Regulatory region cloning and analysis plasmid for bacillus

Novel constructs in plasmids are provided for evaluating the efficiency of expression and secretion of structural genes. The constructs provide for transcriptional and translational regulatory regions, a signal sequence and structural gene, which also may be readily excised and substituted, so as to allow for mixing and matching of regulatory regions, signal sequences and genes to evaluate regions for use in the expression of a desired peptide. Particularly, synthetic regions are provided which may be used with other synthetic regions or wild-type regions. Plasmids are provided for screening Bacillus genomic sequences for regulatory regions, particularly promoters employing a structural gene secreting an enzyme which can produce a product which allows for visual detection.

FIELD OF THE INVENTION 
Plasmids are provided for isolating and evaluating regulatory regions of 
genes in Gram-positive bacteria, particularly Bacillus. Synthetic 
regulatory regions are provided associated with convenient restriction 
sites, whereby various cassettes can be prepared involving at least one of 
a promoter sequence, a ribosomal binding site sequence, and a signal 
sequence functional in Bacillus, where one or more of these regions may be 
substituted by the region to be evaluated. A plurality of restriction 
sites are provided for ease of substitution of one of the regions with a 
different region. 
BACKGROUND OF THE INVENTION 
In the use of genetic manipulation techniques in microorganisms, the genus 
Bacillus has, after E. coli, in recent years also formed the subject of 
extensive investigation. See, for example, Dubnau, in Experimental 
Manipulation of Gene Expression, Academic Press, (1983) 33-51 and Doi, 
Biotechnology and Genetic Engineering (1984) 2:126-155. Bacilli have now 
been used for a long time in the fermentation industry. Bacilli offer 
numerous advantages, such as good growth on inexpensive base materials, 
and in contrast to E. coli, do not produce any endotoxins. Furthermore, 
Bacilli are capable of secreting proteins into the growth medium, in 
particular, certain types of enzymes such as proteases and amylases, 
frequently produced in large amounts by Bacilli. These enzymes may be 
relatively inexpensively and conveniently isolated from the fermentation 
medium. 
Because of the attractiveness of Bacilli as a host for the production of 
homologous or heterologous peptides, it is of substantial commercial 
interest to be able to make use of particular sequences associated with 
transcriptional and translational regulation, which would allow for 
efficient expression and secretion of the peptides of interest. There is, 
therefore, substantial interest in ways for isolating and analyzing these 
sequences from Bacillus or other sources, which would allow for the 
efficient screening of the sequences. 
DESCRIPTION OF THE RELEVANT LITERATURE 
Plasmids from S. aureus are capable of extrachromosomal maintenance in B. 
subtilis (Ehrlich, Proc. Natl. Acad. Sci. USA (1977) 74:1680-1682). 
Various prokaryotic and eukaryotic heterologous proteins have been cloned 
in B. subtilis, usually at low expression levels. See, for example, 
Kovacevic et al., J. Bacteriol. (1985) 162:521-528: Saunders et al., ibid. 
(1984) 157:718-726: Ohmura et al., Third International Conference on 
Genetics and Biotechnology of Bacilli, Stanford, USA (1984); Lundstrom, 
FEBS Letters (1984) 23:65-70, Palva et al., Gene (1983) 22:229-235: 
Lundstrom et al., Virus Res. (1985) 2:69-83: Hardy et al., Nature (1981) 
293:481-483: Mosbach et al., Nature (1983) 302:543-545: Chang et al., NSC 
Ser. (1982) 4:254-261; Williams et al., Gene 1981) 16:199-206 and Flock et 
al., Mol. Gen. Genet. (1984) 195:246-251. 
In order to arrive at an economically acceptable expression level, it is 
necessary to achieve improvements in the cloning system. In this context 
consideration can in particular be given to a modification of the 
promoter, in order to improve the transcription efficiency of the 
heterologous gene, a modification of the ribosomal binding site 
(Shine-Dalgarno), in order to improve the translation efficiency, and/or a 
modification of the signal sequence, in order to improve the secretion of 
the desired heterologous protein product. 
Various proposals have been made for combining synthetic or naturally 
occurring promoters with a gene other than the wild-type gene. See for 
example Williams et al., J. Bacteriol. (1981) 146:1162-1165: Schoner et 
al., Gene (1983) 22:47-57, who describe plasmid pPL 603: Goldfarb et al., 
Nature (1981) 293:309-311 (plasmid pGR 71): Band et al., Gene (1983) 
26:313-315 (plasmid pCPP 3-4): and Donelly and Sonnenshein, J. Bacteriol. 
(1984) 157:965-967 (plasmid pCED 6). 
However, these plasmids have the disadvantage that they are fairly large 
and comprise only 1 or 2 promoter insertion sites. Moreover, they are 
almost all based on chloramphenicol acetyl-transferase as the indicator 
enzyme, and comprise an inducible Shine-Dalgarno sequence (pPL 603) or an 
inactive Shine-Dalgarno sequence (pGR 71). Consequently, promoters can be 
isolated only if a fusion protein is formed with the chloramphenicol 
acetyl-transferase, that is to say if a Bacillus Shine-Dalgarno sequence 
is also co-cloned and the reading frame is in phase with that of the 
chloramphenicol acetyl-transferase. 
For discussion of the Shine-Dalgarno sequence and the initiation codon, see 
Hui et al., EMBO J. (1984) 3:623-629: De Boer et al., DNA (1983) 
2:231-235: Band and Henner, Biochem. Soc. Symp. (1984) 48:233-245: and EPA 
116411. 
Concerns involved with evaluation of regulatory sequences have been 
expressed by Hall et al., Nature (1982) 295:616-618: Shpaer, Nucleic Acids 
Res. (1985) 13:275-289 and Tessier et al., ibid. (1984) 12:7663-7675. 
Stanssens et al., Gene (1985) 36:211-213 describe the effect of alterations 
of the sequence upstream from the Shine-Dalgarno region: Iwakura et al., 
J. Biochem (1983) 93:927-930 describe the construction of plasmid vectors 
employing the dhfr gene: and Hosoya et al., Agricultural and Biological 
Chemistry (1984) 48:3145-3146, describe the construction of a promoter 
cloning vector in P. aeruginosa. 
Ohmura et al., J. Biochem (1984) 95:87-93, describe a B subtilis secretion 
vector system employing the .alpha.-amylase promoter and signal sequence 
region: Enger-Valk et al., Gene (1981) 15:297-305 describe a vector for 
cloning of promoters: Tsoi et al., Genetika (Moscow) (1981) 17:2100-2104 
describe the cloning and expression of promoter fragments of B. 
thuringiensis DNA in E. coli cells: Moran et al., Mol. Gen. Genet. (1982) 
186:339-346 describe nucleotide sequences that signal the initiation of 
transcription and translation in B. subtilis; see also EPA 134048. 
SUMMARY OF THE INVENTION 
Novel DNA sequences and combinations of sequences are provided for the 
isolation of DNA fragments that can function as promoter in Bacilli. Said 
sequences are characterized by a structural gene, including a functional 
signal sequence, and a synthetic ribosomal binding site. The gene product 
encoded by the structural gene is, with the aid of the signal sequence, 
secreted by the host organism. Its activity can be easily assayed for, 
provided that upstream of the ribosomal binding site a DNA fragment is 
inserted, preferably using one of the available unique restriction sites. 
Said sequences are further characterized in that regulatory regions 
concerned with transcription, translation and secretion, and the 
structural gene itself can be conveniently exchanged by the presence of 
unique restriction sites bordering these regions. pPROM 54 is a 
promoterless plasmid useful in the screening of fragments for the presence 
of promoters in proper orientation and spacing.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS 
Novel DNA sequences are provided in which a regulatory domain is provided. 
The domain allows for insertion and exchange of individual functional 
elements of the domain, as well as subunits of a functional element. The 
domain provides for transcriptional and translational regulatory elements, 
including elements affecting such regulation, such as operators, 
enhancers, activators, or the like. Of particular interest is a domain 
which provides for regulation of transcription, regulation of translation, 
both a ribosomal binding site and an initiation codon, a signal sequence 
for secretion and, as appropriate, a structural gene in reading phase with 
the signal sequence, which signal sequence may or may not include a 
processing signal for peptide cleavage. 
The constructs allow for isolation of selected promoter sequences which 
bind to the Bacillus .sigma..sup.55, .sigma..sup.37, .sigma..sup.32, 
.sigma..sup.29, .sigma..sup.28 or other RNA polymerases, provided with 
convenient restriction sites for introduction and excision from a vector. 
Also provided is a Shine-Dalgarno sequence with a plurality of convenient 
restriction sites so as to allow for introduction to and excision from a 
vector. A signal sequence for secretion is provided with convenient 
restriction sites for insertion and excision of the signal sequence, as 
well as insertion downstream and in reading frame with the signal sequence 
of a structural gene of interest. 
A convenient Bacillus replication system is employed, such as the 
replication system pUB110. The vectors which are available will normally 
have a marker for selection, which marker allows for resistance to a 
cytotoxic agent such as an antibiotic, e.g. kanamycin, chloramphenicol, 
tetracycline, streptomycin, etc.; heavy metal, or the like: or 
complementation in an auxotrophic host. One or more markers may be 
present, particularly where a shuttle vector is employed, where the vector 
is capable of replication in two or more hosts. Conveniently, the vector 
may include a replication system for replication in two or more hosts. 
Conveniently, the vector may include a replication system for replication 
in E. coli, so as to allow for cloning and expansion of the DNA after each 
of the manipulative steps involved with the formation of the construct. 
Suitable host microorganisms of the Bacillus species are B. subtilis, B. 
licheniformis, B. amyloliquefaciens and B. stearothermophilus. 
At least one and preferably two of the subject sequences providing for a 
particular regulatory region or signal sequence are employed in 
combination with the sequence to be evaluated. 
The first sequence to be considered is the promoter sequence. This region 
is involved with the binding of the .sigma..sup.X -RNA polymerase wherein 
X intends any of the RNA polymerases indicated previously. The sequence 
may be synthetic or wild-type. One synthetic promoter will have the 
following sequence: 
##STR1## 
The subject synthetic sequence which is recognized by the .sigma..sup.55 
-RNA polymerase has a number of significant features, allowing for the 
individual substitution of the -10 region or the -35 region by digestion 
with restriction enzymes HindIII or XhoI and SstI or digestion with EcoRI 
and HindIII or XhoI, respectively. The entire synthetic promoter may be 
substituted by digestion with EcoRI and SstI. 
A second synthetic promoter, for the .sigma..sup.37 -RNA polymerase will 
have the following sequence: 
##STR2## 
This sequence enjoys similar benefits as described for the .sigma..sup.55 
-RNA polymerase promoter. 
Other promoters which may be used to advantage include wild-type promoters, 
such as the wild-type promoter found in plasmid pPROM 3-4C., deposited at 
the CBS on November 5, 1985, under No. 699.85, where the promoter is a 
chromosomal promoter sequence derived from B. licheniformis. The above 
plasmid is derived from the plasmid pPROM 54, where the insertion of the 
B. licheniformis promoter results in an increase in .alpha.-amylase 
production in B. subtilis of 35%, compared with the plasmid pGB33 carrying 
the original promoter and Shine-Dalgarno sequences. 
Another promoter derived from a bacteriophage promoter sequence is present 
in plasmid pPROM SP02, deposited at the CBS on Nov. 5, 1985, under No. 
698.85. This plasmid is also derived from the plasmid pPROM 54, by 
insertion of a promoter sequence from the bacteriophage sequence derived 
from the plasmid pPL 608, which is described by Williams et al., supra. 
(1981). The insertion of the bacteriophage promoter results in an increase 
of .alpha.-amylase production in B. subtilis of 37% over the natural 
promoter. 
These various promoter sequences may be substituted by any other promoter 
sequence, from any source, where it is intended to determine the 
efficiency of such promoter in a Bacillus host. Thus, promoter sequences, 
or portions of promoter sequences, either synthetic or natural, such as 
the -10 region or the -35 region consensus sequences may be incorporated 
for evaluation, by ligating such DNA sequences to one or more of the other 
sequences provided in accordance with this invention. 
The next region of interest is the Shine-Dalgarno sequence or ribosome 
binding region. For this purpose, a synthetic region may be suitably 
employed having the following sequence: 
##STR3## 
The third sequence which is employed in the subject invention is the signal 
sequence of .alpha.-amylase, which may be conveniently joined to the 
region coding for the mature .alpha.-amylase or to a different gene 
resulting in a hybrid gene. A convenient restriction site is provided 
between the .alpha.-amylase signal sequence and the remainder of the 
.alpha.-amylase gene, so as to allow for substitution of the region coding 
for the mature .alpha.-amylase. 
Each of the fragments which are employed provide for one or more 
restriction sites which allow for introduction and excision of the 
individual fragments. Thus, polylinkers or one or more restriction sites, 
desirably unique restriction sites, are present within and between the 
regions for convenient insertion or excision of sequences. Usually, the 
polylinker will have at least two restriction sites and usually not more 
than about six restriction sites, more usually not more than about four 
restriction sites, frequently unique restriction sites. Exemplary 
restriction enzyme recognition sites have been indicated previously. 
The constructs of the subject invention may be prepared in accordance with 
conventional ways. The substitution by other sequences of the above 
regions may require modification of such other sequences. Modifications 
may include the use of linkers, adapters, in vitro mutagenesis, resection, 
repair, primer repair, or the like, where restriction sites may be 
introduced or removed, termini modified, etc. After each manipulation, it 
will usually be desirable to clone the new construct in a convenient host, 
such as E. coli, isolate the new construct and establish the presence of 
the correct sequence by restriction mapping, sequencing, or the like. Once 
the construct is completed, it may then be transferred to a vector capable 
of replication in a Bacillus host or a shuttle vector may be used for the 
construct, which allows for cloning in E. coli and direct transfer to the 
Bacillus host. 
Of particular interest is the construction of a "fishing" plasmid for 
screening fragments of DNA for promoter regions functional in Bacillus. 
The fishing plasmid has two regions necessary for detection of a promoter 
region. The first region is a promoter screening region and the second 
region is a transformant selection region. The screening region comprises 
in the direction of transcription, a region of from about 4 to 100 bp 
having one or more unique restriction sites, usually not more than about 
6, and lacking any transcriptional initiation activity. Downstream from 
the restriction site region is a ribosomal binding site region of from 
about 5 to 50 bp, including the non-coding nucleotides on either side of 
the Shine-Dalgarno consensus sequence. The sequence may be natural by 
occurring or synthetic. The ribosomal binding region is followed by a 
structural gene having a signal sequence for secretion. The structural 
gene expresses a product which can be readily detected by a simple 
chemical reaction without the possibility of significant interference from 
endogenous host materials. Of particular interest is .alpha.-amylase, 
which can be detected with a combination of amylose and iodine, where 
discharge of the color indicates the expression of .alpha.-amylase and the 
presence of a promoter in the restriction site region. Amylase can be 
conveniently present in the gel nutrient medium and clear halos are 
indicative of expression. 
The second region provides for selection of transformants. This region will 
normally encode a gene imparting antibiotic resistance, so that only 
transformants having the plasmid will survive when grown in medium 
containing an otherwise cytotoxic amount of the antibiotic. Resistance to 
neomycin, tetracycline, penicillin, kanamycin, etc. may be provided with 
the appropriate genes. 
Both the screening and selection regions will be joined to a replication 
system functional in Bacillus. Other functional regions may be present in 
the plasmid, such as a replication system for E. coli for cloning. 
The fishing or screening method will involve fragmenting DNA from a 
Bacillus or other host e.g., virus, which may have regions capable of 
transcriptional initiation in Bacillus. The fragments may be mechanically 
produced or by using one or more restriction enzymes, particularly ones 
that have complementary ends to the restriction sites present in the 
restriction site region. Fragments as small as 20 bp and up to about 5 
kbp, usually 2 kbp, may be obtained for screening, usually from about 50 
bp to 1 kbp. The fragments are inserted into the fishing plasmid in 
accordance with conventional ways. The resulting plasmid library may then 
be transformed into a Bacillus host and the transformants selected by 
means of the antibiotic resistance. 
Surviving Bacillus transformants may then be screened for active promoters 
by contacting clones with amylose and iodine and isolating those clones 
which become clear. 
Transformation of Bacillus may be carried out in accordance with 
conventional ways. See, for example Anagnostopoulos and Spizizen, J. 
Bacteriol. (1961) 81:741-746. Transformants may then be selected in 
accordance with the nature of the marker. 
By employing the subject constructs, structural genes may be evaluated for 
their ability to be expressed and secreted, where regulatory regions and 
the structural genes may be mixed and matched to provide for efficient 
production of the desired product. The production including secretion of a 
peptide may be determined and compared to other regulatory and functional 
sequences. 
The following examples are offered by way of illustration and not by way of 
limitation. 
EXAMPLE I 
Isolation of Chromosomal DNA 
Chromosomal DNA of B. licheniformis, strain T5, deposited at the CBS on 
July 6, 1983, under No. 470.83 (see EPA 134048) was isolated from 3 L of 
cultures which had grown overnight at 37.degree. C., under aeration. The 
cells were centrifuged for 10 min. in a Sorvall GSA rotor at 10,000 rpm, 
suspended in 10 ml of sucrose-Tris buffer which contained 25% by weight of 
sucrose and 50 mM Tris-HC1 at pH 8.0, and lysed by addition of 0.5 ml of 
lysozyme solution (20 mg/ml) and subsequently 15 min. incubation at 
37.degree. C. After addition of 2 ml of EDTA (0.5 M) and 5 min. incubation 
at 0.degree. C., 1 ml of 20% by weight sodium dodecylsulfate (SDS) was 
added. Thereafter, the suspension was extracted with a 1:1 mixture of 
phenol and chloroform. The supernatant water layer was removed and 
carefully overlayered with 2 volume units of ethanol, after which the DNA 
could be isolated with the aid of a glass rod. After dissolution in 
distilled water to which 10 mg/ml ribonuclease had been added, the mixture 
was extracted with 1:1 phenol-chloroform, and the product precipitated 
with 2 parts of ethanol and resuspended in TE buffer (10 mM Tris-HCl, pH 
8.0 and 1 mM EDTA). 
EXAMPLE II 
Isolation of Plasmid DNA 
B. subtilis 1-85, containing plasmid pGB 33, deposited at the CBS under No. 
466.83 (see EPA 134048), was cultured overnight in 1 L of minimal medium 
to which 10 mg/ml neomycin had been added. After centrifuging for 15 min. 
in a Sorvall model GSA rotor at 5,000 rpm and resuspending in 15 ml of 
sucrose-Tris, the cells were lysed and treated with EDTA and SDS (see 
Example I). After addition of NaCl to a final concentration of 1 M, the 
lysate was stored overnight at 4.degree. C. and subsequently centrifuged 
for 15 min. at 12,500 rpm in a Sorvall type SS 34 rotor. The uppermost 70% 
(by volume) of the supernatant liquid was treated for 30 min. at 
37.degree. C. with 20 .mu.g/ml of DNAse-free RNAse, and extracted with a 
1:1 mixture of phenol-chloroform and subsequently with pure chloroform. 
The DNA was precipitated from the extracted supernatant liquid by addition 
of 0.2 part of 5 M NaCl and 0.25 part of 40% by weight polyethylene glycol 
6000, followed by incubation at 4.degree. C. overnight. After 
precipitation and centrifugation (30 min. at 12,500 rpm, Sorvall type SS 
34, the DNA was resuspended in 2-3 ml of TE buffer (see Example I) and 
kept at pH 12.0 for 10-15 min. with the aid of 4N NaOH. Thereafter the pH 
was returned to 8.5 and the mixture was extracted with phenol. After 
precipitation with ethanol, the plasmid DNA was resuspended in a small 
volume of TE buffer. 
EXAMPLE III 
Oligonucleotide Synthesis 
The following oligonucleotide sequences were synthesized with the aid of a 
Biosearch Synthesis Automation Machine and were purified by means of HPLC, 
extraction with phenol-chloroform (1:1) and precipitation with ethanol: 
______________________________________ 
(A) 5'-GATCCAAGGAGGTGAT-3' 
(B) 5'-CTAGATCACCTCCTTG-3' 
(C) 5'-AATTCTTGACAAAGCTTC-3' 
(D) 5'-TCGAGAAGCTTTGTCAAG-3' 
(E) 5'-TCGAGACTGATATAATGAGCT-3' 
(F) 5'-CATTATATCAGTC-3' 
(G) 5'-AATTCAGGATTTATGAAGCTTC-3' 
(H) 5'-TCGAGAAGCTTCATAAATCCTG-3' 
(I) 5'-TCGAGGGAATTGTTTGAGCT-3' 
(J) 5'-CAAACAATTCCC-3' 
______________________________________ 
These oligonucleotides were used for synthesis of the following DNA 
sequences: 
A. Synthetic Shine-Dalgarno Sequence 
The oligonucleotides A and B were kinased by incubating a mixture of 5 
.mu.g of the two oligonucleotides for 1 hour at 37.degree. C. with 60 
.mu.l of 1 mM ATP, 5 .mu.l of 10 x kinase mix (0.5 M Tris-HCl pH 7.0, 0.1 
M MgCl.sub.2, 50 mM dithiothreitol, 1 mM spermidine, 1 mM EDTA) and 3 
.mu.l of T.sub.4 -kinase (Gibco, 10U/.mu.l ) in a total volume of 50 
.mu.l. The kinased oligonucleotides were subsequently annealed by 5 min. 
incubation at 100.degree. C. followed by 30 min. incubation at 65.degree. 
C. After purification with phenolchloroform (1:1) and precipitation with 
ethanol, the DNA was resuspended in a small volume of TE buffer. 
B. Synthetic promoter-sequence recognized by .sigma..sup.55 -RNA polymerase 
Analogously to the description under A, but starting from a mixture of the 
oligonucleotides C, D, E and F, a synthetic promoter sequence recognized 
by .sigma..sup.55 -RNA polymerase was obtained. 
C. Synthetic promoter-sequence recognized by .sigma..sup.37 -RNA polymerase 
Analogously to the description under A, but starting from a mixture of the 
oligonucleotides G, H, I and J, a synthetic promoter sequence recognized 
by .sigma..sup.37 -RNA polymerase was obtained. 
EXAMPLE IV 
Construction of a Shine-Dalgarno/signal sequence construct in plasmid pPROM 
54 
15 .mu.g of pGB 33, isolated from B. subtilis 1-85 (see Example II) was cut 
with the restriction enzyme NdeI, of which the recognition site is located 
precisely between the promoter and the Shine-Dalgarno sequence of the B. 
licheniformis .alpha.-amylase gene, as may be seen from the sequence 
analysis (see FIG. 1). After extraction with phenol-chloroform (1:1) and 
precipitation with ethanol, the digested plasmid-DNA was resuspended in 59 
.mu.l of Ba131 mix (120 .mu.l of 100 mM Tris-HCl pH 8.1, 72 .mu.l of 100 
mM MgCl.sub.2, 72 .mu.l of 100 mM CaCl.sub.2, 120 .mu.l of 1 M NaCl, 156 
.mu.l of H.sub.2 O and 1 .mu.l of Ba131 exonuclease (Gibco 1.2 U/.mu.l)). 
After 3.5 min. incubation at 15.degree. C., the material was again 
extracted with phenolchloroform (1:1) and reprecipitated with ethanol. 
After resuspending, the DNA was digested with the restriction enzyme PstI, 
extracted with phenolchloroform (1:1), precipitated with ethanol and 
resuspended in 20 .mu.l of a ligase mix which contained 20 mM Tris-HCl pH 
7.6, 10 mM MgCl.sub.2, 10 mM dithiothreitol, 0.5 mM ATP, 1 .mu.l E. coli 
phage M13mp10 (digested with the restriction enzymes HincII and PstI) and 
1 .mu.l of T.sub.4 ligase (Boehringer 1 U/.mu.l), after which ligation was 
carried out overnight at 4.degree. C. (see FIG. 2). 
After transformation and selection of white plaques in E. coli, a number of 
recombinant DNA phages were isolated and sequenced with the aid of the 
"dideoxychain terminator" method. In the recombinant most shortened by 
Ba131 (see FIG. 2) the synthetic Shine-Dalgarno sequence (see Example III) 
was inserted after digestion with the restriction enzymes BamHI and XbaI, 
after which the construct was sequenced. This fragment containing the 
Shine-Dalgarno sequence was subsequently excised with restriction enzymes 
EcoRI and PstI (see FIG. 2) and substituted for the EcoRI-PstI fragment 
carrying the original regulation signals of the B. 
licheniformis-.alpha.-amylase gene. The plasmid thus obtained, pPROM 54, 
has a size of about 5.2 kbp. The structure of the plasmid is shown in FIG. 
3. 
The plasmid pPROM 54 in B. subtilis 1A40 (amy.sup.-, lys.sup.-, met.sup.-, 
trp.sup.-) was deposited at the CBS on Nov. 5, 1985 under No. 696.85. 
EXAMPLE V 
Construction of pPROM plasmids which comprise chromosomal promoter 
sequences 
5 .mu.g of chromosomal DNA, isolated from B. licheniformis strain T.sub.5 
(Example I) were cut with RsaI, HaeIII, AluI, HincII and EcoRV and, after 
purification with phenol-chloroform and precipitation with ethanol, were 
ligated to 1 .mu.g of pPROM 54 (Example IV), restricted with SmaI. Another 
portion of chromosomal DNA from B. licheniformis strain T.sub.5 was 
digested with EcoRI under Eco* conditions, purified, precipitated and 
ligated to 1 .mu.g of pPROM 54, linearised with EcoRI. The ligated 
mixtures were transformed into B. subtilis 1A40 (amy.sup.-, lys.sup.-, 
met.sup.-, trp.sup.-) using the method described by Anagnostopoulos and 
Spizizen, J. Bacteriol. (1981) 81:741-746. Transformants were first 
selected for neomycin/kanamycin resistance on minimal agar plates to which 
0.02% (w/v) of casamino acids (Difco) and 10 .mu.g/ml neomycin were added. 
These transformants were subsequently analyzed for the presence of a 
promoter sequence by selection in respect of the capacity achieved for the 
synthesis of .alpha.-amylase, which was done by looking for halos after 
having poured a solution of 0.6% (w/v) of KI and 0.3% (w/v) of I.sub.2 
over the plates. The transformants thus selected were used for 
fermentation production of .alpha.-amylase in comparison with production 
under the influence of the native .alpha.-amylase regulatory region. The 
selected transformants were also used as the source for recombinant DNA 
plasmids. 
One of the selected transformants comprised the plasmid pPROM 3-4C. This 
plasmid in B. subtilis 1A40 (amy.sup.-, lys.sup.-, met.sup.-, trp.sup.-), 
was deposited at the CBS on Nov. 5, 1985 under No. 699.85. 
EXAMPLE VI 
Construction of pPROM plasmids which comprise bacteriophage promoter 
sequences 
5 .mu.g of pPL 608 carrying an SP02 phage promoter fragment of 280 bp 
(Williams et al. J. Bacteriol. (1981) 146:1162-1165), were cut with EcoRI 
and, after purification and precipitation, were ligated to 1 .mu.g of 
pPROM 54, linearized with EcoRI. The ligated mixture was transformed into 
B. subtilis 1A40 (amy.sup.-, lys.sup.-, met.sup.-, trp.sup.-). The 
transformants obtained were selected in the manner described in Example V. 
The selected transformant comprised the plasmid pPROM SP02. This 
transformant was used for fermentative production of .alpha.-amylase in 
comparison with the production under the influence of the native 
.alpha.-amylase gene and also used as a source for the recombinant plasmid 
pPROM SP02. 
The plasmid pPROM SP02 in B. subtilis 1A40 (amy.sup.-, lys.sup.-, 
met.sup.-, trp.sup.-) was deposited at the CBS on Nov. 5, 1985 under No. 
698.85. 
EXAMPLE VII 
Construct PROM plasmids which comprise synthetic promoter sequences 
5 .mu.g of the synthetic promoter obtained by purification and annealing of 
the oligonucleotides C, D, E and F (see Example III) were ligated to 1 
.mu.g of pPROM 54 and digested with Eco RI and Sst I (see FIG. 4.) The 
ligated mixture was transformed into B. subtilis 1A40 (amy.sup.-, 
lys.sup.-, met.sup.-, trp.sup.-). The transformants obtained were selected 
in the manner described in Example V. 
The selected transformant comprised the plasmid pPROM 55s. The transformant 
was used for fermentative production of .alpha.-amylase in comparison with 
the production under the influence of the native .alpha.-amylase gene, and 
also as a source for the recombinant plasmid pPROM 55s. 
The plasmid pPROM 55s in B. subtilis 1A40 (amy.sup.-, lys.sup.-, met.sup.-, 
trp.sup.-) was deposited at the CBS on Nov. 5, 1985 under No. 697.85. 
Analogously to this procedure, but starting from the synthetic 
oligonucleotides G, H, I and J (see Example III), a Bacillus 1A40 
transformant containing the recombinant plasmid pPROM 37S, was obtained, 
which plasmid differs from pPROM 55s in respect of the synthetic promoter 
sequence (compare FIGS. 4 and 5). 
EXAMPLE VIII 
Fermentative Production of .alpha.-Amylase with the aid of genetically 
manipulated Bacillus subtilis strains 
The B. subtilis strains obtained after genetic manipulation as described in 
Examples V, VI and VII, and also the B. subtilis strain with the starting 
plasmid pGB 33, were cultured for 5 days at 37.degree. C. in a liquid 
heart infusion medium made up with 0.4% of Zulkowski starch. The 
.alpha.-amylase was isolated and purified in accordance with standard 
procedures. The quantities of .alpha.-amylase produced, in comparison with 
the original B. subtilis strain having the starting plasmid pGB 33 
(bearing the unmodified .alpha.-amylase gene) are shown in Table I. 
TABLE I 
______________________________________ 
% 
.alpha.-amylase 
compared 
Origin of production 
with 
Promoter Clone No. (TAU/ml) control 
______________________________________ 
B. lich. chromosomal 
DNA x Rsa I pPROM 1.1 48.2 116 
x Rsa I pPROM 2.6 44.3 107 
x Alu I pPROM 11.1 
47.0 113 
x Alu I pPROM 14.3 
43.4 117 
x Hinc II pPROM 17.4 
39.7 96 
x Hinc II pPROM 17.5 
36.4 88 
x Hinc II pPROM 17.6 
43.5 105 
x EcoRV pPROM 23.25 
45.5 110 
x EcoRV pPROM 23.26 
49.3 119 
x EcoRI* pPROM 3-4C 
55.8 135 
pL 608 DNA 
x EcoRI pPROM SPO2 56.8 137 
Synthetic DNA 
pPROM 37s 39.3 95 
pPROM 55s 33.1 80 
Control pGB 33 41.4 100 
______________________________________ 
In accordance with the subject invention, functional sequences can be 
readily isolated and evaluated by substitution or insertion of regulatory 
regions, signal sequences, or structural genes into a designed construct. 
The resulting constructs may then be introduced into a Bacillus host and 
the efficiency of expression and secretion determined. In this manner, 
Bacillus libraries or libraries from other hosts which may have regulatory 
regions functional in Bacillus may be screened for their use in Bacillus. 
Thus, promoters, ribosomal binding sites and signal sequences may be 
evaluated from a wide variety of hosts, such as viruses, microorganisms, 
and the like. 
Although the foregoing invention has been described in some detail by way 
of illustration and example for purposes of clarity of understanding, it 
will be obvious that certain changes and modifications may be practiced 
within the scope of the appended claims.