CA1231314A - Plasmids for foreign gene expression 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 also not 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. The plasmids may additionally include an inducible gene.

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

PLASM S FOX FOREIGN GENRE

EXPRESSION IN B. _UBTILIS

BACKGROUND OF THE INVENTION

Considerable interest exists in the application of genetic engineering techniques for the production of commercially valuable products such as insulin, human and animal growth hormones and enzymes. Much of the work to date has involved use of Escherichia golf as the host into which foreign genetic material is intro-duped. Expression of the genetic material in E. golf results in production of desired products. When combined with growth of genetically engineered cells in culture, it permits production of the desired products in common-Shelley meaningful yields. Unfortunately, use of E.
golf as a host is associated with certain disadvantages.
As a result, alternative hosts, including other bacteria and yeast, are under investigation.
One particularly promising host for commercial applications of genetic engineering is Bacillus subtilis. B. subtilis is a non-pathogenic, gram positive bacterium which is eaten daily by millions of Japanese as part of a fermented soybean product. B. subtilis may be the safest bacterium in which to achieve expression of foreign genes whose products, e.g. interferon, will be purified and sub-sequently injected into humans for at least two reasons.
First, B. subtilis is known to be non-pathogenic.
Secondly, _ golf is known to produce endotoxins which may contaminate genetic products and induce endotoxic shock in humans.

Direct expression in Bacillus subtilis of a gene origin-cling in Escherichia golf has been achieved in a single reported case [Ruin, EM., et at., Gene l0:227-235 (1980)] There the E. golf gene specifying thymidylate synthetase expressed upon integration into the B. subtilis chromosome. However, E. golf genes residing on plasmids typically do not express at the level of genetic function in B. subtilis. [Knelt, I., et at., Mol. con. Gent.
162:59-67 (1978)]. The least complex explanation for the lack of foreign gene expression in B. subtilis is an absence of correct transcription and/or translation.
In vitro transcription studies have indicated that E.
golf RNA polymers is significantly more efficient in initiating transcription from an E. golf promoter than is the B. subtilis RNA polymers [Lee, G., et at., Mol. con. Gent. 180:57-65 (1980)]. It was therefore suspected that inserting an _ golf gene, or other foreign gene, into a segment of DNA known to be efficiently transcribed in B. subtilis might permit functional expression of the foreign DNA.

SUMMARY OF THE INVENTION

A double-stranded DNA plasm id which includes a promoter DNA sequence not derived from B. subtilis plasm id DNA
and a DNA sequence derived from a B. subtilis plasm id is useful for introducing into B. subtilis foreign DNA
which includes a gene coding for the production of a desired product. Preferably, the promoter DNA sequence is not derived from B. sub-tilis. examples of suitable sources of the promoter include bacteriophages SPY and ~105, B. pummels plasm id ply and B. licheniformis.
The plasm id may be linear or circular and may additionally include an inducible gene such as a chloramphenicol inducible gene coding for synthesis of chioramphenicol acetyltransferase.

A double-stranded DNA plasm id which includes a promoter DNA sequence which is not derived from B. subtilis, a DNA sequence derived from a B. subtilis plasm id and foreign DNA which includes a gene coding for the production of a desired product such as a palpated I

and which is capable of expression when the plasm id is introduced into _ subtilis is useful in the production of the desired products for which the foreign DNA codes.

B. subtilis cells which contain the plasmids are novel.
They may be grown in culture to produce commercially valuable materials which can then he recovered.

BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 shows the restriction endonuclease maps of three plasmids, ply, ply and ply. The thickened horizontal line indicates the cloned B. pummels DNA in each plasm id.

Fig. 2 is a diagram of the insertion and orientation of the 1.7 My E. golf tropic fragment in ply. Orientation of the fragment was determined from double digests of ply with Hint II and EcoRI.

Fig. 3 shows induction by chloramphenicol of InGPS specified by ply. BD224 (ply) was grown to the middle of the exponential growth phase in Mix OH containing 100 go per ml of tryptophan. The culture was split and 50 ml samples were withdrawn from each. A sub inhibitory concentration of Cur (0.1 ~ug/ml) was added to one culture, and during subsequent growth, 50 ml samples were periodically withdrawn. Cells in each sample were harvested, processed and assayed for InGPS as described in Hook, SO. and Crawford, IMP., J. Bacterial. 116:685-693 (1973).

Fig. 4 is a diagram of pPL608-TRl. Orientation of the Sty fragment was deduced from Sty and Bgl II double digests.

--'I--DETAILED DESCRIPTION OF THE INVENTION

Plasmids have been created which are useful for the introduction into Bacillus subtilis of foreign DNA
whose nucleic acid sequence includes one or more genes encoding the information necessary for production of desired products, particularly palpated or partially palpated products such as insulin, Thomson, growth hormones, enzymes, antibodies and the various interferon.
These plasmids ale double-stranded DNA 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 B. subtilis plasm id. These plasmids may be linear or circular. However, when used for bacterial transformation, they will generally be circular.
The promoter DNA sequence is preferably not derived from B. subtilis chromosomal DNA, but rather from another source. Numerous suitable sources are available, including the bacteriophages SPY and ~105, the B. pummels plasm id ply and B. licheniformis. The origin of the DNA from a B. subtilis plasm id may likewise vary widely, any B.
subtilis strain being capable of functioning as a source of the plasm id DNA. One example of a suitable source is the B. subtilis plasm id pub.
-These plasmids may additionally include an inducible gene such as a drug inducible gene to facilitate selection of cells carrying the plasm id and increased gene expression in the cells so selected. Illustrative of such inducible genes are a chloramphenicol-inducible gene coding for synthesis of chloramphenicol acetyltransferase. One such gene may be obtained from B. pummels NIB 8600.

Additional plasmids have been constructed which are useful for effecting expression in Bacillus subtilis of DNA which does not naturally occur in B. subtilis and which has a nucleic acid sequence which includes one or I

more genes associated with the production of desired products or the expression of useful properties. These plasmids are double-stranded, deoxyribonucleic acid molecules which include a promoter DNA sequence which is not derived from B. subtilis plasm id DNA, a DNA
sequence derived from a B. subtilis plasm id and foreign DNA having a sequence coding for the production of a desired product. These plasmids may be linear or circular, although they are generally circular when used in bacterial transformation.
The promoter DNA sequence is preferably not derived from B. subtilis chromosomal deoxyribonucleic acid, but from a source other than B. subtilis. Although numerous _ sources are available, suitable sources include bacteria-phases SPY and ~105, the B. pummels plasm id ply and B. licheniformis. Of these, a promoter obtained from SPY is presently preferred.

The DNA derived from a _ subtilis plasm id may be obtained from any such plasm id At present, the preferred source is the B. subtilis plasm id pueblo.

Foreign DNA which includes a gene or genes associated with production of a desired product may be obtained from any appropriate source or may be chemically Cynthia-sized. Examples of suitable foreign DNA sequences - include mammalian genes coding for interferon and genes coding for insulin, enzymes, growth hormones, viral antigens and the like.
The plasmids may also include an inducible gene such as a chloramphenicol-inducible gene coding for the synthesis of chloramphenicol acetyltransferase.

fly Methods for identifying, recovering and purifying the various DNA segments which are portions of the plasmids are known to those skilled in the art as are methods for ligating the segments, transforming bacterial cells, cloning and recovering products synthesized. Accordingly, the methods will only be described by reference to specific embodiments of the invention set forth hereinafter.

Certain of the experimental details which follow were reported in Williams, DIM., Devil, EDGY. and Levitt, P.S., J. Bacteriology, 146:1162-1165, June 1981, and show the state of the art.

DNA preceding structural genes contains regulatory sites essential to gene expression, including sequences necessary for initiation of transcription and translation. Selective expression of genes involved in the developmental process of bacterial sporulation may be partially controlled by the occurrence of sporulation-specific regulatory sequences that participate in modulating sporulation gene expression. A
direct approach to isolate regulatory sequences from DNA was provided by the development of plasm id vectors [Clowns, ARC
Bacterial. Rev. 36:361-405 (1972)] useful for cloning DNA
segments that act as promoters and translation initiation signals in Escherichia golf [An, G., and Frozen JO J.
Bacterial. 140:400-410 (1979); Casadaban, MY et at., J.
Bacterial. 143:971 980 (1980); and Casadaban, M. and Cohen, SUN., J. Mol. Blot. 138:179-207 (1980)]. The potential value of similar cloning vectors for the Bacillus subtilis system prompted screening for a structural gene that was potentially capable of expression in B. subtilis, but appeared to lack those regulatory controls needed for expression. A description follows of the development I

of a plasm id cloning vector that permits direct selection for cloned fragments of DNA which are necessary to the expression of a chloramphenicol acetyltransferase gene in B. subtilis.

Plasm id ply was generated by cloning into the 3-megadalton, neomycin-resistance plasm id pueblo [Gryczan, TO et at., J. Bacterial. 134:318-323 (1978); Sedate, Y., et at., J. Bacterial. 141:1178-1182 (1980~] a 1.44-My, EcoRI-generated fragment from DNA isolated from a chloramphenicol-resistant (Char) derivative of Bacillus pummels NIB 8600, using methods previously described.
[Levitt, P.S. and Keg gins, KIM., Methods Enzymol.
68:342-357 (1979)]. ply (shown in Fig. 1) confers on host cells, e.g., BR151,resistance to both neomycin (10 gel and Cur.

Plasm id ply is a derivative of plus in which the orientation of the Char fragment was reversed by EcoRI
cleavage and ligation. BRIE harboring either pow or ply grew in Puns broth (PUB; Disco Laboratories) containing up to 200 go of Cur per ml (higher levels were not tested). Cells harboring both plasmids produced chloramphenicol acetyltransferase (Table I). BR151(pPL531) or BRl51(pPL600) grown on 5 I of Cur per ml produced approximately 10-fold higher levels of chloramphenicol acetyltransferase than cells grown in the absence of the drug (Table I). Deletion of the Sty fragment (~0.55 My) from ply generated plasm id ply (Fig. l;
Table I). BR151(pPL601) was incapable of growth in PUB
containing 25 go of Cur per ml, although normal growth was observed in 5 go of Cur per my and sparse growth was observed in 10 I of the drug per ml. The specific activity of chloramphenicol acetyltransferase in BR151(pPL601) grown in PUB containing 5 go of Cur per ml was 20-fold lower than the activity produced by BRIE
(ply) (Table I). Reversal of the orientation of the a C~LoRA~p~ENIcoL RESISTANCE PROPERTIES

. Chlora~nenia~ ethyl-Plod Insert Do wit Swallower of erupt transfer go sup act Sax Oh b ( 106~
cry or pP1531 0.04 0.52 200 10pÆ.600 0.09 0~96 2û~
pull Wool 0~03 S
purl 0~04 0~71 ~00 ~03 (Allah 0~04 5 S041 1~3 Decor BLUE 0~334~37 2C~
ply ~54 En pP~10 0~03 0~2~3100 OWE 0~21 Eke* oil ~;P02 0~214~02 kiwi F$~613 0.15 ITS oil B EYE o . 63 21 !110 pP~614 1.0 EKE of En. 0.34 0.68 100 Jo .
method ox arrowhead [arrowhead. PLY, Anal it owe (1976)].

b. Prowl 107 pld~cont~ cells prune grin in Pi ode 5 lug Shea For ml were inlaid into 2 ml of PUB canonry Cur 30 at 5, I 25, 50, 100 or 2~0 ~g/TDl. Lncuba~n ceded or 20 hocus at which time cx~nOE~ratiC~n of Cur allowing Goethe I remrded.

I _ insert in ply or deletion of -the Sty fragment from ply generated ply which, in BRIE, conferred resistance to 200 go of Cur per ml (Table I).

Since ply did confer high-level Char, it was evident that an intact structural gene for chloramphenicol acetyltransferase was present on the cloned EcoRI frog-mint after the internal Sty deletion was made. Moreover, the inability of ply to confer high-level Char sup-gusted that expression in ply was dependent on some property of the vector plasm id pueblo. For example, if the Sty deletion removed a promoter region from the cloned fragment, expression of the gene would be dependent on a pueblo promoter. Hence, chloramphenicol acetyltrans-erase activity would only be expressed when the struck tubal gene was correctly oriented relative to the ox-vernal promoter. In ply, the gene is expressed and therefore the orientation would be correct. In ply, the orientation is incorrect, and the gene is unexpressed or very weakly expressed.
Cloning Bohemia or MboI fragments of Bacillus licheniformis DNA into the Bohemia site on ply failed to permit ply to transform BRIE to Char (selection on tryouts blood ajar base containing 10 go of Cur per ml). Cloning Sty fragments of the same DNA into ply allowed ply to successfully generate numerous Char transform mints of BRIE, suggesting that inserts left of the Sty site of ply (shown in Fig. 1) might promote chloramphenicol acetyltransferase activity. Accordingly, MU the Bohemia proximal EcoRI site on ply was deleted by Bohemia and BgIII digestion, ligation, and transformation of BRIE with selection for Noon. A resulting plasm id which retained a single EcoRI site was designated ply (Fig. l; Table I).

EcoRI* or EcoRI activity [Polisky, BY e-t at., Pro.
Neil. Aged. Sue, U.S.A. 72:3310-3314 (1975)] was used to digest ply, pub, and DNA from B. licheniformis 9945~ and phase ~P02 (~emphill, HUE. and Whitely, HER., Bacterial Rev. 39:257-315 (1975); Levitt, P.S., et at., J. Bacterial. 127:817-828 (1976); Escher, BUM., et at, J. Viral. 28:395-402 (1978); and Yenned, Y., et at., Gene 7:51-68 ~1979)]. Approximately 1 go of EcoRI*- or EcoRI-digested DNA was mixed with 1 go of EcoRI-digested ply in 100 lo The ligated DNA
(approximately 2 go was shaken with 3 X 108 competent [Boil, OF and Wilson, GUY., J. Bacterial. 94:562-570 (1967)] _ subtilis cells for 1 hour and plated directly onto tryouts blood ajar base containing 10 go of Cur per ml. About 200 to 600 Char transform ants were obtained per go of input DNA. Each of more than 200 Char transform ants was neomycin resistant, and each of a total of 83 examined by the single colony lysis-gel electrophoresis procedure contained plasm id having a molecular weight greater than ply. A derivative of I ply containing an EcoRI* or an EcoRI fragment cloned from each of the above DNA species was further examined ply is the designation for a ply derivative con-twining a 1.3-megadalton EcoRI* fragment of pub DNA.
ply was cloned and maintained in BRIAR, a Russ derivative of Brollies, because of the homology between the cloned fragment and a portion of ply. ply conferred on BRIAR the ability to grow in 200 go of Cur per ml, and -the cells produced a high level of chloramphenicol acetyltransferase (Table I). Removal of the insert regenerated a plasm id indistinguishable from ply in size and Cur sensitivity. One of these derivatives of ply from which the insert had been removed was subsequently used to clone EcoRI fragments of ~105 DNA to determine whether the excision process had altered the ability of the chloramphenicol acutely-transfers gene to produce high-level resistance.
There appeared to be no difference between this plasm id and pow in terms of the ability of inserts to promote Char. Relevant data for fragments cloned in ply from EcoRI* digests of ply and SPY and EcoRI digests of s. licheniformis DNA are shown in Table I. In each case, removal of the cloned insert regenerated a plasm id comparable to ply.

EcoRI fragments of B. subtilis DNA were cloned into ply by as procedure differing from the above only in that the transformation recipient was a Russ derivative of BRIE, BRIAR, and the protoplasm transformation system was used [Clang, S. and Cohen, SUN. Mol. con.
Junta 160~ 115 (1979)]. By this procedure, the frequency of Char transform ants was on the order of 1,000 per go of input DNA. Each of more than I trays-formats examined contained ply harboring an insert.

Three classes of DNA fragments were identified on the basis of ability to promote expression of Char when inserted into ply. The EcoRI-generated complex minting fragment previously cloned from B pummels [Keg gins, KIM., et at., J. Bacterial. 134:514-520 (1978);
and Keg gins, KIM., et at., Pro. Neil. Aged. Sat, Use. 75:1423-1427 (1978)] did not promote Char when inserted into ply in either orientation, although tropic complementing activity was expressed in both often-stations. EcoRI fragment F of ~105 DNA [Curly, DO and Garbo, A J. Viral. 3~:789-791 (1980)] promoted Char in one orientation (ply) but not in the reverse orientation (ply). However, the ~105 immunity property of fragment F was expressed in both orientations. Lastly, the EcoRI* fragment cloned from ply into ply [chimera, designated ply (shown in Table IT promoted Char in either orientation. Orientations of all inserts were judged by Hind III cleavage patterns.
Sequences that promote Char expression have been cloned from a variety of DNA sources, and it therefore is likely that these contain regulatory signals common to many genes. Whether these regulatory signals exert their control at -the transcriptional or translational level remains to be determined.

The recombinant vector plasm id, ply, contains an unexpressed CAT gene preceded by a unique EcoRI site and permits the direct cloning of DNA fragments that have promoter activity in B. subtilis. Promoter fragments cloned into the unique EcoRI site result in high level expression of the CAT gene. The cloned promoter in plasm id ply described more fully herein-after is capable of permitting expression of both foreign procaryotic and eucaryotic genes in B. subtilis. ply is therefore the first vector plasm id for B. subtilis capable of expressing cloned procaryotic and eucaryotlc genes.

CLONING E. COLT TRY GENES IN B. SUBTILIS

In order to determine whether splicing a gene not normally expressed in _ subtilis into the cloned CAT gene in ply would allow expression of the foreign DNA, the Hind III site was chosen for cloning because inserts at this location were found to inactivate all CAT activity.

The Hind III generated DNA fragment spanning the F.
golf tropic gene was cloned into ply from HB101 chromosome DNA and pVH5 (See Materials and Methods which hollow for details). Composite plasrnids containing a try insert cloned from chromosome digest (ply) or a digest of pVH5 (ply) were distinguishable by several criteria. Therefore, relevant properties for only ply are described. ply (5.0 My, shown in Fig. 2) complemented mutations in trod, tropic and trpF
genes of B. subtilis, but not mutations in type, trpB, or trap. Removal of the 1.7 My insert from ply by Hind III cleavage and ligation, regenerated the Char phenotype of the vector and deleted the try _ _ F

complementing activity. The 1.7 My cloned insert in ply and ply comigrated with a Hind III fragment of pVH5. Nick translated pVH5 hybridized to the 1.7 My fragment in Hind III digests of ply and 608-3, demonstrating identity among the 1.7 My fragments in the plasmids. In this experiment, approximately 1 go of digested plasm id was subjected to electrophoresis as described previously, [Moorer, R. and Levitt, P.S., J.
Bacterial. 1~3:879-886 (1980)]. Hind III digested DNA
was employed as reference. The gel was blotted to nitrocellulose paper to which was hybridized nick trays-fated pVH5.

In order to test whether sequences in ply were essential to the expression of the cloned E. golf try fragment, the orientation of the fragment was reversed by Hind III cleavage and ligation. Each of three plasmids which were Curs and did not complement _r~2 in BD224 contained the 1.7 My try insert in the reverse orientation as judged by digestions with Hint II and EcoRI endonucleases. Secondly, removal of the 0.2 My promoting fragment from ply by EcoRI
cleavage and ligation resulted in loss of tropic come plementing activity in BD224. Lastly, the 1.7 My try fragment was transferred from ply to the single Hind III site in a joint vector constructed by ligating pBR322 and pueblo at their EcoRI sites. This plasm id complemented the mutation in E. golf strain ISSUE, but did not complement the tropic mutation in BD224 within 36 hour incubation at 37C. Louvre, prolonged 30 incubation (72 hours) of BD224 harboring such composite plasmids did result in sparse growth on tryptophan-free media. Accordingly, sequences present in ply appear essential to expression of the cloned E. golf tropic gene.

CHLOR~lPHENICOL INDUCTION OF E. COLT
tropic EXPRESSION IN PLY
P
The CAT activity specified by ply was 10-fold higher in host cells grown in the presence of Cur than in the same cells grown in drug free broth [Williams, DIM., et at., J. Bacterial. 146:1162-1165 (1981)]. This increase is believed to result from Cur induction of CAT. It was thought therefore that the expression of foreign genes, such as the E. golf tropic fragment, inserted into the CAT gene might also be inducible by Cur. BD224 (ply) was grown to the middle of the exponential growth phase in Mix OH containing 100 gel of tryptophan. The culture was split and a concentration of Cur which is sub inhibitory to BD224, 0.1 gel was added to one. Within 100 mix after addition of Cur to BD224 (ply, a 7-fold increase in the level of the tropic gene product InGPS was detected (as shown in Fig. 3).
BD224 (ply) not exposed to Cur showed no increase in the specific activity of InGPS (Fig. 3). In contrast to the apparent induction of the cloned pi expression by Cur, the expression of the cloned appeared unaffected by the level of tryptophan added to the growth medium. BD224 (ply) grown in Mix OH or Mix OH containing 0.5 gel of tryptophan or 200 gel of tryptophan contained approximately the same level of InGPS.

EXPRESSION OF MOUSE DHFR CLONED IN B. SUBTILIS
In order to test whether ply would allow expression of a cloned eucaryotic gene, the mouse gene specifying DHFR previously cloned in E. golf on a Sty fragment was chosen [Clang, AWOKE., et at., Nature 275:617-624 (1978)].

Mammalian DHFR is resistant to the drug trimethoprim whereas the bacterial enzyme is sensitive. Selection of cells that were capable of expressing the mouse DHFR gene was -therefore based on their resistance to 25 gel of trimethoprim.
pPL608-TRl was a derivative of ply containing to 1.0 My mouse DHFR gene inserted at the P I site (as shown in Fig.
4) and confirmed by agrees gel electrophoresis of Sty and BglII digested ply and pDHFRll which showed the 1.0 My Sty mouse DNA fragment present in both ply and pDHFRll. Each of more than 200 neomycin-resistant transform ants of B. subtilis strain BGSCIS53 generated with pPL608-TRl were resistant to 25 gel of trimethoprim.
Removal of either the promoting fragment from pPL608-TRl (by EcoRI cleavage) on the mouse DHFR fragment (by Sly cleavage) resulted in deleted plasm id forms that no longer specified trimethoprim resistance in strain BGSCIS53.
ply Try confers Char indicating insertion of the fragment of mouse DNA into the Sty site did not inactivate the CAT
gene on ply.
DHFR activity in extracts of ASB298 ~pPL608-TRl) was insensitive to 10 6 M trimethoprim, but was reduced by 93 prevent when 10 6 M methotrexate was present in the reaction mixture (shown in Table II). The sensitivity to methotrexate, and resistance to trimethoprim, are characteristics typical of mammalian DHFR [Clang, AWOKE., et at., Nature 275:617-624 (1978)]. The level of DHFR was similar regardless of where BGSCIS53 (pPL608-TRl) cells were grown in no Cur or 0.1 ug/ml of Cur. These data suggest that expression of a gene inserted at the Sty site is not Cur inducible.

ABLE II
DHFR ACTIVITY IN EXTRACTS OF B. SUBTILIS HARBORING
pPL608-TRlC
Inhibitors Added % Activity Remaining CAM
none 100% 3614 Trimethoprim (10 EM) 100% 3626 Trimethoprim (10 EM) + 7% 247 Methotrexate (10 EM) c. BGSCIS53 (pPL608-TRl) cells were grown in puns broth and harvested during mid exponential growth. Cells were washed and resuspended in 0.1 M KHP04, pi 5.8, and extracts prepared as described for CAT [Skew, WOVE., Methods Enamel.
43:737-755 (1975)~. Assays -For DHFR [Little field, JAW., Pro. Neil. Aged. Sat., USA, 62:88-95 (1969)] were done in duplicate and varied by no more than I percent. DHFR
activity assayed without inhibitors equaled 1.5 units where 1.0 unit will convert lnmole of foliate to tetrahydroFolate per minute at 37C.

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ ply appears to be a valuable vector for achieving foreign gene expression in B. subtilis. The present study demonstrates Cur inducible expression of a foreign procaryotic gene inserted at the Hind III site and expression of the mouse DHFR gene inserted at the Sty site.
Moreover, the promoter fragment within ply is readily removed From the plasm id allowing for the insertion of other promoters that may enhance or reduce the expression of cloned Foreign genes.

., I, The 1.7 My Hind III E. golf try fragment cloned in ply from chromosome digest or from pVH5 was identified by complementation of a genetic defect in the recipient.
The cloned fragment complements mutations in the B.
subtilis try genes D, C, and F. It has been previously shown that two Hind III sites occur within the E. golf try operon flanking the E. golf tropic gene [Crawford, IMP., et at., J. Mol. Blot. 142:489_502 (1980); Nichols, BY et at., J. Mol. Blot. 142:503-517 (1980), and Christie, GEE. and Plait, T., J. Mol. Blot. 142:519-530 (1980)]. One site is located in the beginning of trpB
and the other is approximately in the middle of trod.
The E. golf tropic gene specifies a protein that has two activities, each of which is specified by a separate gene in B. isubtilis, tropic and [Crawford, IMP., Bacterial. Rev. 39:87-120 (1975)]. The E. golf trod citron specifies a protein that also exhibits two activities, each of which is specified by a separate gene in B. subtilis, trig and trod [Crawford, IMP., Bacterial. Rev. 39:87-120 (1975)]. Since the cloned E.
golf fragment complements a mutation in the B. subtilis trod gene, that portion of the _ icoli trod gene corresponding to the B. subtilis trod gene is being expressed. The to_ activity is associated with the car boxy terminal portion of -the trod product, and the corresponding portion of the gene is present on the cloned fragment. The translation initiation cordon necessary for trod expression may reside in the plasm id vector, and may result in the fusion of the amino terminal portion of a vector specified protein (probably CAT) to the car boxy terminal peptize specified by the cloned trod gene.

Expression of the CAT gene in pledge, the tropic gene in ply or the DHFR gene in pPL608-TRl was dependent on the presence of the promoter fragment. Therefore, the promoter fragment likely provides sequences necessary for transcription initiation and that transcription occurs from the promoting fragment through the CAT
structural gene. Insertion of genes between the Sty and Hind III sites on ply presumably places them under control of a strong promoter that is efficiently recognized my B. subtilis RNA polymers.

Production of palpated upon expression of the cloned mouse DHFR gene has been confirmed. Initial molecular weight characterization established that the enzyme DHFR was in fact produced.

In summary, the mouse dihydrofolate reeducates gene and a segment of the Escherichia golf try operon genetically express in Bacillus subtilis when cloned in the novel plasm id ply. The cloned mouse gene confers in-methoprim resistance on B. subtilis and the cloned try fragment complements mutations in the B. subtilis try D
C and F genes. Expression of both cloned fragments is dependent on a promoter present in the vector plasm id.
The E. golf try fragment is cloned in a Eland III site within a chloramphenicol acetyltransferase gene present on ply, and as a result, expression of the E. golf tropic gene product is inducible by chloramphenicol. The mouse gene is inserted at a Sty site preceding the chloramphenicol acetyltransferase gene and its expression is not chloramphenicol inducible. The replication functions and neomycin-resistance of pluck are derived from pub. accordingly, ply is stably maintained at high copy number in B. subtilis.
MATERIALS AND METHODS

Bacteria, plasmids and media Strains of B. subtilis and E. golf used are listed in Table III. Plasmids pub, pCM194, pVH5 and pBR322 have been described [Believer, F., et at., Gene 2:95-113 I

(1977); Gryczan, TO et at., J. Bacterial. 134:318-323 (1978); and Hershfield, V., et at., Pro. ayatollah.
Aged. Sat., U.S.A. 71:3455-3459 (1974)]. pDHFRll is a pBR322 derivative containing a 1.0 My Sty fragment specifying mouse dihydrofolate reeducates [Clang, AWOKE., et at., Nature 275:617-624 (1978)]. Tryouts blood ajar base and puns broth were from Disco. Munich medium has been described [Spizizen, J. Pro. Neil.
Aged. Sat., U.S.A. 44:1072-1078 (1958)]. Minimal ajar for E. golf was My. All incubations were at 37C.
Enzyme assays CAT was assayed as described by Skew [Skew, WOVE., Methods Enzymol. 43:737-755 (1975)]. InGPS was assayed according to the methods of Hook and Crawford [Hook, SO. and Crawford, IMP., J. Bacterial. 116:685-693 (1973)], and DHFR was measured by the procedure of Little field.
[Little field, JAW., Pro. Neil. Aged. Sat., U.S.A.
62:342-357 (1969)]. The Bradford method was used for protein determinations. [Bradford, M., Anal. ~iochem.
72:248-254 (1976)].

press ion plasm id ply The construction of ply is described hereinabove and in Williams, DIM., et Allah J. Bacterial. 146:1162-1165 (1981)]. The plasm id has a mass of about 3.3 My and consists of a major portion of pueblo joined to a 0.8 My segment of B. pummels DNA containing a CAT gene plus a 0.2 My EcoRI* promoter fragment cloned from phase SPY
DNA. The cloned promoter permits expression of the CAT
gene, thereby allowing ply to confer Char on B.
subtilis. Unique restriction sites for Hind III and Sty exist downstream from the promoter (see Fig. 2).
.

I

The specific activity of CAT in BRIE (ply) was 10- to 20-fold higher in cells grown in Cur (5 gel than in cells grown in drug free broth. The molecular size and copy number (approximately 50) were -the same regardless of whether host cells were grown in the presence or absence of Cur. Thus, the increased CAT activity was not due to increased gene dosage. Exposure of BD224 (ply) to a sub inhibitory concentration of Cur (0.1 gel caused a 10-fold increase in the specific activity of CAT within 100 minutes. It therefore appears that CAT is inducible by Cur.

TABLE III
BACTERIAL STRAINS

Organism Strain Relevant properties B. subtilis 168 BRIE tropic Metro Lucy BD224 tropic Thor Russ To trap T20 trpB
T12 trpF
To tropic T22 trod T24 type BGSCIS53 spy _ golf HB101 Troupe ISSUE tropic ___ _ __ _ _ _ _ ___ _ _ ___ __ _ __ _ i CLONING ESCHERICHIA COLT tropic IN B. SUBTILIS
Hind III digests of HB101 chromosome DNA (2 I or plasm id pVH5 (lug) were combined with 0.5 us of Hind III cleaved ply, annealed, ligated [Levitt, P.S. and Keg gins, KIM., Methods Enzymol. 68:342-357 (1979)] and transformed into competent BD224 [Boil, OF and Wilson GUY., J. Bacterial.
94:562-570 (1967)] at 1 gel of DNA. Cells were plated on Mix OH containing lO~ug/ml of neomycin sulfate. Fourteen Try+ transform ants were recovered by cloning from HB101 DNA, and 93 were obtained by cloning from pVH5. ply and ply are Try derivatives of ply containing Try inserts from HB101 and pVH5, respectively. The ability of the cloned try fragment to complement mutations in each of the six _ subtilis try genes was performed as previously described. [Keg gins, KIM., et at., Pro. Natalie. Aged. Sat., U.S.A. 75:1423-1427 (1978)]. Southern transfers, nick translation, hybridizations and agrees gel elec-trophoresis were as previously described [Moorer, R. and Levitt, P.S., J. Bacterial. 143:879-886 (1980)i and Southern, EM., J.
Mol. Blot. 98:503-517 (1975)].

CLONING MOUSE DIHYDROFOLATE
REEDUCATES (DHFR) IN B. SUBTILIS
pDHFRll (l~ug/ml) and ply (lvug/ml) were digested with Sty annealed, ligated and transformed into _ subtilis strain BGSCIS53. Neomycin-resistant transform ants were selected and each was picked to appropriately supplemented Mix OH containing 25~ug/ml of trimethoprim. Approximately 2 percent of the transform ants were trimethoprim resistant.
The plasm id from one such clone, pPL608-TRl, was characterized.

Claims (39)

1. THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
   PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
   
   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 which is not derived from B. subtilis and a DNA sequence derived from a B. subtilis plasmid.
2. 2. A plasmid in accordance with Claim 1 wherein the promoter DNA sequence is derived from bacteriophage SPO2.
3. 3. A plasmid in accordance with Claim 1 wherein the promoter DNA sequence is derived from bacteriophage ?105.
4. 4. A plasmid in accordance with Claim 1 wherein the promoter DNA sequence is derived from B. pumilus plasmid pPL10.
5. 5. A plasmid in accordance with Claim 1 wherein the promoter DNA sequence is derived from B. licheniformis.
6. 6. A plasmid in accordance with Claim 1 wherein the DNA
   sequence from a B. subtilis plasmid is derived from pUB110.
7. 7. A plasmid in accordance with Claim 1 wherein the double-stranded DNA molecule is linear.
8. 8. A plasmid in accordance with Claim 1 wherein the double-stranded DNA molecule is circular.
9. 9. A plasmid in accordance with Claim 1 which additionally includes an inducible gene.
10. 10. A plasmid in accordance with Claim 9 wherein the inducible gene is a chloramphenicol inducible gene coding for production of chloramphenicol acetyltransferase.
11. 11. A plasmid in accordance with Claim 10 wherein chloramphenicol inducible gene is derived from B. pumilus NCIB
    8600.
12. 12. 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 not derived from B.
    subtilis 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.
13. 13. A plasmid in accordance with Claim 12 wherein the promoter DNA sequence is derived from bacteriophage SPO2.
14. 14. A plasmid in accordance with Claim 12 wherein the promoter DNA sequence is derived from bacteriophage ?105.
15. 15. A plasmid in accordance with Claim 12 wherein the promoter DNA sequence is derived from B. pumilus plasmid pPL10.
16. 16. A plasmid in accordance with Claim 12 wherein the promoter DNA sequence is derived from B. licheniformis.
17. 17. A plasmid in accordance with Claim 12 wherein the DNA
    sequence from a B. subtilis plasmid is derived from pUB110.
18. 18. A plasmid in accordance with Claim 12 wherein the double-stranded DNA molecule is linear.
19. 19. A plasmid in accordance with Claim 12 wherein the double-stranded DNA molecule is circular.
20. 20. A plasmid in accordance with Claim 12 which additionally includes an inducible gene.
21. 21. A plasmid in accordance with Claim 12 which additionally includes an inducible gene, wherein the inducible gene is a chloramphenicol inducible gene coding for production of chloramphenicol acetyltransferase.
22. 22. A plasmid in accordance with Claim 21 wherein the chloramphenicol inducible gene is derived from B. pumilus NCIB
    8600.
23. 23. A plasmid in accordance with Claim 12 wherein the foreign DNA codes for production of a polypeptide.
24. 24. A plasmid in accordance with Claim 23 wherein the polypeptide is insulin, growth hormone, the polypeptide portion of interferon, somatostatin or an enzyme.
25. 25. A plasmid in accordance with Claim 12 wherein the foreign DNA is a mammalian gene.
26. 26. Plasmid pPL603 having the restriction map shown in Fig.
    1.
27. 27. Plasmid pPL601 having the restriction map shown in Fig.
    1.
28. 28. Plasmid pPL531 having the restriction map shown in Fig.
    1.
29. 29. Plasmid pPL604 formed by inserting an EcoRI* fragment of pUB110 into pPL603.
30. 30. Plasmid pPL605 formed by inserting an EcoRI* fragment of pPL10 into pPL603.
31. 31. Plasmid pPL608 formed by inserting an EcoRI* fragment of SP02 into pPL603.
32. 32. Plasmid pPL613 formed by inserting an EcoRI fragment of B. licheniformis DNA into pPL603.
33. 33. Plasmid pPL614 formed by inserting an EcoRI fragment of B. licheniformis DNA into pPL603.
34. 34. A method of preparing the plasmid of Claim 1 which comprises obtaining a promoter DNA sequence from a source other than B. subtilis, obtaining a DNA sequence from a B. subtilis plasmid and joining these sequences under suitable conditions.
35. 35. A method of preparing the plasmid of Claim 12 which comprises inserting foreign DNA coding for a desired product into the plasmid of Claim 1 under suitable conditions.
36. 36. A method of introducing into a B. subtilis cell foreign DNA which codes for production of a desired product which comprises transforming said cell with a plasmid in accordance with Claim 12 under suitable transforming conditions.
37. 37. A B. subtilis cell produced by the method of Claim 36.
38. 38. 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 36, growing said cell under suitable conditions permitting expression of said foreign DNA and production of said product and recovering the product so produced.
39. 39. A B. subtilis cell which contains a plasmid in accordance with Claim 1 or Claim 12.