CA1213230A - Plasmids for cloning in b-subtilis - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

A double-stranded DNA plasmid which includes a promoter DNA sequence that is not derived from a B. subtilis plasmid and a DNA sequence derived from a B. subtilis plasmid is useful for introducing into B. subtilis foreign DNA having a nucleic acid sequence which codes for the production of a desired product. Preferably, the promoter sequence is derived from B. subtilis chromosomal DNA. When a foreign DNA sequence having a nucleic acid sequence coding for production of a desired product is introduced into this plasmid, another plasmid is produced which is useful for effecting expression in B. subtilis of the foreign DNA and production of the desired product.

B. subtilis cells transformed with plasmids carrying a gene or genes coding for the production of desired products may be grown in culture and the resulting products recovered.

Description

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' PLASMIDS FOR CLONING IN BACILLUS SUBTILIS

BACKGROUND OF THE INVENTION
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The basis of industrial recombinant DNA applications is production of natural products by a bacterium normally unable to produce them. In this regard, much of the published literature concerning the application of genetic engineering techniques for the production of commercially valuable products such as hormones, vaccines, enzymes and various polypeptides involves use of Escherichia golf as the host into which foreign genes are introduced. Nevertheless, many fermentation en-gingers consider Bacillus subtilis the bacterium of choice for industrial applications of recombinant DNA
technology because of the likelihood of increased yield and decreased toxicity for products made by recombinant B. subtilis organisms.

In Escherlchia golf and other organisms, the use of operon fusions has proved to be a powerful tool for understanding gene regulation. The power of the method is considerable. By putting the expression of a gene whose product is easily measured under the control of a variety of regulatory sites, one can analyze and come pare regulation in a number of systems by means of simple biochemical assays. This is of particular value when the product of the gene whose regulation is of interest is unknown or difficult to measure. The same fusions can be used to generate regulatory mutants, taking advantage of the phenotype provided by the fused structural genes. In addition, expression of a foreign gene in a given host often requires that the gene be transcribed from a promoter site indigenous to the host. This, of course, has important ramifications for many practical applications of recombinant DNA technology.

-2-In order to apply this approach to Bacillus subtilis, novel plasmids, i.e. self-replicating DNA molecules, ; which can be used to direct and regulate expression in B. subtilis have been created. These plasmids permit production in B. subtilis of protein products normally produced by other organisms, including hormones, vaccine components and enzymes. Without such plasmids, express soon of genes for these and other proteins would in most cases be impossible.
, 10 SUMMARY OF THE INVENTION

Plasmids useful for introducing into B. subtilis foreign DNA having a nucleic acid sequence which codes for production of a desired product are double-stranded deoxyribonucleic acid molecules which include a promoter DNA sequence which is not derived from B. subtilis plasm id DNA and a DNA sequence which is derived from a By subtilis plasm id. Preferably, the promoter DNA
sequence is derived from B. subtilis chromosomal DNA.

' When foreign DNA coding for the production of a desired product is incorporated into these plasmids, additional plasmids useful for effecting expression in B. subtilis of the foreign DNA are formed. The resulting plasmids are double-stranded DNA molecules which include the foreign DNA, a promoter DNA sequence nut derived from B. subtilis p]asmid DNA and a DNA sequence derived from a B. sublease plasm id.
The latter plasmids can be introduced into B. subtilis by transformation to produce genetically engineered bacterial cells which produce desired products when grown under suitable conditions. The products can then be recovered.

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: BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 shows the endonuclease restriction maps of various f plasmids constructed in accordance with the teachings 5 of this invention. Distances are measured from the EcoRl site of pB~322. Abbreviations for restriction sites are as follows: Avow, B-BamHl, Bg-B~II, H-Honda, P-PstI, Record Swahili 10 Fig. 2 shows the endonuclease restriction maps of further ¦ , plasmids constructed in accordance with the teachings i of this invention, including the plasm id pMF6.

DETAILED DESCRIPTION OF THE INVENTION
Plasmids have been constructed which are useful for the introduction into B. subtilis of foreign DNA which includes a gene or genes whose expression is associated ; with production of a desired product. The plasmids are l 20 double-stranded DNA molecules constructed in vitro by ;` enzymatic means. They contain a relatively short DNA
sequence necessary for activation of genes in Bacillus subtilis. The plasmids are of two broad types, those -- . . .
in which gene activation is stimulated during growth of 25 the bacterial host and those in which gene activation only occurs after growth ceases and spoxulation begins.
t In the plasmids, the regulatory DNA is attached Jo t longer sequences necessary for replication of the entire ; plasm id in B. subtilis, E. golf, or both.
t _ _ `. 30 Plasmids useful for introducing into B. subtilis foreign DNA, the nucleic acid sequence of which codes for pro-to diction of a desired product, are double-stranded DNA
I` molecules which include a promoter DNA sequence which i'; 35 is not derived from a B. subtilis plasm id and a DNA
sequence which is derived from a B. subtilis plasm id.

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The promoter DNA sequence may be derived from any source ' other -than a B. subtilis plasm id, the only limitation ; being its ability to function as a promoter when intro-Jo duped into B subtilis as part of a plasm id which also includes foreign DNA coding for a desired product.
Suitable sources include other bacteria, e.g. E. golf, phases and viruses.

Although the promoter DNA may be derived from any source, it is preferred that the promoter sequence be derived from B. subtilis chromosomal DNA. In particular, it is preferred that the promoter DNA sequence be derived from a 700 base pair wind III fragment of B. subtilis chromosomal DNA which includes a promoter DNA sequence associated with the tams gene carried on B. subtilis chromosomal DNA. Additional information concerning B.
subtilis chromosomal DNA and various promoters located thereon may be found in Haldenwang, WIG., et at., J.
Bacterial. 142:90-9g (1~80) and Haldenwang, WIG., et at., Cell 23:615-624 ~1981). In particular, Fig. 4 of the former publication shows a genetic and physical map of B. btilis DNA which includes the 700 by wind III
fragment. The disclosures of these publications show the state of the art and provide background information useful for a full understanding of this invention.

In some commercial applications, it may be desirable to haze the foreign DNA coding for the desired product expressed during cell growth. In others, it may be preferable to have the foreign DNA expressed during sporulation. In each case, the promoter DUN sequence should be active during the desired period. It is therefore an aspect of this invention to provide both plasmids active during _ subtilis cell growth and plasmids active during sporulation.

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, The plasmids of this invention also include DNA derived from a B. subtile plasm id which carries information necessary for replication of the hybrid plasm id. Numb Eros suitable plasmids exist including pueblo, pC194 POW, SUE and SUE

Although the plasmids will typically be circular when used to transform B. subtilis cells, they may also be linear.
, 10 Additional plasmids have been constructed which are useful for effecting expression in B. subtilis of foreign DNA having a nucleic acid sequence which codes for production of desired products. These plasmids are double-stranded DNA molecules which include the alone-mentioned foreign DNA, a promoter DNA sequence which is not derived from B. subtilis plasm id DNA and is capable of effecting expression of the foreign DNA when the ? plasm id is present in B. subtilis and a DNA sequence derived from a B. subtilis plasm id.

The promoter DNA sequence may be derived from widely varying sources. However, it is preferred that the promoter sequence be derived from B. subtilis chromoscmal I,- 25 DNA. The presently preferred promoter sequences are described hereinabove. They may be active either during cell growth or during sporulation. The DNA derived from a B. subtilis plasm id is also described hereinabove and may be circular or linear.
The foreign DNA having a nucleic acid sequence coding for production of a desired product may be derived from widely varying sources, including human, animal and plant sources, or it may be chemically synthesized. Examples of desired products, the genes for which may be used in the practices of this invention, include hormones, .

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e.g., insulin, somatostatin and animal growth hormones enzymes, antigens, vaccine components, e.g., viral coat proteins, and numerous other polypeptides.
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5 Particularly useful plasmids include plus, pLS5~Rl, pCEDl, pCED2, pCED3, plus Al and pMF6. The restrict I, lion maps for these are shown in Figs. 1 and 2.
, "pLS5QRl" is a plasm id carrying a regulatory sequence 10 active in growing cells of E. golf and B. subtilis. It ; can be used for initial cloning of foreign genes in E.
golf. The genes can be conveniently inserted next to the regulatory sequence if they are located -on DNA
fragments produced by endonuclease Hind III. Other 15 fragments can also be used after suitable chemical or enzymatic modification.

I, "pCED3" carries the same regulatory sequence as pLS5aRl, but can replicate in both E. golf and B. subtilis. It 20 also accepts fragments of foreign DNA produced by cleavage s with wind III or other suitably modified fragments.

"pLS5-llaRl" carries a regulatory sequence activated in B. subtilis only during sporulation. It is used for initial cloning in E. coil of foreign DNA fragments produced by endonuclqase EcoRl or other suitably modified fragments.

"pMF6" carries the same regulatory sequence a plus-llQRl/ but replicates in B. subtilis. It accepts EcoRl fragments or suitably modified fragments of foreign DNA.

The plasmids of this invention may be prepared by methods known to those skilled in the art. Generally, they are constructed by enzymatic joining or ligation of the various DNA sequences under suitable conditions of ) or ' temperature, time and the like Similarly, methods are known for identifying, recovering and purifying the various NOAH segments which are thereafter combined to form the plasmids; for transforming bacterial cells, including I. subtilis; for cloning transformed cells;
and for recovering desired products particularly polyp peptizes. Accordingly, chose methods will only be described by reference to specific embodiments of the invention set forth hereinafter.
EXPERIMENTAL RESULTS
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CONSTRUCTION AND EXPRESS ION OF pCEDl IN E . COY I
To construct a plasm id useful for cruising operon fusions, the promoter region of the tams gene was cloned as part of a 700 base pair Hind III fragment of B. subtilis DNA
that was ligated to Hind III-cut pBR322. The 700 by fragment was derived from p63 [Hutchison, KIWI. and Halvorson, Hoot Gene 8:267 (1980); and Haldenwang, WIG., et Allah Cell 23:615 (1981)]. The resultant plasm id, plus (shown in Fig. 1), was then digested with EcoRl and transformed into E. golf, selecting for ampicillin-resistance. The deletion plasm id that resulted, termed pLS5~Rl (also shown in Fig. 1), has a single Eland III
; 25 site known to be 85 by downstream from the tams start point for transcription [Haldenwang, WIG., et at., Cell 23:615 (1981)]. A Hind III fragment containing the __ lacy Y and A genes, but no promoter site for these genes, existed on pMBO41, a phenotypically Lacy plasm id.
plus and pMBO41 plasm id DNAs were digested with Hind III, ligated, and used to transform E. golf strain REV
(Luke). Ampicillin resistant transform ants were selected on plates containing the chromogenic substrate Go Blue colonies that appeared were screened for the presence of the pLS5~Rl-lac hybrid plasm id. A transform ant was isolated which harbored a plasm id, pCEDl (shown in Fig. 1), in which the fag genes were inserted downstream

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of the tams promoter. E. golf cells carrying pCEDl grew well on minimal medium with lactose as sole carbon source, indicating reasonably high level expression of the fag genes.

Direct assay of the ~-galactosidase activity indicated , that pCEDl was able to direct synthesis of high amounts of the enzyme (Table I). For comparison, cells carrying pCEDl have a 30-fold higher specific activity than lo those carrying pMBO41, the promoter-less version of the ;? same fag DNA. This high activity is comparable to that .
direct by pMBO40, a pBR322 derivative in which the fag genes are driven by the efficient fag UV5 promoter. A
`, restriction endonuclease map of pMBO41 corresponds to i 15 that of pMBO40 shown in Fig l, except that pMBO40 has an added EcoRl fragment of 205 by. Tins fragment originated in the E. golf chromosome from a mutant strain (W 5) i that shows unregulated expression of the fag operon.
This fragment is from the E. golf fag regulatory region.
Additional information may bye found in Silver stone, AYE., et at., Pro. Neil. Acadia Sat., USE. 66:773 (1970). Values for ~-galactosidase activity given in Table I have been normalized to protein content for later comparison with B. subtilis cells. The finding of 2.3 units per my protein for RV(pCED]), as measured with permeable cells, is equivalent to an activity of 350 units per ml culture in the standard assay of Miller, J., "Experiments in Molecular Genetics" Cold Spring EIarbor Laboratory Cold Spring Harbor, New York p.352 (1972).

pCEDl (lag+) is identical to pMBO41 (Lacy) except that a 29 by EcoRl-Hind III segment of pBR322 has been no-' placed by a corresponding 280 by segment of B. subtilis 35 DNA. This provides circumstantial evidence that the tams promoter is able to direct fag expression in E.
golf. When the fag DNA was inserted into pLS5~Rl in the orientation inverse to that of pCEDl~ a lag+ phenotype ~3~3~

was also observed, suggesting that a promoter site of pBR322 directs transcription left ward from the region of the let genes. This is confirmed by the fact that inverting the lac-pBR322 orientation of pMBO41 leads to the lag+ phenotype and by the direct observation by 'i Tuber, D. and Badgered, H., Pro. Neil. Aged. Sat., U.S.A. OWE (1981) of a transcription initiation site near the beginning of the let genes, from which RNA is copied from the anti-sense strand of the let sequence.
pBR32~, the cloning vector from which plus was derived, normally determines resistance to at least 29 go twitter-cyclone per ml. Since in constructing plus, B. subtilis DNA was cloned into the Hind III site of pBR3~2, a site located within the promoter region for the let genes, it is not surprising that pLS5-containing strains are only resistant to 10 go tetracycline per ml The sub-sequent deletion that formed pLS5~Rl removed 29 by of pBR322 DNA and 420 by of B. subtilis EDNA This led to an unexpected increase in the level of tetracycline-resistance. E. golf cells carrying plural are resistant to 25 I tetracycline per ml. A possible explanation is that the deletion brought together DNA sequences which act as a new promoter for transcription of the let genes.
. ---Alternatively, the deleted DNA may have contained termination sequence or a sequence inhibitory to tams expression. After insertion of fag DNA between the tams promoter and let, the level of resistance determined dropped to 10 gel These perturbations in the level of tetracycline-resistance prevent one from concluding that the only source of fag expression in pCEDl-carrying cells is the tams promoter.

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, TABLE I
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I, EXPRESSION OF ~-GALACTOSIDASE IN GROWING CELLS

, 5Permeabilized cells Strain , Tested CH~3-SDSI'risJToluene Extracts ',, _ < 0.01 - < 0.01 , RV(pCEDl) 2~3 ` 20 E. golf RV(pCED2) 2.3 _ 15 DO100~pMBC~0) 3.0 - 15 DO100(pMBC41) 0.08 - 0.7 ', 15 ` BRIE <0~01 <0.01 BR151(pOED3) 1.5 2.0 B. subtilis BR151(pUB110) ~0.01 <0.01 T~NSFORMATION OF B. SUBTILIS BY pCEDl pCEDl does not replicate in B. subtilis, but contains a 280 by sequence of DNA from the B. subtilis gnome.
This homology was apparently insufficient to generate recombination events that would lead to plasm id integration into the B. subtilis chromosome Even in a congress ion experiment in which competent cells were exposed to 10 go of pCEDl DNA, any Lucy colonies were unable to be detected among transform ants.

The tms-lac was therefore cloned onto pub, a high copy number plasm id which replicates in By subtilis.
; pub contains one BamHl site; linearized pub (4400 by) was exchanged for the small BamHl fragment (1100 by) of pCEDl, creating pCED2 (shown in Fig. 1). The kanamycin resistance coded for by pub is expressed 3231i9 , .
in B. subtilis and E. golf. All kanamycin resistant transform ants of E. golf were ampicillin resistant, lag+, and Tots. The latter was expected since part of the coding sequence of let was removed.

TRANSFORMATION OF B. SUBTILIS BY pCED2 ;, _ When pCED2 was transformed into B. subtilis, many of the initial Kant transform ants were Lacy+ (i.e., blue on XG plates). When these transform ants were purified, however, they proved to be segregate Lacy clones at very high frequency. Plasm id No isolated from such unstable transform ants was of variable size, but was always less than that of pCED2. If transform ants were purified on non-selective medium (i.e., without kanamycin), the plasm id was lost completely.

It was thought that pCED2 might be stabilized in B.
subtilis strain CB20 [Haldenwang, WIG., et at., J., ; Bacterial. 142:90 (1980)]. This strain-contains in its chromosome an integrated plasm id that has some homology with pBR322 component of pCED2. Transformation of this strain with pCED2, however, also resulted in lag+ colonies that segregated Lacy derivatives at high frequency.
Repeated sub culturing of the lag+ colonies led eventually to the isolation of a stably lag+ derivative. Restriction ¦ analysis of the plasm id DNA of one of these stable I transform ants showed that a deletion had occurred. The deletion plasm id, termed pCED3, had lost about 3 Kb of I; DNA at the junction of pueblo and the fag genes (see ¦ 30 Fig. 1). When pCED3 was transformed into E. golf strain REV, the resultant transform ants were phenotypically LacZ+LacY~. The deleted DNA starts beyond the EcoR1 site within the lacy gene, extends through the lacy and A genes, and ends within the pueblo sequence.

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The presence of chromosomal homology in strain CB20 was apparently of no consequence in the stabilization of pCED3.
Transformation of pCED3 in-to B. subtilis strain BRIE gave fully stable transform ants. The stabilization may be due to reduction in the size of the plasm id. It seems more likely to us -that the deletion removes a DNA sequence that is harmful to B. subtilis.
A good candidate for such a sequence is the lacy gene, which codes for a membrane protein. Although B. subtilis strains harboring pCED3 are stably Lacy on plates containing kanamycin, they show rapid loss of both lag+ and Kant phenotypes when grown in the absence of drug.

B-GALACTOSIDASE ACTIVITY IN B. SUBTILIS

Table I compares the activity of B-galaetosidase in growing cells of E. colt and B. subtilis with and without plasmids. Both permeabilized cells and extracts were assayed. It is clear that the presence of pCED3 leads -to halve] expression of B-galac-tosidase in _ subtilis. No detectable activity was found in cells without plasm id or in cells carrying pueblo. Because of a report [Caulfield, MOP., et at., J. Bitterly. 138:345 (1979)]
that a strain of B. sub-tilis designated 1007 has a B~galaetosidase activity inducible by Lutz, a search or such activity in BRIE was conducted. No such activity was found.

In vitro transcription studies of the tams region indicate that the tams gene is only transcribed by the major RNA polymers present in vegetative cells [Haldenwang, WIG., et at., Cell 23:615 ~19~1)]. RNA polymers molecules containing sigma factors that recognize sporulation genes in vitro do not initiate transcription effectively from the tams promoter. By assaying B-galaetosidase in the fusion strain during growth and sporulation, it was found that B-galaetosidase activity 1.3~3~

. ,, decreased five-fold by To (Table II). Activity was , measured in extracts of sonicated cells to avoid the possibility of differential permeabilization of verge-I, native and sporulating cells. The amount of soluble protein liberated by sonication was constant for each ', sample taken after To. The decrease in ~-galactosidase activity during sporulation is consistent with the idea I' that the tams promoter is only poorly active after the end of growth, and the ~-galactosidase protein is subject ,; 10 to normal sporulation-associated protein turnover. The possibility that regulation of ~-galactosidase is at some stop other than transcription has not been clearly ruled out. Experiments are now in progress to demon-' striate that the rates of ~-galactosidase protein and Mona synthesis decrease during sporulation.

TABLE II

EXPRESSION OF ~-GALACTOSIDASE IN GROWING
f 20 AND SPORULATING CELLS OF BRIE (pCED3) l Time of Units of ~-Galactosidase activity harvest per my protein f` 25 -2 3.0 I To 1.4 f To 1.1 To 0.6 f __ _ __ I; .

-- -- _ _ _ _ _ _ _ _ _ The construction of plasmids in which protein coding sequences from E. golf have been inserted downstream from a B. subtilis promoter site has been accomplished.
Although additional work is needed to conclusively SUE

prove that the B. subtilis tams promoter is directing gene expression by these plasmids, that notion is supported by a number of facts. First, the structures of pCED1 and pMBO41, which cause lag+ and lag- phenotypes, respectively are nearly identical. They differ only in the replacement of a 29 by segment of pBR322 DNA by a 280 by segment of B. subtilis DNA. This makes it very unlikely that transcription of the fag genes initiates within any E. coli-derived DOW. In addition, the trays-crip~ion map of pBR322 has been well studied. No pBR322 promoter is known that would direct transcription of ; fag genes in the orientation found in pCEDl stubbier, D.
, and Badgered, H., Pro. Neil. Aged. Sat, U.S.A. 78:167 (1981)].
Expression from the tams promoter in E. golf is not unexpected. The sequence of this promoter site s correlates well with sequences thought to be important for activity of E. golf promoters. This promoter is a strong site for initiation of transcription in vitro by the major vegetative (a55-containing) form of B. subtilis ! RNA polymers [Haldenwang, WIG., et at., Cell 23:615 ~1981)]. Thus, it is not surprising that fag expression I could be detected in B. utilize as well. We would i 25 anticipate that many other coding sequences inserted downstream of the tams promoter would be expressed in By subtilis.
-The site ox translation initiation in B. subtilis for ~-galactosidase coded for by pCED3 is unknown. There are at least three possibilities. The palpated may initiate at the normal lacy site, at the trap site, or at the presumed tams site. In the latter two cases, a fused palpated would be made. Efforts are underway to answer this question by examining the size of the I-galactosidase palpated. The three possible start points are sufficiently far apart that the size of the product should clearly indicate its origin.
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lso underway are examinations of pulse-labelled extracts of B. subtilis cells at various stages of sporulation to measure the rate of synthesis of B-galactosidase at different -times. These results will be correlated with measurements of the rate of synthesis of lacy Mona.

The creation of a tms-lac fusion plasm id that expressed B-galactosidase in both E. golf and B. sub-tilis allows one to use the lag phenotype in searching for promoter mutants.
It will be interesting to compare the effects of particular mutations on gene expression in two hosts.

In addition to the plasmids described here, -there has been developed a promoter-less plasm id, pCED5, that has the lacy gene attached to pBR322 and pueblo. This plasm id was constructed with a deletion internal to the lacy gene, which may be important for plasm id maintenance in B. sub-tilis.
pCED5 can accept a variety of restriction fragments and can serve as a probe for DNA segments that act as promoters in E. golf, s. subtilis or both.

In companion experiments, there has been fused a promoter site for a gene expressed early during sporulation to a segment of the -try operon of B. pummels. Assays of -the expression of these genes in E. golf and B. subtilis indicate -that the promoter is functional.

The enzyme B-galactosidase has been recovered and its identity confirmed in E. golf. Preliminary analytical data would lead one skilled in the art to conclude that -the enzyme is also present in B. sub-tilis.

In other experiments, the hybrid plasm id ply (pC194 prom B. subtilis ligated to pMB9 from E. golf) has been shown to replicate autonomously in E. golf, but transform B. sub-tilis only transiently. Stable transform ants can `.~

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be obtained by introducing fragments of the B. subtilis chromosome into ply. The transform ants arise by homologous recombination 'ceding to integration of the plasm id DUN and duplication of the relevant chromosomal DNA. This approach has been used to map the chromosomal site of particular fragments and to create strains that are deployed for desired markers. In addition, efforts are underway to recover from the integrated state don-natives of ply that replicate in I. golf and carry neighboring B. subtilis chromosomal DNA.

To study the regulation of vegetative and sporulation genes, small fragments (280-420 by) have been cloned that contain various promoters fused to segments of the try operon. Expression of genes from B. pummels to quantitate the activities of these promoters under various physiological conditions can be measured. To prepare DNA templates appropriate for in vitro trays-Croatian studies promoter proximal fragments of verge-native, sporulation and catabolite-controlled genes identified in a By sub tills bank are being sub cloned.

CONSTRUCTION OF pMF6 The 700 by Hind III piece of B. subtilis DNA that had been cloned in pBR322 to make plus was inverted by cutting and relighting. This produced plus. A
deletion step removed about 310 by from the left end, creating ply Creole. This plasm id was ready to accept foreign DNA that could be inserted downstream of the 0.4 gene (sporulation gene) promoter.

The source of foreign DNA was sly [Keg gins, et at., Pro. Neil. Aged. Sat. 75:1423-1427 (1978)], which is a hybrid of pub and a part of the try operon of B.
pummels. These genes were excised and ligated to I

EcoRl-cut pLS5-ll~Rl. By transformation of E. golf, strains were isolated that had acquired the expected plasm id (i.e., pMFl shown in Fig. 2).

An internal Hind III fragment of pMFl that includes the 0.4 gene promoter and the first two genes of the try operon was ligated to Hind III-cut pC194 (a Staphylococcus aureus-B. subtilis plasm id that determines chloramphenicol-resistance). Transformation of B. subtilis yielded strains carrying pMF6 (shown in Fig. 2).

M HODS AND MATERIALS

ENDONUCLEASE DIGESTIONS AND DNA LIGATION
All restriction endonuclease digestions were carried out for 60 minutes at 37C and were terminated by heating for 10 minutes at 65C. Reaction conditions were those suggested by the suppliers of the enzymes. DNA fragments were ligated with To DNA ligate during incubation for 1-2 hours at room temperature or overnight at 13C.

ISOLATION OF PLASM ID DNA

Plasm id DNA was purified from E. golf strains by the following method:

1. Grow 30 ml cultures in enriched minimal medium with antibiotic to select for plasm id carrying cells (under nitrogen at 37C);
2. Harvest cells at 10K, 4C for 10 minutes in 50 ml screw cap Oak Ridge tubes;
3. Wash pellets with 5 ml of 0.05M Trip pi 8.0, vortex and spin at 10K, 4C for 10 minutes;

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4. Resuspend pellets in 0.5 ml of 0.05M Trip pi 8.0 containing 25 percent sucrose. Transfer to small plastic screw cap tubes with Pasteur pipettes;

5. Add 0.12 ml lysozyme (5 mg/ml fresh), mix gently and incubate 5 minutes on ice;

6. Add 0.4 ml Briton lyric mix containing 100 g/ml RNase (from 2 mg/ml, preheated stock) mixing gently while adding. Keep at 4 C under nitrogen;
I, 7. Spin at 20K, 4C for 60 minutes in SUE rotor;
8. Remove supernatant by pouring into 13 X 100 glass screw-cap tube (should get I ml);
9`: Adjust volume to 2 ml with 0.05M Trip pi 8.0;
lb. Add 2 ml of phenol (2x saturated with 0.05M Trip pi 8.0), mix gently to form emulsion and roll at 4-6C for 25 minutes;
11. Separate phases at OK, for 10 minutes at room ', temperature;
12. Remover lower phenol layer leaving behind any I' material at interface;
Jo 20 13. Add 2 ml fresh phenol and repeat step 10;
, 14. Separate phases; remove upper aqueous phase leaving behind interface and place in 20 ml snap-cap ', polypropylene tubes, 15. Extract 2x with 1.5 volumes ethyl ether (in hood).
it 25 Mix and allow to settle each time before removing i upper ether phase Aqueous should change from very cloudy to clear as ether extraction continues;
I To aqueous phase add 1/10 volume (0.2 m]) EM sodium acetate and 4 ml of prechilled 95 percent ethanol.
six well and leave at 20~C under nitrogen;
17. Spin at lox 4C for 20 minutes after removing caps (which will not fit in rotor) and covering with parafilm;
18. Discard supernatant, rinse (do not resuspend) I` 35 pellet with 2 ml cold 95 percent ethanol, then ; place at -70C for 1 hour;

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.,, 19. Repeat steps 17 and 18. Remove last traces of ethanol by placing in vacuum lyophilizer. Replace caps and punch hole in center before placing tubes in desiccator; and 5 20. Dissolve DNA in 0.1 ml of 0.01 TAO and store at ; 4~C
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Briton Lois Mix 50 ml I!' 0.3% Briton X100 0.15 ml stock Briton X100 Jo 10 0.15M Trip pi OWE 7.5 ml lo Trip pi By 0.18M Nay ETA 36 ml 0.25M ETA, pi 8.0 6.35 Ho ' 50 ml Plasm id DNA was purified from By subtilis strains by the method of Gryczan, TO et at., J. Bacterial.
134:318 (1978). In all cases, the purification was followed by equilibrium density gradient centrifugation in sesame chloride containing ethidium bromide.
TRANSFORMATION BY PLASM ID DNA
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Calcium chloride-treated cells of E. golf cells were I¦ transformed with plasm id DNA or ligated DNA as described i US by Cohen, S., et at., Pro. Neil. Aged. Sat., U.S.A.
~9:2110 (owe Competent cells of B. subtilis were prepared as described by Contented S. and Dubnau, D., Mole. con. Gent. 167:251 ~1979). Antibiotic resistant , transform ants were selected after 1.5 hours of growth after exposure to DNA in antibiotic-free medium. Drug concentrations used were per ml 10 go ampicillin, 10 go tetracycline and 5 go kanamycin.

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E. golf cells were made permeable by treatment with SCHICK and sodium dodecyl sulfate (SDS) according to Miller, J., "Experiments in Molecular Genetics", Cold Spring Harbor Laboratory, Cold Spring Harbor, New York p.352 (1972). Permeable cells of B. subtilis were prepared by the cold Tris-toluene method of Fisher, S., et at., In 'Spores VI" (P. Gerhardt, et at., editors), American Society for Microbiology, Washington, DO
p.226 (1975). To make extracts, cultures were concern-treated 100-fold in PM2 buffer [Miller, J., "Experiments in Molecular Genetics", p.3S2 ~1972)] containing phenol-methylsulfonyl fluoride and sonicated for 3-6 minutes in a beaker with a jacket through which ice salt water was continually passed. The sonic ate was centrifuged for 1 hour at 100,000 xg. The supernatant fluid was assayed for ~-galactosidase activity.

~-yalactosidase was assayed as cleavage of o-nitrophenyl ~-D-galactoside by the method of Miller, J., "Experiments in Molecular Genetics", p.352 (1972). One unit of activity produces an increase in absorbency at 420 no of 1Ø Activity it expressed in units per my protein.
For whole cell assays, total cellular protein was measured.

nBTEcTIoN OF LAO PHENOTYPE
_ E. golf and B. subtilis strains were assayed for Lacy phenotype on L ajar containing 5-bromo-4-chloro-3-indolyl-R-D-galactoside [XG; Davies, J. and Jacob, F., J. Mol. Blot. 36:413 (1968)]. Lucy colonies are blue, Lucy colonies are white. To test the Lacy phenotype of E. golf strains, they were plated at 42C on Meg-Convey Ajar containing Melissa [Prestige, LO andPardee, AHAB., Become. Buffs. Act. 100:591 (1965)].
Melibiose is transported by the fag permeate. Lacy colonies are red; Lacy colonies are light pink or white

Claims (40)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A plasmid useful for introducing into B. subtilis foreign DNA, the nucleic acid sequence of which codes for production of a desired product, comprising a double-stranded DNA molecule which includes a promoter DNA
sequence derived from B. subtilis chromosomal DNA and a DNA
sequence derived from a B. subtilis plasmid.

2. A plasmid in accordance with Claim 1 wherein the promoter DNA sequence is derived from a 700 base pair Hind III fragment of B. subtilis chromosomal DNA.

3. A plasmid in accordance with Claim 1 wherein the promoter DNA sequence is associated with the tms gene carried on B. subtilis chromosomal DNA.

4. A plasmid in accordance with Claim 1 wherein the promoter DNA sequence is active in B. subtilis during cell growth.

5. A plasmid in accordance with Claim 1 wherein the promoter DNA sequence is active in B. subtilis during sporulation.

6. A plasmid in accordance with Claim 1 wherein the DNA
sequence from a B. subtilis plasmid is derived from pUB110.

7. A plasmid in accordance with Claim 1 wherein the DNA
sequence from a B. subtilis plasmid is derived from pC194.

8. A plasmid in accordance with Claim 1 wherein the DNA
sequence from a B. subtilis plasmid is derived from PE194.

9. A plasmid in accordance with Claim 1 wherein the DNA
sequence from a B. subtilis plasmid is derived from PSA0501

10. A plasmid in accordance with Claim 1 wherein the DNA
sequence from a B. subtilis plasmid is derived from PSA
2100.

11. A plasmid in accordance with Claim 1 wherein the double-stranded DNA molecule is circular.

12. A plasmid in accordance with Claim 1 wherein the double-stranded DNA molecule is linear.

13. A plasmid useful for effecting expression in B.
subtilis of DNA which is foreign thereto and has a nucleic acid sequence which codes for the production of a desired product, comprising a double-stranded DNA molecule which includes said foreign DNA, a promoter DNA sequence which is derived from B. subtilis chromosomal DNA and is capable of effecting expression of said foreign DNA when the plasmid is present in B. subtilis and a DNA sequence derived from a B.
subtilis plasmid.

14. A plasmid in accordance with Claim 13 wherein the promoter DNA sequence is derived from a 700 base pair Hind III fragment of B. subtilis chromosomal DNA.

15. A plasmid in accordance with Claim 13 wherein the promoter DNA sequence is associated with the tms gene carried on B. subtilis chromosomal DNA.

16. A plasmid in accordance with Claim 13 wherein the promoter DNA sequence is active in B. subtilis during cell growth.

17. A plasmid in accordance with Claim 13 wherein the promoter DNA sequence is active in B. subtilis during sporulation.

18. A plasmid in accordance with Claim 13 wherein the DNA
sequence from a B. subtilis plasmid is derived from pUB110.

19. A plasmid in accordance with Claim 13 wherein the DNA
sequence from a B. subtilis plasmid is derived from pC194.

20. A plasmid in accordance with Claim 13 wherein the DNA
sequence from a B. subtilis plasmid is derived from PE194.

21. A plasmid in accordance with Claim 13 wherein the DNA
sequence from a B. subtilis plasmid is derived from PSA0501.

22. A plasmid in accordance with Claim 13 wherein the DNA
sequence from a B. subtilis plasmid is derived from PSA2100.

23. A plasmid in accordance with Claim 13 wherein the double-stranded DNA molecule is circular.

24. A plasmid in accordance with Claim 13 wherein the double-stranded DNA molecule is linear.

25. A plasmid in accordance with Claim 13 wherein the foreign DNA codes for the production of a polypeptide.

26. A plasmid in accordance with Claim 13 wherein the foreign DNA codes for the production of a polypeptide and wherein said polypeptide is insulin, growth hormone, the polypeptide portion of interferon, somatostatin or an enzyme.

27. Plasmid pLS5 having the restriction map shown in Fig.
1.

28. Plasmid pLS5.DELTA.RI having the restriction map shown in Fig. 1.

29. Plasmid pCED1 having the restriction map shown in Fig. 1.

30. Plasmid pCED2 having the restriction map shown in Fig. 1.

31. Plasmid pCED3 having the restriction map shown in Fig. 1.

32. Plasmid pLS5-11.DELTA.RI having the restriction map shown in Fig. 2.

33. Plasmid pMF1 having the restriction map shown in Fig.
2.

34. Plasmid pMF6 having the restriction map shown in Fig.
2.

35. A method of preparing the plasmid of Claim 1 which comprises obtaining a promoter DNA sequence from B. subtilis chromosomal DNA, obtaining a DNA sequence from a B. subtilis plasmid and joining these sequences under suitable conditions.

36. A method of preparing the plasmid of Claim 13 which comprises inserting foreign DNA coding for a desired product into the plasmid of Claim 1 under suitable conditions.

37. A method of introducing into a B. subtilis cell foreign DNA which codes for the production of a desired product which comprises transforming said cell with a plasmid in accordance with Claim 13 under suitable transforming conditions.

38. A B. subtilis cell produced by the method of Claim 37.

39. A method of producing a desired product which comprises introducing into a B. subtilis cell foreign DNA
which codes for production of said desired product according to the method of Claim 37, growing said cells under suitable conditions permitting expression of said foreign DNA and production of said product and recovering the product so produced.

40. A B. subtilis cell. which contains a plasmid in accordance with Claim 1.