance marker produce spots of resistant growth.

5.

Go back to the original phage suspensions and obtain single plaques of therecombinant phage by standard procedures (p. 277).

6.

For each candidate phage, transfer three single plaques to DNB as in step 1.

7.

Repeat steps 2-4.

8.

Prepare high-titre phage stock from single recombinant phage plaque (p. 278).

Use of glkA counterselection to select deletions from, or loss of,

C31 prophages

Very few counterselectable genes have been described for Streptomyces spp. One of them, glkA,
encoding glucose kinase, has been fairly extensively exploited as a genetic tool. The presence of glkA
renders strains sensitive to the glucose analogue 2-deoxyglucose (the DOGS phenotype) (Hodgson, 1982).
Fisher et al. (1987) found that glkA was deleted from some DOGR mutants of S. coelicolor, and exploited
this in devising a scheme for selecting deletions adjacent to sites of C31 insertion. Basically, thescheme involved constructing an attP-deleted C31 vector, KC573, containing the cloned glkA gene(Fig. 13.4 and p. 524). Insertion of a fragment of S. coelicolor DNA into KC573 allows it to integrate
at the equivalent site in the genome of the glkA-deleted recipient strain, thereby placing glkA at a desired
position in the genome and making the lysogen DOGS. KC573 also contains the selectable tsr and vph
genes in positions that become widely separated upon insert-directed prophage integration, such that vph
is next to glkA at one end of the prophage, and tsr is at the other. Counterselection of glkA (using100mM
DOG in MM containing arabinose or mannitol as carbon source: glucose cannot be used because it
overcomes inhibition by DOG), while retaining selection for tsr, results in deletions extending fromwithin the prophage, through glkA and vph, and out into the flanking DNA (if they do not impair growth).
Such deletions have one end fixed at the fragment used to direct prophage integrations (Fig. 13.4).



&KDSWHU&ORQLQJZLWKSKDJHYHFWRUV

Fig. 13.4 Use of a KC573 derivative in selecting deletions from a defined point in the chromosome.
Buttner et al. (1990) used DOG counterselection to select loss of integrated KC573 derivatives
in a strategy to deliver disrupted genes into the S. coelicolor chromosome (p. 320). In this case, the DNA
inserted into KC573 contained a gene of interest disrupted by a selectable resistance gene. Integrationof the prophage was first selected, then excision was selected with DOG, retaining selection for the
disruption cassette (Fig.14.5, p. 322).
These strategies require the use of a glkA-deleted host (J1668 of Fisher et al., 1987 is often used,because it is Pgl–), so the analysis of the phenotype of the mutants generated in this approach may require
the re-introduction of glkA (the att+ glkA-containing C31 derivative KC895 (Angell et al., 1992), orKC573 itself, may be convenient for this). Alternatively, it may be straightforward to use transformation
with denatured total DNA preparations (p. 235), or transduction (p. 274), to insert the new mutations intoa different genetic background (see also p. 322).

&KDSWHU&ORQLQJZLWKSKDJHYHFWRUVAngell S, Schwarz E, Bibb MJ (1992) The glucose kinase
gene of Streptomyces coelicolor A3(2): its nucleotide
sequence, transcriptional analysis and role in glucose
repression. Mol.Microbiol., 6, 2833-2844.
Benton WD, Davis RW (1977) Screening lambda gt
recombinant clones by hybridization to single plaques in situ.
Science, 196, 180-182.
Brian P, Riggle PJ, Santos RA, Champness WC (1996)
Global negative regulation of Streptomyces coelicolor
antibiotic synthesis mediated by an absA-encoded putative
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Bruton CJ, Guthrie EP, Chater KF (1991) Phage vectorsthat allow monitoring of secondary metabolism genes in
Streptomyces. Bio/Technology, 9, 652-656.
Buttner MJ, Chater KF, Bibb MJ (1990) Cloning,
disruption and transcriptional analysis of three RNA
polymerase sigma factor genes of Streptomyces coelicolor
A3(2). J.Bacteriol., 172, 3367-3378.
Chater KF, Bruton CJ (1983) Mutational cloning in
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Gene, 26, 67-78.
Chater KF, Bruton CJ, King AA, Suarez JE (1982) The
expression of Streptomyces and Escherichia coli drug
resistance determinants cloned into the Streptomyces phage
C31. Gene, 19, 21-23.
Clayton TM, Bibb MJ (1990) Induction of a C31
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coelicolor A3(2). Mol. Microbiol., 4, 2179-2186.
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Fisher SH, Bruton CJ, Chater KF (1987) The glucose
kinase gene of Streptomyces coelicolor and its use in selecting
spontaneous deletions from desired regions of the genome.
Mol.Gen.Genet., 206, 35-44.
Foor F, Morin N (1990) Construction of a shuttle vector
consisting of the Escherichia coli plasmid pACYC177
inserted into the Streptomyces cattleya phage TG1. Gene, 94,
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Foor F, Roberts GP, Morin N, Snyder L, Hwang M,
Gibbons PH, Paradiso MJ, Stotish RL, Ruby CL,
Wolanski B, Streicher SL (1985) Isolation andcharacterization of the Streptomyces cattleya temperate phage
TG1. Gene, 39, 11-16.
Guthrie EP, Chater KF (1990) The level of a transcript
required for production of a Streptomyces coelicolor
antibiotic is conditionally dependent on a tRNA gene.
J.Bacteriol., 172, 6189-6193.
Hahn DR, McHenney MA, Baltz RH (1991) Properties ofthe streptomycete temperate bacteriophage FP43. J.Bacteriol.,
173, 3770-3775.
Hartley NM, Murphy GO, Bruton CJ, Chater KF (1994)
Sequence of the essential early region of C31, a temperate
phage of Streptomyces spp. with unusual features in its lytic
development. Gene, 147, 29-40.
Hodgson DA (1982) Glucose repression of carbon source
uptake in Streptomyces coelicolor A3(2) and its perturbationin mutants resistant to 2-deoxyglucose. J.Gen.Microbiol.,
128, 2417-2430.
Ikeda H, Seno ET, Bruton CJ, Chater KF (1984) Genetic
mapping, cloning and physiological aspects of the glucose
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501-507.
Ishihara H, Nakano MM, Ogawara H (1983) Improved
phage vector system in Streptomyces. Japan.J.Med.Sci.Biol.,36, 299-306.
Isogai T, Takahashi H, Saito H (1980) High frequency
protoplast transfection of Streptomyces parvulus 2297 withactinophage R4 DNA. Agric.Biol.Chem., 44, 2425-2428.



Kawamura F, Saito H, Ikeda Y (1979) A method forconstruction of specialized phage rho 11 of Bacillus subtilis.
Gene, 5, 87-91.
King AA, Chater KF (1986) The expression of the
Escherichia coli lacZ gene in Streptomyces. J.Gen.Microbiol.,
132, 1739-1752.
Kobler L, Schwertfirm G, Schmieger H, Bolotin A,
Sladkova I (1991) Construction and transduction of a shuttle
vector bearing the cos site of Streptomyces phage C31 anddetermination of its cohesive ends. FEMS Microbiol.Lett., 78,
347-354.
Kuhstoss S, Rao RN (1991) Analysis of the integration
function of the streptomycete bacteriophage
C31.
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Kuhstoss S, Richardson MA, Rao RN (1991) Plasmid
vectors that integrate site-specifically in Streptomyces spp.
Gene, 97, 143-146.
Lomovskaya ND, Chater KF, Mkrtumian NM (1980)
Genetics and molecular biology of Streptomyces
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Transduction and transformation of plasmid DNA in
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M, Takahashi H, Shirai M (1996) The sre gene (ORF469)
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Piret JM, Chater KF (1985) Phage-mediated cloning of
bldA, a region involved in Streptomyces coelicolor
morphological development, and its analysis by genetic
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Rausch H, Lehmann M (1991) Structural analysis of theactinophage C31 attachment site. Nucl. Acids Res., 19,5187-5189.
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stimulate transfection of Streptomyces protoplasts.
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derivatives of the Streptomyces temperate phage C31 usefulfor the cloning and functional analysis of Streptomyces DNA.
Gene, 34, 283-292.
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Shimkets LJ, Gill RE, Kaiser D (1983) Developmental cellinteractions in Myxococcus xanthus. Proc. Nat. Acad. Sci.
USA, 80, 1406-1410.
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The complete genome sequence of the Streptomyces
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Ward JM, Janssen GR, Kieser T, Bibb MJ (1986)
Construction and characterisation of a series of multi-copy
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m14tkc.wpd 20 April 2000 Tobias Kieser

Chapter 14

Gene disruption and gene replacement
CONTENTS

Creating null mutants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Method (a). Insertional inactivation via a single crossover . . . . . . . . . . . . . . . . . . . . .
Method (b). Insertional inactivation via double crossing over . . . . . . . . . . . . . . . . . .
Method (c). Insertional inactivation using an in-frame deletion . . . . . . . . . . . . . . . . .
Other factors affecting the choice of approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Polar effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The size of intervals used . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Mutation stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Method of delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Non-replicating E. coli plasmids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Temperature-sensitive replicons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Phage vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Unstable replicons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Dealing with essential genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Introduction of point mutations and other subtle changes . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Counterselection of the delivery vector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
"Heterologous" disruptions and replacements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Choice of resistance markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Selecting for single crossover intermediates during gene replacement . . . . . . . . . . . . . . . . . .
A practical example of gene disruption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Homogenotisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Problems arising from unintended homogenotisation events . . . . . . . . . . . . . . . . . . .
Cloning mutant alleles by homogenotisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Gene replacements involving whole gene clusters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Mutational analysis of transcription units . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

311
312
313
314
316
316
316
316
316
317
318
318
319
319
320
320
323
323
324
324
328
329
330
332
334

Creating null mutants
In the first section of this chapter, we discuss experiments designed to introduce null-mutant
alleles of target genes into the host genome. Before considering delivery systems, we review the threedifferent types of disruption that can be made and the possible advantages and disadvantages of each.

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target gene
chromosome

A

B

C

B

tsr

tsr

A

B

B

C

Fig. 14.1 Insertional inactivation via a single crossover.

Method (a). Insertional inactivation via a single crossoverIn this method, a DNA fragment internal to the gene is inserted into a vector which thenintegrates into the chromosome by a single homologous crossover. This approach results in an integrated
copy of the vector flanked by two mutant alleles of the gene, one truncated at the 30 end and the other atthe 50 end (Fig. 14.1).
It is important to be confident that both the mutant alleles will be non-functional; that is, there
must be essential parts of the gene both upstream and downstream of the internal DNA fragment used
to mediate integration of the vector. There are many examples, for instance E. coli relA (Schreiber et al.,1991), in which partial gene products generated by gene disruption strategies retain one or more of the
known activities of the wild-type protein, making interpretation of data arising from the mutants
uncertain. Be aware that the truncated allele downstream of the vector may be expressed fromtranscription and translation initiation signals contained within the vector.
Mutants generated by a single crossover can be made quickly because they require only the subcloning of an appropriate fragment into a suitable vector and because the primary integration event
generates the mutation directly. Such mutants are therefore very useful for gross phenotypiccharacterisation. However, they can revert to wild-type by excision of the vector via a single crossover,and so have the potential to be unstable, although the level of reversion observed is usually only of theorder of 1% through one round of non-selective growth. Maintenance of selection for the antibiotic
resistance associated with the vector is desirable, but this will not rule out the possibility that some of the

&KDSWHU*HQHGLVUXSWLRQDQGJHQHUHSODFHPHQWchromosomes in a population will lose the mutation through excision (since mycelium carrying such a
population will still be resistant to the antibiotic in most cases). Reversion of simple integration eventsis likely to be a particular problem when the mutation caused by integration is deleterious to growth; inthis case faster-growing revertants are more likely to constitute a significant fraction of the population.
Method (b). Insertional inactivation via double crossing overIn this method, a selectable marker such as an antibiotic resistance gene is put into a cloned copyof the target gene, either as a simple insertion at a single restriction site, or as a replacement for a DNA
fragment carrying all or part of the target gene. The mutant allele thus created, carried on a suitable
vector, is then used to replace the chromosomal copy of the target gene via two crossovers, one on eitherside of the selectable marker, resulting in a stable mutation that cannot revert. These two crossovers may
occur simultaneously, giving the desired product in a single step, or may occur consecutively, proceeding
via a single crossover intermediate in which the vector used to deliver the mutant allele is integrated into
the chromosome (Fig. 14.2). The delivery vector should be marked with an antibiotic resistance gene(different from that inserted into the target gene) so that transformants carrying single and double
crossover events can be distinguished by replica plating. If the double crossover product is to be isolated
in one step, transformants must be selected using the antibiotic corresponding to the resistance gene
inserted into the target gene (hyg in Fig. 14.2), rather than that on the vector (tsr in Fig. 14.2). Selection
for the resistance gene on the delivery vector will eliminate double crossover transformants, but may benecessary if a clean selection cannot be obtained through the resistance gene inserted into the target gene.
If only transformants resulting from single crossovers are isolated (Hyg R, ThioR), they should be grownthrough one or two rounds, selecting only for the antibiotic resistance marker inserted into the target gene(hyg), before plating or streaking spores for single colonies. Colonies carrying the desired second
crossover can then been identified by replica plating (HygR, ThioS).
Transformation with linearised DNA (Oh and Chater, 1997; p. 235), generated by restriction
digestion of a plasmid, can be desirable in these experiments; it increases the percentage of primarytransformants in which double crossover events have occurred because recombination between linearised
DNA and the chromosome requires two crossovers to maintain the integrity of the chromosome.
However, the absolute number of transformants is greatly decreased when introducing linearised DNA,and so its use may not be appropriate if transformation frequencies are low in the strain being used. The
use of linearised DNA would not be appropriate for Method (a), which requires a single crossover event,or for Method (c) (p. 314) unless there is a ready selection for the in-frame deletion phenotype if bothcrossovers occurred simultaneously.
Mutations constructed in this way are marked genetically by the antibiotic resistance gene andcan therefore be moved into new genetic backgrounds by chromosomal transformation (Oh and Chater,
1997) or by crossing (Chapter 7). In contrast, mutations constructed by Method (a) are not particularly
well suited to genetic analysis because their excision seems to be stimulated during crosses (Chater etal., 1985).

&KDSWHU*HQHGLVUXSWLRQDQGJHQHUHSODFHPHQW
tsr

hyg
S

chromosome

1

S

Thio Hyg

2

1

2
tsr
R

R

R

R

Thio Hyg

hyg

2
tsr

Thio Hyg

hyg
1
S

R

Thio Hyg

hyg
Fig. 14.2 Insertional inactivation via double crossing over. Note that thedisruptant at the bottom arises as a result of crossing over on both sides of thetarget gene (shown as Ê and Ë in the figure). The two crossovers may occur
simultaneously, or successively as shown here, in either order.

Method (c). Insertional inactivation using an in-frame deletion
In-frame deletions can be created in several ways:i.
by replacing a restriction fragment internal to the target gene with two complementaryoligonucleotides (e.g. redD; Floriano et al., 1996);ii.
by cloning two non-contiguous restriction fragments carrying the 50 and 30 ends of thetarget gene into a polylinker in the right orientation and reading frame (e.g. actIIORF4; Floriano et al., 1996);iii.
by deleting DNA between two in-frame restriction sites; because of the constraints ofcodon usage in Streptomyces, sites for a given restriction enzyme are very often in thesame reading frame within a gene (an in-frame deletion ( pglY16) was constructedwithin pglY by the deletion of DNA between two SacII sites: Bedford et al., 1995);
iv.
by PCR ( e.g. sigE; Jones et al., 1997).

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tsr

∆

2

1

S

Thio

chromosome

1

2
tsr
R

∆

Thio

2
tsr
R

∆

Thio

1
∆

S

Thio

Fig. 14.3 Insertional inactivation using an in-frame deletion. Note that two
successive crossovers, on either side of the target gene (shown as Ê and Ë in the
figure), are required in order to generate the disruptant at the bottom of the figure.
Unless the in-frame deletion allele has a predictable and easily scorable phenotype, it will benecessary to go through a multi-step process (Fig. 14.3), selecting first for integration of the delivery
vector, growing the transformants non-selectively for one or more generations, and screening single
colonies by replica plating for isolates that have lost the vector. These isolates will be either wild-type(if the vector integrated and excised through the same interval) or will carry the deletion (if the vector
integrated through one interval and excised through the other) . Wild-type and mutant colonies can thenbe distinguished by screening using PCR, colony hybridisation or Southern blotting (pp. 242-248).

&KDSWHU*HQHGLVUXSWLRQDQGJHQHUHSODFHPHQWOther factors affecting the choice of approach
Polar effects
It is essential to consider the context of the gene in the chromosome. If there are genes
downstream of the target gene which may be co-transcribed with it, disruption using Methods (a) or (b)may give rise to polar effects on the expression of those genes and so the phenotypic consequences ofthe disruption cannot be safely attributed to loss of target gene function. It is safe to ignore thispossibility if the downstream gene is oppositely orientated with respect to the target gene, or if one isconfident of a complete absence of readthrough transcription under all conditions. If polar effects area consideration, they can be ruled out if the observed phenotype is complemented in trans by a single
copy of a DNA fragment carrying only the target gene.
The size of intervals used
The frequency of homologous recombination is generally proportional to the lengths of the DNA
fragments used and so, other things being equal, it is desirable to maximise the size of these intervals.
Where double crossover events are sought, it is also desirable to use two flanking intervals of similar size,otherwise the plasmid will tend to integrate and excise through the larger interval. This is particularly
important for in-frame deletions, where there is no positive selection for retention of the deletion allele
on excision of the vector. In experiments designed to inactivate genes via a single crossover - Method(a) - the size of DNA fragment used is obviously constrained by the size of the gene and so this approachis not ideal for small ORFs. Intervals as small as 160 bp have been used in gene replacement experiments(by transformation with single-stranded DNA; Leskiw et al., 1993) but, in general, the use of intervals
smaller than 500 bp is not recommended. In the case of phage-delivered disruptions, 500 bp fragments
are clearly on the lower limit of usable insert size. At this insert size, chromosomal integration events
at IS110 elements, following transposition of IS110 into the phage vector, are detected at a significant
frequency relative to insert-directed integration in S. coelicolor (Chater et al., 1985).
Mutation stability
In general, mutants generated by a single crossover - Method (a) - can be made quickly. Suchmutants are therefore very useful for gross phenotypic characterisation, but because the mutation canrevert through the single crossover excision of the vector, such strains are not ideal if further detailed
analysis is intended. Therefore, as the basis for a longer-term study, a stable mutation constructed by adouble crossover - Method (b) or (c) - is desirable.

Method of delivery
The construct carrying the mutant allele can potentially be introduced into Streptomyces bymating from E. coli, by transformation, by electroporation, or by natural infection with a phage. In strains
for which transformation frequencies are low, the introduction of non-replicating E. coli vectors by

&KDSWHU*HQHGLVUXSWLRQDQGJHQHUHSODFHPHQWtransformation may be precluded because of the relatively low frequency with which they are “rescued”
by immediate integration into the chromosome. Several delivery methods can be used.
Non-replicating E. coli plasmids
E. coli plasmids that cannot replicate in Streptomyces are the delivery vectors of choice in mostcircumstances because of the ease of making the desired construct. Because the delivery vector cannot
replicate after transformation, initial attempts to use this approach were sometimes frustrated by lowtransformation frequencies. However, several methods have been developed which have greatly
facilitated the introduction of non-replicating suicide plasmids from E. coli into Streptomyces. These
are listed briefly below; methods are described in detail on the pages referred to.
(i) Non-replicating plasmids can be introduced into Streptomyces by mating directly from E. coli(p. 249), provided the vector carries oriT (the origin of transfer of plasmid RK2) (Mazodier et al., 1989;
Smokvina et al., 1990; Bierman et al., 1992) and the RK2 transfer functions are supplied in trans by anadditional (oriT–) “driver” plasmid such as pUZ8002 (Sia et al., 1996; J.W. Wilson and D.H. Figurski,
personal communication; Paget et al., 1999). Suitable vectors include pSET151, pKC1132, pOJ260(Bierman et al., 1992), pPM2205 (formerly “pPM925del”; Servant et al., 1993), pIJ6650 (p. 566) and
pPM2208 (formerly “pPM935del”; Servant et al., 1993). pPM2205 and pPM2208 were used in attempts
to disrupt the groEL1 and groEL2 genes of S. albus and the DNA was delivered via mating from E. coli
(Servant et al., 1993). Similarly, the glnA gene of S. viridochromogenes was disrupted using a constructdelivered by mating from E. coli (Hillemann et al., 1993).
pUB307 has also been used as a driver plasmid in mating experiments (Flett et al., 1997).
However, pUB307 carries a fully functional oriT and can therefore transfer itself into Streptomyces, inaddition to the plasmid intended. In principle, this may not be a problem, since pUB307 cannot replicate
in Streptomyces, cannot integrate into the chromosome by legitimate recombination, and has not so far
been detected by Southern analysis in streptomycete ex-conjugants after mating (Flett et al., 1997).
Nevertheless this potential is undesirable and for this reason the use of pUZ8002 (oriT–) is preferable.
When using mating from E. coli to introduce DNA into Streptomyces strains that carry methylspecific restriction systems - see (iii) below - the number of ex-conjugants obtained is greatly (>104-foldin the case of S. coelicolor; Flett et al., 1997) if a Dam– Dcm– Hsd– E. coli strain, such as ET12567(MacNeil et al., 1992), carrying a driver plasmid is used.
(ii) Single-stranded DNA gives transformation frequencies 10- to 100-fold higher than thecorresponding double-stranded molecule (Hillemann et al., 1991). Single-stranded DNA can be derived
from double-stranded vectors carrying a phage-derived single-stranded origin of replication, such as
pDH5 (Hillemann et al., 1991), which carries tsr for selection in Streptomyces. However, it is sometimes
difficult to induce formation of single-stranded DNA with these vectors. Single-stranded DNA can also
be generated conveniently from any double-stranded molecule using sodium hydroxide denaturation
followed by neutralisation, as commonly applied in double-stranded DNA sequencing protocols, and thismethod is also very effective in stimulating transformation frequencies (p. 235; Oh and Chater, 1997).
(iii) For transformation of Streptomyces strains carrying methylation-specific restriction systems,
such as S. avermitilis, S. coelicolor, S. cyanogriseus, S. griseoplanus, S. parvulus, S. rochei (MacNeil,
1988) and S. bambergiensis (Zotchev et al., 1995), plasmids should either be constructed in, or passaged
through, a Dam– Dcm– Hsd– derivative of E. coli such as ET12567 (MacNeil et al., 1992; p. 230).

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If strains are encountered where transformation frequencies are too low to obtain transformantswith non-replicating plasmids and mating from E. coli is also unsuccessful, mutant alleles can bedelivered in other ways.
Temperature-sensitive replicons
Mutant alleles can be delivered to the chromosome using temperature-sensitive replicons. The
most suitable vectors currently available for this purpose are derivatives of the naturally temperaturesensitive plasmid pSG5 (p. 258) from S. ghanaensis (Muth et al., 1989). These plasmids include
pGM160, an E. coli-Streptomyces shuttle vector which greatly facilitates construction. Aftertransformation and initial growth at 28-30o, integration into the chromosome by homologous
recombination with sequences carried on the plasmid is "forced" by placing the strain at 37-39o (a nonpermissive temperature for plasmid replication) while continuing selection for the appropriate antibiotic.
In double crossover experiments - Methods (b) and (c) - one or two rounds of streaking at 37-39o in theabsence of selection for the vector (tsr in the case of pGM160) allow the ready identification of ThioS
colonies from which the plasmid has been lost.
Temperature-sensitive replicons are useful in gene disruption experiments using Methods (b) and(c), but are not satisfactory for insertional inactivation via a single crossover - Method (a) - because, on
returning the mutant strain to growth at 30o, the plasmid can excise from the chromosome and replicate
autonomously; as a consequence, mutants of this type would have to be maintained and analysed at 3739o.
Phage vectors
C31-derived phage vectors deleted for the phage attachment site (attP) have been used very
extensively in single crossover gene disruption experiments (e.g. Chater and Bruton, 1985; Malpartidaand Hopwood, 1986) and occasionally in double crossover gene replacements (Buttner et al., 1990;Potúûková et al., 1995). They provide a mechanism to deliver mutant alleles through natural infection
without protoplasting, provided the strain in question is susceptible to C31. It is simple to assay new
strains for sensitivity to C31 by plaque formation (p. 277) and/or ability to be lysogenised (pp. 294 and303). C31 was used successfully to construct a sigE derivative of S. antibioticus, after attempts tointroduce mutant alleles on non-replicating E. coli plasmids by transformation had proved unsuccessful(Jones et al., 1997), and also to mutate genes of the rapamycin biosynthetic cluster of S. hygroscopicus(ATCC 29253), a strain for which PEG-mediated transformation and conjugal transfer have so far proved
unsuccessful (Lomovskaya et al., 1997). Because lysogenic colonies give rise to plaques when replica
plated to lawns of an indicator strain such as S. lividans (p. 305), it is easy to recover phage during genereplacement and gene disruption experiments. This permits simple verification of the structure ofconstructs after their introduction. It also permits the isolation of alleles and their transduction between
different genetic backgrounds (p. 330).
Probably the most important constraint in using C31-derived vectors for gene disruptions andreplacements is the phage packaging limit. C31 will package up to 42.6 kb stably, leaving up to 6.4 kb
available to accommodate the insert in the type of attP-deleted vectors, such as KC404 (36.2 kb), likely

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to be used in this type of experiment (p. 273). The influence of these constraints on the size of theflanking intervals should be borne in mind.
Unstable replicons
Although they found temperature-sensitive plasmids gave higher frequencies of double crossoverrecombination events, Khosla et al. (1992) also delivered mutant alleles using pIJ4680 (p. 559), a
Streptomyces-E. coli shuttle vector based on an SCP2* derivative that is unstable in Streptomyces because
it lacks the presumed partition function (Lydiate et al., 1985; p. 263). This plasmid is very easily lostfrom mycelium in the absence of selection.
The pIJ101-based shuttle vector pWHM3 (p. 544; Vara et al., 1989) has also been usedsuccessfully for mutant construction, as in the generation of nikkomycin (Möhrle et al., 1995) anderythromycin (Vara et al., 1989) biosynthetic mutants in species in which it is unstable.

Dealing with essential genes
In certain circumstances, it is desirable to demonstrate the essential nature of a gene. The mostrigorous approach to this problem currently in the Streptomyces literature was taken by Vögtli and Cohen(1992) to show that a tRNATyr gene that overlaps the chromosomal integration site (attB) for the plasmid
SLP1 is essential for cell viability in S. lividans. In initial experiments of the "Method (b)" type, the attBsequence was refractory to gene replacement. However, the tRNATyr gene could be deleted from its
natural chromosomal context after a second copy had been introduced into the chromosome at a different
location. Finally, it was shown that the gene could be deleted from the chromosome after a second copy
had been introduced on a highly unstable plasmid. In this strain, the unstable plasmid was uniformly
maintained in the absence of selection, whereas it was completely lost after one round of non-selective
growth in a strain carrying a functional tRNATyr gene in the chromosome.
An alternative approach is to make a construct which can integrate into two separate loci, one
the target gene under test and the other a non-essential gene, preferably with an easily-scorable mutant
phenotype. The non-essential gene acts as an internal control. The two fragments used should beapproximately equal in length. If a large number of transformants are obtained and they all result from
integration into the non-essential control gene, this provides evidence that the target gene (or adownstream gene co-transcribed with it) is essential. This approach was used to demonstrate the essential
nature of the hrdB gene of S. coelicolor (in this case using a C31-based vector, but non-replicating or
temperature-sensitive plasmids could equally have been used). In this experiment, the non-essential
control DNA was part of the gyl operon and integration of the construct at this locus resulted in a glycerol
non-utilising, glycerol-sensitive phenotype. All the lysogens obtained were glycerol mutants, showingthat the hrdB gene of S. coelicolor was essential (Buttner et al., 1990). The same approach was used toshow that the fab (fatty acid biosynthesis) operon of S. coelicolor is essential (Revill et al., 1995).
Similarly, Servant et al. (1993) used non-replicating plasmids carrying mutational fragments of both thegroEL2 and bla genes of S. albus to assess the essential nature of groEL2. The non-essential bla ( lactamase) gene served as an internal control with an easily-scored mutant phenotype.

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A third possible approach would be to adapt for Streptomyces the method used by Joyce andGrindley (1984) to assess the essential nature of the E. coli polA gene. This would be to create an attP–
phage or other integrative vector carrying a deletion internal to the target gene and to select for
integration at the target locus by a single crossover. It is important for this method that integration
through at least one of the intervals carried on the vector should be viable. After non-selective streaking
to identify colonies in which the prophage has been lost, failure to delete the target gene would indicate
either that the resulting deletion strain was not viable or that the desired recombination event did not takeplace. By examining the other product of the excision, the phage released from lysogenic colonies, tosee whether or not they contain both the deleted and wild-type alleles of the gene, it is possible todetermine if the failure to recover a deletion mutant results from its never having been formed or, once
formed, its failure to survive. This experiment could be performed with an attP – phage, a ts plasmid ora non-replicating E. coli plasmid, provided, in the last case, that the products of excision from thechromosome can be recovered for analysis in E. coli after mini-lysate plasmid isolation from
Streptomyces (Chapter 8).

Introduction of point mutations and other subtle changes
In order to study the effects of specific in vitro-generated mutations, such as point mutations, it
is desirable to be able to replace wild-type target genes with mutant alleles in their natural chromosomal
context (Fig. 14.4). This type of experiment is perhaps best accomplished by first replacing all or partof the wild-type gene with an antibiotic resistance marker via Method (b) above. It is then possible toreplace the resistance marker with the point mutant allele using the same approach, screening for loss ofthe chromosomal antibiotic resistance. This approach was first used to make a series of targeted genereplacements in the act cluster of S. coelicolor (Khosla et al., 1992). The actI-ORF3 gene, encoding theact ACP, was first replaced with ermE, conferring lincomycin resistance. The ermE gene was thenreplaced by a series of mutant alleles, most of which were delivered on the temperature-sensitive shuttle
vector pGM160 (p. 318). These experiments were used to: remove the translational coupling between
the ACP gene and the downstream gene; substitute an alanine for the serine believed to be the site ofphosphopantetheinylation of the ACP; substitute a presumptive fatty acid synthase ACP from
Saccharopolyspora. The frequency of double crossovers for the substitution of ermE was 5-100% oftransformants.

Counterselection of the delivery vector
Sometimes in gene replacement or gene disruption experiments, if only the products of single
crossovers are detected among the primary transformants or ex-conjugants, thousands of colonies haveto be screened for loss of antibiotic resistance to identify colonies resulting from the desired second
crossover. This is particularly true if the DNA intervals flanking the mutation in the delivery vector are
small or if the two flanking intervals are very different in size, such that integration and excision tend tooccur through the same interval. To avoid the need for screening, it can be desirable to build into theexperiment a positive selection for the second crossover by incorporating a counterselectable gene into
the delivery vector. An example is the glucose kinase gene (glkA) that confers sensitivity to the glucose
analogue 2-deoxyglucose. Three counterselectable glkA-containing vectors have been constructed for

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ermE
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S

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acp

plasmid carrying
engineered
mutant allele

S

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S

Fig. 14.4 Introduction of a point mutation into a gene (for example acp
for an acyl carrier protein) by two successive double crossovers; the first
replaces the wild-type gene by a marker (ermE, which confers lincomycin
resistance: LinR) and the second replaces the marker by the mutant allele.
use in gene replacement experiments; the phage-derived vector KC573 (Buttner et al., 1990; Fisher etal., 1987; p. 307) used to construct hrdA, hrdC, hrdD and sigF mutants of S. coelicolor (Buttner et al.,
1990; Buttner and Lewis, 1992; Potúûková et al., 1995); the non-replicating E. coli plasmid pIJ2581 (vanWezel and Bibb, 1996) used to construct a tuf3 mutant of S. coelicolor (van Wezel et al., 1995); andpIJ6650 (p. 566), which is derived from pKC1132 (p. 566), thereby allowing the use of glkA
counterselection combined with the ability to transfer the construct by mating from E. coli.
The general approach is illustrated in Fig. 14.5. The experiment must be conducted in a strain
carrying a deletion of glkA, both to allow counterselection of glkA on the vector and to prevent
homologous recombination between the vector and the chromosomal glkA locus. If, after selection oftransformants with Hyg, only single crossover transformants are obtained (HygR, ThioR), they are grownthrough one round selecting only for HygR. Spores are then plated on both 2-deoxyglucose (p. 307) andHyg. Under these circumstances, only colonies in which the delivery vector (carrying glkA) has been lost,leaving the mutant allele (carrying hyg) in the chromosome, will grow.

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selection for Dog

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R

and maintenanceRof HygR∆ glkAS

Dog Thio Hyg

Rhyg
Fig. 14.5 Disruption of a gene by double crossing over, with counterselection of
the delivery vector using 2-deoxyglucose (Dog).
In a variation on the type of gene replacement experiments described above for the act ACP,
point mutations could be introduced at the target locus after it had first been replaced with a cassette
carrying both glkA and an antibiotic resistance marker, for example hyg. Provided these experiments are
conducted in a glkA deletion strain, 2-deoxyglucose can be used as a positive selection for the genereplacement, which should also cause hygromycin sensitivity.
An obvious minor drawback to using glkA as a counterselectable marker in gene replacement
experiments is that the desired replacement must be made in a glkA deletion mutant, available in a varietyof genetic backgrounds in S. coelicolor (J1668, Fisher et al., 1987; J1915, J1916, J1917, Kelemen et al.,
1995; J1501 glkA, van Wezel et al., 1995). However, if the glkA deletion is likely to affect further
analysis, the constructed mutation can easily be moved into a glkA+ background by back-crossing(Buttner and Lewis, 1992), or preferably by transformation with total chromosomal DNA, selecting forthe antibiotic resistance that marks the mutation (Oh and Chater, 1997). The recent advent of generalised
transduction in S. coelicolor and S. lividans (J. Westpheling, personal communication; p. 274) offers analternative means of moving mutations into new genetic backgrounds.

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A second counterselectable gene that has been used successfully in gene replacement experiments
in Streptomyces is rpsL, which encodes ribosomal protein S12 (Hosted and Baltz, 1997). Mutations
mapping to rpsL confer resistance to streptomycin, and Hosted and Baltz (1997) isolated a streptomycinresistant mutant of S. roseosporus. In addition, they cloned the wild-type rpsL gene of the same strain
and incorporated it into a gene-replacement vector. Since the wild-type, streptomycin-sensitive allele of
the gene is dominant, they were able to use streptomycin to counterselect the vector when they used it
to deliver a glnA::hyg insertion into the chromosome of the streptomycin-resistant mutant. Because rpsL
is an essential gene, it was not possible to conduct these experiments in an rpsL deletion mutant, and a
proportion of the primary integration events occurred at rpsL, rather than glnA.
Since the counterselectable S. coelicolor genes for galactose kinase (Fornwald et al., 1987;
Kendall et al., 1987) and sulphate utilisation (Lydiate et al., 1988) have also been cloned, it should bepossible to develop analogous positive selection systems based on 2-deoxygalactose resistance or selenate
resistance. The sacB gene of Bacillus, widely used as a counterselectable marker in other genera, does
not seem to be applicable to S. lividans (Jäger et al., 1992).

"Heterologous" disruptions and replacements
The frequency of homologous recombination decreases sharply when two intervals show lessthan 100% identity. Nevertheless, it is possible to use heterologous DNA to mediate gene disruptions
and replacements, at least between closely-related sequences and strains. Thus the S. hygroscopicus bar
gene was disrupted using a restriction fragment from within the homologous gene from S.
viridochromogenes, pat, which showed 87% sequence identity to bar (Wohlleben et al., 1994); and a 0.5
kb fragment internal to the serine protease B gene of S. coelicolor “Müller” was used to direct disruption
not only of the serine protease B gene itself, but also of the homologous serine protease A gene in thesame strain (Wohlleben et al., 1994). Similarly, the S. coelicolor “Müller” recA gene was partiallyinactivated using a restriction fragment from S. lividans recA (Muth et al., 1997); the bldA39 mutant
allele of S. coelicolor was homogenotised into the S. lividans chromosome (Leskiw et al., 1991); a
deletion allele of the S. lividans ß-galactosidase gene was delivered into the S. coelicolor chromosome(C.J. Bruton, T. Eckhardt and K.F. Chater, unpublished); and internal fragments of the S. coelicolor gyl
operon were used to create Gyl– mutants of S. lividans (C.J. Bruton and K.F. Chater, unpublished). In
none of the last three cases are the sequences from both species available for comparison, but for othergenes, the level of sequence identity between S. coelicolor and S. lividans is approximately 98%.

Choice of resistance markers
See Chapter 21 for maps and other information. For Methods (a) and (b), the antibiotic resistancegenes used must provide a clear selection at single copy number in the chromosome. This requirement
is not trivial and many resistance genes that are tried and tested as selectable markers on replicatingplasmids in Streptomyces can be less than satisfactory at single copy, depending on the exact context.
As a consequence, we have encountered problems such as: inadvertently selecting for tandem duplicationof integrated plasmids (because two copies of the gene provide the level of resistance selected but one
copy does not; p. 234); or finding that a significant percentage of the colonies picked are not transformed

&KDSWHU*HQHGLVUXSWLRQDQGJHQHUHSODFHPHQW

after dropping the level of antibiotic used. In our hands, the aac(3)IV gene, conferring resistance toapramycin, from Klebsiella (Bräu et al., 1984; Allmansberger et al., 1985), seems to provide the cleanest
selection at single copy, but using it to disrupt target genes precludes the use of several very usefulvectors marked with aac(3)IV, either during or after gene disruption (pOJ260, pKC1132, pIJ6650,
pSET152). For this reason, we most often use hyg, conferring resistance to hygromycin, from S.
hygroscopicus (Zalacain et al., 1986). Other genes that have been used successfully for gene disruptions
and gene replacements in Streptomyces include aadA, conferring resistance to both spectinomycin andstreptomycin, from the enteric plasmid R100.1 (Prentki and Krish, 1984); neo from Tn5 (Beck et al.,1982), conferring resistance to kanamycin and neomycin; vph, conferring resistance to viomycin, fromS. vinaceus (Bibb et al., 1985); and ermE from Saccharopolyspora erythraea (Uchiyama and Weisblum,
1985; Dhillon and Leadley, 1990), for which lincomycin, not erythromycin, provides the cleaner selection(at least in S. coelicolor). When using aadA, the frequency of spontaneous spectinomycin-resistantcolonies, at least in S. coelicolor, can be a problem, especially when working in strA backgrounds where
aadA cannot be selected with streptomycin. aadA, aac(3)IV and Tn5 neo have the advantage that theyalso confer resistance on E. coli. vph, hyg and ermE also confer resistance on E. coli if their expression
is driven by an appropriate promoter. Blondelet-Rouault et al. (1997) have constructed well-designed
cassettes for aadA, aac(3)IV, hyg and vph, each of which has been engineered to ensure that it confers
resistance in both E. coli and Streptomyces.
It is best to avoid the use of tsr to disrupt target genes because its almost ubiquitous use as aselectable marker in vectors of all kinds precludes many kinds of subsequent analysis in the mutant.

Selecting for single crossover intermediates during gene replacement
In principle, when a gene is being insertionally inactivated via a double crossover - Method (b) it is desirable to select for the antibiotic resistance marker inserted into the target gene, because selection
for the antibiotic resistance marker on the delivery vector will eliminate double crossover transformants.
However, in constructing a sigE::hyg null mutant of M600 (Paget et al., 1999), the level of hygromycin
resistance conferred by the sigE::hyg allele was so low that it was necessary to select transformants
using the tsr marker on the delivery vector, pSET151. Selection for hygromycin resistance resulted instrains carrying tandem duplications of the integrating plasmid. Prior to screening for the second
crossover event, spores were grown non-selectively; if hygromycin selection had been maintained during
this stage, it would have prevented growth of the desired sigE::hyg null mutant.

A practical example of gene disruption
Because of the large number of variables in gene disruption and gene replacement experiments,
this chapter does not easily lend itself to a defined set of methods. Nevertheless, we thought it useful togive one concrete example; we have chosen an insertional inactivation via double crossing over,introducing a hyg-disrupted mutant allele into S. coelicolor by mating from E. coli, and using 2deoxyglucose counterselection of the delivery vector (Fig. 14.5). The suitability of this particular method
for your own experiments should be determined after reading the remainder of this chapter.

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

1.

Identify a restriction site internal to the target gene into which you will insert theantibiotic resistance cassette. Are you confident that the mutant allele resulting
from insertion at this restriction site will be non-functional?

2.

Clone a 2-4 kb fragment in which the restriction site chosen for insertion of theantibiotic resistance cassette is as central as possible (to ensure flanking intervalsof equal size).

3.

Clone the hyg resistance cassette (p. 485) from the hyg plasmid of BlondeletRouault et al. (1997) or pIJ963 into the chosen restriction site in the sameorientation as the target gene; cloning in the opposite orientation may decreasehyg expression.

4.

Clone the disrupted allele into the counterselectable delivery vector pIJ6650(p. 566).

5.

Introduce the resulting construct into the E. coli strain ET12567 carrying the“driver” plasmid pUZ8002.

6.

Transfer the construct from E. coli ET12567 by mating into S. coelicolor J1915(a glkA deletion derivative of S. coelicolor M145; Kelemen et al., 1995) on MS
agar plates (p. 409), as described on p. 249. Following incubation for 16-20 h
at 30o, overlay each plate with 1 ml water containing 0.5 mg nalidixic acid (tokill E. coli) and 1 mg apramycin (to select for J1915 ex-conjugants in which theplasmid has integrated into the chromosome via a single crossover).

7.

Purify six ex-conjugants by streaking for single colonies on MS agar plates
containing 25 µg ml–1 naladixic acid (to prevent the growth of any viable E. coli
cells carried over from the primary mating plates) and 20 µg ml–1 apramycin (tomaintain selection for the integrated plasmid). Replica plate to Minimal Medium(MM; p. 406) containing 0.5% (w/v) mannitol (as carbon source) and 100mM
2-deoxyglucose (16.4 g l-1) that these single crossover isolates are 2deoxyglucose-sensitive; use J1915 as a 2-deoxyglucose-resistant control andM145 as a 2-deoxyglucose-sensitive control (score plates after 3-4 days). The
counterselection depends on the 2-deoxyglucose-resistant glkA strains being unable to use
glucose as carbon source (see also p. 307); 2-deoxyglucose selection is effective only on minimal
media.

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8.

Prepare chromosomal DNA from each isolate and confirm by Southern blottingthat they have the expected chromosomal structure for single crossover
homologous integration, which can occur either through the interval upstream of
hyg or through the interval downstream. If the intervals flanking the hyg
resistance cassette are not the same length, it is preferable to identify and use exconjugants in which integration has occurred through the smaller interval,
because excision through the larger interval will be favoured and will result in
isolation of the desired mutant. Steps 8 and 9 are usually done in parallel.

9.

Grow two single crossover ex-conjugants from step 7 through one or two rounds
of non-selective growth, thereby permitting plasmid loss. In more detail, use
single colonies from step 7 to seed confluent lawns on MS agar. Harvest sporesand mycelial fragments and seed a second confluent lawn if desired. Is there apossibility that your desired mutant may be bald or non-sporulating? If so, do notfilter your samples through cotton wool. Steps 8 and 9 are usually done in parallel.

10.

Plate a serial dilution of the resulting spores and mycelial fragments on MM
containing 0.5% mannitol (as carbon source) and 200mM 2-deoxyglucose (toselect isolates that have lost the delivery vector). As a control, plate the same
dilution series onto MM containing 0.5% mannitol. In most experiments the rateof spontaneous plasmid loss is no more than 1% per round of non-selective
growth and so the number of colonies obtained in the presence of 2deoxyglucose is usually at least 100-fold lower. Since no hyg selection has been
applied at any stage, the 2-deoxyglucose-resistant colonies should be a mixtureof wild-type revertant colonies, and the desired hyg-insertion null mutants.
Applying hyg selection (for the initial ex-conjugants, or when selecting forplasmid loss with 2-deoxyglucose) can be undesirable because the hyg cassettedoes not always confer significant levels of resistance at single copy. In
addition, the isolation of wild-type revertant colonies gives confidence that nosecond-site mutations have been acquired during the procedure.

11.

Pick off 2-deoxyglucose-resistant colonies and patch or streak on MS agar
(replica plating the primary plates from step 10 is undesirable because nongrowing 2-deoxyglucose-sensitive spores may be transferred from thebackground lawn). Replica plate patches or colonies to:

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i.
ii.
iii.



MS agar;MS agar containing 20 µg ml–1 apramycin;
MS agar containing 10 µg ml–1 hygromycin.

Score the plates after 3-4 days. Exclude any colonies that are still apramycinresistant (2-deoxyglucose-resistant, apramycin-resistant colonies can arise if thedelivery vector is still integrated in the chromosome but the glkA gene becomesdeleted or acquires a mutation).
12.

Hopefully, the remaining colonies will be a mixture of hygromycin-resistantisolates (putative mutants) and hygromycin-sensitive isolates (putative wild-typerevertants). If so, confirm their chromosomal structure by PCR and Southernblot analysis. If all the colonies appear hygromycin-sensitive, confirm thechromosomal structure of 10 or 20 in case the hyg cassette is present but is notconferring significant resistance.
Even if the intervals flanking the hyg cassette in the original constructwere the same length, sometimes the number of wild-type revertants isolated
greatly exceeds the number of null mutants, especially if the null mutation has
a deleterious effect on growth or sporulation.
If you do not obtain the desired mutant, go back to step 10 and plate a
serial dilution of spores and mycelial fragments on MM containing 0.5%
mannitol, 200mM 2-deoxyglucose and 10 µg ml–1 hygromycin. Under these
conditions, only the desired mutants should grow (Fig. 14.5), provided that the
hyg cassette confers significant levels of resistance at single copy. If you still failto obtain the desired mutant, your target gene may be essential (see “Dealing
with essential genes,” p. 319).

13.

Show that you can complement the null mutant phenotype in single copy in trans,
for example by introducing the wild-type allele on the site-specific integrating
vector pSET152 (Bierman et al., 1992; p. 530). Full complementation showsthat the phenotype is caused by loss of target gene function, and not by polar
effects or by an acquired second site mutation.

14.

If the presence of the glkA deletion in the mutant is undesirable, move the hyg
insertion mutation into a preferred genetic background by generalised
transduction (p. 274) or by chromosomal transformation (p. 235) If the hyg
cassette does not confer selectable levels of resistance, it will not be possible tomove the mutation by either of these methods.

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chromosome**= mutation

reciprocal
double crossover*

segregation
(i)

(ii)
*

or
*

Fig. 14.6 Homogenotisation by reciprocal double crossing over followed bysegregation.

Homogenotisation
When a cloned allele is introduced into a strain carrying a different allele of the same gene in thechromosome, homogenotisation can give rise to a strain carrying the same allele in the chromosome andthe cloned DNA. This process can, in principle, occur in at least two different ways. In the first,
homogenotisation by segregation (Fig. 14.6), a reciprocal exchange by homologous double crossing overbetween the two alleles moves the chromosomal allele onto the plasmid and vice versa; the recombinant
plasmid then segregates into a spore (plating unit) together with a non-recombinant chromosome (Fig.
14.6i). Conversely, a non-recombinant plasmid can segregate together with a recombinant chromosome(Fig. 14.6ii). In principle, the multinucleoidal nature of Streptomyces cells should favour suchsegregation events. In the second mechanism, homogenotisation by gene conversion, one allele acts asa template for repair synthesis of the other, giving rise to two identical alleles (Fig. 14.7). This process
is perhaps most likely to occur during vector integration.

&KDSWHU*HQHGLVUXSWLRQDQGJHQHUHSODFHPHQWchromosome*

one allele acts as atemplate for repair
synthesis of the other* mutation
=

(i)

(ii)
*

or
*

Fig. 14.7 Homogenotisation by gene conversion.

Problems arising from unintended homogenotisation events
As shown below, homogenotisation can be extremely useful to isolate mutant alleles and to movethem into different genetic backgrounds to create new isogenic pairs of strains. However, it is importantto keep in mind the unintended possibilities and consequences of reciprocal exchange and geneconversion in any strain constructed to carry two or more different versions of the same gene. For
example, in one experiment, exchange between the chromosomal copy of gylR marked by an XhoI site,
and a second allele lacking the XhoI site introduced on a multicopy plasmid, was monitored by XhoI
digestion of plasmid minilysates. When the plasmid allele was carried in one orientation, approximately
80% of the minilysate DNA had picked up the XhoI-containing chromosomal allele. When the plasmidcarried the insert in the opposite orientation, exchange with the chromosome was undetectable by XhoI
digestion (M. Paget and C.P. Smith, personal communication). It is very important to be aware that thereis the potential for similar events to take place whenever a gene is introduced from one Streptomyces
species into another, as happens, for example, when S. lividans is used as a host for a plasmid or phagecarrying a gene cloned from another streptomycete (provided that S. lividans carries a homologue of thegene).



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S*

chromosome

White Thio

whiG

whiG

tsr

* mutation
=

tsr
*
whiG

whiG

R

Grey Thiohomogenotisation
tsr
*

*
whiG

whiG

R

White Thio

excision of vector*
whiG

recovered as
free phageor plasmid DNA

tsr
Fig. 14.8 Cloning a mutant allele by homogenotisation onto a phage or plasmid vector.

Cloning mutant alleles by homogenotisation
In Figs. 14.8 and 14.9, the whiG gene is used to illustrate the potential of homogenotisation toclone mutant alleles and to move them into different genetic backgrounds (Piret and Chater, 1985;
Méndez and Chater, 1987; Kelemen et al., 1995). These experiments were performed using an attP –phage vector, but the same approach could be taken with non-replicating E. coli plasmids or cosmids
provided the DNA can be recovered in E. coli after mini-lysate plasmid isolation from Streptomyces(Chapter 8), or with autonomously replicating Streptomyces plasmids.
An attP – phage carrying the wild-type whiG locus is introduced by natural infection into a whiG
mutant and ThioR lysogens are selected in which the phage has integrated at the whiG locus byhomologous recombination (Fig. 14.8). Because this strain now carries a functional copy of whiG, it
sporulates and is therefore grey. Propagation of this strain allows the detection of rare WhiG– colonies

&KDSWHU*HQHGLVUXSWLRQDQGJHQHUHSODFHPHQWGrey ThioS

chromosome
whiG

*
whiG

*

= mutation

tsr

tsr
R

*
whiG

whiG

Grey Thiohomogenotisation
tsr
*

*
whiG

whiG

R

White Thioloss of vector
*
whiG

S

White Thio

Fig. 14.9 Replacement of a wild-type by a mutant allele by homogenotisation.

in which homogenotisation has occurred to give rise to a prophage flanked by two copies of the whiG
mutant allele. Phages released from this strain now carry the mutant allele, which can then be introduced
into a new genetic background by a reversal of this process, creating pairs of isogenic strains (Fig. 14.9).
Lysogens are selected in the new genetic background and streaked in the presence of Thio to allow
detection of WhiG– colonies, in which the prophage is again flanked by two copies of the whiG mutant
allele. Streaking these colonies on medium lacking antibiotics allows the growth of rare ThioS WhiG–
colonies, identified by replica plating to Thio, in which the prophage has been lost by homologous
recombination between the duplicate copies of the whiG mutant allele. The structure of the putative newwhiG mutant can then be confirmed by Southern blotting.
The frequency with which homogenotisation events are detected in these kinds of analysis is
likely to vary depending on the exact nature of the experiment, for example the position of the mutation
relative to the ends of the cloned fragment is likely to be influential. In an experiment analogous to the&KDSWHU*HQHGLVUXSWLRQDQGJHQHUHSODFHPHQW

A

A

B

neo

chromosome

B

= may
represent >40kb

tsr

bla

of the chromosome

bla

tsr

R

R

Neo Thio
A

neo

B

A

neo

B

A

B

R

S

Neo Thio+RS

Amp Neo
A

B
tsr

bla

plasmid carrying deleted
DNA can be recovered in

E. coliFig. 14.10 Replacement of a whole gene cluster by double crossing over.
one described above, initial lysogenisation of a bldA mutant with an attP– phage carrying the wild-type
bldA gene gave about 90% Bld+ and 10% Bld– lysogens. Phages released from the Bld– strains were used
to lysogenise a wild-type strain, giving rise to about 1% Bld– lysogens (Piret and Chater, 1985).

Gene replacements involving whole gene clusters
MacNeil et al. (1993) used non-replicating E. coli plasmids in double crossover experiments toremove large, defined parts of the chromosome of S. avermitilis and so to make a deletion map of theavermectin biosynthetic gene cluster. They also showed that, when using non-replicating E. coli plasmids
in Streptomyces, circular plasmid DNA can be recovered from Streptomyces by making a conventional
plasmid mini-lysate and re-transforming an EcoK– (hsdR) strain of E. coli. These plasmids arise in
Streptomyces by low frequency excision from the chromosome. Because the exchange of alleles during

&KDSWHU*HQHGLVUXSWLRQDQGJHQHUHSODFHPHQWdouble crossing over between vector and chromosome is reciprocal, the DNA displaced from thechromosome can be recovered in E. coli as a set of clones complementary to the deletions. MacNeil etal. (1993) refer to this method as gene cluster displacement.
This methodology is likely to be particularly useful in analysing large antibiotic biosynthetic
clusters. In addition, it can be used to isolate all the DNA lying between any two cloned DNA fragments,
provided this interval is small enough that the E. coli vector can support its replication on transfer into
E coli. It can therefore be used to isolate large contiguous intervals of previously uncloned DNA. Usinga pBR322-derived vector, MacNeil et al. (1993) made deletions of >100 kb in the S. avermitilis
chromosome; the largest plasmid successfully recovered in E. coli carried 42 kb of S. avermitilis DNA.
The basic method is illustrated in Fig. 14.10. Two non-adjacent DNA fragments from a genecluster are cloned into an E. coli plasmid with a neomycin (Neo) resistance marker (neo from Tn5) cloned
between them. It is important for these experiments that Tn5 neo confers NeoR in both E. coli andStreptomyces. The plasmid is also marked with the Thio-resistance gene (tsr) for selection in
Streptomyces and bla for selection in E.coli. For deletion formation to work, the fragments in the plasmidmust be in the same relative orientation as they are in the chromosome (Figs. 14.10 and 14.11). Theirorientation with respect to the vector is also important. The desired orientation with respect to the vector
yields a NeoR ThioS deletion strain and allows recovery of the deleted DNA in E. coli; the "wrong"orientation with respect to the chromosome yields a NeoS ThioR deletion strain in which the vector DNA
and tsr replace the deleted DNA, which cannot then be recovered in E. coli. If the relative orientationin the chromosome of the two fragments of DNA is not known, four plasmids should be built in parallel,
reflecting the four possible chromosomal arrangements (-->1 -->2 ; -->1 <--2 ; <--1 -->2 ; <--1 <--2 ; Fig.
14.11). After introduction of the correct construct into Streptomyces, plasmid DNA is recovered froma NeoR ThioR transformant by conventional means and re-introduced into E. coli. Two types oftransformant should be obtained: AmpR, NeoR transformants carrying the plasmid originally introduced
into Streptomyces; and AmpR, NeoS transformants carrying the plasmid that had integrated into theStreptomyces chromosome through one interval but excised through the other and therefore carries thetwo original fragments and all the contiguous chromosomal DNA lying in between as a replacement for
the neo gene (Fig. 14.10). After growing the NeoR ThioR transformant in the presence of Neo alone, NeoR
ThioS colonies carrying the neo gene as a replacement for the deleted DNA can be identified (Fig. 14.10).
If all the DNA for a large gene cluster has been cloned, application of the type of logic describedabove can be used to reassemble the cluster as a contiguous piece of DNA in vivo in cases where suchan experiment is precluded in vitro because of the difficulties of partial digestion and religation whenworking with long stretches of DNA. Malpartida et al. (1990) cloned the entire red gene cluster,
specifying production of the antibiotic undecylprodigiosin, on two only slightly overlapping clones ofS. coelicolor DNA. Two segments of DNA (10.6 and 4.3 kb) from just outside opposite ends of thecluster were cloned side by side in the correct orientation relative to each other in a derivative of the low
copy number plasmid SCP2* and introduced into wild-type S. coelicolor. On propagation of atransformant, a plasmid carrying the entire red cluster on a 38 kb insert arose, presumably by reciprocal
double crossing over with the chromosome. The desired clone was recognised by isolating plasmid DNA
from the wild-type strain and transforming a red mutant which carried a mutation in a gene central to thecluster, not included in the original plasmid.

&KDSWHU*HQHGLVUXSWLRQDQGJHQHUHSODFHPHQW
chromosome

A

B

Construct
A

neo
tsr

B

A

B

A

S

S

R

Neo Thio
deletion

bla

neo

R

Neo Thio
deletion

bla

neo
tsr

ResultB
No deletion

tsr

A

bla

neo

B
No deletion

tsr

bla

Fig. 14.11 Gene replacement of a whole gene cluster by double crossing
over: consequences of four possible orientations of the two flanking
sequences.

Mutational analysis of transcription units
The integration of vectors by a homologous single crossover between the insert and thecorresponding chromosomal interval can be used to analyse the arrangement of transcription units in geneclusters (Chater and Bruton, 1983). The basic principle is illustrated in Fig. 14.12. When the fragment
mediating integration overlaps either the 50 end or 30 end of a transcript, integration will regenerate an
intact transcription unit and will not be mutagenic. However, use of a fragment internal to a transcription
unit is mutagenic since integration does not recreate an intact transcription unit.
To ensure that this kind of experiment is correctly interpreted, it is important that, after
integration, the genes lying downstream of the vector, which are now disconnected from their natural
promoter, are not expressed artificially from a vector promoter reading out into the chromosome. Thiscan be ensured by using a vector in which the cloning site has an efficient transcriptional terminator,
which will lie upstream of the cloned genes. Restriction fragments lying internal to a transcription

&KDSWHU*HQHGLVUXSWLRQDQGJHQHUHSODFHPHQW

A
R

B
R

R

C
R

A



R

R

C

1

3
B

2

1

A

2

3

A

B

A

B

A

B

C

B

C

C

C

Fig. 14.12 Mutational analysis of a transcription unit.
unit may not be mutagenic if the genes lying downstream of the vector do not contribute significantly
to the phenotype. For example, the glycerol utilisation (gyl) operon of S. coelicolor includes adownstream gene, gylX, but mutational cloning analysis did not reveal gylX as part of the operon because
it is not essential for growth on glycerol (Seno et al., 1984; Smith and Chater, 1988).

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gentamicin-(3)-N-acetyl-transferases III and IV.
II.
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Beck E, Ludwig G, Auerswald EA, Reiss B, Schaller H(1982) Nucleotide sequence and exact localization of theneomycin phosphotransferase gene from transposon Tn5.
Gene, 19, 327-336.
Bedford DJ, Laity C, Buttner MJ (1995) Two genes
involved in the phase-variable C31-resistance mechanism ofStreptomyces coelicolor A3(2). J.Bacteriol., 177, 4681-4689.
Bibb MJ, Bibb MJ, Ward JM, Cohen SN (1985)
Nucleotide sequences encoding and promoting expression of
three antibiotic resistance genes indigenous to Streptomyces.
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Bierman M, Logan R, O'Brien K, Seno ET, Rao RN,
Schoner BE (1992) Plasmid cloning vectors for the conjugaltransfer of DNA from Escherichia coli to Streptomyces spp.

Gene, 116, 43-49.
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Pernodet J-L (1997) Antibiotic resistance gene cassettes
derived from the omega interposon for use in E. coli andStreptomyces. Gene, 190, 315-317.
Bräu B, Pilz U, Piepersberg W (1984) Genes for
gentamicin-(3)-N-acetyltransferases III and IV: I. Nucleotide
sequence of the AAC(3)-IV gene and possible involvement of
an IS140 element in its expression. Mol.Gen.Genet., 193,
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that are multiply deficient in the non-essential hrd-encoded
RNA polymerase sigma factors. J.Bacteriol., 174, 5165-5167.
Buttner MJ, Chater KF, Bibb MJ (1990) Cloning,
disruption and transcriptional analysis of three RNA
polymerase sigma factor genes of Streptomyces coelicolor
A3(2). J.Bacteriol., 172, 3367-3378.



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Chater KF, Bruton CJ (1983) Mutational cloning in
Streptomyces and the isolation of antibiotic production genes.
Gene, 26, 67-78.
Chater KF, Bruton CJ (1985) Resistance, regulatory andproduction genes for the antibiotic methylenomycin are
clustered. EMBO J., 4, 1893-1897.
Chater KF, Bruton CJ, Foster SG, Tobek I (1985) Physicaland genetic analysis of IS110, a transposable element ofStreptomyces coelicolor A3(2). Mol. Gen. Genet., 200, 235239.
Dhillon N, Leadlay PF (1990) A repeated decapeptide motif
in the C-terminal domain of the ribosomal RNA methylasefrom the erythromycin producer Saccharopolyspora
erythraea. FEBS Letters, 26, 189-193.
Fisher SH, Bruton CJ, Chater KF (1987) The glucose
kinase gene of Streptomyces coelicolor and its use in selecting
spontaneous deletions for desired regions of the genome.
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Flett F, Mersinias V, Smith CP (1997) High efficiency
intergeneric conjugal transfer of plasmid DNA from
Escherichia coli to methyl DNA-restricting streptomycetes.
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Floriano B, Bibb M (1996) afsR is a pleiotropic but
conditionally required regulatory gene for antibiotic
production in Streptomyces coelicolor A3(2). Mol.Microbiol.
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Brawner ME (1987) Two promoters, one inducible and one
constitutive, control transcription of the Streptomyces lividans
galactose operon. Proc. Natl. Acad. Sci. USA, 84, 2130-2134.
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Hillemann D, Dammann T, Hillemann A, Wohlleben W(1993) Genetic and biochemical characterization of the two
glutamine synthetases GSI and GSII of the phosphenothricylalanyl-alanine producer, Streptomyces viridochromogenes
Tü494. J.Gen.Microbiol., 139, 1773-1783.
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Corynebacterium glutamicum but not in Streptomyces
lividans. J.Bacteriol., 174, 5462-5465.
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

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

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m15tk4b.wpd 14 March 2000 Tobias Kieser

Chapter 15

Reporter systems
CONTENTS
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339
The problems with lacZ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340
Choice of vector: plasmid, phage and transposon systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341
Antibiotic resistance genes as reporters: neo and cat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342
The tyrosinase-encoding operon of S. glaucescens as a reporter system . . . . . . . . . . . . . . . . . 342
The whiE (spore pigment) major operon as a reporter system . . . . . . . . . . . . . . . . . . . . . . . . . 343
The catechol 2,3-dioxygenase determinant, xylE, a readily quantified reporter genefor Streptomyces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343
Detection of xylE expression in situ in colonies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344
Detection of xylE expression in C31 plaques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344
Assay of catechol 2,3-dioxygenase in cell-free extracts . . . . . . . . . . . . . . . . . . . . . . . . . 345
Use of the Vibrio harveyi luxAB genes as a reporter system . . . . . . . . . . . . . . . . . . . . . . . . . . . 346
Reporter systems based on EGFP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 347
A reporter gene encoding a thermostable malate dehydrogenase . . . . . . . . . . . . . . . . . . . . . . 348
Assay of thermostable malate dehydrogenase in cell-free extracts . . . . . . . . . . . . . . . 348
The ampC ( -lactamase) gene as a reporter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349
The lac (secreted -galactosidase) gene of S. lividans as a reporterfor transcription and secretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349
The redD gene of S. coelicolor as an easily scorable reporter of transcription
in S. coelicolor and S. lividans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 350

Introduction
The direct analysis of transcript abundance by techniques such as S1 nuclease protection, reverse
transcriptase-mediated primer extension or, increasingly, by DNA arrays, provides the most reliable
information about the in vivo transcription of particular genes. Similarly, direct analysis of the proteinsencoded by genes of interest, for example by activity measurements, immunological methods or the
global techniques of proteomics, allows post-transcriptional aspects of gene function to be monitored.
However, these techniques can have limitations: they are generally too cumbersome to be used on large
numbers of samples, as in mutant hunts, genetic linkage analysis, or complementation; they may needquite large amounts of material; and they do not always readily permit the study of events that are taking
place in only a small fraction of cells at any given sampling time. To overcome difficulties such as these,
the use of transcriptional and translational fusions to reporter genes was pioneered in the 1970s by
Casadaban and others (Bassford et al., 1980), classically exploiting the lacZ gene of E. coli, and&KDSWHU 5HSRUWHUV\VWHPV

subsequently extended to include a wide range of reporters. The product of lacZ, -galactosidase, hasmany potentially helpful attributes, including very easy, sensitive and quantitative assay, convenient andsensitive tests for expression in colonies (for example, using colorigenic substrates such as 5-bromo-4chloro-3-indolyl- -D-galactoside (X-gal) or fluorigenic substrates such as 4-methylumbelliferyl- -Dgalactoside (MUG)), selectability and counterselectability (Beckwith, 1980). It has therefore foundsustained use over the last two decades in almost all organisms (prokaryotic or eukaryotic, simple orcomplex) in which genetic analysis has been done, except plants (which have significant endogenous galactosidase activity) and, particularly frustratingly, Streptomyces (King and Chater, 1986). In thischapter, we provide documentation and guidance on the use of various reporter systems in Streptomyces.
It is a somewhat painful fact that, after all this time, there is still no Streptomyces reporter system thatcomes close to lacZ in its reliability, convenience and range of applications: the rate of progress in some
aspects of Streptomyces genetics has undoubtedly been limited by this deficiency (see below).

The problems with lacZ
In order to help anyone planning to use lacZ-based reporter systems in Streptomyces, it is worth
briefly enumerating the difficulties. (1) Most streptomycetes possess an extracellular enzyme that has-galactosidase activity. This interferes with colony staining to detect lacZ expression. Mutants lacking
the exoenzyme have been constructed in S. lividans (Eckhardt and Smith, 1986) and S. coelicolor (K. F.
Chater, unpublished), but they do not grow or form aerial mycelium quite as well as their parental strains.
The lac mutations may therefore have unpredictable effects on the expression of other genes. (2)Attempts by King and Chater (1986) to clone lacZ in Streptomyces plasmid vectors were troubled bygenetic instability: the inserted gene suffered deletions at a very high frequency. The gene could bemaintained in C31-derived phage constructs, but its expression was poor: plaques became blue (withX-gal) only on prolonged incubation, and lysogens with lacZ fused to potentially active promoters gavevery low -galactosidase activity. (3) The codon usage of lacZ is very different from that typical of
Streptomyces genes, notably in containing several TTA codons, which are likely to limit translation todifferent extents in different cell types or growth conditions (p. 387). This is because the only tRNA forUUA codons is the product of the bldA gene, which appears to have a developmental role (bldA mutants
lack aerial mycelium and secondary metabolism under most growth conditions) (Leskiw et al., 1991).
As a result of these problems, lacZ does not seem to have been used any further. However, it
is conceivable that a mutant version of lacZ with altered codon usage would prove useful, as has been
the case with the luxAB and gfp systems (pp. 346-347). In addition, some early experiments used the S.
lividans lac gene as a reporter (p. 349).
An early report of successful use of the E. coli galK gene as a reporter in a galK mutant of S.
lividans (Brawner et al., 1985) has not been followed up.

&KDSWHU5HSRUWHUV\VWHPV

An illustration of difficulties encountered in the use of a high copy number promoter-probe
vector
The promoters associated with the whiG and whiE sporulation genes of S. coelicolor A3(2) wereinserted into the high copy number promoter-probe plasmid pIJ4083, in which they caused expressionof the xylE reporter gene (p. 343). Time course analyses of xylE expression during development, citedby Chater (1993), indicated strong dependence of each promoter on whiA, whiB and whiH. However,detailed S1 nuclease protection analysis of mRNA abundance has shown that whiG transcription has
little if any dependence on any of the five whi genes tested (Kelemen et al., 1996), whereas whiE
expression is severely dependent on all of them (Kelemen et al., 1998). Although these xylE results
may eventually be interpretable in terms of the regulatory interplay of whi genes, these data clearly
warn against use of multiple copies of a promoter in evaluating subtle regulatory parameters.

Choice of vector: plasmid, phage and transposon systems
In thinking about the use of reporter genes, it is important to be aware that the genetic situation
used to generate chimaeric transcription units may give rise to misleading results, and that it is generally
worth validating the main conclusions of such analyses by an independent approach, such as direct RNA
analysis. An illustration of this is provided in the box above. High copy number promoter-probe vectors
can be useful for demonstrating promoter activity, and evaluating the effects of major cis-acting
mutations in promoters, but the plasmid copy number may well vary in response to developmental stage,physiological conditions, and genetic background. Moreover, multiple copies of a promoter may
sequester low-abundance regulatory proteins and thereby cause unpredictable secondary effects.
For the biologically more meaningful use of reporter genes in analysing gene expression, it ispreferable to use single copy gene fusions. This may be achieved with vectors based on SCP2*, such as
the xylE vectors pXE4 and pIJ2842. SCP2*-based vectors, because of their low copy number and large
size, are less easy to manipulate than most other vectors, making them less suitable in experiments
involving the analysis of large numbers of plasmids. Another option is to use att+ derivatives of C31,
such as the xylE-containing KC859, pp. 296-299. Here the main limitation is the size of the insert (upto 2.9 kb for KC859). Phage vectors provide a particularly convenient means of moving constructs
between different genetic backgrounds. In a third approach it is convenient to make primaryconstructions in E. coli, using vectors that contain an E. coli replicon, an origin of transfer for conjugal
mobilisation into Streptomyces, and the att-int system of C31 or pSAM2, e.g. gfp-containing plasmid
pIJ8660 (p. 347).
These are all systems in which promoters are cloned upstream of a reporter gene in a promoterprobe vector. Alternatively, reporter genes may be inserted directly into the chromosomal copy of thelocus of interest. One approach is a specialised version of gene disruption by vector integration (p. 312)
in which the disruption vector contains a suitably positioned reporter gene. Vectors include both attdeleted C31 derivatives (e.g. KC860, KC861, KC862; Fig. 13.2, p. 297 and pp. 527-528) and E. coli
plasmids such as pKC1138 (mobilisable by conjugation; p. 566) all containing xylE. The use of suchvectors involves single crossover integration, and may sometimes be marginally complicated by tandem&KDSWHU 5HSRUWHUV\VWHPV

integration of more than one copy of the construct; for example, this has often been observed withintegration events involving the widely used vector pDH5 (Chapter 22), especially if the levels ofthiostrepton used for selection are as high as 50 µg ml–1. Another kind of in situ fusion to reporter genes
is provided by the use of transposons constructed with a reporter gene close to one end of the element(e.g. Tn5099: Hahn et al., 1991; Baltz et al., 1992). Transposon insertions generally cause loss offunction of the gene into which they have inserted, whereas engineered phages or plasmids undergoinginsert-directed single crossover events are not always expected to cause loss of function: in particular,
if the inserted material contains the promoter being studied, the wild-type phenotype is usually retained(p. 312).

Antibiotic resistance genes as reporters: neo and cat
The two reporters that were first successfully used to detect promoter activity in Streptomyces
were cat (from Tn9, encoding chloramphenicol acetyltransferase (cat): pp. 564-565) and neo (from Tn5,
encoding aminoglycoside phosphotransferase: p. 481), in each case present in moderate or high copy
number promoter-probe plasmids such as pSLP124 (Bibb and Cohen, 1982) and pIJ486 (Ward et al.,1986). The main utility of these plasmids is the ease of screening for chloramphenicol or neomycin
resistance conferred by cloned promoters that are active during growth. Most work with these plasmids
has been done in S. lividans hosts. In the case of neo-containing plasmids such as pIJ486 and pIJ487,
the level of resistance bears some relationship to the strength of the promoter, providing a usefuldiagnostic feature. There are simple in vitro assay systems for Cat (Shaw and Hopwood, 1976; Schottel
et al., 1981) and Neo (Ward et al., 1986). Activity may also be detected in situ after non-denaturing
polyacrylamide gel electrophoresis (Reiss et al., 1994).
The Tn5 neo gene has also been introduced into C31 att+ derivatives (KC310 and KC850;
Table 22.1, p. 513), to allow its use at single copy number (as part of a prophage). KC310 derivatives
containing the ermE and aphI promoters from Saccharopolyspora erythraea and S. fradiae bothconferred selectable neomycin resistance when used to lysogenise S. lividans (S. H. Fisher and K. F.
Chater, unpublished). The Tn5 neo gene has also been used successfully in translational fusions in S.
lividans, in the multicopy vector pIJ688 (Kieser et al., 1986).

The tyrosinase-encoding operon of S. glaucescens as a reporter system
The melC operon of S. glaucescens (p. 501) consists of two genes: the second (melC2) encodestyrosinase, and the first (melC1), encodes a protein involved in transferring copper to apotyrosinase(Chen et al., 1992). On media containing CuSO4 (5 mg l–1) and tyrosine (0.3 g l–1), expression of themelC operon leads to dark brown haloes of melanin around colonies, an easily visualised phenotype.
This attribute was exploited in the construction of pMT3010 (based on pIJ486, containing the high copy
number pIJ101 replicon, p. 256) and pMT3020, an E. coli plasmid that contains the site-specificintegrating element IS117 of S. coelicolor A3(2) (Paget et al., 1994). Both vector systems gave
regulatable responses when the S. coelicolor gyl operon promoter was used to drive melC expression.

&KDSWHU5HSRUWHUV\VWHPVThis system may well be useful for mutant isolation and semi-quantitative promoter analysis, but
it is unsuitable for fully quantitative studies because tyrosinase is excreted and therefore difficult tomeasure meaningfully in relation to total cell protein levels. In addition, it should be noted that use of
the IS117 integration system makes it inconvenient to use pMT3020 in S. coelicolor, most strains ofwhich already contains one or two integrated IS117 elements (p. 265). The published experiments havetherefore been done in S. lividans (strain TK64), which does not contain IS117. Usually, transformants
of TK64 with IS117 derivatives contain two tandemly arranged copies of the element (Henderson et al.,
1990; p. 265). Finally, the use of melC at a very low copy number is effective only with strong
promoters.

The whiE (spore pigment) major operon as a reporter system
The grey-brown spore colour of S. coelicolor A3(2) is due to a pigment synthesised via the
polyketide route. The complex biosynthetic pathway involves a cluster of at least eight whiE genes
transcribed as two transcription units from a divergent promoter region (Davis and Chater, 1990; Blanco
et al., 1993). A segment of the rightward operon containing six genes (ORFs II-VII) encodes all the steps
needed to produce a dark brown diffusible pigment, and has been used as a reporter in the vectors pARC1(Horinouchi and Beppu, 1985) and pCLL34 (Feitelson, 1988). Both of these vector systems are basedon the SLP1.2 replicon (p. 265), giving a predicted copy number of about 5-6. Because SLP1 is present
in all S. coelicolor A3(2) derivatives (in an integrated form), its derivatives cannot be used in S.
coelicolor A3(2). Most experiments with these vectors have therefore been done in S. lividans (e.g.
Narva and Feitelson, 1990; Farkašovský et al., 1991).
Synthesis of the pigment does not require the addition of specific substrates, and can take placeon various media. Because the genes originate from a streptomycete they are well expressed, and they
continue to find occasional utility. There is undoubtedly scope for further development of vectors
containing this reporter. The activity of the endogenous whiE cluster of S. lividans does not have asignificant impact on the ease of recognising diffusible brown pigment production, because the spore
pigment as specified by the intact gene cluster is entirely bound to the spore wall and non-diffusible: Yu
and Hopwood (1995). Limitations of the system are that it cannot be meaningfully quantified, and it does
not convey spatial information.

The catechol 2,3-dioxygenase determinant, xylE, a readily quantified reporter genefor Streptomyces
Catechol 2,3-dioxygenase, the product of the xylE gene from the Pseudomonas TOL plasmid (p.
495), converts colourless catechol into 2-hydroxymuconic semialdehyde, which is yellow. Its potential
utility as a reporter system, first recognised by Zukowski et al. (1983), arises from the simplicity of thisassay system, its applicability in situ to colonies and plaques (to give the “Ylo+” phenotype), and theabsence of catechol 2,3-dioxygenase from almost all organisms. Ingram et al. (1989) showed that xylE
was an excellent reporter gene in Streptomyces, with the low copy number SCP2*-based vector pXE4
proving particularly reliable. This led to incorporation of a promoterless xylE gene into a diverse range

&KDSWHU 5HSRUWHUV\VWHPVof genetic contexts including transposons, low and high copy number plasmids, integrating plasmid
vectors, and att+ and att- derivatives of C31 (Tables 13.1 and 13.2, pp. 292-293). One limitation of
xylE, its unsuitability for spatial analysis of gene expression, arises from the diffusible nature of theyellow end-product.
Detection of xylE expression in situ in colonies (Ingram et al., 1989)
Colonies grown on agar medium (preferably a medium with a minimum amount ofbrown/yellow colouration, as this may obscure the Ylo+ phenotype; a replica set ofcolonies will be needed if further subculture is expected, since catechol treatment ofcolonies may cause cross-contamination and reduced viability)
0.5M aqueous catechol (55 g l–1); filter-sterilise; store at 4o in the dark
Aerosol spray
Aluminium foil (or dark bag or box)
30o-37o incubator

1.

Spray the surface of the plate containing colonies with about 0.1-0.3 ml catechol
solution. Use a fume hood for protection against catechol.

2.

Wrap the plate in foil. In the presence of light, and on some media, a brownish colour may
develop in the medium which obscures the Ylo+ phenotype.

3.

Incubate for 5 min to 3 h, depending on the strength of xylE expression, before
scoring the Ylo+ phenotype.

Detection of xylE expression in

C31 plaques (Bruton et al., 1991)

During phage multiplication in plaques, xylE expression may take place either by readthroughfrom phage promoters (providing evidence that xylE is intact; this is seen in KC859-KC861; p. 296) or
because of the promoter activity of a cloned piece of DNA (as in KC862; p. 296). The Ylo+ phenotypeof plaques can often be detected by spraying with catechol as described above for colonies, but better
results are obtained by the following double top-layer procedure.
Spore suspension of S. lividans (c. 109 spores ml–1)
Plaques to be tested
Petri dishes containing 25 ml R2 medium (surface dried, not over-dry: e.g. 10 min inlaminar flow hood. R2 is used, rather than R2YE, to minimise yellow background.)Soft Nutrient Agar (SNA) with and without 2mM catechol, kept molten at 45o in awater bath (2 × 4.5 ml with catechol and 1 x 3 ml without catechol in separate tubes orbottles per set of plaques to be assayed)
Toothpicks or inoculating wire

&KDSWHU5HSRUWHUV\VWHPV



1.

Pour 4.5 ml SNA containing 2mM catechol onto one plate for each set of plaques
and allow to solidify for at least 10 min.

2.

Add a drop of S. lividans spores to each remaining 4.5 ml molten SNA plus 2mM
catechol, and pour as second top layer onto the base plates. Allow to solidify forat least 10 min.

3.

Add a drop of S. lividans spores to each 3 ml aliquot of SNA and pour onto abase plate to form a top layer. Allow to solidify for 10 min.

4.

Stab plaques into the top layers in an orderly array (up to 25 per plate), making
replicates in the top layers without catechol. You can inoculate both plates from asingle stab. Note that if plates with top layers are not used immediately, they should be kept in
the dark, for no more than 1-2 h.

5.

Wrap in aluminium foil to keep out the light, and incubate overnight at 30o,
before scoring the Ylo+ phenotype. In order to avoid expensive colour photography, thediffusible yellow halo round plaques can be photographed as a dark zone in black and white if
a blue filter is used. Use the catechol-free replica for further subculturing of selected phages.

Assay of catechol 2,3-dioxygenase in cell-free extracts
Mycelium from liquid culture, or culture on a cellophane disc (p. 48)
Sample buffer (100mM phosphate buffer pH7.5, 20mM NaEDTA pH8.0, 10% v/v
acetone)Assay buffer (10mM phosphate buffer pH7.5, 0.2mM catechol: 20mM catechol stock
solution prepared in ethanol and stored at -20o)
Bradford reagent and set of protein concentration standards
Triton X-100 (10% aqueous solution)
Ice-water; Sonicator; spectrophotometer; cuvettes (1 cm light path)1.

Suspend mycelium in cold sample buffer in a 1.5 ml Eppendorf tube. Mycelium
scraped from one cellophane disc should be suspended in 0.5 ml sample buffer.

2.

Sonicate on ice, 4 ×15 sec, with 30 sec intervals, avoiding foaming.

3.

Add Triton X-100 solution (5 µl per 50 µl sample, to give 0.1% final
concentration) and keep on ice for 15 min.

4.

Centrifuge for 5 min in microfuge.

5.

Transfer the supernatant to a fresh tube.



6.

&KDSWHU 5HSRUWHUV\VWHPV

Add sample (e.g. 10 µl, 50 µl) to 0.5 ml assay buffer in a prewarmed (30o)cuvette and follow the change in A375. Activity in S. lividans and S. coelicolor appears
to change little during storage of samples at 4o overnight.

7.

Assay samples for protein content by the Bradford reagent.

8.

Using the slope of the linear part of the spectrophotometric output, calculate
specific activity as follows:mU catechol dioxygenase [ nmol min -1 ] =30.03 × ∆ A375
time [min]Use of the Vibrio harveyi luxAB genes as a reporter system
Light emission through the action of luciferase provides the basis of a reporter system themeasurement of which is potentially highly sensitive and linear over many orders of magnitude. Thissystem can provide a significant degree of spatial and temporal information about gene expression. Two
distinct luciferase-encoding reporter systems are used in diverse prokaryotic and eukaryotic organisms.
The work in Streptomyces has always involved the V. harveyi luxAB gene pair (p. 498), because the only
additional component needed for light emission is the volatile aldehyde n-decanal (obtainable from
Sigma). In its gaseous form, n-decanal can readily reach and penetrate even aerial parts of the colony.
The alternative system, the firefly luc gene, encodes a luciferase that can give light only when the nonvolatile co-factor luciferin is taken up by luc-expressing cells, making it less suitable for spatial analysis
of gene expression in Streptomyces.
In pioneering studies by Schauer and co-workers (e.g. Sohaskey et al., 1992), the luxAB genes
were successfully used to obtain a limited amount of spatial and temporal information about gene
expression in Streptomyces, and several luxAB-containing transposon derivatives were constructed andused to generate insertion mutants that had different patterns of light emission. Two papers (Guijarro
et al., 1988; Kormanec et al., 1996) have successfully used the luxAB-containing reporter plasmid
pRS1105 of Schauer et al. (1988).
Two main difficulties have inhibited the wider use of the luxAB system. First, detection of lowlevels of light emission requires sophisticated and expensive light-sensing equipment. Second, the luxAB
genes contain seven TTA codons, with the result that translation of luxAB mRNA depends on the tRNA
encoded by bldA (Leskiw et al., 1991). Thus, luminescence will be dramatically reduced in a bldA
mutant, and any translational effects of natural changes in bldA tRNA abundance may influence lightemission. To circumvent this problem, a TTA-free version of luxAB has been generated (G. R. Brown
and N. Oestreicher, personal communication), and incorporated into new luxAB transposons based on
IS6100 (Tn1771 and Tn1772) and on Tn5 (Tn522); (contact Dr Paul Dyson, Department of Genetics,
University of Wales, Swansea). The TTA-free luxAB cassette is available from the John Innes Centre
as a cassette (contact Celia J. Bruton) and as a single copy number plasmid (pIJ5971) suitable forconstructing promoter-luxAB fusions (contact M.S.B. Paget or M.J. Buttner).

&KDSWHU5HSRUWHUV\VWHPVMethods for detecting or measuring light emission depend on the purpose of the experiment andthe nature of the material. Whole colony luminescence can be measured in a luminometer. Sohaskey
et al. (1991) used the wide end of a standard yellow Pipetman tip to punch a colony from the agar, after
which the colony and its ring of plastic were cut from the rest of the tip with a razor and placed “facedown” on the bottom of a scintillation vial. After placing a filter-paper wick carrying 2 µl n-decanal in
the vial and screwing on the cap, photon output was measured in a luminometer, in the published case,a Turner Designs model 20e, using 30 sec delay following by 45 sec integration. Screening of colonies
on plates was done by placing a 100 mm diameter filter paper disc, carrying 10 µl n-decanal, in the Petri
dish lid and photographing (in the dark) with a Hannamatsu Photonics VIM camera. More recently P.
Herron and P. Dyson (personal communication) have used a microplate luminometer (Anthos Lucy 1),
with 0.2 sec integration. Individual clones are inoculated onto 300 µl solid medium in individual wells.
After, or during, growth each well is exposed to a filter paper (cut to the size and shape of the microplate,and placed above the wells in the lid of the dish) impregnated with n-decanal (c. 1 µl per well) for 5 minat room temperature. After removing the filter paper and lid, the plate is introduced into the luminometerand emissions from each clone are measured. For luciferase measurements during liquid culture (usually
SMM), a 300 µl sample is combined with 1 µl n-decanal in a microplate well, incubated for 5 min atroom temperature and then measured as above. Note: n-decanal inhibits growth of Streptomyces

Reporter systems based on EGFPOne of the most powerful reporter systems to emerge recently, with applications across the wholebiological spectrum, is based on the gfp gene of the jellyfish Aequorea victoria. The product of gfp isa green fluorescent protein, whose fluorescence is an intrinsic property of the mature protein (the natural
excitation maximum is at 395 nm, with emission at 509 nm). A recent review of aspects of gfp relevant
to its use as reporter gene is by Tsien (1998), and many useful aspects of GFP biochemistry and genetics
are provided in the literature supplied by Clontech, Palo Alto, CA, USA, a company specialising in gfp(e.g. Clontech, 1996).
Although several initial attempts to use gfp in Streptomyces were discouraging, more promising
results were obtained by using a synthetic version of the gene in which the codon usage is particularly
suitable for Streptomyces (M.A. Fernández-Abalos, personal communication). Sun et al. (1999) madefurther improvements by incorporating the mutations F64L and S65T (EGFP; p. 499), which reduces
photobleaching, increases fluorescence, shifts the emission peak to one more appropriate for widely-used
FITC filters, and improves solubility (Cormack et al., 1996). This led to construction of pIJ8660, whichcontains many features desirable for a promoter-probe system (Sun et al., 1999):
C
C
C
C
C
C

pUC18 origin of replication for propagation in E. coli;
apramycin resistance gene selectable in E. coli and Streptomyces spp;
oriT (RK2) for conjugal transfer from E. coli to streptomycetes;
C31 att-int region for stable chromosomal integration, ostensibly at single copy number;
EGFP cassette flanked by transcriptional terminators, and containing multiple-cloning site (MCS);
stop codons in all three reading-frames between the MCS and the EGFP start codon to prevent
formation of unwanted translational fusions.

&KDSWHU 5HSRUWHUV\VWHPVAfter cloning a promoter into the pIJ8660 polylinker, using an E. coli host, the DNA isintroduced into the non-methylating, mobilisation-proficient host ET12567/pUZ8002, using kanamycin(100 µg ml–1) and apramycin (100 µg ml–1) to maintain selection for both plasmids, and chloramphenicol(50 µg ml–1) to maintain selection for Tn9 inserted into the dam gene. Transfer from this strain into
streptomycetes by conjugation is described on p. 249.
To analyse EGFP expression, strains are typically grown on a minimal medium (this minimises
background autofluorescence problems), in the angle under a coverslip inserted obliquely into the agar,
prior to fluorescence microscopy. A Zeiss Axiophot photomicroscope fitted with a fluorescein
isothiocyanate (FITC) filter set containing an excitation filter for wavelength 450-490 µm is suitable forthis purpose. Exposure times of 30-100 sec (Kodak Ektachrome Elite 200 film) have been used in ourlaboratory, with a 100× objective. The same microscope, with a 20× objective, can be used to visualise
fluorescence at the whole colony level. For precise spatial information, confocal microscopy (e.g. LeicaTCS NT confocal microscope, 488 nm argon laser, 63× objective, 0.5 NA dry PL FLUOTAR) has beenvery effective (Sun et al., 1999).
A second pIJ8660-like vector, pIJ8668, lacks the C31 att site, so that chromosomal DNA
inserts can be used to direct the resulting fusions to the native chromosomal locations (Sun et al., 1999).
In addition, P. Herron and P. Dyson (Department of Genetics, University of Wales, Swansea) have madetransposons that can generate transcriptional fusions to EGFP, based on Tn5 (Tn502) and IS600 (Tn1761
and Tn1762). They have found that a Leica MZ FLIII fluorescence stereomicroscope, which has a zoom
facility, is convenient for studies at the whole colony level.

A reporter gene encoding a thermostable malate dehydrogenase
To be useful, a reporter gene must usually encode a function absent from the host. However, if
the reporter is only to be used for direct enzyme assays (and not for work on living bacteria), then it may
be possible to inhibit an equivalent activity in the host. An example of this is provided by use of the mdh
gene of Thermus flavus, which encodes a thermostable malate dehydrogenase. A low copy number(SCP2*-based) plasmid, pTMA1, has been designed as a promoter-probe system by Vujaklija et al.
(1991).
Assay of thermostable malate dehydrogenase in cell-free extracts
Washing buffer (4o) (50mM Tris-HCI pH7.5; 5mM EDTA, 50mM NaCl)
100mM potassium phosphate buffer pH7.0
5mM oxaloacetate
1.5mM NADH
Bradford reagent and protein concentration standards
Sonicator; centrifuge or Eppendorf centrifuge (room temperature or refrigerated forcentrifugation at 10,000 × g); 80o water bath; spectrophotometer (30o, 340 nm)
1.

Suspend mycelium from 50 ml culture in 7 ml washing buffer.

&KDSWHU5HSRUWHUV\VWHPV



2.

Sediment on bench centrifuge (e.g. 10 min, 10,000 × g).

3.

Discard supernatant, and resuspend mycelium in 7 ml 100mM potassium
phosphate pH7.0 (on ice).

4.

Sonicate on ice, 4 ×15 sec, with 30 sec intervals, avoiding foaming.

5.

Centrifuge (10,000 × g, 30 min, 4o) to remove debris.

6.

Prepare assay buffer by mixing 1 ml potassium phosphate buffer pH7.0, 0.1 ml
5mM oxaloacetate, 0.3 ml 1.5mM NADH.

7.

Add suitable volume of extract and make up to 3 ml with 100mM potassium
phosphate pH7.0.

8.

Follow A340 at 30o in a spectrophotometer. The extinction coefficient of NADH is
6.22 mM-1 cm-1.

9.

Assay samples for protein content with the Bradford reagent and calculate
specific activity.

The ampC ( -lactamase) gene as a reporter (Forsman and Jaurin, 1987)
The activity of the -lactamase specified by the ampC gene of E. coli (p. 495) can readily andsensitively be detected by the use of chromogenic -lactam substrates, usually nitrocefin (Oxoid Unipath
Ltd., Basingstoke, England), hydrolysis of which gives rise to a reddish-brown pigment. This can bedetected at the colony level by spraying (or, more economically, spotting on) fresh nitrocefin solution(500 µg ml–1), or in cell-free extracts. The SLP1.2-based vector pJAS14, which was developed to exploit
ampC as a reporter, has been used with some success to compare promoter strengths in an S. lividans host(Forsman and Jaurin, 1987), but the utility of the system is compromised in two ways: the SLP1.2
replicon cannot be used in strains of S. coelicolor A3(2) (p. 265), and the ampC gene contains several
TTA codons, rendering it sensitive to possible translational regulation by bldA (Leskiw et al., 1991).

The gal (secreted -galactosidase) gene of S. lividans as a reporter fortranscription and secretion
S. lividans and S. coelicolor both secrete a protein with -galactosidase activity. This protein
is not homologous with E. coli -galactosidase, but its enzymatic activity can be detected by the same
reagents (notably X-Gal or MUG, p. 340).

&KDSWHU 5HSRUWHUV\VWHPVThe S. lividans gal gene was cloned by Eckhardt et al. (1987), and gal– mutants of S. lividans(strains 1326-9R and 1326- gal, p. 496; Eckhardt and Smith, 1987) and S. coelicolor (J1510 and J1511,
gal- deleted derivates of strain M130 and J1501 respectively; K.F. Chater, unpublished) have been
isolated, so the gal gene can be used as a reporter. Stein et al. (1989) constructed an SLP1.2-based
promoter- probe vector, pDS5000, to exploit this possibility in their studies of the regulation of promotersof pIJ101. Two-day-old patches of gal– S. lividans containing pDS5000 derivatives growing on R2YE
were overlaid with 6 ml soft R2YE containing MUG (5 mg ml–1; final concentration in plates 1 mg ml–1)
and examined 15 min later under UV illumination (a conventional 310 nm transilluminator used for gelswith ethidium bromide-stained DNA is suitable). Results were recorded using Polaroid type 55 film
(exposure c. 7 sec, f4.5, yellow filter).

The redD gene of S. coelicolor as an easily scorable reporter of transcription i nS. coelicolor and S. lividans
The mycelium-based red pigment undecylprodiosin (Red; p. 95) is conspicuously
produced by S. coelicolor and S. lividans when the pathway-specific activator gene redD is expressedsufficiently (Bibb, 1996). After deleting redD from the S. coelicolor chromosome, Red production can
only be activated by expressing a cloned copy of redD. Deletion of the actII-ORF4 gene needed toactivate red/blue actinorhodin production makes Red production very easy to score. Using such a redD
actII-ORF4 double mutant, M512 (p. 423), as host (G.P. van Wezel, J. White and M.J. Bibb,
unpublished) have successfully used a cloned promoterless redD gene as a very effective, semiquantitative indicator of the promoter activity of cloned DNA fragments. Two vectors have been
described, both of which can be propagated in E. coli. In pIJ2527, the medium copy number SCP2*based pHJL401 provided the Streptomyces replication origin, whereas pIJ2585 is based on pSET152(Fig. 11.7, p. 262) and can be transferred by conjugation from E. coli into Streptomyces, where it
integrates at the C31 attB site. Vegetatively active promoters cloned in these vectors cause colonies
to be red as soon as they are visible, to a degree that depends on promoter strength. The vectors can also
be used effectively in S. lividans, because on most media it produces little or no red pigment. Thissystem should be valuable in the isolation and genetic study of both cis- and trans-acting mutations.

Baltz RH, Hahn DR, McHenney MA, Solenberg PJ (1992)
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&KDSWHU5HSRUWHUV\VWHPVChater KF (1993) Genetics of differentiation in
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Hopwood DA (1990) Transposition of IS117 (theStreptomyces coelicolor A3(2) mini-circle) to and from acloned target site and into secondary chromosomal sites.
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Chater KF, Buttner MJ (1996) The positions of the sigma
factor genes, whiG and sigF, in the hierarchy controlling thedevelopment of spore chains in the aerial hyphae ofStreptomyces coelicolor A3(2). Mol.Microbiol., 21, 593-603.
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

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

&KDSWHU 5HSRUWHUV\VWHPV

m16tk9c.wpd April 20, 2000 Tobias Kieser

Chapter 16

RNA methods
CONTENTS
General precautions when working with RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 354
Harvesting Streptomyces cultures for RNA isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 354
Isolation of RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 354
Isolation of RNA using modified Kirby mix, phenol/chloroform extraction
and DNase I treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355
Isolation of RNA using CsCl gradients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357
Isolation of RNA using SDS and hot phenol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359
Storage of RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360
Assessing the quantity and quality of RNA preparations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360
Spectrophotometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360
Agarose gel electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361
High resolution S1 nuclease mapping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363
General strategies for making probes for high resolution S1 nuclease mapping . . . . 363
Notes of caution in probe construction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366
Specific activity of the probe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366
How much probe to add to each hybridisation reaction . . . . . . . . . . . . . . . . . . . . . . . . 367
High resolution S1 nuclease mapping of the 30 ends of transcripts . . . . . . . . . . . . . . . 367
Hybridisation solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 368
The practicalities of high resolution S1 nuclease mapping . . . . . . . . . . . . . . . . . . . . . . 369
Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 370
Generating sequencing ladders for high resolution S1 nuclease mapping . . . . . . . . . 371
Interpretation of results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371
Low resolution S1 nuclease mapping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 372
Primer extension mapping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373
Northern blotting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 374
In vitro transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 374
Streptomyces RNA polymerase purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 377
Standard purification of Streptomyces RNA polymerase . . . . . . . . . . . . . . . . . . . . . . . 377
Purification of histidine-tagged RNA polymerase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 382
Preparation of DNA-cellulose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383



&KDSWHU51$PHWKRGVGeneral precautions when working with RNA
Always wear disposable gloves during RNA work to protect your samples from RNase on yourskin. Although it is not necessary to treat disposable plasticware differently for RNA work, it isadvisable to prepare and keep separate stocks of Eppendorf tubes, Gilson tips, etc exclusively for RNA
experiments and to be sure that they were not handled without gloves before autoclaving. Bake all
glassware at 180o for 4 h before use and keep separate stocks of glass pipettes for RNA work. As an
extra precaution, keep separate stocks of isopropanol and ethanol for RNA work, decanted frompreviously unopened supplies of the solvents, in "virgin" bottles.
We used to treat aqueous solutions used in RNA work with diethylpyrocarbonate (DEPC) toinactivate any RNases present. However, because of the extremely toxic nature of DEPC, we haveabandoned its use and now simply autoclave distilled water and aqueous solutions twice. We have notnoticed any reduction in the quality of the RNA preparations as a result.
For your own safety, make sure that you wear disposable gloves and safety glasses whenhandling modified Kirby mix and phenol/chloroform.

Harvesting Streptomyces cultures for RNA isolation
To minimise physiological shock to the mycelium, it is desirable to harvest cultures quickly. For
liquid cultures lacking high concentrations of sucrose, a 2 min spin at 10,000 rpm is sufficient. Liquid
cultures can also be harvested quickly by filtration onto cellulose acetate filters (e.g Sartorius Cat. No.
11106-47-ACN). The filter and mycelium can be placed directly into the Universal bottle containing the
Kirby mix and glass beads and the filter can subsequently be removed after vortexing. Surface cultures
should be grown on cellophane discs (p. 48) and can be harvested quickly with a spatula; plates should
be dried before inoculation to prevent the formation of wet patches on the surface of the cellophane
which inhibits development. When setting up a developmental time course using a large number ofplates, development must be as synchronous as possible between individual plates; it is therefore
advisable to set up an excess, so that any aberrant plates may be excluded.

Isolation of RNA
Three different protocols for RNA isolation are given below. To minimise physiological shockto the mycelium and to maximise the integrity of the RNA, it is preferable to break the mycelium open
immediately after harvesting, instead of partially protoplasting the cells first, because the effect ofprotoplasting on RNA expression and turnover is unknown. However, it is also important to maximise
the extent of lysis to ensure that RNA from all the cells in the culture is present in representative amounts
in the final preparations. This is particularly a concern when dealing with differentiating, surface-growncultures containing different cell types. Accordingly, three alternatives are given at step 2 in both the“modified Kirby mix” and the CsCl procedure for RNA isolation. In addition, an alternative procedurethat incorporates very harsh lysis conditions is given on p. 359.

&KDSWHU51$PHWKRGVIsolation of RNA using modified Kirby mix, phenol/chloroform extraction and DNase I treatment
This procedure is based on that of Kirby et al. (1967) with extensive modifications by S.N.
Covey and C.P. Smith (personal communication). The mycelium is homogenised in the presence ofdetergent and a chelating agent to disrupt nucleic acid-protein complexes and efficiently denature allproteins, which are subsequently removed by extraction with phenol. Total nucleic acid is recovered byisopropanol precipitation and the DNA is selectively degraded with RNase-free DNase I.
Lysozyme solution (2 mg ml–1 in P buffer) - optional
Phenol mixture (phenol, 500 g; 8-hydroxyquinoline, 0.5 g, equilibrated with 50mM
Tris-HCl pH8.3)
Modified Kirby mix (1% (w/v) sodium-triisopropylnaphthalene sulphonate (TPNS,
detergent, from Phase Separations Ltd, Deeside, U.K.), 6% (w/v) sodium 4-amino
salycilate (chelating agent), 6% (v/v) phenol mixture, all made up in 50mM Tris-HCl
pH8.3)
Phenol/chloroform (phenol mixture: chloroform: isoamyl alcohol, 50:50:1)
3M sodium acetate, pH6"10x" DNase I buffer (0.5M Tris-HCl pH7.8, 0.05M MgCl2)
RNase-free DNase I (e.g. Pharmacia 27-0514-01)
Isopropanol; 80% ethanol
Autoclaved 20 ml Universal bottles with c. 14 g 4.5-5.5 mm glass beads; baked
beaker; disposable polypropylene tubes with caps (we recommend the individuallywrapped "Falcon 2006" 17 mm × 100 mm tubes)
Sonicator, centrifuge

1.

Harvest mycelium. Then proceed to 2a, 2b or 2c.

2a.

Immediately transfer the mycelium into a 20 ml Universal bottle (cooled on ice
before use) containing 5 ml modified Kirby mix and c. 14 g 4.5-5.5 mm glass
beads and vortex vigorously for 3-4 min. It is important that the vortexing gives goodagitation as evidenced by the glass beads advancing well up the side of the bottle.

2b.

Immediately transfer the mycelium into a baked glass beaker containing 5 ml
modified Kirby mix and disrupt by sonication (MSE Soniprep 150, 9.5 mm
diameter probe; six burst of 30 sec with 30 sec pauses; 0o). Transfer thesonicated material to a 20 ml Universal bottle (cooled on ice before use)
containing c. 14 g 4.5-5.5 mm autoclaved glass beads and vortex vigorously for
3-4 min. It is important that the vortexing gives good agitation as evidenced by the glass beads
advancing well up the side of the bottle.

2c.

Resuspend the mycelium in 3 ml lysozyme solution and incubate at 30o until
protoplast formation just starts (usually around 10 min). Add the mycelium to&KDSWHU51$PHWKRGV

a 20 ml Universal bottle (cooled on ice before use) containing 3 ml double
strength modified Kirby mix and c. 14 g 4.5-5.5 mm glass beads andimmediately vortex vigorously for 3-4 min. It is important that the vortexing gives goodagitation as evidenced by the glass beads advancing well up the side of the bottle.

3.

Add 5 ml phenol/chloroform (room temperature) and vortex for a further 2-3
min. Material can be stored frozen at this stage; this is useful if there are many samples.

4.

Using a 10 ml pipette, transfer the mixture to a polypropylene tube (leaving theglass beads behind) and centrifuge at 7,000 rpm for 10 min at 4o.
Transfer the upper (aqueous) phase to a fresh polypropylene tube; take carebecause the interface material may "jump up". Add 3 ml phenol/chloroform
mixture, vortex 1 min and separate phases as in step 4.

5.

6.

Repeat step 5 until there is no significant amount of material at the phaseinterface. The number of phenol/chloroform extractions required depends on the way in which
the mycelium was grown; solid phase cultures and minimal medium cultures usually require only
two extractions whereas rich medium cultures require more. NB. It is often necessary to top up
the volume of the aqueous phase with distilled water during repeat extractions, to prevent severe
yield losses.

7.

Transfer the aqueous phase to a fresh polypropylene tube and precipitate the total
nucleic acid by adding 1/10 volume 3M sodium acetate (pH6) and one volume ofisopropanol; mix and leave 30 min on ice.

8.

Centrifuge at 7,000 rpm for 10 min at 4o and discard the supernatant.

9.

Re-dissolve the pellet in 0.5 ml distilled water (this may take some time) and reprecipitate with sodium acetate and isopropanol. Without this second precipitation,
traces of phenol sometimes inhibit the DNase I digestion in step 11.

10.

Rinse the pellet with 5 ml 80% ethanol; air dry the pellet and re-dissolve in 0.5
ml distilled water. Remove 2 × 5 µl samples and label them total nucleic acid(TNA). Taking a TNA sample for comparison with the final RNA sample (Fig. 16.1, p. 361)
is useful if you are preparing RNA for the first time, but is unnecessary otherwise.

11.

To the total nucleic acid preparation from step 10, add 1/10 volume "10x" DNase
I buffer (dilute with 1x DNase I buffer if solution is very viscous) and 7.5 U
RNase-free DNase I and incubate for 1 h at 37o.

&KDSWHU51$PHWKRGV



12.

Add an equal volume of phenol/chloroform mixture; vortex mix and separate thephases.

13.

Remove the aqueous phase to a fresh tube and repeat step 12.

13.

Recover the aqueous phase; add 1/10 volume 3M sodium acetate and precipitateby adding an equal volume of isopropanol.

14.

Pellet the RNA by centrifugation; rinse with 5 ml 80% ethanol, air dry and redissolve in 0.5 ml distilled water. Remove 2 × 5 µl samples and label them"RNA."

15.

Store the RNA at -70o as an aqueous solution or as an isopropanol precipitatep. 360). Use the samples of TNA and RNA removed during the procedure toassess the quantity and quality of RNA by a combination of spectrophotometry
and agarose gel electrophoresis (p. 360).

Isolation of RNA using CsCl gradients
Glišin et al. (1974) described a one-step procedure for isolating RNA using high-speed caesium
chloride centrifugation. The procedure is based on the fact that RNA is the densest cellular
macromolecule. By layering a cell homogenate onto a dense caesium chloride solution, followed by highspeed centrifugation, the RNA is specifically pelleted, the DNA bands at the interface and the proteins(including RNase) actually migrate in the opposite direction. A modified procedure (Hobbs et al., 1992;
Hindle and Smith, 1994) employing the caesium chloride centrifugation method in combination with partof the modified Kirby mix procedure is described below. Many people prefer this procedure because
neither extensive phenol/chloroform extraction nor DNase I treatment are required. RNA purified by
CsCl is generally cleaner than RNA prepared by repetitive phenol/chloroform extraction. For example,
it will be free of polysaccharides that can co-purify with RNA using standard methods. This isparticularly important if mycelium is grown in medium containing the dispersal agent Junlon (Hobbs etal., 1989; p. 51), and potentially for mycelium grown in “production media.”
Lysozyme solution (2 mg ml–1 in P buffer) - optional
Phenol mixture (phenol, 500 g; 8-hydroxyquinoline, 0.5 g, equilibrated with 50mM
Tris-HCl pH8.3)
Solid CsCl (baked in a 180o oven for 4 h)
5.7M CsCl (made up with double-autoclaved water)
Modified Kirby mix (1% (w/v) sodium-triisopropylnaphthalene sulphonate (TPNS,
detergent, from Phase Separations Ltd, Deeside, U.K.), 6% (w/v) sodium 4-amino
salycilate (chelating agent), 6% (v/v) phenol mixture, all made up in 50mM Tris-HCl
pH8.3)
Phenol/chloroform (phenol mixture: chloroform: isoamyl alcohol, 50:50:1)
3M sodium acetate pH6&KDSWHU51$PHWKRGVIsopropanol
80% ethanol
Centrifuge tubes, e.g. Sorvall SS-34 centrifuge tubes (sterilised by autoclaving or
treated as SW41 tubes: below)
Beckman SW41 centrifuge tubes (boiled in 0.1% SDS/0.1M NaOH for 10 min,drained, then rinsed with RNase-free water)
Disposable polypropylene tubes with caps (e.g. "Falcon 2006" tubes)
Autoclaved Universal bottles with c. 14 g 4.5-5.5 mm glass beads; baked beaker

1.

Harvest mycelium. Then proceed to 2a, 2b or 2c.

2a.

Immediately transfer the mycelium into a 20 ml Universal bottle (cooled on ice
before use) containing 5 ml modified Kirby mix and c. 14 g 4.5-5.5 mm glass
beads and vortex vigorously for 3-4 min. It is important that the vortexing gives goodagitation as evidenced by the glass beads advancing well up the side of the bottle.

2b.

Immediately transfer the mycelium into a baked glass beaker containing 5 ml
modified Kirby mix and disrupt by sonication (MSE Soniprep 150, 9.5 mm
diameter probe; six bursts of 30 sec with 30 sec pauses; 0o). Transfer thesonicated material to a 20 ml Universal bottle (cooled on ice before use)
containing c. 14 g 4.5-5.5 mm autoclaved glass beads and vortex vigorously for
3-4 min. It is important that the vortexing gives good agitation as evidenced by the glass beads
advancing well up the side of the bottle.

2c.

Resuspend the mycelium in 3 ml lysozyme solution and incubate at 30o until
protoplast formation just starts (usually around 10 min). Add the mycelium toa 20 ml Universal bottle (cooled on ice before use) containing 3 ml double
strength modified Kirby mix and c. 14 g 4.5-5.5 mm glass beads andimmediately vortex vigorously for 3-4 min. It is important that the vortexing gives goodagitation as evidenced by the glass beads advancing well up the side of the bottle.

3.

Add 5 ml phenol/chloroform (room temperature) and vortex for a further
2-3 min. Material can be stored frozen at this stage; this is useful if there are many samples.

4.

Using a 10 ml pipette, transfer the mixture to a polypropylene tube (leaving theglass beads behind) and centrifuge at 7,000 rpm for 10 min at 4o. Transfer theupper (aqueous) phase to a fresh polypropylene tube; take care because theinterface material may "jump up".

&KDSWHU51$PHWKRGV



5.

Add water to give a final volume slightly in excess of 8 ml. Transfer 8 ml to apolypropylene tube containing 3.2 g CsCl. Invert to dissolve CsCl.

6.

Add 3.5 ml 5.7M CsCl to SW41 tubes, balance, and mark the position of thecushion and the number of the tube at the bottom.

7.

Gently layer (using a Pasteur pipette) the RNA-containing solution onto the CsCl
cushion by allowing the sample to run down the sides of the tube. Leave a c. 2
mm gap at the top of the tube. Check balance and spin at 30,000 rpm for 14-18
h at 20o.

8.

Using a Pasteur pipette, remove the supernatant down to the mark of the cushion.
Change pipette and continue removing the supernatant. Change pipette once
more before all the supernatant is removed. This is to ensure that DNA, which remains
at or above the cushion, does not contaminate the samples.

9.

With a hot scalpel cut most of the tube off to leave about 1-1.5 cm of tube wall.
Remove all supernatant with a yellow tip, then wash the pellet with 0.5 ml 80%
ethanol. Remove all ethanol and allow pellet to air dry.

10.

Resuspend pellet in 0.2 ml water and transfer to a screw-capped Eppendorf tube.
Occasionally a white, insoluble precipitate comes down with the RNA during
centrifugation. This can be removed by phenol/chloroform extraction followedby isopropanol precipitation. The pellet is then washed in 80% ethanol andresuspended in water as before. Remove 2 × 5 µl samples and assess thequantity and quality of the RNA by a combination of spectrophotometry andagarose gel electrophoresis (p. 360).

Isolation of RNA using SDS and hot phenol (Kelemen et al., 1996)
Lysozyme solution (2 mg ml–1 in P buffer)
Phenol mixture (phenol, 500 g; 8-hydroxyquinoline, 0.5 g, equilibrated with 50mM
Tris-HCl pH8.3)
Phenol/chloroform (phenol mixture: chloroform: isoamyl alcohol, 50:50:1)
10%(w/v) SDS
0.5M EDTA pH8.0
3M sodium acetate, pH6
Isopropanol; 80% ethanol
Disposable polypropylene tubes with caps (e.g. "Falcon 2006" tubes)
30E and 60E water baths

&KDSWHU51$PHWKRGV

1.

Harvest mycelium.

2.

Resuspend mycelium in a Universal in 6 ml lysozyme solution. Incubate at 30o
until protoplast formation just starts (usually around 10 min).

3.

Add 2 ml 0.5M EDTA and 1 ml 10% SDS. Place the samples at 65o, add 3 ml
phenol/chloroform and vortex briefly. Incubate at 65o for 5 min, then vortex
vigorously for 3-4 min. Extra care is required at this stage when handling hot
phenol/chloroform. Material can be stored frozen at this stage; this is useful if there aremany samples.

4.

Using a 10 ml pipette, transfer the mixture to a polypropylene tube andcentrifuge at 7,000 rpm for 10 min at 4o.

5.

Complete RNA purification using steps 5-16 of the “modified Kirby mix”
protocol (pp. 356-357).

Storage of RNA
RNA should be stored at -70o, either dissolved in water or as an isopropanol precipitate. Storageas an isopropanol precipitate protects the RNA from RNase and the suspensions are homogeneous when
vortexed, allowing a defined amount of RNA to be removed for co-precipitiation with the probe.
However, many people keep their RNA dissolved in water without any apparent degradation during longterm storage. For S1 nuclease protection experiments (p. 363), RNA stocks dissolved in water can bedried down directly with the probe: the resulting pellets redissolve much more easily in the hybridisationbuffer than precipitated RNA pellets.

Assessing the quantity and quality of RNA preparations
Spectrophotometry

1.

Dilute one of the 5 µl samples of TNA and one of RNA, each in 495 µl water.

2.

Measure the A260 and A280 of both samples in a quartz cuvette. To assess thegeneral quality of the RNA samples the A260:A280 ratio is calculated. An RNA
preparation essentially free of protein (and residual phenol) has a ratio of 1.902.05, whereas a ratio lower than 1.9 indicates protein contamination. A ratio
greater than 2.10 generally indicates severe degradation.

&KDSWHU51$PHWKRGVFig. 16.1. Appearance of TNA (total nucleic acid) and RNA on 1.5% agarose TBE gels.

3.

Calculate the concentrations of TNA and RNA assuming:
1 A260 unit = 40 µg ml–1
(Although DNA has a slightly different molar extinction coefficient from that of RNA, TNA is
predominantly RNA and so the same assumption can be used for both.)

Agarose gel electrophoresis
RNases are both abundant and very stable. In addition, gel tanks are routinely contaminated with
RNase through addition of the enzyme to miniprep digests. Degradation of RNA during gelelectrophoresis is therefore a very common problem, leading to a much more pessimistic assessment of
the quality of RNA samples than is actually warranted. To avoid these problems, the agarose, 1 × TBE
buffer and loading dye used for RNA work should be autoclaved and kept separate from stocks used forroutine DNA analysis. It is also most important that the gel tank, the plate on which the gel is poured
and the comb used to form the wells are soaked in 100mM NaOH for more than two hours, then rinsed
in water before use.
Another way to avoid degradation of RNA during electrophoresis is to incorporate sodium
iodoacetate (RNase inhibitor) (Aldrich, Cat. No. 16,054-7) in the agarose to a final concentration of
10mM. It is added as a powder after the agarose has been cooled to 50-600, before pouring the gel.



&KDSWHU51$PHWKRGV

5’genometranscript

3’

5’
3’

3’
5’
5’
3’

probe

3’
* 5’
hybridisation of
RNA and probe3’
undigested
hybrid5’
DNAdigested
hybrid

3’

RNA

* 5’
S1 nuclease

5’
3’

3’

* 5’
denature andrun on asequencing gel

Fig. 16.2. Mapping the 50 ends of transcripts.
Dilute the second 5 µl samples of TNA and RNA to a concentration of approximately 0.25 µgµl , add ¼ volume 5 × neutral loading dye (50% (w/v) sucrose, 1% (w/v) Orange G dye, 5mM EDTA;
autoclaved once) and load approximately 0.75 µg of each on a 1.0-1.5% agarose/TBE gel with 5 mm
slots. The appearance of RNA preparations on agarose gels is the best way to assess their integrity before
S1 mapping or primer extension experiments. Because total RNA is <5% mRNA, only stable RNA (16S,
23S, and 5S rRNAs and tRNA) can be seen clearly on the gels. For a good preparation of RNA preparedby the Kirby mix procedure (Fig. 16.1) you should see:–1

1. a total absence of chromosomal DNA in the RNA preparation;
2. a 23S rRNA band that is more intense than the 16S rRNA band; the 23S rRNA ispreferentially degraded, so in poor preparations of RNA the 16S band will be more intensethan the 23S band;
3. an absence of bands between the 23S and 16S bands and below the 16S band (these bands
result from degradation of the 23S and 16S rRNAs which are cleaved preferentially at
specific sites).

&KDSWHU51$PHWKRGVDepending on exactly how the TNA/RNA is prepared, you may see a ladder of one to three bandsof decreasing intensity above the 23S rRNA band (Fig. 16.1). These are rRNA aggregation products andthey are quite normal. RNA prepared by the caesium chloride method forms relatively heterogeneous
aggregates and therefore looks smeary on neutral agarose gels. Samples prepared by the caesium
chloride method should therefore be run on denaturing agarose gels.

High resolution S1 nuclease mapping
If the aim of the work is to identify the 50 ends of transcripts, in most situations high resolution
S1 mapping can be used without the need for prior analysis by other techniques such as low resolution
S1 mapping, Northern blotting or promoter probing. The technique involves hybridisation between a
specific mRNA molecule and the complementary DNA strand from which it is transcribed; the 50 end of
the DNA strand is radiolabelled and the 30 end extends beyond the transcription initiation site (Fig. 16.2).
Digestion with single-strand specific S1 nuclease yields a double-stranded RNA:DNA hybrid, the DNA
component of which extends from its radiolabelled end (the "datum" point) within the transcription unit
to the site at which the transcript initiated (Fig. 16.2). Electrophoresis of the DNA component of the S1protected hybrid against a suitable sequence ladder allows the precise (within 1-2 nucleotides)
determination of the transcriptional start site. S1 nuclease mapping can be used quantitatively, provided
that the amount of probe used in the hybridisation greatly exceeds the amount of the transcript. This may
not be the case if a very abundant species is being quantified, such as an rRNA transcript (Clayton,1989).
General strategies for making probes for high resolution S1 nuclease mapping
Very short mRNA:DNA hybrids are unstable; therefore, in the absence of prior information
about the positions of potential promoters, construct a probe in which the labelled site is at least 70 bp
downstream of the translation start site, to ensure a minimum overlap between the probe and the mRNAof 70 bp (note that "leaderless" transcripts, initiating at, or within a few nucleotides of the translation
start codon are unusually frequent in Streptomyces; Janssen, 1993). If the hybrids observed turn out to
be too large to assign transcription start points accurately, the experiment can be repeated with a second
probe in which the datum point has been moved closer to the 50 ends of the transcripts.
To determine the position of the transcription start point (50 end) for a given mRNA at highresolution, the probe should be uniquely radiolabelled on the 50 end of the strand complementary to themRNA (the "bottom" strand, if the gene is represented in the conventional manner running 50 --> 30 left
to right; Fig. 16.2). The probe is uniquely radiolabelled in order to avoid ambiguity in the interpretationof the results; if a probe is labelled on both 50 ends, any hybrid detected could, in principle at least, arise
from protection of either strand. High resolution S1 mapping probes can be generated in several ways.
1. By PCR. PCR provides a simple and convenient way to make probes of high specific activity.
In this approach, the downstream primer is phosphorylated using -32P ATP and T4 polynucleotide
kinase. The probe is then generated by PCR using the radiolabelled downstream primer and an
unlabelled upstream primer, thereby generating a uniquely labelled probe. Because oligonucleotides canbe phosphorylated with high efficiency, PCR-generated primers usually have high specific activity. The&KDSWHU51$PHWKRGV
promoterBam HI

XbaI
Bam HI

plasmid
clone

Xba I

Bam HI

5’ - PP - 5’

XbaI

alkaline phosphatase5’ - HOOH - 5’T4 polynucleotide
Xba I

32
kinase + γ - P ATP

32

5’ - P32
P - 5’
Xba I

5’ - P32
P - 5’

Fig. 16.3. Phosphorylating a restriction fragment.
downstream primer is also used to generate the appropriate sequencing ladder.
2. By phosphorylating a restriction fragment. This method requires that the DNA to be labelled
is free of contaminating RNA because the enzyme used, T4 polynucleotide kinase, preferentially labels
the 50 ends of RNA over DNA. Probably the most reliable, if old-fashioned, way of making suitable
DNA is to purify the plasmid through two CsCl-ethidium bromide gradients (Chapter 8). RNase
treatment of plasmid preparations makes the problem of RNA contamination worse, not better, because
it generates an even higher molar excess of RNA 50 ends over DNA 50 ends. Generally, restriction sites
having 50 overhangs are used, as they radiolabel more efficiently than blunt or 30 recessed ends.
In the example shown in Fig. 16.3, the probe is to be a BamHI-XbaI fragment, uniquely labelledon the 50 end of the BamHI site. A plasmid carrying the gene of interest is digested with BamHI and the

&KDSWHU51$PHWKRGV



5’

5’
3’

3’

P2

P1

5’

3’
3’
5’

5’
3’

3’

* 5’
pUC

probe

hybridisation of
RNA and probe

3’ P1 transcript
5’
DNA

5’
*
S1 nuclease

3’
5’
3’

3’
* 5’

3’ P2 transcript

5’

DNA

P1 hybridS1 nuclease

3’
5’
3’
probe

* 5’
3’

* 5’

P2 hybrid

exp
probe-probe

reannealing
autoradiograph"full-length protection"arising from theof final gel

P2 transcript
P1 hybrid

Fig. 16.4. Utility of probes carrying a non-homologous tail.
digest is treated with alkaline phosphatase to remove the phosphates from the 50 ends of the resulting
restriction fragments. After inactivation of the alkaline phosphatase by two rounds of phenol/chloroform
extraction, the digest is treated with T4 polynucleotide kinase and -32P ATP, so that all the 50 ends
become radiolabelled. In order to generate a probe that is uniquely radiolabelled, the DNA is cleaved

&KDSWHU51$PHWKRGVwith a second enzyme, XbaI. The radiolabelled double digest is then separated by agarose gelelectrophoresis and the probe DNA is recovered. It is therefore important to confirm in advance that theprobe fragment is well separated from other fragments in the final double digest, to avoid crosscontamination. A sample of the probe, denatured but otherwise untreated, should be run along with theputative hybrids on the final gel, to ensure that the probe is free of contamination.
Notes of caution in probe construction
1. If a probe fragment is labelled on both ends, but one end corresponds to a site in an E. coli
vector or polylinker, then it should be impossible for that end to be protected by Streptomyces RNA.
Therefore, in principle, any hybrids obtained should be derived from protection of the other end. In
practice, we recommend that a second cut is made, removing any possibility of misinterpretation.
2. Some people have attempted to generate uniquely labelled probes in the following way.
Using the basic scheme in Fig. 16.3 as an illustration, they cut with BamHI and treat the digest withalkaline phosphatase. Then they inactivate the phosphatase, digest the DNA with XbaI, isolate thefragment from a gel, and treat the isolated fragment with T4 polynucleotide kinase and -32P ATP.
Because the 50 terminal phosphate at the XbaI site was not removed (because the XbaI digest was done
after the alkaline phosphatase treatment), it is assumed that no label will be incorporated at that end of
the fragment. This is not a valid assumption, for two reasons: because T4 polynucleotide kinase will
catalyse an “exchange reaction" with 50 ends that are phosphorylated; and because a certain percentage
of 50 ends lose their phosphates during manipulation, even without alkaline phosphatase treatment.
3. If a restriction fragment having one 50 projecting end and one 50 recessed (or blunt) end isradiolabelled after phosphatase treatment, never assume that the level of incorporation of 32P into the 50
recessed (or blunt) end will be insignificant. Depending on the exact conditions used, radiolabelling of
50 recessed (or blunt) ends can be poor to very efficient, but it is never negligible. Unless a second cut
is performed to create a uniquely labelled probe, there is the potential for ambiguity in the interpretationof hybrids, especially keeping in mind that a very abundant transcript might protect the “wrong" strandof the probe, while the transcript of interest might be far less abundant.
4. Because a detectable amount of probe-probe reannealing is common in S1 mapping
experiments, it can be difficult to distinguish between this artifact and “full-length protection” of theprobe by a transcript originating upstream of the end of the probe. To differentiate between probe-probe
reannealing and full-length protection, it is useful to incorporate a non-homologous tail at the 30 end of
the radiolabelled strand of the probe (Fig. 16.4). This can be done simply by designing the upstream
primer to fall in vector or polylinker sequences if making a PCR probe (for example using the universal
or reverse primer with pUC derivates), or by making the second cut within the vector or polylinker, if
a phosphorylated restriction fragment is being used (the two PvuII sites in pUC18/19 are often useful inthis respect). As a result of this, hybrids arising from “full-length protection” will be shorter than probeprobe reannealing artifacts, because of digestion of the non-homologous tail. Ensure that the tail is longenough to distinguish the two; for example a 15 bp tail on 600 bp probe will not be sufficient.

&KDSWHU51$PHWKRGVSpecific activity of the probe
It is the specific activity of the probe that determines the strength of signal obtained, not theabsolute number of counts added to the hybridisation. It is vital therefore that the probe is end-labelled
to high specific activity. Values between 7.5 × 105 and 1.5 × 106 Cerenkov cpm pmole–1 are typical for
uniquely labelled probes generated by phosphorylating DNA restriction fragments, but higher values areusually obtained using the PCR approach.
How much probe to add to each hybridisation reaction
Estimate the probe concentration by running a little on an agarose gel with a standard of known
concentration. For hybridisation with 40 µg RNA, use c. 30 fmole of probe. This is equivalent to 10 ng
of a 0.5 kb double-stranded DNA fragment, 20 ng of a 1 kb fragment, etc. This will constitute a
sufficient molar excess of probe over transcript to drive all of the transcript into a hybrid and provide
approximately quantitative data, unless the transcript is abundant, such as an rRNA transcript (Clayton,1989).
High resolution S1 nuclease mapping of the 30 ends of transcripts
To determine the position of the 30 end of a transcript, the same logic and method is applied,
except that the probe must now be labelled on the 30 end of the strand complementary to the mRNA (the"bottom" strand in Fig. 16.5). Again, the size of the hybrid obtained corresponds to the distance between
the labelled site and the end (the 30 end in this case) of the transcript. The only important differences inthis kind of analysis are the methods for generating the probe and the sequencing ladder.
The probe. 30 end labelled probes cannot be generated by PCR and are most easily made by"filling in" a restriction half site using Klenow and the appropriate [ -32P] dNTP. Therefore, a restriction
site having a 30 recessed end should be used. As before, it is important that the double-stranded probe
is not labelled on both 30 ends, so a second cut may be necessary.
The sequencing ladder. Because the datum point in these experiments is a 30 end, an appropriate
sequencing ladder cannot be generated enzymatically (because DNA synthesis proceeds in the 50 to 30
direction), but the Maxam and Gilbert T+C and G+A reactions can be used (p. 394). If only anapproximate localisation of the 30 end is required, the hybrid can be run against a set of size standards
such as a heat-denatured, radiolabelled HpaII digest of pBR322.
Hybridisation solution
The best hybridisation solution for S1 nuclease mapping contains sodium trichloroacetate(NaTCA), rather than formamide, for two main reasons: the window between Tm(DNA:RNA) andTm(DNA:DNA) (i.e. probe-probe reannealing) is large enough in NaTCA that all hybridisations can be
carried out at 45o, regardless of probe length and G+C content (Murray, 1986), making it unnecessary
to determine the melting temperature of the DNA probe experimentally; 40 µg of RNA is readily soluble
in 20 µl of NaTCA buffer whereas the same quantity of RNA dissolves very slowly in formamide buffer.

1.

Dissolve 2.53 g PIPES in approximately 90 ml distilled water, then add 1.67 ml
0.5M EDTA. Adjust to pH7.0 with 5M NaOH, then autoclave.



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5’genometranscript

3’

5’
3’

3’
5’
5’
3’ *

probe

3’
5’
hybridisation of
RNA and probe

RNA
undigested
hybrid5’3’
3’ *5’ DNAS1 nuclease

digested
hybrid

5’
3’ *

3’
5’
denature andrun on asequencing gel

Fig. 16.5. Mapping the 30 ends of transcripts.

2.

Dissolve 93.1 g NaTCA (Aldrich Cat. No. 19,078-0) in the PIPES/EDTA
solution. Make up to a final volume of 167.4 ml with sterile distilled water.
There is no need to readjust the pH. The final solution is 3M NaTCA, 50mM PIPES, 5mM
EDTA, pH7.0. N.B. Aldrich NaTCA is not usually 100% pure and so the number of grams
added should be adjusted accordingly. For example, if the chemical is 97% NaTCA, add 96 g.

3.

Store as 100 µl samples at -70o and thaw only once.

Should Aldrich cease to manufacture NaTCA, a protocol for making it is described by
Summerton (1983), although this is demanding and can be dangerous without the appropriate chemistrylaboratory facilities.
The practicalities of high resolution S1 nuclease mapping
Total RNA preparation and 32P end-labelled test DNA fragments
3M sodium acetate pH6
Isopropanol; 80% ethanol
Sodium TCA hybridisation solution (stored in aliquots at -70o and thawed only once)
5x S1 digestion buffer ("5x" is 1.4M NaCl, 150mM sodium acetate pH4.4, 22.5mM zincacetate, 100 µg ml–1 partially-cleaved, denatured, non-homologous DNA; stored at -20o)
S1 nuclease (e.g. Pharmacia 27-0920-01 or Boehringer 818 330)
S1 termination solution (2.5M ammonium acetate, 0.05M EDTA)
Carrier tRNA, 5 mg ml–1.

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

1.

If the RNA is stored dissolved in water, mix 40 µg RNA and 30 fmole probe
together and dry down in a vacuum centrifuge. If the RNA is stored as an
isopropanol precipitate, co-precipitate 40 µg RNA and 30 fmole probe. Spin
5 min in microcentrifuge and discard supernatant. Rinse pellet with 80%
ethanol, spin, remove all supernatant and air dry pellet (vacuum drying
precipitated pellets seems to make them more difficult to re-dissolve).

2.

Add 20 µl NaTCA hybridisation solution and carefully dissolve the pellet byvortexing and pipetting (this will take longer if the pellet was generated by
precipitation - step 1). Heating the tube will speed up the solubilisation. Dissolving thepellet completely is crucial; failure to do so results in a substantial background signal caused by
the non-denatured duplex DNA.

3.

Briefly centrifuge to bring everything to the bottom of the tube. Place tubes ina 70o water bath, submerged up to their necks and tightly capped. Incubate for
10-15 min. Microcentrifuge tubes can be held firmly in racks by first placing some heatresistant tape (e.g. autoclave tape) across the holes and then forcing the tubes through.

4.

Transfer rapidly to a second water bath equilibrated to 45 o, ensuring that the
tubes are again submerged up to their necks.

5.

Incubate at 45o for 2-15 h, keeping the lid on the water bath.

6.

Treating each sample sequentially, forcefully dispense 300 µl chilled 5 × S1
digestion buffer containing 100 units S1 nuclease, while the bulk of the sample
tube is still submerged in the water bath. Mix immediately on a vortex mixer
and place in ice. In high resolution S1 mapping, loss of signal requires only the removal of
the label from the probe in the DNA:RNA hybrid duplex (this is not true of low resolution Sl
mapping in which the hybrids are detected by Southern blotting; p. 371). Because the ends ofhybrids "breathe," it is important to use neither excessive amounts of S1 nuclease nor longer
periods of digestion at 37o, and to ensure that all samples are treated the same to allow quantitative
comparison between them.

7.

Incubate samples at 37o for 45 min.

8.

Add 75 µl S1 termination solution; vortex mix.

9.

Add 10 µg carrier tRNA; vortex mix.



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10.

Add 400 µl isopropanol; leave 10 min on ice, then spin 10 min.

11.

Rinse pellets in 80% ethanol and vacuum dry. Then proceed to 12a or 12b.

12a.

Resuspend S1 hybrids in S1 loading buffer [80% (v/v) formamide, 10mM
NaOH, 1mM EDTA, 0.1% (w/v) xylene cyanol, 0.1% (w/v) bromophenol blue]and denature by heating to 90-100o for 1-2 min. Run on a conventional 6%
polyacrylamide-urea sequencing gel, alongside an appropriate sequencing ladder(p. 370).

12b.

If the expected S1 nuclease protected hybrid is too large for analysis on apolyacrylamide-urea sequencing gel, an agarose gel can be used instead (see“low resolution S1 nuclease mapping”, steps 13-14, p. 372). Afterelectrophoresis, soak the gel twice in at least 3 volumes of 7% (w/v)
trichloroacetic acid for 30 min to fix the bands, place the gel on pre-wetted
Whatman 3MM paper, cover with cling film and place on a gel dryer for 30-60
min at about 60o.

13.

Expose the gel to X-ray film or phosphorimager plate.

Controls
It is always advisable to include a negative control in S1 mapping experiments. As the main
reason for doing this is to detect probe reannealing or S1-digestion artifacts, tRNA is often used.
Alternatively, the control RNA could come from another organism such as E. coli, or ideally from thesame Streptomyces strain grown under conditions in which the gene is not expressed, or an otherwise
isogenic strain in which the gene has been deleted.
Additionally, a sample of the probe, denatured but otherwise untreated, should be run along withthe putative hybrids on the final gel, to ensure that the probe is free of contamination.
Generating sequencing ladders for high resolution S1 nuclease mapping
To assign a transcription start point for a given promoter, it is necessary to compare the mobilityof the protected DNA fragment generated in the S1 nuclease protection assay with an appropriate DNA
sequencing ladder. This can be generated in two ways.
i. A sequencing ladder can be generated enzymatically using an oligonucleotide primer having
the same 50 end as the radiolabelled probe. If PCR was used to make the probe, then the downstream
primer can be used directly for this purpose. To ensure that an enzymatically-generated sequencing
ladder is exactly comparable with the S1 nuclease-protected DNA fragment, it is best to radiolabel theoligonucleotide primer on the 50 end and generate the sequencing ladder with a “cold” extension,

&KDSWHU51$PHWKRGVavoiding analogue nucleotides such as7-deaza GTP. This eliminates any differences in mobility thatmight arise from an absence of a phosphate on the 50 end of the primer or from the incorporation of
analogue nucleotides; the only difference between the S1 nuclease-protected fragment and thecorresponding band in the sequencing ladder will be the absence of the 30 OH on the chain- terminating
dideoxy nucleotide in the latter (S1 nuclease-generated DNA fragments have a 50 phosphate and a 30 OH).
An appropriate sequencing ladder cannot be generated enzymatically if a 30 end-labelled probe
is being used to identify the 30 end of a transcript (p. 367).
ii. A sequencing ladder can be generated by subjecting the radiolabelled S1 nuclease probe tochemical sequencing (Maxam and Gilbert, 1980; the A+T and G+C reactions will suffice; p. 394).
Maxam and Gilbert chemical sequencing ladders lack the final nucleotide (so, for example, thebands appearing in a Maxam and Gilbert G track do not terminate in a G but in the preceding nucleotide)and carry phosphates on both the 50 and 30 ends. Therefore, the S1 nuclease-generated hybrids run 1-1.5
nucleotides above the "equivalent" molecule in the sequencing ladder, and this should be taken into
account when making assignments.
Interpretation of results
In comparing transcription start point assignments made by different methods (for example
primer extension vs S1 nuclease mapping), it should be borne in mind that S1 nuclease can leave a short
single-stranded overhang of 1-3 nucleotides depending on the severity of the digestion conditions used.
Inverted repeat sequences can give rise to hairpin loop structures in the probe. Because S1 is anendonuclease, it can cleave these loops to give rise to specific bands which could be misinterpreted ascorresponding to the 50 ends of mRNA species. It is therefore advisable to be aware of any inverted
repeat sequences within the sequence covered by the probe, and to compare their positions with thoseof signals detected in the S1 nuclease mapping experiments. Generally speaking, artifactual signals
arising in this way will correspond to the base of the loop.

Low resolution S1 nuclease mapping
Low resolution S1 mapping is a method to study the transcriptional organisation of a group ofgenes in which, because of their large size, the S1 nuclease-protected DNA fragments have to be run onan agarose gel. In this technique, the probe used is non-radioactive and, after agarose gel electrophoresis,
the sizes of the hybrids are determined by conventional Southern blotting. The interpretation of data
derived in this way is different from the interpretation of data derived from high resolution experiments
using end-labelled probes. In high resolution mapping, the RNA must protect the radiolabelled end of theprobe to generate a signal, whereas in low resolution experiments, any part of either strand of the probe
can generate hybrids that will be detected in the final Southern blot. Thus a combination of probes is used
to define the positions of the transcribed DNA sequences. The choice of probes for low resolution S1
nuclease mapping is reviewed in depth by Smith (1991), and a good practical example of low resolution
S1 nuclease mapping is the analysis of the transcriptional organisation of the gyl operon by Smith andChater (1988).

&KDSWHU51$PHWKRGV

5 × neutral loading dye (50% (w/v) sucrose, 1% (w/v) orange G dye, 5mM EDTA;
autoclaved once)
AgaroseTBE
Solutions for Southern transfer and hybridisation

1-12. Complete steps 1-11 of the high resolution S1 mapping protocol (p. 369-370)
using 40 µg RNA, but using a non-radioactive restriction fragment as the probe.
Use 30 fmole of probe, equivalent to 20 ng of a 1 kb double-stranded DNA
fragment, 40 ng of a 2 kb fragment, etc.
13.

Dissolve final pellets in 10-20 µl of 1 × neutral loading dye (do not heat
denature).

14.

Electrophorese DNA:RNA hybrids alongside suitable double-stranded DNA size
standards on a standard TBE-agarose gel. At this resolution the DNA:RNA hybrids
have the same mobility as the duplex DNA counterparts.

15.

Soak gel in Southern denaturation solution for 15 min to equilibrate in high salt.

16.

Rinse gel briefly in water and soak gel in Southern neutralisation solution for 30
min.

17.

Transfer DNA to hybridisation membrane, hybridise with an appropriate probe
and wash filter.

18.

Expose to X-ray film or phosphorimager plate.

Primer extension mapping
Primer extension is an alternative method for determining the positions of the 50 ends oftranscripts which relies on the ability of reverse transcriptase to extend 50 end-labelled oligonucleotides
annealed to mRNA species. Transcription start points are assigned by comparison with an enzymaticsequencing ladder generated with the same oligonucleotide used in the primer extension. This method
is much less work than S1 mapping, but the quality of results in Streptomyces work is rather variable,
depending on the exact transcript being mapped. The distance between the oligonucleotide and the endof the transcript (the length of extension) is very important, particularly in experiments with GC-richStreptomyces RNA, because secondary structure in the transcript limits the efficiency of primer
extension. The oligonucleotide should therefore lie within a few tens of bp of the transcription startpoint. Therefore, primer extension is often more useful to examine transcription from a promoter whose

&KDSWHU51$PHWKRGVstart point has already been determined, rather than for primary transcript mapping. Because
oligonucleotides are annealed to total RNA, extension products which do not derive from the intended
transcript are sometimes obtained. It is therefore highly desirable to include, as a control, RNA isolated
from the same Streptomyces strain under conditions in which the gene is not expressed, or an otherwise
isogenic strain in which the gene has been deleted. For a full discussion of the pros and cons of primer
extension in comparison with high resolution S1 nuclease mapping, see Smith (1991).
10 × SB Buffer (100mM Tris-HCl pH7.4, 600mM NH4Cl, with 60mM 2-mercaptoethanol
added after autoclaving)
100mM Mg-acetate
1.5mM dNTP stock solutions
AMV reverse transcriptase (Boehringer-Mannheim)

1.

In a 0.5 ml Eppendorf tube, mix:
2 µl 10 × SB Buffer
10-40 µg RNA
1 pmol 50 radiolabelled oligonucleotide primer
Make up to 20 µl with water2.

Incubate at 90o for 5 min, then transfer the tubes to a 75o waterbath, submerged
up to their necks and tightly capped, and allow the RNA and oligonucleotide toanneal by turning the waterbath off. When the temperature has fallen to 50 o, spin
the tube briefly to collect all the liquid at the bottom and place on ice.
Microcentrifuge tubes can be held firmly in racks by first placing some heat-resistant tape (e.g.
autoclave tape) across the holes and then forcing the tubes through.

3.

Mix on ice:
1 µl 10 × SB buffer
1.5 µl 100mM Mg-acetate
1.5 µl 5mM dTTP
1.5 µl 5mM dCTP
1.5 µl 5mM dGTP
1.5 µl 5mM dATP
0.5-1U AMV reverse transcriptase
Make up to 10 µl with water4.

Add 5 µl of the annealing mix and incubate at 37-42o for 30 min.



5.

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Add 10 µl loading buffer (80% (v/v) formamide, 10mM NaOH, 1mM EDTA,
0.1% (w/v) xylene cyanol, 0.1% (w/v) bromophenol blue), boil for 2 min andload 3-5 µl on a 6-8% sequencing gel with an appropriate sequencing ladder.

Northern blotting
In Northern blotting, total RNA is separated by agarose gel electrophoresis, transferred to amembrane and probed with a labelled DNA fragment. This method can give an approximate picture of
the number and sizes of transcripts originating from a particular cloned genomic region, but the resultsof Northern blots when working with prokaryotic RNA are often unsatisfactory and we usually find that
low and high resolution S1 nuclease mapping yield more conclusive answers. There are several reasonsfor this. (1) In prokaryotes, total RNA has to be used in Northern blots because there is no procedure topurify mRNA selectively. The substantial amounts of stable RNA bound to the filter somehow preventthe probe from hybridising to mRNA transcripts which co-migrate with one of the rRNA species. (2)Because S1 nuclease mapping employs liquid hybridisation, it is more sensitive than the Northernblotting which employs solid phase hybridisation. (3) RNA is more vulnerable to nuclease degradationin Northern blot analysis than in S1 nuclease mapping. (4) In S1 nuclease mapping, only that portion of
the transcript overlapping the probe has to be intact to give rise to the appropriate signal, whereas inNorthern blotting the entire transcript must be intact. (5) In general, prokaryotic mRNAs have a very
short half life in vivo.

In vitro transcription
Hybrids identified in high resolution S1 nuclease mapping or primer extension experiments may
represent genuine transcription start points, but they may also represent processing or degradation oflarger species. As part of a strategy to confirm the identification of a promoter, the simple techniqueof in vitro transcription is very useful (keeping in mind that not all promoters will be active in a purified
in vitro system lacking transcription activators etc, and also that some transcripts observed in purified
systems are in vitro artifacts). The major drawback is that Streptomyces RNA polymerase is notavailable commercially and so must be purified in the laboratory (p. 377).
Heparin (5 mg ml–1 in water)
BSA (bovine serum albumin, RNase-free, 50 mg ml–1)
100mM DTT (replace regularly)
100mM EDTA
Glycerol (sterilised by autoclaving)
3M sodium acetate pH6
Isopropanol
80% ethanol[ -32P]-CTP

&KDSWHU51$PHWKRGV

2 × Master Mix

Weigh out:

1.21 g Tris base
9.3 mg Na2EDTA
487.2 mg MgCl2.6H2O

1.

Adjust to 35 ml with sterile H2O.

2.

Adjust to pH7.9 with HCl.

3.

Add 25 ml 4mM potassium phosphate pH7.5.

4.

Adjust to pH7.9.

5.

Adjust to 70 ml final volume.

6.

Filter sterilise and store in 5 ml aliquots at -20o.

RTP mix: RTP mix is an equal mixture of: 30
mg ml–1 rATP, 30 mg ml–1 rGTP, and 30 mg
ml–1 rUTP
CTP mix: 15 mg ml–1 rCTP

Ribonucleoside triphosphate stocks should bepurchased as ready-made and pH-adjusted
solutions (for example from Pharmacia). If you
are preparing your own RTP solutions fromsolid stocks, make them up in water and adjust
to pH7.0 with Tris base solution as described bySambrook et al. (1989).

Precipitation mix
tRNA (10 mg ml–1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 µl
3M sodium acetate pH6.0 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 µl
Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 µl

1.

Prepare reaction mix (fresh) by mixing the following (enough for 10 reactions):
2 × Master Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115 µl
BSA (RNase-free, 50 mg ml–1) . . . . . . . . . . . . . . . . . . . . . . . . . 6 µl
100mM DTT (replace regularly) . . . . . . . . . . . . . . . . . . . . . . . 6 µl
100mM EDTA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 µl
Glycerol (sterilised by autoclaving) . . . . . . . . . . . . . . . . . . . . 80 µl
Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 µl



2.

&KDSWHU51$PHWKRGV

Mix:
7 µl
25 µl
5 µl

water
reaction mix
DNA solution (containing 0.5 pmole of the restriction fragment template)and incubate at 30o for 2 min.
3.

Add 1 µl (0.1 units) RNA polymerase and incubate at 30o for 5 min.

4

Add 1 µl (=l0 µCi) [ -32P]CTP (>600 Ci mmole–1; e.g. NEN Cat. No. NEG
508X) and 1 µl RTP mix and incubate at 30o for 2 min.

5.

Add 1 µl 5 mg ml–1 heparin (replace this solution regularly) and incubate at 30ofor 5 min.

6.

Add 2 µl CTP mix and incubate at 30o for 10 min.

7.

Stop on ice, add 10 µl precipitation mix and 60 µl isopropanol. Mix and leave
on ice at least 10 min.

8.

Spin and discard the supernatant.

9.

Redissolve the pellet in 180 µl water, add 20 µl 3M sodium acetate pH6.0 and
200 µl isopropanol. Mix and leave on ice at least l0 min. This second round of
precipitation is simply to remove additional unincorporated label. If this is not a concern, thepellet can be washed with 80% ethanol, air-dried and prepared for loading after the firstprecipitation.

10.

Spin and discard supernatant.

11.

Wash pellet with 80% ethanol, air dry, and redissolve in 10 µl run-off loading
buffer (80% formamide, 1mM EDTA, 0.1% (w/v) xylene cyanol, 0.1% (w/v)
bromophenol blue). (N.B. Avoid any loading buffer containing NaOH which
would hydrolyse the RNA!)

12.

Split the samples in two or into three (to allow for repeat gels - do not repeatedly
heat the same aliquot); heat denature one aliquot of each sample at 90o for 2 min
and run on a standard 6% polyacrylamide/urea sequencing gel with appropriate
radiolabelled DNA size markers. The mobility of RNA and single-stranded DNA
molecules of the same length on denaturing polyacrylamide gels is comparable. Accordingly,

&KDSWHU51$PHWKRGV

we use heat-denatured, radiolabelled digests of suitable plasmids as size standards. A HpaII
digest of pBR322 gives a good range of markers between 76 and 622 nucleotides.

Streptomyces RNA polymerase purification
Two procedure are given below: a classical protocol and one based on histidine tagging of the
0 subunit of RNA polymerase.
Standard purification of Streptomyces RNA polymerase
This protocol - Buttner and Brown (1985); Buttner et al. (1988); Brown et al. (1992) - is a minor
modification of the procedure developed by Burgess and Jendrisak (1975) for the purification of the E.
coli enzyme. It works for a wide range of Gram-positive and Gram-negative bacteria and therefore willalmost certainly be applicable to all actinomycete genera. The most significant difference between the
Burgess and Jendrisak (1975) protocol and the one given below is the substitution of the original Bio-Gel
A5 gel filtration column with a Superdex 200 (or Superose 6) FPLC gel filtration column (Pharmacia)
as the second chromatographic step. If you have access to an FPLC machine this substitution is certainly
worthwhile.
This purification can be completed in 2 days, excluding the final dialysis. There is no need to
do any RNA polymerase assays (which are time consuming) or run SDS gels during the procedure. In
the original method (Burgess and Jendrisak; 1975), a NaCl gradient was used to elute protein from the
DNA cellulose column, but this does not result in a very significant increase in RNA polymerase
purification. The extensive purification of RNA polymerase achieved by DNA-cellulose affinity
chromatography occurs primarily through the loss of the proteins which do not bind to the column. The
step elution recommended here is quick and, because all the protein eluted from the DNA cellulose
column is pooled, no assays are required.
DNA-cellulose or heparin-sepharose column
Superdex 200, Superose 6 or Bio-Gel A5 gel filtration column
High purity ammonium sulphate
10% (v/v) polyethyleneimine (PEI; see note to step 5)
Phenylmethylsulphonyl fluoride (PMSF)
French press or sonicator; conductivity meter; peristaltic pump; fraction collector;
chart recorder; absorbance monitor1.

Prepare in advance (see p. 378 for the composition of the buffers):
0.5 litre TGED + 0.2M NaCl
0.5 litre TGED + 0.5M NaCl
0.5 litre TGED + 1M NaCl
0.25 litre TGED + 2M NaCl
0.25 litre TGED (10% glycerol) no salt (for redissolving the ammonium sulphatepellets prior to the DNA cellulose column)

&KDSWHU51$PHWKRGV

1 litre TGED (10% glycerol) + 0.15M NaCl (for washing the DNA cellulose column
and for the salt gradient)0.25 litre TGED (10% glycerol) + 1M NaCl (for the DNA cellulose salt gradient)
0.5 litre Grinding Buffer
2 litres Storage Buffer
10 ml Superdex 200 Resuspension Buffer
1 litre Superdex 200 Running Buffer
Grinding Buffer
50mM Tris-HCl pH7.9
5% (v/v) glycerol
2mM EDTA
0.233M NaCl
1mM 2-mercaptoethanol
0.1mM DTT

TGED
10mM Tris-HCl pH7.9
5% (v/v) glycerol (10% where specified)
0.1mM EDTA
0.1mM DTT

Storage Buffer
l0mM Tris-HCl pH7.9
50% (v/v) glycerol
0.1mM EDTA
0.5mM DTT
0.1M NaCl
10mM MgCl2

Superdex 200 Resuspension Buffer
50mM NaH2PO4 pH7.0
150mM NaCl
5% (v/v) glycerol
0.1mM EDTA
0.1mM DTT

Superdex 200 Running Buffer
50mM NaH2PO4 pH7.0
5% (v/v) glycerol
0.1mM EDTA
0.1mM DTT
0.5M NaCl

2.

Prepare 1 ml of 23 mg ml–1 PMSF in 100% ethanol. Add PMSF to a final
concentration of 23 µg ml–1 (1 in 1000 dilution) to the Grinding Buffer and the
TGED + 0.2M NaCl immediately before use (because the half-life of PMSF inwater is only 12 h). Pre-equilibrate the DNA-cellulose column in TGED +0.15M NaCl (10% glycerol) at c. 20 ml h–1. Then proceed to 3a or 3b.

3a.

Resuspend 40-50 g mycelium in 200 ml grinding buffer containing PMSF.
Disrupt by passage through a French pressure cell at 15,000 lb in–2 (10.5×106kg m-2).

&KDSWHU51$PHWKRGV



3b.

Dissolve 0.5 g of lysozyme in 200 ml Grinding buffer containing PMSF. Afterthe lysozyme has dissolved, use the buffer to resuspend 40-50 g mycelium.
Disperse on a stirrer and then incubate at 30o. Remove onto ice at the earliest
sign of lysis (usually after 15-20 min). Allow to cool to 5-6o on ice and thensonicate using a wide probe at full-scale deflection. Pass from one flask on ice
to another on ice in small samples of c. 40 ml, sonicating in a small plastic
beaker. Each sample should only be sonicated until the temperature reaches 10 o
at most; probably only 15-20 sec. This should be repeated backwards andforwards until the viscosity of the suspension is reduced to that of a free-flowing
liquid; this usually takes three passes. After each pass make sure the temperatureof the suspension has returned to well below 10o before making the next pass.

4.

Add 266 ml TGED + 0.2M NaCl + PMSF and spin in four 250 ml centrifuge
bottles at 12,000 rpm at 4o for 20 min. When removing the supernatant it is best
to leave behind the last 20 ml of fluid over the pellet to avoid contamination,then re-spin to recover all supernatant fluid. Discard the pellets.

5.

Measure the volume of the supernatant and add, dropwise on a stirrer in the coldroom, 3.5 ml of 10% (w/v) Polyethylenimine (**PEI) per 100 ml supernatant.
Leave stirring for 10 min then spin in 250 ml bottles at 5,000 rpm for 90 sec (PEI
precipitated protein pellets very easily and is very difficult to resuspend if
compacted by prolonged centrifugation). If the RNA polymerase has come outof solution the supernatant fluid will be crystal clear. If not, add more PEI (onefifth of the original addition) and try again. **PEI (also known as Polymin P) is soldby Sigma (Catalogue No. P3143) as a 50% (w/v) aqueous solution (extremely viscous). Before
use it should be titrated with concentrated HCl to pH7.9 (this will require a large volume of theacid), then diluted to 10% (w/v) and clarified by filtration (if needed). Some papers recommendthat the PEI is dialysed extensively against distilled water before use; if this step is included, it
must be done before dilution, otherwise its osmotic potential will cause substantial dilution
during dialysis (it can even burst dialysis bags if too constrained!).

6.

Discard the supernatant and resuspend each of the four pellets in 100 ml TGED
+ 0.5M NaCl. Break up the pellets using a 60 ml syringe (no needle) and thenleave stirring in the cold room for 15 min. RNA polymerase remains in thepellet at this salt concentration.

7.

Spin in 250 ml bottles at 5,000 rpm for 90 sec. Discard the supernatant andresuspend each of the four pellets in 50 ml TGED + 1M NaCl using a 60 ml&KDSWHU51$PHWKRGV

syringe (no needle) and then leave to stir for 20 min in the cold room during
which time the RNA polymerase will come out of the pellet into solution.
8.

Remember this time it’s the supernatant you want. Pellet the insoluble material
at 12,000 rpm for 10 min. Measure the supernatant volume and precipitate theRNA polymerase by adding 35 g enzyme grade ammonium sulphate per 100 ml
initial volume**. Add the ammonium sulphate a few grams at a time and allow
to dissolve before adding the next amount. After all is dissolved, leave stirring
for a further 20 min. Check that the ammonium sulphate has not affected the pH
and if it has, adjust to pH7.9 with 1M NaOH; usually it makes little or nodifference to the pH. Split the suspension between 8 × 50 ml SS-34 centrifuge
tubes and spin for 30 min at 17,000 rpm at 4o. Discard supernatant and respin
tubes for 5 min at 5,000 rpm to remove the last traces of the ammonium sulphate
solution supernatant. This is important because the next step is to resuspend in
buffer containing no salt. **If you do not have a conductivity meter (step 9), instead ofprecipitating the RNA polymerase with ammonium sulphate (step 8), dialyse the supernatant
extensively against TGED [10% glycerol] + 0.15M NaCl before going directly to step 10.

9.

Resuspend each of the eight pellets in 7 ml TGED (10% glycerol), pool them ina beaker and measure the conductivity. Keep diluting the protein solution with
TGED (10% glycerol) until the conductivity is equal to that of TGED (10%glycerol) + 0.15M NaCl (the buffer in which the DNA-cellulose column isequilibrated). The conductivity of solutions is very sensitive to temperature so
all buffers must be at the same temperature for valid comparison.

10.

Respin the solution at l0,000 rpm for l0 min at 4o to remove particulate matterthat would otherwise clog the column. Load the material onto a 25 ml doublestranded DNA-cellulose column at 20-25 ml h–1. Many laboratories substitutecommercially prepared heparin-sepharose as the affinity matrix at this point. We use (doublestranded) DNA cellulose because it is cheap, disposable, and very easy to make on a bulk scalein the laboratory (p. 383).

11.

Wash the column with TGED (10%glycerol) + 0.15M NaCl at 20-25 ml h–1 until
the A280 trace returns to baseline (usually 3-4 h).

12.

Step-elute the protein from the column with TGED (l0% glycerol) + 1M NaCl
at 20-25 ml h–1. A 0.15-1.0M NaCl gradient can be used at this point, but does not result ina very significant increase in RNA polymerase purification. The extensive purification of RNA

&KDSWHU51$PHWKRGVpolymerase achieved by DNA-cellulose affinity chromatography occurs primarily through theloss of the proteins which do not bind to the column.

13.

Pool all the protein-containing fractions eluted from the column. Measure thevolume and precipitate by the gradual addition of 35 g enzyme-grade ammonium
sulphate per 100 ml initial volume, on a stirrer in the cold room. Check the pH
and adjust to pH7.9 with 1M NaOH if necessary. Leave stirring for a further 20
min after all of the ammonium sulphate has dissolved.

14.

Split between 4 × 30 ml Corex tubes and spin in rubber support sleeves at 9,000rpm for a minimum of 30 min at 4o. Respin for 2 min to remove all traces of theammonium sulphate solution supernatant. Each of the 4 pellets should be c. 1
cm in diameter and will contain c. 5 mg of protein, most of which (c. 70%) is
RNA polymerase. Because the loading capacity of the Superdex 200 gel
filtration column is small, the material has to be run in four separate batches,
each corresponding to one 5 mg pellet.

15.

Pre-equilibrate the Superdex 200 column on the FPLC (Pharmacia) in Superdex
200 Running Buffer. Resuspend one pellet in 200 µl of Superdex 200
resuspension buffer (although the pellet is large, it will dissolve in such a small
volume). Then spin in an Eppendorf tube in an Eppendorf centrifuge for 10 min
at 4o and transfer the soluble material into a fresh tube (the centrifugation is toremove particulate matter that would otherwise clog the Superdex 200 column).
Inject the sample onto the Superdex 200 column via a "200 µl superloop" andrun the column in Superdex 200 Running Buffer at a flow rate of 0.18 ml min–1
and collect 0.5 ml fractions. Identify fractions of interest by running aliquots ofprotein-containing fractions on SDS-polyacrylamide gels.

16.

Samples are dialysed for a minimum of 24 h (36 h if **Eppendorf dialysis isbeing used) in Storage Buffer. Alternatively, simply add an equal volume ofglycerol and mix. Store enzyme at -20o. **It is often desirable to dialyse and store thefractions from the Superdex 200 column separately because this matrix partially resolves anumber of different holoenzymes (Buttner et al., 1988; Brown et al., 1992). This canconveniently be done by collecting the fractions directly in screw-capped Eppendorf tubes andthen performing “Eppendorf dialysis.” Remove the black O ring from the lids of the tubes witha pair of pointed forceps and use the O rings to secure small squares of dialysis tubing across themouths of the tubes. Place the tubes in a polystyrene float and invert onto the surface of a

&KDSWHU51$PHWKRGV

container of Storage Buffer. Because the interface surface across the mouth of the tube is small
it is necessary to invert the tubes at regular intervals to obtain good mixing.

17.

After the experiment is over, wash the DNA cellulose column in TGED + 2M
NaCl to clean the column. When the remaining bound protein has eluted suchthat the A280 trace has returned to baseline, re-equilibrate the column with TGED
(l0% glycerol) + 0.15M NaCl and then store the matrix at -20o in the same
buffer. We have generally avoided long term storage of DNA-cellulose columns
in the cold room.

Purification of histidine-tagged RNA polymerase
Following the work of Kashlev et al. (1993) in E. coli, Babcock et al. (1997) constructed strainsof S. coelicolor and S. lividans which express a modified rpoC gene (encoding the 0 subunit of RNApolymerase) carrying six histidine codons at the 30 end. RNA polymerase suitable for in vitrotranscription work can be purified from these strains using Ni-NTA agarose affinity chromatography
followed by heparin-Sepharose chromatography (Babcock et al., 1997; Brawner et al., 1997).
5 ml HiTrap heparin-sepharose column (Pharmacia 17-0407-01)
Ni-NTA agarose (Qiagen).

1.

Equilibrate 4 ml of Ni-NTA agarose (Qiagen) in buffer I (10mM Tris-HCl
pH7.9, 0.1M NaCl, 1mM -mercaptoethanol, 5% (v/v) glycerol) containing
2.5mM imidazole.

2.

Resuspend the cell pellet (c. 15-20 g) in a minimal volume of buffer I and disrupt
by passage through a French pressure cell at 15,000 lb in–2 . Remove cell debris
by centrifugation at 15,000 × g for 10 min.

3.

Add the equilibrated Ni-NTA agarose to the supernatant and stir in the cold
room. Optimal binding is achieved within one hour.

4.

Load the Ni-NTA agarose-protein suspension onto a 15-30 ml column and wash
twice with 3-6 ml of buffer I + 20mM imidazole + 0.1M NaCl.

5.

Elute the RNA polymerase with 2 ml of buffer I + 300mM imidazole + 0.1M
NaCl.

&KDSWHU51$PHWKRGV



6.

Immediately load the eluate onto a 5 ml HiTrap heparin-sepharose column(Pharmacia LKB Biotechnology) previously equilibrated with buffer H (10mM
Tris-HCl pH7.9, 0.lmM dithiothreitol, 5% (v/v) glycerol). Wash the column
with 15 ml buffer H + 0.1M KCl. Elute the bound RNA polymerase with a 0.3M
to 0.65M KCl gradient in 20 ml of buffer H.

7.

Identify fractions containing RNA polymerase by SDS gel electrophoresis or byin vitro run-off transcription assays. Add an equal volume of glycerol, mix, andstore fractions at -20o.

Preparation of DNA-cellulose
This procedure allows preparation of DNA cellulose as cheaply as possible.

1.

Buy crude calf thymus DNA (e.g. Sigma Catalogue No. D1501) and purify it
extensively by repeated phenol/chloroform extraction, followed by ethanol
precipitation and ethanol washes. Redissolve the DNA in l0mM Tris-HCl(pH7.9) to a final concentration of 2 mg ml–1.

2.

Mix 24 ml calf thymus DNA with 8 g cellulose to form a wet paste and leave to
dry overnight at room temperature.

3.

Place in 50o oven for 3 days. Remove daily and grind the material to form asmooth powder.

4.

Lyophilise overnight, then resuspend in 10mM Tris-HCl pH7.9 and allow the
DNA-cellulose to settle. Decant the supernatant, including very fine particulatematter ("finings"). Add more buffer to the DNA-cellulose and repeat thisprocess several times. This allows any unbound DNA to be lost and the removalof the finings helps the flow characteristics of the matrix (cellulose has poor flow
characteristics, particularly in buffers containing 10% (v/v) glycerol).

5.

Assay the amount of DNA bound to the matrix. Take a sample of matrix andspin it down. Remove the supernatant and vacuum dry the pellet. Measure thedry weight of the pellet and then release the bound DNA by boiling it for 10 min
in water in a screw-capped Eppendorf tube. Cool the tube, spin out the matrix

&KDSWHU51$PHWKRGVand measure the volume and A260 of the supernatant. Calculate the amount ofDNA bound to the matrix assuming:
1 A260 unit = 40 µg ml–1 (the boiled DNA is single-stranded).
On average, 30-40% of the DNA becomes bound to the DNA (2-3 mg DNA g–1
cellulose).
6.

Store the freshly-prepared matrix frozen at -20o, either in l0mM Tris-HCl pH7.9
or equilibrated in the buffer used for chromatography (TGED (10% glycerol) +0.15M NaCl.)

Babcock MJ, Buttner MJ, Keler CH, Clarke BR, Morris
RA, Lewis CG, Brawner ME (1997) Characterization of therpoC gene of Streptomyces coelicolor A3(2) and its use todevelop a simple and rapid method for the purification of
RNA polymerase. Gene, 196, 31-42.
Brawner ME, Mattern SG, Babcock MJ, Westpheling J(1997) The Streptomyces galP1 promoter has a novel RNA
polymerase recognition sequence and is transcribed by a newform of RNA polymerase in vitro. J.Bacteriol., 179, 3222-31.
Brown KL, Wood S, Buttner MJ (1992) Isolation andcharacterization of the major vegetative RNA polymerase of
Streptomyces coelicolor A3(2): renaturation of a sigma
subunit using GroEL. Mol.Microbiol., 6, 1133-1139.
Burgess RR, Jendrisak JJ (1975) A procedure for the rapid,
large-scale purification of Escherichia coli DNA-dependent
RNA polymerase involving Polymin P precipitation andDNA-cellulose chromatography. Biochemistry, 14, 4634-8.
Buttner MJ, Brown NL (1985) RNA polymerase-DNAinteractions in Streptomyces. In vitro studies of a S. lividans
plasmid with S. coelicolor RNA polymerase. J.Mol.Biol., 185,
177-88.
Buttner MJ, Smith AM, Bibb MJ (1988) At least three
RNA polymerase holoenzymes direct transcription of the
agarase gene (dagA) of Streptomyces coelicolor A3(2). Cell,
52, 599-607.
Clayton TM (1989) Inhibition of transcription in
Streptomyces coelicolor A3(2) on induction of thebacteriophage C31. PhD thesis, University of East Anglia,
Norwich.
Glišin V, Crkvenjakov R, Byus C (1974) Ribonucleic acid
isolated by cesium chloride centrifugation. Biochemistry, 13,2633-7.
Hindle Z, Smith CP (1994) Substrate induction andcatabolite repression of the Streptomyces coelicolor glycerol
operon are mediated through the GylR protein.
Mol.Microbiol., 12, 737-745.
Hobbs G, Frazer CM, Gardner DCJ, Cullum J, Oliver SG(1989) Dispersed growth of Streptomyces in liquid culture.
Appl.Microbiol.Biotechnol., 31, 272-277.
Hobbs G, Obanye AIC, Petty J, Mason JC, Barratt E,
Gardner DCJ, Flett F, Smith CP, Broda P, Oliver SG(1992) An integrated approach to studying regulation of
production of the antibiotic methylenomycin by Streptomyces
coelicolor A3(2). J.Bacteriol., 174, 1487-1494.
Janssen GR (1993) Eubacterial, archaebacterial, andeucaryotic genes that encode leaderless mRNA. In Baltz,
R.H., Hegeman, G.D. and Skatrud, P.L. (eds.), Industrial
Microorganisms: Basic and Applied Molecular Genetics.
American Society for Microbiology, Washington, DC, pp. 5967.
Kashlev M, Martin E, Polyakov A, Severinov K, Nikiforov
V, Goldfarb A (1993) Histidine-tagged RNA polymerase:
dissection of the transcription cycle using immobilized
enzyme. Gene, 130, 9-14.
Kelemen GH, Brown GL, Kormanec J, Potúûková L,
Chater KF, Buttner MJ (1996) The positions of the sigma
factor genes, whiG and sigF, in the hierarchy controlling thedevelopment of spore chains in the aerial hyphae ofStreptomyces coelicolor A3(2). Mol.Microbiol., 21, 593-603.
Kirby KS, Fox-Carter E, Guest M (1967) Isolation of
deoxyribonucleic acid and ribosomal ribonucleic acid frombacteria. Biochem.J., 104, 258-62.
Maxam AM, Gilbert W (1980) Sequencing end-labelled
DNA with base-specific chemical cleavages. Meth.inEnzymol., 65, 499-560.
Murray MG (1986) Use of sodium trichloroacetate andmung bean nuclease to increase sensitivity and precision
during transcript mapping. Anal.Biochem., 158, 165-70.
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular
Cloning: a Laboratory Manual, 2nd ed. Cold Spring Harbor,
Cold Spring Harbor, NY.
Smith CP (1991) Methods for mapping transcribed DNA
sequences. In Brown, T.A. (ed.) Essential Molecular Biology:
A Practical Approach. Oxford University Press, Oxford, Vol.
2, pp. 237-252.
Smith CP, Chater KF (1988) Cloning and transcription
analysis of the entire glycerol utilization (gylABX) operon ofStreptomyces coelicolor A3(2) and identification of a closely
associated transcription unit. Mol.Gen.Genet., 211, 129-137.
Summerton J, Atkins T, Bestwick R (1983) A rapid method
for preparation of bacterial plasmids. Anal.Biochem., 133, 7984.

m17tk2b.wpd 14 March 2000 Tobias Kieser

Chapter 17

Production and secretion of proteins by
Streptomyces
CONTENTS

Transcription initiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 385
Translation initiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 386
Signal peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 387
Codon usage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 387
Regulated expression systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 388
Culture conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 388
Levels of expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 388
A selection of plasmids suitable for intracellular expression . . . . . . . . . . . . . . . . . . . . . . . . . . 389
Plasmids suitable for secretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 390
Optimising expression of Streptomyces genes in E. coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 391
Changing the codon usage at the 50 end of a coding region . . . . . . . . . . . . . . . . . . . . . 391
E. coli vectors that have been used to over-express streptomycete genes . . . . . . . . . . 391
Among the streptomycetes, the readily manipulable S. lividans has been used extensively as an
expression host for a wide variety of genes from diverse sources (for detailed reviews see Molnar, 1994;
Gilbert et al., 1995; Binnie et al., 1997). This reflects the availability of well-established plasmid-based
expression systems, developed fermentation technology and, for secreted proteins, a low level ofendogenous protease activity (Engels and Koller, 1992). Here, we make a few general points about theexpression of genes in streptomycetes, describe several purpose-built expression vectors, and refer thereader to the excellent reviews quoted above for further details. We also comment on the expression of
streptomycete genes in E. coli.

Transcription initiation
In recent years, much effort has gone into defining the transcription start sites of many
Streptomyces genes (Strohl, 1992; Bourn and Babb, 1995). It has revealed a high degree of promoter
sequence heterogeneity, much of which probably reflects the occurrence of multiple
factors.
Nevertheless, there are promoter sequences that show a high degree of similarity to the consensus
sequence for promoters recognised by the major and essential factors of other bacteria, notably E. coli



&KDSWHU3URGXFWLRQDQGVHFUHWLRQRISURWHLQVE\6WUHSWRP\FHV( 70) and Bacillus subtilis ( A) and that are probably recognised by hrdB, which is essential in S.
coelicolor, and its orthologues in other Streptomyces species.
Frequently, the aim of expressing a gene in S. lividans will be to produce as much protein as
possible. Thus a strong promoter is desirable. ermE*p (Schmitt-John and Engels, 1992; Bibb et al.,1994) and the promoter of the S. longisporus STI-II gene (encoding a secreted protease inhibitor;
Fornwald et al., 1993), have proved particularly effective for heterologous genes, while the thiostreptoninducible tipA promoter has proved highly successful for the over-expression of streptomycete genes (J.
White and M.J. Bibb, unpublished). tipAp requires the TipAL protein for induction (Holmes et al.,1993), which is not present in all streptomycetes. Moreover, tipAp is not inducible in all strains of S.
lividans (1326, TK20 and TK24 are inducible, TK19 and TK21 (p. 425) are not; C.J. Thompson,
personal communication).

Translation initiation
High rates of protein synthesis also require efficient ribosome binding sites for translation
initiation. Ribosome binding sites of mRNAs are usually characterised as sequences that occur 5-9
nucleotides (nt) upstream of the translational start codon and that show complementarity to sequences
close to the 30 end of 16S rRNA:
30 end of 16S RNA:mRNA:

30 UCUUUCCUCCACUAG- - - - - 50
50- - - - - - GGAGG- - - - --AUG/GUG/UUG--- 30

Analysis of a large number of such sequences (Strohl, 1992) reveals that complementarity can
extend from 3 to 7 nt and is generally centred on the mRNA sequence GGAGG. The extent ofcomplementarity is similar to that found in E. coli, but somewhat less than is generally observed in B.
subtilis, where the 30 end of 16S rRNA is identical to that found in S. lividans (Bibb and Cohen, 1982).
As in other bacteria, most streptomycete genes use AUG as translation start codon. Analysis of 1735
protein coding sequences present in the partial S. coelicolor genome sequence (p. 424) revealed that 61%
started with AUG, 36% with GUG and 3% with UUG (K. Rutherford, personal communication). In
contrast, when 100 randomly chosen E. coli genes were analysed, only 3% started with GUG, and 97%
with AUG. Although little is known about translational initiation in streptomycetes, the mRNAs derived
from the elongation factor EF-Tu gene (tuf1) and tipA, each of which is capable at single copy of giving
rise to protein levels that are readily detected on Coomassie-stained polyacrylamide gels of total protein
extract, are likely to possess efficient ribosome binding sites (the tuf1 and tipA mRNAs possess regionsof complementarity of 50AGGAGG30 and 50AGAA(G)GGAG30, respectively). Remarkably, somestreptomycete mRNAs have no conventional ribosome binding site and transcription initiates at, or one
nt before, the translation start codon (Janssen, 1993; Bibb et al., 1994). At least one such transcript (thatof the aphI gene of S. fradiae) can yield high levels of protein (Janssen et al., 1989), suggesting thatsequences 30 of the translation initiation codon can provide efficient ribosome recognition.

&KDSWHU3URGXFWLRQDQGVHFUHWLRQRISURWHLQVE\6WUHSWRP\FHV

Signal peptides
Often it is desirable to produce the expressed gene product in a secreted form, particularly if thatreflects its normal fate and there is a need to establish disulphide bridges to attain the appropriate tertiary
structure for biological activity (the redox potential of the cytoplasm often prevents this). An additional
advantage is that the expressed gene product can usually be separated from the cells by simple filtrationor centrifugation, and will frequently be the predominant protein in the culture filtrate. Streptomycetes
in general produce many extracellular enzymes as part of their saprophyic life-style, and they have thecapacity to secrete large amounts of protein. Unless the cloned gene encodes a suitable signal sequenceof its own, the protein to be made will have to be fused to a signal sequence for secretion. Signal
peptides in streptomycetes conform to the general rules established for other bacterial secretion systems,
although they are generally longer than those found in E. coli and B. subtilis. A short stretch of positively
charged hydrophilic amino acids at the amino terminus is usually followed by a long -helical core ofhydrophobic amino acids, a helix-breaking residue (frequently Pro) and a short carboxy-terminal
sequence motif containing small amino acids that is recognised and cleaved by a signal peptidase. In
general, amino acid substitutions and deletions have confirmed the functional importance of these motifs
for efficient protein secretion in streptomycetes (Park et al., 1991; Hale and Schottel, 1996; Page et al.,
1996; Fass and Engels, 1996; Lammertyn and Anné, 1998). Several streptomycete signal sequences havebeen used to secrete heterologous proteins in S. lividans (Molnar, 1994; Gilbert et al., 1995; Binnie etal., 1997).

Codon usage
The high G+C content of streptomycete DNA results in an extremely biased codon usage that
may be indicative of the relative abundance of isoaccepting tRNAs, and which results in the infrequent
use of A+T-rich codons (Wright and Bibb, 1992). Particularly rare examples are the leucine codons CTA
and TTA (Table 18.1, p. 400). TTA codons generally occur in Streptomyces genes that are likely to be
expressed late in the developmental cycle, and there is evidence to suggest that the tRNA required fortranslation of TTA codons (the product of the bldA gene) is limiting in vegetatively growing cultures(Leskiw et al., 1993), although other experiments failed to detect any such limitation in liquid-growncultures (Gramajo et al., 1993). Consequently, attempts to achieve high-level expression of genes
containing one or more TTA codons should be undertaken with caution.
Despite the biased codon usage of Streptomyces genes, little attention has been paid to changing
the codon usage at the 50 end of heterologous genes with lower G+C content prior to their attempted
expression in streptomycetes, where the population of cognate tRNAs is likely to be more biased.
However, one attempt to do so, for mouse tumour necrosis factor, resulted in reduced levels of mRNAstability and protein yield (Lammertyn et al., 1996). (For the expression of Streptomyces genes in E. coli,changing the amino terminal codons has proved beneficial; p. 390.)&KDSWHU3URGXFWLRQDQGVHFUHWLRQRISURWHLQVE\6WUHSWRP\FHVRegulated expression systems
Although constitutive high-level expression of a cloned gene may be acceptable, it may benecessary to regulate expression for particular experimental needs, or to prevent deleterious effects oncell growth. Of the many regulated promoters that have been identified in streptomycetes, three havebeen incorporated into expression vectors that require the addition of exogenous compounds for
induction - tipAp induced by addition of thiostrepton (Smokvina et al., 1990; Kuhstoss and Rao, 1991;
Takano et al., 1995), gylCABp induced by glycerol (Hindle et al., 1994) and mcrABp induced bymitomycin C (August et al., 1996). In contrast, the system developed by Kataoka et al. (1996) istemperature-inducible (see pITS107, p. 389). Different levels of expression can be obtained with both
tipAp and mcrABp by altering the level of inducer; for the gyl system, this is achieved by using gylCAB
promoters of varying inherent strength from different streptomycetes.
While a high copy number plasmid is likely to yield maximal levels of production, it is importantto remember that it may not be tightly regulated. Thus in experiments where regulation is crucial, a low
copy number or integrative vector may be preferable (for example, the integrative tipAp vector pIJ8600;
Sun et al., 1999).

Culture conditions
Achieving high levels of protein production often involves the empirical determination of themost suitable growth medium and conditions, including medium composition, oxygen supply,
temperature, and agitation. Where the protein of interest is secreted, degradation by extracellular
proteases can be reduced by the addition of protease inhibitors, and Casamino acids were found to inhibit
the activity of a protease produced by stationary phase cultures of S. lividans (Fornwald et al., 1993;
Brawner, 1994).

Levels of expression
Although in many cases growth has been confined to shake flasks with little attempt to optimise
fermentation conditions, the levels of production of cloned gene products have often been very
encouraging (Gilbert et al.,1995). For streptomycete regulatory proteins produced intracellularly, levels
as high as 40% of total cell protein have been obtained (J. White and M.J. Bibb, unpublished results);
comparable levels of synthesis of some of these proteins in E. coli led to their total incorporation into
insoluble inclusion bodies. While it is possible to solubilise the inclusion bodies, there is no guaranteethat attempted renaturation will lead to significant levels of active protein. Whether this higher level ofsolubility is a general characteristic of proteins made in streptomycetes remains to be seen. For asecreted derivative of the human sCD4 receptor, over 300 mg l–1 were observed in the culture supernatant(Fornwald et al., 1993). The expression rate and product characteristics (including protein solubility,
proper formation of disulphide bonds, and bioactivity) were much better than those found for the same
protein produced in E. coli or in animal cell culture.

&KDSWHU3URGXFWLRQDQGVHFUHWLRQRISURWHLQVE\6WUHSWRP\FHVA selection of plasmids suitable for intracellular expression
Maps of many of the plasmids are in Chapter 22.
pIJ6021 (p. 539; Takano et al., 1995) derives from the high copy number pIJ101 (p. 256)
derivative pIJ486. It contains the thiostrepton-inducible tipAp followed by a multiple-cloning site, bothflanked by transcriptional terminators. It carries kan for selection of transformants, and tsr to allow forthiostrepton induction of tipAp. Optimal conditions for induction should be determined for each gene
expressed.
pIJ4123 (p. 539; Takano et al., 1995) is similar to pIJ6021 but contains a sequence encoding an
N-terminal His-tag, allowing for the production of fusion proteins that can be purifed on a Ni affinity
column. A similar system has been developed by Enguita et al. (1996).
pIJ4090 (p. 535; M. J. Bibb, unpublished) derives from the high copy number (50-100) pIJ101
derivative pIJ486 (p. 533; Ward et al., 1986) that contains the constitutively active ermE* promoter. It
carries tsr for selection of transformants.
pMT3206 (p. 535; F. Amini, M. S. B. Paget, G. Bucca and C. P. Smith, personalcommunication). A derivative of the high copy number (50-100) pIJ101 derivative pIJ680 (p. 534)
containing the glycerol-inducible gylp1/p2 promoters. xylE lies downstream of gylp1/p2, potentiallyallowing for the detection of cloned fragments by “insertional inactivation”. The expression cassette isflanked by fd and mmr transcriptional terminators, and the vector contains tsr for the selection oftransformants.
pITS107 (Kataoka et al., 1996) is a derivative of the multicopy pIJ101 derivative pIJ486 (p. 533)
with a temperature-inducible promoter/repressor cassette (p. 504) from pSN22. Induction ratios of 100
and 25 were observed with temperature shifts from 28o to 37o and 32o, respectively. The plasmid
contains tsr for the selection of transformants.
pANT849 (p. 537) and its derivatives (pANT857, pANT859, pANT863 and pANT864; W.
Strohl, personal communication) are pIJ702-based expression vectors that contain the snpA promoterwith transcriptional activity at least 50 times that of melC1p of pIJ702 (pp. 501, 536). Preliminary
experiments in S. lividans indicate highest levels of activity in mid-to-late exponential phase cultures.
It is possible to construct translational fusions by using the SphI, EcoRI, or BglII sites of pANT849, orby using restriction sites in pANT863 and pANT864. These vectors have been used to over-express
anthracycline biosynthetic genes in S. lividans and actinorhodin biosynthetic genes in S. galilaeus(Dickens et al., 1996; Dickens and Strohl, 1996).
pIJ8600 (p. 531; Sun et al., 1999) a derivative of the integrative plasmid pSET152 (p. 530;
Bierman et al., 1992) that contains the C31 attachment site and oriT, and can thus be transferred by
conjugation into streptomycetes from E. coli. It contains the thiostrepton-inducible tipAp followed bya multiple-cloning site, both of which are flanked by transcriptional terminators. It carries apr (aac(3)IV,

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encoding apramycin resistance) for the selection of exconjugants or transformants, and tsr to allow forthiostrepton induction of tipAp.
pPM927 (p. 563; Smokvina et al., 1990) is an integrative plasmid that contains the pSAM2(p. 265) attachment site for stable site-specific integration and can be transferred into streptomycetes
from E. coli by conjugation (p. 249). It contains the thiostrepton-inducible tipAp which is preceded bya transcriptional terminator. It carries spc/str (aadA), and tsr for selection of transformants.
pHM6-11 (Motamedi et al., 1995). This is a series of integrative expression vectors that containthe strong constitutive ermE* promoter, a synthetic ribosome binding site and multiple-cloning site,
followed by a transcriptional terminator. pHM6,8,9 and 11 contain the S. coelicolor IS117 (p. 265),
permitting stable site-specific integration in many different streptomycetes. pHM9, 10 and 11 also
contain oriT, permitting conjugation from E. coli (p. 249). All the vectors contain hyg for selection in
streptomycetes.
pMT3226 (p. 532; F. Amini, M. S. B. Paget, G. Bucca, C. P. Smith, personal communication).
An integrative vector based on pSET152 (p. 530) with the same expression features as pMT3206 (above).
oriT allows conjugation from E. coli (p. 249), and the plasmid can be selected in E. coli and in
streptomycetes with apramycin.
pKT02 (p. 532; van Mellaert et al., 1998). An integrative vector that carries tsr for selection oftransformants, and ampicillin resistance for selection in E. coli. Integration is mediated by the int andattP regions of the S. venezuelae phage VWB, and occurs towards the 30 end of a putative arginine tRNA
gene recognising the codon AGG.
pKC1064 (Kuhstoss and Rao, 1991) is an E. coli-Streptomyces (SCP2*-based) shuttle vector
containing the thiostrepton-inducible tipAp. It carries aac(3)IV for selection of transformants in E. coli
and streptomycetes, and tsr to allow for thiostrepton induction of tipAp. The presence of oriT allows
conjugation from E. coli to Streptomyces spp (p. 249).
pMCXpress (http://www.cbs.umn.edu/asirc/database/veclist/pMCXpress.html; August et al.,1996) is a Streptomyces-E. coli shuttle plasmid selectable with kanamycin. It contains an NdeI site ina multiple-cloning sequence permitting production of His-tagged fusion proteins. The mcrA promoter
is inducible with 25 µg ml–1 mitomycin C.

Plasmids suitable for secretion
pAX5a and pAX8 are bifunctional E. coli-streptomycete expression vectors based on the E. coli
replicon pUC19 and the pIJ101-based streptomycete replicons pIJ680 (pAX8) and pIJ702 (pAX5a) (J.W.
Engels, personal communication). pAX5a contains the constitutive ermE*p, and pAX8 the thiostreptoninducible tipAp. For for selecting transformants in streptomycetes, pAX5a contains tsr and pAX8 aneomycin resistance gene (and tsr for induction of tipAp). Both plasmids contain the tendamistat signal

&KDSWHU3URGXFWLRQDQGVHFUHWLRQRISURWHLQVE\6WUHSWRP\FHVsequence for protein secretion (as well as the tendamistat coding sequence), followed by a transcription
terminator.

Optimising expression of Streptomyces genes in E. coli
Changing the codon usage at the 50 end of a coding region
Although overall codon usage does not appear to limit the expression of a heterologous gene in
E. coli severely (unless expressed at a very high level and containing AGA and/or AGG codons
recognised by a rare arginine tRNA; Brinkmann et al., 1989), the efficiency with which a ribosome clears
the translation start site may influence the level of protein production (Liljenstrom and von Heijne,
1987). Consequently, when expressing streptomycete genes in E. coli, the codon usage at the 50 end of
the coding region (usually encompassing codons 2-10) has sometimes been changed to incorporatesynonymous codons that are found in highly expressed E. coli genes. At least in some cases thisincreased the amount of protein produced without apparently affecting the level of mRNA (e.g. Gramajo
et al., 1991). Many E. coli expression vectors have NdeI or NcoI sites positioned downstream of an
efficient ribosome binding site to allow high-level expression of a native (as opposed to a fusion) protein.
Consequently, the high-level expression of a streptomycete gene in E. coli may require the insertion ofan NdeI or NcoI site at a position corresponding to the translational start codon; given the flexibility
afforded by PCR, changing the first few codons of the gene at the same time often involves little, if any,
additional work and may markedly increase the level of protein produced.
E. coli vectors that have been used to over-express streptomycete genes
Several expression systems utilising a variety of promoters have been used to over-produce
Streptomyces proteins in E. coli, including vectors (Robbins et al., 1981), pUC18 derivatives (Smith
and Owen, 1991), and T7 RNA polymerase based vectors (e.g. Gramajo et al., 1991). The latter have
proved particularly successful, with several offering the additional facility of His-tagging for subsequent
protein purification.

August PR, Rahn JA, Flickinger MC, Sherman DH (1996)
Inducible synthesis of the mitomycin C resistance geneproduct (MCRA) from Streptomyces lavendulae. Gene, 175,
261-267.
Bibb MJ, Cohen SN (1982) Gene expression in
Streptomyces: Construction and application of promoterprobe plasmid vectors in Streptomyces lividans. Mol. Gen.
Genet., 187, 265-277.
Bibb MJ, Findlay PR, Johnson MW (1984) The
relationship between base composition and codon usage inbacterial genes and its use in the simple and reliable
identification of protein coding sequences. Gene, 30, 157166.
Bibb M J, White J, Ward J M and Janssen G R (1994).
The mRNA for the 23S rRNA methylase encoded by theermE gene of Saccharopolyspora erythraea is translated in
the absence of a conventional ribosome-binding site. Mol.

Microbiol. 14, 533-45.
Bierman M, Logan R, O’Brien K, Seno ET, Rao RN,
Schoner BE (1992) Plasmid cloning vectors for the conjugaltransfer of DNA from Escherichia coli to Streptomyces spp.
Gene, 116, 43-49.
Binnie C, Cossar JD, Stewart DI (1997) Heterologous
biopharmaceutical protein expression in Streptomyces.
Trends.Biotechnol., 15, 315-320.
Bourn WR, Babb B (1995) Computer assisted identificationand classification of streptomycete promoters.
Nucleic.Acids.Res., 23, 3696-3703.
Brawner ME (1994) Advances in heterologous gene
expression by Streptomyces. Curr.Opin.Biotechnol., 5, 475481.
Brinkmann U, Mattes RE, Buckel P (1989) High-level
expression of recombinant genes in Escherichia coli isdependent on the availability of the dnaY gene product. Gene,&KDSWHU3URGXFWLRQDQGVHFUHWLRQRISURWHLQVE\6WUHSWRP\FHV

85, 109-114.
Dickens ML, Strohl WR (1996) Isolation andcharacterization of a gene from Streptomyces sp. strain C5
that confers the ability to convert daunomycin to doxorubicinon Streptomyces lividans TK24. J.Bacteriol., 178, 3389-3395.
Dickens ML, Ye JS, Strohl WR (1996) Cloning,sequencing, and analysis of aklaviketone reductase from
Streptomyces sp. strain C5. J. Bacteriol., 178, 3384-3388.
Engels JW, Koller K-P (1992) Gene expression andsecretion of eukaryotic foreign proteins in Streptomyces. In
Murray, J.A.H. (ed.), Transgenesis - Applications of Gene
Transfer. John Wiley and Sons, New York, pp. 32-53.
Enguita FJ, de la Fuente JL, Martin JF, Liras P (1996) An
inducible expression system of histidine-tagged proteins in
Streptomyces lividans for one-step purification by Ni2+
affinity chromatography. FEMS Microbiol.Lett, 137, 135-140.
Fass SH, Engels JW (1996) Influence of specific signal
peptide mutations on the expression and secretion of the amylase inhibitor tendamistat in Streptomyces lividans.
J.Biol.Chem., 271, 15244-15252.
Fornwald JA, Donovan MJ, Gerber R, Keller J, Taylor
DP, Arcuri EJ, Brawner ME (1993) Soluble forms of thehuman T-cell receptor CD4 are efficiently expressed by
Streptomyces lividans. Bio/Technology, 11, 1031-1036.
Gilbert M, Morosoli R, Shareck F, Kluepfel D (1995)
Production and secretion of proteins by streptomycetes.
Crit.Rev, Biotech., 15, 13-39.
Gramajo HC, White J, Hutchinson CR, Bibb MJ (1991)
Overproduction and localization of components of thepolyketide synthase of Streptomyces glaucescens involved in
the production of the antibiotic tetracenomycin C.
J.Bacteriol.,173, 6475-6483.
Gramajo HC, Takano E, Bibb MJ (1993) Stationary-phaseproduction of the antibiotic actinorhodin in Streptomyces
coelicolor A3(2) is transcriptionally regulated.
Mol.Microbiol., 7, 837-845.
Hale VA, Schottel JL (1996) Mutational analysis of the
Streptomyces scabies esterase signal peptide.
Appl.Microbiol.Biotechnol., 45, 189-198.
Hindle Z, Smith CP (1994) Substrate induction andcatabolite repression of the Streptomyces coelicolor glycerol
operon are mediated through the gylR protein. Mol.
Microbiol., 12, 737-745.
Holmes DJ, Caso JL, Thompson CJ (1993) Autogenoustranscriptional activation of a thiostrepton-induced gene inStreptomyces lividans. EMBO J., 12, 3183-3191.
Janssen GR (1993) Eubacterial, archaebacterial, andeukaryotic genes that encode leaderless mRNA. In Baltz, R.
H., Hegeman, G. D., and Skatrud, P. L. (eds.), Industrial
Microorganisms: Basic and Applied Molecular Genetics.
American Society for Microbiology, Washington DC, pp. 5967.
Janssen GR, Ward JM, Bibb MJ (1989) Unusualtranscriptional and translational features of theaminoglycoside phosphotransferase gene (aph ) from
Streptomyces fradiae. Genes Dev., 3, 415-429.
Kataoka M, Tatsuta T, Suzuki I, Kosono S, Seki T,
Yoshida T (1996) Development of a temperature-inducible
expression system for Streptomyces spp. J.Bacteriol., 178,
5540-5542.
Kuhstoss S, Rao RN (1991) A thiostrepton-inducible
expression vector for use in Streptomyces-spp. Gene, 103, 9799.
Lammertyn E, Anné J (1998) Modifications of Streptomyces
signal peptides and their effects on protein production andsecretion. FEMS Microbiol.Lett., 160, 1-10.

Lammertyn E, Van Mellaert L, Bijnens AP, Joris B, Anne
J (1996) Codon adjustment to maximise heterologous gene
expression Streptomyces lividans can lead to decreasedmRNA stability and protein yield. Mol.Gen.Genet., 250, 223229.
Leskiw BK, Mah R, Lawlor EJ, Chater KF (1993)
Accumulation of bldA-specified transfer RNA is temporally
regulated in Streptomyces coelicolor A3(2). J.Bacteriol., 175,
1995-2005.
Liljenstrom H, Von Heijne G (1987) Translation rate
modification by preferential codon usage - intragenic position
effects. J.Theoret.Biol., 124, 43-55.
Molnár I (1994) Secretory production of homologous andheterologous proteins by recombinant Streptomyces - what
has been accomplished? In Murooka, Y. and Imanaka, T.
(eds.), Recombinant Microbes for Industrial and Agricultural
Applications. Marcel Dekker, Inc., New York, pp. 81-104.
Motamedi H, Shafiee A, Cai SJ (1995) Integrative vectors
for heterologous gene expression in Streptomyces spp. Gene,
160 , 25-31.
Page N, Kluepfel D, Shareck F, Morosoli R (1996) Effectof signal peptide alterations and replacement on export ofxylanase A in Streptomyces lividans.
Appl.Environ.Microbiol., 62, 109-114.
Park JS, Horinouchi S, Beppu T (1991) Characterization of
the leader peptide of an endo-type cellulase produced by analkalophilic Streptomyces strain. Agri.Biol.Chem., 55, 17451750.
Robbins PW, Wirth DF, Hering C (1981) Expression of the
Streptomyces enzyme endoglycosidase H in Escherichia coli.
J.Biol.Chem., 256, 10640-10644.
Schmitt-John T, Engels JW (1992) Promoter constructions
for efficient secretion expression in Streptomyces lividans.
Appl.Microbiol.Biotechnol., 36, 493-498.
Smith MCM, Owen CE (1991) Three in-frame N-terminallydifferent proteins are produced from the repressor locus of the
Streptomyces bacteriophage C31. Mol.Microbiol., 5, 28332844.
Smokvina T, Mazodier P, Boccard F, Thompson CJ,
Guerineau M (1990) Construction of a series of pSAM2based integrative vectors for use in actinomycetes. Gene, 94,
53-59.
Strohl WR (1992) Compilation and analysis of DNA
sequences associated with apparent streptomycete promoters.
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Sun J, Kelemen GH, Fernandez-Abalos JM, Bibb MJ(1999) Green fluorescent protein as a reporter for spatial andtemporal gene expression in Streptomyces coelicolor A3(2).
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Construction of thiostrepton-inducible, high-copy-number
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genome into Streptomyces venezuelae and construction of a
VWB-based integrative vector. Microbiol., 144, 3351-3358.
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Chapter 18

Analysing Streptomyces DNA
CONTENTS
DNA sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393
Alternative and additional Maxam and Gilbert base-specific reactions forsequencing end-labelled DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 394
Sequence analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 395
Identifying protein coding regions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 395
FRAME analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 395
Codon preference . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 397
Hidden Markov model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 398
Codon usage tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 398
Accessing Streptomyces (actinomycete) sequences in the databases . . . . . . . . . . . . . . 398
Design of oligonucleotides for use as probes and PCR primers . . . . . . . . . . . . . . . . . . . . . . . . 402
PCR conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 402

DNA sequencing
The strategies used in sequencing Streptomyces DNA are no different from those employed for
DNA from other organisms, although the high average G+C content (over 70%) often poses problems
that are seldom encountered with DNA samples of more even base composition.
Most sequencing of Streptomyces DNA is accomplished using the dideoxy chain termination
method (Sanger, 1977), although the method of Maxam and Gilbert (1980) still finds application(particularly in the precise determination of transcriptional start sites by S1 nuclease mapping; e.g.
Chakraburtty et al., 1996). A problem that is encountered with both of these methods is the relatively
frequent occurrence of "compressions" within sequence ladders. A region of the sequence fails to resolve
properly and intense bands are observed, often in all tracks, that may represent several compressed
nucleotides. These "compressions" result from regions of secondary structure that fail to denature under
the running conditions, reflecting the high G+C content of Streptomyces DNA. To try to reduce thisproblem, sequencing gels are often run at high temperature, taking care not to crack the glass plates. The
conditions employed need to be determined empirically for each type of gel system, but as a guide an air
temperature next to the glass plates of c. 55o has proved useful in our hands. Systems with temperature
controlled backing plates are convenient and reproducible.
If the chain termination method is employed, substitution of dGTP with dITP will replace the
G:C base-pairs with the weaker I:C; alternatively, dGTP can be replaced with 7-deazaGTP, potentially&KDSWHU$QDO\VLQJ6WUHSWRP\FHV'1$

avoiding the problem altogether. In general, dITP is particularly good at resolving the number of bases
in a compression, while 7-deazaGTP gives better discrimination between alternative bases.
Compressions may also be resolved by including 25-50% formamide in the gel to assist in denaturation;
however, the general use of formamide is to be avoided because the quality of the sequence ladders asa whole is markedly reduced. It is highly desirable, if not obligatory, to determine the sequence of bothstrands of a DNA fragment. This often resolves the problems of secondary structure since thecompressed region generally occurs at slightly different positions on opposite strands.
The provision of sequencing kits and improved DNA polymerases (e.g. T7 DNA polymerase(Sequenase) and Taq polymerase) has greatly facilitated sequencing projects, most of which are now
conducted on automated sequencers using protocols optimised by the manufacturers. Sequencing
reactions are frequently carried out using the Polymerase Chain Reaction (PCR) in a procedure known
as cycle sequencing. Currently, we use the ABI PRISMTM BigDye Terminator Cycle Sequencing Kit
containing AmpliTaqR DNA Polymerase FS and the conditions recommended by the supplier (Perkin
Elmer; p. 418). Good template preparation is crucial for high quality sequencing. Most kit
manufacturers provide protocols for template preparation that have proven effective under their particular
reaction conditions (but see p. 402 for a PCR protocol for template preparation that has proved effectivewith Streptomyces DNA). With automated sequencing, as with manual sequencing, the quality ofsequence obtained with single-stranded DNA tends to be superior to that from double-stranded templates,
but whether this justifies the additional work involved in template preparation is debatable; it may dependon the sequencing strategy. The protocols and strategies used for the S. coelicolor genome sequencing
project can be obtained at http://www.sanger.ac.uk/Teams/Team51/protocols/.
Alternative and additional Maxam and Gilbert base-specific reactions for sequencing end-labelled
DNA
These reactions have been used in addition, or as alternatives, to those shown on p. 529 of thearticle by Maxam and Gilbert (1980) and are given in the same format:
A>C
1 µl carrier DNA (l mg ml–1)
10 µl 32P-labelled DNA
100 µl 1.2N NaOH, lmM EDTA
Total volume = 111 µl
80o, 11 min
2 µl 10 mg ml–1 tRNA
150 µl 1N acetic acid
750 µl ethanol
Continue as G, p. 529 of Maxamand Gilbert (1980).

G+A
1 µl carrier DNA (l mg ml–1)
10 µl 32P-labelled DNA
8 µl 50mM Na citrate, 0.lmM EDTA, pH4
4 µl water
Total volume = 23 µl
80o, 8 min
2 µl 10 mg ml–1 tRNA
250 µl 0.3M Na acetate, pH 7
750 µl ethanol
Continue as G, p. 529 of Maxamand Gilbert (1980).

The G+A reaction has proved more reliable than that of Maxam and Gilbert. The A>C reaction
has frequently proved useful to confirm a sequence when one reaction has not worked well, and is

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

recommended in addition to the standard reactions on p. 529 of Maxam and Gilbert (1980). The
following reaction times have allowed readings of sequences >350 bp (assuming good labelling obviating the need for an intensifying screen - and good gels) but may require local adjustment:
G
G+A
A>C
TC
C

1.5 min
8 min (for the Na citrate protocol)
11 min
2.5 min
2.5 min

The following loading orders have proved most useful when reading the gels:
G G+A A>C T+C C

or

G G+A T+C C A>CSequence analysis
Once a nucleotide sequence has been determined, one of the first objectives is to identify open
reading frames (ORFs) that are likely to encode proteins (here an ORF is defined as a nucleotide
sequence that lacks potential translation stop codons). While in nucleotide sequences of low andintermediate G+C content, the occurrence of an ORF of any substantial length is likely to be highly
indicative of a protein-coding sequence, the paucity of A+T- rich triplets in high G+C streptomycete
DNA frequently produces extensive ORFs in most or all of the six possible reading frames. The frequentlack of readily recognisable ribosome binding sites (Strohl, 1992; p. 386) exacerbates the problem.
Consequently a method is required that can confidently predict protein coding regions, and potential
sequencing errors, particularly those resulting in frameshifts.
Identifying protein coding regions
FRAME analysis
The high G+C content of streptomycete DNA results in a highly biased codon usage (Wright andBibb, 1992). Since there are codons ending in G or C for all 20 amino acids specified by the genetic
code, most codons in streptomycete genes end in G or C (the average G+C content of codon position 3
in 64 streptomycete genes was 92%, with a range of 81-98; M.J. Bibb, unpublished results). In contrast,
there are 12 amino acids whose codons all contain an A or T at position 2; consequently, the G+C content
found at codon position 2 of streptomycete genes generally lies between 40% and 60% (the average for64 genes was 51%, range 44-58%). Finally, since there are seven generally rarely used amino acids thathave only A or T at codon position 1, the G+C content at this site lies between the other two (averagefor 64 genes 70% G+C, range 64-75%). Consequently, protein coding sequences in DNA of high G+C
content are characterised by an asymmetric G+C distribution across codons of the form
intermediate:low:high. (Note that this asymmetry immediately tells us the reading frame and directionof the gene.) This distribution forms the basis of the FRAME program (Bibb et al., 1984) which analyses
sequences using a moving window of triplets, the number of which is defined by the user (usually 50).
The window moves three nucleotides (nt) at a time across the sequence. After each move, the average

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Streptomyces cacaoi beta-lactamase
1440 bases, bandwidth = 50 triplets (1-1440)
N3<
N2<
N1<
N3>
N2>
N1>
100

75

50

25

0
0

100

200

300

400

500

600

700
800
Base Number

900

1000

1100

1200

1300

1400

Fig. 18.1 FRAME analysis of a nucleotide sequence. The extent of the known coding region is shownby an arrow superimposed on the FRAME plot.
G+C content for each of the three triplet positions in the chosen number of triplets ahead (the “window”)
is plotted against the nt number. In Fig. 18.1, the FRAME plot starts in an untranslated sequence andthen encounters a protein coding sequence, BLA, starting at nt 271 in reading frame 1. Note that thethree lines start diverging approximately 150 (50 × 3) nt before the ATG start codon because the window
covers the sequence ahead, and that the non-random G+C distribution “equilibrates” once the trailingedge of the window reaches the start codon at nt 271. Similarly, the three lines start to merge
approximately 150 nt before the end of the coding region (around nt 1070) since that is when the window
begins to encounter sequences beyond the end of BLA; they merge completely around the position (nt1215) where the trailing edge of the window leaves the BLA coding sequence. The three lines
representing the G+C content at each of the triplet positions come to an end at the position when theleading edge of the window reaches the end of the sequence (i.e. 150 nt from the end of the sequence,at nt 1290 in this example).
The reading frame of the ORF can be determined from the relative position of the three lines thatindicate G+C content. In the example shown, the line with intermediate G+C content (corresponding tocodon position 1) is the thinnest line, while the line with lowest G+C content is of intermediate thickness(corresponding to codon position 2). This indicates that the ORF goes from left to right, and that it lies
in reading frame N1>, which is shown by the thinnest line above the FRAME plot going from left toright. Putative ATG and GTG start codons are indicated by large and small >, respectively, and stopcodons by |. If the ORF predicted from the G+C distribution is not accompanied by appropriately
positioned start and stop codons, then there is likely to be an error in the sequence. The effect of a

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Streptomyces cacaoi beta-lactamase with a 1 bp deletion at position 750
1439 bases, bandwidth = 50 triplets (1-1439)
N3<
N2<
N1<
N3>
N2>
N1>
100

75

50

25

0
0

100

200

300

400

500

600

700
800
Base Number

900

1000

1100

1200

1300

1400

Fig. 18.2 As Fig. 18.1 but with a 1 bp deletion artificially introduced at nucleotide position 750.
frameshift mutation in BLA on the FRAME plot is shown in Fig. 18.2, where a single base (nt 750) has
been deleted from the sequence. Note that the reading frame shifts from N1> to N3>, and that the
FRAME plot starts to detect the frameshift 150 nt (3 × 50 triplets) before it occurs in the sequence. The
FRAME plot “re-equilibrates” once the trailing edge of the moving window passes position nt 750. The
numbering of the six reading frames and the corresponding distributions of G+C content across codons
are shown in Fig. 18.3.
Note that the codon usage at the beginning of a gene may not always be typical of the gene asa whole. This may be because codon usage at the beginning of the gene may influence the rate oftranslation initiation and hence the level of gene expression (p. 391). Nevertheless, FRAME analysis
generally gives unambiguous predictions of protein-coding regions, and is particularly useful for
identifying frameshift errors, which with practice can be localised to within a few nucleotides of thesequencing mistake.
The version of FRAME that we use is written in Fortran77, runs on a VAX, and requires thecommercial UNIRAS package for graphical output. A similar version, FRAMEPLOT, has been
implemented by Ishikawa and Hotta (1999) and is available on the internet at
http://www.nih.go.jp/~jan/cgi-bin/frameplot-2.36.pl, but it plots the data in a slightly different mannerfrom that described above. However, it does allow the user to carry out immediate database searcheswith potential sequences identified by FRAMEPLOT, an extremely useful feature. A different graphicalrepresentation of the FRAME analysis is provided by the PC version of Shields et al. (1992).

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Codon preference
An alternative to FRAME analysis for the identification of protein-coding regions is to assesscodon preference using one of the commonly available sequence analysis packages e.g. the
CODONPREFERENCE program found in the UWGCG suite of programs. This can be accomplishedusing standard bacterial codon usage tables found in most of the packages, or by applying codon usage
data derived from streptomycete genes. Although both identify protein-coding regions, use of
Streptomyces codon usage tables generally gives a clearer prediction.
Hidden Markov model
A further strategy for gene identification involves the development of a hidden Markov model.
The method attempts to model codons and their frequencies in genes, as well as patterns found inintergenic regions (e.g. the Shine-Dalgarno motif characteristic of ribosome binding sites) to locate
protein coding genes (for an example of how this was used in E. coli, see Krogh et al., 1994). This
technique is also used by the Sanger Centre to localise protein-coding sequences in the emerging S.
coelicolor genome sequence (p. 424 and below).
Codon usage tables
Codon usage for 100 streptomycete genes (ranging in G+C content from 61% to 80%; M.J. Bibb,
unpublished) is given in Table 18.1. All of the sequences were analysed using FRAME and no apparent
frameshifts were observed.
Updated codon usage tables for many Streptomyces species can be found at
http://www.dna.affrc.go.jp/~nakamura/S.html.
Accessing Streptomyces (actinomycete) sequences in the databases
A genome sequencing project for S. coelicolor is now in progress, with a hoped-for completion
date of late 2000. Details can be found at http://www.sanger.ac.uk/Projects/S_coelicolor/). This projectis based on the ordered cosmid library of Redenbach et al. (1996). Raw data (contigs >1000 nt) are
updated on the web site daily and can be accessed via FTP and interrogated by a dedicated BLAST
server; when the sequence for each cosmid is finished and annotated the information is submitted to the
EMBL database. A comprehensive ACEDB-type database for the project (SCODB) is available (p. 424).
In addition to this genome sequencing project, there are many more streptomycete and actinomycete
sequences in the EMBL (http://www.embl-heidelberg.de/Services/) and Genbank(http://www.ncbi.nlm.nih.gov/) databases. These can be most readily accessed via BLAST databasesearches and Entrez on the internet site of the National Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/Web/Search/). When searching for sequences of homologues of genes ofinterest, perusal of the Mycobacterium leprae, M. tuberculosis and M. bovis genome sequences(http://www.sanger.ac.uk/Projects/M_leprae, /M_tuberculosis and /M_bovis), actinomycetes withrelatively high G+C content, may prove particularly useful, both as a source of genes for hybridisation
probes and for sequence information that can be used in the design of oligonucleotides for use as probes
or PCR primers.

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Reading frame:

N1> N2> N3>
N2< N3< N1<

Sequence position:

1

2

3

4

5

6

.

Triplet position:

1

2

3

1

2

3

.

Distributionof lines
N1>
N1>

N2>
N2>

N3>
N3>

N1<
N1<

N2<
N2<

N3<
N3<

Sequence position:

1

2

3

4

5

6

.

Fig. 18.3. Determination of reading frame from a FRAME plot. The vertical bars to the left indicate
all possible intermediate:low:high G+C distributions and their corresponding reading frames. For
example, the distribution at the top indicates that triplet position 1 has an intermediate G+C content,
position 2 is low, and 3 high. This indicates that the coding sequence is transcribed from left to right,
and that the first codon position corresponds to triplet position 1; the appropriate reading frame isfound in one of the two thinnest horizontal lines, line N1>, above the FRAME plot. The fifth
distribution down has a high G+C content at triplet position 2, low at 3 and intermediate at 1. This
indicates that this coding sequence is transcribed from right to left, and that the first codon position
corresponds again to triplet position 1; the appropriate reading frame is found in one of the twothinnest horizontal lines, line N2<, above the FRAME plot. Appropriately positioned potential
translational start (> or >) and stop codons (|) should then be present on N1> and N2<, respectively.
Reading frames N1> and N1<, N2> and N2<, and N3> and N3<, respectively, occur in the sameregisters, but on opposite strands. To the right are shown diagrammatic representations of the six
possible types of FRAME plot corresponding to each of the reading frames.

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

Table 18.1 Codon usage in 100 Streptomyces genes
Number of codons
F
F
L
L

TTT
TTC
TTA
TTG

14
859
13
78

S
S
S
S

TCT
TCC
TCA
TCG

19
658
40
460

Y
Y
*
*

TAT
TAC
TAA
TAG

35
660
4
15

C TGT
C TGC
* TGA
W TGG

33
241
81
475

L
L
L
L

CTT
CTC
CTA
CTG

61
1147
9
1673

P
P
P
P

CCT
CCC
CCA
CCG

47
724
27
911

H
H
Q
Q

CAT
CAC
CAA
CAG

49
708
50
764

R
R
R
R

CGT
CGC
CGA
CGG

188
1163
95
974

I ATT
I ATC
I ATA
M ATG

45
933
36
506

T
T
T
T

ACT
ACC
ACA
ACG

46
1335
50
618

N
N
K
K

AAT
AAC
AAA
AAG

34
699
43
693

S
S
R
R

AGT
AGC
AGA
AGG

49
455
29
133

V
V
V
V

70
1403
62
972

A
A
A
A

GCT
GCC
GCA
GCG

95
2390
175
1413

D
D
E
E

GAT
GAC
GAA
GAG

93
1892
325
1516

G
G
G
G

GGT
GGC
GGA
GGG

247
1825
254
516

GTT
GTC
GTA
GTG

% synonymous codon usage
F
F
L
L

TTT
TTC
TTA
TTG

1.6
98.4
0.4
2.6

S
S
S
S

TCT
TCC
TCA
TCG

1.1
39.1
2.4
27.4

Y
Y
*
*

TAT
TAC
TAA
TAG

5.0
95.0
4.0
15.0

C TGT
C TGC
* TGA
W TGG

12.0
88.0
81.0
100.0

L
L
L
L

CTT
CTC
CTA
CTG

2.0
38.5
0.3
56.1

P
P
P
P

CCT
CCC
CCA
CCG

2.7
42.4
1.6
53.3

H
H
Q
Q

CAT
CAC
CAA
CAG

6.5
93.5
6.1
93.9

R
R
R
R

CGT
CGC
CGA
CGG

7.3
45.0
3.7
37.7

I ATT
I ATC
I ATA
M ATG

4.4
92.0
3.6
100.0

T
T
T
T

ACT
ACC
ACA
ACG

2.2
65.2
2.4
30.2

N
N
K
K

AAT
AAC
AAA
AAG

4.6
95.4
5.8
94.2

S
S
R
R

AGT
AGC
AGA
AGG

2.9
27.1
1.1
5.2

V
V
V
V

2.8
56.0
2.5
38.8

A
A
A
A

GCT
GCC
GCA
GCG

2.3
58.7
4.3
34.7

D
D
E
E

GAT
GAC
GAA
GAG

4.7
95.3
17.7
82.3

G
G
G
G

GGT
GGC
GGA
GGG

8.7
64.2
8.9
18.2

GTT
GTC
GTA
GTG

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Table 18.1 Continued
Overall usage (frequency per thousand)
F
F
L
L

TTT
TTC
TTA
TTG

0.45
27.51
0.42
2.50

S
S
S
S

TCT
TCC
TCA
TCG

0.61
21.07
1.28
14.73

Y TAT
Y TAC
* TAA
* TAG

1.12
21.13
0.13
0.48

C TGT
C TGC
* TGA
W TGG

1.06
7.72
2.59
15.21

L
L
L
L

CTT
CTC
CTA
CTG

1.95
36.73
0.29
53.57

P
P
P
P

CCT
CCC
CCA
CCG

1.51
23.19
0.86
29.17

H
H
Q
Q

CAT
CAC
CAA
CAG

1.57
22.67
1.60
24.47

R
R
R
R

CGT
CGC
CGA
CGG

6.02
37.24
3.04
31.19

I ATT
I ATC
I ATA
M ATG

1.44
29.88
1.15
16.20

T
T
T
T

ACT
ACC
ACA
ACG

1.47
42.75
1.60
19.79

N
N
K
K

AAT
AAC
AAA
AAG

1.09
22.38
1.38
22.19

S
S
R
R

AGT
AGC
AGA
AGG

1.57
14.57
0.93
4.26

V
V
V
V

2.24
44.93
1.99
31.13

A
A
A
A

GCT
GCC
GCA
GCG

3.04
76.54
5.60
45.25

D
D
E
E

GAT
GAC
GAA
GAG

2.98
60.59
10.41
48.55

G
G
G
G

GGT
GGC
GGA
GGG

7.91
58.44
8.13
16.52

GTT
GTC
GTA
GTG

Nucleotides: 93681; codons: 31227; mol% G+C: 70.9; mol% G: 33.9; mol% C: 37.0;
mol%A: 15.6; mol% T: 13.5.
The amino acid composition of the 100 proteins encoded by the genes in Table 18.1 is shown in
Table 18.2. For a discussion of how changes in base composition can influence the amino acid content
of a protein, see Wright and Bibb (1994).
Table 18.2 Amino acid composition of 100 Streptomyces gene products
Number of residues(out of 31127)% composition

F
L
I
M
V

873
2981
1014
506
2507

2.8
9.6
3.3
1.6
8.1

S
P
T
A

1681
1709
2049
4073

5.4
5.5
6.6
13.1

Number of residues(out of 31127)% composition

Y
H
Q
N
K
D
E

695
757
814
733
736
1985
1841

2.2
2.4
2.6
2.4
2.4
6.4
5.9

C
W
R
G

274
475
2582
2842

0.9
1.5
8.3
9.1

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Design of oligonucleotides for use as probes and PCR primers
The design of degenerate oligonucleotides as hybridisation probes or as PCR primers will clearly
be influenced by the high G+C content of the genome. In practice, where the third codon position showsdegeneracy for all four bases, we incorporate only the appropriate G and/or C, not A and/or T, to reduce
the level of degeneracy. The inclusion of I at such positions tends to yield unacceptably high levels of
spurious hybridisation/annealing. When designing PCR primers, potential mismatches close to the 30 endof the oligonucleotide are avoided if possible to increase the probability of correct annealing. Careshould be taken to avoid inter- and intra-primer complementarity, and potential base-pairing, which are
more likely to occur in G+C rich sequences. Where there is flexibility in the location of the primer on
a genomic sequence, PCR primers that are as close to 50 mol% AT as possible should be designed; thiswill reduce the level of spurious annealing and the chances of inter- and intra-primer complementarity.
Primers should generally be at least 18 bases long to ensure good hybridisation and to reduce thepossibility of hybridisation to spurious secondary sites. A number of computer programs have been
written to assist in primer design (e.g. the program PRIME from the GCG package); see also
http://alces.med.umn.edu/webprimers.html for a primer selection program.

PCR conditions
Several companies now supply PCR kits, with different DNA polymerases and their own
optimised reaction conditions and buffers (the contents of which are often not completely specified). For
many users, purchase of such a kit will prove most convenient. Alternatively, it is possible to purchasee.g. Taq polymerase and make your own buffer. The following reaction conditions are routinely used:
Final concentration or amountPrimers . . . . . . . . . . . . . . . . . . . . . . . . 40 pMol of eachTemplate DNA . . . . . . . . . . . . . . . . . . . . . . . . 10-50 ng
Taq polymerase . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 u
Tris-HCl pH8.3 . . . . . . . . . . . . . . . . . . . . . . . . . . 10mM
KCl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50mM
dNTP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200µM
MgCl2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5mM
Gelatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.1%
Total volume . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 µl

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

Cycle conditions

1.
2.

3.

Denaturation: 95o, 5 min.
PCR - 30 cycles of:denaturation at 95o for 30 sec
primer annealing at Tm-4o for 30 sec (Tm = 4 × (G+C) + 2 × (A+T))
extension at 72o for 1 min.
Final extension: 72o, 10 min.

Most primers produce good signal intensity and minimal noise when the MgCl2 concentration
is approximately 2mM. However, some primers perform better at different magnesium concentrations,and it may be necessary to vary it between 0.5mM and 8mM to obtain optimal results.
Although not always a problem, PCR templates with a high G+C content or stable secondary
structure may amplify inefficiently, if at all. The addition of co-solvents can often alleviate this problem.
Glycerol (5-10%), dimethylsulphoxide (DMSO; 1-10%, often 5%) and formamide (5-10%) have all beenused to good effect with high G+C DNA (Pomp and Medrano, 1991; Wong et al., 1991; Filichkin andGelvin, 1992; Korge et al., 1992; Moreau et al., 1994, 1997). All of these co-solvents lower the Tm of
the DNA template, but may also stimulate or inhibit polymerase activity (depending on the temperatureand the polymerase used).
See the PCR jump station at http://www.horizonpress.com/gateway/per.html for further
information.

Bibb MJ, Findlay PR, Johnson MW (1984) The
relationship between base composition and codon usage inbacterial genes and its use in the simple and reliable
identification of protein coding sequences. Gene, 30, 157166.
Chakraburtty R, White J, Takano E, Bibb MJ (1996)
Cloning, characterization and disruption of a (p)ppGpp
synthetase gene (relA) from Streptomyces coelicolor A3(2).
Mol.Microbiol., 19, 357-368.
Filichkin SA, Gelvin SB (1992) Effect of dimethyl sulfoxide
concentration on specificity of primer matching in PCR.
Biotechniques, 12, 828-830.
Ishikawa J, Hotta K (1999) FramePlot: a new
implementation of the Frame analysis for predicting proteincoding regions in bacterial DNA with a high G plus C
content. FEMS Microbiol.Lett., 174, 251-253.
Korge BP, Gan SQ, Mcbride OW, Mischke D, Steinert
PM (1992) Extensive size polymorphism of the humankeratin-10 chain resides in the C-terminal V2 subdomain due
to variable numbers and sizes of glycine loops.
Proc.Natl.Acad.Sci.USA, 89, 910-914.
Krogh A, Mian IS, Haussler D (1994) A hidden Markov
model that finds genes in Escherichia coli DNA. Nucleic
Acids Res., 22, 4768-4778.
Maxam AM, Gilbert W (1980) Sequencing end-labelled
DNA with base specific chemical cleavages. Meth.Enzymol.,65, 449-560.
Moreau A, Duez C, Dusart J (1994) Improvement of GCrich template amplification by inverse PCR. Biotechniques,17, 232-234.
Moreau A, Duez C, Dusart J (1997) GC-rich template
amplification by inverse PCR. DNA polymerase and solvent
effects. Methods Mol.Biol., 67:47-53, 47-53.
Pomp D, Medrano JF (1991) Organic solvents as facilitatorsof polymerase chain-reaction. Biotechniques, 10, 58-59.
Redenbach M, Kieser HM, Denapaite D, Eichner A,
Cullum J, Kinashi H, Hopwood DA (1996) A set of ordered
cosmids and a detailed genetic and physical map for the 8 Mb
Streptomyces coelicolor A3(2) chromosome. Mol.Microbiol.,21, 77-96.
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing
with chain-terminating inhibitors. Proc.Natl.Acad.Sci.USA,74, 5463-5467.
Shields DC, Higgins DG, Sharp PM (1992) GCWIND - a
microcomputer program for identifying open reading frames
according to codon positional G+C content.
Comput.Appl.Biosci., 8, 521-523.
Strohl WR (1992) Compilation and analysis of DNA
sequences associated with apparent streptomycete promoters.
Nucleic Acids Res., 20, 961-974.
Wong HC, Ting Y, Lin HC, Reichert F, Myambo K, Watt
KWK, Toy PL, Drummond RJ (1991) Genetic organization
and regulation of the xylose degradation genes in
Streptomyces rubiginosus. J.Bacteriol., 173, 6849-6858.
Wright F, Bibb MJ (1992) Codon usage in the G+C-richStreptomyces genome. Gene, 113, 55-65.



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m19tkb.wpd 13 March 2000 Tobias Kieser

Chapter 19

Media, buffers and suppliers
CONTENTS
Agar Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 406
Minimal medium (MM) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 406
Complete medium (CM) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 407
Hickey-Tresner agar (HT agar) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 407
R2 Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 408
R2YE Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 408
R5 Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 409
Mannitol soya flour medium (MS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 409
Supplemented minimal medium, solid (SMMS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 410
MMT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 410
Difco nutrient agar (DNA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 411
Oxoid nutrient agar (ONA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 411
Soft nutrient agar (SNA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 411
L agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 411
Liquid media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 412
Yeast extract-malt extract medium (YEME) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 412
Tryptone soya broth (TSB) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 412
Difco nutrient broth (DNB) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 412
L broth (LB) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 412
Supplemented liquid minimal medium (SMM) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 413
Minimal liquid medium (NMMP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 413
Labelling medium for Streptomyces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 414
2 X YT medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 414
Growth factor supplements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 415
Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 415
P (protoplast) Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 415
T (transformation) buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 416
L (lysis) buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 416
TE Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 417
SM Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 417
1
20 × SSC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 417
Addresses of suppliers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 418

&KDSWHU0HGLDEXIIHUVDQGVXSSOLHUVAgar MediaWe generally prepare agar media and sterilise them in 200 ml (or sometimes 100 ml) quantitiesin 250 ml Erlenmeyer flasks. It saves time if the agar is weighed dry into each flask and the solublecomponents are made up in batches of several litres and added to the agar, because this saves melting theagar before dispensing into the flasks. After sterilisation the flasks can be kept for months and the
medium re-melted just before use. Often sterile solutions of supplementary components are added at thispoint. We prefer plastic foam plugs for the flasks (e.g. 5 x 3.5cm foam bungs from Hospital andLaboratory Supplies), especially because the prepared medium can then be melted rapidly in a microwave
oven (cotton wool plugs ignite in the microwave); otherwise a 90o water bath or small autoclave is used.
The media described below, and the various sterile solutions to add to them, can all be sterilised by
autoclaving at 121o for 15 min, unless otherwise stated.
Agar is a notoriously variable material. This affects such Streptomyces features as antibiotic
production, sporulation and carbon source utilisation. We therefore test a manufacturer’s new batch
rigorously and try to ensure a continuing source of the same batch. We use different agars for different
purposes. For example, for SMMS we currently use 1% Lab M agar (from London Analytical and
Bacteriological Media, Ltd.) whereas for MM we use “Iberian agar” (from T. M. Duché and Sons Ltd.);
this agar gives good sporulation and generally permits the testing of auxotrophic requirements; it is also
inexpensive! For R2 and its variations (R2YE and R5) a particularly strong gel is needed and we use
2.2% Difco Bacto agar (now D1490 from Beckton Dickinson).
Minimal medium (MM) (Hopwood, 1967)

L-asparagine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 g
K2HPO4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 g
MgSO4.7H2O . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 g
FeSO4.7H2O . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.01 g
Glucose (added after autoclaving) . . . . . . . . . . . . . 10 g
Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
Distilled water . . . . . . . . . . . . . . . . . . . . . . . . . . 1000 ml
Dissolve the ingredients, except agar and glucose (or other carbon source), in the
distilled water, adjust to pH7.0-7.2 and dispense 200 ml into 250 ml Erlenmeyer flasks
each containing 2 g agar. Close the flasks and autoclave. At time of use, re-melt themedium and add 4 ml of a 50% solution of glucose to each flask, together with any
required growth factor for auxotrophy at normal levels.
In studies of carbon source utilisation, glucose can be replaced by alternative
carbon sources such as glycerol (5 ml l–1) or arabinose (5 g l–1). L-asparagine is thenreplaced by (NH4)2SO4 (1 g l–1) as nitrogen source to avoid confusion caused by theutilisation of asparagine as carbon source. For optimal aerial growth and sporulation,
mannitol (5 g l-1) is often preferable to glucose as carbon source.

&KDSWHU0HGLDEXIIHUVDQGVXSSOLHUVComplete medium (CM) (Hopwood, 1967)

K2HPO4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
NaCl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 g
MgSO4.7H2O . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 g
DifcoBacto-peptone . . . . . . . . . . . . . . . . . . . . . . . . . 2 g
Difco yeast extract . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g
Difco Casaminoacids . . . . . . . . . . . . . . . . . . . . . . 1.5 g
1L-histidine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 mg
1
L-proline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 mg
1
L-tryptophan . . . . . . . . . . . . . . . . . . . . . . . . . . 50 mg
1
L-tyrosine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 mg
2
Yeast nucleic acid hydrolysate . . . . . . . . . . . . . . . 5 ml
3
Vitamin solution (added after pH adjustment) . . . . . . . 1 ml
Glucose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 g
Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
Distilled water . . . . . . . . . . . . . . . . . . . . . . . . . . 1000 ml
1

These amino acids are added to ensure an adequate supply for relevant auxotrophs; if thesemarkers are not in use, any or all of the amino acids can be omitted.
2
Boil 2 g yeast nucleic acid (e.g. “ribonucleic acid, from yeast”, BDH) in 15 ml 1N HCl for 10
min; boil 2 g yeast nucleic acid in 15 ml 1N NaOH for 10 min; mix the two solutions, adjust to
pH6, filter through filter paper while hot and make up to 40 ml with water. Do not sterilise.
3
Riboflavine 100 mg; nicotinamide 100 mg; p-aminobenzoic acid 10 mg; pyridoxine HCl 50 mg;
thiamine HCl 50 mg; biotin 20 mg; water 100 ml. Autoclave (115o, 10 min). Add after pH
adjustment.

Dissolve all the ingredients, except agar and vitamin solution, but including
glucose, in the distilled water, adjust to pH7.2 with 1N HCl, add vitamin solution andpour 200 ml into 250 ml Erlenmeyer flasks each containing 2 g agar. Close the flasks
and autoclave.
Hickey-Tresner agar (HT agar)

Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g
Yeast extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g
Beef extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g
NZamine A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 g
Dextrin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
CoCl2·6H2O . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 mg
Distilled water . . . . . . . . . . . . . . . . . . . . . up to 1000 ml

&KDSWHU0HGLDEXIIHUVDQGVXSSOLHUV

R2 Medium (Okanishi et al., 1974; Hopwood and Wright, 1978)

Make up the following solution:
Sucrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103 g
K2SO4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.25 g
MgC12.6H2O . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.12 g
Glucose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
Difco Casaminoacids . . . . . . . . . . . . . . . . . . . . . . 0.1 g
Distilled water . . . . . . . . . . . . . . . . . . . . . . . . . . . 800 ml
Pour 80 ml of the solution into 250 ml Erlenmeyer flasks each containing 2.2 g
Difco Bacto agar. Close the flasks and autoclave. At time of use, remelt the medium
and add to each flask the following autoclaved solutions in the order listed:
KH2PO4 (0.5%) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 ml
CaCl2.2H2O (3.68%) . . . . . . . . . . . . . . . . . . . . . . . . 8 ml
L-proline (20%) . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 ml
TES buffer (5.73%, adjusted to pH7.2) . . . . . . . 10 ml
1
Trace element solution . . . . . . . . . . . . . . . . . . . 0.2 ml
NaOH (1N) (unsterilised is OK) . . . . . . . . . . . . 0.5 ml
2
Required growth factors for auxotrophs . . . . 0.75 ml
1

Trace element solution (l–1):
ZnCl2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 mg
FeCl3.6H2O . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200 mg
CuCl2.2H2O . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 mg
MnCl2.4H2O . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 mg
Na2B4O7.10H2O . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 mg(NH4)6Mo7O24.4H2O . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 mg

2

For stock solutions, see p. 415

R2YE Medium (Thompson et al., 1980)

Make up in the same way as R2, but add 5 ml of Difco yeast extract (10%) toeach 100 ml flask at time of use.

&KDSWHU0HGLDEXIIHUVDQGVXSSOLHUV

R5 Medium
This is an alternative way of making R2YE. It obviously saves time at the bench. For most
purposes it seems satisfactory, but there is some folklore suggesting that R2YE made by the traditional
process gives more reproducible results.
Make up the following solution:

Sucrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103 g
K2SO4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.25 g
MgCl2.6H20 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.12 g
Glucose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
Difco Casaminoacids . . . . . . . . . . . . . . . . . . . . . . 0.1 g
Trace element solution . . . . . . . . . . . . . . . . . . . . . . 2 ml
Difco yeast extract . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
TES buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.73 gDistilled water to . . . . . . . . . . . . . . . . . . . . . . . . 1000 ml
Pour 100 ml of the solution into 250 ml Erlenmeyer flasks each containing
2.2 g Difco Bacto agar. Close the flasks and autoclave. At time of use, re-melt themedium and add to each flask in the order listed:
KH2PO4 (0.5%) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 ml
CaCl2.2H2O (5M) . . . . . . . . . . . . . . . . . . . . . . . . 0.4 ml
L-proline (20%) . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 ml
NaOH (1N) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.7 ml
1
Any required growth factors for auxotrophs 0.75 ml
1

For stock solutions, see p. 415

Mannitol soya flour medium (MS) Hobbs et al. (1989). Sometimes referred to as “SFM”.

Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g
Mannitol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g
1
Soya flour . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g
Tap water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1000 ml
1

Use soya flour from a health food shop or supermarket, not the expensive material froma laboratory supplier.

Dissolve the mannitol in the water and pour 200 ml into 250 ml Erlenmeyer
flasks each containing 2 g agar and 2 g soya flour. Close the flasks and autoclave twice(115o, 15 min), with gentle shaking between the two runs.

&KDSWHU0HGLDEXIIHUVDQGVXSSOLHUVSupplemented minimal medium, solid (SMMS)
This is our preferred medium for the production of the S. coelicolor antibiotics. Make up thefollowing solution and adjust to pH7.2 with 5N NaOH.

Difco Casaminoacids . . . . . . . . . . . . . . . . . . . . . . . . 2 g
TES buffer . . . . . . . . . . . . . . . . . 5.73 g (25mM final)
Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1000 ml
Pour 200 ml of the solution into 250 ml Erlenmeyer flasks each containing 3 gof Lab M agar. Close the flasks and autoclave. At time of use, re-melt; add to eachflask:
NaH2PO4+K2HPO4 (50mM each) . . 2 ml (1mM final)
MgSO4 (1M) . . . . . . . . . . . . . . . . . . . 1 ml (5mM final)
Glucose (50% w/v) . . . . . . . . . . . . 3.6 ml (50mM final)
Trace elements (as in SMM) . . . . . . . . . . . . . . . 0.2 ml
Growth factors for auxotrophs (p. 415) . . . . . . 1.5 ml
MMT (for tyrosinase expression and melanin production)

Supplement 200 ml MM with the following sterile solutions:
Difco Casaminoacids (30%) . . . . . . . . . . . . . . . . . . 4 ml
1
Tiger Milk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 ml
L-tyrosine (0.75%) . . . . . . . . . . . . . . . . . . . . . . . . 10 ml
1

Tiger Milk (Per 100 ml)
L-arginine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 750 mg
L-cystine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 750 mg
L-histidine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1000 mg
DL-homoserine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 750 mg
L-leucine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 750 mg
L-phenylalanine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 750 mg
L-proline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 750 mg
Adenine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150 mg
Uracil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150 mg
Nicotinamide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 mg

Sterilise by autoclaving at 115o, 15 min.

&KDSWHU0HGLDEXIIHUVDQGVXSSOLHUV

Difco nutrient agar (DNA)Place 4.6 g Difco Nutrient Agar in each 250 ml Erlenmeyer flask and add 200
ml distilled water. Close the flasks and autoclave.

Oxoid nutrient agar (ONA)

Place 5.6 g of Oxoid Nutrient Agar in each 250 ml Erlenmeyer flask and add 200
ml distilled water. Close the flasks and autoclave. Note that ONA contains 0.5% (w/v)
added NaCl.

Soft nutrient agar (SNA)

Difco Nutrient Broth powder . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 g
Agar, Difco Bacto . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
Distilled water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1000 ml
Autoclave to dissolve the agar. Dispense in suitable containers and re-sterilise
by autoclaving.

L agarAgar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
Difco Bacto tryptone . . . . . . . . . . . . . . . . . . . . . . . 10 g
NaCl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
Glucose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g
Distilled water . . . . . . . . . . . . . . . . . . . . . up to 1000 ml
Dissolve the ingredients, except agar, in the distilled water and pour 200 ml into
250 ml Erlenmeyer flasks each containing 2 g agar. Close the flasks and autoclave.

&KDSWHU0HGLDEXIIHUVDQGVXSSOLHUV

Liquid media
Yeast extract-malt extract medium (YEME)

Difco yeast extract . . . . . . . . . . . . . . . . . . . . . . . . . . 3 g
Difco Bacto-peptone . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
Oxoid malt extract . . . . . . . . . . . . . . . . . . . . . . . . . . 3 g
Glucose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
Sucrose . . . . . . . . . . . . . . . . . . . . . . . 340 g (34% final)
Distilled water . . . . . . . . . . . . . . . . . . . . . up to 1000 ml
After autoclaving, add:
MgCl2.6H2O (2.5M) . . . . . . . . . 2 ml/litre (5mM final)
For preparing protoplasts, also add:1

Glycine (20%) . . . . . . . . . . . . 25 ml/litre (0.5% final)

1

Different Streptomyces strains may need different concentrations of glycine; 0.5% isbest for S. lividans and S. coelicolor.

Tryptone soya broth (TSB)

Oxoid Tryptone Soya Broth powder (CM129 . . . 30 g
Distilled water . . . . . . . . . . . . . . . . . . . . . . . . . 1000 ml

Difco nutrient broth (DNB)

Difco Nutrient Broth powder . . . . . . . . . . . . . . . . . . 8 g
Distilled water . . . . . . . . . . . . . . . . . . . . . . . . . . 1000 ml
L (Lennox) broth (LB)

Difco Bacto tryptone . . . . . . . . . . . . . . . . . . . . . . . 10 g
Difco yeast extract . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
NaCl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
Glucose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g
Distilled water . . . . . . . . . . . . . . . . . . . . . . . . . . 1000 ml

&KDSWHU0HGLDEXIIHUVDQGVXSSOLHUVSupplemented liquid minimal medium (SMM) (Strauch et al., 1991)

PEG 6000 (BDH 6.1% w/v in distilled water) 81.9 ml
MgSO4·7 H2O (24 g l–1) . . . . . . . . . 2.5 ml (5mM final)
TES buffer (0.25M, pH7.2) . . . . . 10 ml (25mM final)
NaH2PO4+ K2HPO4 (50mM each) . . 1 ml (1mM final)
Glucose (50% w/v) . . . . . . . . . . . . . . . . . . . . . . . . . 2 ml
Antifoam 289 (Sigma A 5551) (1% w/v) . . . . . . . . 1 ml
1Trace element solution . . . . . . . . . . . . . . . . . . 0.1 ml
Difco Casaminoacids (20% w/v) . . . . . . . . . . . . . . 1 ml
Total . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 ml
Glycine (20%) . . . . . . . . . . . . . . . 25 ml l-1 (0.5% final)
1

Make a fresh trace element solution (0.1 g l–1 each of ZnSO4.7H2O, FeSO4.7H2O,
MnCl2.4H2O, CaCl2.6H2O and NaCl) every 2-4 weeks; store at 4o.

Each of the solutions, with the exception of the trace element solution (which isfilter-sterilised), is autoclaved separately, then mixed in the order given.

Minimal liquid medium (NMMP) (Hodgson, 1982)
This medium is used to obtain dispersed growth, with specific carbon sources.

Make up the following solution:(NH4)2SO4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 g
Difco Casaminoacids . . . . . . . . . . . . . . . . . . . . . . . . 5 g
MgSO4.72O . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 g
PEG 6000 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 g
1
Minor elements solution . . . . . . . . . . . . . . . . . . . . 1 ml
Distilled water . . . . . . . . . . . . . . . . . . . . . . . . . . . 800 ml
1

Minor elements solution
ZnSO4.72O . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g
FeSO4.7H2O . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g
MnCl2.4H2O . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g
CaCl2, anhydrous . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g

Dispense in 80 ml aliquots and autoclave; then see overleaf.

&KDSWHU0HGLDEXIIHUVDQGVXSSOLHUVAt time of use, add:
2

NaH2PO4/K2HPO4 buffer (0.1M, pH6.8) . . . . . . 15 ml
Carbon source (20%) . . . . . . . . . . . . . . . . . . . . . 2.5 ml
Any required growth factors . . . . . . . . . . . . . . . 2.5 ml
Spore inoculum (a heavy inoculum, usually
pre-germinated: see pp. 50-54)
2

Mix 0.1M solutions of NaH2PO4 and of K2HPO4 to give pH of 6.8.

Labelling medium for Streptomyces

This medium, which contains no purines or pyrimidines, is suitable for in vivolabelling of Streptomyces DNA with radioactive thymidine, and yields mycelium which
lyses easily with lysozyme.
Difco Casaminoacids . . . . . . . . . . . . . . . . . . . . . . 24 g
1
Glycine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
Sucrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340 g
Distilled water to . . . . . . . . . . . . . . . . . . . . . . . . 1000 ml
1

Some Streptomyces strains may need more than the 0.5% glycine which is suitable forS. coelicolor and S. lividans.

Dissolve the components in 500 ml water and make up to 1000 ml. Dispense,and sterilise by autoclaving. After autoclaving, add:
MgCl2.6H20 (2.5 M) . . . . . . . . . . . . . . . . . . . . . 2 ml l–1

2 X YT medium

Difco Bacto tryptone . . . . . . . . . . . . . . . . . . . . . . . 16 g
Difco Bacto yeast extract . . . . . . . . . . . . . . . . . . . . 10 g
NaCl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1000 ml

&KDSWHU0HGLDEXIIHUVDQGVXSSOLHUV

Growth factor supplements
Stock solutions (or suspensions in the case of poorly soluble compounds) are made in water,
sterilised by autoclaving and stored at room temperature (or at 4o for long periods). Add 1.5 ml to 200
ml of growth medium.
CompoundStock solution

Working concentration(mg ml–1)(µg ml–1)

Histidine

10

50

1

Other amino acids

7.5

37

Adenine, guanine, thymine, uracil

1.5

7.5

Vitamins

0.1

0.5

1

We use cystine for cysteine auxotrophs.

Buffers
P (protoplast) buffer (Okanishi et al., 1974; Hopwood and Wright, 1978)

Make up the following basal solution:
Sucrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103 g
K2SO4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.25 g
MgCl2.6H20 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.02 g
1
Trace element solution . . . . . . . . . . . . . . . . . . . . . 2 ml
Distilled water to . . . . . . . . . . . . . . . . . . . . . . . . . 800 ml
1

See p. 408 (the same as in R2 and R2YE)

Dispense in 80 ml aliquots and autoclave. Before use, add to each flask in order:
KH2PO4 (0.5%) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 ml
CaCl2.2H20 (3.68%) . . . . . . . . . . . . . . . . . . . . . . . 10 ml
TES buffer (5.73%, adjusted to pH7.2) . . . . . . . 10 ml&KDSWHU0HGLDEXIIHUVDQGVXSSOLHUV

T (transformation) buffer (Thompson et al., 1982)

Mix the following sterile solutions:
Sucrose (10.3%) . . . . . . . . . . . . . . . . . . . . . . . . . . 25 ml
Distilled water . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 ml
1
Trace element solution . . . . . . . . . . . . . . . . . . . 0.2 ml
K2SO4 (2.5%) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 ml
1

See p. 408 (the same as in R2 and R2YE)

To 9.3 ml of the above solution add:
CaCl2 (5M) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 ml
1
Tris-maleic acid buffer . . . . . . . . . . . . . . . . . . . 0.5 ml
1Make up a 1M solution of Tris and adjust to pH8.0 by adding maleic acid.

For use, add 3 parts by volume of the above solution to 1 part by weight of PEG
1000, previously sterilised by autoclaving.

L (lysis) buffer (Thompson et al., 1982)

Mix the following sterile solutions:
Sucrose (10.3%) . . . . . . . . . . . . . . . . . . . . . . . . 100 ml
TES buffer (5.73%, adjusted to pH7.2) . . . . . . 10 ml
K2SO4 (2.5%) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 ml
1Trace element solution . . . . . . . . . . . . . . . . . . . 0.2 ml
KH2PO4 (0.5%) . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 ml
MgCl2·6H2O (2.5M) . . . . . . . . . . . . . . . . . . . . . . . 0.1 ml
CaCl2 (0.25M) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 ml
1

See p. 408 (the same as in R2 and R2YE)

This stock solution keeps indefinitely. Just before use dissolve lysozyme in asample of the solution at a concentration of 1 mg ml-1 and sterilise by filtration.

&KDSWHU0HGLDEXIIHUVDQGVXSSOLHUVTE buffer

Concentration

Suggested stock
solution

Volume of stock
solution per litre

Tris-HCl

10mM

2M (pH8)

5 ml

Sodium EDTA

1mM

0.25M (pH8)4 ml

Mix the stock solutions and make up to volume with distilled water. If needed
sterile, autoclave at 10× concentration and dilute with sterile water.

SM buffer
Note that this buffer, for handling phages (Chapters 12 and 13) differs from standard SM buffer.

Tris-HCl (1M, pH7.5) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 ml
Mg2SO4 (1M) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 ml
NaCl (5 M) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 ml
Gelatine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g
Distilled water to . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1000 ml
Dissolve the gelatine by autoclaving in 10 ml of water and add to the othercomponents. Sterilise by autoclaving.

1

20 × SSC

NaCl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175.32 g
Trisodium citrate . . . . . . . . . . . . . . . . . . . . . . . 88.23 g
Distilled water to . . . . . . . . . . . . . . . . . . . . . . . 1000 ml
1

20 × SSC has 3M NaCl and 0.3M sodium citrate. It is usually diluted for use as e.g.
1 × SSC, 0.1 × SSC (1 × SSC has 0.165M Na+).

&KDSWHU0HGLDEXIIHUVDQGVXSSOLHUV

Addresses of suppliers(See http://www.microbiol.org/vlmicro/vl_prod.htm for other suppliers)
ABI PRISM TM BigDye Terminator Cycle Sequencing Kit
http://www.pebio.com/ab/about/dna/dnaseq/bdterm.html
Agar
Difco Bacto
Beckton Dickinson Microbiology Systems, Sparks, MD21152, USA (http://www.bdms.com/)
Iberian
T.M. Duché and Sons Ltd, Ford Lane, Pendleton, Manchester M6 6PD
Lab M
London Analytical and Bacteriological Media Ltd, Topley House, Wash Lane, Bury BL9 6AY, UK
AmpliTaq Gold
http://www.pecorporation.com/news/pcr05.html
Bead-beater
Bespoke Products, Bartlesville, OK, USA (http://www.stratech.co.uk/beadbeat.html)
In UK: Stratech Scientific Ltd, 61-63 Dudley Street, Luton, Befordshire LU2 0NP, UK.
“Bijou” bottles (7 ml glass bottles with polypropylene caps: BJL01)
Astell Scientific, Powerscroft Road, Sidcup, Kent, UK.
Cellophane discs (75 mm diameter 352P)
Cannings Packaging Ltd, Avonmouth Way, Avonmouth, Bristol BS11 9D2, UK.
Dynabeds DNA DIRECTTM kit
http://www.dynal.no/DynalWeb.nsf/htmlmedia/molecular.biology.html
In UK: Dynal UK, 10 Thursby road, Croft Business Park, Bromborough, Wirral, Merseyside L61 3PW,
UK.
Easi-GridTM
Jencons, Cherrycourt Industrial Estate, Stanbridge Road, Leighton Buzzard, Bedfordshire LU7 8UA.
(http://www.jencons.co.uk/)
Fluorescent-coupled antibodies
Jackson ImmunoResearch Laboratories, P.O. Box 9, 872 West Baltimore Pike, West Grove, PA 09390,
USA (http://www.jacksonimmuno.com/). In UK: http://www.stratech.co.uk/jackson.htm

&KDSWHU0HGLDEXIIHUVDQGVXSSOLHUVFoam bungs
Hospital and Laboratory Supplies, PO Box 25, Ilford, Essex, UK.
High Speed Homogeniser System and zirkonium-silica beads
Cole-Parmer Instruments Co, 625 East Bunker Court, Vernon Hills, IL 60061, USA (http://www.coleparmer.com/main.htm). In UK: Unit 3, River Brent Business Park, Trumpers Way, Horwell, London
W7 2BR, UK.
JunlonHoneywell and Sein Ltd, Greenfield House, 69/73 Manor Road, Wallington, Surrey SM6 0BP, UK.
Lyophiliser
Edwards High Vacuum International, Manor Royal, Crawley, Sussex W RH10 2LW, UK
PAP hydrophobic marking pen
Electron Microscopy Sciences, P.O. Box 251, 34 Morris Road, Fort Washington, PA 19034, USA(http://www.emsdiasum.com/ems/). (Also supplier of gelatin capsules, etc.)
Petri dishes (square: NUNC)
http://nunc.nalgenunc.com/
Phosphor-screen
Molecular Dynamics: http://www.moleculardynamics.com/
Polyethylene glycol (PEG)
NBS Biologicals, 14 Tower Square, Huntingdon, Cambridgeshire PE18 7DT, UK.
Potter homogeniser
e.g. http://www.glascol.com/
Slow Fade Kit
Molecular Probes: http://www.molecularprobes.com
Spin X filter system
COSTAR, Cambridge MA 02139, USA.
Stainless steel springs
Alliance Spring Co., 44-46 Queensland Road, London N7 7AR UK.
“Universals” (25 ml glass bottles with polypropylene caps: BJ L004)
Astell Scientific, Powerscroft Road, Sidcup, Kent, UK.

&KDSWHU0HGLDEXIIHUVDQGVXSSOLHUVUltra-Turrax Turbomixer
Diatech Equipment Co, P.O. Box 58468, Cincinatti, OH 45258, USA(http://www.diatechequipment.com/).
Velvet (for replica plating)E.g. Braemar Midnight Blue Velvet, untreated (no water or stain-repellant chemicals), 122 cm wide.
Lister Home Furnishings, Manningham Mills, Heaton Road, Bradford BD9 48H, UK.

Hobbs G, Frazer CM, Gardner DCJ, Cullum JA, Oliver
SG (1989) Dispersed growth of Streptomyces in liquid
culture. Appl.Microbiol.Biotechnol., 31, 272-277.
Hodgson DA (1982) Glucose repression of carbon source
uptake in Streptomyces coelicolor A3(2) and its perturbationin mutants resistant to 2-deoxyglucose. J.Gen.Microbiol.,
128, 2417-2430.
Hopwood DA (1967) Genetic analysis and genome structurein Streptomyces coelicolor. Bacteriol.Rev., 31, 373-403.
Hopwood DA, Wright HM (1978) Bacterial protoplast
fusion: recombination in fused protoplasts of Streptomyces
coelicolor. Mol.Gen.Genet., 162, 307-317.
Okanishi M, Suzuki K, Umezawa H (1974) Formation andreversion of streptomycete protoplasts: cultural conditions

and morphological study. J.Gen.Microbiol., 80, 389-400.
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular
Cloning: a Laboratory Manual, 2nd ed. Cold Spring Harbor,
Cold Spring Harbor, NY.
Strauch E, Takano E, Baylis H, Bibb MJ (1991) The
stringent response in Streptomyces coelicolor A3(2). Mol.
Microbiol., 5, 289-298.
Thompson CJ, Kieser T, Ward JM, Hopwood DA (1982)
Physical analysis of antibiotic-resistant genes from
Streptomyces and their use in vector construction. Gene, 20,
51-62.
Thompson CJ, Ward JM, Hopwood DA (1980) DNA
cloning in Streptomyces: resistance genes from antibioticproducing species. Nature, 286, 525-527.



PWNEZSG$SULO7RELDV.LHVHU

Chapter 20

Genome maps and genetically marked strains
CONTENTS
S. coelicolor A3(2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 423
Genetic/physical map . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 423
Genetically marked strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 423
Genome sequencing project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 424
S. lividans 66 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 425
Genetic/physical map . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 425
Genetically marked strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 425
Genetic differences between S. coelicolor A3(2) and S. lividans 66 . . . . . . . . . . . . . . . . . . . . . . 426
S. griseus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 430
S. ambofaciens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 433
S. rimosus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 433
Plasmids SCP1 and SLP2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 433

We have provided genetic/physical maps of the chromosomes of S. coelicolor A3(2), S. lividans
66, S. griseus IFO 3237, S. ambofaciens DSM 40697 and S. rimosus R6. These appear to be the only
species for which such information exists. S. coelicolor A3(2) is given prominence because of the more
extensive map data available, its status as the academic “model” strain for much genetic work, and toprovide a background for a genome sequencing project currently in progress. We have tried to collectdata for the other species, but realise that our coverage of them is inevitably incomplete, especially forthe last three, because they are not extensively studied at Norwich. For them, we have not attempted tolist any useful, genetically marked strains.
We also include a restriction map of the large, linear SCP1 plasmid of S. coelicolor A3(2), bothbecause of its special interest as a rare example of a Streptomyces plasmid that encodes antibiotic
production, and because it represents 4% of the genome; and of SLP2, because it shares an end with theS. lividans chromosome.

&KDSWHU*HQRPHPDSVDQGJHQHWLFDOO\PDUNHGVWUDLQV

C

960

E 690

I 290 L140
H

G480

115

M

110

C1650

G

F610

280

J200
E
820

Streptomyces
coelicolor

B

1770

"IS1373" bldG

H

8 Mb

380

Q
10

D900

A

A

2100

1500

D820
OK
16

190

N
45

F530
P11

B 1300Fig. 20.1 Combined genetic and physical map of the S. coelicolor M145 chromosome. The positionsof markers on the outside of the circle come from their locations on the cosmid contig (Redenbach etal., 1996), assuming equal average lengths for each segment (unique or overlapping segment). Markers
inside the circle have been mapped only genetically (Hopwood and Kieser, 1990); their appropriate
positions relative to the physically mapped markers were determined by interpolation. Sizes of the AseIand the DraI fragments are in kb. , chromosomal ‘telomeres’ (Lin et al., 1993); ñ, oriC. Reprinted
with permission from Redenbach et al. (1996); copyright (1996) Blackwell Sciences Ltd.

&KDSWHU*HQRPHPDSVDQGJHQHWLFDOO\PDUNHGVWUDLQV

S. coelicolor A3(2)Genetic/physical map
The genetic map evolved over more than 30 years before being converted to a combined
genetic/physical map by the PFGE studies of Kieser et al. (1992). The circular map was converted toa linear one by the studies of Lin et al. (1993). Much more detail was added by the construction of acontig of overlapping cosmid clones and the physical mapping of many genes to the contig by Redenbach
et al. (1996). This version of the map is in Fig. 20.1. With the progress of the sequencing project for
the S. coelicolor A3(2) chromosome (see below) this map is becoming increasingly out of date, but it
may nevertheless be useful as a reference point.
Genetically marked strains
There are thousands of genetically marked derivatives of S. coelicolor A3(2). Table 20.1
includes a series of commonly used strains, with three different chromosomal genotypes and carrying
the SCP1, SCP2 and SCP2* plasmids in various combinations.
Other selected strains are listed below.
1258
2708
2709
J1501
J1508
J1700
J1681
J1929
M480
CH999
M510
M511
M512
M520
M521
M522

proA1 hisC9 argA1 cysD18 uraA1 strA1 NF (SCP2*?)
proA1 hisA1 argA1 cysD18 uraA1 SCP1– SCP2–
proA1 hisA1 argA1 cysD18 uraA1 strA1 SCP1– SCP2–
hisA1 uraA1 strA1 pgl-1 SCP1– SCP2–
hisA1 uraA1 strA1 pgl-1 NF SCP2–
hisA1 uraA1 strA1 pgl-1 bldA39 SCP1– SCP2– (from J1501)
hisA1 uraA1 strA1 pgl-1 bldA::ermE SCP1– SCP2– (from J1501)
pglY16 SCP1– SCP2– (from M145)
glkA SCP1– SCP2– (from M145)
proA1 argA1 redE60 act::ermE SCP1– SCP2–
redD (from M145)
actII-ORF4 (from M145)
actII-ORF4 redD (from M145)
redD (from M600)
actII-ORF4 (from M600)
actII-ORF4 redD (from M600)

The first three strains, which carry a standard set of markers, are often useful for preliminary mappingof a new mutation. Other strains, carrying further markers, are then chosen for more precise mapping,depending on the results of the preliminary crosses. Some other useful strains that have been used
repeatedly carry mutations in the pgl (= C31S), glkA, bldA and/or pathway specific antibiotic regulatory
genes (actII-ORF4 for actinorhodin and redD for undecylprodigiosin, the Red antibiotic).



&KDSWHU*HQRPHPDSVDQGJHQHWLFDOO\PDUNHGVWUDLQV

Table 20.1 Sets of S. coelicolor strains of different plasmid status
Chromosomal markers
Plasmid status
None

hisA1 uraA1 strA1

argA1 proA1 cysD18

SCP1– SCP2–

1

M145, M600,
MT1101

2

M130

M124

SCP1– SCP2+

J800

1190

A700

SCP1– SCP2*

M144

M110

M107

SCP1+ SCP2–

J841

M146

M138

SCP1+ SCP2+

1147

104

2278

SCP1+ SCP2*

-

M111

M108

NF (SCP2+)

-

-

2612

3

J801

A317

-

NF (SCP2*?)

1

These are three independently-derived, prototrophic, plasmid-free strains withsomewhat different physiological characteristics. M145 is the source of the DNA forthe cosmid library (Redenbach et al., 1996) and hence for the genome sequencing
project. M600 has been used in Norwich (e.g. Chakraburtty and Bibb, 1997) and
MT1101 in Manchester (Colin Smith, personal communication).
2
M130 is pglts
3
Most NF strains in the culture collection are SCP2*Genome sequencing projectA project to sequence the entire S. coelicolor chromosome, funded by the UK Biotechnology andBiological Sciences Research Council and the Wellcome Trust, began on 1 August, 1997 and should becomplete by late 2000. This is a cosmid-by-cosmid project, using the clones that form the ordered libraryof Redenbach et al. (1996), with additional cosmids to fill gaps and deal with a few problems concerning
cosmid overlaps that have been identified. The cosmids are curated at the John Innes Centre by Helen
Kieser (helen.kieser@bbsrc.ac.uk) and sequencing and annotation are done at the Sanger Centre on the
Wellcome Genome Campus at Hinxton, Cambridge. Each cosmid is sequenced first by a “shotgun”
phase, in which up to 1000 M13 clones are sequenced at random. This is followed by a directed phasein which any gaps are closed and the sequence is finished to an accuracy of well over 99.99%. Finishedcosmids are analysed for protein coding and other sequences, which are annotated using the Artemis toolof the Sanger Centre and deposited in the public databases. Before this stage, all sequences >1000 bp
are updated daily on the website for the project (http://www.sanger.ac.uk/Projects/S_coelicolor) which
includes a dedicated BLAST server that can be used to interrogate the entire sequence, finished and&KDSWHU*HQRPHPDSVDQGJHQHWLFDOO\PDUNHGVWUDLQV

unfinished. The sequences are available by FTP. A graphical overview of each cosmid is accessible
from the project home page.
A customised ACEDB-type database for the project, called SCODB, has been developed byAndreas Wietzorrek and is constantly updated. ACEDB is a mouse-driven program for displaying andanalysing most types of “objects” encountered in molecular biology, such as genes, genetic maps, contig
and other physical maps, clones, sequences, pictures and publications. It is primarily developed to run
under the Unix operating system, using X-Windows for graphics. In order to use SCODB you need the
ACEDB software and the SCODB data. Both are available by anonymous FTP. Contact
http://www.sanger.ac.uk/Software/Acedb. This site contains versions for various types of UNIXcomputers (“platforms”). Currently, SCODB uses version 4_5 which is not the latest. The program
requires relatively little space. The compressed binary files (e.g. bin.alpha.4_5.tar.Z) are 6-10Mb. Pleasecontact your computer support for local advice. Feel free to contact Andreas Wietzorrek at the John
Innes Centre, Norwich (wietzorr@bbsrc.ac.uk) for advice specific to SCODB.

S. lividans 66
Genetic/physical map
Fig. 20.2 shows the chromosome map of the ZX7 derivative of S. lividans 66, compared with thatof S. coelicolor A3(2) and with the S. lividans wild-type.
Genetically marked strains
Here is a short list of some useful S. lividans derivatives. The list includes four prototrophs with
SLP2 and SLP3 in all four combinations, and a group of SLP2– SLP3– strains carrying chromosomal
markers. JT46 carries the rec-46 mutation which reduces inter-plasmid recombination and can stabilise
recombinant clones (Tsai and Chen, 1987). ZX7 carries a c. 100 kb deletion that removes the dnd locus
responsible for the DNA methylation that renders S. lividans DNA susceptible to in vitro degradation(Zhou et al., 1988), as well as the determinant of resistance to phage HAU3. ZX64 is a well-sporulatingstrain that is an excellent recipient for DNA during transformation or conjugation; the dndA locus isdisrupted by insertion of aadA (spc, str). TK503 carries Tn4560 in the chromosome ready for delivery
onto plasmids. M252 is a chloramphenicol-sensitive strain used for the expression of cloned
chloramphenicol resistance genes (Bibb and Cohen, 1982).
1326
TK21
TK64
3198
TK79
ZX7
ZX64

SLP2+ SLP3+
TK19 SLP2– SLP3+
TK20 SLP2+ SLP3–
SLP2– SLP3+
TK23 spc-1 SLP2– SLP3–
TK24 str-6 SLP2– SLP3–
pro-2 str-6 SLP2– SLP3–
TK54 his-2 leu-2 spc-1 SLP2– SLP3–
his-2 leu-2 ura-6 ath-8 str-6 SLP2– SLP3–
ath-8 leu-2 rib-9 SLP2– SLP3–
JT46 pro-2 str-6 rec-46 SLP2– SLP3–
S––
pro-2 str-6 rec-46 dnd HAU3 SLP2 SLP3
pro-2 str-6 rec-46 dndA::aadA
TK503 Tn4560
M252 cmlS&KDSWHU*HQRPHPDSVDQGJHQHWLFDOO\PDUNHGVWUDLQV

rrn
gyl
rrn

E3 485
B

L

I 290

975

140

I2145

G 480

H

M

115

E 690

C

960

110

C1650

G

280

VI 150

IV
700

D

III

V

J

1700

ura-6

his-2
met-7
leu-2
pro-2

Class 1 AUDs
AUD 2 ( mer )

615

F610

H1

200

300

C
710

E

820

225

S. coelicolor
B

ZX7

30

VII

Linear genomesc. 8000 kb

1770

G
270

II

H

380

dnaA

S. lividans dndA
(non-modified DNA,stable during PFGE)

IS117

leuA

rrn
whiB
bldA
galK

ZX7

E2 5152500

cysC,D
red
bldB
Ia
whiG
1400
whiE

cys-8
spc-1
str-6

145

I1

Q10

A

A

2100

1500

Aa
1300

ilv-3

E1
rrn

Ib

D

525

Aa

1300

1400

argG
Class 2 AUD"AUD 5.7" = AUD 1

D820

900

IS117

L

15

16

F

O

F 530

K

190

355

N P

11

K39

B1300

10

45

H2

225

80

J

70

M

J2

A1000
b

2

N

actIII

S. lividans 66(wild-type)

dndA
A1000
b

φ HAU3 R

Fig. 20.2. Physical map of the chromosome of S. lividans strain ZX7 compared with that of S. coelicolor
A3(2). The outer arc shows the AseI-A region of the S. lividans wild-type (wt) chromosome.

Genetic differences between S. coelicolor A3(2) and S. lividans 66
Table 20.2 lists some of the significant differences between these two closely related strains.

&KDSWHU*HQRPHPDSVDQGJHQHWLFDOO\PDUNHGVWUDLQVTable 20.2 Genetic differences between S. coelicolor A3(2) and S. lividans 66
Chromosome ends
S. coelicolor$60 kb TIR (Kieser et al., 1992)
S. lividans
31 kb TIR (includes Tn4811)

Amplifiable DNA elements/unstable genes
S. coelicolor
AUD1: 1 kb-4.7 kb-1 kb repeat; because of the lack of the 4.7 kb duplication, AUD1 is usually notamplified
AUD2: there is a mercury resistance in some S. coelicolor strains but only one copy of IS1373, nota complete AUD2; amplification has not been observed (Nakahara et al., 1985)
AUD3: sequences homologous to AUD3 are present in S. coelicolor (Kieser et al., 1992) but
amplification of AUD3 has never been observed
AUD4: not present in S. coelicolor (J. Altenbuchner, personal communication)
Unstable chloramphenicol resistance (Freeman and Hopwood, 1978; Flett and Cullum, 1987)
Unstable Arg– phenotype (Flett et al., 1987)
S. lividans
AUD1: 1 kb-4.7 kb-4.7 kb-1 kb repeat (Volff et al., 1996)
AUD2: 92 kb element encoding mercury resistance; direct repeats at the end are formed by
IS1373; the element is unstable and is deleted (along with SLP3) in SLP3– strains (Eichenseer and
Altenbuchner, 1994; Volff and Altenbuchner, 1997)
AUD3: 24 kb amplifiable class I element (Altenbuchner et al., 1988)
AUD4: 8.2 kb amplifiable class I element (Altenbuchner et al., 1988; AF072709)
4.3 kb class I element (Betzler et al., 1997)
AUDType1: 70 kb region within 300 kb from one chromosomal end in which class I amplifications
arise (Rauland et al., 1995)
RES1 amplification and deamplification are connected to chloramphenicol resistance versus
sensitivity (Danilenko et al., 1986)
Unstable chloramphenicol resistance (Dittrich et al., 1991)
Unstable tetracycline resistance (Dittrich and Schrempf, 1992)
Unstable argG (Altenbuchner and Cullum, 1984)(continued)



&KDSWHU*HQRPHPDSVDQGJHQHWLFDOO\PDUNHGVWUDLQV

Table 20.2 (continued)
Mobile genetic elements
S. coelicolor
IS110 (1.55 kb): at least three copies, of which two have been mapped to the chromosome; J1501
lacks an unmapped copy (Chater et al., 1985)
IS117 (the 2527 bp mini-circle): two integrated copies, together with the CCC form at #0.1 copy
per chromosome; J1501 has only one copy (Lydiate et al., 1989)
IS118 (c. 1 kb): $ 2 copies in chromosome (variable between strains) and one copy on SCP2 (T.
Kieser, unpublished)
IS281-like element (1.4 kb): one copy (Sladkova, 1986)
IS466 (1628 bp): two copies in the chromosome and one on SCP1 (Kinashi et al., 1992)
S. lividans
IS113 (2 kb): one copy (Henderson, 1988)
IS493 (1.6 kb): three copies (Solenberg and Burgett, 1989)
IS281-like element (1.4 kb): one copy (Sladkova, 1986)
Tn4811 (5.4 kb): one copy in each TIR of the chromosome and one on SLP2 (Chen et al., 1993)
IS1372 (1304 bp element): five to seven copies (Fischer et al., 1996)
IS1373 (846 bp element): two copies flanking mercury resistance of AUD2 (Volff and
Altenbuchner, 1997)

DNA modification that renders DNA subject to in vitro degradation
S. coelicolor
No
S. lividans
Yes. A mutant (ZX1), and derivatives of it, lack the dnd locus (Zhou et al., 1988)

Plasmids
S. coelicolor
SCP1: 350 kb, linear low copy number (Kinashi et al., 1987)
SCP2: 31.4 kb, circular, low copy number; SCP2* is a mutant of SCP2 with enhanced chromosome
mobilising ability (Bibb and Hopwood, 1981)
SLP1INT: 17.2 kb, integrated plasmid (Bibb et al., 1981; Omer et al., 1988); may be responsible forlow level recombination between SCP1– SCP2– strains
SLP4: unknown structure (Hopwood et al., 1983)(continued)

&KDSWHU*HQRPHPDSVDQGJHQHWLFDOO\PDUNHGVWUDLQV

Table 20.2 (continued)
S. lividans
SLP2: 50 kb, linear, low copy number (Chen et al., 1993)
SLP3: unknown structure (Hopwood et al., 1983). Loss of SLP3 in e.g. TK20 and TK21 (and theirderivatives) is correlated with a large deletion that also removes AUD2

Restriction-modification systems
S. coelicolor
A very strong methylation-dependent restriction system is present. DNA from the non-methylating
ET12567 strain of E. coli (dam– dcm– hsdS–) can be introduced into S. coelicolor (MacNeil, 1988)

S. lividans
There is a weak methylation-dependent restriction (MacNeil, 1988; Orekhov et al., 1989)

Relations with phages
S. coelicolor
Shows “phage growth limitation” (Pgl+) for C31 (Chinenova et al., 1982). C31 does not form
plaques on wild-type S. coelicolor A3(2), but lysogenisation is possible. J1501 is one of many
Pgl– derivatives. M130 is temperature-sensitive for Pgl (Laity et al., 1993; Bedford et al., 1995).
C31 lysogens are very stable. Sensitive to HAU3.

S. lividans
Pgl–. C31 lysogens (at least with KC300) are rather unstable. Resistant to HAU3; mutant ZX1(dnd–) is sensitive (Zhou et al., 1994)

Photoreactivation of UV lesions
S. coelicolor
No (Harold and Hopwood, 1970)
S. lividans
Yes (Harold and Hopwood, 1970)(continued)



&KDSWHU*HQRPHPDSVDQGJHQHWLFDOO\PDUNHGVWUDLQV

Table 20.2 (continued)
Antibiotics known to be produced
S. coelicolor
Actinorhodin and -actinorhodin (ACT); undecylprodigiosin and three other prodiginines (RED);
calcium-dependent antibiotic (CDA); methylenomycin when SCP1 is present (pp. 93-97)
S. lividans
Same as S. coelicolor but at least the first three are produced only under particular nutritional
conditions

Diffusible agarase (Dag)
S. coelicolor
Yes, except in SCP1NF strains: insertion of NF caused a deletion of DNA including dagA (Kendall
and Cullum, 1984; Kieser et al., 1992)
S. lividans
No
Improved sporulation on R2YE medium with added Cu2+ (2µM)
S. coelicolor
No
S. lividans
Yes (T. Kieser and R. E. Melton, unpublished)

Inducible MLS resistanceS. coelicolor
Not known
S. lividans
Yes (Jenkins et al., 1989)

S. griseus
As mentioned on p. 32, several different streptomycin-producing strains of S. griseus have beenused in genetic work. Fig. 20.3 shows a genetic/physical map of the chromosome of a derivative ofstrain IFO3237.

&KDSWHU*HQRPHPDSVDQGJHQHWLFDOO\PDUNHGVWUDLQV





&KDSWHU*HQRPHPDSVDQGJHQHWLFDOO\PDUNHGVWUDLQV

&KDSWHU*HQRPHPDSVDQGJHQHWLFDOO\PDUNHGVWUDLQV
rrn
RP2

rrn



leuA
rrn

dnaA
rrn

G

H
V
VI

C2

I
VII

IV

RP3

B
III

A
F

att -pSAM2
I
K

II

D

C1
E

recA
otc

J

Ase I Dra I

J

Dra I Ase I

Fig. 20.5 Physical map of the 8 Mb S. rimosus R6 chromosome. RP2 and RP3 are prophages; otc is thecluster of genes for oxytetracycline biosynthesis (Pandza et al., 1977). Kindly supplied by J. Cullum.

S. ambofaciens
Physical maps of the chromosomes of three strains of S. ambofaciens were constructed by
Leblond et al. (1996). Fig. 20.4 shows the map for S. ambofaciens DSM 40697 and its comparison with
S. coelicolor A3(2).

S. rimosus
Two isolates of S. rimosus, the producer of oxytetracycline, have been studied. A chromosomal
linkage map was established for the Pfizer strain by Friend and Hopwood (1971) and for the Zagreb
strain by Alaûevií et al. (1973). Fig. 20.5 shows a physical map of the chromosome of S. rimosus R6,
the strain studied by the Zagreb group.

Plasmids SCP1 and SLP2
Physical maps of these two plasmids are in Figs. 20.6 and 20.7.

&KDSWHU*HQRPHPDSVDQGJHQHWLFDOO\PDUNHGVWUDLQV

&KDSWHU*HQRPHPDSVDQGJHQHWLFDOO\PDUNHGVWUDLQV





&KDSWHU*HQRPHPDSVDQGJHQHWLFDOO\PDUNHGVWUDLQVAlaûevií M, Strašek-Vešligaj M, Sermonti G (1973) The
circular linkage map of Streptomyces rimosus.
J.Gen.Microbiol., 77, 173-185.
Altenbuchner J, Cullum J (1984) DNA amplification andunstable arginine gene in Streptomyces lividans 66.
Mol.Gen.Genet., 195, 134-138.
Altenbuchner J, Eichenseer C, Brüderlein M (1988) DNA
amplification and deletion in Streptomyces lividans. In
Okami, Y., Beppu, T, and Ogawara, H. (eds.), SeventhInternational Symposium on Biology of Actinomycetales.
Japan Scientific Press, Tokyo, pp. 139-144.
Bedford DJ, Laity C, Buttner MJ (1995) Two genes
involved in the phase-variable C31-resistance mechanism ofStreptomyces coelicolor A3(2). J.Bacteriol., 177, 4681-4689.
Betzler M, Tlolka I, Schrempf H (1997) Amplification of a
Streptomyces lividans 43 kb DNA element causes
overproduction of a novel hypha- and vesicle-associatedprotein. Microbiol., 143, 1243-1252.
Bibb MJ, Cohen SN (1982) Gene expression in
Streptomyces: construction and application of promoter-probe
plasmid vectors in Streptomyces lividans. Mol.Gen.Genet.,
187, 265-277.
Bibb MJ, Hopwood DA (1981) Genetic studies of thefertility plasmid SCP2 and its SCP2* variants in Streptomyces
coelicolor A3(2). J.Gen.Microbiol., 126, 427-442.
Bibb MJ, Ward JM, Kieser T, Cohen SN, Hopwood DA(1981) Excision of chromosomal DNA sequences from
Streptomyces coelicolor forms a novel family of plasmids
detectable in Streptomyces lividans. Mol.Gen.Genet., 184,230-240.
Chakraburtty R, Bibb MJ (1997) The ppGpp synthetasegene (relA) of Streptomyces coelicolor A3(2) plays aconditional role in antibiotic production and morphologicaldifferentiation. J.Bacteriol., 179, 5854-5861.
Chater KF, Bruton CJ, Foster SG, Tobek I (1985) Physicaland genetic analysis of IS110, a transposable element ofStreptomyces coelicolor A3(2). Mol. Gen. Genet., 200, 235239.
Chen CW, Yu T-W, Lin Y-S, Kieser HM, Hopwood DA(1993) The conjugative plasmid SPL2 of Streptomyces
lividans is a 50 kb linear molecule. Mol.Microbiol., 7, 925932.
Chinenova TA, Mkrtumian NM, Lomovskaya ND (1982)
Genetic study of a novel phage resistance character in
Streptomyces coelicolor A3(2). Genetika, 18, 1945-1952.
Danilenko VN, Starodubtseva LI, Navashin SM (1986)
Regulation and expression of kanamycin andchloramphenicol resistance determinants in Streptomyces
lividans 66. In Szabo, G., Biro, S. and Goodfellow, M. (eds.),
Biological, Biochemical and Biomedical Aspects of
Actinomycetes. Akademiai Kiado, Budapest, pp. 79-81.
Dittrich W, Schrempf H (1992) The unstable tetracycline
resistance gene of Streptomyces lividans 1326 encodes aputative protein with similarities to translational elongation
factors and Tet(M) and Tet(O) proteins. Antimicrob.Agents
Chemother., 36, 1119-1124.
Dittrich W, Betzler M, Schrempf H (1991) An amplifiableand deletable chloramphenicol-resistance determinant ofStreptomyces lividans 1326 encodes a putativetransmembrane protein. Mol.Microbiol., 5, 2789-2797.
Eichenseer C, Altenbuchner J (1994) The very large
amplifiable element AUD2 from Streptomyces lividans 66 has
insertion sequence-like repeats at its ends. J.Bacteriol., 176,
7107-7112.
Fischer J, Maier H, Viell P, Altenbuchner J (1996) The useof an improved transposon mutagenesis system for DNA
sequencing leads to the characterization of a new insertion
sequence of Streptomyces lividans 66. Gene, 180, 81-89.

Flett F, Cullum J (1987) DNA deletions in spontaneous
chloramphenicol-sensitive mutants of Streptomyces coelicolor
A3(2) and Streptomyces lividans 66. Mol.Gen.Genet., 207,
499-502.
Flett F, Platt J, Cullum J (1987) DNA rearrangements
associated with instability of an arginine gene in S. coelicolor
A3(2). J.Basic Microbiol., 27, 3-10.
Freeman RF, Hopwood DA (1978) Unstable naturally
occurring resistance to antibiotics in Streptomyces.
J.Gen.Microbiol., 106, 377-381.
Friend EJ, Hopwood DA (1971) The linkage map ofStreptomyces rimosus. J.Gen.Microbiol., 68, 187-197.
Harold RJ, Hopwood DA (1970) Ultraviolet-sensitive
mutants of Streptomyces coelicolor I. phenotypiccharacterisation. Mut.Res., 10, 427-438.
Henderson D (1988) Molecular and genetic analysis of the
Streptomyces coelicolor A3(2) mini-circle. Ph.D Thesis,
University of East Anglia, Norwich.
Hopwood DA, Kieser T (1990) The Streptomyces genome.
In Drlica, K. and Riley, M. (eds.), The Bacterial
Chromosome. American Society for Microbiology,
Washington,D.C., pp. 147-162.
Hopwood DA, Kieser T, Wright HM, Bibb MJ (1983)
Plasmids, recombination and chromosome mapping in
Streptomyces lividans 66. J.Gen.Microbiol., 129, 2257-2269.
Jenkins G, Zalacain M, Cundliffe E (1989) Inducible
ribosomal RNA methylation in Streptomyces lividans,conferring resistance to lincomycin. J.Gen.Microbiol., 135,
3281-3288.
Kendall K, Cullum J (1984) Cloning and expression of an
extracellular-agarase from Streptomyces coelicolor A3(2) in
Streptomyces lividans 66. Gene, 29, 315-321.
Kieser HM, Kieser T, Hopwood DA (1992) A combined
genetic and physical map of the Streptomyces coelicolor
A3(2) chromosome. J.Bacteriol., 174, 5496-5507.
Kinashi H, Shimaji M, Sakai A (1987) Giant linear plasmids
in Streptomyces which code for antibiotic biosynthesis genes.
Nature, 328, 454-456.
Kinashi H, Shimaji-Murayama M, Hanafusa T (1992)
Integration of SCP1, a giant linear plasmid, into theStreptomyces coelicolor chromosome. Gene, 115, 35-41.
Laity C, Chater KF, Lewis CG, Buttner MJ (1993) Genetic
analysis of the C31-specific phage growth limitation (Pgl)system of Streptomyces coelicolor A3(2). Mol.Microbiol., 7,329-336.
Leblond P, Fischer G, Francou F-X, Berger F, Guérineau
M, Decaris B (1996) The unstable region of Streptomyces
ambofaciens includes 210 kb-terminal inverted repeats
flanking the extremities of the linear chromosomal DNA.
Mol.Microbiol., 19, 261-271.
Lin Y-S, Kieser HM, Hopwood DA, Chen CW (1993) The
chromosomal DNA of Streptomyces lividans 66 is linear.
Mol.Microbiol., 10, 923-933.
Lydiate DJ, Ashby AM, Henderson DJ, Kieser HM,
Hopwood DA (1989) Physical and genetic characterizationof chromosomal copies of the Streptomyces coelicolor minicircle. J.Gen.Microbiol., 135, 941-955.
MacNeil DJ (1988) Characterization of a unique methylspecific restriction system in Streptomyces avermitilis.
J.Bacteriol., 170, 5607-5612.
Nakahara H, Schottel JL, Yamada T, Miyakawa Y,
Asakawa M, Harville J, Silver S (1985) Mercuric reductase
enzymes from Streptomyces species and group B
Streptococcus. J.Gen.Microbiol., 131, 1053-1059.
Omer CA, Stein D, Cohen SN (1988) Site-specific insertionof biologically functional adventitious genes into theStreptomyces lividans chromosome. J.Bacteriol., 170,
2174-2184.

&KDSWHU*HQRPHPDSVDQGJHQHWLFDOO\PDUNHGVWUDLQV

Orekhov AV, Strokina IV, Furs AR (1989) Restriction of
two-replicon shuttle Escherichia coli-Streptomyces plasmids
in Streptomyces lividans strain 66. Genetika, 25, 614-25.
Pandza K, Pfalzer G, Cullum J, Hranueli D (1997)
Physical mapping shows that the unstable oxytetracycline
gene cluster of Streptomyces rimosus lies close to one end of
the linear chromosome. Microbiology, 143, 1493-501.
Rauland U, Glocker I, Redenbach M, Cullum J (1995)
DNA amplifications and deletions in Streptomyces lividans
66 and the loss of one end of the linear chromosome.
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Redenbach M, Kieser HM, Denapaite D, Eichner A,
Cullum J, Kinashi H, Hopwood DA (1996) A set of ordered
cosmids and a detailed genetic and physical map for the 8 Mb
Streptomyces coelicolor A3(2) chromosome. Mol.Microbiol.,21, 77-96.
Sladkova IA (1986) Physical mapping of IS281 ofStreptomyces. Molekyularnaya Biologiya, Moscow, 20, 10791083.
Solenberg PJ, Burgett SG (1989) Method for selection oftransposable DNA and characterization of a new insertion
sequence, IS493, from Streptomyces lividans. J.Bacteriol.,

171, 4807-4813.
Tsai JF-Y, Chen CW (1987) Isolation and characterizationof Streptomyces lividans mutants deficient in intraplasmid
recombinatioin. Mol.Gen.Genet., 208, 211-218.
Volff J-N, Altenbuchner J (1997) High frequency
transposition of IS1373, the insertion sequence delimiting theamplifiable element AUD2 of Streptomyces lividans.
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Volff J-N, Eichenseer C, Viell P, Piendl W, Altenbuchner
J (1996) Nucleotide sequence and role in DNA amplificationof the direct repeats composing the amplifiable element
AUD1 of Streptomyces lividans 66. Mol.Microbiol., 21,1037-1047.
Zhou X, Deng Z, Firmin JL, Hopwood DA, Kieser T(1988) Site-specific degradation of Streptomyces lividans
DNA during electrophoresis in buffers contaminated withferrous iron. Nucl.Acids Res., 16, 4341-52.
Zhou X, Deng Z, Hopwood DA, Kieser T (1994)
Characterization of HAU3, a broad-host-range temperate
Streptomyces phage, and development of phasmids.
J.Bacteriol., 176, 2096-2099.



&KDSWHU*HQRPHPDSVDQGJHQHWLFDOO\PDUNHGVWUDLQVChapter 21

Maps of DNA fragments
CONTENTS
Conventions used for the restriction maps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 440
List of restriction endonucleases, recognition sites and isoschizomers (Table) . . . . . . 441
Lists of genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 442
Alphabetical list of resistance and indicator genes, and other DNA
fragments (Table) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 442
List of genes grouped according to function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 446
Resistance genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 446
Counterselectable markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 449
Indicator genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 449
Other DNA fragments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 449
Resistance genes grouped according to antibiotic class . . . . . . . . . . . . . . . . . . . . . . . . 450
Aminoglycosides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 450
Bialaphos, phosphinothricin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 450
Bleomycin, phleomycin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 450
Chloramphenicol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 450
Gyrase inhibitors, novobiocin, ciprofloxacin . . . . . . . . . . . . . . . . . . . . . . . . . . 450
Hygromycin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 450
Macrotetrolides, nonactin, tetranactin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 450
MLS (macrolide, lincosamide and streptogramin B) resistance . . . . . . . . . . . 451
Puromycin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 451
Spectinomycin, streptomycin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 451
Streptothricins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 451
Tetracyclines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 451
Thiostrepton and analogues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 451
Viomycin, capreomycin B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 451
Other resistances . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 451
Antibiotics, antimetabolites, and suppliers (Table) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 452
Maps of DNA fragments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 456
Resistance genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 456
Counterselectable markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 493
Indicator genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 495
Other DNA fragments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 502
We describe DNA fragments encoding resistance and indicator genes, as well as other functions
useful for vector construction, gene disruption and replacement, and promoter probing. Also included
are potentially useful resistance genes shown to function in streptomycetes but not yet used extensively

&KDSWHU 

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0DSVRI'1$)UDJPHQWVOpen Box, sequence unknown

Black line, sequenced region

Transcription
startpoint
Signal peptide
cleavage site

EcoRI

1.30

Unique site

PstI

1.40

Multiple sites

Species

BclI*

1.60

Site Dam or Dcm methylated

Mr=

PstI

1.72

(SalI)

1.85

Parentheses indicate that
not all sites are shown(BamHI/BglII)

Junction that cannot becleaved by either enzyme

Name
FunctionAC=
EMBL/GenBank
Accession number

Terminator

Ter

Shaded box, DNA fragment from different origin

Fig. 21.1 Key to the restriction maps and figure conventions

as selective markers for cloning. Streptomyces promoters are rarely active in E. coli, but E. coli
promoters are generally expressed in Streptomyces (Chapter 17). Genes containing the TTALeu codon,
very rare in streptomycetes, are poorly expressed in bldA strains lacking the corresponding tRNA
(Leskiw et al., 1991); the numbers of TTA codons in each gene are listed in the captions to the restriction
maps.

Conventions used for the restriction maps
The symbols on the linear restriction maps are explained in Fig. 21.1. The sites shown on themaps were chosen subjectively from a list of commercially available enzymes. Enzymes that cut the
fragment once, commonly used enzymes, and enzymes that might be useful for determining theorientation of cloned fragments are shown preferentially. The map captions list enzymes with no site
in the fragment and also sites that occur several times in the fragment but are not shown on the map. For
this information only the sites listed in Table 21.1 are considered.

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Table 21.1 List of restriction endonucleases, recognition sites and isoschizomers
Only the enzymes in this list are mentioned in all the figure captions if the enzyme does not cut or if
there are many sites that are not shown on the maps. Underlining of isoschizomers indicates different
cleavage specificity. *, sites protected by Dam or Dcm methylation in wild-type E. coli K; lower caseletters indicate flanking sequences that are required for methylation but not for recognition.
http://vent.neb.com/rebase/ contains up-to-date information about all known restriction endonucleases.
AatII

GACGT0C

NcoI

C0CATGG

Bsp19IAflII

C0TTAAGNdeI

CA0TATG

FauNDIApaI*GGGCC0C(a/t)gg

NotI

GC0GGCCGC

CciNI

ApaLI

G0TGCAC

Alw44I, VneI

NruI*

gaTCG0CGA

Bsp68IAseI

AT0TAATAsnI, PshBI, VspI

NsiI

ATGCA0T

BamHI

G0GATCC

PstI

CTGCA0G

BclI*

T0GATCA

PvuI

CGAT0CG

BglII

A0GATCT

PvuII

CAG0CTG

ClaI*

gAT0CGATSacI

GAGCT0C

Ecl136II, EcoICRI,
Psp124BI, SstIDraI

TTT0AAA

SacII

CCGC0GG

Cfr42I, KspI,
Sfr303I, SstIIEcoRI

G0AATTCSalI

G0TCGACEcoRV

GAT0ATC

Eco32IScaI

AGT0ACT

Acc113I

FspI

TGC0GCA

Acc16I, AviII, NsbI

SmaI

CCC0GGG

Cfr9I, PspAI, XmaCI,
XmaI

HindIII

A0AGCTTSpeI

A0CTAGT

AclNI, BcuI

HpaI

GTT0AAC

BstHPI, KspAI

SphI

GCATG0C

BbuI, PaeI

KpnI

GGTAC0C

Asp718I, Acc65I

SspI

AAT0ATT

MluI

A0CGCGTStuI

AGG0CCTNaeI

GCC0GGC

MroNI, NgoAIV,
NogMIVXbaI*

T0CTAGAtc

NarI

GG0CGCC

BbeI, EgeI, EheI,
Mly113I, SfoI

XhoI

C0TCGAG

BfrI, BspTI, Bst98I,
MspCI, Vha464I
Bsp120I,
PspOMI

BsiQI, FbaI, Ksp22I

BanIII, Bsa29I, BscI,
BseCI, Bsp106I,
BspDI, BspXI,
Bsu15I

EcoT22I, Mph1103I,
Ppu10I, Zsp2I

BspCI, Ple19I

AatI, Eco147I,
Pme55I, SseBI

PaeR7I, Sfr274I

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Lists of genes
Table 21.2 Alphabetical list of resistance and indicator genes, and other DNA fragments
The names of commonly used genes are printed in bold
Gene

Function

OriginAccession nos

Maps

aac(3)IV

Apramycin acetyltransferase

Klebsiellapneumoniae

X99313

p. 465

aacC1

Gentamicin acetyltransferase

E. coli Tn1696

U12338
X15852
U04610p. 466

aacC7

Paromomycin
acetyltransferase

S. rimosus

M22999p. 467aacC8

Neomycin acetyltransferase

S. fradiae

M55426

p. 467aacC9

Neomycin acetyltransferase

Micromonospora
chalcea

M55427

p. 467

aadA

Spectinomycin/ streptomycin
adenyltransferase

Pseudomonas plasmid
R100.1

M60473
K02163

pp.468, 469

ampC$-lactamase

E. coli chromosome

V00277

p. 495aph(4)

Hygromycin
phosphotransferase

Klebsiellapneumoniae

V01499p. 464

aphD

Streptomycin
phosphotransferase

S. griseus

X05647

p. 487aphE

Streptomycin
phosphotransferase

S. griseus

M37378

p. 487aphI

Aminoglycosidephosphotransferase

S. fradiae

X02394
K00432

p. 478

ard1

Aminoglycoside antibiotic
A201A resistanceS. capreolus

X84374--bar

Bialaphos resistanceS. hygroscopicus

X05822

p. 471$gal$-galactosidase

S. lividans

M17359p. 496bla$-lactamase

E. coli pBR322,
pUC19

J01749M77789p. 572
p. 568ble

Bleomycin resistance(bleomycin binding)

E. coli Tn5

U00004

p. 481

bleSh

Bleomycin binding

Streptoalloteichus
hindustanus--

p. 473

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GeneFunction

OriginAccession nos

Maps

blmA

Bleomycin binding

S. verticillus

L26954/5

p. 473

blmB

Bleomycin acetyltransferase

S. verticillus

L26955

p. 473cacCapreomycin
acetyltransferase

S. capreolus

U13077

p. 474

carA

Carbomycin efflux

S. thermotolerans

M80346--carB

23S rRNA methylase

S. thermotolerans

D31821
M16503--cat

Chloramphenicolacetyltransferase

E. coli Tn9
pACYC184
pBR325

V00622
X06403
L08855

pp. 564-565

catSa

Chloramphenicolacetyltransferase

S. acrimycini--

p. 475

cmlSl

Chloramphenicol efflux

S. lividans

X59968

p. 491

cmlv

Chloramphenicol export

S. venezuelae

U09991

p. 488cph

Capreomycin
phosphotransferase

S. capreolus

U13078--cpt

Chloramphenicol
phosphotransferase

S. venezuelae

U09991

p. 488creCurromycin resistanceS. hygroscopicus

M28599--drrA,B

Daunorubicin resistanceS. peucetius

M73758U18082--drrC

Daunorubicin resistanceS. peucetius

L76359

--

EGFP
(gfp)

Green fluorescent protein

Aequorea victoria

U76561

p. 499ermE

23S rRNA dimethylase

Saccharopolyspora
erythraea

M37378
X51891

p. 459ermSF

Old name for tlrA RNA
N-methyltransferase

S. fradiae

M19269--galK

Galactokinase

E. coli

D90714

p. 497

glkA

Glucose kinase

S. coelicolor

X65932
X98363

p. 493grmMp

16S rRNA methylase

Micromonospora
purpurea

M55520p. 460grmMr

16S rRNA methylase

Micromonospora
rosea

M55521

p. 460

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GeneFunction

OriginAccession nos

Maps

gylR

Repressor of glycerol operon

S. coelicolor

X14188p. 503gyrB

Novobiocin resistant gyrase

S. sphaeroides

Z17304

p. 463hur

Hydroxyurea resistance(tested in E. coli only)

S. aureofaciens

M81739--hyg

Hygromycin
phosphotransferase

S. hygroscopicus

X99315p. 485kamA

16S rRNA methylase

S. tenjimariensis

D13170--kamB

16S rRNA methylase

S. tenebrarius

M64625p. 462kamC

16S rRNA methylase

Saccharopolyspora
hirsuta

M64626

p. 462kan

16S rRNA methylase

S. kanamyceticus

M27488--kan

16S rRNA methylase

Micromonospora
echinospora--

pp. 461, 359

kgmB

16S rRNA methylase

S. tenebrarius

S60108p. 461km

Aminoglycosidephosphotransferase

E. coli Tn903,
pACYC177,
pUC4K

J01839V00359V00621

p. 479lmrA

Lincomycin efflux

S. lincolnensis

X59926--lmrB

23S rRNA methylase

S. lincolnensis

X62867--lrm

23S rRNA methylase

S. lividans

M74717--

luxA,B

Luciferase

Vibrio harveyi

X58791

p 498

mdh

Malate dehydrogenase

Thermus flavus

X54073--

mcrA,B

Mitomycin C resistanceS. lavendulae

L29247

p. 492mel

Other tyrosinases

S. venezuelae
S. lincolnensis
S. galbus

M20422
X95703
X95705--

melC1,C2

Tyrosinase

S. antibioticus

M11582

p. 501

melC1,C2

Tyrosinase

S. glaucescens

M11302

p. 501mer

Mercury resistanceS. lividans

X65467--mmr

Methylenomycin efflux

S. coelicolor

M18263--

mtrA,B

Mithramycin resistance,
mtrA, ATP-binding,mtrB membrane protein

S. argillaceus

U43537--

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GeneFunction

OriginAccession nos

Maps

myrB

23S rRNA methylase

Micromonospora
griseorubida

E07944
D14532--

nat1

Nourseothricin
acetyltransferase

S. noursei

S60706 X73149

p. 476neo

Aminoglycosidephosphotransferase

E. coli Tn5

U00004

p. 481

nmr

Neomycin resistanceS. cyanogenus
pSB24.2

X03756
M32513--nonR

Macrotetrolide nonactin,
tetranactin efflux

S. griseus

M75853

p. 489nsh

23S rRNA methylase,nosiheptide resistanceS. actuosus

U75434

p. 457

oleA,B

Oleandomycin resistanceS. antibioticus

L36601--oleC

Oleandomycin efflux

S. antibioticus

L06249--oriT

Origin of transfer

Plasmid RP4=RK2

L27758 (nt50590-51384)

p. 502otrA

Oxytetracycline
sequestration(similar to EF Tu)

S. rimosus

X53401
I32939p. 491otrB

Oxytetracycline efflux

S. rimosus

AF061335--

pac

Puromycin acetyltransferase

S. alboniger

X76855

p. 477pat

Phosphinothricin
acetyltransferase

S. viridochromogenes

A02804

p. 471

ptr

Multidrug resistanceS. pristinaespiralis

X84072--

pur8

Puromycin resistanceS. alboniger

X76855

p. 477

rphS1

Ribostamycin
phosphotransferase

S. ribosidificus

M22126

p. 479rpsL

Ribosomal protein (Str s)

S. roseosporus

U60191

p. 494spcN

Spectinomycin
phosphotransferase

S. flavopersicus

U70376

p. 486

sph

Streptomycin
phosphotransferase

S. glaucescens

X78976

p. 487srmB

Spiramycin efflux

S. ambofaciens

X63451--ter(fd)Transcriptional terminatorE. coli phage fd

V00602

p. 503tet

Tetracycline efflux

E. coli pBR322

J01749p. 572

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GeneFunction

OriginAccession nos

Maps

tetSl

Tetracycline efflux

S. lividans

M74049p. 491

tipA

Thiostrepton-inducible
proteinS. lividans

Y08949--tlrA

N-methyltransferase

S. fradiae

M19269--tlrB

Methyltransferase

S. fradiae

AF055922
AJ009971--

tlrC

Tylosin efflux resistanceS. fradiae

M57437--tlrD

Tylosin constitutive 23S
rRNA monomethylase

S. fradiae

X97721--

tnrB2,3Tetronasin efflux

S. longisporoflavus

X73633

p. 489traR

Transfer gene regulator

pSN22, S. nigrifaciens

D14281

p. 504tsr

23S rRNA methylase

S. azureus

X54219X02392

p. 457

tsr

Thiostrepton resistanceS. laurentii

L39157--vph

Viomycin
phosphotransferase

S. vinaceus

X02393
X99314

p. 483xylE

Catechol-2,3-dioxygenase

Pseudomonas

U03992
J01845

p. 495

List of genes grouped according to function
This is the order of the restriction maps starting on p. 456. Names of frequently used genes are
printed bold in this list. The page numbers refer to the restriction maps. Sequence accession numbers
are in the alphabetical list of all genes (p. 442).
Resistance genes
There is also a list of resistance genes grouped according to antibiotic class (p. 450). Note that
hygromycin, spectinomycin and streptomycin are listed separately from the "main" aminoglycosides.
rRNA methylases
23S rRNA is methylated before it is incorporated into the 50S ribosome subunit, explaining thelong expression times needed before selection can be applied. 16S rRNA is methylated afterincorporation into the 30S ribosomal subunit. The subject is reviewed by Cundliffe (1989) andsummarised in Fig. 21.2 (p. 448).

&KDSWHU 

m21tk.wpd 16 March 2000 Tobias Kieser

S. azureus tsr

S. fradiae tlrA
Sacch. erythraea ermE

Thiostrepton resistanceDimethylation, MLS resistance

23S rRNA

1067

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2058

S. lividans lrm
S. caelestis clr(Monomethylation, lincosamide resistance)

ermE
lrm

kan
grm
kgmB

NH 2

kam

N1 6

5

2

4

3

N
7

grm Micromonospora
Gen and Kan resistance

HO

8
9

N

N
5’

A

kamA S. tenjimariensis
Apra, Neo and Kan resistance

H2C O
4’

1’

3’

2’

HO

OH

tsr

H 2N

G

N1 6

5

2

4

3

N

N
7
8

1405

N

H2C 5’ O
4’

1408

9

1’

3’

2’

HO

OH

kan S. kanamyceticus
Gen and Kan resistancekamB + kgmB S. tenebrarius
Apra, Neo, Kan and Gen resistanceFig 21.2. rRNA methylases. The 23S rRNA is methylated before incorporation into theribosome. The ermE product dimethylates adenine 2056 at the 6 position, etc. The 16S rRNA
methylases confer gentamicin or apramycin resistance depending on the guanine bases that is
methylated.
23S rRNA A1067 O2’methylases (thiostrepton resistance): tsr (p. 457), nsh (p. 457)
23S rRNA A2058 N6 dimethylation (MLS resistance): ermE (p. 459), tlrA
23S rRNA A2058 N6 monomethylation (lincosamide resistance): carB, lmrB, lrm, mdmA, myrB, tlrD
16S rRNA A1405 N1 methylation (gentamicin and kanamycin resistance): grmMp (p. 460),
grmMr (p. 460), kgmB (p. 461), kan ( Micromonospora echinospora, selection in pIJ6021).
16S rRNA A1408 N7 methylation (apramycin, neomycin and kanamycin, resistance): kamA,
kamB (p. 462) kamC (p. 462)
Acetyltransferases
Aminoglycoside: aac(3)IV (apramycin; p. 465), aacC1 (gentamicin; p. 466),
aacC7 (paromomycin; p. 467), aacC8 (neomycin; p. 467), aacC9 (neomycin; p. 467)
Bialaphos, phosphinothricin: bar (p. 471), pat (p. 471)
Bleomycin, phleomycin: blmB (p. 473)
Capreomycin: cac (p. 474)
Chloramphenicol: cat, catSa (p. 475)
Nourseothricin: nat1 (p. 476)
Puromycin: pac (p. 477)

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Table 21.3 Examples of cross-resistance between aminoglycosides

Levels of antibiotic resistance (µg ml -1) of S. lividans or
S. coelicolor containing resistance genes.

<1<1

Geneticin (G418)

#10

>50

50

#10

<1

Gentamicin

<5

<25

<25

<25

>200

10-30

Kanamycin

<30

<50

400

<50

>400

>1000

Neomycin B

<1

<5

200

10

<5

>30

Paromomycin

<10

<5

>100

>5

<2

>10

Sisomicin<3-10

Tobramycin

<10

1

50

rRNA A1408
methylase<1

kamB 16S>100

A1405methylase<1

grm 16S rRNA

Apramycin

phosphotransferase>100

aphI S. fradiae<25

phosphotransferase<25

neo (Tn5)<25

acetyltransferase<25

aacC1 gentamicinaac(3)IV apramycin

Amikacin

Aminoglycoside 1

acetyltransferase

No resistance gene

The values are approximate guidelines and the precise levels of resistancedepend on the medium, the promoter and the copy number of the resistancegene. Bold type indicates high level resistance, useful for selection.

>300

>100
>300

>100

There is no cross-resistance between these antibiotics and hygromycin, spectinomycin andstreptomycin.

m21tk.wpd 16 March 2000 Tobias Kieser&KDSWHU 

0DSVRI'1$)UDJPHQWVAdenyltransferase
Spectinomycin, streptomycin: aadA (p. 468, 469)
Phosphotransferases
Aminoglycoside: aphI (p. 478), km (p. 479), neo (p. 481), rphS1 (p. 479)
Capreomycin, viomycin: cph, vph (p. 483)
Chloramphenicol: cpt (p. 488)
Hygromycin: aph(4) (p. 464), hyg (p. 485)
Spectinomycin: spcN (p. 486)
Streptomycin: aphD, aphE (p. 487), sph (p. 487)
Antibiotic efflux systems (Reviewed by Marshall and Piddock, 1997)
Lincosamides: carA, lmrA, oleB, oleC, srmB, tlrC
Chloramphenicol: cmlSl (p. 491), cmlv (p. 488)
Methylenomycin: mmr
Multidrug (pristinamycin etc.): ptr
Nonactin nonR: (p. 489),
Puromycin: pur8 (p. 477)
Tetracycline: otrB, tet
Tetronasin: tnrB2 (p. 489), tnrB3 (p. 489)
Antibiotic binding
Bleomycin: ble (p. 481), bleSh (p. 473), blmA (p. 473)
Tetracycline: otrA (p. 491), tetSl (p. 491)
Other resistances
Curromycin: cre; Daunorubicin: drrA,B,C; Hydroxyurea: hur; Mercury: mer; Mithramycin: mtrA,B;
Mitomycin C: mcrA,B (p. 492); Novobiocin: gyrB (p. 463)

Counterselectable markers
glkA (glucose kinase; p. 493), rpsL (ribosomal protein S12; p. 494)

Indicator genes
ampC (p. 495), $gal (p. 496), EGFP (p. 499), galK (p. 497), luxAB (p. 498), mdh, melC1,C2 (p. 501),
mel (p. 501), xylE (p. 495)
Other DNA fragments
oriT (p.502 ), terfd (p. 503), gylR (p. 503), tipA, traR (pSN22; p. 504)



&KDSWHU 

0DSVRI'1$)UDJPHQWVResistance genes grouped according to antibiotic classSee also alphabetical list of antibiotics p. 452; alphabetical list of all genes p. 442. Frequently
used genes are printed in bold.
Aminoglycosides(Note: hygromycin, streptomycin, and spectinomycin are listed separately in alphabetical order;
aminoglycosides tend to be more effective on low salt media.)
Amikacin, apramycin, butirosin, geneticin (G418), gentamicin, kanamycin, neomycin B, paromomycin,
ribostamycin, sisomicin, tobramycin, kasugamycin (atypical aminoglycoside). See Table 21.3 (p. 448)
for examples of cross-resistance between aminoglycosides.
16S rRNA A1405 N1 methylation (gentamicin, kanamycin, tobramycin resistance): grmMp (p. 460),
grmMr (p. 460), kan (p. 539), kgmB (p. 461)
16S rRNA A1408 N7 methylation (apramycin, kanamycin, neomycin, paromomycin, tobramycin
resistance): kamA, kamB (p. 462) kamC (p. 462)
Aminoglycoside acetyltransferases: aac(3)IV (p. 465), aacC1 (p. 466), aacC7 (p. 467), aacC8 (p. 467),
aacC9 (p. 467)
Aminoglycoside phosphotransferases: aphI (p. 478), km (p. 479), neo (p. 481), rphS1 (p. 479)
Bialaphos, phosphinothricin
Acetyltransferases: bar (p. 471), pat (p. 471)
Bleomycin, phleomycin
Bleomycin, phleomycin acetyltransferases: blmB (p. 473)
Bleomycin binding: ble (p. 481), blmA (p. 473), bleSh (p. 473)
Chloramphenicol
Chloramphenicol acetyltransferases: cat, catSa (p. 475)
Chloramphenicol phosphotransferases: cpt (p. 488)
Chloramphenicol efflux resistance: cmlSl (p. 491), cmlv (p. 488)
Gyrase inhibitors, novobiocin, ciprofloxacin
Resistant gyrases: gyrB (p. 463)
Hygromycin
Hygromycin phosphotransferases: aph(4) (p. 464), hyg (p. 485)

Macrotetrolides, nonactin, tetranactin
Efflux resistances: nonR (p. 489), tnrB2 (p. 489), tnrB3 (p. 489)

m21tk.wpd 16 March 2000 Tobias Kieser&KDSWHU 

0DSVRI'1$)UDJPHQWV

MLS (macrolide, lincosamide and streptogramin B) resistance(macrolides: carbomycin, erythromycin, oleandomycin, spiramycin, tylosin; lincosamides: lincomycin,
clindamycin)
23S rRNA A2058 N6 dimethylation (MLS resistance): ermE (p. 459), tlrA
23S rRNA A2058 N6 monomethylation (lincosamide resistance): carB, clr, lmrB, lrm, myrB, tlrD
Lincosamide efflux resistance: carA, lmrA, oleC, srmB, oleB, oleC, tlrB, tlrC
Puromycin
Puromycin acetyltransferase: pac (p. 477)
Puromycin efflux resistance: pur8 (p. 477)
Spectinomycin, streptomycin
Spectinomycin, streptomycin adenyltransferase: aadA (p. 468,469)
Spectinomycin phosphotransferase: spcN (p. 486)
Streptomycin phosphotransferases: aphD (aph(6)) (p. 487), aphE (p. 487), sph (p. 487)
Ribosomal protein S12: rpsL (counterselectable, dominant streptomycin sensitivity p. 494)
Streptothricins(grisin, nourseothricin, racemomycin, streptothricin)
Nourseothricin acetyltransferase: nat1 (p. 476)
Tetracyclines
Chlortetracycline, minocycline, oxytetracycline, tetracycline
Efflux resistances: otrB, tet.
Antibiotic binding (EF Tu-like protein): otrA (p. 491), tetSl (p. 491)
Thiostrepton and analogues(peptide antibiotics: berninamycin, nosiheptide, siomycin, sporangiomycin, thiopeptin, thiostrepton.)
23S rRNA A1067 O2’methylases: tsr (p. 457), nsh (p. 457)
Viomycin, capreomycin B(peptide antibiotics)
Capreomycin acetyltransferase: cac (p. 474)
Capreomycin, viomycin phosphotransferases: cph, vph (p. 483)
Other resistances
ampC (p. 495), bla, hur, mcrA,B (p. 492), mer, mtrA,B, myrB, ptr

&KDSWHU 

0DSVRI'1$)UDJPHQWV

Table 21.4. Antibiotics, antimetabolites, and suppliers
Name (Synonyms)

Class, properties

Suppliers1 and relative price 

Amikacin

Aminoglycoside

Sigma Apramycin

Aminoglycoside

Duchefa, Sigma 

Bialaphos

Glutamine synthetase inhibitor,
herbicide

Meiji Seika, Duchefa 

Blasticidin S

Used as fungicide against rice
blast

Invitrogen, CAYLA

Bleomycin

Cross-resistance withphleomycin and zeocin

Boehringer, Calbiochem,
Duchefa, Sigma 

Butirosin

Aminoglycoside

Sigma, Duchefa 

Capreomycin

Peptide, cross-resistance withviomycin

Sigma Carbomycin(Magnamycin®)

Macrolide

PfizerChloramphenicol

Antibacterial

Sigma, Duchefa, etc. 

Ciprofloxacin

Gyrase inhibitor

Clindamycin

Semi-synthetic lincosamide

Sigma 

Daunomycin/daunorubicin

Anthracycline, anti-cancer
agent

Sigma 

Destomycin

Aminoglycosideused as feed additive

Erythromycin

Macrolide

Fortimicin

Aminoglycoside

Fosfomycin (phosphomycin)

Antibacterial

Sigma Fusidic acid

Antibacterial

Sigma Geneticin (G418)

Aminoglycoside

Boehringer, Clontech,
Gibco/BRL, Invitrogen,Sigma, A.G. Scientific 

Gentamicin

Aminoglycoside

Boehringer, Calbiochem,
Duchefa, Sigma 

Hydroxyurea

Antineoplastic agent

Sigma Sigma, Duchefa m21tk.wpd 16 March 2000 Tobias Kieser&KDSWHU 

0DSVRI'1$)UDJPHQWVName (Synonyms)

Class, properties

Suppliers1 and relative price 

Hygromycin B

Best selection on low-salt
media, light sensitive

A.G. Scientific Boehringer,
Calbiochem, Clontech,
CAYLA, Duchefa,Invitrogen,Sigma Kanamycin

Aminoglycoside, best selected
on low-salt media

Boehringer, Calbiochem,
Clontech, Duchefa, Sigma Kasugamycin

Aminoglycoside

Sigma Lincomycin

Lincosamide, used in
preference to erythromycin to
select ermE in S. coelicolor

Sigma, Duchefa Lividomycin

Aminoglycoside, too toxic for
medical use

Methylenomycin

Cyclopentane

Minamycin

Macrolide

Mitomycin C

Anticancer agent

Boehringer, Calbiochem,
Duchefa, Sigma 

Nalidixic acid

Gyrase inhibitor;
streptomycetes are naturally
resistant, E. coli is sensitive

Duchefa, Sigma 

Neomycin

Aminoglycoside

Calbiochem, Sigma Nonactin

Macrotetrolide(no medical use)

Sigma 

Nosiheptide

Peptide, similar to thiostrepton

Nourseothricin

Similar to streptothricin

Novobiocin

Gyrase inhibitor

Boehringer, Serva Oleandomycin(Oleandocyn®)

Macrolide

Serva Pfizer 

Pactamycin

No medical use

Paromomycin

Aminoglycoside

Sigma, Duchefa Penicillins

Most streptomycetes arenaturally resistant topenicillins

Many

Phleomycin

Similar to bleomycin

Sigma, CAYLA 

Phosphinothricin

Glutamine synthetase inhibitor

Duchefa &KDSWHU 

0DSVRI'1$)UDJPHQWVName (Synonyms)

Class, properties

Suppliers1 and relative price 

Puromycin

Nucleoside

Calbiochem, Clontech, Sigma,
CAYLA, A.G. ScientificRacemomycin

Streptothricin type; active
against mycobacteria

Ribostamycin

Aminoglycoside

Sigma Rifampicin

Light-sensitive

Boehringer, Duchefa, Sigma

Siomycin

Peptide, similar to thiostrepton

Sisomicin

Aminoglycoside

Sigma Spectinomycin

Antibacterial

Boehringer, Duchefa, Sigma Spiramycin

Macrolide

Sigma Streptogramin B(Synercid®)

Antibacterial

Rhône-Poulenc Rorer

Streptomycin

Antibacterial

Calbiochem, Duchefa Streptothricin

Similar to nourseothricin

Tetracycline

Light-sensitive

Tetranactin

Macrotetrolide (see nonactin)

Tetronasin

Polyether (feed additive)

Thiopeptin

Peptide, similar to thiostrepton

Thiostrepton

Peptide; poor selection on MS(=SFM)

Calbiochem 
Sigma

Tobramycin

Aminoglycoside

Duchefa, Sigma 

Tuberactinomycin

Similar to viomycin

TylosinMacrolide

Sigma Viomycin(Viocin, Florimycin)

Peptide; cross-resistance withcapreomycin IA, B; mostactive on low-salt media

Sigma 
Research Diagnostics

ZeocinSimilar to bleomycin; use
25 µg ml-1 in low-salt medium
for E. coli

Invitrogen
CAYLA (Footnote on following page)

Calbiochem, Boehringer,
Duchefa, Sigma m21tk.wpd 16 March 2000 Tobias Kieser&KDSWHU 

0DSVRI'1$)UDJPHQWVFootnote to Table 21.4
1

Suppliers of antibiotics: the blocks () indicate the relative prices of the antibiotics; some of theprices are very high only because the quantities sold are small and the demand for the substances is low.
Samples for research may be obtainable from the antibiotic producers directly.
A.G. Scientific Inc. 6450 Lusk Blvd Suite E102, San Diego, CA 92121, Telephone:+1 619-452-9925
Toll-free: +1-877-452-9925 Fax: +1 619-452-9926 Email: customerservice@agscientific.com,
http://www.agscientific.com/
Boehringer, http://biochem.boehringer-mannheim.com/HomePage.htm, http://193.197.95.199/
Calbiochem, http://www.calbiochem.com/
Clontech, http://www.clontech.com/clontech/Catalog/CellBio/Antibiotics.html
CAYLA, ZI Montaudran - BP4437, 5 rue Jean Rodier, F-31405 Toulouse Cedex 4, France
http://www.cayla.com/ Email orders: orders@cayla.com,
Tel: +33 (0)562 71 69 39, Fax orders: +33 (0)562 71 69 30
Duchefa, Duchefa Biochemie, P.O. Box 2281, NL-2002 CG Haarlem, The Netherlands.
Phone: +31 23 5319093, FAX: +31 23 5318027, Email: DUCHEFA@PI.NET.
In the UK Duchefa products are distributed by Melford Laboratories Ltd, Bildeson Road,
Chelsworth, Ipswich IP7 7LE, UK. Tel. +44 1449 741178, Fax +44 1449 741 1217,
Email: Melford@ibm.net
GIBCO/BRL = Life Technologies, http://www.lifetech.com/homepage.html
Invitrogen, http://www.invitrogen.com/
Pfizer: http://www.pfizer.com/main.html
Research Diagnostics Inc Email:ResearchD@aol.com; http://www.researchd.com/index.htm.
Pleasant Hill Road Flanders NJ 07836 Fax: +1 973 584 0210
Rhône-Poulenc Rorer, = Aventis: http://www.aventis.com/com/
Sigma, http://www.sigma.sial.com/sigma/catalog.htm
http://pipeline2.sial.com/pubcatalog.nsf
http://pipeline2.sial.com/pubsigma.nsf/$$Search?OpenForm

&KDSWHU 

0DSVRI'1$)UDJPHQWV

Maps of DNA fragments
Resistance genes
23S rRNA methylases
tsr, 23S A1067 rRNA methylase from S. azureus, thiostrepton resistance
50 µg ml-1 thiostrepton is used on all media (stock, 0.5 µg ml-1 in DMSO). Lower resistance wasobserved for a chromosomally integrated tsr by Henderson et al. (1990). Selection is poor on MS(=SFM) (strong background growth). Allow at least 12 h for expression after protoplast transformation.
Spontaneously thiostrepton resistant strains often have mutations in rplK (=relC) (Ochi et al., 1997).
When "real" thiostrepton-resistant transformants are very infrequent, low numbers of pseudo-resistantcolonies may cause confusion. Often these are sensitive on re-testing by replica plating to another platecontaining thiostrepton.
Gram-negative bacteria are resistant because thiostrepton is not taken up. A thiostreptonpermeable E. coli strain, SCC1431, has been isolated and >100 µg ml-1 thiostrepton resistance wasobserved after transformation with pDH5 (K. Brocklehurst, personal communication).
BamHI fragment has no sites for: AseI, BglII, EcoRI, HindIII, KpnI, PstI, SacI, SphI, XbaI, XhoI. The
sequenced region, additionally, has no sites for: AflII, ApaLI, DraI, HpaI, NcoI, NotI, NsiI, ScaI, SpeI,SspI, StuI. Several sites for: AatII, HincII, NaeI, NruI, PvuI. No TTA codon.
Convenient sources: pIJ39, BamHI fragment in pBR322 (Thompson et al., 1982a); pIJ702, BclI
fragment; pIJ2581, tsr+glkA (van Wezel and Bibb, 1996).
AC=X02392, X54219. Cloning: Thompson et al. (1980, 1982a). Sequence: Bibb et al.
(1985b). Promoters: Janssen and Bibb (1990). Mode of action: Thompson et al. (1982b); Bechthold
and Floss (1994). Thiostrepton-induced gene expression: Murakami et al. (1989). Other thiostrepton
resistance genes: Sulphomycin resistance (tsr homologue) from S. viridochromogenes ssp. sulfomycini
ATCC29776 (Nakanishi et al., 1986); nsh (p. 457; Malina and Robert-Gero, 1992).
nsh, 23S A1067 rRNA methylase from S. actuosus, nosiheptide and thiostrepton resistance
AC=M32744 (Dosch et al., 1988; Li et al., 1990, 1991; Dary et al., 1989). ORF699 is a regulator fornsh expression. No sites for: AflII, ApaI, ApaLI, AseI, BclI, BglII, ClaI, DraI, EcoRI, EcoRV, HindIII,
HpaI, KpnI, NcoI, NdeI, NotI, NruI, NsiI, ScaI, SpeI, SspI, XbaI, XhoI. Several sites for: AatII, FspI,
NaeI, NarI, SacII, SalI, SmaI, StuI. No TTA codon.

m21tk.wpd 16 March 2000 Tobias Kieser

ORF699&KDSWHU 

BamHI

1

BamHI

1

DraIII

53

SalI

61

PleI

130

SphI

224

AgeI

338

MluI

436

SacI

551

PmlI
BstEII
PflMI

675
712
730

PleI

899

BstEII

tsr

nsh
Nosiheptide

StuI*

1002

BfaI

1055

PvuI
PvuII
XmnI

1312
1323
1349

S. actuosus
23S rRNA
A-1067 methylase
Mr=29183



0DSVRI'1$)UDJPHQWV

Esp3I

1610

Bpu1102I

1787

AC=U75434

PleI

1800

Eco47II
AscI

1843
1870

StuI
StuI

1885
1950

DraIII

2039

BsaBI

2069

PstI

2321

Thiostrepton
S. azureus
23S rRNA
A-1067 methylase
Mr=29000
AC=X54219, X02392

201

SalI

251

ApaI
SmaI

307
326

BclI*

434

1

NdeI
EarI

452
488

19
55

XcmI
SacII
ClaI

634
666
683

201
233
250

AgeI

850

417

PvuI
HincII

873
878

440
445

EcoRV

956

523

SalI

1070

637

PvuII 1199
BspEI* 1255
EcoNI 1306

766
822
873

BclI*
HincII
PvuI

1489
1530
1612

1056
1097
1179

FspI
MluI
BsaI
BglI
NarI

1675
1699
1750
1770
1778

BamHI

1790



&KDSWHU 

0DSVRI'1$)UDJPHQWV

ermE, gene for constitutively expressed 23S rRNA A2058 N6-dimethylase from Saccharopolyspora
erythraea conferring MLS resistance
20 µg ml-1 lincomycin is best for selection in S. lividans and S. coelicolor because, unlike erythromycin,
it does not induce the S. lividans lrm gene (23S rRNA A2058 N6-monomethylase; Jenkins and Cundliffe,
1991) which confers low level macrolide and high-level lincosamide resistance. 100 µg ml-1
erythromycin can be used for S. coelicolor. ermE expression and MLS resistance in E. coli (200 µg ml-1
erythromycin) depends on readthrough from vector promoters.
The DNA sequence for the restriction map was taken from X51891 except for positions 52-95
which are from M11200. The polylinker sequences are from pIJ2925 and pUC19. No sites for: AflII,
ApaI, AseI, BclI, BsaBI, ClaI, DraI, EcoRV, HpaI, MluI, NcoI, NdeI, NsiI, ScaI, SpeI, SspI, StuI, XhoI.
Several sites for: ApaLI, BamHI, BglII, FspI, HindIII, KpnI, NaeI, NarI, PvuI, SalI, SmaI, StyI. No
TTA codon.
Convenient source: pIJ4026 (source of BglII fragment shown; Bibb et al., 1985a). The ermE
promoter is present in the expression vector pIJ459; pIJ4090 contains the ermE* up-promoter mutation(p. 389).
Cloning: Thompson et al. (1982a; 1982c). Promoters: Bibb et al., (1985a); see erratum Gene38, E357-E368; Bibb et al., 1994). Sequence: Uchiyama and Weisblum (1985); Dhillon and Leadlay(1990). Mode of action: Thompson et al. (1982b); Katz et al. (1987). Use in transduction:
McHenney and Baltz (1991).

16S rRNA methylases
These methylases give very high level resistance to kanamycin (>1000 µg ml-1) and to otheraminoglycosides. There are two types of 16S rRNA methylases which modify different bases (Fig. 21.2;
p. 447) and give different resistance patterns. See Table 21.3 (p. 448) for information about crossresistance between aminoglycosides. Only G1405 N7 rRNA methylase genes grm and kan, which confer
gentamicin resistance, are currently in use as selective markers; they are tolerated on high copy number
pIJ101 derivatives and high resistance levels are also obtained with single copies integrated into theS. coelicolor chromosome. The methylases also confer resistance to E. coli, provided that the genes are
expressed from vector promoters.

m21tk.wpd 16 March 2000 Tobias Kieser&KDSWHU 

0DSVRI'1$)UDJPHQWV

BglII
PstI
HindIII

1
5
13

SphI
Sse8387I

19
24

PstI
SalI

25
31

XbaI

37

BamHI
SmaI
KpnI
ApaLI

43
48
52
213

Tth111I 265
PmlI
276
BamHI

326

BsiWI

598

HindIII

639

Esp3I
PvuII

786
807

ermE
Erythromycin
Lincomycin
Saccharopolyspora

SalI

906

StyI

934

SalI
FspI

939
961

Mr=41708

AscI
FokI

967
1052

AC=M11200, X51891

FspI

1069

NotI

1297

NruI

1361

StyI

1400

SmaI

1467

SacII

1625

ApaLI

1664

BsaI

1682

erythraea

23S rRNA G-2058
dimethylase

pIJ4026

KpnI

1711

SacI
EcoRI

1717
1723

BglII

1732

&KDSWHU 



grmMp
Gentamicin
Kanamycin
Micromonospora
purpurea
16S A-1405
rRNA methylase
Mr=30542

0DSVRI'1$)UDJPHQWV

BamHI-0.52kb

XXXX

1

Bsu36I
PflMI
NarI
SrfI

140
193
219
227

SmaI
BglII

228
300

RsrII

393

BglII

477

BamHI

-2.1kb

XXXX
SspI
AgeI

1
76
93

BamHI

102

PflMI

189

NarI

215

SrfI
SmaI

223
224

BglII
NarI

296
304

ApaLI

421

BglII

473

BsaI

565

BsiWI
AatII
BbsI

649
697
714

BstEII

795

grmMr

BsiWI
XhoI
AatII
BbsI

653
674
701
718

BstEII
PvuI

799
830

EcoRI

897

EarI
BspEI

960
964

BspMI

1069

Gentamicin
Kanamycin
Micromonospora
rosea
16S A-1405
rRNA methylase
Mr=30722

PvuI

826

NheI

874

PvuI

883

EarI

956

BspMI 1065

1405

SmaI

1197

NarI
ApaLI

1307
1327

BglII
BamHI

1342
1378

SfaNI

1187

ApaLI
BglII
BamHI

1323
1338
1374

N7 rRNA methylases (gentamicin/kanamycin resistance)

grmMp from Micromonospora purpurea
AC=M55520 (Kelemen et al., 1991; Thompson et al., 1985).
No sites for: AflII, ApaI, AseI, BclI, ClaI, DraI, EcoRV, FspI, HindIII, HpaI, KpnI, MluI, NcoI, NdeI,
NotI, NruI, NsiI, PstI, PvuII, SacI, SacII, ScaI, SpeI, SphI, SspI, StuI, XbaI.
Several sites for: BglII, NaeI, NarI, SalI, SmaI. No TTA codon.
grmMr from Micromonospora rosea
AC=M55521 (Kelemen et al., 1991).
No sites for: AflII, ApaI, AseI, BclI, ClaI, DraI, EcoRI, EcoRV, FspI, HindIII, HpaI, KpnI, MluI, NcoI,
NdeI, NotI, NruI, NsiI, PstI, PvuII, SacI, SacII, ScaI, SpeI, SphI, StuI, XbaI, XhoI.
Several sites for: ApaLI, BamHI, BglII, NaeI, NarI, PvuI, SalI. No TTA codon.

m21tk.wpd 16 March 2000 Tobias Kieser&KDSWHU 

0DSVRI'1$)UDJPHQWV

BamHI -0.16kb

1

kgmB

ApaI
SfiI

51
52

BfaI

171

SalI

322

NaeI
SacII
EcoNI
BglII

377
408
417
458

SalI

526

StyI

986

EcoRI

1063

DrdI

1131

BfaI
SalI

1168
1193

FspI

1314

BstEII
SphI

1438
1452

EcoRI

SacI
KpnI
SmaI
BamHI

Gentamicin
Kanamycin
S. tenebrarius
16S A-1405
rRNA methylase
Mr=31 000
AC=S60108

SphI
HindIII

pLST314kgmB from S. tenebrarius
AC=S60108 (Holmes and Cundliffe, 1991).
No sites for: AflII, ApaLI, AseI, BamHI, BclI, ClaI, DraI, EcoRV, HindIII, HpaI, KpnI, NcoI, NdeI,
NotI, NruI, NsiI, PstI, PvuI, PvuII, SacI, ScaI, SmaI, SpeI, SspI, StuI, XbaI, XhoI. Several sites for:
AatII, MluI, NarI, SalI. No TTA codon.
kan from Micromonospora echinospora
E. Takano (personal communication); selective marker in pIJ6021 (p. 539).
20 µg ml-1 kanamycin or gentamicin for selection of S. lividans on R2YE or MM agar. No resistance toneomycin or ribostamycin. No sites for:AatII, ApaI, BamHI, BclI , ClaI, EcoRI, EcoRV, HindIII, HpaI,
KpnI, MluI, NcoI, NruI, PstI, SacI, SacII, ScaI, SphI, StuI, XbaI. Several sites for: AvaI, AvaII, BalI,
BglII, BstEII, NarI, PvuII, RsaI.

&KDSWHU 

0DSVRI'1$)UDJPHQWV

1

BsaI
XbaI
SphI

15
19
31

FspI

34

SphI

AatII

90

BglII

190

BspMI

235

BglI
NaeI
EagI
PvuII
ApaI

379
379
381
416
426

NarI

491

AgeI

96

AatII

122

HphI

181

StyI

201

BclI*
NotI

435
460

PleI

506

EcoNI
NarI
MscI

556
578
600

SalI

628

AatII

647

FspI

716

HphI

794

BamHI

846

kamB
Apramycin
Kanamycin
NeomycinS. tenebrarius
16S A-1408
rRNA methylase
Mr=16759

kamC
Apramycin
Kanamycin
Neomycin
Saccharopolyspora
hirsuta
16S A-1408
rRNA methylase
Mr=16757

567
BsaI
Eam1105I 578
604
AgeI
SmaI

656

BssHII
DrdI
SalI
RsrII
MluI

687
706
718
725
740

SmaI

775

KpnI

850

16S A1408 N1 rRNA methylases (apramycin, kanamycin, neomycin)
kamB from S. tenebrarius
AC=M64625 (Holmes et al., 1991).
No sites for: AflII, ApaI, ApaLI, AseI, BamHI,
BclI, ClaI, DraI, EcoRI, EcoRV, FspI, HindIII,
HpaI, KpnI, NcoI, NdeI, NotI, NruI, NsiI, PstI,
PvuI, SacI, SacII, ScaI, SpeI, SspI, StuI, XbaI,
XhoI. Several sites for: SmaI. No TTA codon.

kamC from Saccharopolyspora hirsuta
AC=M64626 (Holmes et al., 1991).
No sites for: AflII, ApaI, ApaLI, AseI, BamHI,
BglII, ClaI, DraI, EcoRI, EcoRV, HindIII, HpaI,
KpnI, MluI, NaeI, NcoI, NdeI, NruI, NsiI, PstI,
PvuI, PvuII, ScaI, SmaI, SpeI, SspI, StuI, XhoI.
Several sites for: AatII, FspI, SacI, SacII. No
TTA codon.

&KDSWHU 

m21tk.wpd 16 March 2000 Tobias Kieser

BglII

0

EcoRI
KpnI

KpnI

0.03

1.05

PvuII
1.9
BamHI 2.05

gyrB
Novobiocin

2.5

BglII



0DSVRI'1$)UDJPHQWV
AvrII
BfaI

77
78

PvuII

231

BamHI

337

PmlI
BsiWI

355
414

SmaI

427

PflMI*
SacI

465
481

BglII

755

AgeI

773

MscI*

914

BbsI

1035

PstI

1177

resistant
DNA gyrase
S. spaeroides

PstI

3.6

FspI

1445

BglII

3.6

Eco57I

1456

PstI

3.6

FspI
SmaI

1526
1553

BglII

1667

ScaI

1791

BamHI

4.3

BglII

Mr=74568

PstI

1795

ApaLI

1843

4.8

Eco47II 2126
2177
NcoI
BstEII 2208

PvuII

6.0

ClaI

6.1

XbaI

7.2

MluI
BpmI

2360
2410

SmaI
BamHI

2521
2537

PstI

HindIII
PstI
BglII

7.3

gyrB, gene for novobiocin resistant DNA gyrase from S. sphaeroides
The 7.3 kb BglII fragment is from the pIJ487 derivative pLST18 (MboI partial inserted into BamHI site).
AC=Z17304 (Thiara and Cundliffe, 1988; 1989).
Selection of S. lividans clones with 750 µg ml-1 novobiocin. (MIC of S. lividans without gene:
20 µg ml-1.) The sequenced region has no sites for: AflII, ApaI, AseI, BclI, ClaI, DraI, EcoRI, EcoRV,
HindIII, HpaI, KpnI, NarI, NdeI, NotI, NruI, NsiI, PvuI, SpeI, SphI, SspI, StuI, XbaI, XhoI.
Several sites for: AatII, BamHI, BglII, FspI, NaeI, PstI , SacII, SalI, SmaI. No TTA codon.



&KDSWHU 

0DSVRI'1$)UDJPHQWV
SalI

PstI

1

?

BsiWI
NaeI
XmnI

352
386
396

SacI

545

BstEII 574

aac(3)IV
Apramycin
Geneticin
Acetyltransferase
Klebsiella
Mr=32000

NarI
BglI

789
827

MluI 1057
XhoI 1070
EarI 1126
EcoNI 1132
BsmI 1266
BstXI 1283
SacI

1296

AatII 1550

aph(4)

SacI
BspHI
PmlI
Eco57I

1
5
64
92

HphI

247

AatII
Esp31

254
293

EcoRI
BspMI
PstI
NcoI/StyI
PvuI
RsrII
NdeI

474
535
564
583
592
638
680

SacII

1008

HincII

1065

ScaI

1199

BfaI

1319

EcoRI 1770

Hygromycin

PstI

1860

NcoI

1879

Phosphotransferase

NdeI

1976

Mr=42000

SacII

2304

ScaI

2495

Bpu1102I 1381

BglII

2783

HphI

1484

BglII

1542

aac(3)IV-aph(4) operon from Klebsiella pneumoniae
The map was derived from the overlapping sequences AC=X99313 (aac(3)IV) and AC=V01499 aph(4).
In the sequenced region there are no sites for: AflII, ApaI, AseI, BamHI, BclI, ClaI, DraI, EcoRV,
FspI, HindIII, HpaI, KpnI, NotI, NruI, NsiI, PvuII, SalI, SmaI, SpeI, SphI, SspI, StuI, XbaI. Several sites
for: ApaLI, PstI, SacI. No TTA codon in either gene.
aph(4), hygromycin phosphotransferase
Expression of hygromycin resistance in E. coli (200 µg ml-1) and Streptomyces (600 µg ml-1) depends onvector promoters. aph(4) is also an important selection marker for plant transformation. (See also hyg
of S. hygroscopicus, p. 485.) The 1542 bp SacI -BglII fragment containing aph(4) has no sites for:AflII, ApaI, AseI, BamHI, BclI, ClaI, DraI, EcoRV, FspI, HindIII, HpaI, KpnI, MluI, NaeI, NarI,
NotI,NruI, NsiI, PvuII, SalI, SmaI, SpeI, SphI, SspI, StuI, XbaI, XhoI. Several sites for: ApaLI. No TTA
codon. AC=V01499 Cloning: Rao et al. (1983). Sequence: Gritz and Davies (1983); Kaster et al.
(1983). Expression in S. ambofaciens: Kuhstoss and Rao (1983).

m21tk.wpd 16 March 2000 Tobias Kieser&KDSWHU 
STOP
T4 Ter

0DSVRI'1$)UDJPHQWV
EcoRI
SmaI
BamHI
HindIII
DraI
AseI

68
102

BsaBI*

252

BsiWI
NaeI
XmnI

336
370
380



1
6
11

32

Paac

aac(3)IV
Apramycin
Acetyltransferase
Mr=3200
Klebsiella
AC=X99313

T4 Ter
STOP

SacI

529

BstEII

558

NarI
BglI

773
811

BspMI

918

MluI
XhoI
Eco47II
EarI
EcoNI
HincII
BsmI
BstXI
Bpu1102I

1041
1054
1076
1110
1116
1193
1250
1267
1278

SacI

1281

PmlI

1344

AseI
DraI
HindIII
BamHI
SmaI
EcoRI

1683
1717
1753
1774
1779
1784

Omega aac(3)IVaac(3)IV, aminoglycoside acetyltransferase inacivating apramycin and geneticin (G418)
See Table 21.3 (p. 448) for cross-resistances. 50-100 µg ml-1 apramycin or geneticin are used forselection of E. coli, S. lividans and S. coelicolor clones. aac(3)IV gives increased kanamycin andgentamicin resistance in E. coli (Davies and O'Connor, 1978; Rao et al., 1983; Stanzak et al., 1986).
Sequence of the promoter region: Kaster et al. (1983). Convenient source of gene: Omega aac(3)IV(=SaacC4) cassette cloned into the E. coli vector pHP45 AC=X99313 (Blondelet-Rouault et al., 1997).
A NcoI cassette containing aac(3)IV is described by Paradkar and Jensen (1995).

&KDSWHU 

0DSVRI'1$)UDJPHQWV

0
BamHI
PstI
0.021
HindIII 0.024

PvuII

1.0

1
6
12
18
24
30
35
39
45

BamHI
NaeI

1
26

HindIII
SphI
PstI
SalI
XbaI
BamHI
SmaI
KpnI
SacI

SpeI

144

BsrI

112

AatII
NruI

231
296
FokI

211

FspI

445

PvuII

573

SphI
XmnI
BbsI
BsaI

713
779
830
919

DraIII
HincII

999
1032I

int
SphI 1.15

Tth111I 373

584
BglII
BsaBI* 585

aacC1
Gentamicin
Acetyltransferase
E. coli
Mr=19442

BglII

Tth111I

1352

PleI

1436

BglII

EcoRV

1757

2.25
SacII
2 adjacent sites

NotI

1848

HindIII 2.575
2.579
PstI

BpmI

1963

SalI 2.589

2149
BspHI
2162
BstEII
Bpu1102I 2198

BamHI

2.6
(aadA)

HindIII

715

EcoRV

778

EagI

833

SacI
KpnI
SmaI
BamHI
XbaI
SalI
PstI
SphI
HindIII

880
886
890
895
901
907
913
919
925

1563

2.0

EcoRV 2.2

HphI

2319

pUCGM

aacC1, gentamicin acetyltransferase gene from Tn1696 (Tn21 family)
Gentamicin selection: 15 µg ml-1 for E. coli, 5-20 µg ml-1 for Streptomyces. The aacC1 gene has also
been used for promoter probing (Labes et al., 1997). The 2.6 kb BamHI fragment is from pWKR209(Wohlleben et al., 1988). The 2324 bp BamHI-HindIII fragment is from AC=X15852 (Wohlleben etal., 1989). int, Tn1696 integrase; aadA, spectinomycin-streptomycin adenyltransferase (p. 468).
No sites for: AflII, ApaI, AseI, BamHI, BclI, ClaI, DraI, EcoRI, HpaI, KpnI, MluI, NarI, NcoI, NdeI,
NsiI, PstI, PvuI, SacI, SalI, ScaI, SmaI, SspI, StuI, XbaI, XhoI. Several sites for: ApaLI, SacII. 2 TTA
codons in aacC1. Vectors containing aacC1: pGM11, pGM160, pSIT151.
aacC1 HindIII fragment from pUCGM
AC=U04610 (Schweizer, 1993). No sites for: AatII, AflII, ApaI, ApaLI, AseI, BamHI, BclI, BglII, ClaI,
DraI, EcoRI, EcoRV, FspI, HindIII, HpaI, KpnI, MluI, NaeI, NarI, NcoI, NdeI, NotI, NruI, NsiI, PstI,
PvuI, PvuII, SacI, SacII, SalI, ScaI, SmaI, SpeI, SphI, SspI, StuI, XbaI, XhoI. Several sites for: BamHI,
KpnI, PstI, SacI, SacII, SalI, SmaI, SphI, XbaI. 2 TTA codons in aacC1.

&KDSWHU 

m21tk.wpd 16 March 2000 Tobias Kieser

SacII

4

SfiI
PvuI
FspI
MscI
BstXI

10
41
160
176
179

NcoI

185

EcoNI

228

AlwI

246

StuI

312

SmaI

356

RsrII

412

ApaI

434

ApaLI

441

NcoI

491

523

SmaI

551

AatII

667

NaeI

848

aacC7
Paromomycin

117
133
166
167

BsaI
SfaNI
Eam1105
BsrI

DrdI

aacC8



0DSVRI'1$)UDJPHQWV

BfaI

453

XhoI

532

ApaLI

571

PvuI

590

Bpu1102I

771

aacC9
Neomycin

Neomycin

AgeI
Bsu36I

27
50

AvrII
StyI
SmaI
XhoI

131
131
163
175

BsaI

352

NdeI
MscI
BstXI
BsiWI
EcoNI
Eco47II
BglI

506
523
526
566
579
618
624

Acetyltransferase

Acetyltransferase

Acetyltransferase

PstI

689

S. rimosus

S. fradiae

Micromonospora chalcea

EagI

771

Mr=30389

Mr=30945

AatII
HincII
HpaI
EcoRV

841
886
886
919

NarI
SacII
BbsI
Eco57I
DraIII

1036
1040
1072
1108
1120

NruI

980

Mr=31070

SalI

1105

SacII

1229

1279
BstBI
Tth111I 1291
SalI

1297

BclI*

1357

RsrII
SmaI

1478
1489

BsiWI

1058

AatII
HincII
SalI

1111
1114
1114

AlwI

1205

EcoNI
EarI

1245
1246

AlwI

1286

FokI

1298

aacC7, paromomycin acetyltransferase gene from S. rimosus forma paromomycinus
Resistance to 120 µg ml-1 paromomycin in S. lividans on MM. Apramycin, geneticin (G418),
gentamicin, kanamycin, lividomycin, neomycin, netilmycin, sisomicin, and tobramycin are acetylated
in vitro and cross-resistance may occur. Amikacin, butirosin and kasugamycin are not acetylated. (S.
rimosus also contains a paromomycin phosphotransferase which acts synergistically with theacetyltransferase.) No sites for: AflII, ApaLI, AseI, BamHI, BglII, ClaI, DraI, EcoRI, EcoRV, HindIII,
HpaI, KpnI, MluI, NdeI, NotI, NsiI, PstI, PvuII, SacI, ScaI, SpeI, SphI, SspI, XbaI, XhoI. Several sites
for: ApaI, NarI, NcoI, SalI, SmaI. No TTA codon. AC=M22999 (Perez-Gonzalez and Jimenez, 1984;
Lopez-Cabrera et al., 1989; Perez-Gonzalez et al., 1989).
aacC8, neomycin acetyltransferase from S. fradiae
The aphI neomycin phosphotransferase gene (p. 478) is from the same S. fradiae strain. Selection with
10 µg ml-1 neomycin. AC=M55426 (Salauze et al., 1991). No TTA codon.
aacC9, neomycin acetyltransferase from Micromonospora chalcea
Selection with 10 µg ml -1 neomycin. AC=M55427 (Salauze et al., 1991). 2 TTA codons.

&KDSWHU 



STOP
T4 Ter

0DSVRI'1$)UDJPHQWV

EcoRI
SmaI
BamHI
HindIII
DraI
AseI

1
6
11
32
68
102

BsaBI*
SphI
AlwNI
XmnI

252
300
316
366

BglI

402

EagI
BbsI

409
417

ApaLI

449

BsaI

506

BglII

PleI
913
BstEII 1042
BclI*
1080I
BpmI
1113

aadA
Spectinomycin
Streptomycin
Adenyltransferase
E.coli R100.1
Mr=29332

T4 TerSTOP

Omega fragment

ApaLI

1

EcoRI
11
SacI
17
KpnI
23
SmaI
27
BamHI
32
XbaI
38
SalI,HincII 44
SphI
52
AlwNI
68
XmnI
118

1157

BglI

154

EagI
BbsI

161
169

ApaLI

201

BsaI

258

BspHI

634

BstEII
BclI*
BpmI

794
832
865

ApaLI

909

Bpu1102I 942
BspMI
990
PvuI
1012

Bpu1102I 1190
BspMI 1238
PvuI
1206

BglI

1508

BglI

1260

NaeI
BssHII

1531
1606

NaeI
BssHII

1283
1358

BspHI

1589

AseI
DraI
HindIII
PstI

1728
1762
1798
1806

BglII

1810

AseI
DraI
HindIII
BamHI
SmaI
EcoRI

1976
2010
2046
2067
2072
2077

T4 TerSTOP

pIJ2059

aadA, spectinomycin-streptomycin adenyltransferase gene from R100.1
The gene is expressed, presumably from its own promoter, in E. coli and in Streptomyces. Double
selection (Table 21.4, p.470) greatly reduces the number of spontaneous mutations.
Omega fragment from pHP45S The aadA gene is flanked by T4 transcriptional terminators and there
are STOP codons in all reading frames. AC=M60473 (Prentki et al., 1991; Prentki and Krisch, 1984).
No sites for: AatII, AflII, ApaI, BglII, ClaI, EcoRV, FspI, HpaI, KpnI, MluI, NarI, NcoI, NdeI, NotI,
NruI, NsiI, PstI, PvuII, SacI, SacII, SalI, ScaI, SpeI, SspI, StuI, XbaI, XhoI. Several sites for: ApaLI,
AseI, BamHI, DraI, EcoRI, HindIII, SmaI. 1 TTA codon. The aadA gene of the Omega fragment has
been replaced by an apramycin resistance, a hygromycin resistance or a viomycin resistance (pp. 465,
485, 483; Blondelet-Rouault et al., 1997).
aadA from pIJ2059. SphI-HindIII sequence of Omega fragment inserted into pIJ2925; M.J. Buttner(unpublished). No sites for: AatII, AflII, ApaI, ClaI, EcoRV, FspI, HpaI, MluI, NarI, NcoI, NdeI, NotI,
NruI, NsiI, PvuII, SacII, ScaI, SpeI, SspI, StuI, XhoI. Several sites for: ApaLI, BglII. 1 TTA codon.

&KDSWHU 

m21tk.wpd 16 March 2000 Tobias Kieser

T7
STOP
T4 TeraadASpectinomycin
Streptomycin
Adenyltransferase
E.coli R100.1
Mr=29332

T4 TerSTOP

SP6

pIJ4642

0DSVRI'1$)UDJPHQWV
SalI
PstI
BglII
XbaI
HindIII

1
3
35
41
47

BamHI
SacI
SmaI
HindIII
XhoI

1
10
15
21
27

EcoRI

33

BsaI
AseI
HindIII
DraI
AseI

39
60
104
140
174

BamHI
SacI
AseI

407
417
451

BsaBI*
SphI
AlwNI
XmnI

324
372
388
438

BsaBI*
SphI
AlwNI
XmnI

601
649
665
715

BglI

474

BglI

751

EagI
BbsI

481
489

EagI
BbsI

758
766

ApaLI
BsaI

521
578

ApaLI
BsaI

798
855

BstEII
BclI*
BpmI

1114
1152
1185

BstEII
BclI*
BpmI

1391
1429
1462

ApaLI

1229

ApaLI

1506

ter(fd)Bpu1102I 1262
BspMI 1310
PvuI
1332

Bpu1102I 1539
BspMI 1587
1609
PvuI

BglI

1580

BglI

1857

NaeI

1603

NaeI

1880

BssHII

1678

BssHII

1955

AseI
DraI
HindIII

2048
2082
2118

AseI
SacI
BamHI

2328
2326
2336

AseI
XhoI
HindIII
SmaI
SacI
BamHI

2333
2399
2405
2409
2412
2425

HindIII
XbaI
BglII
PstI
SalI

2696
2702
2708
2740
2742

ter(fd)pIJ4676

aadA from pIJ4642: Omega fragment with outward-reading SP6 and T7 promoters (T. Kieser etal., unpublished)No sites for: AatII, AflII, ApaI, ApaLI, AseI, BclI, BglII, ClaI, DraI, EcoRI, EcoRV, FspI, HpaI, KpnI,
MluI, NaeI, NarI, NcoI, NdeI, NotI, NruI, NsiI, PstI, PvuI, PvuII, SacII, SalI, ScaI, SpeI, SphI, SspI, StuI,
XbaI. Several sites for: ApaLI, AseI, DraI, HindIII. 1 TTA codon.
aadA from pIJ4676 aadA flanked by ter(fd) (T. Kieser and R.E. Melton, unpublished).

&KDSWHU 

0DSVRI'1$)UDJPHQWV

Table 21.4 Selection for the aadA spectinomycin/streptomycin adenyltransferase geneµg ml-1 antibiotic used for selection
StrainE. coli

S. lividans

Medium

LB

MM

R2YE

R2YE overlay

Spectinomycin

20-10050100

100200400

Streptomycin

25

10

50

30

Bialaphos and phosphinothricin resistance genes
The phosphinothricin acetyltransferase genes bar and pat confer resistance to the herbicidal andbactericidal glutamine synthetase inhibitors bialaphos and phosphinothricin in Streptomyces, E. coli, andalso in plants. S. coelicolor contains a cryptic bar homologue that conferred resistance when cloned in
pIJ702 (high copy number). AC=M62753 (Bedford et al., 1991). Selection of resistant Streptomyces
clones: 50 µg ml-1 bialaphos or phosphinothricin in MM.
bar, bialaphos resistance gene from S. hygroscopicus
The 2.1 kb PstI-BamHI fragment shows DNA present in pBG4 or pBG3 (Thompson et al., 1987). The
sequenced region has no sites for: AatII, AflII, ApaLI, AseI, BclI, ClaI, DraI, EcoRV, HpaI, MluI,
NarI, NcoI, NdeI, NotI, NruI, NsiI, PvuI, ScaI, SpeI, SspI, StuI, XbaI, XhoI. Several sites for: KpnI,
NaeI, SmaI, SphI. No TTA codon. AC=X05822 (Thompson et al., 1987). Cloning: Murakami et al.
(1986). The fragment from pIJ4104 lacks bacterial expression signals. AC=X17220 (White et al.,1990).
pat, phosphinothricin acetyltransferase from a spontaneous phosphinothricin-resistant
S. viridochromogenes
The 4 kb BamHI fragment is from pEB2 (Strauch et al., 1988). The sequenced part has no sites for:AflII, ApaI, ApaLI, AseI, BamHI, BclI, ClaI, DraI, EcoRI, EcoRV, FspI, HindIII, HpaI, KpnI, MluI, NarI,
NdeI, NotI, NruI, NsiI, PstI, PvuI, PvuII, SacII, SalI, ScaI, SpeI, SphI, SspI, XbaI, XhoI. Several sites
for: BglII, NaeI. No TTA codon. AC=M22827 (Strauch et al., 1988; Wohlleben et al., 1988).

m21tk.wpd 16 March 2000 Tobias Kieser&KDSWHU 

0DSVRI'1$)UDJPHQWV





&KDSWHU 

0DSVRI'1$)UDJPHQWV

Bleomycin resistance genes from S. verticillus
In the centre of the figure is the 6.3 kb SacI fragment of pMSA-1 (pIJ702 derivative) conferring
resistance to >600 µg ml-1 bleomycin on S. coelicolor (200 µg ml-1 was used for selection). blmA, blmB,
and ORF6 contribute to the high resistance. AC=L26955 (Calcutt and Schmidt, 1994). Cloning:
Sugiyama et al. (1994b). Acetyltransferase: Sugiyama et al. (1994a). No sites for: AflII, AseI, BglII,
ClaI, DraI, EcoRV, HindIII, HpaI, KpnI, NdeI, NruI, NsiI, PstI, PvuI, ScaI, SpeI, SphI, SspI, StuI, XbaI,
XhoI.
blmA, bleomycin binding protein
The EcoRI-BamHI fragment confers resistance to 400 µg ml-1 bleomycin. No sites for: AflII, ApaLI,
AseI, BamHI, BclI, BglII, ClaI, DraI, EcoRI, EcoRV, FspI, HindIII, HpaI, KpnI, MluI, NdeI, NotI, NruI,
NsiI, PstI, PvuI, PvuII, SacI, SalI, ScaI, SpeI, SphI, SspI, StuI, XbaI, XhoI. Several sites for: ApaI, NaeI,
SacII, SmaI. No TTA codon.
blmB, bleomycin acetyltransferase
The 3 kb BamHI fragment confers resistance to 400 µg ml-1 bleomycin (note, phleomycin is notacetylated in vitro by BlmB). No sites for: AflII, ApaLI, AseI, BamHI, BglII, ClaI, DraI, EcoRV,
HindIII, HpaI, KpnI, MluI, NdeI, NotI, NruI, NsiI, PstI, PvuI, SacI, ScaI, SpeI, SphI, SspI, StuI, XbaI,
XhoI. Several sites for: AatII, ApaI, BclI, FspI, NaeI, NarI, SacII, SalI, SmaI. No TTA codon.

bleSh, gene for bleomycin binding protein from Streptoalloteichus hindustanus
The inserts from the plasmids pUT56 and pUT58 have no bacterial expression signals.
AC=X52869 (Drocourt et al., 1990). No sites in core region (black bar) for: AflII, AseI, BclI, ClaI,
DraI, EcoRI, FspI, HindIII, HpaI, KpnI, MluI, NarI, NruI, NsiI, PstI, PvuI, PvuII, SacI, SalI, ScaI, SpeI,
SphI, SspI, StuI, XbaI, XhoI. No TTA codon.

m21tk.wpd 16 March 2000 Tobias Kieser&KDSWHU 

0DSVRI'1$)UDJPHQWV

ORF 6





&KDSWHU 

0DSVRI'1$)UDJPHQWV
PstI
SfiI*
BsiWI

1
9
59

NheI
BfaI

167
168

XmnI
PleI
SacII

266
279
303

ApaI

506

BsaBI*
BstEII

557
599

AatII

633

SacI
ApaLI
HincII

651
687
714

PvuII

795

PvuI

1078

EcoNI

1124

DraIII

1239

Esp3I

1337

NheI
BfaI
AatII

1387
1388
1421

NcoI
SphI

1583
1594

cac
Capreomycin
Acetyltransferase
S. capreolus

cac, capreomycin acetyltransferase gene from S. capreolus
Cac, is active against all four components of the capreomycin complex but viomycin is not acetylated.
vph does, however, give partial resistance to capreomycin (p. 482). Introducing cac on a pIJ101
derivative into S. lividans increased the MIC from 20 to >1000 µg ml-1 capreomycin in NE agar
consisting of 10 g l-1 glucose, 2 g l-1 yeast extract, 2 g l-1 Casaminoacids and 1 g l-1beef extract, pH7(Thiara and Cundliffe, 1995). No sites for: AatII, AflII, ApaI, ApaLI, AseI, BamHI, BclI, BglII, ClaI,
DraI, EcoRI, EcoRV, FspI, HindIII, HpaI, KpnI, MluI, NaeI, NarI, NcoI, NdeI, NotI, NruI, NsiI, PstI,
PvuI, PvuII, SacI, SacII, SalI, ScaI, SmaI, SpeI, SphI, SspI, StuI, XbaI, XhoI. Several sites for: AatII,
NaeI, SmaI. No TTA codon. AC=U13077.

m21tk.wpd 16 March 2000 Tobias Kieser&KDSWHU 

0DSVRI'1$)UDJPHQWV

BamHI

BclI*
SmaI
AvaI
SacII

SacII

AvaI
SmaI

SacII
SacII

catSa

AvaI

Chloramphenicol
Acetyltransferase
S. acrimycini
Mr=24908

SacII

198

EarI

277

SmaI

337

Bpu1102I 399

AvaI

SmaI

SfiI

AlwI

419

PvuI
PmlI
ApaLI
BsiWI
AatII
EcoNI
BspMI
HincII
DrdI
SfaNI
BbsI

436
449
497
502
525
575
581
606
651
674
690

AlwI

983

SrfI

1122

BclI*

BamHI

SmaI

1123

ApaI

1149

SmaI

1152

PleI
BclI*

1166
1198

catSa, chloramphenicol acetyltransferase gene from S. acrimycini
Selection of clones using 50 µg ml-1 chloramphenicol in all media using the chloramphenicol
hypersensitive (<1µg ml-1) S. lividans mutant M252 (Bibb and Cohen, 1982). (S. lividans and S.
coelicolor have a natural unstable chloramphenicol resistance pp. 20, 427.)
The 2.7 kb BamHI fragment is from the pIJ61 derivative pIJ871. Cloning of this fragment into
an E. coli vector and selection for chloramphenicol resistance resulted in deletion of the upstream region,
indicating the presence of a transcriptional terminator.
The sequenced region has no sites for: AflII, AseI, BamHI, BglII, ClaI, DraI, EcoRI, EcoRV,
FspI, HindIII, HpaI, KpnI, MluI, NcoI, NdeI, NotI, NruI, NsiI, PstI, PvuII, SacI, SalI, ScaI, SpeI, SphI,
SspI, StuI, XbaI, XhoI. Several sites for: NaeI, NarI, SacII, SmaI. No TTA codon. Cloning: Gil et al.
(1985). Sequence: Murray et al. (1989); the sequence is not in the EMBL database. Expression froma leaderless mRNA: Wu and Janssen (1997).

&KDSWHU 

0DSVRI'1$)UDJPHQWV
BamHI

nat1
Nourseothricin
Acetyltransferase
S. noursei
Mr=2056661

Eam1105I
ApaI
SalI, HincII

32
51
67

KpnI
SmaI

232
248

FokI

278

Tth111I
BbsI

297
303

StyI

370

PleI
BsiWI

432
451

DrdI
BssHII
NruI
NarI
BpmI
BstEII

528
569
586
612
630
635

HincII

645

MluI

664

FokI

676

SphI
BfaI

760
773

BamHI

1.12

nat1, nourseothricin acetyltransferase gene from S. noursei
The nat1 gene cloned on pIJ702 increased the resistance of S. lividans from 5 to 10,000 µg ml-1. The
sequenced region has no sites for: AatII, AflII, ApaLI, AseI, BamHI, BclI, BglII, ClaI, DraI, EcoRI,
EcoRV, FspI, HindIII, HpaI, NcoI, NdeI, NotI, NsiI, PstI, PvuI, PvuII, SacI, SacII, ScaI, SpeI, SspI, StuI,
XbaI, XhoI. Several sites for: NaeI. No TTA codon. AC=X73149 (Krügel et al., 1988, 1993).
Streptothricin is similar to nourseothricin and the resistance gene stat can be used as selection marker
for Leishmania (Horinouchi et al., 1987).

m21tk.wpd 16 March 2000 Tobias Kieser&KDSWHU 

pacPuromycin
Acetyltransferase
S. alboniger
Mr=21531

dmpM



0DSVRI'1$)UDJPHQWV
XmnI

16

NotI

181

AatII

296

BstEII

388

AatII

794

NcoI

901

BamHI

1030

XmnI
AlwI*
EcoNI
BspMI

1
35
38
70

NotI
AgeI
FokI

161
173
187

Tth111I

273

AatII
SmaI

276
283

BsiWI
SalI
RsrII
BstEII
EarI

290
342
349
368
387

BamHI

1548

PmlI

1621

StuI

563

NotI
NotI

1629
1666

MscI*

607

BssHII
BsaI

689
714

AatII

774

NcoI

881

SmaI

902

ApaLI

2149

BsaBI* 2371
NotI

2456

BamHI

2598

pur8

BamHI

2720

Puromycin
Efflux
Mr=51851

PvuI
NruI*

2838
2841

BclI*

3105

BamHI

3228

AlwNI

3412

BamHI

3458

BamHI 1006

EcoRV 3502
NcoI

3519

Eco57I 3536

EcoRI

4.2

pac, puromycin acetyltransferase gene, and pur8, puromycin export resistance gene form S.
alboniger Both genes confer >500 µg ml-1 puromycin resistance on S. lividans. The resistance levels
are medium-dependent and highest in strains containing both genes. No known cross-resistance to otherantibiotics for both genes. Selection of clones in the puromycin-sensitive S. lividans strain CP2 using
100 µg ml-1 puromycin (overlay of R2YE plates). (Strain 1326 requires 200 µg ml-1.) The pac gene canbe used for selection in mammalian cells (Vara et al., 1986). The sequenced region of the 4.2 kb
XmnI-EcoRI fragment has no sites for: AflII, ApaI, AseI, BglII, ClaI, DraI, EcoRI, FspI, HindIII,
HpaI, KpnI, NdeI, NsiI, PstI, PvuII, SacI, ScaI, SpeI, SphI, SspI, XbaI, XhoI. No TTA codon in pac andpur8. The 1 kb pac fragment (right) has no sites for: AflII, ApaI, ApaLI, AseI, BamHI, BclI, BglII, ClaI,
DraI, EcoRI, EcoRV, FspI, HindIII, HpaI, KpnI, MluI, NaeI, NdeI, NruI, NsiI, PstI, PvuI, PvuII, SacI,
ScaI, SpeI, SphI, SspI, XbaI, XhoI. Several sites for: AatII, NarI, SacII, SmaI. No TTA codon.
AC=X76855, M25346. Acetyltransferase: Lacalle et al. (1989); Vara et al. (1985). pur8: Tercero etal. (1998); Vara et al. (1988).

&KDSWHU 
BamHI

PvuII
SacII
PstI

SacII
SacII

aphI
Neomycin
Phosphotransferase
S. fradiae
Mr=29000

0DSVRI'1$)UDJPHQWV
1

28
31

BsaI

185

BssHII

231

NaeI

302

Esp3I
NcoI

371
397

DraIII

444

BamHI

607

370

891
978

1131
SacI
BamHI 1225
PstI
SacI
SmaI
BstEII
SalI
XbaI
KpnI
SacII
KpnI

1482
1527
1608
1622
1773
1819
1889
1931
1980

BstEII
SalI

2221
2291

SmaI
BstEII

BglI
PmlI

2890

T

EcoNI

863

PstI

864

PflMI*

896

Tth111I
NcoI
SmaI
BstEII
AatII

939
953
990
1004
1020

SalI

1155

XbaI

1201

KpnI
BsaI

1271
1288

KpnI

1362

BamHI 3430

aphI, neomycin phosphotransferase gene from S. fradiae
Note, ribostamycin resistance (MIC 250 µg ml-1 in S. lividans/pIJ385) but no kanamycin resistance.
Resistance is medium-dependent: 1 µg ml-1 neomycin for selection on MM, 10 µg ml-1 for R2YE. The
3.4 kb BamHI fragment has no sites for BclI, BglII, ClaI, EcoRI, EcoRV, HindIII, PvuII, SphI, XhoI.
The sequenced region has no sites for: AflII, ApaLI, AseI, BclI, BglII, ClaI, DraI, EcoRI, EcoRV, FspI,
HindIII, HpaI, MluI, NdeI, NotI, NruI, NsiI, PvuI, PvuII, ScaI, SpeI, SphI, SspI, StuI, XhoI. Several sites
for: ApaI, KpnI, NarI, NcoI, SacI, SacII. No TTA codon. Vectors containing aphI: pIJ61, pIJ680.
AC=K00432, X02394. Cloning: Thompson et al. (1980, 1982c). Sequence: Thompson and Gray
(1983). Transcription and promoter sequence: Janssen et al. (1989). Terminator: Pulido and Jimenez(1987).

&KDSWHU 

m21tk.wpd 16 March 2000 Tobias Kieser

km
Kanamycin
Neomcycin
Phosphotransferase
Tn903
Mr=30721



0DSVRI'1$)UDJPHQWV

EcoRI
BamHI
SalI,HincII
PstI

1
9
15
21

BspHI

97

XhoI
BanII
NruI
ClaI

177
230
234
269

RsaI
SmaI
EcoNI
SspI

411
451
492
502

PvuI

576

rphS1

BamHI
PmlI
BssHII
Bpu1102I

1
32
87
91

ApaI

99

StuI

213

XmnI
AvrII

309
335

BfaI

336

DraIII
PleI

336
433

ApaI

469

SacI

475

SacII

647

EcoNI

822

Ribostamycin
Phosphotransferase
S. ribosidifiscus

HindIII

697

AseI

770

DrdI

898

PflMI

871

BspMI

1003

Sau96I

1131

StuI*

1201

PstI
SalI,HincII
BamHI
EcoRI

1261
1267
1273
1277

Mr=29011

ApaI

1103

PvuII
XbaI*

1141
1160

BfaI

1161

Esp3I

1172

SmaI

1.66 kb

km, kanamycin, neomycin phosphotransferase gene from Tn903 (E. coli)
The fragment shown is from pUC4K AC=X06404 Pharmacia (Taylor and Rose, 1988). The km gene isalso in pACYC177. No sites for: AatII, AflII, ApaI, ApaLI, BclI, BglII, DraI, EcoRI, EcoRV, FspI,
HpaI, KpnI, MluI, NaeI, NarI, NcoI, NdeI, NotI, PvuII, SacI, SacII, ScaI, SpeI, SphI, XbaI. Several sites
for: BamHI, NsiI, PstI, SalI. 6 TTA codons.
rphS1, ribostamycin phosphotransferase gene from S. ribosidificus
S. lividans containing rphS1 is highly resistant to ribostamycin (MIC 750 µg ml-1) and less resistant toneomycin B (MIC 20 µg ml-1). No sites for: AflII, ApaLI, AseI, BclI, BglII, ClaI, DraI, EcoRI, EcoRV,
FspI, HindIII, HpaI, KpnI, MluI, NdeI, NotI, NruI, NsiI, PstI, ScaI, SmaI, SpeI, SphI, SspI, XhoI. Several
sites for: AatII, ApaI, NaeI, NarI, NcoI, PvuI, SalI. No TTA codon. Cloning: Murakami et al. (1983).
AC=M22126 (Hoshiko et al., 1988).

&KDSWHU 

0DSVRI'1$)UDJPHQWV

neo, aminoglycoside phosphotransferase gene, and ble, gene for bleomycin binding protein fromTn5 (E. coli)
The neo gene confers resistance to neomycin, kanamycin and geneticin, but almost no resistanceto apramycin and gentamicin (p. 447). The neo marker of Supercos1 is routinely used for selection in
S. coelicolor (Redenbach et al., 1996). In pIJ486 and pIJ487 it is used as indicator gene for promoter
probing (Ward et al., 1986).
Bleomycin selection has not been used widely in Streptomyces because of the high cost of theantibiotic; phleomycin or zeocin are cheaper alternatives.
The Tn5 streptomycin resistance gene (str) is incomplete on the HindIII-BamHI fragment shown.
It is not expressed in E. coli, and it has not been used in Streptomyces.
The 1.9 kb HindIII-BamHI fragment has no sites for: AatII, AflII, ApaI, ApaLI, AseI, BamHI,
ClaI, DraI, EcoRI, EcoRV, HindIII, HpaI, KpnI, MluI, NdeI, NotI, NsiI, PvuI, SacI, SacII, ScaI, SpeI,SspI, StuI, XbaI. Several sites for: NaeI, NarI, PstI, PvuII. No TTA codon in neo. AC=U00004.
The FD Neo-S cassetteThis has a strong artificial promoter which is active in Streptomyces and E. coli (Denis and Brzezinski,1991). No sites for: AatII, AflII, ApaI, ApaLI, AseI, BclI, DraI, EcoRV, HpaI, MluI, NdeI, NotI, NruI,
NsiI, PvuI, SacII, ScaI, SmaI, SpeI, SspI, StuI, XbaI. Several sites for ClaI. No TTA codon. The FD
Neo-S cassette is part of pFD660 AC=AF000429 (Denis and Brzezinski, 1992).

&KDSWHU 

m21tk.wpd 16 March 2000 Tobias Kieser

neo
APH II
Mr=29053



0DSVRI'1$)UDJPHQWV

HindIII

1

AlwNI

103

BfaI

185

PvuII
BsaBI*

228
315

BglII
BclI*

320
325

BsaBI

339

EagI

390

DrdI

505

PstI

533

MscI
FspI

564
584

PvuII

588

Tth111I

599

SphI
NcoI

885
916

RsrII

999

EcoRI
SacI
KpnI
BamHI
NheI
XhoI
BglII
ClaI*

neo
Mr=29053

1
7
13
21
31
36
83
90

BsaBI*
EagI

106
157

NarI
DrdI
MscI
FspI
PvuII
Tth111I
HphI

250
272
331
351
355
366
437

NcoI
StyI
NaeI
RsrII

683
683
751
766

ClaI*

943

SalI
PstI
SphI
HindIII

957
963
969
975

ble
Mr=14065

BstBI

1165

SmaI
NruI

1321
1337

AlwnI
PvuII

1350
1350

PstI

1456

SalI
1489
BspHI 1546
DraIII 1580
Eco47II 1594
XhoI
1637
BsmI
1696
BstEII 1716
BglI
1767

(Str)

BamHI 1861

Tn5

FD Neo-S&KDSWHU 

0DSVRI'1$)UDJPHQWV

vph, viomycin phosphotransferase gene from S. vinaceus
Clean selection of Streptomyces and E. coli clones is possible with $30-50 µg ml-1 viomycin on MM orDNA, respectively. The level of viomycin resistance depends critically on the amount of salt in themedium; more viomycin is required at higher salt concentrations; R2YE and L agar require much higher
viomycin concentrations.
Vph also phosphorylates capreomycins except capreomycin 1B, which is present in variable
small amounts in commercial capreomycin (complex) preparations. If necessary, viomycin can be
replaced by commercially available capreomycin provided that it is a batch with a low capreomycin 1B
content. The useful concentration range of capreomycin is narrow: 25-50 µg ml-1 for R2YE and 2.5-10µg ml-1 in YEME (Lee et al., 1987).
Expression of vph from the commonly used 2 kb BamHI or 942 bp SphI fragments depends ontranscriptional readthrough from vector promoters. It seems that a weak promoter is sufficient for highlevel resistance even when the gene is present at low copy number, and non-expression has not beenreported in any situation in Streptomyces. Viomycin resistance has been expressed in Streptomyces andE. coli (e.g. transcribed from P tet of pBR322).
In the 2 kb BamHI fragment there are no sites for: BclI, BglII, ClaI, EcoRV, HindIII, KpnI,
XbaI, XhoI. The sequenced region has no sites for: AflII, ApaI, ApaLI, AseI, BamHI, BclI, BglII, ClaI,
DraI, EcoRI, EcoRV, FspI, HindIII, HpaI, KpnI, MluI, NcoI, NdeI, NruI, NsiI, PstI, PvuII, ScaI, SpeI,SspI, StuI, XbaI, XhoI. Several sites for: SacI, SmaI, SphI. No TTA codon.
Vectors with vph: pIJ101: pIJ364, pIJ699; SCP2*: pIJ920, pIJ4231, Tn4560; MC31: KC400,
KC401, KC857, KC858, KC860. Convenient sources for the vph gene: pIJ36 BamHI fragment(Thompson et al., 1982a). vph-oriT cassette on HindIII or BamHI fragment (Servant et al., 1993).
Cloning: Thompson et al. (1980, 1982c). AC=X02393 (Bibb et al., 1985b). Promoters: Janssen
and Bibb (1989). Suppressible mutations: Paradiso et al. (1987).
Svph
The viomycin resistance is expressed in Streptomyces and in E. coli from the aadA promoter of theOmega fragment (p. 468). No sites for: AflII, ApaI, BclI, BglII, ClaI, EcoRV, FspI, HpaI, KpnI, MluI,
NcoI, NdeI, NruI, NsiI, PstI, PvuI, PvuII, SspI, StuI, XbaI, XhoI. Several sites for: AseI, DraI, HindIII,
SmaI, SphI. No TTA codon. AC=X99314 (Blondelet-Rouault et al., 1997).

&KDSWHU 

m21tk.wpd 16 March 2000 Tobias Kieser

0DSVRI'1$)UDJPHQWV

PvuII PvuII
SalI
SalI
SacII SstIISTOP
T4 Ter

PvuII PvuII

BamHI
SphI

1
43

vph
Viomycin
Phosphotransferase
S. vinaceus

Mr=29000

SacI
SacII
BstEII
SalI

610
647
661
727

SphI

985

SacI

1114

BamHI

1

SphI
NaeI
NaeI
SmaI

43
60
131
142

Eam1105I

161

SmaI

233

NotI
AatII

238
268

NarI

334

DrdI

453

SacI

610

EcoRI
SmaI
BamHI
HindIII
DraI
AseI

1
6
11
32
68
102

BsaBI*

252

SphI

300

XmnI
BbsI
ApaLI
BsaI

366
417
449
506

SphI
SmaI

1090
1189

PaadA

Eam1105I 1208
PflMI
1233
SmaI

1280

NotI
AatII
NarI
BsmI
DrdI

1285
1315
1381
1424
1500

SacI
1657
SacII
1694
BstEII
1708
SalI, HincII 1774
Tth111I
1783

SacII
647
BstEII
661
SalI, HincII 727
Tth111I
736

BpmI
AgeI
PstI

SalI

1370

1500

BstEII 1670

BpmI

886

AgeI

953

SphI

1002

PvuI

1072

SacI

1114

T4 Ter
STOP

SphI

2049

BspMI

2073

AseI
DraI
HindIII
BamHI
SmaI
EcoRI

2490
2524
2560
2581
2586
2591

Omega-vph
BamHI 2000

1933
2000



&KDSWHU 

0DSVRI'1$)UDJPHQWV

hyg, hygromycin phosphotransferase gene from S. hygroscopicus
The first 277 bp of the fragment shown (BamHI-MboI fragment) are from within the ORF of the aphI
gene (p. 478) and have no known function. Expression of hygromycin resistance in E. coli (50 µg ml-1)
depends on transcriptional readthrough from vector promoters. The level of resistance depends on theamount of salt in the medium; media with a high salt content require more hygromycin. Note that
hygromycin is light-sensitive.
No sites for: AflII, ApaLI, AseI, ClaI, DraI, EcoRV, FspI, HindIII, HpaI, MluI, NcoI, NdeI,
NsiI, PvuI, PvuII, ScaI, SpeI, SspI, StuI, XbaI, XhoI. Several sites for: ApaI, BglII, NaeI, NarI, SacII,
SmaI. 1 TTA codon. AC=X03615 (Zalacain et al., 1986). Cloning: Malpartida et al. (1983).
Expression in E. coli and assay: Zalacain et al. (1987). Convenient source: pIJ963 (Lydiate et al.,1989). See also aph(4) of Klebsiella (p. 464).
Vectors containing hyg: pIJ4231, pIJ698, pIJ941, pIJ4696, pIJ4697 (IS117), pJAR4, pJOE829,
PM8 (phage), xylE-hyg cassette (p. 495).
Shyg
AC=X99315 (Blondelet-Rouault et al., 1997). hyg inserted into the Omega fragment (p. 468).
The hygromycin resistance is expressed from Plac in Streptomyces and in E. coli.
No sites for: AflII, BglII, EcoRV, FspI, HpaI, KpnI, MluI, NcoI, NdeI, NsiI, PstI, PvuI, PvuII,
ScaI, SpeI, SphI, SspI, StuI, XbaI, XhoI. Several sites for: ApaI, AseI, BamHI, DraI, EcoRI, HindIII,
NarI SmaI, SacII. 1 TTA codon.

&KDSWHU 

m21tk.wpd 16 March 2000 Tobias Kieser

Phyg

hyg
Hygromycin

BglII

1

SphI
PstI

13
19

DrdI

73

(Sau3AI)

277

PflMI*

314

DrdI
SacII

404
431

NruI

452

EcoRI
SacI
BclI*

583
619
701

BspEI

732

BsiWI
BglI

761
800

SacII

810

DraIII
BbsI

970
1018

SacII

1068

Phosphotransferase
S. hygroscopicus
Mr=37021

EcoRI
SmaI
BamHI
HindIII
DraI
AseI

1
6
11
32
68
102

BsaBI*

252

PflMI*

426

DrdI
NruI

516
564

STOP
T4 Ter

Plac

Phyg

EcoRI

695

SacI

731

BclI*
BsiWI
BglI

813
873
912

Bbs
BspMI
BstEII
SalI
AatII

1130
1287
1330
1343
1355

BsaI

1529

SmaI

1622

NotI

1654

SmaI

1688

hyg
Hygromycin
Phosphotransferase
S. hygroscopicus

Mr=37000T

0DSVRI'1$)UDJPHQWV

BstEII
SalI
AatII

1218
1231
1243

BsaI

1417

BspEI*
SmaI

1449
1510

NotI

1542

Bpu1102

1764

SmaI
ApaI
ApaI

1576
1579
1636

ClaI

1853

BspHI

2090

AseI
DraI
HindIII
BamHI
SmaI
EcoRI

2229
2263
2299
2320
2325
2330

Ter

Bpu1102I 1652
BamHI 1739

pIJ963

SmaI

1744

KpnI

1748

BglII

1759

T4 TerSTOP

Omega-hyg

&KDSWHU 

0DSVRI'1$)UDJPHQWV

SphI
XmnI
BglII
EcoRI
DrdI
BsiWI
AgeI
Esp3I

1
1
61
126
128
177
194
205
246
278

MluI
HincII

454
458

PflMI

530

spcN

BstXI

589

Spectinomycin
Phosphotransferase
S. flavopersicus

BspEI
NaeI

645
677

NarI

768

NruI

865

EagI

955

AatII

996

BsaI

1182

BpmI

1264

PvuII

1364

BamHI

Mr=36090
AC=U70376

spcN, spectinomycin phosphotransferase from S. flavopersicus
Selection of S. lividans clones in MM containing 100 µg ml -1 spectinomycin.
The 1364 bp fragment has no sites for: AflII, ApaI, AseI, BclI, ClaI, DraI, EcoRV, FspI, HindIII,
HpaI, KpnI, NdeI, NotI, NsiI, PstI, PvuI, SacI, SspI, StuI, XbaI, XhoI. Several sites for: ApaLI, NcoI,
SacII, SmaI. No TTA codon. AC=U70376 (Lyutzkanova et al., 1997).

m21tk.wpd 16 March 2000 Tobias Kieser&KDSWHU 

0DSVRI'1$)UDJPHQWVStreptomycin phosphotransferases
sph, from S. glaucescens
Selection with 50 µg ml -1 streptomycin. The sph gene of S. glaucescens is frequently deleted together
with the chromosome end (Hintermann et al., 1984). The sequenced region has no sites for: AatII,
AflII, ApaI, ApaLI, AseI, BamHI, BclI, BglII, ClaI, DraI, EcoRI, EcoRV, FspI, HindIII, HpaI, KpnI,
MluI, NaeI, NarI, NcoI, NdeI, NotI, NruI, NsiI, PstI, PvuI, PvuII, SacI, SacII, SalI, ScaI, SmaI, SpeI,
SphI, SspI, StuI, XbaI, XhoI. Several sites for: FspI, MluI, NaeI, NarI, SacII. 1 TTA codon.
AC=X05648 (Hintermann et al., 1984; Vögtli and Hütter, 1987).
Several other streptomycin phosphotransferase genes have been sequenced. S. griseus contains
two phosphotransferases, AphD and AphE, which phosphorylate hydroxyl groups on the streptidine andon the N-methylglucosamine part of streptomycin, respectively (Heinzel et al., 1988).

&KDSWHU 

0DSVRI'1$)UDJPHQWV

cmlv

KpnI

1

EcoRI

154

BclI*

395

MscI
BstXI
SrfI

443
446
475

SmaI

476

BspHI*

491

StuI*

806

PmlI

1258

BstEII

1411

Chloramphenicol
EffluxS. venezuelae
Mr=43811

cpt
Chloramphenicol
Phosphotransferase
Mr=18784

BclI*

1425

BpmI

1478

SalI

1517

AlwI
Eco47II

1674
1719

ApaLI

1853

SalI

1883

AgeI
2105
Eam1105I 2113
SmaI

2144

AscI

2125

BspMI

2266

SacI

2350

cmlv, chloramphenicol efflux resistance, and cpt, chloramphenicol phosphotransferase gene fromS. venezuelae
cmlv and cpt together gave resistance to 25-50 µg ml -1 chloramphenicol in S. lividans M252.
No sites for: AflII, AseI, BamHI, BglII, ClaI, DraI, EcoRV, FspI, HindIII, HpaI, MluI, NcoI, NdeI, NotI,
NsiI, PstI, PvuII, SacII, ScaI, SpeI, SphI, SspI, XbaI, XhoI. Several sites for: AatII, ApaI, BclI, NaeI,
NarI, NruI, PvuI, SalI, SmaI. No TTA codon. AC=U09991 (Mosher et al. 1990, 1995).

&KDSWHU 

m21tk.wpd 16 March 2000 Tobias Kieser

BglII

BbsI
NotI
AgeI

SfiI
AatII
RsrII

nonR
NonactinS. griseus
Mr=30610

1

223
280
307

549
606
650

BsiWI

801

PflMI

878

PvuI

HincII

0DSVRI'1$)UDJPHQWV
XbaI

1

KpnI

83

Esp3I

184

PvuII

207

SacI
SphI
SmaI

229
281
377

HindIII

551

DrdI

651

ApaLI

1335

NcoI

1716

PvuII

1796

tnrB2
Tetronasin
EffluxS. longisporoflavus
Mr=32278

1168

1347

tnrB3
Tetronasin
Mr=35251

EcoNI

1517

AlwNI
NdeI
Bpu1102I
SacI

1719
1746
1769
1772

AscI
BstXI
PleI
BamHI

Bpu1102I 1987

PvuI

2217

1888

XhoI

2628

2049
2070
2072

MluI

2829

KpnI

2844

nonR, nonactin efflux resistance gene from S. griseus
No sites for: AflII, ApaLI, AseI, BamHI, BclI, BglII, ClaI, DraI, EcoRI, EcoRV, HindIII, HpaI, KpnI,
MluI, NcoI, NruI, NsiI, PstI, PvuII, SacII, SalI, ScaI, SpeI, SphI, SspI, XbaI, XhoI. Several sites for:
ApaI, FspI, NaeI, NarI, SmaI, StuI. No TTA codon. AC=M75853 (Plater and Robinson, 1992).
tnrB2,3, tetronasin efflux resistance gene from S. longisporoflavus
Selection in S. lividans TK64 with 10 µg ml-1 tetronasin in medium contiaining 0.2 M NaCl. S. lividans
TK24 seems to be naturally resistant to tetronasin (H. Lewis, personal communication). No sites for:
AatII, AflII, ApaI, ApaLI, AseI, BamHI, BclI, BglII, ClaI, DraI, EcoRI, EcoRV, FspI, HpaI, NdeI, NotI,
NruI, NsiI, PstI, ScaI, SpeI, SspI, StuI. Several sites for: AatII, KpnI, NaeI, NarI, PvuII, SacII, SalI.
No TTA codon in either ORF. AC=X73633 (Linton et al., 1994). Review about related polyether
antibiotics: Birch and Robinson (1995).



&KDSWHU 

0DSVRI'1$)UDJPHQWV

cmlSl, chloramphenicol efflux resistance gene from S. lividans
Clones confer resistance to 200 µg ml-1 chloramphenicol on a chloramphenicol-sensitive S. lividans
derivative M252.
No sites for: AflII, ApaI, AseI, BamHI, BglII, ClaI, DraI, EcoRI, EcoRV, HindIII, HpaI, KpnI,
MluI, NdeI, NotI, NsiI, PstI, PvuI, PvuII, ScaI, SpeI, SspI, StuI, XbaI, XhoI. Several sites for: AatII,
BclI, NaeI, NarI, NcoI, NruI, SacII, SalI. No TTA codon. AC=X59968 (Dittrich et al., 1991).

Tetracycline resistances
Tetracycline resistance can be conferred by three different mechanisms: efflux resistances (e.g. tet of
pBR322 and tetB of S. rimosus), ribosomal protection by proteins similar to EF-G and EF-Tu (e.g otrAof S. rimosus and tetSl of S. lividans), and tetracycline modification which was found only in Bacteroides
strains (Salyers et al., 1990).
otrA, oxytetracycline resistance, ribosomal protection, from oxytetracycline-producing S. rimosus
Selection of clones in an oxytetracycline-sensitive S. rimosus mutant using 25 µg ml-1 oxytetracycline.
S. lividans has a similar, genetically unstable, tetracycline resistance gene (Dittrich and Schrempf, 1992).
S. coelicolor also has a natural tetracycline resistance.
No sites for: AatII, AflII, ApaI, ApaLI, AseI, BamHI, BclI, BglII, ClaI, DraI, EcoRI, EcoRV,
FspI, HindIII, HpaI, KpnI, MluI, NaeI, NarI, NcoI, NdeI, NotI, NruI, NsiI, PstI, PvuI, PvuII, SacI, SacII,
SalI, ScaI, SmaI, SpeI, SphI, SspI, StuI, XbaI, XhoI. Several sites for: AatII, ApaI, ApaLI, BclI, FspI,
MluI, NaeI, SacII, SmaI. No TTA codon. AC=X53401 (Butler et al., 1989; Doyle et al., 1991).
tetSl, tetracycline resistance, ribosome protection, unstable gene of S. lividans
No sites for: AflII, AseI, BclI, BglII, ClaI, DraI, EcoRI, EcoRV, HindIII, HpaI, MluI, NcoI,
NdeI, NruI, NsiI, ScaI, SpeI, SspI, XbaI. Several sites for: AatII, ApaI, ApaLI, BamHI, NaeI, NarI, PvuI,
SacII, SalI, SmaI, SphI. No TTA codon. AC=M74049 (Dittrich and Schrempf, 1992).

m21tk.wpd 16 March 2000 Tobias Kieser&KDSWHU 

0DSVRI'1$)UDJPHQWV





&KDSWHU 

0DSVRI'1$)UDJPHQWV

SphI

BglII

1

BssHII
ApaI

125
150

MscI

309

XhoI

484

S. lavendulae

PmlI
AatII

797
854

Mr=48351

NotI

940

NcoI

1295

Tth111I
StuI

1704
1737

BssHII
SphI

2090
2097

FspI

2100

BssHII

2169

XhoI

2294

mcrA
PvuII

Mitomycin C

BamHI

BamHI

mcrB
Mitomycin C
Mr=11504

NcoI

StuI
SphI

MluI
2330
Eam1105I 2343
ApaI

2497

SmaI
PvuI
BspHI

2587
2610
2690

SphI

2896

ClaI
SstI

mcrA, B, mitomycin C resistance genes from S. lavendulae
10 µg ml-1 mitomycin C in R2YE for selection of clones in S. lividans (MIC >100 µg ml-1).
The sequenced fragment has no sites for: AflII, ApaLI, AseI, BamHI, BclI , BglII, ClaI, DraI,
EcoRI, EcoRV, HindIII, HpaI, KpnI, NdeI, NruI, NsiI, PstI, PvuII, SacI, ScaI, SpeI, XbaI. Several sites
for: ApaI, NaeI, NarI, SacII, SalI, SphI, XhoI. No TTA codon in either ORF. AC=L29247 (August etal., 1994).

m21tk.wpd 16 March 2000 Tobias Kieser&KDSWHU 

0DSVRI'1$)UDJPHQWVCounterselectable markers
Two genes, glkA and rpsL, have been used forcounterselection in Streptomyces (pp.307-308 and
320-323). The sacB gene was shown not to givesucrose resistance in S. lividans (Jäger et al., 1992).
Selenate resistance (see below) is also potentially
useful.

KpnI

1

SphI
SalI
XbaI

26

BsaI
SfiI

376
388

SalI

415

MluI

570

glkA
GlucoseKinaseS. coelicolor

glkA, glucose kinase of S. coelicolor: SmaI-KpnI
fragment from pIJ2581
AC=X98363 (van Wezel and Bibb, 1996). Strains
lacking glucose kinase are resistant to the nonutilisable sugar 2-deoxyglucose (Dog). Thesestrains cannot utilise glucose as sole carbon source,
but grow and sporulate well on 0.5% arabinose.
Spontaneously Dog-resistant S. coelicolor strainsare easily isolated on MM+mannitol+100 mM(16.4 mg ml-1, expensive) Dog; they often havedeletions in glkA (Fisher et al., 1987). No sites for:
AatII, AflII, ApaI, ApaLI, AseI, BamHI, BclI, BglII,
BsaBI, ClaI, DraI, EcoRI, EcoRV, FspI, HindIII,
HpaI, NcoI, NdeI, NotI, NruI, NsiI, PstI, PvuI,
PvuII, SacI, SpeI, SspI, StuI, XhoI. Several sites
for: NaeI, NarI, SacII, SalI. No TTA codon.
Vectors containing glkA: pIJ2581, pIJ6650,
KC573.

DraIII 1015

tsr

SalI
SalI

1048
1084

SmaI
NdeI

1184
1207

ClaI

1438

AgeI
PvuI

1605
1628

EcoRV

1711

SalI

1825

PvuII
BspEI
EcoNI

1954
2010
2061

XbaI

2250

SalI
SphI

HindIII
EcoRV

EcoRI
PstI
SmaI
BamHI 2300
SpeI
XbaI

NotI
BstXI
SacI

2336



&KDSWHU 

rpsL

0DSVRI'1$)UDJPHQWV
BglII

5

MscI

125

AlwI
PflMI
AlwNI
PvuII
BpsEI
NaeI
Tth111I
XhoI
MluI

259
262
263
266
273
282
290
320
353

SfaNI
SmaI
HphI
Sse8387I
PstI

429
451
455
469
470

SacII

544

BglI
NarI
BsmI

549
602
623

BglII

640

Ribosomal
protein S12
S. roseosporus
Mr=17455
AC U60101

rpsL from S. roseosporus
Ribosomal protein S12 giving dominant streptomycin sensitivity. Most spontaneously streptomycin
resistant mutants of S. coelicolor had a point mutation in rpsL. Several different single amino acidchanges gave streptomycin resistance and some greatly increased antibiotic biosynthesis (Shima et al.,1996). AC=U60101 (Hosted and Baltz, 1997). No sites for: AatII, ApaI, ApaLI, AseI, BamHI, BclI,
BsaBI , ClaI, DraI, EcoRI, EcoRV, FspI, HindIII, HpaI, KpnI, NcoI, NdeI, NotI, NruI, NsiI, PvuI, SacI,
SalI, ScaI, SpeI, SphI, SspI, StuI, XbaI. Several BglII sites. No TTA codon.
Selenate resistance
Spontaneous selenate resistant mutants of S. coelicolor have been isolated on MM without sulphates andcontaining 5mM selenate (toxic) and 0.2 mM cysteine as sulphur source. Selenate resistant mutants were
often auxotrophic for cysteine, allowing two-way selection. Several genes of sulphur biosynthesis canbe affected (Lydiate et al., 1988).

&KDSWHU 

m21tk.wpd 16 March 2000 Tobias Kieser(Sau3AI)

0DSVRI'1$)UDJPHQWV
1

BglII
PstI

HindIIIter(fd)

1
5

13

ClaI

19

EcoRI
SacI
KpnI
SmaI
BamHI
XbaI

44
50
56
60
65
71

BamHI

332

AseI

388

SmaI

484

PleI

75

ampC

XhoI

645

BglI
EarI

86
124

Cephalosporin

FokI

775

BspHI
PvuII

136
171

ß-lactamase
E. coli

974

Eco57I

267

Eco571
HindIII
DraI
AlwNI

1062
1065
1120
1208

BpmI
NdeI

378
397

PstI

1326

AlwNI

672

AseI

xylE
PstI

1637

KpnI
BclI

1704
1730

AseI

1806

EcoNI
PvuI
PflMI
RsrII

1824
1946
1961
1981

SmaI

2062

BspEI
DrdI
MluI
MunI

Catechol 2,3dioxygenase
Pseudomonas
putidaMr=35156

SalI,HincII 719
755
BspEI
804
BbsI

2439
2582
2676
2735

AseI

2799

EarI

2850

PstI

3021

ClaI

852

EcoRV

855

AgeI

971

PvuII

993

BsmI

1024

BspHI

1042

DrdI

1055

PstI
PleI

1074
1105

BglII

1480

to

Indicator genes (see Chapter 15)
ampC, promoterless $-lactamase gene from the E. coli chromosome
xylE, catechol 2,3-dioxygenase from Pseudomonas putida
Fragment with terminator to from pIJ4083 (Clayton and Bibb, 1990). No sites for: AflII, ApaI, ApaLI,
AseI, BclI, DraI, FspI, HpaI, MluI, NarI, NcoI, NotI, NruI, NsiI, PvuI, SacII, ScaI, SpeI, SphI, SspI, StuI,
XhoI. Several sites for: AatII, ClaI, NaeI, PstI, PvuII. No TTA codon. AC=V01161 (Zukowski et al.,1983). Assay: pp. 344-346 (Sala-Trepat and Evans, 1971). Plasmid vectors: Altenbuchner et al. (1992);
Ingram et al. (1989). Phage vectors: p. 296-297; Bruton et al. (1991). pKC1064 contains a usefulcassette with a promoterless xylE gene and a hyg gene for selection (Kuhstoss and Rao, 1991).



&KDSWHU 

0DSVRI'1$)UDJPHQWV
PvuI

BstEII

PstI
BalI

0
0.2

NarI
XmnI

1.47

PvuII

1.65

PvuII

2.10

XmnI
AatII

β gal
ß-galactosidase
S. lividans

Mr=130 000

BamHI 2.80
StuI

3.20

FokI
PvuII

BstEX 3.70

BclI*

4.60

MluI
PvuII
MluI

5.15
5.22
5.30

NcoI

5.40

MluI

5.50

SphI

6.40

$gal, promoterless gene for secreted $-galactosidase from S. lividans
The sequenced region has no sites for: AflII, ApaI, ApaLI, AseI, BamHI, BclI, BglII, ClaI, DraI,
EcoRI, EcoRV, FspI, HindIII, HpaI, KpnI, MluI, NcoI, NdeI, NotI, NruI, NsiI, PstI, SacI, SacII, SalI,
ScaI, SmaI, SpeI, SphI, SspI, StuI, XbaI, XhoI. Several sites for: NaeI. AC=V00622 (Eckhardt andSmith, 1987; Eckhardt et al., 1987).

&KDSWHU 

m21tk.wpd 16 March 2000 Tobias Kieser

PvuII

1

SnaBI

47

EcoRV
NruI
BspEI
NarI

139
150
157
243

BclI*

518

338

galT

SfaNI

412

Uridyltransferase

BclI*

518

P2

Galactokinase
ClaI*

736

AgeI
FspI
PvuI
MluI
BpmI

865
930
947
960
1015

BstBI

1084

Mr=

HpaI

1364

FokI

1387

BsmI

1491

HpaI
PvuII

1611
1617

NruI
BspEI

92
103

Bpu1102I

511

SalI

661

BglII

779

ApaLI
EcoRI

973
1036

P1

ApaLI

galK
E.coli

0DSVRI'1$)UDJPHQWV

galE

PvuII

1144

Eco57I
BspHI

1224
1320

SalI

1426

BstXI
ScaI
NcoI

1492
1578
1605

SalI

1757

BclI*

1767

SmaI

2029

SacII

2146

MluI

2244

Esp3I

2483

SalI

2530

SacII
PvuII

2705
2770

EarI

2871

SalI
SmaI
SalI

2949
2956
3036

PmlI

3124

Epimerase

galK
Galactokinase
S.lividans

SalI

3269

SfiI

3283

SacII

3288

PuvI

3335

SacII
SacII
SacII

3356
3398
3418

NheI

3541

galK of E. coli and of S. lividans
The E. coli galKI gene has been used for promoter probing using a $-galactosidase-deficient S. lividans
mutant (Brawner et al., 1985).



&KDSWHU 

0DSVRI'1$)UDJPHQWV
BglII
PstI

1
5

HindIII 13
SphI
19
27
SalI
33
XbaI
BamHI 39
BsiWI 127

PstI

399

PstI

729

luxA
Luciferase
Vibrio harveii
Mr=40,146

BspHI* 880
SspI

1145

XmnI 1201
AseI 1249

luxB
Luciferase
Mr=36,432

luxAB, genes for NADP-dependent luciferase from
Vibrio harveyi
AC=X58791 (pp. 346-347).
The sequenced region has no sites for: AatII, AflII,
ApaI, ApaLI, DraI, KpnI, NaeI, NarI, NcoI, NdeI, NotI,
PvuI, SacI, SmaI, SpeI, StuI, XhoI. Several sites for:
NruI, PstI, SspI. 7 TTA codons.

SspI
EcoRI

1252
1308

BclI*
SnaBI
AlwnI
BglI

1326
1363
1445
1480

NsiI
HpaI
MluI
FspI

1685
1747
1757
1779

ScaI
SacII

2110
2150

ClaI

2209

ClaI

2390

? BglII

2540

EcoRV 2550
SacI
2600
EcoRI
BglII

2602
2612

A TTA-free form of luxAB, made by site-directed mutagenesis (G.L. Brown, C.J. N. Oestreicher and
K.F. Chater, unpublished) has been incorporated into a promoter-probe vector (pIJ5971; M.S.B. Paget,
personal communication) and into promoter-trap transposons (P. Herron and P. Dyson, personalcommunication; p. 346). pIJ5971 was used successfully by B. Aigle et al. (submitted for publication).

m21tk.wpd 16 March 2000 Tobias Kieser&KDSWHU 

0DSVRI'1$)UDJPHQWV

HindIII
SphI
PstI
SalI

1

XbaI

BamHI
SmaI
KpnI
NcoI

39
53

BsrI

666

NotI

776

EGFP
Green Fluorescent Protein
Mr=22000

Aequorea victoria

XbaI

EcoRI
StuI
SpeI
BglI
ApaI

791
849

DrdI

973

870

ApaLI
NdeI 1063
EGFP, enhanced green fluorescent protein from pEGFP
AC=U76561 (Clontech, www. clontech.com). This is a synthetic mutant form of the natural gfp from
Aequorea victoria which gives increased light emission. Excitation is at 488 nm (argon laser) andemission is maximal at 507 nm (green). The codon usage has been optimised for high GC organisms(Cormack et al. 1996). No sites for: AatII, AflII, AseI, BclI, BglII, ClaI, DraI, EcoRV, FspI, HpaI, MluI,
NaeI, NarI, NruI, NsiI, SacI, SacII, ScaI, SspI, XhoI.
Promoter-probe vectors pIJ8630, pIJ8655, pIJ8660, and pIJ8668 (pp.347-384, 531, 571), containing
EGFP, have been constructed by Sun et al. (1999). Attempts to make MC31 vectors containing EGFP,
equivalent to xylE-containing phages (pp. 296-297), were unsuccessful (C.J. Bruton, personalcommunication).



&KDSWHU 

0DSVRI'1$)UDJPHQWV

melC1,C2 operon of S. antibioticus
The dotted horizontal line in melC1 indicates the signal cleavage site. Tyrosinase requires Cu2+
and the activity is inducible by L-methionine (Katz and Betancourt, 1988). Spontaneous melaninnegative mutants of pIJ702 occur with c. 1% frequency. Some have mutations in the melC1,C2 operon,often removing the BglII site which is in a region with an inverted repeat. Other melanin-negative
mutations seem to occur spontaneously in the S. lividans host, giving white colonies from which intact,
melanin-producing pIJ702 can be isolated by transformation.
No sites for: AflII, ApaI, ApaLI, BamHI, ClaI, DraI, EcoRI, EcoRV, HindIII, HpaI, KpnI, NcoI, NdeI,
NotI, NruI, NsiI, PstI, PvuII, ScaI, SpeI, SspI, StuI, XbaI, XhoI. Several sites for: AatII, BclI, MluI,
NarI, SalI. No TTA codon.
The following vectors contain the melC operon: pIJ702, pMT660, pB48, pB83, pMT3010,
pIJ943, pWOR126.
Cloning: Katz et al. (1983). Sequence: AC=M11582 (Bernan et al., 1985) . Promoter: Leu etal. (1989); Function of melC1: Chen et al. (1992); Leu et al. (1992); Tseng et al. (1990). Genetic
instability of melC operon in S. antibioticus: Yu and Chen (1993). Expression in E. coli: della-Cioppa
et al. (1990); Sugiyama et al. (1990); Tseng et al. (1990). Use as indicator for plasmid recombination:
Tsai and Chen (1987); Chen et al. (1987); Kieser et al. (1989).
Other melanin operons have been cloned from S. glaucescens and S. lavendulae (Kawamoto etal., 1993) and used to make promoter probe vectors.
RC3 cassette form the melC1,C2 operon of S. glaucescens
The RC3 cassette containing the melC1,C2 operon of S. glaucescens without promoter has beenused for promoter-probing (Paget et al., 1994). ter(mmr) is the terminator from the methylenomycin
resistance gene (Neal and Chater, 1987). ter(fd) is described on p. 502. AC=Y00457, M11302 (Huberet al., 1985, 1987). No TTA codon in melC1 and melC2.

m21tk.wpd 16 March 2000 Tobias Kieser&KDSWHU 

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EcoRI

1

AvaI

(HpaII)

oriT

HinfI

RP4=RK2(HpaII)
StyI(HpaII)
StyI

SalI
PstI

Other DNA fragments
oriT of plasmid RP4=RK2
The c. 800 bp PstI fragment containing oriT was constructed by Labigne-Roussel et al. (1987). It
contains bp 50598-51376 of RP4 (AC=L27758). oriT is within a 107 bp HpaII fragment that includes
the HinfI site (Guiney and Yakobson, 1983). None of the commonly used restriction endonucleases
listed in Table 21.1 (p. 441) cleaves within the PstI-SalI fragment. (See p. 267 for a list of vectors
containing oriT.)fd terminator, bidirectional terminator of the filamentous E. coli phage fd
The 332 bp Sau3AI fragment of phage fd (AC=V00602) regenerates a BglII site at one end and a BamHI
site at the other. The fragment has a weak promoter activity reading towards the BglII site. The
fragment is not cleaved by any of the restriction enzymes listed in Table 21.1 (p. 441).
Several other terminators have been used for vector construction or shown to function in Streptomyces:
8 to (xylE vectors, etc), mmr terminator (RC3 cassette; p. 501), hyg terminator, M29 terminator.

m21tk.wpd 16 March 2000 Tobias Kieser&KDSWHU 

gylR

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SmaI

1

Esp3I

60

BspEI
PmlI
BstEII

114
153
191

AgeI

255

XhoI
NotI

354
363

MscI
FspI
EcoNI
PvuII
ApaI

439
466
473
522
580



S. coelicolor

Repressor
Mr=27641
No TTA codons

Ter

gylC

BspMI 704
BstXI
742
Tth111I 774

BfaI

1094

XmnI
ClaI
PvuI

1151
1199
1224

MluI

1319

SmaI

1392

KpnI
StuI
SfiI
BspHI

1472
1527
1528
1587

NruI

1656

gylR expression cassette
gylC is the first gene of the gyl operon. It is induced by glycerol provided that the growth medium does
not contain glucose which induces catabolite repression (Hindle and Smith, 1994; Paget et al., 1994;
Seno and Chater, 1983; Smith and Chater 1988a, 1988b).

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EcoRI
SacI
KpnI

1
7
13

SmaI
BamHI
PstI

17
22
33

NcoI

47

SmaI

56

NruI
XmnI

235
267

BgIII

428

BsaI

477

MscI

734

traR
pSN22
S. nigrifaciens

PtraR

P tra

MscI

950

Psp1406

961

BamHI

971

XbaI
SalI
Sse8387

977
983
988

PstI

SphI
HindIII

989

995
1001

Temperature-sensitive expression cassette derived from pSN22
This cassette encodes a temperature-sensitive TraR repressor protein that controls the expression of Ptra
Induction is by temperature shift from 28 to 37E (Kataoka et al., 1996).
tipA promoter cassette
Regulation of PTipA by thiostrepton requires a functional tipA gene in the host (pp. 119, 386). Several
expression vectors contain the tipA promoter.

m21tk.wpd 16 March 2000 Tobias Kieser&KDSWHU 

0DSVRI'1$)UDJPHQWV

Altenbuchner J, Viell P, Pelletier I (1992) Positive selection
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and analysis of a locus (mcr) involved in mitomycin C
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Bechthold A, Floss HG (1994) Overexpression of thethiostrepton-resistance gene from Streptomyces azureus in
Escherichia coli and characterization of recognition sites ofthe 23S rRNA A1067 2’-methyltransferase in the guanosine
triphosphatase center of 23S ribosomal RNA. Eur.J.Biochem.,
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Gritz L, Davies J (1983) Plasmid-encoded hygromycin B
resistance: the sequence of the hygromycin B phospho-

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&KDSWHU 

0DSVRI'1$)UDJPHQWV

transferase gene and its expression in Escherichia coli and
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RNA methylase gene from Streptomyces tenebrarius which
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T (1987) Nucleotide sequence of the streptothricin
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Nucl.Acids Res., 15, 4227-4240



&KDSWHU 

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

&KDSWHU 

0DSVRI'1$)UDJPHQWV

m22tk425.wpd 25 April 2000 Tobias Kieser

Chapter 22

Maps of plasmids, transposons and phage
genomes
CONTENTS
Conventions used for the restriction maps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 512
Lists of all restriction maps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 512
Phage C31 and its derivatives (Table) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 513
Integrating plasmids derived from C31 and phage VWB (Table) . . . . . . . . . . . . . . 514
Plasmid maps grouped according to the Streptomyces replicon (Table) . . . . . . . . . . . 514
E. coli plasmids (Table) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 517
Transposable elements (Table) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 518
Restriction maps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 519
Phage C31 and its derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 519
Integrating plasmids derived from C31 and phage VWB . . . . . . . . . . . . . . . . . . . . . 530
Plasmid maps grouped according to the Streptomyces replicon . . . . . . . . . . . . . . . . . . 533
pIJ101 derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 533
pRES1 derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 545
pJV1 derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 546
pSG5 derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 548
SCP2* derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 553
pSAM2 and SLP1 derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 563
E. coli plasmids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 564
Transposable elements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 573
IS117 derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 573
IS493 derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 575
Tn4556 derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 578
Tn5 derivative . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 581
The restriction maps in this chapter have been collected over a long time, helped by contributions
from many colleagues. The accuracy of the map information is variable. A few vectors have been fully
sequence; the maps of others have been predicted from the known sequences of the parental DNA
fragments and the construction method. Most of the maps rely, however, partly or completely on theestimates of fragment sizes determined by agarose gel electrophoresis. Experience shows that restriction
maps, even when based on sequences, may contain errors. Also vectors may suffer rearrangements
during storage. It is therefore recommended to confirm essential vector features before use.



Chapter 0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHVConventions used for the restriction maps
The maps are grouped according to the Streptomyces replicon or transposon functions.
Restriction sites indicated on maps have been selected subjectively. Where possible, all the sites areindicated for the restriction endonucleases shown; exceptions are indicated. The maps of restriction andindicator genes in Chapter 21 may contain additional sites which are not indicated on the vector maps.

Scale

100 bp and 1 kb intervals are marked

Bold

Unique sites, cloning sites in replacement vectors, and sites unique to the right arms of phage
vectors (pp. 522-529).

C

Recommended cloning sites*

Sites known to be protected by E. coli dam or dcm methylation/

Junctions between fragments, destroying both sites indicated()Endonuclease for which not all sites are shown

Indicator for boundary between left and right phage arms

List of all restriction maps
General considerations about cloning are in Chapter 9.
Plasmids and their use for gene cloning are described in Chapter 11, and a list of vectors for special
purposes is on p. 267.
Phages are introduced in Chapter 12 and phage vectors are described in Chapter 13.
The uses of transposons is described in Chapter 6.
Vectors and methods for gene disruption and gene replacement are introduced in Chapter 14.
In the following tables bold names and page numbers indicate phages or plasmids for which there is a
restriction map.

&KDSWHU0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV

Table 22.1 Phage

C31 and its derivatives

Name

Phage functions

Markers and comments

C31 whole genome
AC=AJ006589

Wild-type, c+ att+ int+

Wild-type phage, with location of
C3

Page number
291, 519

C31 genes of the early Wild-type, c+ att+ int+
regionWild-type phage520

C31,rightmost sites in Wild-type, c+ att+ int+
the left arm

Wild-type phage521

C31, right arm and int Wild-type, c+ att+ int+
gene

Wild-type phage, with locations ofnamed deletions

522

KC210

c+ att+ int+

tsr (KC859 minus xylE PstI fragment

292

KC211

c+ att-int

aac(3)IV (replacing xylE BglII-PstI
fragment of KC857)

293

KC212

c+ att+ int+aac(3)IV (left PstI end of KC211
joined by StyuI site-containing linker
to rightmost SphI end of C31)

292, 294, 295

KC300

c+ att+ int+

vph (Chater et al., 1982)

294, 295

KC301

c+ att+ int+

tsr (Chater et al., 1982)

294, 295

KC304

c+ att+ int+

tsr vph292, 523

KC310

c+ att+ int+

vph, promoterless neo

342

KC401

C3 att-int

vph

293

KC466

c+ att-int

vph gyl0A,B0

524

KC505

C3 att-int

tsr vph293, 526

KC515

c+ att-int

tsr vph293, 525

KC516

c+ att-int

tsr vph293, 525

KC517

c+ att-int

vph

293

KC518

C3 att-int

tsr vph293, 526

KC573

c+ att-int

tsr vph glkA

524

KC850

c+ att+ int+

tsr, promoterless neo

342

KC857

c+ att-int

tsr vph xylE

293 ,297, 527

KC858

c+ att-int

tsr vph xylE

293, 297

KC859

c+ att+ int+

tsr vph xylE ter(fd) ter(to)297, 523

KC860

c+ att-int

tsr vph xylE

KC861

c+ att-int

tsr xylE

293, 297,308,
527
293, 528



Chapter 0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV

NamePhage functions

Markers and comments

KC862

c+ att-int

tsr xylE

293, 528PM8

c+ att-int

tsr hyg ter(fd)293, 529

Table 22.2 Integrating plasmids derived fromPage number

C31 and phage VWB(pPM927, a pSAM2-derived integrating vector, is listed in Table 22.3)
Name

Integration function,
E coli repliconMarkers etc

Page number

pIJ5971

C31, pUC18

aac(3)IV oriTRK2
luxA,B (without TTA codons)

pIJ8600

C31, pUC18

aac(3)IV oriTRK2 tipAp

389, 531

pIJ8630

C31, pUC18

aac(3)IV oriTRK2 EGFP

531

pIJ8660

C31, pUC18

aac(3)IV oriTRK2 EGFP

531

pKTO2

VWB, pIC20R

pMT3226498

tsr bla390, 532

C31, pUC18

aac(3)IV oriTRK2 gylR gylAp xylE

390, 532

pOJ436

C31, pBR322

aac(3)IV, cos, oriTRK2530

pOJ444

C31, P1

aac(3)IV neo P1(pac,loxP) oriTRK2

530

pSET152

C31, pUC18

aac(3)IV lacZ oriTRK2

530

Table 22.3 Plasmid maps grouped according to the Streptomyces replicon (Additional vectors containing
different Streptomyces replicons are described in Hopwood et al. (1987)
Name

Replicon(s)Markers etc

Page number

pIJ101

pIJ101

Wild-type plasmid, Tra+

21,126, 153,
254, 256

pANT1200

pIJ101, pUC

tsr bla snpAp snpR538

pANT1201

pIJ101, pUC

neo bla snpAp snpR538

pANT1202

pIJ101, pUC

aac(3)IV bla snpAp snpR538

pANT849

pIJ101

tsr snpAp snpR

389, 537

pANT850

pIJ101

aphI snpAp snpR

389, 537

pANT851

pIJ101

aphI snpAp snpR

389, 537

pHZ1351

pIJ101, ColE1

tsr bla sti

542

pHZ1358

pIJ101, ColE1

tsr bla sti oriTRK2 cos

542

pHZ1271

pIJ101, ColE1

tsr bla kan tipAp His6

540

pHZ1272

pIJ101, ColE1

tsr bla kan tipAp

540

&KDSWHU0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV



Name

Replicon(s)Markers etc

Page number

pIJ364

pIJ101

tsr vphpIJ4090

pIJ101

tsr neo ermEp*

389, 535

pIJ4123

pIJ101

tsr kan tipAp His6

539

pIJ459

pIJ101

tsr neo ermEp

534

pIJ487

pIJ101

tsr neopIJ6021

pIJ101

tsr kan tipAp

pIJ680pIJ101

tsr aphI534

pIJ699

pIJ101, p15A

tsr neo vph

216, 218, 541

pIJ702

pIJ101

tsr melC

536

pJOE829

pIJ101

hyg

541

pJOE875

pIJ101

tsr melC541

pMT3010

pIJ101

tsr melC342, 536

pMT3206

pIJ101

tsr gylR gylAp xylE

389, 535

pMT660pIJ101 (ts)tsr melC

pRS1105

pIJ101

tsr neo luxAB

346, 543

pSKO2

pIJ101, pUC8

tsr bla galK543

pUWL series

pIJ101, pUC18

tsr bla lacZ

544

pWHM3

pIJ101, pUC19

tsr bla lacZ

319, 544

pRES1

pRES1

Wild-type plasmid

pRES18/19

pRES1, pUC18/19

tsr bla lacZ

pJV1

pJV1

Wild-type plasmid, Tra+

pB45

pJV1

hyg

547

pB48

pJV1

hyg melC

547

pB83

pJV1

tsr melC547

pFD666

pJV1, ColE1, M13

neo cos

546

pWOR126

pJV1

tsr melC

546

pSG5

pSG5

Wild-type plasmid

257

pCZA185

pSG5, pBR322

tsr bla oriTRK2

551

534

256, 342, 533
388, 539

536

545

126, 258



Chapter 0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV

Name

Replicon(s)Markers etc

Page number

pGM11

pSG5

neo (aacC1)

548

pGM16

pSG5

tsr ble549

pGM160pSG5, pUC

tsr aacC1 bla

318, 320, 550

pGM17

pSG5

neo ble catSa

549

pGM9

pSG5

tsr neo ble

548

pHZ132

pSG5

tsr vph bla cos oriTRK2550

pKC1139

pSG5, pBR322

aac(3)IV lacZ oriTRK2

551

pSIT151

pSG5, pBR322

neo aacC1 Tn1792 oriTRK2

119, 120

pUCS30pSG5, pBR322

tsr neo ble aadA tipAp oriTRK2

119, 552

SCP2*

SCP2*

Wild-type plasmid, Tra+

pCJR29SCP2*, pUC19

tsr bla actII-ORF4

558

pHJL302SCP2*, pUC19

tsr bla553

pHJL401SCP2*, pUC19

tsr bla262, 350, 553

pIJ4231SCP2*

hyg vph attBIS117 Tra+

pIJ4680

SCP2*, p15A

aadA tsr bla Tra+

319, 559

pIJ698SCP2*, p15A

tsr hyg neo vph Tra+

218, 260, 557

pIJ80

SCP2*

aphI Tra+

261, 559

pIJ903SCP2*, pBR327

tsr bla Tra+

260, 554

pIJ920SCP2*

vph Tra+260, 555

pIJ921SCP2*

vph Tra+555

pIJ922

SCP2*

tsr Tra+260, 554

pIJ940SCP2*

tsr hyg Tra+

260, 556

pIJ941SCP2*

tsr hyg Tra+

260, 556

pIJ943

SCP2*

tsr melC239, 261, 562

pJAR4

SCP2*, pBR322

hyg cos cos cos Tra+

261, 560

pKC1064

SCP2*, pUC

tsr aac(3)IV oriTRK2 tipAp390

pKC1218

SCP2*, pUC

aac(3)IV lacZ oriTRK2

268, 561

pKC505SCP2*aac(3)IV cos cos cos

261, 560

pOJ446SCP2*, pBR

aac(3)IV cos cos cos oriTRK2

126, 154, 217,258-262

266

562

&KDSWHU0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV



Name

Replicon(s)Markers etc

Page number

pRM5

SCP2*, pBR322

tsr bla actII-ORF4

261, 557

pTMA1

SCP2*

tsr mdh Tra+

348, 561

pSAM2

pSAM2

Wild-type plasmid, Tra+

216, 264, 265

pPM927

pSAM2 int, pBR

tsr aadA oriTRK2 tipAp

390, 563

pCAO106

SLP1, p15A

km bla263

pCAO170

SLP1, p15A

tsr bla km

263

pIJ61

SLP1

tsr aphI

263,265, 563

Table 22.4 E. coli plasmids (The maps show sites that are rare in Streptomyces DNA)
Name

Replicon(s)Markers etc

Page number orreferencepACYC177

p15A

km bla263, 564

pACYC184

p15A

cat tet564

pIJ666p15A

cat neo565

pIJ1117

p15A

cat, no AseI site

Kieser et al.
(1992)

pIJ4642

p15A

cat aadA

218, 565

pKC1250

p15A

aadA lacZ oriTRK2566

pANT840

pUC19

bla lacZ

568

pBluescript

pUC

bla

568

pBR322

pBR322

bla tet572

pBR327

pBR327

bla tet572

pCJR24

pUC19

tsr bla actII-ORF4

567

pDH5

pUC19, f1

tsr bla lacZ

pHM10A

pBR322

hyg bla ermEp oriTRK2

pIJ2581

ColE1, f1

tsr bla glkA

pIJ2925

pUC19

bla

568

pIJ6650

pUC18

aac(3)IV oriTRK2 glkA

566

230, 570
390, Motamedi etal. (1995)
321, 571



Chapter 0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV

Name

Replicon(s)Markers etc

Page number orreference

pIJ8668

pUC18

aac(3)IV oriTRK2 EGFP

pIJ8671

pUC18

tsr aac(3)IV oriTRK2567

pKC1132

pUC18

aac(3)IV lacZ oriTRK2566

pKC1138

pUC18

aac(3)IV xylE lacZ oriTRK2566

pOJ260pUC18

aac(3)IV lacZ oriTRK2566

pPM2208

pBR

tsr aadA oriTRK2

pSET151

pUC19

tsr bla xylE lacZ oriTRK2

pSupercos 1

ColE1

neo bla cos cos

pSupercos 1 after
packaging

ColE1

neo bla cos

569

pUC19

pUC19

bla lacZ568

pVE1011

pBR322

tsr bla570

pVE616

pBR322

tsr bla cos

569

348, 571

317, 571
570
216, 218, 236,
569

Table 22.5 Transposable elements (See p. 428 for S. coelicolor and S. lividans elements)
Name

Transposase, RepliconMarkers etc

IS110 AC=Y00434

IS110

Wild-type element

316

IS117 AC=X15942

IS117

Wild-type element

265, 266

pHM11A

IS117, pBR

hyg ermEp

Motamedi et al.
(1995)

pIJ4201

IS117, pBR327

tsr neo bla

573

pIJ4210

IS117, pBR327

tsr neo bla

573

pIJ4696

IS117, pBR327

hyg bla574

pIJ4698

IS117, pBR327

tsr aadA bla (neo)574

pIJ4700

IS117, pBR327

aadA bla

574

IS493 AC=M28508

IS493

Wild-type element

IS493 derivatives, summary diagram

Page number

117, 575
576

&KDSWHU0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV



Name

Transposase, RepliconMarkers etc

Page number

Tn5096
pCZA163

IS493, pUC19

aac(3)IV bla

577

Tn5099-10, pCZA258

IS493, pSG5

aph(4) bla oriTRK2

577

Tn5100-4,
pCZA276

IS493, pUC19

aph(4) bla oriTRK2

577

Tn4556 AC=M29297

Tn4556

Wild-type element578

Tn4560Tn4556vph578

pUC1169

Tn4560, pMT660

vph tsr

112, 579

Tn5351

Tn4556vph luxAB

580

Tn5353

Tn4556

neo luxAB580

Tn5 AC=U00004

Tn5

neo ble str

Tn5493,
pJOE2577

Tn5, pSG5, pUC

tsr bla neo

121, 581

Restriction maps
Phage

C31 and its derivatives

The black boxes on pp. 520 and 521 mark the early region and the int gene. See Table 22.1
(p. 513) for a list of the phages and for cross-references to other chapters where the properties of thedifferent phage vectors are explained.
Complete sequence of C31 AC=AJ006589 (Smith et al., 1999).
Key references for the structure and function of C31: Bruton et al. (1991); Chater et al. (1981a,
1981b); Hendrix et al. (1999); Howe and Smith (1996a, 1996b); Ingham et al. (1993, 1994, 1995);
Ingham and Smith (1992); Smith and Owen (1991); Smith et al. (1992); Thorpe and Smith (1998);
Wilson et al. (1995); Wilson and Smith (1998).



Chapter 0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV

&KDSWHU0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV

C31 genes of the early region and rightmost sites in the left arm of C31 (useful for determining thesizes of restriction fragments after cloning).



Chapter 0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV

&KDSWHU0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV





Chapter 0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV

&KDSWHU0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV





Chapter 0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV

&KDSWHU0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV





Chapter 0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV

&KDSWHU0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV





Chapter 0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV

lacZ

ori
pUC18

aac(3)IV
int

pSET152

ØC31

5.5 kb

oriT

ØC31

RK2

attP

T7

Apramycin

T3
cos

int
ØC31

pOJ436
attP
ØC31

cos
cos

11.1 kb

ori
ColE1

oriT
RK2

ØC31

loxP

aac(3)IVpac
P1

Apramycin

oriTRK2
26.48 EcoRI

rep
P1l

pOJ44435.3 kb

loxP

neo
Tn5

rep P1p

Integrating plasmids derived from

C31 and phage VWB.

pSET152, pOJ436, and pOJ444 are oriT RK2 plasmids containing aac(3)IV, the apramycin resistancegene (Bierman et al., 1992). pSET152 is one of the most frequently used vectors in our laboratory.
pOJ436 is a cos vector. We are not aware of anybody having tried pOJ444, which is designed for use
with the P1 packaging system.

&KDSWHU0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHVIntegrating vectors constructed by Sun et al. (1999). tfd and to are transcriptional terminators from phagefd and , respectively. The SmaI site labelled with an asterisk is not unique in the vectors.
pIJ8600. tipAp expression vector. (See also pIJ8671, p. 567, which lacks the attP and int functions.)
pIJ8630 and pIJ8660. Promoter-probe vectors containing the promoterless EGFP (green fluorescentprotein) as indicator (pp. 347, 499).



Chapter 0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV

pMT3226. Integrating expression vector using the gylR cassette for glycerol-induced gene expression(pp. 388, 503; F. Amini, M.S.B. Paget, G. Bucca and C.P. Smith, personal communication). pMT3206(p. 535) is a similar but autonomously replicating plasmid.

pKT02. Integration vector derived from phage VWB from S. venezuelae. The plasmid integrates intotRNAArg (AGG). Integration is less efficient than with the 31-derived vectors (Van Mellaert et al.,1998).

&KDSWHU0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV

end rep

4572

BglII 45

4563
4557

EcoRI• 44
SacI• 43

4551
4547

rep
pIJ101

KpnI 42GGATCT
SmaI 414542 BamHI• 40
BfaI 394537
XbaI• 38
4536
4530
4524

ter

SalI 37
PstI 36

ori
pIJ101

SphI 34

4512 HindIII• 33
4504
4500

pIJ4876215 bp4523 Sse8387I 35
4518

start
start rep
rep

neo

PstI 32
BglII 31
Start neo

tsr

start
start tsr
end neo

End tsr

Plasmid maps grouped according to the Streptomyces repliconpIJ101 derivatives
The restriction map of the wild-type plasmid is on p. 254. Note that all the pIJ101-derived
vectors lack the transfer function, and most of them also lack sti and therefore accumulate ss DNA(p. 256).
pIJ487, containing the promoterless neo gene, is probably the most used multicopy vector ( pp. 256, and342). pIJ486 differs from pIJ487 only in the orientation of the BglII fragment containing the cloning sites(Ward et al., 1986). Note that there is no BamHI site in the rep gene. The position where the site wasis shown in parentheses.
A weak promoter in the fd terminator fragment gives a baseline kanamycin resistance of c.
-1
1 µg ml in S. lividans on MM. The level of resistance depends on the amount of salt in the medium.
Insertion of random DNA fragments upstream of the neo gene frequently results in selectable kanamycin(and neomycin) resistance. This can be used for monitoring the success of shotgun cloning experiments.
Multiple sites for: AatII, NaeI, NarI, NcoI, NruI, PvuI, PvuII, SmaI. No sites for: AflII, ApaLI, DraI,
HpaI, MluI, NsiI, ScaI, SpeI, SspI, StuI.



Chapter 0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV

rep
pIJ101

pIJ364

ori
pIJ101

5.6 kbvph
rep
pIJ101aphI

3955 XbaI

tsr

pIJ6805.3 kbori
pIJ101

tsr

rep
pIJ1014927 BglII
EcoRI
SacI
4906 KpnI

ter

pIJ459fd

PermE

6633 bpori
pIJ1014637 BamHI

XbaI
SalI
PstI
SphI
HindIII
PstI
BglII
4589 BclI

neo
tsr

pIJ364 is segregationally and structurally very stable and is particularly suitable for cloning into the tsr
gene (Kieser et al., 1982).
pIJ680 is also structurally and segregationally stable (Hopwood et al., 1985); it is the parent of
pMT3010 (p. 536).
pIJ459, an expression vector, is a derivative of pIJ487 containing the strong, constitutive ermE promoter(p. 386) driving neo expression (M.J. Bibb, unpublished). pIJ4090 (p. 535) contains the stronger, mutant
ermE promoter, ermEp*.

&KDSWHU0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHVpIJ4090. Expression vector like pIJ459 (p. 534) but containing the mutant ermE* promoter withincreased constitutive activity (p. 389; M.J. Bibb, unpublished).
pMT3206. Expression vector using the gylR cassette for glycerol-induced gene expression (pp. 388, 489,
503) (F. Amini, M.S.B. Paget, G. Bucca and C.P. Smith, personal communication). pMT3226 (p. 532)
is an integrating vector containing the same expression cassette.



Chapter 0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV

end rep

rep
pIJ101start rep
start melC1

melC1

pIJ702ori

5685 bp

pIJ101

end melC1
start melC2

melC2

tsr
start tsr
end melC2
end tsr

rep
pIJ101

t fd

ori
pIJ101melC2Tyrosinase5425 PstI

pMT30107251 bp

melC14729 SphI

t mmr

tsr

XbaI
BamHI
XhoI
BamHI

4675 EcoRI

pIJ702. Insertion into the BglII, SacI or SphI sites abolishes tyrosinase activity conferred by the S.
antibioticus melC operon. Note that tyrosinase requires copper for activity (Katz et al., 1983). No sites
for: AflII, ApaLI, DraI, EcoRI, HindIII, HpaI, NsiI, ScaI, SpeI, SspI, StuI, XbaI. Multiple sites notshown: AatII, MluI, NaeI, NarI, NruI, PvuI.
pMT660. pIJ702 derivative with point mutation, probably in the rep gene, causing temperaturesensitivity for replication (Birch and Cullum, 1985). pUC1169 (p. 579), containing Tn4560, is based on
pMT660.
pMT3010. Promoter-probe vector using the S. glaucescens melC operon as indicator. tfd and tmmr aretranscriptional terminators from phage fd and from the methylenomycin resistance gene of plasmid SCP1,
respectively (Paget et al., 1994).

&KDSWHU0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV



rep
pIJ101

snpR

pANT849ori

5343 bp

pIJ101

PsnpA
3671 XhoI3659 ClaI

3652 SphI•
EcoRI•
BglII•XbaI•
SacI•
HindIII•
DraI•
SpeI•
HpaI•
3610 MluI•

tsr

rep
pIJ101snpR

4167 ClaI

4160 SphI•
4154 EcoRI•
4148 BglII•
4131 DraI•
4126 SpeI•
4122 HpaI•
4105 EcoRI•

PsnpA

pANT850ori

5851 bp

pIJ101

aphI

rep

(tsr)

pIJ101

snpR

pANT851ori

5851 bp

pIJ101

PsnpA
4167 ClaI

4160 SphI•
4148 BglII•
4135 HindIII•

4131 DraI•
4126 SpeI•
4122 HpaI•

aphI

(tsr)

4102 HindIII•

pANT849, pANT850, and pANT851. Expression plasmids using the small neutral protease SnpA
promoter which is regulated by SnpR (Lampel et al., 1992). AC=M86606. Reporter protein levels up
to 50-fold greater than those from the melC1 promoter have been observed with these vectors (DeSantiand Strohl, manuscript in preparation). Uses of pANT849: Dickens et al. (1996a, 1996b, 1997).



Chapter 0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV

pANT1200, pANT1201 and pANT1202. Bifunctional expression vectors containing the snpA promoter.
The three vectors differ in the selective marker for Streptomyces (aphII is the neo gene from Tn5). See
previous page for description and references. The terminator is from the methylenomycin resistancegene.

&KDSWHU0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV

BglII 7.80

pIJ6021 and pIJ4123 (Takano et al., 1995). Expression vectors containing the thiostrepton-inducible
tipA promoter. Note that the host must contain a functional tipA gene for the thiostrepton induction tofunction; see pp. 119, 386 for information about tipAp induction, and p. 389 for a description of theplasmids.



Chapter 0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV

BglII 10.1

pHZ1272 and pHZ1271 are derivatives of pIJ6021 and pIJ4123 (p. 539) containing the pBR322
replication function and bla gene (Z. Deng, personal communication).

&KDSWHU0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV



rep
pIJ101ter

ori
pIJ101

neo

pIJ6997.12 PvuII

SacII 2.38

tsr

9.5 kb

ter

vph
ori

Ptet

p15A

3925 HindIII
SacI
XhoI

BglII•
BfaI•
XbaI*•
EcoRV•ClaI•

rep
pIJ101EcoRI

BamHI•
3876 SalI

pJOE8294728 bpori
pIJ101hyg

pIJ699. Positive selection vector. Self-religation after excision of the "stuffer" fragment creates a c. 350
bp perfect palindrome from the inverted fd terminator (ter) sequences, rendering the plasmid "inviable".
For efficient positive selection, digest the plasmid e.g. with DraI and BglII and gel purify the 5 kb vector
fragment. This prevents recloning of the stuffer fragment and deletion of the terminator sequences by
BglII star activity. Note that the p15A E. coli replicon is replaced in the process of cloning and theresulting hybrid plasmids replicate in Streptomyces only. The neo gene can be selected in E. coli
(50 µg ml-1 kanamycin in L agar) but not in Streptomyces (Kieser and Melton, 1988).
pJOE875. Positive selection vector using non-viability of palindromes similar to pIJ699 (Altenbuchneret al., 1992).
pJOE829. Plasmid without tsr (J. Altenbuchner, personal communication). No sites for: AflII, ApaLI,
AseI, DraI, HpaI, KpnI, MluI, NdeI, NsiI, PvuII, ScaI, SpeI, SphI, StuI. Multiple sites not shown: ApaI,
NaeI, NarI, NruI, PvuI, SmaI.



Chapter 0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV

PstI

ori
ColE1

bla
tsr

pHZ1351

SspI

8.0 kbori

sti

pIJ101

pIJ101

BamHI
T7 T3
NotI
EcoRI
EcoRI
ClaI NotI

rep
pIJ101

PstI
XbaI
PstI

PstI
HindIII

cos

bla

oriT
oriT
RP4

BglII
PstI
SalI
BclI*

ori
ori

RP4

ColE1
ColE1

pHZ1358
tsr

SalI

10.7 kb

EcoRV

ori oripIJ101
pIJ101

ClaI
BclI*

AseI

rep
rep

pIJ101
pIJ101

SacII

sti

(neo)
(neo)
Tn5
Tn5

sti
pIJ101
pIJ101

ApaI

PstI
BclI*
BglII
EcoRI
BclI*
SalI

SmaI
SmaI

SmaI
XhoI

pHZ1351. Highly unstable Sti+ vector with a wider host range than other pIJ101 derivatives, useful forgene replacements (K. Bao and Z. Deng, personal communication).
pHZ1358. Derivative of pHZ1351 containing oriT RK2 andcommunication).

cos (K. Bao and Z. Deng, personal

&KDSWHU0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV



7.18 XbaI•
SphI
PstI
XbaI•

BamHI•
BglII•
KpnI
SacI
EcoRI
XbaI•
7.10 SalI

rep
pIJ101

ter

ori

fd

pIJ101PL

6.75 PstI

luxA

pRS1105

BclI 2.19

8.8 kb

tsr
luxB
neo

k

t RI

ori
pIJ101

rep

galK

pIJ101

E. coli

k

ori

pSKO2

pUC8

8.5 kbbla
tsr

pRS1105. Vibrio harveyi luxAB promoter-probe plasmid derived from pIJ487 (p. 533). The plasmid
suffers from structural instability (Schauer et al., 1988; Sohaskey et al., 1992a, 1992b; see also pIJ5971
p. 498).
pSKO2. E. coli galK promoter-probe plasmid (Brawner et al., 1985, 1997).



Chapter 0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV

rep
pIJ101

ori
pUC19

ori
pIJ101pWHM3
7.2 kb

bla
tsr
lacZ

pWHM3 (Vara et al., 1989). Bifunctional derivative of pIJ486 allowing blue/white selection.
Recommended by D.F. Brolle for general cloning purposes.

pUWL series (Wehmeier, 1995). Bifunctional derivative of pIJ101 allowing blue/white selection. Four
plasmids containing the replication region and the tsr gene excised from pIJ699 ligated to pUC18. The
bla gene is for selection in E. coli. Blue/white screening is possible for inserts in the cloning sites inlacZ . The four plasmids contain the multi-cloning sites of pUC18, pUC19 (unique cloning sites for
HindIII, SphI, PstI, XbaI, BamHI, KpnI, SacI, and EcoRI), pBluescript KS, or pBluescript SK (uniquecloning sites for SacI, BstXI, XbaI, BamHI, PstI, EcoRI, HindIII, and KpnI), respectively.

&KDSWHU0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV

pRES1 derivatives
pRES18/19 (Ishikawa et al., 1996). Bifunctional plasmids derived from the 4.8 kb cryptic S. griseus SS1429 plasmid pRES1 and pUC18/19. tsr and bla for selection in Streptomyces and E. coli, respectively.
Blue/white screening in E. coli. Single cloning sites for EcoRI, SacI, SmaI, BamHI, XbaI, PstI, SphI, and
HindIII.



Chapter 0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV

SP6>

<T7

T

T
rrnC

trpA

neo
SP

rep

pFD666

pJV15251 bp

ori
ColE1

ori

ori

pJV1

M13

cos

rep
pJV1

pWOR126tsr

7.2 kbori
pJV1
1.56 kb

pJV1 derivatives
pJV1 has been fully sequenced AC=U23762 and analysed in great detail (p. 258). The replication
functions are very similar to those of pIJ101 but the transfer functions are more like those of pSN22(Servin-Gonzalez, 1993, 1996; Servin-Gonzalez et al., 1995). pJV1 seems to have a wider host-range
than pIJ101, and it is more stable in Saccharopolyspora erythraea.
pFD666 AC=AF000429. Shuttle cosmid vector, also suitable for generating single-stranded DNA. T,
terminators flanking the cloning sites (Denis and Brzezinski, 1992). For S. lividans transformation
overlay with 5 mg kanamycin per plate (200 mg ml-1 final concentration).
pWOR126 (Bailey et al., 1986).

&KDSWHU0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV

pB45. BamHI-KpnI fragment from pJV1. Unique sites are marked with an asterisk. The BglII site has
been used for cloning (L. Servin-Gonzalez, personal communication). pB45 has been used for making
polyketide libraries (Xue et al., 1999).
pB48. Derivative of pB45 containing the 1.5 kb BclI fragment of pIJ702 inserted into the BglII site. The
BglII site in melC1 is for cloning; insertion prevents tyrosinase expression as in pIJ702. Unique sites aremarked with an asterisk (L. Servin-Gonzalez, personal communication).
pB83. Plasmid similar to pIJ702 but with pJV1 replicon. Unique sites are marked with an asterisk(L. Servin-Gonzalez, personal communication).



Chapter 0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV

tsr
ble

ori

pGM9

pSG5

neo

6.20 kb

rep
pSG5

ter
rep
pSG5

aacC1
(Gentamicin)

pGM11
5.30 kb

ori
pSG5

neo

pSG5 derivatives
All derivatives of pSG5 are naturally temperature-sensitive for replication. The replication region ofpSG5 has been sequenced AC=X80774, and a low frequency of plasmid transfer has been observed evenwith plasmids lacking the pSG5 traB gene; a map of pSG5 is on p. 257 (Maas et al., 1998; Muth et al.,1995).
pGM9 and pGM11. Note that aacC1 of pGM11 is not expressed because it lacks a promoter (Muth etal., 1989).

&KDSWHU0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV



ble

tsr

P

pGM16
5.10 kbori
pSG5

rep pSG5

catSa

ble

Chloramphenicol

neo

pGM17

5.08 PstI

6.80 kb
ori
pSG5

rep
pSG5

pGM16 and pGM17 differ in the selection markers. The promoter (P) in pGM16 is from the neo geneof Tn5 (Wohlleben et al., 1994).



Chapter 0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV
7.55 SacI
7.54 KpnI
7.54 SmaI

7.53 BamHI•
7.53 XbaI•
7.52 (SalI)
7.51 PstI
7.51 SphI
7.50 HindIII

full size

aacC1

rep

(Gentamicin)

pSG5

pGM1607.78 kb

ori
pSG5

tsr
bla

oriT
rep
pSG5

cos

ori
pSG5

pHZ132

7.61 SphI

tsr

10.1 kbvph
bla
ori
pAT153

pGM160 (Muth et al., 1989) and pHZ132 (Hu et al., 1994) are bifunctional pSG5 derivatives.

&KDSWHU0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV



.

lacZ
.?

oriT
RK2

aac(3)IV

pKC1139

Apramycin

6.5 kbori
psG5

6.85 SacI
KpnI
SmaI

BamHI•XbaI•(SalI)
6.80 PstI

oriT
rep

RK2

pSG5

pCZA185ori

7.1 kb
bla

pSG5

tsr

pKC1139 (Bierman et al., 1992) and pCZA185 (Solenberg and Baltz, 1994). Bifunctional oriT RK2
vectors for gene replacement and transposon delivery. (The pBR322 origins of replication are notindicated on the figures.)

Chapter 0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV

tsr
ori

oriT

pSG5

rep
pSG5

pUCS30

ori
pBR32215.4 kb

neo
ble
PtipA fd

tnp terpUCS30, a tipAp-driven transposon delivery plasmid (Smith and Dyson, 1995).

&KDSWHU0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV



tsr
bla

pHJL302
5.1 kb

ori

ori

pUC19

SCP2*
(1000 copies)

Plac

2.72 HindIII•

NarI 2.51

SphI

PstI•
SalI/HincII

XbaI•
BamHI•
SmaI•
KpnI

tsr

SacI•
2.67 EcoRI•

bla

pHJL401

BclI 1.49

5.9 kb

ori
pUC19

Plac

ori
SCP2*
(10 copies)

3.52 HindIII•
SphI

PstI•
SalI/HincII

XbaI•
BamHI•
SmaI•
KpnI

SacI•
3.47 EcoRI•

SCP2* derivatives
A map of SCP2* and summary diagrams of many vectors are on pp. 258-262.
pHJL302 has an extremely high copy number, and pHJL401 has an intermediate copy number. Bothplasmids are transfer-deficient and lack the SCP2* stability function (Larson and Hershberger, 1986).



Chapter 0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHVori

pBR327

ori

bla

SCP2*

Origin!!!

Stability

pIJ90325.8 kb

tsr
Transfer

Stability

ori
SCP2*

pIJ92224.0 kb

Transfer

tsr

traA

pIJ903 is a bifunctional derivative that has been used to study the SCP2* transfer functions using
Tn4560, and pIJ922 has been used for cloning large DNA fragments (Lydiate et al., 1985; Malpartidaand Hopwood, 1984).

&KDSWHU0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV



ori
SCP2*
Stability

pIJ92026.5 kbvph
Transfer

traA

ori
SCP2*
Stability

pIJ92126.5 kbvph

Transfer

traA

pIJ920 and pIJ921 contain the vph marker in opposite orientations (Lydiate et al., 1985).



Chapter 0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV

ori
SCP2*

Stability

pIJ94024.9 kbtsr
hyg

Transfer

31 SphI

35 KpnI
34 BclI
33 KpnI
32 BclI

1 XhoI
2 XbaI
3 BamHI30 PvuII
4 BglII
29 BclI

28 KpnI

OriginSCP2*

27 BclI

Stability

5 XbaI

pIJ9416 SphI

7 SstI

25.0 kb

8 BamHI/BclI

tsr

9 BclI

10 ClaI

26 SphI
11 EcoRV

12 PvuII

13 BclI

14 BamHI/BclIhyg

Transfer

15 BamHI/BglII

16 BclI
17 SstI
18 EcoRI
19 BamHI/MboI
25 SstI
24 BclI

20 PstI
23 KpnI
22 SphI

21 SphI

pIJ940 and pIJ941 (Lydiate et al., 1985).

&KDSWHU0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV



ter
neo

ori

vph

SCP2*

ori
p15A

ter

pIJ698

Stability

29.9 kb

SCP2*

Transfer
SCP2*

tsr
hyg

BamHI/MboI

tsr

actII-ORF4(activator)
KR

pRM5

KS
NsiI

CLF

ori-SCP2*

ACP
ARO
CYC

ori-ColE1
bla

pIJ698 is a replacement vector. Note that the p15A E. coli replicon is lost on cloning (Kieser andMelton, 1988). Its use by Ryding (1995) and Ryding et al. (1998) is described on pp. 261-262.
pRM5, containing part of the S. coelicolor actinorhodin PKS gene cluster, was specifically designed forthe regulated expression of combinations of polyketide synthase genes. Note that pRM5 lacks the SCP2*transfer and stability functions (McDaniel et al., 1993). pCJR29 (p. 558) is another expression plasmid
for PKS genes.



Chapter 0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV

pCJR29 (Rowe et al., 1998) is similar to pRM5 (p. 557) but lacks the KS, CLF, ACP, ARO and CYC
actinorhodin biosynthesis genes. (See also pCJR24, p. 567, which lack the SCP2* replication functions.)
Xue et al. (1999) constructed and used three similar plasmids with SCP2* or pJV1 replicons, and apSET152 derivative ( C31 integration functions) for preparing combinatorial libraries of polyketides.

&KDSWHU0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV



aphI
Neomycin

ori
SCP2*

pIJ80
19.2 kb
Transfer

ori
Replication

pBR327blatsr

pIJ4680
23.5 kbter
aadA
spc/strter
Transfer

traA

pIJ80 is an unstable plasmid that has been used as an incompatible replicon to eliminate wild-type
SCP2* derivatives from S. coelicolor and S. lividans (Thompson et al., 1980).
pIJ4680 is a positive selection vector similar to pIJ698 (p. 557) but it lacks the SCP2* stability region
and the pBR327 origin is not lost on cloning. This plasmid has been used for making gene replacements(p. 319) (Khosla et al., 1992).



Chapter 0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV

ori
cos

colE1

aac(3)IV
Apramycin

ori

cos

SCP2*

pKC505cos

18700 bp

(aphI)(Transfer, 5.10- 5 )

cos

cos
Transfer
SCP2*

cos

hyg

pJAR4
21.9 kb

ori
SCP2*

pKC505 (Rao et al., 1987; Richardson et al., 1990) and pJAR4 (Tercero et al., 1992) arevectors containing the SCP2* replication functions.

cosmid&KDSWHU0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV



BglII 1.69

EcoRI•SacI(KpnI)(SmaI)

BamHI•XbaI(SalI)PstI(SphI)

HindIII• 1.75
PstI 2.05
BglII 2.06
SacI 2.35

ter
mdh

BclI/BamHI 2.85

BglII 3.24

ori
SCP2*

Stability

pTMA1
25.7 kb

tsr
Transfer

lacZ
ori
pUC18

ori
SCP2*

pKC1218
5.8 kbaac(3)IV
Apramycin

oriT
RK2

pTMA1 is a promoter-probe vector using the thermostable malate dehydrogenase gene (mdh) of Thermus
flavus as indicator (Vujaklija et al., 1991). The malate dehydrogenase assay is described on p. 348.
pKC1218 (Bierman et al., 1992). oriT RK2 vector that replicates autonomously in Streptomyces.



Chapter 0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV

cos

ori
SCP2*

pOJ44610.4 kb

cos
cos

oriT
RK2

tsr

ori
SCP2*

melC1
melC2
Tyrosinase

pIJ94320.6 kb
Stability

pOJ446 (Bierman et al., 1992). oriT RK2 cosmid vector.
pIJ943 (Hopwood et al., 1985). SCP2 vector for insertional inactivation of the mel genes, similar to
pIJ702 (p. 536).

&KDSWHU0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV



Replication

aphI

pIJ61

11.14 BstEII

14.8 kb

BamHI/BclI 3.58

tsr

Transfer
Replication

oriT
RK2

xis
int
pSAM2

pPM927attP
pSAM2

13.0 kbori
pBR322

tsr
PtipA

ter
fd

aadA
spc/str

pSAM2 and SLP1 derivatives
Maps of parental plasmids are on pp. 263 and 264, and their properties are described on p. 265.
pIJ61. Autonomously replicating plasmid (Thompson et al., 1982).
pPM927. Integrating vector; no autonomous replication in Streptomyces (Smokvina et al., 1990).



Chapter 0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV

bla

pACYC177
3941 bp

ori
p15A

km

cat

ori

pACYC184

p15A

4245 bp

tet

E. coli plasmids
pACYC177, AC=X06402, and pACYC184, AC=X064023 (Rose, 1988).
Note that the chloramphenicol acetyltransferase in E. coli gives a colour reaction on rich agar containing
2-3 µg ml-1 of the rosaniline dye crystal violet (Proctor and Rownd, 1982). Tetracycline- resistant E. coli
can be counterselected using 2 mg ml-1 fusaric acid (Maloy and Nunn, 1981).

&KDSWHU0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV(NheI joined to XbaI)(BglII joined to BamHI)(BamHI filled andενδjoined
ααδΑto HindIII)

ter

SP6

lacZ
aadA
Spc/Str

pIJ4642ori
pACYC184

6.17 kb
T7

cat
ter(PvuII joined to filled BamHI)(BamHI joined to BglII)

ter

ter
fd

fd

neo

lacZ

pIJ666

ble
SmaI 1665

6716 bp

ori
p15A

cat

pIJ4642 and pIJ666 are E. coli positive selection vectors based on the lethality of uninterrupted
palindromes which are formed by the 350 bp inverted fd terminator sequences (Kieser and Melton,
1988). Similar plasmids have been constructed by Elhai and Wolk (1988) and by Altenbuchner et al.
(1992).



Chapter 0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV

lacZ

ori
pUC18

xylE

lacZ

aac(3)IV
Apramycinori

pKC1138

p15A

oriT

6.6 kbRK2

pKC1250
3.8 kboriT RK2

aadA
Spc/Str

lacZ
ori
pUC18

lacZ
oriT
RK2

pOJ260ori
pUC18

3.5 kbglkA
Glucosekinaseaac(3)IV
Apramycin

pIJ6650
4.7 kbaac(3)IV
Apramycin

oriT
RK2

pKC1138, pOJ260, pKC1250 and pIJ6650 are suicide vectors for Streptomyces containing oriT RK2
for conjugation from E. coli to Streptomyces (Bierman et al., 1992).
pKC1132 was derived from pOJ260 by deletion of the KpnI-SpeI fragment which contains the DraI site
(Bierman et al., 1992).
pIJ6650 is a derivative of pKC1132 (see above) containing a 1.2 kb SmaI-BclI glkA fragment of
S. coelicolor (p. 493) inserted into the BglII site. pIJ6650 is used frequently for making genereplacements (M. Paget, personal communication); see also pIJ2581 (p. 571).

&KDSWHU0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHVpIJ8671. Suicide vector containing tipAp flanked by transcriptional terminators, for expressing genes
inserted into the host chromosome by homologous recombination (J. Sun, personal communication).
pCJR24. E. coli plasmid derived from pUC19 containing tsr and bla for selection in Streptomyces andE. coli, and actII-ORF4 and actinorhodin promoters for the regulated expression of antibiotic
biosynthesis genes in Streptomyces (Rowe et al., 1998). (See also pCJR29, p. 558 and pRM5, p. 557.)



Chapter 0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV

T7
SacI 653
BstXI
SacII
NotI
EagI
XbaI
SpeI
BamHI
SmaI
PstI
EcoRI
EcoRV
HindIIIClaISalI, HincII
XhoI
ApaI
KpnI 755
T3

ori
f1

bla

pBluescript II KS+

2217 AseI

2961 bp

ori
ColE1

HindIII 233
SphI
PstI
Sse8387I
SalI, HincII
XbaI
BamHI
SmaI
KpnI
SacI
EcoRI 284

BglII

PstI
HindIII 233
SphI
SalI, HincII
XbaI
BamHI
SmaI
KpnI
SacI
EcoRI 284

lacZ
ori

pUC19
2686 bp

BglII

lacZ

bla

ori

pIJ2925
2686 bp

bla

SphI

PstI
Bgl II

Hin dIII

Acc I
Hin cII
Sal I

Xba I

Sma I
BamHI

Sst I
Kpn I

Eco RI

Bgl IIATGAC ATG ATT ACC AGATCT GCA GCC AAG CTT GCA TGC CGG TCG ACT CTA GAG GAT CCC CGG GTA CCG AGC TCG AAT TCC CCA GAT CTG AGC TTG
T
R
S
A
A
K
L
A
C
R
S
T
L
E
D
P
R
V
P
S
S
N
S
P
D
L
S
L
M I

pBluescript II KS+ AC=X52329 (Alting-Mees and Short, 1989), is sold by Stratagene. See Mayer(1995) for similar plasmids with kanamycin or tetracycline resistance.
pIJ2925. pUC18 derivative with a multi-cloning site (AC=X69147) flanked by BglII and shown under
the plasmid map (Janssen and Bibb, 1993).
pUC19 AC=X02514 (Yanisch-Perron et al., 1985).
pANT840 (W.R. Strohl, personal communication) is a pUC19 derivative useful for cloning Streptomyces
genes in E. coli. Multi-cloning site: EcoRI, SacI, KpnI, XmaI, BamHI, XbaI, BglII, XhoI, StuI, BclI, ClaI,
ApaI, StyI, NcoI, NheI, NotI, MluI, NruI, SpeI, XbaI, SalI, HincII, AccI, PstI, SphI, HindIII.

&KDSWHU0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV



cos
bla

cos

cos
ori
SV40

SuperCos1

ori
ColEI

7937 bp(6857+1080bp)

neo

ApaLI 352
DraI 444

7748 DraIcos (delete)cos

BamHI 1/7932
7905 NotI
7899 EcoRI
7874 ClaI

ScaI 541

NotI 18
EcoRI 26
AatII 101

PvuI 652

PstI 778
AseI 848

DraI 1136
DraI 1155

cos
bla

ori
SV40

ApaLI 1598

Super1ori
ColEI

6852 bp4942 PstI

4863 SphI

4791 SphI

4654 SfiI4610 StuI
4591 HindIII

neo

PvuII 3241
SmaI 3270

SuperCos 1 AC=M99566 is sold by Stratagene (Evans et al., 1989). pSupercos 1 was used for theordered S. coelicolor A3(2) library (p. 424; Redenbach et al., 1996). The neo marker can be selected in
S. coelicolor (pp. 218,236).
Super1 is pSupercos 1 as it is found in the cosmid clones after packaging into phage heads, lacking the
DNA between the cos sites.
pVE616 is a cosmid vector containing tsr and a BamHI, DraI, BglII, HpaI, DraI, PstI multi-cloning site
MacNeil et al. (1992).



Chapter 0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV

lacZ

ori
pUC19

oriT
RK2

pSET151bla

6.2 kbEcoRI 0.19
SacI
KpnI
XmaI
AvaI

SmaI
BamHI
XbaI

xylE
tsr

SalI, AccI

HincII
PstI
SphI
HindIII 0.24

ori

lacZ

f1

bla

AseI 0.39
PvuII 0.45
AseI 0.45

AlfIII 0.62

lacI

pDH5

ori
pUC19

4.65 kbtsr

pDH5 is suitable for making single-stranded DNA using phage superinfection (Hillemann et al., 1991;
Bierman et al., 1992).
pSET151 contains oriT RK2 for conjugation from E. coli to Streptomyces (Hillemann et al., 1991;
Bierman et al., 1992).
pVE1011 is a pBR322 derivative containing tsr and BamHI, HpaI and BglII cloning sites close together
and has been used for the efficient creation of chromosomal deletions and for in vivo cloning of large
DNA fragments (Chapter 14; MacNeil et al., 1993).
Other E. coli plasmids containing tsr and different polylinkers have been constructed by MacNeil et al.
(1992) and by Gewain et al. (1992).

&KDSWHU0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV

pPM2208 is a derivative of pPM925 (Smokvina et al., 1990) which lacks most of the pSAM2 int geneand thus no longer integrates site-specifically into the Streptomyces chromosome (P. Mazodier, personalcommunication; Servant et al., 1993).
pIJ8668 is an E. coli plasmid containing the promoterless EGFP gene (p. 499) and the apramycin
resistance gene (aac(3)IV) for selection in E. coli and Streptomyces (Sun et al., 1999).
pIJ2581. A vector containing the counterselectable glkA gene for making gene replacements (pp. 307308, 320-322; van Wezel and Bibb, 1996). (See also pIJ6650, p. 566.)



Chapter 0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV

bla

tet

pBR3273274 bp

ori
ColE1

bla

tet

pBR3224361 bpori

pBR322 AC=J01749 (Bolivar et al., 1977; Sutcliffe, 1979; Watson, 1988).
Reviews about pBR322: Balbas et al. (1986, 1988).
pBR327 AC=L08856 (Soberon et al., 1980). Note that pBR327 has a copy number higher than pBR322
but lower than pUC18/19.

&KDSWHU0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV



attM
ORF1

ori
pBR327(ApaLI)

pIJ4201

6119 ΣαχΙΙ

8168 bp

neo

bla
bla

tsr

ORF2,3

ORF1

ori
pBR327

pIJ42108172 bp

tsr

attM

(neo)

Transposable elements
IS117 derivatives
IS117 from S. coelicolor, missing from S. lividans, integrates into specific sites. The uses ofIS117 derivatives are explained on pp. 265-267. A map of IS117 is on p. 266.
pIJ4201 and pIJ4210 differ in the orientation of the resistance genes which is important because neo
from Tn5 lacks its own promoter (Lydiate et al., 1986, 1989).



Chapter 0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV

ORF2,3

bla

ORF1

SmaI 874

ori

pIJ4696

pBR322

ApaI
XhoII
MluI

SacI 1892

7538 bp

SacII

PmaCI 984

attM

hyg

bla

ORF2,3

ORF1

ori
pBR327attM

pIJ4698
10793 bp

(neo)

tsr
ter

ter

aadA

bla

ORF2,3

ORF1

ori
pBR327

pIJ4700
7829 bp

attM

aadA

pIJ4696, pIJ4698, and pIJ4700 (Bhatt and Kieser, 1999; Smokvina et al., 1994) are IS117 derivatives.
pIJ4698 can be used as a positive selection replacement vector similar to pIJ4642 (p. 565).
Other IS117 derivatives have been constructed by Motamedi et al. (1995) and by Paget et al. (1994).

&KDSWHU0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV
Bsu36I

13

BsmI

277

PvuII

458

PvuII
PstI

555
598

Bst1107I
BstXI
SacI

610
623
655

SphI
StyI
BbsI

731
750
759

PmlI

860

BsaI
DraIII
BspMI

944
952
974

PvuII

1089

XmnI
PvuI

1150
1185

SfiI
BglI
NaeI

1288
1289
1294

SacII

1432



ORF A
Mr=19249

ORF B
Mr=27497

PstI

1455

ClaI
NruI
DrdI

1476
1518
1522

IS493 1641 bp 66% G+C
3 copies in S. lividans
IS493 derivatives
IS493 from S. lividans and its application for transposon mutagenesis are described on pp. 117-118. A
summary figure showing IS493 derivatives is on the next page. Examples of vectors containing someof these elements are on p.577. Note that these transposons cannot be used in S. lividans because of thethree resident copies of IS493. AC=M28508 (Solenberg and Burgett, 1989).



Chapter 0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV

aph(4)PL

aph(4)

PL

P

xylE

xylE

PL

PL

ORFA

aac(3)IV

ORFA

ORFB

ORFA

ORFB

aph(4)ORFA

ORFB

aph(4)

ORFA

ORFB

ORFB

Summary of IS493 derivatives. Note that Tn5099-10 and Tn5100-4 have mutations that increase thetransposition frequency in Streptomyces (Baltz et al., 1992; Hahn et al., 1991; Solenberg and Baltz,
1991, 1994; Solenberg and Burgett, 1989). PL, phage promoter which is expressed in E. coli and in
Streptomyces.

&KDSWHU0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHVEcoRI 396
KpnI

SmaI
BamHI
XbaI
PstI
SphI
HindIII
BglII
NsiI
XbaI 461
PstI 651

bla
ORFB

pCZA1634315 AlwNI

5784 bpori

aac(3)IV

puC19

Apramycin

Plac

bla

ori
ColEI

Tn5096

oriT

pCZA258
6900 bp

P lac

Tn5099-10

aph(4)
Hygromycin

bla

oriT

ori
pUC19

pCZA276

ORF A

7000 bp

P lac

Tn5100-4

ORF B

aph(4)
Hygromycin

pCZA163. E. coli plasmid containing Tn5096 (Solenberg and Burgett, 1989).
pCZA258. E. coli plasmid containing oriT RK2 and Tn5099-10 (Solenberg and Baltz, 1994).
pCZA276. E. coli plasmid containing oriT RK2 and Tn5099-4 (Solenberg and Baltz, 1994).



Chapter 0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV

Tn4556 derivatives
The properties of these elements are described on p. 111-117.
Tn4556. Wild-type element without resistance marker AC=M29297 (Siemieniak et al., 1990).
Tn4560 Chung (1987). pUC1169 containing Tn4560 is on the next page.

&KDSWHU0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV

14259 SfiI
SmaI 32

13969 SacII
13942 SmaISamI 227SalI 483
FspI 527

13041 SamI
MunI 1451
SfiI 1477

Eam1105 1738
MscI 1770
Bpu1102 1872
12345 AlwnI

SacII 1970

rep

11830 SalI

pIJ101

IR

tnpA10864 SalI

ori
pIJ101SalI 2825

tsr

pUC1169

SalI 3331

14310 bp

SalI 3598

IR

res

SalI 4143
SacII 4160
FspI 4226

tnpR

vph

BstXI 4735

8528 SalI

7755 SalI
7675 SacII

SalI 6590

pUC1169. pMT660 (p. 536) containing Tn4560 (Chung, 1987).



Chapter 0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV

Tn5351 and Tn5353. Tn4556 derivatives containing luxAB for promoter probing (Sohaskey et al., 1992a,
1992b).

&KDSWHU0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV



rep
ori

pSG5

pSG5

ter
IR

tsr
neo

pJOE2577

Tn5

9.7 kbIR

SacI 2.45

tnpA
Tn5

Pmer
bla

Tn5 derivative
pJOE2577. Mini Tn5 derivative Tn5493 containing tsr on a naturally temperature sensitivepSG5-derived delivery vector (Volff and Altenbuchner, 1997). A protocol for the use of pJOE2577 is
on pp. 121-122. Note that the transposase will be lost with the vector after transposition.



Chapter 0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHV

Altenbuchner J, Viell P, Pelletier I (1992) Positive selection
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

Chapter 0DSVRISODVPLGVWUDQVSRVRQVDQGSKDJHVSmith MC, Burns RN, Wilson SE, Gregory MA (1999)
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,QGH[$IDFWRUWR$PPRQLXPDFHWDWH



Index
A
A-factor 17, 27
aac(3)IV (apramycin acetyltransferase)
442, 447, 450, 464-465 (maps)
cross-resistance 448 (table)
on vectors 292, 293, 513-514, 516518
aacC1 (gentamicin acetyltransferase)
442, 447, 450, 466 (map)cross-resistance 448 (table)
on vectors 120, 268, 548 (map), 549(map)

Actinorhodin 430
assay 93
gene cluster
act promoters 557 (map), 558(map), 567 (map) see also actIIORF4
expression in S. parvulus 29
expression in S. galileus 389
gene replacements 320, 322
non-coding segment 18
on pRM5 557 (map)production by S. coelicolor 93
production by S. lividans 95, 224
in pocks 153
structure 94

aacC7, aacC8, aacC9 (aminoglycosideacetyltransferases) 442, 447,
450, 467 (maps)

Adriamycin (doxorubicin) 13

aadA (spectinomycin/streptomycin
adenyltransferase) 442, 449,
451, 468-469 (maps)
on vectors 268, 390, 516-518
selection of 241, 324, , 470

Aerial mycelium see also bld
development 16, 17, 19 (figure),
48, 49diagnostic of actinomycetes 2
inhibition in pocks 153, 239, 255
microscopy 64
preparation 54-55

ABI PRISM sequencing kit 394, 418(supplier)
Acetonefor precipitating protein 87
in catechol 2,3-dioxygenase assay
345
mixing with chloroform causes
explosions 164
Acid phenol/chloroform 172, 187(recipe)
Acidophilic streptomycetes 6
Aclacinomycins, biosynthesis 26
Acridines, causing DNA
rearrangements 20
actII-ORF4, activator gene 350,
on vectors 557 (map), 558 (map),
567 (map)strains containing 423
Actinomadura spp., as pathogens 6
Actinomyces spp., as pathogens 6
Actinomycin 12
binding to DNA 162
Activated charcoal, in dialysis of DNA
165
Actinoplanes teichomyceticus,
teichoplanin producer 15

Agar 418 (suppliers)
antibiotic production on 16
as carbon source 106
as dispersant in liquid medium 51
cultures on 47-49
for microscopy 64, 70
preservation 58
media 406-411
Agar block technique, for isolating
antibiotic non-producing
mutants 106
Agaraseproduced by S. coelicolor 45, 223,430
to remove agar 51
Agarose, LMP for PFGE 198, 200-203
Agarose gel electrophoresis
of DNA 196-197 (pictures)
DNA degradation during 22, seealso dnd
effect of G+C content 212
interpretation of partial digests
215
PFGE 198-205
preparative agarose gelelectrophoresis 175
quantitation of DNA 175
staining of DNA 167
standard gels 195-198 (pictures)of RNA 361 (picture), 361-363
Agarose, Ni-NTA, for purifying
histidine-tagged proteins 382
AG50W-X8 (ion exchange resin) 182
Algae, as producers of secondary
metabolites 11
Alkaline lysis, for plasmid isolation
171, 172, 195-198
agarose gel picture 196-198
and CsCl centrifugation 174
procedures 184-186, 187
Allele ratios, in linkage mapping 148,
150, 152
in NF × SCP1- crosses 129 (figure)Alumina, for cell-free extracts 76
AMCA-S stain 67, 69, 70
Amikacin 452
resistance 450, 467
cross-resistance 448 (table)
Amino acidbiosynthesis 9
catabolism 9
composition of proteins 83, 401
Aminoglycosides 13-15, 452-454
resistance 450
cross-resistance 448 (table)
maps 460-462, 464-469, 478-481
marker genes 442-445
rRNA methylases 446, 447(figure)transferases 447, 449
aml (alpha amylase), regulatory
sequences, involved in glucose
repression 8
Ammoniumas nitrogen source
for actinorhodin production 93
in minimal media 406, 413(recipe)in selective isolation ofStreptomyces species 44
assimilation 8
inhibitor of polynucleotide kinase
165
repression 9, 16,
Ammonium acetate, in DNA isolation
165, 173
solutions 205

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Ammonium sulphate, in RNA
polymerase isolation 380-381
ampC ( -lactamase) 442, 449, 495(map)
as reporter 349
Amplificationof chromosomal DNA segments 20,
24, 26, 28, 31, 427
of E. coli vectors 172
AmpliTaq DNA Polymerase FS 394
AmpliTaq Gold 248, 418 (supplier)
Ampoules, for lyophilisation 59(supplier)
Amycolatopsis
DNA base composition 18
gene exchange 125
taxonomy 3
Amycolatopsis mediterranei
gene exchange 127
linkage mapping 31, 138
rifamycin production 14, 31
transformation 31, 231
Amycolatopsis methanolica
plasmids 31
transformation 231
Amycolatopsis orientalis
transformation 31
vancomycin production 15, 31
Animals, as producers of secondary
metabolites 11
Anion exchange chromatography, inplasmid isolation 175, 189-190
Anthracyclines 12, 13, 452, see also
Daunomycin/daunorubicin
genes 27, 389

24-32, 212,
isolation of 222, 224, 291
replacements in 333
vectors for expression 557 (map),
558 (map), 567 (map)non-producing mutants
effects of bldA 17
isolation 106
produced by actinomycetes see
tables11, 12-15
production 7, 10-11, 50
by S. coelicolor and S. lividans
93-95, 430
effect of rpsL (strA) mutations220
effects of plasmid copy number
217
improvement of 23, 100in agar-grown cultures 16, 48,406
in competition 16
in liquid 16, 50
in pocks 153, 155
in soil 10-11
plasmid-encoded 255, 422
regulation of 16, see also actIIORF4
useful 12-15
properties 12-15, 452-455
suppliers 455
used in Streptomyces genetics see
table 452-455
Antibodies
for clone recognition 222
for phage SV1 inactivation 275
for spore isolation from soil 44
in immunofluorescence microscopy
66-69, 418 (supplier)in immunogold labelling 73
Anticoccidial agents 13, 15

Antiamoebal agent 14

Antifoam 50, 51, 52, 413 (recipe)

Antibiotic resistanceaminoglycoside cross-resistance 448(table)determining level of 219
genes see table 442-446
markers as reporters 342
medium dependence 172, 219, 470
spontaneous mutation to 104, 220
to detect transformants 239-240,
323, 324
delayed expression 221
for selecting lysogens 304-307
in colony hybridisation 244, 247
in conjugation from E. coli 250
in gene disruption 323-324
table of concentrations 241

Antihelminthic agent 13

Antibiotics see also individual names
biosynthetic gene clusters 16, 21,

Antimycin A 12
Antiparasitic agents 12, 13
Antiserum see Antibodies
Antitumour agents 12, 13, 14
aph(4) (hygromycin
phosphotransferase) 442, 449,
450, 464 (map)on vector 577 (map)aphD, aphE (streptomycin phosphotransferases) 442, 449, 451,
487 (maps)
aphI (aminoglycoside phospho-

,QGH[

transferase) 442, 449, 450, 478(map)lack of leader sequence 212
on vectors 514-517
selection of 241 (table)
cross-resistance 448 (table)
Apramycin 452
resistance 442, 447, 450, see also
aac(3)IV
cross-resistance 448 (table)
to select recombinants 241(table), 250, 327, 348
Apurinic site endonuclease activity see
Exonuclease III 193
Arabinose, as carbon sourcein glkA counterselection 307, 493
in minimal medium 406
ard1 (aminoglycoside resistance) 442,
447
argG (argininosuccinate synthetase),
unstable gene 20, 300, 426(map), 427
Argininebiosynthesis 9
metabolism 9
unstable biosynthetic gene see argG
Arginine salts agar, for selective
isolation of Streptomyces 44
Arginine tRNA
gene on phage VWB 390
rare codons for 391
AseI
in PFGE 199, 203, 422 (map), 426(map), 431-433 (maps)
rarity of sites 212
recognition site 441
transposons with rare sites 111
Asparagine, as carbon or nitrogensource 406 (recipe)
AT-specific dye 167, 175 see also
Hoechst 33258
ATP, -32P, for 5’ end labelling 246(recipe)
att sites, of integrating plasmids andphagesof IS117 266 (map)of C31 see C31, attP vectorsof pSAM2 264 (map), 265, 390
on vectors 267, 573-574 (maps)of SLP1 263 (map) 265
attB site essential 319
on vectors 263 (map)tandem integration 225
of VWB phage, on vectors 290, 532(map)

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attM see att site of IS117

BamHI
for making libraries 214
frequency of sites in Streptomyces
DNA 212
recognition site 441

Attenuation, of gene expression 9, 26
AUD elementsof S. ambofaciens 432 (map)of S. coelicolor 426 (map), 427, 432(map)of S. lividans 110, 117, 426 (map),
427-429
Autoclaving
autoclaved PEG 8000 acquires
growth inhibitor on standing 51
for mannitol soya flour medium 409
for RNA work 354
of cellophane discs 64
of velvets for replicating 135
skim milk, for lyophils 59
starch must be autoclaved withmedium 51
toothpicks, to remove nucleaseactivity 163
Auxotrophic mutants
isolation 104-105
by NTG mutagenesis 101
by transposon mutagenesis 111,
119
selecting for selenate resistance494
use
as markers in crosses 45, 131,
133, 141
choice of agar 406
for microscopy 70
supplement levels 415 (recipe)
Avermectin 12
gene cluster 25, 332
Azide, sodium, for storing
chromatography columns 182(recipe)
B
Bacillus
immunofluorescent staining 66
in vivo phage packaging 299
ribosome binding sites 386
sacB 323
sigma factor 386
signal peptides 387
taxonomic position 2
Bacillus mycoides, as indicator formethylenomycin 95
Bacillus subtilis, in competition with
Streptomyces 16
Bacteriophages see Phages
Balanced crosses 137, 153
Balanced growth 50
Bambermycin 12

bar (bialaphos resistance) 442, 471(map)
disruption using pat 323

349, 387, 440
ble (bleomycin resistance) 442, 449,
450, 481 (map), see also bleSh;
blmA, blmBon vectors 268, 548, 549, 519

Bead beater, for cell-free extracts 76,
92, 418 (supplier)

Bleomycin 12, 452, see also
Phleomycin; Zeocin
resistancecross-resistance with phleomycin
and zeocin 452
genes 31, 442, 443, 447, 449,
450, 473 (maps), 481 (map), seealso ble; bleSh; blmA, blmB

Beckman rotors, maximal speeds for
CsCl solutions 181 (table)

bleSh (bleomycin resistance) 442, 449,
450, 473 (map)Bennett’s agar 118 (recipe)

blmA, blmB (bleomycin resistances)443, 447, 449, 473 (maps)

Base composition, of DNA see Codon
usage; G+C

Berninamycin, resistance 451-galactosidase
gene of S. lividans 323, 442, 449,
496 (map)
as reporter 349-350
in plants 340
in Streptomyces 340-lactamase 25, 349 see also ampC;
bla
clavulanic acid as inhibitor 12

Boiling method, for plasmid isolation
171, 190-191
Branching, of mycelium 17, 49
Brij-58, detergent 175, 201
Bromocresol green, alkali-stableloading dye 187 (recipe)
Bromoperoxidases 25

bld genes/mutants see also bldA
crosses between 130
effect on glucose repression 8
effects on antibiotic production 16
homogenotisation 323, 332
isolation 106
roles in sporulation 17, 19 (figure)S. coelicolor strains carrying 423
storage 44-45

Buffers see the following entries
Cacodylate
DNase I
GlucoseHE
HEPES
KinaseL (lysis)
Loading buffer
NDS
P (protoplast)
PBS
PCR
S1 digestion
SB
SET
SM
SSC
STET
T (transformation)
TAE (Tris-acetate-EDTA)
TBE (Tris-borate-EDTA)TE
TE25S/TE25-sucrose
TEGR
TES
TESLR
TGED
TM
Thiourea
Tris-HCl
Tris-maleic acid

bldA, and TTA codons 17, 340, 346,

Bung, foam 419 (supplier)

BfrIin PFGE 199
recognition site 441
Bialaphos 12, 452 see also
Phosphinothricinbiosynthesis 27, 31
resistance gene 31, 442, 447, 450,471 (map), see also bar; pat
Bijou bottles 45, 47, 418 (supplier)Birnboim plasmid isolation procedure184-187
Bisbenzimide see Hoechst 33258
bla ( -lactamase)of E. coli 442, 568 (map)on vectors 514-519
of S. cacaoi, FRAME analysis 396
BLAST, database search 398, 424
Blasticidin S 452


Butirosin 452
resistance 450, 467
Butylcycloheptylprodiginine 95
structure 94
Butyrolactones 16, 31
C

C.m.a (chromosome mobilisingability), of plasmids 126(table), 127, 136-137, 140, 255
in plate-crosses 152

Calcium-dependent antibiotic (CDA)
93, 95structure 94

CD4, receptor protein 388

Candicidin 12

Cell-free extracts 75-77, 345, 348-349

Capreomycin 452, 482
resistance 443, 447, 449, 451, 454,
474 (map), see also caccross-resistance with viomycin
452

Cellophane overlays 48-49, 223, 418(supplier)
for assay of catechol 2,3-dioxygenase345
for isolating DNA 162
for isolating RNA 354
for microscopy 64, 70
germination on 50, 54-55

carA, carB (carbomycin resistance)
443, 447, 449, 451

cac (capreomycin acetyltransferase)
443, 447, 451, 474 (map)

Carbapenem, biosynthesis 26

Cacodylate buffer, for electron
microscopy 70 (recipe)

Carbomycin 452
biosynthesis 30
resistance 443, 451, see also carA,
carB

Caesium chloride
concentration, for DNA precipitation
using ethanol 165
detergents, choice of 163
for removing polysaccharides from
DNA 182-183
genomic DNA, purification 167
harvesting gradients 167, 181-182
in RNA isolation 357-359, 363
increases protoplast transformation
frequency 221
maximal rotor speeds 181 (table)
phage purification, large-scale 282285
plasmid purification 174-175, 176(figure)choice of lysis method 171
common problems 176
low-speed centrifugation ofsamples, to remove debris 174
removal of small RNA molecules
173
standard CsCl EB gradientcentrifugation procedure 178
unreliable, for coupledtranscription-translation 79
preparation of gradients 178-182,
179 (table), 180 (figure)removal by dialysis 165, 182
storage of DNA in 167
use of AT-specific dye see Hoechst
33258
used in transformation of
Amycolatopsis, Nocardia andSaccharopolyspora
Calciumin nuclease activation 79
in phage adsorption 278
in proteinase activation 163
in protoplast fusion 156
in soil structure 44
in spore germination 6
in transformation 231

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Carbon catabolite repression (CCR) 78, 503
of amino acid catabolism 9
Carbon monoxide, utilisation by S.
thermoautotrophicus 10
Carbon replicas, for electron
microscopy 70
Carboxy-methyl-cellulose, as
dispersant in liquid medium 51
Casaminoacids, to enhance growthand/or germination 50, 52-54,
410 (recipes), 413 (recipe), 414(recipe)cat (chloramphenicol acetyltransferase)
443, 447, 450, 564 (map)as reporter 342
on vectors 565-566 (maps)
Catechol 2,3-dioxygenase 296, 343,
see also xylE
assay in cell-free extracts 345-346
detectionin colonies 344
in plaques 344-345
catSa (chloramphenicol acetyltransferase) 443, 447, 450, 475(map)
on vectors 268, 549 (map)
CCC plasmid DNA
isolation 171-193
of integrating plasmids 265
of IS117 225, 428
on electrophoresis 195-198
on PFGE 204
precipitation by PEG 166
transformation by denatured 235
CCR see Carbon catabolite repression
ccrA1 of S. coelicolor, in catabolite

repression 8

Cellulases
production by Streptomyces 6, 27
can damage dialysis membranes
165
to remove carboxy-methyl-cellulose
51
Cellulose-binding protein 29
Centrifugation
CsCl EB, standard procedure 178,
see also Caesium chloride
equivalent conditions for different
rotors 183
in DNA isolation, conditions 162,
176, 181, 183, 185
in RNA isolation 176
low-speed, to remove debris before
ultracentrifugation 174
maximal rotor speeds for CsCl
gradients 181
to purify spores 44
Cephamycin C 12
biosynthesis 25, 32
Cesium chloride see Caesium chloride
Cetyltrimethyl ammonium bromide see
CTABChainia 4
CHAPS 86
Charcoal see Activated charcoal
Chelating agentsdesferrioxamine 13
in cell-free extracts 76
in RNA isolation 355
to select phage deletions 273, 290
to select transducing phages 274,281-282
Chelex-100, ion-exchange resin, forisolating Streptomyces from soil
44
Chi-squared test 143
chi63 promoter, regulatory sequences
involved in glucose repression
7-8

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Chitinagar, for selective isolation ofStreptomyces 44
biosynthesis (target of nikkomycin)
14degradation 6
Chloramphenicol 12, 452
biosynthesis 31
for amplification of plasmids 172
resistance 219, 342, 425, 443, 447,
449, 450, 475 (map), 488(map), 564 (map), see also cat;
catSa; cmlSl; cmlv; cpt
instability in Streptomyces 20,
427
Chloroform
in actinorhodin isolation 93
in DNA isolation 164
salting out procedure for genomic
DNA isolation 169-171
in DNA storage 167
in phage storage 218, 226
Chlortetracycline 12
resistance 451
Ciprofloxacin 452
resistance 450
Chromosome, of Streptomyces see
Linearity; oriC; Telomeres
Circularity, of linkage maps 140
Clavulanic acid 12
biosynthesis 25
Clindamycin 452
resistance 451
Cloning, general considerations 212226
clr (lincosamide resistance) 447(figure), 451
cmlSl (chloramphenicol resistance)
443, 449, 450, 491 (map)cmlv (chloramphenicol resistance) 443,
449, 450, 488 (map)Codon usage 387, 400-401 (table), 499
see also bldA; G+C; TTA
CODONPREFERENCE 398
effects on distribution of restriction
sites 314
FRAME analysis 395-397, 399(figure)of EGFP 347
optimising for gene expression 391
Cohesive ends see also cos; C31
annealing of phage cos ends seen on
agarose gels 274
generated by restriction enzymes
441

in transformation of linearised
plasmids 232
Cointegrates, of plasmids
created by transposition 110, 111,
119, 120 (figure), 121
of pIJ101 derivatives 256
of SCP2* 115, 213, 262
Colloidal chitin agar, for selective
isolation of Streptomyces 44
Colony hybridisation 218, 222, 242248
Combinatorial biosynthesis, ofpolyketides 23, 262
vectors for 557(map), 558 (map)Complementation
extracellular, in differentiation 17
in clone isolation 222, 295
antibiotic biosynthetic genes 2731, 217, 224
using C31 vectors 292
using SuperCos-1 218
using Tra+ SCP2* vectors 261262
in gene disruption 327
in heteroclones and heterokaryons
136in phage genetics 273
Complete medium (CM) 407 (recipe)
for auxotroph isolation 104-105
for crosses 130, 152
for growth from lyophils 60

using hybridisation 226
for cloning antibiotic production
genes 224
of pIJ101 256of plasmid vectors 217
of pSG5 259
of SLP1.2 172
SCP2*-derived vectors withincreased copy number 262(maps), 263
Corynebacterium, taxonomy 3cos sitescos, on vectors 514-516, 518, seealso Cosmid; SuperCos 1of Streptomyces phages
and self-ligation 274
in DNA packaging 218, 273, 299
of C31, in in vivo transduction
299
Cosmidcloning 162, 214, 216
SuperCos 1, library of S. coelicolor
218
for genome mapping 141, 218,422 (map), 423
for genome sequencing 398, 424,
see also Genome projecttransformation 232, 236-237
vectors
lambda 267 (list), 546 (map), 560(maps), 562 (map), 569 (maps)
Streptomyces phages 299, 300
Cotransduction 275

Confocal microscopy 73, 348
Conjugal transfer of plasmids
between species 21, 217
detection by pock formation 153155, 239, 255, see also Pocks
genetic determination 255, see alsokil; kor; tra; traR
in soil 21
into Streptomyces from E. coli see
Escherichia coli; oriTof integrating plasmids 20, 265
to detect transposition 111, 114-117
Conjugation, in Streptomyces 125 see
also C.m.a.
and linkage mapping 137-152
Copperfor tyrosinase activity 240, 342, 536
in induction of sporulation 430
staining of proteins 90
Copy number
and colony hybridisation 222
and gene expression 388
and promoter probing 341, 343
low copy number vectors
analysing low copy number clones

Cotransformationof IS117 265
of plasmids 221
Cotton wool
filters
for preparing lysogens 304
for spores and protoplasts 46(figure)for harvesting Streptomyces spores
45
plugs ignite in microwave ovens 406
Counterselection
fusaric acid, for tetracycline
resistance 564
in gene replacement 320-323
practical example 324-325
in transposon delivery 113
markers 220, 449, 493-494 (maps),
571
using glkA on C31, to selectdeletions 307-308
glkA plasmid vectors 566, 571(map)using rpsL 323



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Coupled transcription-translation 7883
Covalently closed circular see CCC
plasmid DNA
Coverslip
cultures for microscopy 64, 348
for cutting agarose blocks 203, 205
cph (capreomycin phosphotransferase)
443, 449, 451
cpt (chloramphenicol
phosphotransferase) 443, 449,
450, 488 (map)cre (curromycin resistance) 443, 449
m-Cresol, in phenol 164
CRM (growth medium forelectroporation) 237 (recipe)
Crossovers
in gene disruption/replacement see
Gene disruption andreplacement
in linkage mapping
multiple 145-152
single and double, in merozygotes
139 (figure)
CTAB (detergent), in DNA isolation
162, 164, 170-171, 173
Curing, of plasmids
pIJ101 derivatives, using ethidium
bromide 256
temperature-sensitive plasmids 112113, 120
to confirm that phenotype depends
on plasmid clone 224-225
Curromycin, resistance 443, 449
Cy3 fluorescent stain 66-69
Cycloheximide, as fungicide, in
selective isolation ofStreptomyces 44
Cycloserine 12
D
2D PAGE 83-90, 85 (figure)dag (diffusible agarase), of S.
coelicolor 430
Dam methylation, of E. coli DNA 213,
see also ET12567
effect on mutation frequency in E.
coli 226
effect on transformation of E. coli
221
protection of restriction sites 214,440 (figure), 441, 512
DAPI stain, for DNA 65

Daptomycin 12
Databaseof genes see ScoDB
of proteins 84, 90
Daunomycin/daunorubicin 12, 452
biosynthesis 27, 29, 31,
resistance 443, 449, see also drrA,B,
drrC
Dcm methylation, of E. coli DNA 213,
see also ET12567
methyl-specific restriction, in
Streptomyces 317, 429
protection of restriction sites 214,440 (figure), 441, 512

,QGH[

blotting
not required for nick-translated
probes 244
not used for colony hybridisation
using Whatman 541 filters 247
Deoxycholate
for plasmid isolation 175
in PFGE sample preparation 201
Deoxygalactose, for counterselection
323
Deoxyglucose see also glkA
for counterselection 320-322, 324327
to select glkA mutants 307-308

DE52 resin, Whatman, for purifying
oligonucleotides 246

Deoxysugar genes, isolated byheterologous probing 224

7-DeazaGTP, in sequencing reactions
371, 393-394

DEPC (diethylpyrocarbonate) 354

Decanal, for luxA,B detection 346-347
Deletion(s)creating in-frame 314-316, 320
FRAME analysis, to detectframeshifts 397 (figure)in avermectin gene cluster 332-334,
570
in phage vectors 273, 290, 513(table)of chromosomal DNA 20, 26, 218
in S. lividans ZX7 425, 426(map)of AUD2 of S. lividans 429
using glkA on C31, to selectdeletions 307-308
of plasmid DNA 219, 265
Denaturation, of DNA see also
Denatured DNA
alkaline, for plasmid isolation 184192, 194
boiling method, for plasmid isolation
190
by phenol 193
for generating radioactive probes
242, 242, 303
for transformation 218, 232, 235237
in Northern blotting 376
irreversible denaturation of CCC
plasmid DNA 172, 174, 176,
196
Denatured DNA
as standard for S1 mapping 367
for transformation 128, 232, 235237
in agarose gel electrophoresis 195198 (pictures)in S1 digestion buffer 368 (recipe)
Denhardt’s solution for Southern

Dephosphorylation, to prevent selfligation 221
Depurination, by acid phenol 193
Desferrioxamine 12
biosynthesis 29
Destomycin 452
Detergentsin DNA isolation 163, 175
in PFGE 199
in protoplast isolation 58
in RNA isolation 355
in spore isolation 44
Developmental biology, of
Streptomyces 17-18, 19(figure), 64,
see also bld; whi
DH5, E. coli endA strain 172
Diagnostic media, for analysing crosses
133-135
supplement concentrations 415
Dialysis see also Microdialysis
in DNA isolation 165, 174
in RNA polymerase isolation 379,
381
Diaminopimelic acid, in actinomycete
taxonomy 2
Diatomaceous earth, for “Jurassic”
plasmid purification 194
Diazomethane, mutagenic product of
NTG decomposition 103
Difco nutrient agar (DNA) 441(recipe)for phage propagation 278, 304
Difco nutrient broth (DNB) 412(recipe),QGH['LIFRQXWULHQWEURWKWR(U\WKURP\FLQ

for phage storage 59
Dihydrostreptomycin, biosynthesis 26
Dimeric plasmids
monomerisation during replication of
RCR plasmids 255
position of bands in agarose gels
195, 196-197 (figures)
Dimethylsulphoxide see DMSODispersants, used in liquid medium 51(table)PEG 6000 in SMM and NMMP 413(recipes)
Dispersed growth, in liquid 49-50, 413(recipe for NMMP)
dispersants 51 (table)
dITP, in sequencing reactions 394
DMSO
for spore preservation 45
in PCR 403
in protoplast fusion 156
to dissolve thiostrepton 241, 456
DNA fragments, containing usefulgenes 439-504 (maps)
DNA homology, in actinomycete
taxonomy 5
DNA see also Codon usage; G+C
analysis see Agarose gelelectrophoresis; FRAME
analysis; Hidden Markov; PCR;
Pulsed-field gel electrophoresis;
Sequencing
cloning of, general considerations
212-226, see also Vectors
on phage vectors 290-308
on plasmid vector 253-269
degradation in vitro see dnd
effects of Streptomyces antibiotics on
12-14
extraction from soil, to monitor
growth 21
homology, in Streptomycestaxonomy 5
introduction into Streptomyces 230250, see also Electroporation;
Transduction; Transformation
isolation protocols
genomic 162-171
phage 282-287
plasmid 171-195
lesions caused by mutagens 100,
101, 102
modification see Dam; Dcm; dnd;
Restriction-modification
phage DNA, properties 273-274
rearrangement see Amplification;
Deletion(s)recombinant DNA, safety guidelines
33
single-stranded see Denatured
DNA;
Single-stranded DNA
size fractionation, importance inlibrary construction 215
staining with DAPI 65
synthesis
in mycelium 17,
on germination 54
unmethylated
from ET12567 218, 230, see also
Dam; Dcm; Hsd
from PCR 230
DNA-cellulose, for RNA polymerase
purification 377-384
DNase
DNase I buffer 355 (recipe)in DNA isolation 162-163
in RNA isolation 355-356
dnd (DNA degradation locus) 426(map), 428
S. lividans strains 425
dNTP concentrations
for PCR 248, 402 (recipe)
for primer extension 372
Doxorubicin (adriamycin) 13
DraI
in PFGE 199, 203, 422 (map), 426(map), 431-433 (maps)
rarity of sites 212
recognition site 441
transposons with rare sites 111449, 499 (map)on vectors 268 (list), 531 (map), 571(map)
use as reporter 347-348
EGTA, in coupled transcriptiontranslation 78, 82
Electron microscopy 70-73
Electrophoresis see Agarose gelelectrophoresis; 2D PAGE;
PAGE; Pulsed-field gelelectrophoresis; SDS-PAGE
Electroporation 29, 31, 130, 230, 237239
Elloramycin, biosynthesis 29
Embden-Meyerhof-Parnas (glycolysis)
pathway 7
endA (endonuclease A), strains forplasmid isolation 172, 193
RNase activates EndA 177
tRNA inhibits EndA 174
End-labelling of DNA see Kinase
Endonuclease A see endA
Enrichment
for auxotrophic mutants 104
in isolating organisms from soil 44
in phage isolation 281
Entner-Doudoroff pathway 7
Entry disadvantage, of plasmids 126,
127

Drying, of agar plates see Laminarflow hood

Enzyme production 23
optimising 385-391

drrA,B, drrC (daunorubicin resistance)
443, 449

Epoxy resin, for embedding
Streptomyces colonies 70, 71

Duracryl®, for 2D gel electrophoresis
86 (supplier)

Eppendorf dialysis 381

Dynabeads DNA DIRECTTM kit, for
PCR 248, 481 (supplier)ermE (erythromycin resistance) 443,
447 (figure), 451, 459 (map),see also ermEp/ermE*p
as marker for gene replacement 320321, 324
lack of mRNA leader 212
lincomycin for selection 324

E
E. coli see Escherichia coli
Easi-GridTM, for master plates 133,418
(supplier)
Ecology, of Streptomyces 5-6
EDTA, pH8 206 (recipe)
EF-Tu gene (tuf1), efficiency oftranslation 386
EGFP (green fluorescent protein)443,

Eppendorf tubes, engraving 187

ermEp/ermE*p 386
on vectors 389, 390, 535 (map),
517-518
ermSF see tlrA
Erythromycin 13, 452
biosynthesis 32, 319
resistance 451, see also ermE(U\WKURP\FLQWR)UHH]HHWFKLQJ

inducible lrm in S. lividans 219,
458
selection 241 (table)
Escherichia coli
bifunctional plasmid vectors 219,
261, 267 (list), 319, 514-517
chromosome circularity 140
cloning transposon-tagged DNA
111, 121
conjugation from 30, 32, 230, 249250, see also ET12567; oriT
vectors 267 (list)
Dam or Dcm methylation see Dam;
Dcm
endA, in DNA isolation see endA
E. coli strains 172, 193
expression of Streptomyces markers
212, 458, 470, 482, 484, 500
expression of Streptomyces proteins
391
lacZ ( -galactosidase) 339
lethal zygosis 153
non-methylating mutant see
ET12567
plasmid amplification 255
plasmid DNA isolation 171, see also
endA
plasmids 517-518 (table)
promoters, expressed in
Streptomyces 440
pSAM2, integration in 265
recovery of replicons bytransformation of 225, 226,
320, 332-333
relA (ppGpp synthetase) 312
resistance markers of see aacC1;
ampC; bla; ble; cat; galK; km;
neo; tetribosome binding sites 386
RNA polymerase 377
signal peptides, for protein secretion
387
suicide vectors with Streptomyces
markers 218-219, 222, 236,
267
in gene disruption 317-318, 321
thiostrepton-sensitive strain 456transposon mutagenesis in 110
vectors with blue/white selection
269 (list), 544, 545
Essential genes, disruption 319-320,
327
ET12567, non-methylating E. coli 213,
218, 429
containing pUZ8002 219, 325, 348
for conjugation with Streptomyces249-250for cosmid transformation of S.
coelicolor 236-237
genotype 230

Ethanolfor DNA precipitation 165-166, 173from CsCl solutions 182
for spermine extraction 184
Ethidium bromide
and chromosome rearrangements 20
in agarose gel electrophoresis 195,
215
in PFGE 204
in plasmid curing 256
in plasmid isolation 174-175, 176(figure)for removing sample impurities
192-193
standard CsCl EB procedure 178183
removal of 182

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Feulgen stain, replaced by fluorescent
stains 65
Filter sterilisationof catechol solution 344
of phage suspensions 278
of pyrophosphate solution 304
of trace element solution 413
Filter tubes, for preparing spore andprotoplast suspensions 46(figure)FITC (fluorescein isothiocyanate) stain
66-69
optical filters for 67, 348
Fixatives see also Glutaraldehyde
for electron microscopy 71, 72
for light microscopy 64, 67-68

Ethyl methanesulphonate, as mutagen
101

FK506 (tacrolimus) 13

Extracellular agarase 223

Florimycin 454

Extracellular -galactosidase 340

Fluctuation test, for determining
spontaneous mutation
frequencies 111

Extracellular enzymes
in soil 6
inhibition of secreted protease 388
removal of 76, 163,
secretion
signal peptides 387
vectors 390
Extracellular signalling molecules see
also A-factor; butyrolactonesin bld cascade 17, 106
Exonuclease III
apurinic site endonuclease activity
193
nicking and deletion mapping 176
Extraction buffer, for spore cell-free
extracts 76, 77
F
f1 (filamentous phage) origin, forgenerating single-stranded
DNA 230, 517
fab (fatty acid biosynthesis genes) 26,
319, 320
Falcon® tubes 91, 355
Fatty acids
biosynthesis 26, 319, 320
in taxonomy of Streptomyces 2
fd phage terminator see ter(fd)Feedback inhibition, of amino acidbiosynthesis 9
Fertility see C.m.a.; NF strains

Fluorescence microscopy 65, 66-70,
348
film for 70
filters for 67
with EGFP 73, 347-348
Fluorescent-coupled antibodies 65-70,
418 (supplier)
Foam bungs, for agar media 406, 419(supplier)
Folin-Ciocalteu’s reagent, inhibition by
PEG 51
Formamidein DNA sequencing 394
in PCR 403
in S1 mapping 367
Fortimicin 13, 452
biosynthesis 32
Fosfomycin 13, 452
Four-on-four procedure, for linkage
mapping 137, 141-147
FP43 phage, transduction by 27, 117,
128, 300
FRAME analysis, for gene finding395-399
Frankia, taxonomy 3Freeze dryer 59-60, 419 (supplier), seealso Lyophilisation
Freeze-etching, for electron microscopy
70

,QGH[)UHH]LQJWR*OXWDPLQHV\QWKHWDVHFreezing
causing DNA damage 274
of non-sporulating mutants 44-45
of spore suspensions 47, 58
to recover antibiotics from agar 48
French press, for cell-free extracts 76,
81, 377, 382

Gel filtration chromatography, inplasmid purification 174
Gelatin capsules, for electron
microscopy 70, 419 (supplier)Germination of spores 17
affecting storage 47
for electroporation 238
for reproducible liquid cultures 50,
53-54
in aerial mycelium 48
in soil 5-6

Gelatin, in PCR 402
gfp see EGFP

Fructose-PTS 7

Geldanamycin
biosynthesis 28
in soil 11

FtsZ protein 66
Fungi
as producers of antibiotics 11
inhibited by antibiotics in soil 11
inhibition using chloroform 226
selective inhibition of 44
trans-sulphurase pathway 9
Fusaric acid, to counterselecttetracycline resistance 564
Fusarium spp., antagonised by
Streptomyces 11
Fusidic acid 452
G
g force see Centrifugation
G+C content, of DNA 2, 18, 212, 401,
see also Codon usage
and dye binding 167, 175, see also
Hoechst 33258
effect on codon usage 387
FRAME analysis 395-399
effect on density 174, 176 (figure),
179 (table)
effect on melting temperature 212
effect on migration in gels 195, 204
effect on PCR 402-403
effect on restriction site frequencies
199
effect on sequencing 393, 395
of Streptomyces phages and plasmids
18
gal (galactose operon of Streptomyces),
DNA sequences involved inglucose repression of 8
Galactose catabolism 7, see alsogalactosidase, galKelectrophoresis; SDS-PAGE-galK (galactokinase), of E. coli 340,
443, 449, 497 (map)on vectors 268, 543 (map)Gamma-actinorhodin 93
structure 94
Gamma-butyrolactones see
Butyrolactones
Gel electrophoresis see Agarose gelelectrophoresis; 2D PAGE;
PAGE; Pulsed-field gel

Gene conversion 329 (figure), see also
Homogenotisation
Gene disruption and replacement 2431, 311-327
counter-selection of delivery vector
320, see also glkA; rpsL
dealing with essential genes 319
heterologous 323
homogenotisation 328-332
in S. azureus 299
of whole gene clusters 332-334
practical example 324-328
size of homologous DNA 316
unstable plasmids for 268 (list)
using denatured DNA 235-236
using C31 318
vectors for 316-319, see also
Suicide plasmids
Gene libraries see also Cosmid
assessing quality 226
construction 214-215
transfer to other strains byconjugation 217
Genera, of actinomycetes
antibiotic production 10
DNA base composition 18
host-range of phages and plasmids
215, 300
phylogenetic tree (16S rRNA) 3(figure)taxonomy 2-3
Geneticin (G418) 452
resistance 450, 467, 480, see also
aac(3)IV
cross-resistance 448 (table)

Glass beads
for harvesting dry spores 45
for preparing cell-free extracts 76-77
boiling of 76, 77
in RNA isolation 354-358
to disperse growth in liquid 50
Glass knives, for thin sections 71
glkA (glucose kinase) 423, 443, 449,
493 (map), see also S. coelicolor M480
as counterselective marker 220, 307308, 320-322, 325
in glucose repression 7-8
on vectors 513, 517, 524 (map),
566,
571 (map)
glnA (glutamine synthetase), of S.
viridochromogenes, disruption
317, 323
Glucoseas buffer for pH12.5 191
as carbon source 406
catabolism 7, see also EmbdenMeyerhof-Parnas pathway;
Entner-Doudoroff pathway;
Hexose monophosphate shuntlack of glucose-PTS in Streptomyces7
not metabolised by S. clavuligerus 7
released from starch on autoclaving
51
repression see also Carbon
catabolite repression (CCR)of agarase 223
of gylR 503
Glucose isomerase, genetic engineering
23

Genome project, for S. coelicolor 394,
398, 424-425

Glucose kinase, in carbon catabolite
repression 7-8, see also glkA

Gentamicin 13, 452
resistance 442, 443, 450, 458, 460,
461, see also aacC1
concentrations for selection 241
cross-resistance 448 (table)

Glutamate:2-oxyglutarate transaminase(GOGAT), in nitrogenassimilation 8

Gentian violet, as counterstain 64
Geosmin 6

Glutamate synthetase (GS), in nitrogenassimilation 8

Germicidal UV lamp, for mutagenesis
102

Glutamine synthetase
genes 27

Glutamate dehydrogenase (GDH), in
nitrogen assimilation 8*OXWDPLQHV\QWKHWDVHWR,PSUHVVLRQSUHSDUDWLRQV
disruption 319, 323, 335
inducible promoters 342, 388, seealso gylR
on vectors 389, 532 (map), 535(map)
promoter and regulatory sequences
involved in glucose repression
8
S1 mapping, example 371

target for bialaphos/phosphinothricin
12, 452, 453
Glutaraldehyde, as fixativefor electron microscopy 70 (recipe)
for light microscopy 66, 67 (recipe)
Glutathione, replaced by mycothiol in
actinomycetes 10
Glycerolas carbon source 7, 406
catabolism see gyl; gylR
for microscopical mounts 64
for stabilising cell-free extracts 76,
80, 378for storing frozen mycelium 118,
201
for storing frozen spores 44-45, 47,
53, 58in electroporation 237
in PCR 403
inducing restriction enzyme “star”
activity 218
Glycerol-sensitive mutants 319
storage in DMSO 45
Glycine, in lysis by lysozyme 168, 412(recipe)
Glycogenin colony development 17-18
metabolism 24
staining in electron microscopy 72
Green fluorescent protein (GFP), fordetecting gene expression 347,
see also EGFP
Grids
for electron microscopy 71-73
for master plates 132, 133, 418(supplier)haemocytometer 233
Grinder see Homogeniser
Griseusin, biosynthesis 27
Grisin, resistance 451
grmMp, grmMr (aminoglycosideresistance) 443, 447 (figure),
450, 460 (maps)
cross-resistance 448 (table)

gylR (repressor of glycerol operon) 8,329, 444, 449, 503 (map)on vectors 532 (map), 535 (map)Gyrase, target of ciprofloxacin,
nalidixic acid and novobiocin
14, 452-453, see also gyrB
gyrB (gyrase -subunit, novobiocin
resistant) 444, 449, 450, 463(map)
H
Hemi-methylated DNA, and plasmid
replication in E. coli 221
Haemocytometer, for counting
protoplasts 233
Heat shockfor spore germination 53
genes 24, see also groEL
Heparinin in vitro transcription 374
in RNA polymerase purification
377, 380, 382
HEPES buffer
for agarose gel electrophoresis 197
in electroporation 238
in in vitro transcription-translation80
in PFGE 201

Hoechst 33258 (bisbenzimide), ATspecific DNA stain 167, 175,
179 (table)
Homogeniser
High Speed Homogeniser System
54, 419 (supplier)Potter 118, 419 (supplier)teflon 163
Ultra-Turrax Turbomixer 54, 420(supplier)
Homogenotisation 222, 224, 226, 328331
in phage cloning 295
Hpr protein, in sugar transport 7
hrd (sigma factor genes), of S.
coelicolor
disruption of hrdA, hrdC, hrdD 321,
386
hrdB, as example of an essential
gene 319
Hsd methylation of E. coli DNA 213
HT (Hickey-Tresner) agar 407 (recipe)hur (hydroxyurea resistance) 444, 449
HV agar, for selective isolation ofStreptomyces 44
Hybrid antibiotics 23, 24, 26, 48, 224,
see also Combinatorial
biosynthesis
Hybridisation solution, for S1 mapping367-368
Hydroxylamine, as mutagen 101
Hydroxyquinoline
in DNA isolation 164, 168
in RNA isolation 355, 357, 359

Herbicidal agents see Bialaphos;
Phosphinothricin

Hydroxyurea 452
resistance 444, 449

Heteroclones 136, 140, 141hyg (hygromycin phosphotransferase)
444, 449, 450, 485 (map)for gene disruption 313-314, 321322
on vectors 293, 514-518

Heterokaryons 136, 142
Hexose monophosphate shunt 7
Hickey-Tresner (HT) agar 121, 122,
407 (recipe)

groEL (chaperonins) of S. albus,
disruption 317, 319

Hidden Markov model, for gene
finding 398

Growth factor supplements 415(recipe)High Speed Homogeniser System 24,
419 (supplier)

GTE buffer, in immunofluorescent
staining 67

Histidine, catabolism 9

Guanidine thiocyanate, for “Jurassic”
plasmid purification 194

Histidine-tagged recombinant proteins
RNA polymerase 382
vectors for 389, 390, 391, 539-540(maps)gyl (glycerol operon of Streptomyces)

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Hygromycin 13, 453
resistance 324, see also hyg; aph(4)cross-resistance 448
to select recombinants 241 (table)
I
Immunofluorescence staining 66-70
Immunogold labelling 66, 73
Immunomagnetic capture, of spores 44
Impression preparations, formicroscopy 64

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In-frame deletions, generation of 314316
Inclusion bodies, of recombinant
proteins 388
Infilling, partial for ligating MboI and
SalI ends 215 (figure)
Instabilityof lacZ 340
of lysogens 295of plasmids 216, 219, 256segregational 110 see also
Curing; pMT660; pSG5
structural 223, 453
of Streptomyces genome 20, 24, 26,
28, 29, 427, 500, see also argG;
Chloramphenicol; tetSl
int genes, of integrating plasmids andphagesof C31 see C31
of pSAM2 264 (map), 265
of SLP1 265
on vectors 563
of VWB phage 390
on vectors 390, 532
INT, tetrazolium stain, for respiratory
activity 66
Integrating plasmids 514 (list), seealso pSAM2; SLP1International Streptomyces Project(ISP) 2
Interspecific gene transfer
and safety guidelines 33
in soil 21
restriction barriers 22
Interspecific mating 28, 30, 128
by protoplast fusion 29
Inverted repeats see also Positive
selection vectors
as terminators 212
at chromosome ends 20, 427, 428
in promoter regions 8
in S1 mapping 371
of transposons 111, 117, 119, 120,
121
SCP1 TIRs 434
Iodide, sodium, for “Jurassic” plasmid
purification 194

IS6 family 119

K

IS110, of S. coelicolor 428, 518(sequence)causing vector integration 316

Kalafungin, biosynthesis 30

IS113, of S. lividans 428
IS117, mini-circle, of S. coelicolor 21,
216, 218, 225, 266 (map), 428
host-range 215, 266-267
vectors 265-267, 342-343, 390, 518(list), 573-574 (maps)



kamA, kamB, kamC (aminoglycosideresistances) 444, 447 (figure),
450, 462 (maps)
cross-resistance 448 (table)kan (aminoglycoside resistance) 444,
447 (figure), 450, 461
on vectors 539-540 (maps)

Kanamycin 13, 453
resistance 268 (list), 447 (figure,list), 450
IS281-like element, of S. coelicolor andcross-resistance 448 (table)
S. lividans 428
on transposons 119-122
selection of neo gene 226, 236IS466, of S. coelicolor 428
237, 241, 546
IS493, of S. lividans 21, 265, 428, 575
to select recombinants 241 (table)(map)
for transposon mutagenesis 117-118 Kasugamycin 453
resistance 450
vectors 518-519 (list), 576-577(maps)KC phage vectors see also KC vectors,
IS118, of S. coelicolor 261, 428

IS1372, of S. lividans 428
IS1373, of S. lividans 110, 117, 422,
427, 428
IS6100, of Mycobacterium fortuitum
21
for transposon mutagenesis 119-120
vectors 120 (map), 346, 348
Isoelectric focusing 83-89
Isopropanol
for DNA isolation 165-166, 173,
185
for in vitro transcription 374
for RNA isolation 354-360
in S1 mapping 368-370
Isoschizomers, of restriction enzymes
441
ITP see dITP
J
J medium, for radiolabelling of proteins
90 (recipe)
J prefix see S. coelicolor, strains
Jadomycin, biosynthesis 31
JHJ-1 phage, of Saccharopolyspora
272

Iodoacetate, sodium, as RNase inhibitor
JT prefix see S. lividans, strains
361
Ion exchange resin, for isolating
Streptomyces from soil 44

Junlon, as dispersant in liquid medium
51, 357, 419 (supplier)Iron
chelation 13, see also
Desferrioxamine
regulation of gene expression 29

Jurassic preps, for plasmid isolation
171, 175, 194-195

listhigh-capacity c deletion derivatives
293 (table)
ligation conditions 294
lists of special purpose vectors 267268
tables of C31-based cloning
vectors 513-514
att+ 292
att– 293
functions, markers and location ofmaps 513-516
xylE-containing C31 vectors 297(maps)
KC vectors, list see also KC phage
vectors
KC210 292 (table),
KC211 268, 293 (table)
KC212 268, 292 (table), 294, 295,
304, 305
KC300 294, 295, 304, 429
KC301 294, 295, 304, 305
KC304 292 (table), 305, 523 (map)
KC310 268, 294, 342
KC400 482
KC401 268, 290, 292 (table), 293(table), 294, 482
KC404 318
KC466 268, 524 (map)
KC505 218, 268, 290, 292 (table),
293 (table), 294, 526 (map)
KC515 290, 291, 293 (table), 294,
306, 525 (map)
KC516 291, 293 (table), 294, 525(map)
KC517 268, 293 (table), 513
KC518 218, 268, 290, 292 (table),
293 (table), 294, 526 (map)KC573 268, 493, 524 (map).&YHFWRUVWR/\VRJHQV

use of glkA counterselection
308 (figure), 321
KC850 268, 310, 513
KC857 291, 293 (table), 296, 298,
482, 527 (map)multi-cloning site 297 (map)KC858 268, 293 (table), 296, 298,
482, 513
multi-cloning site 297 (map)KC859 268, 292 (table), 294, 296,
298, 341, 344, 523 (map)multi-cloning site 297 (map)
KC860 268, 293 (table), 298, 341,
344, 482, 527 (map)multi-cloning site 297 (map)
KC861 268, 293 (table), 298, 341,
344, 528 (map)multi-cloning site 297 (map)
KC862 268, 293 (table), 296, 298,
341, 344, 528 (map)
KC895 308
kgmB (aminoglycoside resistance) 444,
447 (figure), 450, 461 (map)kil (lethal gene), on plasmids 254 (mapof pIJ101), 255, 256
Kinase (T4, polynucleotide), for endlabelling DNA 165, 173, 246,363-366
buffer 246 (recipe)
Kirby mix
for isolating chromosomal DNA
162, 163, 168-169 (recipe)
for isolating RNA 354, 355-356,
357-358
Kitasatoa, synonymy with
Streptomyces 4
Kitasatosporia, synonymy with
Streptomyces 2, 3
km (aminoglycosidephosphotransferase) 444, 449,
450, 479 (map)on vectors 263 (maps), 564 (map)kor (kil override gene), on plasmids
254 (map of pIJ101), 255, 256,
264 (map of pSAM2)
L
L (lysis) buffer 57, 415 (recipe)
L agar 237, 411 (recipe),and viomycin selection 482
L broth (LB) 249-250, 412 (recipe)
Labelling
medium for in vivo DNA labelling414
of DNA probes for colony
hybridisation 246

of proteins 90-92
lac ( -galactosidase of S. lividans)
340, 349-350
on vector 268
lacZ ( -galactosidase) of E. coli
as reporter gene for Streptomyces
296, 339-340
vectors allowing blue/white selectionin E. coli 269 (list), 514-518
Laminar flow hoodfor drying agar plates
after plating spores 47
for growth on cellophane 49
in conjugation from E. coli 250
in spot-transformation 236
R2YE transformation plates 233
for harvesting spores 45

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Liposomes, for protoplasttransformation/transfection
128, 301
Liquid cultures
antibiotic production in 16, 106
for DNA isolation 172
for in vivo labelling 90-92
for physiological studies 49-52
for RNA isolation 354
inoculation with vegetative
mycelium 223
lack of mating in 125
loss of prophages in 295
luciferase measurements in 347
media 412-414 (recipes)
sporulation in 58

Lasalocid 13
biosynthesis 28

Liquid nitrogenfor preparing cell-free extracts 77,
163
for storing cultures 55, 58, 76

Lead citrate, for staining in electron
microscopy 71-72

Lithium chloride, to precipitate RNA
173, 206 (recipe)

Leaderless transcripts 363, 386, 475

Lividomycin 453
resistance 487

Leaky growth, of auxotrophs 105, 131,
133

LMP (low melting point) agarose, for
PFGE 198, 200-203

Lennox broth (LB) 249, 412 (recipe)
Lichens, as producers of secondary
metabolites 11
Life cycle, of Streptomyces 5, 17, 19(figure)2D PAGE analysis of 84
Ligation, of DNA
conditions for 216, 220-221, 226,
232
in phage cloning 294, 301-302
Lignin, degradation 30, 31
Lincomycin/lincosamides 13, 452, 453
biosynthesis 28
for selection of ermE 241, 320, 321,
424, 458
resistance 444, 445, 447 (figure),
449, 451, see also lrm; lmrA;
lmrB; myrB
selection of recombinants 241(table)Lincosamides see Lincomycin
Linear plasmids 20-21, 25, 30, 156,
198, 204, see also SCP1; SLP2
Linearity, of Streptomyces
chromosomes 20, 138-141, 145
genome maps 129, 422, 426, 431,
432, 433
Linkage mapping 137-152

lmrA, lmrB (lincosamide resistances)444, 447, 449, 451
Loading buffer
for in vitro transcription 376
for in vitro transcription-translation
79 (recipe)
for RNA 362 (recipe)
for S1 mapping 370 (recipe)
LR White epoxy resin, for electron
microscopy 70
lrm (lincosamide resistance) 444, 447(figure), 451, 458
Ltz (lethal zygosis) 153, 155, 239 see
also Pocks; spd
Luciferase genes see luxAB
Luminometer, for measuring luxAB
expression 347
luxAB (luciferase) 444, 449, 498 (map)as reporter 346-347
for spatial expression in colonies 73
on vectors 268 (list), 543 (map),
580 (map)
TTA-free version 346, 498
Lyophilisation 59-60, 419 (supplier)of phages 276
Lysogens
as sources of phages 21, 272

,QGH[double lysogens 213, 290, 292, 295,
299
of FP43 300
preparation 294-295, 303-307
stability 298, 429
Lysozyme
for cell-free extracts 76, 379
for preparing protoplasts 56-58
in colony hybridisation 243, 245,
248
in DNA isolation 163, 414
in electroporation 237
in immunofluorescent staining 6668
in PFGE 199, 201
in RNA isolation 355, 358, 360
inhibiting PCR 222
Lytic cycle, of phages 298, 303
detecting promoter activity inplaques 296
M
M prefix see S. coelicolor and S.
lividans, strains
Magnesiumin acid phenol extraction 193
in DNase activity 163
in PCR 403
in phage adsorption 277, 278 (table)
in soil structure 44
Magnetic beads, in immunomagnetic
capture 44
malR (maltose regulator), in glucose
repression 8
Mannitol, as carbon sourcein glkA counterselection 307, 325327, 493
in minimal medium 406
Mannitol soya flour (MS)agar medium 409 (recipe)
for good sporulation 44
poor selection of thiostrepton
resistance 456
used in conjugation from E. coli
249
used in gene disruption 325-327
liquid medium, for protein labelling
92
Master plates
grids for 132 (figure), 133, see also
Easi-GridTM
in plate crossing 152, 261
preparation of 133
replicas from 133, 135 (figure), 136
Maxam and Gilbert sequencing
reactions 367, 371, 393-395(recipes)/\VRJHQVWR06
mcr (methyl-specific restriction systemof E. coli) 111
mcrA,B (mitomycin resistance) 444,
492 (maps)
inducible promoter 388, 390
mdh (malate dehydrogenase) 444, 449,
as reporter 348-349
on vectors 561 (map)Melanin biosynthesis 24 see also
melC1, C2, mel; Tyrosinase
melC1, C2, mel (tyrosinase) 444, 500,501 (maps)
as reporter 342, 343
detection 240
on vectors 268 (list), 515-516
promoter strength 389
mer (mercury resistance of S. lividans)see also Mercury resistanceon genome map 444
promoter 121
Mercury resistance 427, 428, see alsomer
not present on Tn4556 111

calichiamicin producer 32
fortimicin production 13, 32
gentamicin producers 13
rosariamicin producer 32
taxonomy 3transcriptional regulation 32
transformation 32,
Microscopical methods 63-73 see also
Confocal microscopy;
Electron microscopy;
Fluorescence microscopy;
Phase-contrast microscopy;
Scanning electron microscopy
Microwave oven, melting soft nutrient
agar 240
unsuitability of cotton bungs 406
Milbemycin 13
Minamycin 453
Mini-circle see IS117
Minimal liquid medium (NMMP) 50,
413 (recipe)
Minimal medium (MM) 406 (recipe)

Merodiploids 159

Minocycline, resistance 451

Merozygosity, and map circularity 138

Mithramycin 13
resistance see mtrA,B

Methionine
and induction of tyrosinase 500
biosynthesis 9
35
S-labelled
for coupled transcriptiontranslation 79, 82-83
for 2D gels 85 (picture), 89-92

Mitomycin C 13, 453
resistance 28, see also mcrA,B
to induce mcrA,Bp 388, 390
to induce prophages 272, 299
MLS resistance 241(table), 430, 447(figure), 451, 458

8-Methoxypsoralen + NUVmutagenesis by 101

mmr (methylenomycin resistance) 444
terminator 389, 500, 502

Methyl methanesulphonate, as mutagen
101

MMT medium, to visualise tyrosinase
activity 240, 410 (recipe)

Methyl-specific restriction see
Restriction of methylated DNA

MNNG see NTG

Methylene blue, as counter-stain 64,
66

Mobile genetic elements see IS and Tn
prefixMobilisable plasmids see oriT

Methylenomycin 453
production 21, 95, 114, 430
resistance 21, see also mmr
structure 94
MG medium, for radiolabelling
proteins 90-91
Microdialysis, in DNA isolation 165,188
Micromonospora spp.
antibiotic production 10
antibiotic resistances see aacC9;
grmMr; grmMp; kan; myrBbase composition of DNA 18

Mobilising plasmids for oriT see
pUB307; pUZ8002
Monensin 13
Monoclonal antibodies
in immunostaining 73
in selective isolation of Streptomyces
spp. 44
MOPS buffer 91, 189
mrr (methyl-specific restriction systemof E. coli) 111
MS see Mannitol soya flour agar



PWU$%WR2' RSWLFDOGHQVLW\,QGH[

mtrA,B (mithramycin resistances) 444

NDS buffer, for PFGE 199 (recipe)

Nocardia spp., as pathogens 6

MUG, indicator for lacZ 340, 349, 350

NE agar, for testing capreomycin
resistance 474

Nocardia lactamduranscephamycin production 12, 32
transformation 32, 231

Multicopy vectors 256-265
Mutanolysin, for lysing mycelium 163
Mutagenesis 99-106
by NTG 99, 101, 103-104
by NUV plus 8-methoxypsoralen
101by transposons 21, 24, 109-122, 217
by UV 101, 102-103
clustered mutations 101
expression of mutations 100
in strain improvement 23
localised, using generalised
transduction 274-275
optimal level 99-100
Mutational cloning 222, 224
analysis of transcription units 298,
334-335
choice of C31 vectors 291-292
Mycelium, vegetative see also Aerial
mycelium; Branchinggrowing 49-50, see also Cellophane
overlays; Liquid cultures;
Nitrocellulose filters
harvesting 51, 81, 162, 454
lysingfor cell-free extracts 75-76
for DNA isolation 163, 172-173
protoplasting 56-58
staining 64-69
storing 57, 163

NEF agar, for spore preparation 78(recipe)neo (neomycin/kanamycin resistance ofTn5) 10, 324, 445, 447(figure), 480-481 (maps)
cross-resistance 448 (table)
as reporter 268, 342
in gene disruption 332-334
on SuperCos-1 218, 226, 236
on vectors 268 (list), 513-519
to select recombinants 241 (table)
Neomycin 14, 453
producer 26
resistance 241, 342, 390, 447(figure), 450, 462, see also
aacC8; aacC9; aphI; kamA;
kamB; kamC; kan; km; neo;
nmr
cross-resistance 448 (table)
medium dependence 219, 241
Netilmycin, resistance 467
NF (SCP1NF) strains of S. coelicolor
423 (list), 424 (table)
instability of pIJ101 derivatives in110lack of extracellular agarase in 223,430
recombination in 126, 127, 129
transposon mutagenesis of 113-114

Mycobacterium spp.
as pathogens 6
genome sequences of M. bovis, M.
leprae and M. tuberculosis 398

Nikkomycin 14
biosynthesis 30, 319

Mycobacterium fortuitum see IS6100

Nitrocellulose filtersfor colony hybridisation 242-343
for growing mycelium 48
for plaque hybridisation 303
to prepare phage lysates 276
to remove ss DNA 172
VMWP, for microdialysis 165

Mycobacterium smegmatis
Streptomyces integrating elements in
215, 255, 265-267
taxonomy 3
Mycothiol, in disulphide reduction 10
myrB (lincosamide resistance) 445
Myxococcus, prophage insertion 295
N

Nitrocefin, for -lactamase assay 349

Nitrogen fixation, by S. thermoautotrophicus 10
Nitrogen utilisation, by Streptomyces
8, see also Ammonium
in antibiotic production 16, 93
in minimal medium 406
in selective isolation media 44
non-utilising mutants 106

Nocardia lurida, ristocetin producer
14
Nocardia uniformis, nocardicin
producer 14
Nocardicin 14
Nonactin 453
biosynthesis 27
resistance 27 see also nonR
nonR (nonactin resistance) 445, 489(map)
Northern blotting, limited utility in
Streptomyces 374
Nosiheptide 14, 453 see also
Thiostrepton
resistance see nsh; tsr
Nourseothricin 453
resistance see nat1
Novobiocin 14, 453
resistance 450, see also gyrBnsh (nosiheptide resistance) 445, 447,
451, 457 (map)
NTG (N-methyl-N0-nitro-Nnitrosoguanidine, MNNG),
mutagenesis by 99-101, 103104
Nuclease see also DNase; RNase
micrococcal, for coupledtranscription-translation 79
on toothpicks 163
NUNC, square Petri dishes 77, 419(supplier)
NUV (near UV, 365 nm), for 8methoxypsoralen mutagenesis 101
Nystatin 14
as fungicide, for selective isolationof Streptomyces 44
Onat1 (nourseothricin resistance) 445,476 (map)

NMMP (minimal liquid medium) 50,
413 (recipe)

OC (open circular) plasmid DNA
ethidium bromide fluorescence 175
on agarose gels 195, 196-197(pictures)
position in CsCl gradient 176(figure), 183removal by acid phenol extraction
193
removal by alkaline denaturation
191

Natamycin 13

nmr (neomycin resistance) 445

OD (optical density)

N-methyl-N0-nitro-N-nitrosoguanidine
see NTG
Nalidixic acid 453
to counterselect E. coli 249-250,
325

Nitroquinoline oxide (4-nitroquinoline1-oxide), as mutagen 101

,QGH[2' RSWLFDOGHQVLW\ WR & SKDJH

OD260/280 for estimating
DNA concentration 167
RNA concentration 360
OD450, for estimating spore
concentration 50, 52, 53
OD600, for growth curves 200, 202,450
of NADH 349
of S. coelicolor antibiotics 93, 95
oleA,B, oleC (oleandomycin resistance)
445
Oleandomycin 14, 453
biosynthesis 24
resistance 445, see also oleA, B,
oleC
Omega (antibiotic resistance) fragments
aac(3)IV 465 (map)aadA 468 (map)hyg 485 (map)vph 483 (map)Open circular see OC plasmid DNA
Operons, in Streptomyces 212
analysis of 334-335
polar effects 109, 298
Ordered cosmid library, of S. coelicolor
see Cosmid
ori, of plasmid replication/replication
regionpIJ101 254 (map), 254-256
pSAM2 264 (map), 265
pSG5 120, 257 (map)
SCP2* 259 (map) 266, 350
SLP1 263 (map)oriC, of S. coelicolor, bidirectional
replication from 20, 422 (map)oriT, of RK2/RP4, in transfer toStreptomyces 128, 219, 221,249-250, 317, 445, 502 (map)vectors 267 (list), 513-519
Osmium tetroxide, in electron
microscopy 70
otrA, otrB (oxytetracycline resistance)
445, 449, 451, 491 (map)pac (packaging site of phages) 299300
on vector 530 (map)pac (puromycin acetyltransferase) 445,
451, 477 (map)
Pactamycin 453
PAGE, see also SDS-PAGE
non-denaturing, for detecting
phosphotransferase activity 342
Pal6 phage, divalent cations foradsorption 278
Palindromic inviability 218, 541(map),
557 (map), 565 (maps)
PAP marking pen 67, 68, 419(supplier)Paromomycin 14, 453,
resistance 442, see also aacC7
cross-resistance 448 (table)
Partial digestion, with restriction
enzymes 214-215, 333
pat (phosphinothricin acetyltransferase)
445, 472 (map)similarity to bar 323
Pathogenesis, by actinomycetes
animal pathogens 6
plant pathogens 6, see also S.
scabies
PBS buffer, in immunofluorescent
staining 67 (recipe)
PCR 402-403
buffer 402-403 (recipe)
effect of base composition 212
in DNA sequencing 394
probes for S1 mapping 363, 366367
to generate in-frame deletions 314
to generate unmethylated DNA 230
to identify transformants 222, 248
Penicillin 453
enrichment 104
Periodic acid, in electron microscopy
72

Over-mutagenesis 100
Oxoid nutrient agar (ONA) 411(recipe)for CDA detection 95
unsuitable for phage assays 277

Petri dishes, square (NUNC) 419(supplier)
PFGE see Pulsed-field gelelectrophoresisPpgl (phage growth limitation, of S.
coelicolor) 213, 276, 295, 304,
429
isolation of mutants 106, 314
strains 423

P (protoplast) buffer 415 (recipe)

Phages, of Streptomyces see also

Oxytetracycline 14
biosynthesis 30, 433
resistance 445, see also otrA, otrBFP43; JHJ-1; Pal6; 448;
C31; HAU3; R4; S14;
SAt-1; SF1; SH5; SV1; TG1;
VP5, VP7; VWB
as vectors 292-293 (tables), 513-514(table), 519-529 (maps)
as alternatives to plasmids 217218, 341
cloning procedures 290-308
for gene disruption 318-319
for homogenotisation 330-331
list of special purpose vectors
267-269
deletion mutants 273
DNA, base composition 18
DNA, preparation 282-287
host range 272, see also pgl;
Restriction-modificationindustrial contaminants 273
isolationfrom lysogens 272
from soil 272, 279-281
lytic and temperate 272, see also att
sites; int genes
occurrence 21-22
packaging 273, see also cos
plaque assay 277
preparation of high-titre stocks 278279
preservation/storage 59, 274 (DNA),
276
relevance to host genetics 271, seealso Transduction
restriction mapping 273
single-plaque isolation 278transducing phage selection 281-282
Phase-contrast microscopy 64, 66
to monitor protoplast formation 57
to monitor spore breakage 76
Phenetic characters, in Streptomycestaxonomy 2, 4, 5
Phenolin DNA isolation 164
in RNA isolation 355, 357, 359
Phenol/chloroform
in DNA isolation 164, 365
in RNA isolation 355, 357, 359
Phenotypic lag, in mutation expression
101
Phenotypic resistance, to phages 272
Phenoxazinone synthase 73
448 phage, divalent cations foradsorption 278
C31 (phage) 290, see also pgl
c (repressor gene) 290-294, 296, 303
cloning scheme 290
cos 290in vivo transduction 299

& SKDJH WR3ODVPLGVZLWKSUHIL[Sdetection of xylE expression inplaques 344
divalent cations, for adsorption 278
DNA base composition 212
genetic analysis of 273
genome 290, 291 (map), 519-522(maps)
glkA, to select loss of prophages ordeletions in lysogens 307-308
host-range 215
in vivo packaging, for transduction299
int 265, 300, 522 (map)
integration functions 265
ligation conditions 294
lysogens, preparation of 294-295,
303-307
maximising insert frequency 294
mutants 273, 275
packaging limits 216, 218, 273, 318
plaque hybridisation 303
sequence 519
shotgun cloning 290-291
stability of lysogens 294-295, 429
storage 276transcription 291, 521 (maps)
transfection of protoplasts 301-302
vectors see KC prefix and PM8 forindividual vectors
attP vectors, for transfer from E. coli
249, 341, 350, 389
development of 290
for cloning by complementation292
for gene disruption 318-319
for gene fusions 296-299, 341
for homogenotisation 330-331
for mutational cloning 291-292
integrating 216, 265, 268, 300
lack of EGFP-containing 499
maps 523-529
promoter-probe vectors 296-299,
341
tables 292, 293, 513, 514
yield from plaques and plates 277,
286
HAU3 (phage)
phasmid vector 300
resistance of S. lividans 425, 426,
429
Phleomycin 14, 453
resistance 447, 450, see also
Bleomycin; Zeocin
Phosphinothricin 453, see also
Bialaphos
resistance gene 445, 447, 450, 471(maps), see also bar; pat
Phosphoenolpyruvate:phosphotransferase system (PTS) 7
Phosphor screen 89, 419 (supplier)

Photoreactivation, of UV lesions 102,429
pIJ see Plasmids with prefix p
Plantsas producers of secondary
metabolites 11-galactosidase of 340
diseases, caused by Streptomyces 6
protection, by antibiotics 14, 15transformation 464, 470

,QGH[

transformation of protoplasts 232236
transposon mutagenesis of 111, 114117

Plasmids with prefix p
p15A (E. coli plasmid, compatible
with pBR322)
plasmid amplification 172
on vectors 263, 515-517
pACYC177 263, 300, 479, 564(map)
pACYC184 184, 218, 263, 443,
Plaque assay 277
444, 564 (map)
Plaque formation 272, 275 see also pgl
pANT840 517, 568
pANT849 268, 389, 537 (map)
Plaque hybridisation 303
pANT850 268, 537 (map)pANT851 268, 537 (map)
Plaque isolation 278
pANT857/859/863/864, expression
Plasmids, of E. coli 564-572 (maps),
vectors 268 (list), 389
see also oriT
pANT1200/1201/1202 267 (list),
268, 538 (maps)
Plasmids, of Streptomyces see also
pARC1
343
Linear plasmids; Plasmids with
pAT98 300
prefix p
pAX5a, pAX8 390
as prophages 22
pB45 268, 547 (map)as template in coupled transcriptionpB48 268, 500, 547 (map)translation 79
pB83 500, 547 (map)as vectors 514-517 (tables), 530-563
pBG3, pBG4 470(maps)
pBluescript 568 (map)for intracellular expression 398pBR prefix see also bla; pBR322;
390
pBR325; pBR327; pBR328
for secretion 390-391
amplification using
general criteria for choice 217chloramphenicol or
218, 341
spectinomycin 172
list of special purpose vectors
in vectors 514-518
267-269
pBR322 572 (map)base composition 18
derivatives as suicide plasmids
causing c.m.a. 126-127
333,
curing 224-225
inearly C31 derivatives 290
DNA518, 572 (map)isolation 171-195
pBR325 443
on agarose gel electrophoresis
pBR327 572 (map)195-198
in SCP2* vectors 260- 262 (maps)
on PFGE 204
in IS117 vectors 573-574
mobilising plasmids for oriT see
pBR328,5 kb ladder 204
pUB307; pUZ8002
pCAO106 263 (map), 267, 268, 517
non-transmissibe plasmids
pCAO170 263, (map), 267, 517
pIJ101 vectors 256
pCAO200 263 (map)pUZ8002 for mobilising oriTpCJR24 268, 567 (map)vectors 219, 249, 317, 348
pCJR29 267, 268, 558 (map)of S. coelicolor and S. lividans 428pCLL34 343
429 see also SCP1; SCP2;
pCZA126 110
SCP2*; SLP1; SLP2; SLP3;
pCZA163 267, 577 (map)
SLP4
pCZA185 267, 268, 551 (map)stabilisation by transposons 109
pCZA186 117-118
structure and properties 20, 21, 253pCZA258 267, 577 (map)256
pCZA276 267, 577 (map)temperature-sensitive see pMT660;
pDH5 222, 230, 265, 269, 317, 342,
pSG5
456, 570 (map)transduction of 128, 299-200
pDS5000 268, 350
transfer see also Ltz; Pocks; trapEB2 470by protoplast fusion 128
pEGFP 499
detection by pocks 153-156
pFD666 258, 267, 480, 546 (map)frequency 137, 152

,QGH[3ODVPLGVZLWKSUHIL[S FRQWLQXHG S*0WRS6%

pGM9 268, 548 (map)pGM11 268, 466, 548 (map)
pGM16 268, 549 (map)pGM17 268, 549 (map)
pGM160 267, 268, 318, 320, 466,
550 (map)
pHJL197 262 (map)
pHJL210 262 (map)
pHJL302 262 (map), 263, 267, 553(map)
pHJL401 262 (map), 263, 267, 350,
576, 533 (map)
pHM10A 267, 517
pHM11A 267, 268, 518
pHM6-11 390
pHZ132 267, 268, 550 (map)pHZ1271/1272 267, 268, 540(maps)
pHZ1351/1358 256, 267, 268, 542(maps)
pIC20R 514
pIJ39 456
pIJ61 263 (map), 265, 475, 478, 563(map)
pIJ80 261 (map), 268, 559 (map)pIJ101 21, 254 (map)c.m.a. 126, 159
co-integrates with SCP2* 262
copy number 172
host-range 215
kil/kor 254, 256
no insertions of Tn4560 114
pocks 239
promoters 350
replication 255, 256
spreading functions 153, 254
stability increased in S. lividans
JT46, 216, 219, 257
sti function 256
vectors 267, 514-515
pIJ110 from S. parvulus 156
pIJ303 225, 256
pIJ364 482, 534 (map)pIJ365 299
pIJ385 478
pIJ408 from S. glaucescens 156,214
pIJ459 268, 458, 534 (map)pIJ486 226, 238, 239, 268, 342,
389, 480, 484, 533 (map), 544
pIJ487 256, 268, 342, 480, 484, 533(map)
pIJ666 565 (map)pIJ673 21
pIJ680 256, 389, 390, 478, 534(map)
pIJ688 342
pIJ698 218, 260 (map), 261, 267,
268, 484, 557 (map)pIJ699 216, 218, 267, 268, 482, 541(map)
pIJ702 110, 239, 254 (map), 256,

300, 389, 470, 476, 500, 536(map), 547
pIJ860 256
pIJ871 475
pIJ903 114, 260 (map), 267, 554(map)
pIJ916 239, 261
pIJ920 260 (map), 268, 482, 555(map)
pIJ921 268, 555 (map)pIJ922 260 (map), 261, 554 (map)pIJ940 260 (map), 261, 556 (map)pIJ941 260 (map), 261, 484, 556(map)
pIJ943 239, 261 (map), 500, 562(map)
pIJ963 325, 484, 485 (map)pIJ1117 517
pIJ2059 468 (map)pIJ2527 268, 350
pIJ2581 268, 321, 456, 493, 571(map)
pIJ2585, containing redD 268, 350
pIJ2842 341
pIJ2923 260
pIJ2925 458, 468, 568 (map)pIJ4026 458, 459 (map)pIJ4083 268, 341, 495
pIJ4090 268, 319, 458, 535 (map)pIJ4104 470
pIJ4123 268, 389, 539 (map)pIJ4201 267, 268, 573 (map)pIJ4210 267, 268, 574 (map)pIJ4231 260 (map), 266 (map), 267,
268, 482, 484, 516
pIJ4642 218, 268, 469 (map), 565(map)
pIJ4676 469 (map)pIJ4680 267, 268, 319, 559 (map)pIJ4696 267, 268, 574 (map)pIJ4698 267, 268, 574 (map)pIJ4700 267, 268, 574 (map)pIJ5971 267, 268, 346, 498
pIJ6021 268, 389, 539 (map)pIJ6650 267, 317, 321, 324, 325,
566 (map)
pIJ8301 300
pIJ8600 267, 268, 389, 531 (map)pIJ8630 267, 268, 531 (map)pIJ8660 267, 268, 347-348, 388,531 (map)
pIJ8668 267, 268, 348, 571 (map)pIJ8671 267, 531, 567 (map)pITS107 389
pJAR4 261, 267, 268, 484, 560(map)
pJAS14 349
pJOE829 268, 484, 541 (map)pJOE875 268, 541
pJOE2577 121-122, 267, 581 (map)pJV1, from S. phaechromogenes 29,
258
c.m.a. 126

entry disadvantage 126
host-range 255
sequence 258
vectors 558
pKC505 261 (map), 267, 268, 560(map)
pKC531 262 (map)pKC575 262 (map)pKC731 300
pKC796 300
pKC1064 267, 268, 390, 495, 516
pKC1132 267, 317, 321, 324, 566
pKC1138 269, 341, 566 (map)pKC1139 267, 268, 269, 551 (map)pKC1218 262 (map), 267, 268, 269,
561 (map)
pKC1250 267, 269, 566 (map)pKTO2 267, 268, 390, 532 (map)pKU112 110
pKU306 260 (map)pLST18 463
pLST314 (map)pMCXpress 390
pMEA300 231
pMSA-1 472
pMT603 261 (map)pMT660 110, 112, 115, 256, 268,
500, 519, 536 (map), 579
pMT3010 268, 342, 500, 536 (map)pMT3020 268, 342, 343
pMT3206 268, 289, 535 (map)pMT3226 267, 268, 390, 532 (map)pOJ260 267, 269, 317, 324, 566(map)
pOJ436 267, 268, 530 (map)pOJ444 267, 268, 530 (map)pOJ446 267, 268, 562 (map)pPM925 571
pPM927 267, 268, 390, 563 (map)pPM2205 317
pPM2208 267, 268, 317, 571 (map)pRES1 545 (map)pRES18/19 267, 269, 545 (maps)
pRHB101 300
pRHB106 117, 300
pRM5 261 (map), 262, 267, 268,
557 (map)
pRS1105 268, 346, 468, 543 (map)pSAM2 20, 264 (map), 265
att site on S. rimosus chromosome
433
discovery of 156
integration at att site 216, 265,300
integration in E. coli 265
random integration 265
replication of 255, 265
tandem integration 225
use in Mycobacterium 215, 255,
265
vectors 341, 563 (map)pSB24.2 445

,QGH[

3ODVPLGVZLWKSUHIL[SWR3XURP\FLQ

pSCP103 (SCP2* derivative) maps
258, 260-262
pSET151 222, 265, 267, 268, 269,
317, 324, 570 (map)pSET152 267, 268, 269, 324, 327,
350, 389, 390, 530 (map), 558
pSG5, from S. ghanaensis 220, 257(map)
copy number 259
for gene disruption 220, 260, 318
for transposon delivery 110, 117118, 121, 224, 260
host-range 255, 259
lack of pocks 259
no insertions of Tn4560 114
replication 259
vectors 515-516, 519
pSIT151 119-121, 466, 516
pSKO2 543 (map)pSKO3 267, 268
pSLA2 20
pSLP124 342
pSN22, from S. nigrifaciens 255
similarities to pIJ101 and pJV1
258
traR see traR
pSupercos-1 267, 569 (map)pTMA1 268, 348, 561 (map)pUB307 ( self-transmissible oriT
mobilising plasmid) 250, 317
pUC4K 444, 479
pUC18/19
pUC19 568 (map)in vectors 262 (maps), 514-519
pUC1169 111-117, 579 (map)pUCGM 466 (map)pUCS30 119, 267, 268, 552 (map)pUT56/58 472
pUWL series 267, 269, 544 (maps)
pUZ8002 (non-transmissible oriT
mobilising plasmid) 219, 249,
317, 325, 348
pVE1011 518, 570
pVE1043 260 (map)pVE616 267, 518, 569
pVE616 267, 518, 569
pWHM3 267, 269, 319, 544 (map)pWKR209 466
pWOR126 500, 546 (map)pXE4 268, 341, 343
Plastic tubes, disposable, for protoplastpreparation 56
Plate-crosses 47, 152
Plating out spore suspensions 47, 131
Platinomycin, biosynthesis 29
PM8, C31 vector 293, 529 (map)PMSF
in PFGE 200 (recipe)in RNA polymerase isolation 377379

Pocks, formed by conjugative plasmids
21, 153-155, 154 (pictures),
255, see also spd
lack of by pSG5 259
to recognise transformants 115, 239
visualisation of 47, 48, 153, 155
Poly-L-lysine, in DAPI staining 65
Polyclonal antibodies
cross-reaction to Streptomyces
proteins 73
in immunomagnetic capture 44
Polyethylene glycol (PEG) 419(supplier)
as dispersant in liquid media 51
for DNA precipitation 166, 173
in protoplast fusion 156-157
in protoplast transfection 301-302
in protoplast transformation 231236
Polyketide synthase, immunogold
staining of 73
Polyketides see also Hybrid antibiotics
genes 24-32, 224, 547, 557, 558
producers 12-15
Polyoxins 14
Polysaccharides, in DNA isolation
164, 173, 174, 176, 182
CTAB procedure 170-171
Positive selection vectors 218,
268 (list)
Potassium acetate
3M for DNA precipitation 207(recipe)in plasmid isolation 165, 184-186
solubility in ethanol 165
Potato scab disease 6531, 533, 536, 543, 561 see also
Reporter systems
Promoters, of Streptomyces 7-8, 212213, 225, 385-386, see alsochi63; ermEp/ermE*p; gyl;mcrAB; melC; snpAp; pSN22;
tipAp
in in vitro transcription 374
regulated promoters 388, 389-390(list)
shut down, by C31 infection 296
whiE/whiG 341
Pronase E, in PFGE 199
Prophages
induction of 272, 299
loss of 295
using glkA 307-308
plasmids as prophages 22
Propidium iodide
as counterstain for nucleoids 66, 67,
69
in plasmid isolation 175
Propionibacterium, taxonomy 3
Protease see also Pronase E;
Proteinase K; Subtilisin
inhibition of secreted, by
casaminoacids 388
inhibitor 386 see also PMSF
low level of extracellular in S.
lividans 385
promoter see snpAp
Protein kinases, in regulating antibiotic
biosynthesis 16
Protein phosphorylation 7, 16
Proteinase K
in DNA isolation 163
in PFGE 199

Potter homogeniser 118, 419 (supplier) Protoplasts
fusion 25, 29, 30, 31, 156-158
ppGpp, in growth regulation 17
genetic analysis by 158
pra (regulatory gene of pSAM2) 264
in strain improvement 23(map)
preparation 56-58
regeneration 232
Preservationtransformation 26, 28, 31, 32, 231,of phages 58, 276
232-236of strains 58
Pseudomonas spp. see also aadA; xylE
Primer extension mapping 372-374
in rhizosphere 6
Pristinamycin 14,
ptr (multidrug resistance) 445, 451
biosynthesis 29
resistance 449 see also ptr
Pulsed-field gel electrophoresis (PFGE)
18, 111, 197, 198-205
Prodiginines see
Butylcycloheptylprodiginine;
pur8 (puromycin resistance) 445, 449,
Undecylprodigiosin
451, 477 (map)
Proline catabolism 9
Promoter-probe vectors 226, 298, 341,

Puromycin 14, 454
biosynthesis 24, 224

,QGH[resistance 445, 451, see also pac;
pur8
Pyrophosphate see also Chelating
agentsin preparing phage lysogens 304
to select transducing phages 281282
Q
Qiagencolumns for plasmid purification 79,
171, 173, 176, 189-190
Ni-NTA agarose 382
R
R2 medium 408 (recipe)
for transfection 301
for visualising pocks 155, 239
R2YE medium 408 (recipe)
antibiotic concentrations in 241(table), 470, 482
for crosses 130
for protoplast fusion 157
for transfection 301
for transformation 232
for visualising pocks 155, 239, 255
sporulation on 430
without sucrose, for plating C31
306
R4 phage
divalent cations, for adsorption 278
g mutants 275
in vivo packaging 299
induction by mitomycin C 272
restriction 276transduction 299
transfection 301
vectors 300
R5 medium 409 (recipe), see also
R2YE medium
Racemomycin 454
resistance 451
Radiolabelling see Labelling
Rapamycin 14
biosynthetic genes 28, 318
RCR see Rolling circle replication
Rearrangement, of DNA
by transposon insertion 109
of clones 225
on storage 511
rec-46 mutation
effect on clone stability 216, 219
on S. lividans strains 425
recA, disruption 323
Recombination frequency see C.m.a.;
NF strains



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red cluster, of S. coelicolor 333, 426
Red antibiotic see Undecylprodigiosin
redD (regulator of red cluster) 423
as reporter 350
on vectors 268 (list)
Reduced arginine starch salts agar, inselective isolation ofStreptomyces 44

single-stranded DNA to overcome
231, 249
Restriction endonucleases/enzymes
for libraries 214
for PFGE 111, 119, 202
produced by Streptomyces 22, 212,
213
sites 441 (table)
frequency of 212
star activity 174, 218

Refractive index (RI), of CsCl solutions
Restriction, of methylated DNA 22,
178, 179 (table), 180
230, see also ET12567; mcr;
Renaturation
mrrof DNA
Restriction-modification 22, 24, 26,
cot curves 162
213, 230, 275-276
kinetics for taxonomy 18
of S. coelicolor and S. lividans 429
of proteins 388
rep (replication) genes of plasmids
255, see also Rolling circle
replicationof pIJ101 254 (map), 256
of pSAM2 264 (map), 265
of pSG5 257 (map), 260
of SCP2* 259 (map)Reverse transcriptase, for primer
extension mapping 372, 373

Replica plating see also Velvet
for analysing crosses 133, 135-136
for isolating mutants 106
for quantifying plasmid transfer 137
in sib-selection 223

Rhizosphere, colonisation by
Streptomyces 6, 11

Reporter systems 339-350
choice of marker 340
vectors 268 (list), 296, 341-342
RES1 element, of S. lividans 427
Resistance genes see also individualgenes
as part of antibiotic gene clusters
224
choice ofas markers 219-220
as reporters 342
for gene disruption 223-224
expression/selection, after
transformation 221, 239-241(table)in tests of lysogeny 306
listed alphabetically 442-446 (table)
listed by class 450-451
listed by mechanism 446-449
maps 456-494
of Gram-negative bacteria 110
on vectors 513-519
Resistant mutants
isolation 104
usually recessive 158
Restriction barriers 213, 230
heat treatment to overcome 230
restriction-deficient mutants 230,
275

Rhizobia, streptomycin-resistant, in soil
11
Rhizoctonia, antagonised by
Streptomyces 11

Rhodococcus, taxonomy 3
Ribosome binding site 212, 394, 398,
see also Leaderless transcripts
sequence of 3’ end of 16S rRNA 386
Ribostamycin 454
resistance 450, 461, 478, see also
rphS1
Rifampicin 454
Rifamycin 14
biosynthesis 31
Ristocetin 14
RK2 plasmid, transfer functions see
also oriT
on mobilising plasmids see
pUB307; pUZ8002
RNA polymerase
in in vitro transcription 374
purification 377-383
T7 391target for rifamycin 14RNAharvesting cultures for isolation 354
in CsCl-ethidium bromide
centrifugation 176
isolation 51, 355-360
methods 354-377
precautions in handling 354
quantity and quality control 360(picture), 360-363

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removal from DNA 162, 166, 173,
189
rRNA
genes 29
methylases 446, 447 (figure)phylogenetic tree (16S rRNA) 3(figure)sequence 386
storage 360
RNase
avoidance in probe preparation 364
avoidance in RNA isolation 354,
361
in DNA preparation 162, 166, 173,
285, 364
inhibition by iodoacetate 361
reduction of transformation
frequencies 232
Rolling circle replication (RCR), of
plasmids 174, 255
of pIJ101 254
of pSAM2 265
of pSG5 257
Rotors, for DNA isolation 174,
181 ( table), 183 (figure),
185 (table), 191
RP4 see RK2
rphS1 (ribostamycin
phosphotransferase) 445, 479(map)
rpsL (strA, ribosomal protein) 445
counterselection 323
mutations and antibiotic production
220
S
S1 mapping 341
formamide in 367
high resolution 363-371
inverted repeats 371
loading buffer 370 (recipe)
low resolution 371-372
PCR probes 363, 366-367
S1 digestion buffer 368 (recipe)
size standards 367
TCA buffer 367-368 (recipe)
S14 phage, divalent cations foradsorption 278
S. achromogenes, DNA amplification24
S. acidoscabies, causing potato scab 6
S. acrimycini see also catSa
genetic mapping 24, 138
sporulation in liquid 56
S. actuosus, nosiheptide producer 14,
see also nsh

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S. albidoflavus
sporulation in liquid 56
taxonomy 5

S. bambergiensis
bambermycin producer 12
methyl-specific restriction 317

S. alboniger see also pac; pur8
puromycin production 14, 24

S. bikiniensis
genetic mapping 25
inhibiting Salmonella in soil 10taxonomy 4, 27

S. albus
expression of foreign genes 24
genetics of SalI 24, 276
heat shock genes 24, 317, 319
inhibition by S. lividans 291
salinomycin producer 15
sporulation in liquid 56
taxonomy 4, 5
S. ambofaciens see also pSAM2; srmB
expression of aph(4) 464
genetic instability 24
genetic mapping 24, 138
genome map 18, 20, 199, 342
PFGE 199
spiramycin production 15, 24
transposon mutagenesis 24, 117
S. antibioticus see also melC; oleA,B,
oleC
gene disruption 318
genetic instability 24
hexose monophosphate shunt 7
melanin synthesis 24
oleandomycin production 14, 24
stringent response 24
taxonomy 4
S. anulatus, taxonomy 5
S. argillaceus see also mtrA,B
mithramycin producer 13
S. atroolivaceus, taxonomy 4
S. aureofaciens see also hur(chlor)tetracycline production 12,
15, 25
bromoperoxidases 25
glycogen metabolism 24lack of GDH:GOGAT couple 8sigma factors 24
taxonomy 4
S. avermitilis
avermectin production 12, 25
DNA modification 22, 25, 179, 199,
204, 317
gene replacement 332-333
genetic mapping 25, 127, 138
methyl-dependent restriction 22,
213, 317
transposition in 112
S. azureus see also SAt-1
coupled transcription-translation 78
lysogeny 25
thiostrepton
production 15
resistance 25, see also tsr

S. cacaoi-lactamase gene 25, 396-397
polyoxin producer 14
S. caelestis see clr
S. caespitosus, mitomycin C producer
13
S. capreolus see ard1; cac; cph
S. cattleya 300, see also argG; TG1
phagethienamycin producer 15
S. chartreusis, as cloning host 25
S. chromofuscus, taxonomy 4
S. chromogenus, taxonomy 4
S. chrysomallus, protoplast fusion 25
S. cinnamonensis
monensin producer 13
polyketide synthase genes 25
S. clavuligerus
cephamycin biosynthesis 25
clavulanic acid production 12, 25
genetic mapping 25
lack of GDH:GOGAT couple 8
lack of glucose utilisation 7
linear plasmid 25
protoplast formation 200
stringent response 25
taxonomy 4
S. coelicolor A3(2) see also S.
coelicolor A3(2) strains
2D PAGE of proteins 84, 85
database 90
agarase production 92, 106, 223,430
amplifiable DNA 427
antibiotic production 16, 93-95,
410-436, see also Actinorhodin;
Calcium dependent antibiotic;
Methylenomycin;
Undecylprodigiosin
conditions for 50-52
antibiotic resistance 219, see also
mmr
selection 241, 448
unstable 20, 427
c.m.a. 126, 127, 217
carbon catabolite repression 7-8
clumped growth in liquid 50
clustered mutagenesis 101

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colony hybridisation 243
counterselection using glkA 307,322
coupled transcription-translation 78
developmental biology 17-19 see
also bld; whi
fructose PTS 7
FtsZ protein 66
gene disruption 319, 321, 323, 335
genetic differences from S. lividans
426-430
genomeproject 394, 398, 424-425
structure 18, 129 (map), 140, 141,422 (map), 423
glycerol utilisation see gyl; gylR
growth for DNA isolation 162, 199
growth temperature 106
heteroclones 141
lack of attB for R4 phage 300
life cycle 19
linkage mapping 138, 140
lysogens of C31 294-295
mobile genetic elements 21, 428, seealso IS110; IS117
ordered cosmid library 141, 218,
236, 569
phage growth limitation see pgl
photoreactivation, lack of 102, 429
plasmids 20, 429, see also SCP1;
SCP2; SCP2*; SLP1
pocks 154, 256
protoplast fusion 156, 157-158
protoplasting, conditions for 57, 412
prototrophic plasmid-free strains, forphysiological studies 424
reporter genes
problems with lacZ 340
whiE, redD 343, 350
restriction of methylated DNA 22,
213, 230, 236, 317, 429
spore germination 53
spore-shaped bodies in liquid 56
sporulation, ectopic, induced bywhiG 56
sporulation on MS agar 44
strains, genetically marked 423, 424
see also S. coelicolor A3(2)strainstransduction 22, 128, 274
transformation, by denatured DNA
231, 235
transformation/transfection
frequencies 232
transposon mutagenesis of NF strains
113-114
S. coelicolor A3(2) strains 423-424(lists)
CH999 ( act, red) 423
J1501 350, 423, 428, 429
2D protein gel 85
for phage cloning 294
transformation of 232

J1501 glkA 322
J1510/J1511 ( -gal-) 350
J1668 ( glkA) 308, 322
J1915/J1916/J1917 ( glkA) 322
M145 422 (map), 424
cosmid library 141, 236
doubling time 50
genome sequence 195transformation frequencies 220,
232
M130 424, 429
M152 (host for redD) 350, 423
S. coelicolor Müller
gene disruption 323
oxidative stress 25
S. curacoi, polyketide synthase genes
26
S. cyaneus, taxonomy 4, 5
S. cyanogenus see nmr
S. cyanogriseus, methyl-specificrestriction 317
S. diastaticus, taxonomy 4
S. espinosus, transformation 26
S. exfoliatus, taxonomy 4
S. flavopersicus see spcNbiosynthesis 26
resistance see sph
fatty acid biosynthesis 26
genetic mapping 26, 127, 138, 146
hybrid antibiotics 26
pIJ408 156, 214
taxonomy 4tetracenomycin biosynthesis 26
S. graminifaciens, streptogramin
producer 15
S. granaticolor, sporulation andgermination 54
S. griseoflavus, taxonomy 4
S. griseofuscus
as non-restricting cloning host 27,
213, 272
fructose-PTS 7
gene expression in 27
transduction by FP43 27, 300
transposon mutagenesis 117-118
S. griseoplanus, methyl-specificrestriction 317
S. griseoruber, taxonomy 4
S. griseoviridis
antibiotic production in soil 11
taxonomy 4

S. flavovirescens, isolation from trees 6 S. griseus
A-factor 17, 27
S. fradiae see also Tn4556
candicidin producer 12
hybrid antibiotics 26
daunorubicin biosynthesis 27
mutagenesis 26, 101
gene expression 27
neomycin producer 14
genetic recombination 27
neomycin resistance 26, see also
genome map 18, 27, 431 (map)aacC8; aphI
griseusin biosynthesis 27
protoplast fusion 26, 29
nonactin
restriction-modification 22, 26
biosynthesis 27
taxonomy 4resistance 27 see also nonR
transduction 26, 274
pRES1 545translation 26, 386
protoplast fusion 30
tylosinsporulation in liquid 27, 56
production 15, 26
streptomycin
resistance see tlrA-D
production 12, 27
urdamycin biosynthesis 26
resistance see aphD; aphE
taxonomy 5, 32
S. fulvissimus, taxonomy 4
S. halstedii
S. fulvoviridis, cabapenem producer 26
cellulases 27
spore pigment 27
S. galbus, mel 444
taxonomy 5
S. galileus
S. hygroscopicus
aclacinomycin biosynthesis 26
balanced growth 50
expression of act genes 389
bialaphos
hybrid antibiotics 26
production 12, 27
S. ghanaensis see pSG5
resistance see bar
curromycin
resistance see cre
S. glaucescens see also melC
FK506
producer
13
amplification and deletion of DNA
geldanamycin production 11, 28
26
gene disruption 318
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glutamine synthetases 27
hygromycin
producer 13
resistance see hyg
inhibiting fungi in soil 11
milbemycin producer 13
rapamycin production 14, 28, 318
restriction modification 275
taxonomy 5transduction 27
validamycin producer 15
S. ipomoea, causing sweet potato pox
6
S. jumonjinensis, transformation 28
S. kanamyceticus
glucose repression 8
interspecific mating 28
kanamycin
producer 13
resistance see kan
S. kasugaensis, transformation 28
S. lasaliensis, lasalocid production 13,
28
S. laurentii, tsr gene 446
S. lavendulae
genetic instability 28
melanin operon 500
mitomycin C resistance 28 see also
mcrA,B
streptothricin producer 15
taxonomy 4, 5
S. levoris
restriction-modification 275
valinomycin biosynthesis 28
S. lincolnensis
coupled transcription-translation 78
gene amplification 28
mel 444
lincomycin
production 13, 28
resistance see lmrA; lmrB
S. lividans 66 see also S. lividans 66
strains
amplifiable DNA 427
antibiotic production 85, 224, 430
antibiotic resistance 219, see also
cmlSl; lrm; tetSl
selection 241, 448
unstable 20, 427
c.m.a. 126
chromosome deletions 20
clumped growth in liquid 50
colony hybridisation 243
copper induction of sporulation 430
coupled transcription-translation 7883
DNA modification 22, 197, 199,

428, see also dnd
electroporation 130
expression of cloned proteins 385386, 387, 308
fructose-PTS 7
genetic differences from S.
coelicolor 426-430
genome structure 18, 140, 204, 425,
426 (map), 427
growth, for DNA isolation 162, 199
heterologous gene disruption 323329
heterologous gene expression in 28,
30
host for transfection 291, 301-302
IS117, circular DNA in 225
lack of attB for R4 phage 300
linkage mapping 138, 159
lysogens
detection 303-307, 318
instability 295
melanin production by melC 240
mercury resistance see mer
methylation-dependent restriction429
MLS resistance, inducible 430
mobile genetic elements 428, seealso IS493
monoclonal antibody for
immunogenetic capture 44
PFGE 204
photoreactivation 102
plasmid discovery in 127
plasmid transfer in soil 21
plasmids 429, see also SLP2; SLP3loss from 256
pocks, indicator for 153-155, 239,
256, 259
protoplast fusion 157-158
protoplasting, conditions for 57, 412
reporter genes
problems with lacZ in 340
use in 340, 342-345, 349-350
restriction, lack of 213, 272
rRNA sequence 386
spore-shaped bodies in liquid 56
sporulation on MS agar 44
strains, genetically marked 425 see
also S. lividans 66 strains
taxonomy 5transduction 128, 274, 322
transformation/transfection
frequencies 232
transposon mutagenesis in 110, 114115, 119-122
S. lividans 66 strains 425 (list)
1326 (wild type) 119, 386, 425, 426(map)1329-9R ( -gal-)/1326- -gal 350
JT46 (rec-46) 425
lack of tipAp induction in 119
plasmid stability in 216, 257, 425
M252 (chloramphenicol sensitive)

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425, 475, 488, 490
TK19 425
tipAp induction in 386
TK20/TK21 425
deletion of AUD2 429
tipAp induction in 386
TK23 425
indicator for pocks 153, 155
TK24 425
indicator for pocks 153, 155tetronasin resistant 489
tipAp induction in 386
TK64 425
lack of tipAp induction in 119
selection of tnrB2,3 489
to study IS117 343
TK503 (chromosomal Tn4560) 114
ZX1 (dnd) 22, 425, 428
sensitive to HAU3 429
tipAp induction 119
ZX7 (dnd) 22, 425, 426 (map)plasmid stability in 216
ZX64 (dnd) 22, 425
plasmid stability in 216
S. longisporoflavus
taxonomy 4tetronasin resistance see tnrB2,3
S. longisporus, STI-II proteaseinhibitor gene, promoter 386
S. lydicus, taxonomy 4
S. microflavus, taxonomy 4
S. narbonensis, interspecific protoplast
fusion 29
S. nataensis, natamycin producer 13
S. nigrifaciens see pSN22; traR
S. niveus, novobiocin producer 14
S. nogalater, taxonomy 4
S. noursei
nystatin producer 14
nourseothricin resistance see nat1
plasmids 29
S. olivaceoviridis
cellulose-binding protein 29
taxonomy 4
S. olivaceus
elloramycin biosynthesis 29
hybrid antibiotics 26
S. orchidaceus, cycloserine producer
12
S. parvulus
expression of act genes 29
isolation from trees 6
lysogeny 29
methylation-specific restriction 317
pIJ110 156

,QGH[6SDUYXOXVWR6DOPRQHOODW\SKLPXULXPSCP2* in 258
SLP2 pocks 154 (picture)transfection 56, 301

S. setonii
carotenoid biosynthesis 30
lignin degradation 30

S. peucetius
daunomycin/doxorubicin
production 12, 13, 29
resistance see drrA,B; drrC

S. somaliensis, as human pathogen 6

S. phaechromogenes see also pJV1
taxonomy 4transduction 274

S. sparsogenes, isolation from trees 6

S. pilosus, desferrioxamine production
13, 29S. sphaeroides see gyrBS. platensis, platinomycin biosynthesis
29

S. tendae
nikkomycin production 14, 30
tendamistat expression in S. lividans
30

S. plicatus, chi63 promoter 7-8
S. pristinaespiralis
multidrug resistance see ptr
pristinamycin production 14
S. purpurascens, hybrid antibiotics 26
S. reticuli, genetic instability 29
S. ribosidificus see rphS1
S. rimosus see also aacC7
antifungal activity in soil 11
dispersed growth in liquid 50, 130
electroporation 29, 130, 237-238
genetic mapping 29, 138
genome map 433
heteroclones 141
heterokaryons 136
interspecific mating 28, 30
lysogeny 29
oxytetracycline
production 14, 30
resistance see otrA, otrB
paromomycin producer 14
rRNA genes 29
sporulation in liquid 56
SRP1 plasmid 127
taxonomy 4
S. rochei
antifungal activity in soil 11
linear plasmids 20, 30
methyl-specific restriction 317
taxonomy 4
S. roseochromogenes, plasmids 30
S. roseosporus see also rpsL
daptomycin producer 12
sporulation in liquid 56
S. roseus, taxonomy 4
S. rubiginosus, xylose operon 7
S. scabies
causing potato scab 6, 11
zinc regulation of esterase 30

S. sp. strain C5, daunomycin
biosynthesis 31

S. spectabilis, spectinomycin producer
15

S. tenebrarius see also kamB; kgmB
tobramycin producer 15



S. violaceoniger, taxonomy 4, 5
S. violaceoruber
methylenomycin production 93
taxonomy 5
S. violaceus, taxonomy 4
S. virginiae
coupled transcription-translation 78-butyrolactones 31
virginiamycin producer 15
S. viridochromogenes
bialaphos
biosynthesis 31
resistance 31, see also pat
disruption of glnA 317
sporulation in liquid 56
taxonomy 4
tsr homologue 456
S. viridosporus, lignin degradation 31

S. tenjimariensis see also kamAprotoplast fusion 30
S. thermoautotrophicus
growth on carbon monoxide andhydrogen 10
nitrogen fixation 10
S. thermotoleranscarbomycin
biosynthesis 30
resistance see carA; carBhybrid antibiotics 24
S. thermovulgaris, taxonomy 4
S. toyocaensis, conjugation,transduction, transformation 30
S. venezuelae
chloramphenicol
production 12, 31
resistance see cmlv; cpt
genetic mapping 31, 138
jadomycin biosynthesis 31
mel 444
phages see also SV1; VWB
in soil 272
plasmids SVP1, SVP2, SVP3 127
sporulation in liquid 56transduction 22, 31, 128, 274-275,
282
S. verticillatus, mitomycin C producer
13

S. wadayamaensis, transformation 31
sacB, as counterselective marker 323
Saccharopolyspora
DNA base composition 18
gene exchange in 125
phage JHJ-1 272
taxonomy 3transduction by FP43 300
Saccharopolyspora erythraea
erythromycin
production 13, 32, see also
Erythromycin
resistance see ermE
gene disruption 32, 319
replication of pJV1 in 258, 546
SEP1 plasmid 127transformation 32
Saccharopolyspora hirsuta see also
kamC
polyketide synthase genes 32
Saccharopolyspora spinosaconjugation from E. coli 32, 249
spinosyn producer 15transformation by modified DNA 32
Safety guidelines, for genetic
manipulation 33
SalI
frequency of sites 212
genetics of 24, 276
ligation into MboI sites 215
recognition site 441

S. verticillus
bleomycin
producer 12
resistance 31 see also blmA; blmB Salinomycin 15
phleomycin producer 14
Salmonella spp.
S. vinaceus see vph
inhibited by soil antibiotics 10
S. violaceolatus, transfer of plasmids in Salmonella typhimurium
soil 21circular chromosome 138

6DOPRQHOODW\SKLPXULXPWR6PD,

localised mutagenesis 275
Salting out
to isolate chromosomal DNA 169
sap genes of S. coelicolor
SapB, as morphogen 17
sapC, sapD,E, on SCP1 434 (map)Sarkosylin DNA isolation 163, 174
in PFGE 201
SAt-1 phagecloning with 299
induction by mitomycin C 272, 299
SB buffer, for primer extension
mapping 373 (recipe)
Scanning electron microscopy 70
ScoDB, S. coelicolor genomic database398, 425, see also Genome
project
SCP1, linear plasmid of S. coelicolor
428, 434 (map), see also
Methylenomycin; mmr; NF
strainsc.m.a. 126-127
homology with SCP2 258, 434
IS466 on 428
strains containing 423, 424
SCP2/SCP2* 258-259 (maps), 428
c.m.a. 126
copy number 217
high copy mutants 263
entry disadvantage 126
homology with SCP1 258, 434
incompatibility 213
IS118 on 428
Ltz caused by 154 (picture), 239
on PFGE 204
replication 255
sequence 260
strains containing 423, 424
transposition to 111, 113, 114-117
vectors 217, 260-262 (maps), 266(map), 267, 516-517 (table),553-562 (maps)
containing reporter genes 341,
343, 348
in gene disruption/replacement
264, 319, 333
SDS (sodium dodecyl sulphate)
in DNA isolation 163, 174
in PFGE 199, 201
in phage isolation 286
in RNA isolation 359, 360
in selective isolation of Streptomycesfrom soil 44
to detect protoplasts 58
SDS-PAGE, for protein separation 8390

Secondary antibodies, for
immunofluorescence 66, 67, 69
Secondary metabolism 10-11, see also
Antibiotics
ammonium repression 9and differentiation 17-18
change of carbon metabolism 7
metabolites of S. lividans 95
Sectoring, to detect transposition 117118, 121
Selective isolation, of Streptomycesfrom soil 43-44
Selective markers, for cloning 219-220
see also Resistance genes
Selective media
for analysing crosses 131, 134, 141
for isolating Streptomyces 44

,QGH[

387, 391, 440
Siliconised
flasks for spore germination 52
glass wool for 5’ end labelling 246
Silver staining
for glycogen 72
for proteins 89
Single-plaque isolation, of phages 278
Single-stranded DNA
conjugal transfer from E. coli 249
for transformation 230-231, 316,
317, see also Denatured DNA
vectors to generate 546, 570
in DNA isolation 172, 174, 176
in plasmid replication 21, 255, seealso stion electrophoresis 195-198

SEP1 plasmid, of Sac. erythraea 127

Siomycin 454
resistance 451

Sephacryl S-100, for gel filtration ofplasmid DNA 174

Sisomicin 454
resistance 448, 450, 467

Sephadex
damage by cellulase 165
for gel filtration of plasmid DNA
174

Size standards
for PFGE 204
for proteins 81
for S1 mapping 367

Sepharose 6B-CL, for gel filtration ofplasmid DNA 174

Skim milk, for lyophils 59

Sequenase (T7 DNA polymerase) 394

Slide cultures, for microscopy 64, 67,
68

Sequencing, of DNA 393-395, 424
analysis 395-402

Slow Fade Kit 67, 69, 419 (supplier)Sequencing project, for S. coelicolor
see Genome project
Serine-threonine protein kinase 16
SET buffer 169 (recipe)
SF1 phage 111

SLP1/SLP1.2 263 (map), 265
c.m.a. 126
copy number 172
incompatibility 214, 215, 265
integration 265, 319
pocks and discovery 156
vectors 263 (maps) 267, 349, 350,
517 (table), 563 (map)

SFM medium see Mannitol soya flour
agar

SLP2, linear plasmid of S. lividans 435(map)
c.m.a. 126
SH5 phage, restriction-modification
entry disadvantage 126
275
lack of transposition of Tn4560 111
Shine Dalgarno sequence see Ribosome
pocks 153, 154 (picture)binding sitestrains containing 425
Tn4811 428
Sib-selection 223
sigE (sigma factor gene), null mutants
318, 324
Sigma factor, of RNA polymerase 17,24, 56, 217, 225, see also hrd;
sigE; whiG

Sigma mode of replication see Rolling
circle replication
Signal peptide, for protein secretion

SLP3, plasmid of S. lividans 127, 153,
427, 429
pocks 154 (picture)strains containing 425
SLP4, plasmid of S. coelicolor 428
SM buffer 417 (recipe)
SmaI
frequency of sites 212
recognition site 441

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SMM see Supplemented liquid
minimal medium
SMMS see Supplemented minimal
medium, solid
SNA see Soft nutrient agar
snpAp (protease gene promoter) 389
on vectors 537-538 (maps)
Soak-outs, for single-plaque isolation
278
Sodium4-aminosalicilate, in Kirby mix 163,
166
acetate3M for DNA precipitation 207(recipe)for plasmid precipitation 192, 207(recipe)pH4, for acid phenol extraction
193 (recipe)
stimulating protoplasttransformation 221
azide, for storing chromatography
columns 182 (recipe)
chloride 206 (recipe)
citrate see SSC
concentration in hybridisation probes
247
concentration in SSC 247
deoxycholate, detergent 175
dodecylsulphate see SDS
hydroxide 207 (recipe)
iodide see Iodide
iodoacetate, as RNase inhibitor 361
laurylsulphate see SDS
pyrophosphate
as chelating agent for selecting
transducing phages 181
for 50 end labelling ofoligonucleotides 246
for inactivating C31
in hybridisation solution 245
TCA buffer, for S1 mapping 367368 (recipe)
tri-isopropylnaphthalene sulphonate(TPNS), detergent 163, 168,357
trichloroacetate (NaTCA), in S1
hybridisation solution 367-368(recipe)vapour lamp, for avoiding
photoreactivation in UV
mutagenesis 102
Soft nutrient agar (SNA) 411 (recipe)
melting in microwave oven 240
Sonication
for cell-free extracts 76-77, 379
for growth dispersal 52
for shearing DNA 215
in RNA isolation 355, 358



Sorvall rotors
equivalent centrifugation conditions
183 (figure)maximal speeds for CsCl solutions
181 (table)

Springs, stainless steel 49, 50, 52, 419(supplier)Soyabroth see Tryptic soy broth, Difco;
Tryptone soya broth, Oxoid(TSB)
flour see Mannitol soya flour (MS)SRP1 plasmid, of S. rimosus 127

spcN (spectinomycin
phosphotransferase) 445, 449,
451, 486 (map)spd (plasmid spread genes) 153, 254,
255, 264
Spectinomycin 15, 454
for plasmid amplification 172
resistance 241, 446, 448, 449, 451,
see also aadA; spcN
to select transformants 241, 470
Spermidine 174, 246
Spermine, for DNA precipitation 166,
174, 186
sph (streptomycin phosphotransferase)
445, 449, 451, 487 (map)Spin X microcentrifuge filter unit 76,
419 (supplier)srmB (spiramycin resistance) 445, 449,
451

ss DNA see Single-stranded DNA
SSC buffer 417 (recipe)
SspI
in PFGE 111, 199
recognition site 441
Staining
for glycogen in electron microscopy
72-73
for light microscopy 64
DAPI staining, for nucleoids 65
immunofluorescent staining 6670
tetrazolium staining, forrespiratory activity 66
Star activity, of restriction enzymes
174, 218
Starch, as dispersant in liquid medium
51
Start codons 386
Sterilin tubes 57 (supplier)
STET buffer 190 (recipe)

Spinosyns 15
Spiramycin 15, 454
biosynthesis 24
resistance 445, 451
Sporangiomycin, resistance 451
Spore germinationfor electroporation 237
in culture 50-54
in soil 5-6
Spore suspensions, preparation 44, 55
Sporescell-free extracts from 76-77
development 19
electron microscopy 70
in soil 5
mutagenesis 101
pigment 27, see also whiE
preparation of suspensions 44-47,
54-55
selective isolation 43-44
Sporulation agar 54
Spot-transformation, of protoplasts
236
Spreaders for plating out
protoplasts 158
spores 47, 133sti (strong incompatibility) of pIJ101
213, 254 (map), 533
Sti+ vectors 542
use in plasmid curing 113
Storage see also Lyophilisationof DNA 167, 274, 276
of ligation mixtures 226
of mycelium 163, 172of phages 218, 226of protoplasts 58
of RNA 360
of spores 47of transfection mixtures 302
of whi and bld strains 44-45
RNA polymerase storage buffer 378
strA see rpsL
Strain improvement 23
Streptoalloteichus hindustanus see
bleSh
Streptogramins 15, 454
resistance see MLS
Streptomyces, definition of genus 2, 4,
5
individual species see S.
Streptomycin 15, 454
biosynthesis 27, 431



6WUHSWRP\FLQWR7KLRVWUHSWRQ

producing strains 27, 32, 430
production in soil 10-11
resistance 449, 451, see also aadA;
aphD; aphE; rpsL; sph
phenotypic lag 101
recessive 136
rpsL (strA) mutations
antibiotic production 220
rpsL as counterselection marker
323
to select transformants 241, 448,
470
Streptosporangium
Stm. fragile, recovery from soil byimmunomagnetic capture 44
taxonomy 3, 4, 5
Streptothricin 15, 454
resistance 451
Stringent response 24, 25, see also
ppGpp
Strong incompatibility (Sti) of pIJ101
see sti
Subtilisin, in PFGE 199, 201
Sucrose
10.3% solution
for resuspending protoplasts 57
for washing mycelium of fragile
strains 163
34% solution, for plasmid isolationby alkaline lysis 177
as dispersant in liquid medium 51
DNA precipitation from sucrose
solutions 174
gradients, in DNA isolation 175
in CRM liquid medium forelectroporationin J medium, for seed culture 90(recipe)in labelling medium 414
in P buffer 415 (recipe)in R2 medium 408 (recipe)in R5 medium 409 (recipe)in T and L buffer 416 (recipe)in YEME medium, for protoplastpreparation 56, 412 (recipe)
loading dye, for RNA gels 362(recipe)not catabolised by S. coelicolor andS. lividans 50
resistance see sacB

Superinfection, of lysogens 213
Supplemented liquid minimal medium(SMM) 50, 52, 413 (recipe)
Supplemented minimal medium, solid(SMMS) 410 (recipe)
SV1 phage, of S venezuelae,transduction by 128, 275, 282
Sweet potato pox, caused by S.
ipomoea 6T
T (transformation) buffer 416 (recipe)
T4 terminator 468, 469, 483, 485
Tacrolimus see FK506
TAE (Tris-acetate-EDTA) buffer
gel picture 196
recycling 196
Taq polymerase
in DNA sequencing 394
in PCR protocol 402
inhibition by lysozyme 222
Taxonomy, of actinomycetes 2-5
TBE (Tris-borate-EDTA) buffer forelectrophoresis
0.5 ×, for PFGE 203, 204
1 ×, autoclaved, for RNA work 361
containing thiourea 197
effect on migration of CCC DNA
196
TCA (trichloroacetic acid), S1
hybridisation buffer 367-368(recipe)TE buffer 417 (recipe)
TE25S/TE25-sucrose buffer
for PFGE 199 (recipe)in DNA isolation 168, 170

,QGH[

Terminators, of transcription 212, 213,
502, see also fd phageterminator; mmr
of aphI 478
utility in vectors 225, 292, 298, 334,
347, 389-390
TES (tris[hydroxymethyl]methyl-2aminoethanesulphonic acid)bufferfor germinating spores 53
in L and P buffers 415 (recipe), 416(recipe)in R2/R5 agar 408 (recipe), 409(recipe)in Supplemented minimal medium(SMM and SMMS) 410(recipe), 413 (recipe)
TESLR buffer, for plasmid isolation
187 (recipe)tet (tetracycline resistance) 445, 449,
451
on vectors 517
Tetracenomycin, biosynthesis 26
Tetracycline 15, 454
biosynthesis 25
resistance 449, 451, 490, see also
otrA; otrB; tet; tetSl
unstable in S. lividans 427, 490
Tetrazolium staining, for respiratory
activity 66
Tetronactin 454
resistance 445, 446, 450
Tetronasin 454
resistance see tnrB2,3
tetSl (tetracycline resistance of S.
lividans) 446, 449, 451, 491(map)
TG1 phage 300

TEGR buffer, for plasmid isolation
191, 192 (recipe)

TGED buffer, for RNA polymerase
purification 378 (recipe)

Teichoplanin 15

Thermoactinomyces vulgaris,transformation 128

Telomeres, of Streptomyces
chromosomes 201, 204, 422
Temperate phages 22, 272, 274, seealso FP43; C31; HAU3;
SAt-1; R4; SF1; SV1; TG1

Suicide plasmids, for genedisruption/replacement 218,
220, 267 (list), 317

Temperature-inducible promoter, of
pSN22 388, 389, 504 (map)SuperCos-1 216, 218, 236, 241, 569(maps)

Tendamistat 30
signal sequence 390-391

Superdex 200, for RNA polymerase
purification 377
running buffer 378 (recipe)ter(fd) (major terminator of phage fd)
296, 298, 445, 502
on vectors 292, 293, 513, 514

Thermus flavus see mdh
Theta replication, of plasmids 20, 255
Thienamycin 15
Thin sections, for electron microscopy
70-73
Thiocarbohydrazide (TCH), forglycogen staining 72
Thiopeptin 454 see also Thiostrepton
resistance 451
Thiostrepton 15, 454

,QGH[

7KLRVWUHSWRQWR7UDQVSRVRQV

inducible promoter see tipAp
resistance 25, 241, 451 see alsonsh; tsr
reduced level 267
to select transformants 240, 241,456
vectors without 268 (list)
Thiourea, in electrophoresis buffer
197, 199, 204
Tiger Milk 240, 410 (recipe)
tipA (thiostrepton-inducible protein)
386, 446
tipAp (thiostrepton-inducible promoter)
119-120, 388, 504
on vectors 514-517
TIR (terminal inverted repeat) see
Inverted repeats
TK prefix see S. lividans 66, strains
tlrA (tylosin resistance) 446, 447(figure), 451
tlrB, tlrC, tlrD (tylosin resistance) 446,
447, 449, 451
TM see Tris-maleic acid buffer
Tn3 111
Tn5 481 (map), 519
mutagenesis of S. lividans 121-122,
see also Tn5493
transposition into cloned
Streptomyces DNA 110
Tn10, transposition into cloned
Streptomyces DNA 110
Tn502 (Tn5 derivative containing
luxAB) 346
Tn522 (Tn5 derivative containing
EGFP) 348
Tn610, of Mycobacterium, source ofIS6100 119
Tn1696 see aacC1
Tn1761/Tn1762, containing EGFP
348
Tn1771/Tn1772, containing luxAB 346
Tn1792 from Mycobacterium,mutagenesis in S. lividans 119121

111-117
in TK503 425transposition in Mycobacterium 215
Tn4593, possible integrating vector
218
Tn4811, of S. lividans and SLP2 427,
428, 435 (map)Tn5096 (IS493 derivative) 576-577(maps)
delivery by FP43 phage 300
mutagenesis of S. griseofuscus 117118
Tn5099 (IS493 derivative containingxylE) 342, 576 (map)Tn5099-10 (IS493 hypertransposing
derivative) 117, 576-577(maps)
Tn5099-7 (IS493 derivative) 576(map)
Tn5100-4 (IS493 hypertransposing
derivative) 117, 576-577(maps)
Tn5351/Tn5353 (Tn4556 derivatives
containing luxAB) 580 (maps)
Tn5493 (Tn5 derivative) 581 (map)mutagenesis of S. lividans 121-122
tnrB2,3 (tetronasin resistance) 446,
449, 450, 489 (maps)
Tobramycin 15, 454
resistance 450, 467
cross-resistance 448 (table)
Toluidine blue stain, for Streptomyces
hyphae 64
Toothpicks, nucleases on 163
TPNS (tri-isopropylnaphthalenesulphonate), detergent in Kirby
mix 163, 168 (recipe), 355(supplier), 357
tra, transfer genes of Streptomyces
plasmids 153, see also
Conjugal transfer of plasmids;
kil; kor; oriT; RK2
of pIJ101 254 (map), 256
of pSAM2 265 (map)of pSN22 504 (map)of SCP2* 259 (map), 261 (maps)

Tn4556 of S. fradiae 519, 578 (map)see also Tn4560
as integrating vector 265
derivatives, for mutagenesis in
Streptomyces 110, 111-117

Trace mineral/element solutionsfor MG labelling medium 91
for R2, R2YE, R5, L, P and T
buffer 408,
for SMM and SMMS 413
for NMMP 413

Tn4560 578 (map), see also pUC1169
for mutagenesis in Streptomyces

Tramlines, as indicators of Ltz 137,

153, 155
Trans-sulphurase pathway 9
Transcription see also Coupledtranscription-translation;
Northern blotting; Primer
extension mapping; Reporter
systems; S1 mappingin C31 296, 521 (map)in vitro 374-377
initiation 385-386
methods of assay 339
termination 212
Transduction 274-275, see also FP43;
SV1
in S. coelicolor 128, 322
in S. fradiae 26
in S. griseofuscus 27
in S. hygroscopicus 27
in S. toyocaensis 30
in S. venezuelae 31, 128, 282
in Streptomyces 128
of ermE 458
of plasmids 299
selection for 281
Transfection, of Streptomyces
protoplasts 231, 232, 301-302
Transfer genes, of plasmids see
Conjugal transfer of plasmids;
traTransformation 221
natural occurrence in actinomycetes
128of Streptomyces protoplasts 26, 28,
30, 31, 32, 221, 231, 316-318
by cosmids 216, 218, 236-237
by denatured DNA 128, 232,
235-236, 308
by single-stranded DNA 230-231,317
conditions for 156, 176
protocols 232-237
Translation see also bldA; Leaderlesstranscripts
and codon usage 212, 391, 397
fusions 342
initiation 386
Transposons 518-519 see Tn prefix
for individual transposons
hypertransposing 118, 576 (maps)
in Streptomyces 21, 110
inducible 119
integration via 216, 218, 265
maps 573-580
mutagenesis 21, 24, 28, 109, 122
non-Streptomyces 110
vectors for delivery 268 (list), 551552
vectors for reporter genes 341, 342,
346, 348



WUD5 UHJXODWRURIWUDQIHUJHQHV WR:*$traR (regulator of transfer genes) see
also kilof pSG5 257 (map), 259
of pSN22 446, 449, 504 (map)Trehalose, in spores 18
Tri-isopropylnaphthalene sulphonate
see TPNS
Triparental mating
for oriT mobilisation 250
predicting rounds of recombination
125
Tris acetate EDTA buffer see TAE
bufferTu), ribosome binding site 386
Tween 80, as wetting agent 44
Two-dimensional (2D) gel
electrophoresisof plasmid DNA 196-197
of proteins 83-90
Tylosin 15, 454
biosynthesis 26
resistance 446, 451, see also tlrA-D
Tyrosinase see also mel; melC1, C2
expression of 410, 500
extracellular 343

Tris-borate-EDTA buffer see TBE
bufferTyrosinebiosynthesis 9
in Tiger Milk 410

Tris-HCl buffers 208 (recipe)U

Tris[hydroxymethyl]methyl-2-aminoethanesulphonic acid see TES
bufferUltra-Turrax Turbomixer 54-55, 420(supplier)Tris-maleic acid buffer
for NTG mutagenesis 103 (recipe)in T (transformation) buffer 416(recipe)
Triton-X100
as wetting agent for spores 44
in DNA isolation 175
tRNA see also bldA; TTA codons
as carrier in transcript mapping 368370, 375
genes, as att sites for integratingplasmids and phages 20, 214,
265, 319, 390, 532
in colony hybridisation 244
in Maxam and Gilbert sequencing
394
inhibitor of EndA 174
on agarose gels 361 (picture), 362
on CsCl gradients 173, 176rare arginine tRNA in E. coli 391
Tryptic soy broth, Difco 58, 237
Tryptone soya broth, Oxoid (TSB)
412, (recipe)
Tryptophan, biosynthesis 9
TSB see Tryptone soya broth, Oxoid
412 (recipe)tsr (thiostrepton resistance) 446, 447(figure), 451, 457 (map)
TTA codons see also bldA
frequency 387, 400-401
in DNA fragments 440
in reporter genes 340, 346, 349
Tuberactinomycin 484
tuf1 (translation initiation factor EF-

Ultramicrotome, for thin sections 71
Unbufferedphenol, acid 172, 187 (recipe)
potassium acetate 186, 207, 286
sodium acetate 169, 191, 193, 207
Undecylprodigiosin (Red) see also
Butylcycloheptylprodiginine;
red cluster; redDbinding to DNA 162
production 93, 95, 430
in pocks 153
structure 94

,QGH[

Vacuum filtration, to harvest mycelium
162
Vacuum tester, for lyophils 59-60
Validamycin 15
Valine dehydrogenase, in ammonium
repression 9
Valinomycin, biosynthesis 28
Vancomycin 15, 31
Vectors
choice of 215-220
for intracellular expression 289-290
for secretion 390-391
for transposon delivery 110-111
list of special purpose vectors 267269
phage vectors
C31-derived 290-299, 318, 292293 (tables), 513-514 (table),523-529 (maps)
other phage systems 299-300
plasmid vectors 514-519 (tables),530-580 (maps)
for reporter systems 341-342
properties of parent replicons
253-267
preparation of DNA 214
phage DNA 282-287
plasmid DNA 171-195
Velvet, for replica plating 135, 420(supplier)Vibrio harveyi see luxAB

Universals (bottles) 419 (supplier)

Viocin 454

Uranyl acetate, for electron microscopy
71-72

Viomycin 454
resistance 241, 451, see also vph
cross-resistance 454
to select transformants 115, 241,
482

Urdamycin, biosynthesis 26
UUA codons see TTA codons
UV (ultraviolet light) see also NUVfluorescence of Hoechst 33258 ATspecific DNA stain 175
harvesting CsCl EB gradients 181
in generalised transduction 282
in protoplast fusion 157
induced mutations 101, 102, 103
photoreactivation 102, 429
lack of Streptomyces phage induction
by 172
nicking of CCC DNA 254
sensitive mutants 106
to detect MUG ( -galactosidase)
fluorescence 350
V

Virginiamycin 15
VP5, VP7 phages, divalent cations foradsorption 278
vph (viomycin phosphotransferase)
446, 449, 451, 474, 483 (map)in transposon mutagenesis 111-116
on vectors 268, 292-293, 513, 516,
519
VWB phage 390, 530, 532 (map)W
Water
DEPC treatment 354
Millipore Super Q 78
twice autoclaved for RNA work 354

Vacuum drying see also Lyophilisation
WGA (wheat germ agglutinin), for cellof DNA 173, 369
wall staining 66-69
ammonium acetate evaporates 165

,QGH[Whatman papers, for colony
hybridisation 243-247, 247248

X

whi (white aerial mycelium)
growth on cover slips, formicroscopy 64
isolation 106
roles in sporulation 17, 18, 341, 426
storage 44

xis (excisionase) of integratingplasmids 20, 264, 265

whiE (spore pigment)
as reporter 343
promoter 341
whiG (sigma factor) 217
effect on ectopic sporulation 56
homogenotisation 330-331
promoter 341

X-gal 340, 349

xylE (catechol 2,3-dioxygenase) 446,
449, 495 (map)assay of product 345-346
expression in colonies 344
expression in plaques 344-345
fusions 296-299, 341, 343-346
on vectors 268 (list), 291-292, 293,296-299, 513-515, 518

medium 56, 412 (recipe)
Ylo phenotype see catechol 2,3dioxygenase; xylE
YT medium 52, 249, 414 (recipe)
Z
Zeocin 454, 480, see also Bleomycin;
Phleomycin
Zincregulation of esterase 30
staining of proteins 90

Xylose utilisation, by S. rubiginosus 7

Zirconium-silica beads, for growth ofmycelium 92

Y

ZX prefix see S. lividans 66, strains

YEME (yeast extract malt extract)
Wizard columns 194, 248

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