Transducing phages of Actinomycetales

The present invention is directed to isolated transducing phages, methods of isolating transducing phages, and methods of using transducing phages including, for instance, transferring at least one nucleic acid fragment from a donor microbe to a recipient microbe, and producing a secondary metabolite from a microbe. The transducing phages typically have a broad host range, and transduce microbes in the Order Actinomycetales, in particular in the Family Streptomycetaceae, including Streptomyces coelicolor, Streptomyces lividans, Streptomyces venezuelae, Streptomyces avermitilis, and Saccharopolyspora erythraea. The transducing phages can be specialized transducing phages or generalized transducing phages.

BACKGROUND OF THE INVENTION
 It would be difficult to overestimate the contribution generalized
 transduction has made to the study of prokaryote biology since the
 discovery of phage P22 in Salmonella in the early 1950s. The use of
 generalized transducing phages for strain construction, fine structure
 mapping, and genetic manipulation have played major roles in the genetic
 analysis of Salmonella and E. coli. One of the most important applications
 of generalized transduction has been to facilitate the cloning of genes
 identified by transposon generated mutations. The use of generalized
 transduction in combination with transposon mutagenesis to clone genes
 involved in morphogenesis has been invaluable in the study of sporulation
 in Bacillus subtilis.
 Streptomyces are Gram-positive soil bacteria of special interest for two
 reasons. First, their mycelial growth mode and sporulation cycle are among
 the most dramatic examples of prokaryotic morphological differentiation.
 They grow vegetatively as multicellular, multinucleoid, branching hyphae
 that penetrate and solubilize organic material in the soil forming a
 mycelial mass. In response to environmental signals (a process that
 requires cell--cell communication mediated by diffusible substances), they
 initiate a cycle of differentiation that begins with the production of
 aerial hyphae that septate into uninucloid compartments that give rise to
 spores. Second, during the initiation of morphological development they
 produce a large number of secondary metabolites, including most of the
 natural product antibiotics used in human and animal health care. Because
 of its unique biology, Streptomyces offers special advantages for the
 study of how morphogenesis is initiated. The question of how cells within
 multicellular organisms sense changes in their environment and communicate
 that information to each other is of fundamental importance to the study
 of developmental biology. In spite of their interesting biology and
 commercial importance, relatively little is known about the gene
 expression pathways that regulate morphological development or antibiotic
 biosynthesis.
 A major limitation in the study of Streptomyces is that the typical genetic
 approaches for recovering genes identified by chemically induced mutations
 have been difficult to implement in Streptomyces. Because relatively few
 genetic markers exist in Streptomyces, fine structure mapping is not
 possible. Cloning by complementation is slow and tedious. Transformation
 of plasmid libraries constructed in either E. coli or Streptomyces is
 extremely inefficient and the libraries are often incomplete.
 Transposition systems have been developed in Streptomyces but they have
 not proved to be effective for insertional mutagenesis. This is in part
 due to the use of temperature sensitive plasmid vectors as transposon
 delivery systems. Plasmid curing is not effective and exposure to high
 temperatures is mutagenic in itself. This has resulted in a high
 background of mutations not caused by transposition. Thus, it has not been
 possible to determine whether a mutant phenotype was caused by transposon
 insertion into a gene of interest until the candidate gene was cloned,
 thereby permitting complementation analysis and directed disruption
 studies. This is not only time consuming and laborious, it is often a
 futile exercise because of the high background of extraneous mutations.
 It has long been recognized that an efficient system for generalized
 transduction is needed to make transposon mutagenesis an effective genetic
 tool in Streptomyces. However, generalized transducing phages have not
 been characterized in species that can serve as genetic model systems.
 Attempts by many workers over the years to isolate generalized transducing
 phages for Streptomyces coelicolor have been uniformly unsuccessful, as
 have been attempts to transduce markers by the most extensively studied
 lytic actinomycete phages fC31, VP5, and R4. Generalized transduction has
 been demonstrated in Streptomyces venezuelae. This involved transduction
 of several markers including genes for cholemphenicol production. This was
 thought, however, to be an anomaly and somehow specific to Streptomyces
 venezuelae since the approaches used to identify transducing phages for
 Streptomyces venezuelae did not work for Streptomyces coelicolor.
 Subsequent to the publication of much of the work describing these
 intraspecific generalized transducing phages of Streptomyces venezuelae
 and Streptomyces olivaceus, a report was authored by one of the
 investigators that had taken part in many of the studies. In this report
 titled "Generalized Transduction in Streptomyces Species," (Stuttard, In:
 Genetics and Molecular Biology of Industrial Microorganisms, Hershberger,
 et al., (eds.), pp. 157-162, ASM, Washington, D.C. (1989)) he reported "a
 possibly significant lack of success with Streptomyces coelicolor and
 Streptomyces lividans." The author hypothesized "that some essential host
 function(s), possibly expressed in few potential host strains, may be
 required for lytic growth of" generalized transducing particles. If such
 host functions are required, then generalized transducing phages will not
 be isolated that transduce those strains lacking the essential host
 functions. The author concludes that "generalized transducing phages for
 Streptomyces coelicolor and Streptomyces lividans remain as elusive as
 ever."
 In the recent past there has been a significant increase in the
 identification of antibiotic resistant microbes. However, the
 identification of new antibiotics has not kept pace with the occurrence of
 antibiotic resistant microbes. Accordingly, there has been a significant
 increase in human and animal morbidity and mortality due to infectious
 diseases. Thus, there is a need for new antibiotics. As mentioned above,
 Streptomyces, and other microbes, produce secondary metabolites. Many of
 these secondary metabolites are natural product antibiotics used in human
 and animal health care. It has recently become possible to use recombinant
 genetic techniques to modify the metabolic pathways of microbes to result
 in the synthesis of new natural product antibiotics, often referred to as
 new natural products or non-natural products, having new activities. A
 limitation to this is, for instance, the need for appropriate vectors to
 carry large DNA fragments, and the ability to efficiently move DNA into
 appropriate hosts (see, for instance, Cane, D. E. et al., (1998) Science,
 282, 63-68). Thus, there is a need and significant advantage to developing
 genetic techniques of microbes that synthesize natural product
 antibiotics.
 SUMMARY OF THE INVENTION
 The present invention is directed to a method of isolating a transducing
 phage, preferably, a generalized transducing phage. The method includes
 combining a sample containing a transducing phage with a microbe forming a
 first phage-microbe mixture, and incubating the first phage-microbe
 mixture at a temperature of less than 28.degree. C. to form a first plaque
 comprising a generalized transducing phage. The invention includes a phage
 isolated using this method.
 Another aspect of the invention is a method of isolating a transducing
 phage, preferably, a generalized transducing phage, involving phage DNA.
 The method includes combining a sample containing generalized transducing
 phage DNA with a microbe forming a phage DNA-microbe mixture and
 incubating the phage DNA-microbe mixture at a temperature of less than
 28.degree. C. to form a first plaque comprising a transducing phage.
 Another method of the invention is a method of transferring at least one
 nucleic acid fragment from a donor microbe to a recipient microbe. The
 method includes providing an isolated transducing particle comprising a
 nucleic acid fragment from a donor microbe, combining the transducing
 particle with a recipient microbe to result in a transducing
 particle-recipient microbe mixture, and incubating the transducing
 particle-recipient microbe mixture at a temperature of less than
 28.degree. C. to form a transduced recipient microbe comprising a nucleic
 acid fragment from the donor microbe. This method can also be used to
 produce a secondary metabolite from a microbe. When a secondary metabolite
 is to be produced, the method further includes providing conditions
 effective for the recipient microbe to produce a secondary metabolite. The
 invention also includes a microbe prepared by this method, and a secondary
 metabolite produced by this method.
 The invention is also directed at an isolated generalized transducing phage
 that can transfer at least one nucleic acid fragment from a donor microbe
 to a recipient microbe, wherein the frequency of transduction is at least
 about 10.sup.-7, and wherein the transduction of the recipient microbe
 occurs at less than 28.degree. C.
 A "phage" is able to inject a nucleic acid fragment into a host microbe. A
 type of phage is a "transducing phage." When a transducing phage infects a
 host microbe and replicates, two types of particles can result. One type
 of particle produced during the replication process is a "phage particle."
 As used herein, a phage particle contains a phage nucleic acid fragment
 and can infect another microbe and replicate, and can therefore be used as
 a transducing phage. The second type of particle is a "transducing
 particle." As used herein, a transducing particle contains at least one
 nucleic acid fragment derived from the host microbe. This distinction is
 important with respect to the discussion of superinfection killing herein.
 Thus, as used herein, the term phage is used generically to encompass
 phage that contain a phage nucleic acid fragment (i.e., a phage particle)
 or at least one nucleic acid fragment derived from a host microbe (i.e., a
 transducing particle).
 Transducing particles retain the ability to inject a nucleic acid fragment
 into a microbe. A microbe that is the recipient of a host microbe nucleic
 acid fragment from a transducing particle is said to be "transduced," and
 is referred to herein as a "transductant."

DETAILED DESCRIPTION OF THE INVENTION
 The present invention is directed to isolated transducing phages and
 methods of isolating transducing phages. The present invention is also
 directed to methods of using phages including, for instance, transferring
 at least one nucleic acid fragment from a donor microbe to a recipient
 microbe, and optionally producing a secondary metabolite from a microbe.
 The transducing phages can be specialized transducing phages or
 generalized transducing phages. Preferably, they are generalized
 transducing phages.
 A phage can include a phage nucleic acid fragment (i.e., a nucleic acid
 fragment containing at least a portion of a phage genome) wrapped in a
 protein coat. In nature, phages are not capable of growth outside
 microbial cells. A phage adsorbs to a microbial cell via the proteins in
 the coat and injects the nucleic acid fragment into the microbial cell.
 The phage nucleic acid fragment is replicated, transcribed, and the
 transcipts are used to produce protein for the production of new phage
 particles, i.e., more phage. Transducing phages are phages capable of
 generating two types of particles. One type of particle, a transducing
 particle, contains a nucleic acid fragment other than a phage nucleic acid
 fragment, e.g., a nucleic acid fragment from a host microbe. The second
 type of particle, a phage particle, contains only a phage nucleic acid
 fragment (i.e., it does not include a nucleic acid fragment from the host
 microbe).
 When the transducing phage is a generalized transducing phage, the nucleic
 acid fragment present in a transducing particle can originate from
 different areas of the genomic DNA present in the donor, or can originate
 from a plasmid present in the donor. When the transducing phage is a
 specialized transducing phage, the nucleic acid fragment present in a
 transducing particle typically originates from one specific area of the
 genomic DNA present in the donor.
 A transducing particle can be produced naturally, i.e., it is the result of
 the infection and subsequent lysis of a microbe infected with a
 transducing phage. A transducing particle can also be produced using
 artificial methods, including, for instance, in vitro packaging of
 fragmented genomic DNA. As used herein, "isolated" phage, phage particle,
 or transducing particle refers to a phage separated from its natural
 environment. Preferably, an "isolated" phage, phage particle, or
 transducing particle is a phage, phage particle, or transducing particle
 that is separated from microbes and other phage, as opposed to essentially
 free from agar, cellular debris, and other impurities.
 The phage of the present invention, preferably a generalized transducing
 phage, can transfer at least one nucleic acid fragment from a donor
 microbe to a recipient microbe. A recipient microbe that has received at
 least one nucleic acid fragment from a transducing particle is referred to
 as transduced. A "nucleic acid fragment" as used herein refers to a
 polymeric form of nucleotides of any length, either ribonucleotides or
 deoxynucleotides, and includes both double- and single-stranded DNA (both
 genomic and plasmid) and both double- and single-stranded RNA. A
 polynucleotide fragment may include both coding and non-coding regions
 that can be obtained directly from a natural source (e.g., a microbe), or
 can be prepared with the aid of recombinant or synthetic techniques.
 Significantly and preferably, the phage of the present invention transduce
 at a temperature of less that 28.degree. C. More preferably, transduction
 occurs at, in increasing order of preference, less than about 28.degree.
 C., less than about 25.degree. C., less than about 23.degree. C., and less
 than about 21.degree. C. It is expected that the lower limit of the
 temperature at which transduction occurs is about 16.degree. C. The low
 temperature is advantageous because it allows a significant increase in
 the frequency of transduction. The ability of the phage to cause
 transduction, preferably generalized transduction, at increased
 frequencies at a temperature of less that 28.degree. C. was unexpected.
 Without intending to be limiting, it is believed that the decreased
 temperature of transduction causes decreased superinfection, particularly
 superinfection killing, of a transduced recipient. Other methods to
 decrease superinfection, particularly superinfection killing, are
 described herein.
 Typically, the phage of the present invention transduce a recipient microbe
 at a frequency of transduction of at least about 10.sup.-7 (i.e., one
 transduced recipient per 10.sup.7 phage). "Frequency of transduction"
 refers to the number of transduced recipients (i.e., transductants) per
 phage particle after exposing a recipient strain to phage. Preferably,
 transduction occurs at, in increasing order of preference, at least about
 10.sup.-6, at least about 10.sup.-5, and at least about 10.sup.-4. It is
 estimated that as high as about 10.sup.-3 can be achieved.
 Preferably, the donor and recipient microbes are members of different
 families, more preferably, members of different genera, even more
 preferably, members of different species, and most preferably, members of
 the same species. This is referred to in the art as having a broad host
 range.
 Preferably, the Families are of the Order Actinomycetales. Preferably, the
 Families include Mycobacteriaceae, Actinomycetaceae, Streptomycetaceae,
 and Actinoplanaceae, more preferably, Streptomycetaceae. Preferably, the
 microbe is a spore, a mycelial fragment, a germling, a protoplast, or
 mixtures thereof. Many Actinomycetales naturally grow as a filament of
 cells. A mycelial fragment is a portion of this filament. A germling is a
 spore that is beginning germination as determined by the appearance of
 germ tubes on a spore. Preferably, members of the family Streptomycetaceae
 that can be transduced by the phage are Streptomyces and
 Saccharopolyspora. Examples of members the genus Streptomyces include
 Streptomyces coelicolor, Streptomyces lividans, Streptomyces venezuelae,
 and Streptomyces avermitilis. An example of members of the genus
 Saccharopolyspora includes Saccharopolyspora erythraea.
 The phage of the present invention, preferably a generalized transducing
 phage, can be isolated by combining a sample containing a transducing
 phage with a microbe forming a first phage-microbe mixture and incubating
 the first phage-microbe mixture to form a first plaque comprising a
 transducing phage. Preferably, the incubation temperature is less than
 28.degree. C. Typically, the plaques formed by the phage of the present
 invention are clear or turbid. A plaque refers to an area, typically but
 not necessarily in a solid or semi-solid bacteriological medium,
 containing phage and lysed microbes. Typically, a plaque will also contain
 unlysed microbes that may or may not be infected with a phage nucleic acid
 fragment. The lysed microbes have been lysed by infection of a phage
 nucleic acid fragment, subsequent replication of the phage within the
 microbe, and then release of the replicated phage into the surrounding
 area by lysis of the microbe. Typically, a plaque containing predominantly
 lysed microbes appears to be clear (i.e., no microbes are visible), while
 a plaque containing unlysed microbes appears as turbid (i.e., microbes are
 visible). The invention is further directed to a phage prepared by this
 method. Preferably, phage prepared by this method is an isolated phage.
 The microbe that is used to isolate a phage of the present invention can be
 chosen from different families as described herein. Preferably, the
 microbe is an Actinomycetales.
 Typically, a source of divalent cations is present during a portion of the
 incubation of the isolation process. Preferable divalent cations include
 transition metals and main group metals, and more preferably, calcium and
 magnesium. The sample containing a transducing phage, preferably a
 generalized transducing phage, can be obtained from the lithosphere and
 hydrosphere, including, for instance, soil, water, organic material,
 decomposing organic material, or volcanic ash. Preferably, a transducing
 phage is obtained from a composition that includes soil or volcanic ash,
 more preferably, soil.
 Optionally, the isolation method includes separating the phage from the
 lithosphere or hydrosphere prior to combining the sample containing a
 generalized transducing phage with a microbe. For instance, separating the
 phage from the lithosphere can include combining a sample containing the
 phage, preferably a soil sample, with a diluent, preferably including
 water, to form a slurry and removing particles that are heavier than the
 phage. Particles heavier than the phage can be removed by, for instance,
 centrifuging the slurry. The sample can be further manipulated to remove
 microbes. Preferably, microbes are removed by filtration. Alternatively,
 microbes can be removed by adding an organic solvent, preferably
 chloroform, to the sample containing the phage.
 The isolation method can include (and typically does include) separating
 the phage from the plaque to form isolated phage. For instance, the phage
 can be separated from the plaque by combining the first plaque with a
 microbe to form a second phage-microbe mixture and incubating the second
 phage-microbe mixture, preferably at a temperature of less than 28.degree.
 C., to form a second plaque containing a transducing phage. This step can
 be repeated as many time as is necessary, preferably about three times, to
 form an isolated phage. This process is typically referred to as plaque
 purification.
 An alternative method of isolating a transducing phage includes isolating
 phage DNA from a sample containing a transducing phage. This DNA can be
 combined with a microbe to form a phage DNA-microbe mixture and incubating
 the phage DNA-microbe mixture at a temperature of less tan 28.degree. C.
 to form a plaque comprising a transducing phage. Preferably, the microbe
 is an Actinomycetales, more preferably a Streptomycetaceae, most
 preferably a Streptomyces. Preferably, the microbe is a protoplast for
 this method of isolation.
 The present invention is also directed to a method of transferring at least
 one nucleic acid fragment from a donor microbe to a recipient microbe.
 Preferably, the donor and recipient are Actinomycetales, more preferably a
 Streptomycetaceae, most preferably a Streptomyces. A method of such a
 transfer includes providing an isolated transducing particle comprising a
 nucleic acid fragment from a donor microbe. The transducing particle can
 be combined with a recipient microbe to result in a transducing
 particle-recipient microbe mixture, and the transducing particle-recipient
 microbe mixture incubated, preferably at a temperature of less than
 28.degree. C., to form a transduced recipient microbe, where the
 transduced recipient microbe contains a nucleic acid fragment from the
 donor microbe. The invention is further directed to a microbe prepared by
 this method.
 The method of transferring at least one nucleic acid fragment can further
 include reducing superinfection, preferably superinfection killing, of the
 transduced recipient microbe. Superinfection of a transduced recipient
 refers to a recipient containing a nucleic acid fragment from a phage
 particle and a nucleic acid fragment from a transducing particle. The
 presence of phage DNA from the phage particle will typically result in
 lysis of the recipient. This is referred to as superinfection killing. It
 is advantageous to reduce superinfection, preferably superinfection
 killing, of a transduced recipient to increase the frequency of
 transduction.
 Superinfection can be reduced by treating the transducing particle (which
 is typically in a suspension containing phage particles) prior to
 combining it with the recipient microbe. Preferably, the transducing
 particle-phage particle mixture is treated by exposing it to ultraviolet
 radiation. Without intending to be limiting, it is believed that the
 ultraviolet radiation inactivates the particles present. Since there is
 typically many more phage particles relative to transducing particles,
 more phage particles are inactivated. In general, appropriate conditions
 for using ultraviolet radiation include the time of exposure, the distance
 of the particles from the ultraviolet source, and the media the particles
 are in. Such conditions vary but can be easily determined by a person of
 skill in the art. Preferably, the wavelength is about 250 nm to about 270
 nm, and more preferably about 250 nm to about 260 nm. Preferably, the
 intensity is about 1.9 mW/cm.sup.2 /s to about 2 mW/cm.sup.2 /s, and more
 preferably it is 2 mW/cm.sup.2 /s.
 Superinfection can also be reduced by treating the transduced recipient
 microbe with a chelator. Chelators useful in the present invention include
 citrate and ethylene glycerol-bis(.beta.-aminoethyl ether
 N,N,N',N',-tetraacetic acid (EGTA)). Preferably, the chelator is a source
 of citrate, such as sodium citrate. Chelators are preferably used at a
 concentration that inhibits the ability of a particle to adsorb to a
 microbe, but does not significantly negatively affect the viability of the
 microbe. This concentration typically varies depending on the chelator
 used, but can be easily determined by a person of skill in the art.
 Typical concentrations of citrate are from about 1 mM to about 50 mM,
 preferably about 10 mM. Superinfection can also be reduced by combining
 low temperature and a chelating agent, or low temperature and ultraviolet
 radiation, or all three.
 An isolated transducing particle that includes a nucleic acid fragment from
 a donor microbe can be obtained by several methods. For instance, an
 isolated phage, preferably a transducing phage, can be combined with a
 donor microbe to form a phage-donor microbe mixture. This phage-donor
 microbe mixture can be incubated, preferably at less than 28.degree. C.,
 to form transducing particles. Alternatively, a transducing particle can
 be produced using artificial methods, for instance, in vitro packaging.
 Preferably, the isolated transducing particle is provided in a suspension
 of phage comprising isolated transducing particles. In general, the higher
 the concentration of transducing particles that are combined with a
 recipient microbe, the higher the probability of forming a transduced
 recipient microbe that contains a nucleic acid fragment from the donor
 microbe. Preferably, the concentration of the transducing particles in the
 suspension of phage is, in increasing order of preference, at least about
 1 in 10.sup.8 (1 transducing particle in 10.sup.8 phage particles), at
 least about 1 in 10.sup.7, at least about 1 in 10.sup.6, at least about 1
 in 10.sup.5, at least about 1 in 10.sup.4, and at least about 1 in
 10.sup.3.
 A nucleic acid fragment from a donor microbe can contain a non-coding
 region, a coding region or a portion thereof, or a mixture thereof.
 Preferably, the nucleic acid fragment from a donor microbe includes at
 least one coding region. A "coding region" is a linear form of nucleotides
 that typically encodes a polypeptide, usually via mRNA, when placed under
 the control of appropriate regulatory sequences (e.g., a promoter). The
 boundaries of a coding region are generally determined by a translation
 start codon at its 5' end and a translation stop codon at its 3' end, or a
 transcriptional start site at the 5' end and a translational stop codon or
 a transcriptional stop site at the 3' end.
 A coding region may encode a polypeptide or a transcript (i.e., an RNA
 transcript) that is involved in the synthesis of a metabolite, or
 polypeptides that impart antibiotic resistance or catalyze the synthesis
 of an antibiotic (e.g., lincomycin, or rifampicin). A metabolite includes
 primary metabolites (i.e., the products or intermediates of a primary
 metabolic pathway), and secondary metabolites (i.e., products or
 intermediates of a secondary metabolic pathway). As used herein,
 "metabolic pathway" includes primary metabolic pathways and secondary
 metabolic pathways. A "polypeptide" as used herein refers to a polymer of
 amino acids and does not refer to a specific length of a polymer of amino
 acids. Thus, for example, the terms peptide, oligopeptide, protein,
 structural protein (e.g., one of several polypeptides in a multimeric
 complex) and enzyme are included within the definition of polypeptide. A
 polypeptide can be involved in, e.g., the catalysis of a product or
 intermediate, or the transport or anchoring of a product or intermediate.
 A polypeptide can also be involved in, e.g., holding a multimeric complex
 together, or post-synthesis steps of a product, e.g., transport of a
 product.
 Using the methods of the present invention, a recipient microbe can be
 transduced so that it expresses polypeptides not produced by the recipient
 microbe prior to transduction. Alternatively, a recipient can be
 transduced so that it expresses a polypeptide in different amounts
 (increased or decreased) than the microbe could prior to transduction.
 This method is expected to allow the construction of microbes that have
 altered metabolic pathways. This is sometimes referred to in the art as
 "metabolic engineering." For instance, the transduced recipient microbe
 can produce a metabolite, a secondary metabolite, or a polypeptide at a
 different level, either higher or lower, than is produced by the recipient
 microbe prior to transduction. A nonlimiting example of this is the
 transduction of a recipient to produce increased levels of acetyl-CoA. In
 this transduced recipient producing increased levels of acetyl-CoA it is
 further expected that the amounts of products of metabolic pathways that
 use acetyl-CoA will be increased.
 The present invention can be used to transfer at least one nucleic acid
 fragment containing a coding region that encodes a marker, including, for
 example, one that can complement a mutation present in a recipient or
 encodes an antibiotic. When a marker that complements a mutation is
 transferred to a recipient, preferably the transduction of the marker
 occurs at a frequency that is greater than the normal mutation rate (i.e.,
 reversion frequency) for a marker. For example, as shown in the Examples,
 the reversion frequency of the arg mutation in a recipient strain is &lt;1 in
 10.sup.+10, and the transduction frequency of a functional arg marker is
 greater than the reversion frequency.
 Another aspect of the present invention is directed to a method of
 producing a product or an intermediate of a metabolic pathway from a
 microbe. Preferably, the method produces a secondary metabolite.
 Preferably, the donor and recipient are Actinomycetales, more preferably a
 Streptomycetaceae, most preferably a Streptomyces.
 The method of producing a product or an intermediate of a metabolic
 pathway, preferably a secondary metabolite, from a microbe is similar to
 the method of transferring at least one nucleic acid fragment from a donor
 microbe to a recipient microbe, as described herein. When producing a
 secondary metabolite, the method can include providing conditions
 effective for the recipient microbe to produce the secondary metabolite.
 The secondary metabolite produced by the transduced recipient can be
 produced by the donor. The secondary metabolite produced by the transduced
 recipient microbe can be produced by the recipient microbe prior to
 transduction. Preferably, if the secondary metabolite is the same as one
 produced by the donor or recipient microbe prior to transduction, the
 transduced recipient produces a secondary metabolite at a higher level
 than is produced by the donor microbe or the recipient microbe prior to
 transduction.
 Alternatively and significantly, it is anticipated that the methods of the
 present invention will allow for the production of secondary metabolites
 that are not produced by the recipient microbe prior to transduction or by
 the donor microbe, i.e., new secondary metabolites. New secondary
 metabolites are often referred to in the art as new natural products, or
 non-natural products. The concept of the production of secondary
 metabolites that are not produced by the recipient microbe prior to
 transduction or by the donor microbe is typically referred to in the art
 as combinatorial biosynthesis.
 The present invention is illustrated by the following examples. It is to be
 understood that the particular examples, materials, amounts, and
 procedures are to be interpreted broadly in accordance with the scope and
 spirit of the invention as set forth herein.
 EXAMPLES
 These examples detail the isolation of the first generalized transducing
 phages for Streptomyces coelicolor, the most genetically well
 characterized strain of this important bacterial genus. Phages ranging in
 size from approximately 25 kb to more than 60 kb were shown to transduce a
 number of markers at frequencies from 10.sup.-5 to 10.sup.-8. Transduction
 is apparently general since markers were transduced from locations around
 the entire chromosome. Co-transduction of several markers predicts linkage
 that is in good agreement with data obtained from genetic mapping by
 conjugal mating. An important aspect of the invention was the
 establishment of conditions that severely reduce superinfection killing
 during selection of transductants. It is expected that generalized
 transduction will provide an important genetic tool for the study and
 manipulation of this organism.
 Streptomyces coelicolor phages DAH4, DAH5, and DAH6 (ATCC Accession Numbers
 203877, 203878, and 203879, respectively) and Streptomyces avermitilis
 phages JSN1, JSN2, and JSN3 (ATCC Accession Numbers 203874, 203875, and
 203876, respectively) were deposited with the American Type Culture
 Collection, 10801 University Blvd., Manassas, Va., 20110-2209, USA, on
 Mar. 25, 1999. The deposits were made under the Budapest Treaty on the
 International Recognition of the Deposit of Microorganisms for the
 Purposes of Patent Procedure.
 Example 1
 Experimental Procedures
 Bacterial strains and culture conditions. Bacterial strains used in this
 study are listed in Table 1. Spore stocks were made from strains grown on
 MYM (Brawner et al., (1985) Gene, 40, 191-201). To prepare spore stocks,
 bacteria were streaked for isolated colonies on MYM media and incubated
 and 30.degree. C. for 4 days. An isolated colony was picked and spread on
 MYM plates and incubated at 30.degree. C. for 4 days or until spores were
 visible. The spores were removed with a cotton swab and stored at
 -20.degree. C. Antibiotics used in the experiments described herein and
 the concentrations are listed in Table 2.
 TABLE 1
 Bacterial Strains and Culture Conditions
 SPECIES STRAIN GENOTYPE SOURCE
 Streptomyces A3(2) WT John Innes Centre
 coelicolor Norwich, UK
 Streptomyces J2402 M145, prototrophic K. Chater
 coelicolor SCP1.sup.- SCP2.sup.- John Innes Centre
 whiB::hyg Norwich, UK
 Streptomyces J1258 proA1 hisC9 argA1 K. Chater
 coelicolor cysD18 uraA1 strA1 John Innes Centre
 Norwich, UK
 Streptomyces J2408 M145, prototrophic K. Chater
 coelicolor SCP1.sup.- SCP2.sup.- John Innes Centre
 whiH::ermE Norwich, UK
 Streptomyces YU105 proA1 argA1 J. Nodwell
 coelicolor redE60 McMaster
 act::ermE whiE::hyg University
 Hamilton, Ontario
 Streptomyces BldK::.OMEGA. bldK::str/spc J. Nodwell
 coelicolor McMaster
 University
 Hamilton, Ontario
 Streptomyces J222 uraA1 rifA K. Chater
 coelicolor John Innes Centre
 Norwich, UK
 Streptomyces J2709 proA1 hisC9 K. Chater
 coelicolor argA1 uraA1 John Innes Centre
 Norwich, UK
 Streptomyces J1258 proA1 hisC9
 coelicolor arga1 cysD18
 uraA1 strA1 NF
 Streptomyces 1326 WT John Innes Centre
 lividans Norwich, UK
 Streptomyces TK64 proA1 John Innes Centre
 lividans Norwich, UK
 Streptomyces 10712 WT C. Stuttard
 venezuelae Dalhousie
 University
 Halifax, NS, Canada
 Streptomyces JW1100 pdx C. Stuttard
 venezuelae Dalhousie
 University
 Halifax, NS, Canada
 Streptomyces JW1400 rib J. Westpheling
 venezuelae Athens, GA
 Streptomyces 32172 WT C. Denoya
 avermitilis Pfizer
 Groton, CT
 Streptomyces CD1251 ermE C. Denoya
 avermitilis Pfizer
 Groton, CT
 Saccharopoly- 2338 WT C. Denoya
 spora erythraea Pfizer
 Groton, CT
 TABLE 2
 Antibiotics
 STRAIN ANTIBIOTIC CONCENTRATION
 J222 Rifampicin 50 .mu.g/ml
 J2402 Hygromycin 100 .mu.g/ml
 J2408 Lincomycin 150 #g/ml
 Erythromycin 75 .mu.g/ml
 YU105 Hygromycin 100 .mu.g/ml
 Lincomycin 150 .mu.g/ml
 Erythromycin 75 .mu.g/ml
 CD1251 Erythromycin 5 .mu.g/ml
 BldK::.OMEGA. Spectinomycin 50 .mu.g/ml
 J1258 Streptomycin 15 .mu.g/ml
 Isolation of phage. Approximately 25 grams of top soil, collected in
 plastic vials, was incubated with 15 mls of Actinomycete Phage Buffer
 (APB, 4 mM Ca(NO.sub.3).sub.2, 10 mM Tris HCL, 0.005% gelatin) (Vats, S.
 et al., (1987) J. Bacteriol. 169, 3809-3813) overnight at room temperature
 on a rocking shaker. The mixture was centrifuged at 3,000 rpm for 10
 minutes and the supernatant was passed through a 0.45 .mu.m cellulose
 acetate filter (Nalgene, Rochester, N.Y.). The phage-containing filtrate
 was stored at 4.degree. C. To detect phage, 100 .mu.l of filtrate was
 added to Streptomyces coelicolor spores diluted to approximately 10.sup.7
 cfu/ml. Cfu refers to colony forming unit. The mixture was added to 4 ml
 of Nutrient Soy (Difco, Detroit, Mich.) (Nutrient Soy contains 0.3% beef
 extract, 0.5% peptone) and 0.7% agar (NSA, also referred to as "top agar")
 and poured over Nutrient Agar (Difco, Detroit, Mich.) plates (Nutrient
 Agar contains 0.3% beef extract and 0.5% peptone) and 1.5% agar, 4 mM
 Ca(NO.sub.3).sub.2, and 0.5% Dextrose (referred to as "NCG plates"). Agar
 was obtained from Difco. Plates were incubated at 25.degree. C. for 3 days
 and examined for turbid plaques.
 Phage were isolated by three rounds of plaque purification. From the top
 agar individual plaques were picked with a toothpick and streaked onto a
 lawn of spores (10.sup.7 cfu) that had been spread on Nutrient Agar. The
 plates were incubated at 25.degree. C. for 3 days. This process was
 repeated twice to generate a lawn of isogenic plaque-purified phage. A
 starter lysate was made by adding 2 ml of APB to the lawn of
 plaque-purified phage. A sterile glass rod was used to scrape the top agar
 from the underlying agar plate which was then transferred to a sterile
 centrifuge tube, vortexed, and centrifuged at 10,000 RPM for 10 minutes to
 clarify lysate from cell debris. The phage-containing supernatant (lysate)
 was then transferred to a sterile tube and stored at 4.degree. C.
 Preparation of phage stocks. Phages were propagated on donor strains by
 standard agar-layer techniques (Sambrook, (1989) Molecular Cloning: A
 Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, p. 2.65)
 with APB used as phage diluent. The phage stocks were prepared by adding
 100 .mu.l of the starter lysate to 10.sup.7 cfu spores of the appropriate
 donor strain. This mixture was then added to 4 ml of NSA and poured over
 NCG plates. A total of 10 plates per phage were made. The plates were
 incubated at 25.degree. C. for 5 days. The phage lawns were harvested by
 adding 2 ml of APB to the first plate only of each phage 10 plate set, and
 the NSA transferred to the next plate. This process was repeated with the
 top agar transferred from plate to plate in series. The phage lawns from
 all 10 plates were then transferred to a centrifuge tube, vortexed, and
 centrifuged as above. Phage lysates were purified by filtration through a
 0.45 Nalgene cellulose acetate filter. Each phage lysate was titered
 (i.e., the number of phage determined) by diluting the phage in APB and
 spotting 20 .mu.l of each dilution onto lawns (10.sup.7 cfu) of spores on
 NSA. The "titer" of a lysate is the number of plaque forming units (pfu)
 per ml of lysate.
 Preparation of germlings. Spores were incubated at 50.degree. C. for 10
 minutes in 0.05 M TES buffer (TES:
 N-tris(Hydroxymethyl)methyl-2-aminoethanesulfonic acid), pH 7.2 (Hopwood,
 et al., (1985) Genetic Manipulation of Streptomyces--A Laboratory Manual,
 The John Innes Foundation, Norwich, UK, pp. 8-9). An equal volume of 2X
 germination broth (GB) (2X GB: 1% yeast extract, 1% casaminoacids, and
 0.01 M CaCl.sub.2) was added, and the germlings were incubated at
 30.degree. C. for 2 hours, centrifuged for 5 minutes at 6,000 rpm and
 resuspended in water. At 2 hours, a sample is removed and examined using a
 light microscope to determine if the spores are beginning germination. The
 appearance of germ tubes from the spores indicates germination. The
 culture is considered germlings when about 80% of the spores show short
 germ tubes.
 Adsorption assay. Germlings were prepared as described above. At 2 hours,
 the germlings were centrifuged and resuspended in MYM broth, and incubated
 at 30.degree. C. for an additional 4 hours. A 100 .mu.l sample of
 germlings (about 10.sup.5) was taken once each hour from 0 to 6 hours. The
 germling samples were added to phage (at a concentration of 10.sup.5
 pfu/ml) and incubated for 30 minutes at room temperature to allow for
 adsorption. Each mixture was then centrifuged for 5 minutes at 10,000 rpm
 to pellet germlings and any adsorbed phages. The titer of free phage
 remaining in the supernatant was determined by diluting the supernatant in
 APB and spotting 20 .mu.l of each dilution onto a lawn of about 10.sup.7
 spores on NSA.
 UV irradiation. A kill curve was established for each phage by exposing
 phage suspended in APB (10.sup.6 pfu/ml) to ultraviolet (UV) light
 (250-260 nm) at an intensity of 2 mW/cm.sup.2 /s and sampling at 10
 seconds, 20 seconds, and 30 seconds. The samples containing the phage were
 approximately 6 inches from the UV light (Sylvania, Danners, Mass.). Phage
 were subsequently diluted in APB, and phage titers were determined by
 spotting 20 .mu.l of each dilution onto a lawn of 10.sup.7 S. coelicolor
 A3(2) spores on NSA as described herein.
 Inactivation of phage with citrate. To determine phage sensitivity to
 sodium citrate, phage was diluted in APB and titered by spotting 20 .mu.l
 of each dilution onto a lawn of 10.sup.7 Streptomyces coelicolor spores on
 NSA containing 10 mM sodium citrate. Plates were incubated at either
 22.degree. C. or 30.degree. C. for 3 days and examined for plaques.
 Genetic transduction assays. High titer (approximately 10.sup.10 pfu/ml)
 phage lysates were prepared on donor strains as described herein (see
 Preparation of phage stocks), added to recipient germlings and incubated
 at room temperature for 30 minutes, then spread on supplemented minimal
 glucose medium (Hopwood, et al., (1985) Genetic Manipulation of
 Streptomyces--A Laboratory Manual, The John Innes Foundation, Norwich, UK,
 p. 223) or NCG containing antibiotic and incubated at for 5-7 days
 22.degree. C. Minimal glucose medium was supplemented with 0.02% trace
 elements solution. Trace elements solution (100%) contains 0.004%
 ZnCl.sub.2, 0.02% FeCl.sub.3, 0.001% CuCl.sub.2, 0.001% MgCl.sub.2, 0.001%
 Na.sub.2 B.sub.4 O.sub.7, and 0.001% (NH.sub.4).sub.6 Mo.sub.4 O.sub.24
 (Hopwood, et al., (1985) Genetic Manipulation of Streptomyces--A
 Laboratory Manual, The John Innes Foundation, Norwich, UK, p. 235).
 Transduction frequencies were calculated as the number of colonies
 obtained after incubation for 5-7 days per pfu added to the recipient
 strain. To prevent superinfection killing, the phage were either
 irradiated with UV to 0.1% survival using the established kill curve
 described herein prior to their addition to germlings, or the
 phage-germlings mixture was plated on medium that contained 10 mM sodium
 citrate.
 Southern hybridization analysis. Chromosomal DNA is purified from
 Streptomyces using the protocol for rapid small scale isolation of total
 DNA (Hopwood, et al., (1985) Genetic Manipulation of Streptomyces--A
 Laboratory Manual, The John Innes Foundation, Norwich, UK, pp. 72-74).
 Briefly, total DNA is isolated as follows. A single colony is picked and
 used to inoculate 50 ml of YEME broth (0.3% yeast extract, 0.5% bacto
 peptone, 0.3% malt extract, 1% dextrose, 34% sucrose, and 5 mM MgCl.sub.2)
 which is then incubated 30.degree. C. for 40 hours. The cells are
 harvested by centrifugation at 6,000 rpm for 10 minutes. The resulting
 pellet is then resuspended in 5 ml of SET buffer (75 mM NaCl, 25 mM EDTA,
 pH 8.0, and 20 mM Tris pH 7.5). Lysozyme (1 mg/ml final concentration), is
 added to the pellet suspension and incubated at 37.degree. C. for 1 hour,
 at which time Proteinase K (final concentration 56 .mu.g/ml) and sodium
 dodecyl sulfate (SDS, final concentration 1%,) is added to the suspension
 and incubated at 55.degree. C. for 2 hours. After incubation, NaCl (final
 concentration 0.8 M) is added. The resulting mixture is then extracted
 once with an equal volume of phenol and once with an equal volume of a 1:1
 mixture of phenol/chloroform. To the aqueous phase, Proteinase K (final
 concentration 1.5 mg/ml) and 500 mg of sarkosyl are added and the aqueous
 phase is incubated overnight at 37.degree. C. The solution is then
 extracted again with an equal volume of a 1:1 mixture of
 phenol/chloroform, and then again with an equal volume of chloroform.
 Next, to precipitate the DNA, 0.1 volume of 3 M sodium acetate and 0.6
 volume of 2-propanol are added. The DNA can then be spooled onto a sterile
 glass rod and suspended in about 1 ml of 10 mM Tris, pH 8.0. The DNA is
 stored at 4.degree. C.
 Genomic DNA is digested with the restriction enzymes DraI and AseI
 (Boehringer Mannheim, Indianapolis, Ind.) following the manufacturer's
 instructions. The resulting DNA fragments are separated by electrophoresis
 on a 0.8% agarose gel. The resolved DNA fragments are transferred to a
 nitrocellulose membrane using technics well known to the art (Sambrook,
 (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor
 Laboratory Press, pp. 9.31-9.55). Southern hybridizations use either a
 hygB gene probe or an ermE gene probe.
 The hygB gene is isolated from pUH19b (obtained from Richard Seyler,
 University of Georgia) by cutting the plasmid with NcoI (New England
 BioLabs, Beverly, Mass.) liberating a fragment containing the hygB gene.
 The fragment is gel-purified by separation by electrophoresis on 0.8%
 agarose gel. The hygB fragment (determined by size) is cut out of the gel,
 and the DNA eluted using a Promega (Madison, Wis.) Wizard DNA Purification
 System. An ermE gene probe is obtained and used in Southern hybridization
 analysis.
 The probes are labeled at the 5' end with [.alpha.-.sup.32 P] dATP using
 techniques well known to the art. Prehybridization and hybridization
 buffers consist of the following (final concentrations given): 6X SSC (1X
 SSC is 0.15 M NaCl, 0.015 M sodium citrate), 5X Denhardt's solution (50X
 Denhardt's: 10 grams/liter Ficoll Type 400, 10 grams/liter
 polyvinylpyrrolidone, 10 grams/liter bovine serum albumin Fraction V),
 0.1% SDS, 10 mM potassium phosphate, pH 7.2, and 250 mg/ml salmon sperm
 DNA. Prehybridization is for 2 hours at 55.degree. C., and hybridization
 is overnight at 55.degree. C. with about 50 pmol of radiolabelled probe.
 Hybridization is followed by 3 consecutive washes at room temperature for
 15 minutes each in a solution containing 2X SSC and 0.1% SDS, followed by
 3 consecutive washes at 37.degree. C. for 15 minutes each in a solution
 containing 1X SSC and 0.1% SDS. Kodak X-Omat scientific imaging film is
 used for autoradiography.
 Phage DNA isolation and characterization. Phage DNA was prepared by the
 method of Hopwood, et al., ((1985) Genetic Manipulation of Streptomyces--A
 Laboratory Manual, The John Innes Foundation, Norwich, UK, pp 99-102) with
 the following modifications. Lysates were centrifuged at 25,000 rpm for 90
 minutes at 4.degree. C. to sediment phage. Phage pellets were resuspended
 in RNAase solution (50 .mu.g/ml in APB, the RNAse was obtained from Sigma
 (St. Louis, Mo.)), incubated at 37.degree. C. for 20 minutes followed by
 the addition of 80 .mu.l of a 10% SDS solution and incubation at
 70.degree. C. for 30 minutes. One hundred .mu.l of 8 M ammonium acetate
 was added and the mixture was incubated for 15 minutes on ice, then
 centrifuged 10 minutes at 4.degree. C. The supernatant was extracted with
 phenol, (1 volume supernatant:1 volume phenol), extracted with 1 phenol:1
 chloroform (1 volume supernatant:1 volume phenol:chloroform), and
 extracted with chloroform (1 volume supernatant:1 volume chloroform). The
 nucleic acid was precipitated with ethanol. Digestion of DNA with for
 instance BamHI, DraI, AseI, EcoRV, and ScpII (Boehringer Mannheim,
 Indianapolis, Ind.) was carried out following the manufacturers
 instructions and separated on 0.8% agarose gel.
 Results
 Most wild type phage isolated from soil were found to be temperature
 sensitive for lytic growth on Streptomyces coelicolor. Twenty-six soil
 samples from ten different locations around Athens, Ga. were collected and
 extracted with phage buffer. Samples of the extracts were tested for the
 presence of plaque forming units at 30.degree. C. Nine phages, assumed to
 be different from each other because of differences in plaque morphology,
 were purified. All nine phages formed turbid plaques and yielded lower
 titer lysates (10.sup.5 to 10.sup.7) as compared to the same phage grown
 at 30.degree. C. which formed clear, large plaques and yielded higher
 titer lysates (10.sup.8 to 10.sup.10). The turbidity of a turbid plaque
 was due to cells within the plaque that are not lysed by other phage in
 the plaque. It was distinguished from other clear plaques (plaques in
 which all the bacteria in the region are killed and lysed) because of the
 turbid center. A comparison of phage titers generated from Streptomyces
 coelicolor at 25.degree. C. and 30.degree. C. (Table 3) indicates that the
 phage are naturally temperature sensitive for lytic growth.
 TABLE 3
 Temperature Sensitivity of Phage Isolated from Soil.
 The titer of each phage was determined at 22.degree. C. and 30.degree. C.
 in the presence and absence of citrate.
 22.degree. C. 22.degree. C. 30.degree. C. 30.degree. C.
 Phage -Citrate +Citrate -Citrate +Citrate
 DAH1 1 .times. 10.sup.5 0 2.5 .times. 10.sup.7 500
 Turbid plaques Clear plaques Turbid plaques
 DAH2 3 .times. 10.sup.6 10 2.5 .times. 10.sup.7 4 .times.
 10.sup.5
 Turbid plaques Turbid Clear plaques Turbid plaques
 plaques
 DAH3 5 .times. 10.sup.6 300 1.5 .times. 10.sup.9 5 .times.
 10.sup.7
 Turbid plaques Turbid Clear plaques Turbid plaques
 plaques
 DAH4 2.5 .times. 10.sup.7 400 5 .times. 10.sup.9 5 .times.
 10.sup.6
 Turbid plaques Turbid Clear plaques Turbid plaques
 plaques
 DAH5 1 .times. 10.sup.5 30 4 .times. 10.sup.7 5 .times.
 10.sup.7
 Turbid plaques Turbid Clear plaques Turbid plaques
 plaques
 DAH6 1 .times. 10.sup.5 75 1 .times. 10.sup.6 5 .times.
 10.sup.3
 Turbid plaques Turbid Clear plaques Turbid plaques
 plaques
 Phage inactivation reduces superinfection killing. The release of large
 numbers of phage from infected cells during growth leads to
 superinfection. Superinfection refers to a recipient containing a nucleic
 acid fragment from a phage particle and a nucleic acid fragment from a
 transducing particle. Superinfection typically leads to the killing of
 transductants. This is referred to as superinfection killing and the
 amount of superinfection killing that occurs has a dramatic effect on the
 number of transductants recovered. To reduce superinfection killing of
 transductants, several methods were investigated to inhibit phage
 infection.
 As shown in FIG. 1, exposure of the phage particles to UV light resulted in
 a sharp decrease in phage titer. All of the Streptomyces phages tested
 were sensitive to inactivation by UV at doses and times similar to those
 used for mutagenesis of phage P1. While UV light was effective at
 preventing phage infection, its potential mutagenic effects on DNA within
 transducing particles made it a less than desirable method for phage
 killing.
 Citrate is a chelator of divalent metal ions and has been shown to prevent
 phage adsorption at concentrations that do not affect the growth of
 bacterial cells (Vats, S. et al., (1987) J. Bacteriol. 169, 3809-3813). To
 test for sensitivity to citrate, phage were titered on NCG plates with and
 without sodium citrate. No plaque forming units were visible after 1 day
 at 22.degree. C. Three days after plating, small, turbid plaques
 (10.sup.2) were visible indicating some phage adsorption. However, the
 number of plaques is significantly less than on control plates without
 citrate. Dilutions of cells plated on the same medium showed no effect on
 the viability of Streptomyces coelicolor.
 Genetic transduction in Streptomyces coelicolor is efficient and
 generalized. Each newly isolated phage was examined for its ability to
 mediate transduction. Transduction assays were performed at 22.degree. C.
 to reduce lytic growth of the phage. To reduce superinfection, two
 different methods were used: 1) the phage were irradiated prior to
 addition to germlings; or 2) the phage-germling mixture was plated on
 medium containing 10 mM sodium citrate. Transduction of several
 auxotrophic and drug resistance markers in Streptomyces coelicolor was
 examined for each phage. Surprisingly and unexpectedly, transduction of
 several markers at frequencies ranging from 10.sup.-4 to 10.sup.-8 cfu/pfu
 was detected (Table 4). The markers transduced are positioned around the
 entire chromosome and the frequencies of transduction are similar
 suggesting that transduction is generalized. Transduction is efficient as
 the transduction frequencies are at least 3 orders of magnitude higher
 than the reversion frequency of the recipient strain (see Table). Also,
 these frequencies are well within the range of frequencies reported for
 other transducing phages. For example, P22 (a well established transducing
 phage of Salmonella) transduces markers at frequencies ranging 10.sup.-4
 to 10.sup.-8.
 The ability of phage to mediate transduction was determined in other
 Streptomyces spp. (Streptomyces avermitilis, Streptomyces lividans, and
 Streptomyces venezuelae). Surprisingly and unexpectedly, intraspecific
 transduction was observed at frequencies of about 10.sup.-5 for
 Streptomyces avermitilis, about 10.sup.-4 to about 10.sup.-6 for
 Streptomyces lividans, and about 10.sup.-6 to about 10.sup.-8 for
 Streptomyces venezuelae. Particularly surprising was the observed
 intergeneric transduction between Saccharopolyspora erythraea and
 Streptomyces avermitilis (Table 4).
 In Table 4, the germling only control is the recipient with no phage added.
 This control indicates the reversion frequency of the strain, i.e., how
 often one would expect to see spontaneous revertants. The phage only
 control is the phage with no recipient strain added. This is a test for
 contamination of phage lysates.
 Auxotrophic markers: proA1 means that the recipient strain cannot grow
 unless the media is supplemented with proline or a transducing phage
 provided the recipient cell with the appropriate gene from the donor
 strain. This is true for hisC9, argA1, and uraA1 as well; recipient will
 not grow without supplemented histidine, arginine, or uracil unless the
 phage provided the cell with the appropriate genes from the donor strain.
 Antibiotic resistance: rifA1 means that the strain is resistant to
 rifampicin and therefore, will grow in the presence of rifampicin. Strains
 without a rifA1 genotype are sensitive to rifampicin and can only grow if
 a transducing phage has provided the appropriate gene from the donor
 strain. This is the same for all antibiotic markers. strA confers
 resistance to streptomycin, hygB confers resistance to hygromycin and ermE
 confers resistance to erythromycin and lincomycin.
 TABLE 4
 Transduction
 RECIPIENT
 STRAIN
 SELECTED REVERSION
 TRANSDUCTION
 DONOR STRAIN PHAGE RECIPIENT STRAIN MARKER FREQUENCY
 FREQUENCY
 A. Intraspecific Transduction
 Streptomyces coelicolor
 J222, uraA1 rifA1 NF DAH2 J2709,pro his arg cys ura Arginine +
 3 .times. 10.sup.-7
 J222, uraA1 rifA1 NF DAH2 Arginine +
 0
 J222, uraA1 rifA1 NF DAH4 J2709,pro his arg cys ura Arginine +
 3 .times. 10.sup.-6
 J222, uraA1 rifA1 NF DAH4 Arginine +
 0
 J222, uraA1 rifA1 NF DAH5 J2709,pro his arg cys ura Arginine +
 3 .times. 10.sup.-6
 J222, uraA1 rifA1 NF DAH5 Arginine +
 0
 J222, uraA1 rifA1 NF DAH6 J2709,pro his arg cys ura Arginine +
 5 .times. 10.sup.-6
 J222, uraA1 rifA1 NF DAH6 Arginine +
 0
 J2709,pro his arg cys ura Arginine + &lt;1 in
 10.sup.+10
 J222, uraA1 rifA1 NF DAH2 J2709,pro his arg cys ura Histidine +
 8 .times. 10.sup.-7
 J222, uraA1 rifA1 NF DAH2 Histidine +
 0
 J222, uraA1 rifA1 NF DAH4 J2709,pro his arg cys ura Histidine +
 5 .times. 10.sup.-5
 J222, uraA1 rifA1 NF DAH4 Histidine +
 0
 J222, uraA1 rifA1 NF DAH5 J2709,pro his arg cys ura Histidine +
 3 .times. 10.sup.-7
 J222, uraA1 rifA1 NF DAH5 Histidine +
 0
 J222, uraA1 rifA1 NF DAH6 J2709,pro his arg cys ura Histidine +
 7 .times. 10.sup.-6
 J222, uraA1 rifA1 NF DAH6 Histidine +
 0
 J2709,pro his arg cys ura Histidine + &lt;1 in
 10.sup.+10
 J222, uraA1 rifA1 NF DAH2 J2709,pro his arg cys ura Proline +
 3 .times. 10.sup.-6
 J222, uraA1 rifA1 NF DAH2 Proline +
 J222, uraA1 rifA1 NF DAH4 J2709,pro his arg cys ura Proline +
 3 .times. 10.sup.-6
 J222, uraA1 rifA1 NF DAH4 Proline +
 0
 J222, uraA1 rifA1 NF DAH5 J2709,pro his arg cys ura Proline +
 7 .times. 10.sup.-6
 J222, uraA1 rifA1 NF DAH5 Proline +
 0
 J222, uraA1 rifA1 NF DAH6 J2709,pro his arg cys ura Proline +
 5 .times. 10.sup.-6
 J222, uraA1 rifA1 NF DAH6 Proline +
 0
 J2709,pro his arg cys ura Proline + &lt;1 in
 10.sup.+10
 J222, uraA1 rifA1 NF DAH2 A3(2), WT Rifampicin
 5 .times. 10.sup.-6
 resistance
 J222, uraA1 rifA1 NF DAH2 Rifampicin
 0
 resistance
 J222, uraA1 rifA1 NF DAR4 A3(2), WT Rifampicin
 3 .times. 10.sup.-6
 resistance
 J222, uraA1 rifA1 NF DAR4 Rifampicin
 0
 resistance
 J222, uraA1 rifA1 NF DAH5 A3(2), WT Rifampicin
 7 .times. 10.sup.-6
 resistance
 J222, uraA1 rifA1 NF DAH5 Rifampicin
 0
 resistance
 J222, uraA1 rifA1 NF DAH6 A3(2), WT Rifampicin
 5 .times. 10.sup.-6
 resistance
 J222, uraA1 rifA1 NF DAH6 Rifampicin
 0
 resistance
 A3(2), WT Rifampicin &lt;1 in
 10.sup.+10
 resistance
 Streptomyces avermitilis
 CD1251 JSN 31272, WT Lincomycin
 4 .times. 10.sup.-5
 resistance
 CD1251 JSN Lincomycin
 0
 resistance
 CD1251 JSN3 31272, WT Lincomycin
 3 .times. 10.sup.-5
 resistance
 CD1251 JSN3 Lincomycin
 0
 resistance
 31272, WT Lincomycin &lt;1 in
 10.sup.+9
 resistance
 Streptomyces venezuelae
 10712, WT SV1 JW1400, rib Riboflavin +
 0
 10712, WT SV1 Riboflavin +
 0
 10712, WT BTH JW1400, rib Riboflavin +
 0
 10712, WT BTH Riboflavin +
 0
 10712, WT MRT JW1400, rib Riboflavin +
 7 .times. 10.sup.-8
 10712, WT MRT Riboflavin +
 0
 JW1400, rib Riboflavin + &lt;1 in
 10.sup.+10
 10712, WT SV1 JW1100, pdx Pyridoxal +
 0
 10712, WT SV1 Pyridoxal +
 0
 10712, WT BTH JW1100, pdx Pyridoxal +
 2.5 .times. 10.sup.-8
 10712, WT BTH Pyridoxal +
 0
 10712, WT MRT JW1100, pdx Pyridoxal+
 2.times. 10.sup.-6
 10712, WT MRT Pyridoxal +
 0
 JW1100, pdx Pyridoxal+ &lt;1 in
 10.sup.+10
 Streptomyces lividans
 1326, WT DAH2 TK64 Proline +
 3 .times. 10.sup.-6
 1326, WT DAH2 Proline +
 0
 1326, WT DAH3 TK64 Proline +
 2 .times. 10.sup.-6
 1326, WT DAH3 Proline +
 0
 1326, WT DAH4 TK64 Proline +
 4 .times. 10.sup.-5
 1326, WT DAH4 Proline +
 0
 1326, WT DAH5 TK64 Proline +
 1 .times. 10.sup.-6
 1326, WT DAHS Proline +
 0
 1326, WT DAH6 TK64 Proline +
 1 .times. 10.sup.-6
 1326, WT DAH6 Proline +
 0
 TK64 Proline + &lt;1 in 10.sup.+10
 B. Interspecific Transduction
 Saccharopolyspora JSN1 31272, WT Lincomycin
 4.times. 10.sup.-4
 erythraea, WT resistance
 Saccharopolyspora JSN1 Lincomycin
 0
 erythraea, WT resistance
 Saccharopolyspora JSN2 31272, WT Lincomycin
 3 .times. 10.sup.-6
 erythraea, WT resistance
 Saccharopolyspora JSN2 Lincomycin
 0
 erythraea, WT resistance
 Saccharopolyspora JSN3 31272, WT Lincomycin
 4.times. 10.sup.-4
 erythraea, WT resistance
 Saccharopolyspora JSN3 Lincomycin
 0
 erythraea, WT resistance
 31272, WT Lincomycin &gt;1 in
 10.sup.+9
 resistance
 Co-transduction confirms linkage established by conjugal mating for several
 genetic markers. To examine co-transduction (i.e., the transfer of two
 genetic markers at the same time) of markers by these phage, genes
 identified by mutations that had been previously mapped using conjugal
 mating or physical mapping were used in combination with each other and
 with drug resistance markers that had been introduced into known locations
 within the chromosome. As shown in Table 5, co-transduction was observed
 at frequencies that are in good agreement with previously reported
 linkage.
 TABLE 5
 Cotransduction
 RECIPIENT
 STRAIN
 RECIPIENT SELECTED REVERSION
 TRANSDUCTION SCREENED COTRANSDUCTION
 DONOR STRAIN PHAGE STRAIN MARKER FREQUENCY
 FREQUENCY MARKER FREQUENCY
 YU105, pro arg red DAH5 bldK::.OMEGA. Lincomycin 2
 .times. 10.sup.-8 Spectinomycin 100%
 act::ermE whiE::hyg resistance
 sensitivity
 YU105, pro arg red DAH5 Lincomycin
 0
 act::ermE whiE::hyg resistance
 YU105, pro arg red DAH6 bldK:.OMEGA. Lincomycin 3
 .times. 10.sup.-8 Spectinomycin 100%
 act::ermE whiE::hyg resistance
 sensitivity
 YU105, pro arg red DAH6 Lincomycin
 0
 act::ermE whiE::hyg resistance
 bldK::.OMEGA. Lincomycin &lt;1 in 10.sup.+9
 resistance
 argA1 cysD18 uraA1 DAH5 J222 Streptomycin 2
 .times. 10.sup.-6 Rifampicin 50%
 straA1 NF resistance
 sensitivity
 argA1 cysD18 uraA1 DAH5 Streptomycin
 0
 straA1 NF resistance
 argA1 cysD18 uraA1 DAH6 J222 Streptomycin 1
 .times. 10.sup.-5 Rifampicin 100%
 straA1 NF resistance
 sensitivity
 argA1 cysD18 uraA1 DAH6 Streptomycin
 0
 straA1 NF resistance
 J222 Streptomycin &lt;1 in 10.sup.+9
 resistance
 Physical analysis of transductants confirms DNA transfer. To examine the
 physical location of transduced markers in the recipient chromosome,
 Southern hybridization experiments are performed. DNA is isolated from S.
 coelicolor strains J2402, which contains a hygB insertion into the whiB
 gene, and J2408, which contains an ermE insertion into the whiH gene.
 Phage grown on strain J2402 is added to J2408 germlings, and cells that
 are resistant to both hygromycin (encoded by hygB) and lincomycin (encoded
 by ermE) are isolated from cultures at a frequency above the reversion
 frequency. No doubly resistant cells are recovered from control cultures
 (no phage added) in any experiment, which strongly suggests that the phage
 provide the recipient cells with the gene that confers resistance to
 hygromycin from the donor strain. To confirm that the transductants
 contained the transferred drug resistance gene, DNAs isolated from
 randomly selected transductants are analyzed by Southern blotting using
 the hygB drug resistance gene probe. DNA hybridization patterns of the
 transductants is the same as those of the donor strain, while no
 hybridization is seen with the recipient strain. A gene probe for the ermE
 gene present in the recipient strain hybridizes with the DNA from the
 transductants, but does not hybridize with the donor strain. These
 experiments demonstrate that chromosomal DNA from the donor strain is
 transferred by the phage to the transductant strain and integrated in the
 host chromosome.
 Transduction was not detected when assays were performed at 30.degree. C.
 These phages were exceptional in their ability to grow lytically at
 30.degree. C., the growth temperature typically used to incubate S.
 coelicolor and in fact most streptomycete strains. If lawns with only a
 few plaques were incubated at 30.degree. C., the entire lawn was lysed in
 a few days. Extensive superinfection killing resulting from this active
 lytic growth might explain the failure to detect transduction in many
 Streptomyces species. In fact, when transduction assays were performed
 exactly as described above but at 30.degree. C., no transductants were
 detected. It is very likely that superinfection killing does, in fact,
 reduce or eliminate the ability of transductants to survive.
 Preliminary physical characterization of the transducing phages reveals
 that they are different from each other. To determine the size of the
 phage genomes, nucleic acid was extracted from four phage, DAH2, DAH4,
 DAH5, and DAH6, using a standard approach for Streptomyces phages
 (Hopwood, et al., (1985) Genetic Manipulation of Streptomyces--A
 Laboratory Manual, The John Innes Foundation, Norwich, UK, pp. 99-102). In
 all cases the nucleic acid isolated from the phages was digested with
 several restriction enzymes thus indicating that it is double stranded
 DNA. The genome sizes were estimated using digestion with restriction
 endonucleases and separation of fragments by agarose gel electrophoresis.
 The size of DNA isolated from DAH2, DAH4, DAH5, and DAH6 was about 60
 kilobases (kb), 45 kb, 45 kb, and 25 kb, respectively. The differences in
 size strongly suggest that there are at least three different types of
 phage. Moreover, the differences in transduction frequencies and the
 differences in plaque morphology between DAH4 and DAH5 strongly suggest
 that these two phages are not the same phage, despite the similar size of
 the DNA. Thus, DAH2, DAH4, DAH5, and DAH6 are each unique.
 The complete disclosures of all patents, patent applications, publications,
 and nucleic acid and protein database entries, including for example
 GenBank accession numbers and EMBL accession numbers, that are cited
 herein are hereby incorporated by reference as if individually
 incorporated. Various modifications and alterations of this invention will
 become apparent to those skilled in the art without departing from the
 scope and spirit of this invention, and it should be understood that this
 invention is not to be unduly limited to the illustrative embodiments set
 forth herein.