Transformation of Bacillus subtilis and Escherichia coli by a hybrid plasmid pCD1

153 Gene, 1 (1977) 153--167 © Elsevier/North-Holland Biomedical Press, Amsterdam --Printed in The Netherlands

TRANSFORMATION OF BACILLUS 8UBTILI8 AND ESCHERICHIA C O L I B Y A HYBRID PLASMID pCD1

(Bacteriophage ¢3T; cloning; DNA-DNA hybridization; thymidylate synthetase; thy P3 gene transfer, restriction endonuclease) CRAIG H. DUNCAN, GARY A. WILSON and FRANK E. YOUNG

Department o f Microbiology, and of Radiation Biology and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, N. Y. 14642 (U.S.A.) (Received October 15th, 1976) (Accepted October 27th, 1976)

SUMMARY

The gene thyP3 from Bacillus subtilis bacteriophage • 3T was cloned in the plasmid pMB9. The resulting chimeric p]asmid, pCD1, is effective in transforming both Escherichia coli and Bacillus subtilis to thymine prototrophy. The activity of the thyP3 gene produc.t, thymidylate synthetase, was assayed and found to be 9 times greater in a ~ransformed strain of Escherichia coli than in a ~3T lysogen of Bacillus subtilis. The physical location of restriction sites has been determined for two related plasmids pCD1 and pCD2. Hybridization studies clearly indicate that the plasmid gene responsible for Thy* transformation is the gene from the bacteriophage ~3T. The lack of restriction in this transformation process is consistent with our previous studies using bacterial DNA in heterospecific exchanges indicating that the nucleotide sequence surrounding the gene is the dominant factor in determining interspecific transformation.

INTRODUCTION

Bacteria are constantly exposed to crude lysates of deoxyribonucleic acid in vivo or to high concentrations of purified DNA in vitro. While DNAmediated transformation occurs readily among closely related species or between different isolates of the same species it is rare to obtain transformation between widely separated species. Thus, Haemophilus influenzae (Schaeffer, Abbreviation: SDS, sodium dodeeyl sulfate.

154 1956), Streptococcus pneumoniae ( Chen and Ravin, 1966), Neisseria meningitidis (Catlin and Cunningham, 1961) and Bacillus subtilis (Marmur et al., 1963) can be transformed with DNA extracted from different species (interspecific or heterologous transformation). However, the efficiency of this process is very low as compared to the homologous cross, Once the heterologous DNA is incorporated by the recipient cell to form an intergenote, subsequent transformation of the original recipient strain by intergenotic DNA is highly efficient (Wilson and Young, 1972, Wilson and Young, 1973). Four major hypotheses have been proposed to explain the inefficiency of heterospecific transformation: (1) inefficien; recombination due to inhomology of the nucleotide sequences in the region of synapse; (2) restriction of the donor DNA by endonucleases in the recipient cell; (3) death of the recipient cell from induction of lysogenic viruses or integration of heterologous DNA and, (4) post-synaptic repair processes that alter the genetic information in the donor strand. Because experimental data (Chilton and McCarthy, 1969; Wilson and Young, 1973) indicated that the nucleotide sequence around the synaptic region was the dominant factor in determining the outcome of transformation with heterologous DNA, it is conceivable that the heterologous DNA integrated in a plasmid could function in widely divergent organisms. The successful translation of a gene encoding B-lactamase from Staphylococcus aureus in a chimeric plasmid in E. coil (Chang and Cohen, 1974) provided support for this contention. Rather than shot~gun chromosomal DNA into plasmids derived from various enterobacteriaceae, we elected to use the gene (thyP3) encoding thymidylate synthetase from the Bacillus subtilis bacteriophage ~3T. Infection with this virus results in the conversion of thymine requiring auxotrophs to prototrophy (Tucker, 1969). Studies in our laboratory have demonstrated that thyP3 and • 3T integrate into the terminal region of the chromosome of B. subtilis be. tween the two bacterial genes that regulate the biosynthesis of thymine (Young et al., 1976, Williams and Young, 1976). The thyP3 is an attractive gene for cloning in t2~.eB. subtilis system because it can be easily isolated from purified bacteriophage particles. In addition, the transforming activity of the gene survives treatment with either the s i t , specific endonucleases BamHI or EcoRI (Graham et al., 1976), and can be purified from fragment A of a BamHI digest of phage DNA by agarose gel electrophoresis (Thomas et al., 1976), thus eliminating many of the phage specific genes associated with the thyP3 gene. Therefore we selected the E. coil plasmid pMB9, and the thyP3 gene in bacteriophage • 3T for these studies. The investigations described in this communication resulted in the construction of a chimeric plasmid that can be propagated in K coil and transform Thy" K coli or Thy- B. subtilis to Thy +. Surprisingly there was no significant restriction of the donor DNA from E. coil by B. subtilis.

155 MATERIALS A N D METHODS

Bacterial strains, plasmids and viruses The chimeric plasmids were studied in two principal strains: B. subtilis strain RUB830 carryingpheA, trpC2, thyA and thyB, and E. coli strain C600. AThy- mutant of the latter strain was isolated following growth in the presence of trimethoprim and thymine (Stacey and Simson, 1965). B. subtilis strains were grown on tryptose blood agar base or Spizizen's minimal agar with 22 mM glucose as the carbon source and supplemented with the auxotrophic requirements of the strain as described previously (Young and Wilson, 1974). K coil C600 (provided by S. Cohen) was cultured in L-broth (1% Bacto tryptone, 0.5% NaC1 and 0.5% yeast extract pH 7.0). Bacteriophage cb3T was propagated in B. subtilis 168 strain BR151 (lys-3 trpC2 metBlO) or induced by mitomycin C from a lysogen (strain RUB1617 carrying gtaB) as described by Williams and Young (1976). Plaque assays were done on lawns of strain BR151 in a 2 m l semisolid agar overlay on tryptose blood agar plates. The E. coli plasmid pMB9 (constructed in the l~boratory of H. Boyer) that contains a single EcoRI site and carries a gene for tetracycline resistance was generously provided by Dr. T. Mania~is. Genetic analyses Bacterial DNA was isolated from B. subtilis as described by Yasbin et al. (1975). ~he plasmids pMB9 and pCD1 were propagated in strains C600 and C600 Thy" respectively and amplified by the addition of chloramphenicol (100/zg/ml). The plasmids were purified by density gradient centrifugation in the presence of ethidium bromide (Clewell and Helinski, 1969 ). Radioactive plasmid was prepared by growing K coli C600 Thy'/pCD1 in 1% Neopeptone broth (Difco)containing 1% glucose, 25 ~g/ml tetracycline, and 5 mCi radioactive H33~PO4 per liter. Procedures for the amplification and isolation of plasmid DNA were the same a~ for non-radioactive preparations. DNA concentrations were estimated by the diphenylsmine method (Giles and Myers, 1965). Transformation of K coli was accomplished by the method of Cohen et al., (1972). The procedures for development of competence, for transfection and for transformation of/3. subtilis were described previously (Boylan et al., 1972; Yasbin et al., 1975). Since the time of maximal competence for transformation with chromosomal DNA was similar to that for transformation with plasmid DNA (E. Hailparn and F.E. Young, unpublished observations) no special procedures were used for transformation with pCD1 DNA. Formation o f chimeric plasmids A limit digest of DNA from plasmid pMB9 and bacteriophage ~3T was performed by incubation of the preparations with EcoRI (Wilson et al., 1974). A 5/~g sample of the limit digest of pMB9 was mixed with 5/~g of the limit digest of ~3T and 10/d of T4 ligase (Miles Laboratories) in a total volume of"

156

105 pl of 50mM Tris (hydroxymethyl) aminomethane (Tris) buffer pH 7.7 containing 10 mM MgCI2, 0.2 mM ATP; 1.0 mM dithiothreitol, and I mM spermine (T. Maniatis, personal communication). After 15 min at 0°C the temperature was shifted to 15°C for 4 h. The reaction was then terminated with 20 pl of I M EDTA (pH 7.5) and extracted for 18 hr with 7X volume of phenol containing 0.2% 8-hydroxyquinoline that had been previously saturated with 0.15 M Tris buffer containing 0.1 M EDTA (pH 8.0). The aqueous phase was dialyzed against 2 changes of 10 mM Tris buffer, pH 7.5 containing 1raM EDTA and 20 mM NaCI. A I pg sample of this mixture was used to transform E. coli C600Thy-. Two Thy ÷ Tet R clones were isolated on minimal agar containing 10 pg/ml tetracycline from a mixture that yielded 2000 Thy-Tet s transformants when plated on L plates containing 100 pg/ ml thymine and 20 pg/ml tetracycline. These two yielded plasmid DNA. One of the plasmids was designated pCD1.

Hybridization of pCD1 with bacteriophage 4,3T Non-radioactive pCD1 DNA was digested with EcoRI to produce a limit digest. Samples of the limit digest of pCD1 and bacteriophage ~3T were electrophoresed in agarose ethidium bromide gels, denatured and eluted onto a cellulose nitrate filter as described by Southern (1975). A limit digest of s2p pCD1 DNA was electrophoresed in a second agarose-ethidium bromide gel, denatured and eluted at right angles onto the same cellulose nitrate filter under similar conditions (as developed by C. Hutchinson, III.) Renaturation was accomplished during elution of the DNA from the second gel over a period of 18 h at 65°C in 0.6 M NaCI containing 60 mM sodium citrate (4 X SSC) and 0.1% SDS. Hybridization was detected by exposure of the cellulose nitrate filter or' X-ray film. Endonuclease and electrophoresis of DNA T4 ligase was provided by Miles Laboratories. The purification procedures for EcoRI (Wilson et al., 1974 ), BamHI (Wilson and Young, 1975), and BglII (Wilson and Young, 1976) were described previously. Enzyme reactions were done in TMM buffer (6 mM Tris pH 7.5 containing 6ram MgCI2 and 6 mM ~-mercaptoethanol). The electrophoresis of DNA was done on a Blaircraft vertical slab gel apparatus with 1% agarose with TPE buffer (0.04 M Tris pH 7.7 containing 0.036 M K2HPO4, 0.001 M EDTA, and 0.5 pg/ml ethidium bromide). The molecular weights of nuclease fragments were estimated by comparison of their mobility with the mobility of the known molecular weight fragments of an EcoRI, BamHI digest of Adenovirus-2 DNA (Carel Mulder, personal communication). For a 1% gel, mobility was inversely proportional to the logarithm of molecular weight up to 3 megadaltons. Thymidylate synthetase assays Thymidylate synthetase assays were performed according to Wahba and

157

Friedkin (1962). Although this assay is not suitable for crude extracts of K coli Thy ÷, it is reproducible and linear with protein up to 1 mg/ml for C600 Thy-/pCD1 and RUB 830 (4,3T). Cells for this assay were grown in L broth containing 20/~g/ml tetracycline or penassay broth (antibiotic assay medium number 3, Difco)for K coli or B. subtilis strains respectively. All assays were done at 30°C in a Gflford 2400 S recording spectrophotometer. Tetrahydrofolate and dUMP were purchased from Sigma. Protein concentrations were determined by the method of Lov~T et al. (i951). RESULTS

Isolation and characterization o f pCD1 Previom work in our laboratory (Young et al., 1976; Graham et al., 1977) and in others (Ehrlich et al., 1976) established that an EcoRI limit digest of bacteriophage 4,3T DNA still retained Thy ÷ transforming activity, albeit with a 200-fold reduction when compared with intact phage DNA. Because thyP3 resides on a very large (36 md) BamHI fragment (Thomas et al., 1976), we chose to clone the smaller EcoRI piece. Accordingly, pMB9 and bacteriophage • 3T DNA were digested with EcoRI, the reaction terminated, the fragments ligated, and then incubated with a Thy- strain of E. coil C600 as described in MATERIALS AND METHODS. The recombinant plasmid, pCD1, thus generated will transform E. coli C600 Thy- to both Tet R and Thy ÷ simultaneously. Of 500 Tet R transformants, all were fotmd to be Thy ÷ when replica plated onto minimal agar.

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Fig.1. Transformation ofB. subtilis RUB830 to Thy ÷ prototrophy. DNA from purified • 3T bacteriophage particles (v . . . . v), plasmid pCD1 (o-----o) and a strain of B. subtilis thyA thyB thyP3* (~ -- - - - - ~) was used to transform B. subtilis RUB830 under standard conditions (see MATERIALS AND METHODS).

158

To determine the transforming efficiency of the cloned thyP3 gene in B. subtilis, pCD1 DNA isolated from E. coli was used to transform a competent culture of strain RUB 830. This Thy + transforming activity was compared with the Thy÷ transforming activity of bacteriophage ~3T DNA and prophage DNA (Fig 1). It is surprising that transformation occurs with such a high degree of efficiency considering that the thyP3 gene is surrounded by heterologou~ DNA in pCD1 and propagated in E. coli. The linear relationship of transformants with incre~ing DNA concentration suggests that one molecule of pCD1 DNA is sufficient to transform the recipient to Thy ÷. It is conceivable that the high efficiency of transformation of B. subtilis is due to the physical state of the plasmid DNA. Because it has been shown in E. coli that closed circular DNA is 4--5 orders of magnitude more efficient in transformation than linear molecules (P.C. Wensink, personal communication), we treated pCD1 DNA with site-specific nucleases designed to linearize or alter the molecular weight (Fig 2). The corresponding biological activities (Table I) demonstrate that pCD1 DNA cleaved with EcoRI is approx. 12% as efficient as intact DNA in Thy ÷ transforming activity. Linear molecules, produced by BamHI cleavage at a site at least 0.8 md from the thyP3 gene, were only slightly less efficient than circular DNA. As expected, digestion with both nucleases results in the same biological activity as does digestion with EcoRI alone. TABLE I T R A N S F O R M A T I O N O F RUB830 with pCD1 DNA Three ~g of pCD1 D N A was digested for 5.5 h at 37°C using approx. 10 units o f E c o R I or B a m H I endonuclease in a 50 , ! reaction m i x t u r e as described in M A T E R I A L S A N D METHODS. 25 ~1 were assayed by agarose gel electrophoresis (see Fig. 2 ) a n d 25 ul were used to transform B. subtilis R U B 8 3 0 to T h y ÷ in a one ml t r a n s f o r m a t i o n mixture. Viability was 9.5 • 107 per ml and using saturating levels of bacterial DNA, 1.6 • l 0 s Trp ÷ transformants were obtained. Enzyme

Thy+ transformants/108 cells

Efficiency ratio

None BamHI EcoRI B a m H I + EcoRI

6,890 6,340 857 494

1.00 0.92 0.12 0.07

Thymidylate synthetase activity in transformed cells In order to determine the efficiency of expression of the foreign gene, thy P3, in E. coil, we assayed thymidylate synthetase activity in transformed cells (Table II). The enzyme activity is approx, 9-fold greater in E. coli C600 Thy-/pCD1 as compared with RUB 830 (~3T). It is impossible to measure the thymidylate synthetase activity in crude extracts of Thy ÷ E. coil C600 due to the naturally occurring inhibitor. However, when one compares the

159

I

2

, 3

4

Fig.2. Agarose gel electrophoresis of pCD1 DNA. Plasmid pCD1 was digested with endonuclease BamHI (2) EcoRI (3) and BamHI and EcoRI (4) and compared to undigested pCD1 DNA (1) by agarose gel electrophoresis. Assay conditions described in legend to '£able I. The 0.40 md fragment is not shown (4) on this gel.

160 specific activity obtained by Wahba and Friedkin (1962) for partially purified enzyme from E. coil K12, with that in strain C600 Thy-/pCD1, there is a 4-fold higher level in the latter strain. TABLE II THYMIDYLATE SYNTHETASE ACTIVITY OF CRUDE EXTRACTS aSpecific activity is expressed as nanomoles tetrahydrofolate oxidized/h/mg protein at 30°C. The reaction mixture contairted 0.1 umole ,,L-tetrahydrofolate, 15 pmoles formaldehyde, 25 umoles MgClz, 100 pmoles 2-mercaptoethanol, 50 pmoles Tris buffer (pH 7.5), and various amounts of crude extract (0.5 to 2 mg protein) in a final volume of 1.1 ml. The reaction mixture was equilibrated to 30°C and started by adding 0.1 pmole deoxyuridine 5'-monophosphate tdUMP). The change in absorbance (A) at 340 nm was compared to a reference cuvette lacking dUMP. bThymidylate synthetase cannot be assayed in crude extracts of this strain (see MATERIALS AND METHODS). This value is that given by Wahba and Friedkin (1962). Strain B. B. E. E.

Specific activity a

subtilis RUB830 subtilis RUB830 (~3T) coil C600 Thy-/pCD1 coli K12 Thy +

<1 45 380 90 b

Location of the endonuclease sites Locations of EcoRI, BglII, and BamHI restriction sites were determined by analysis of single and combined enzyme digests of pCD! (Fig 3). A photograph of an ethidium bromide agarose gel showing the nuclease patterns of ¢3T, pCD1, and the parent plasmid pMB9, along with the Adenovirus-2 BamHI EcoRI molecular weight markers is shown in Fig. 4. The molecular weight of the larger EcoRI fragment and retention of a BamHI site within

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Fig.3. Physical Maps of pCD1 and pCD2.

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161

1 2 3 4 5 6 7 8 9 1 0 1 1

Fig.4. Agarose ethidium bromide gel electrophoresis of DNAs. In each reaction, 1 ~g of DNA was incubated with the appropriate nucleases, mixed with sucrose, layered on a 1% agarose gel, and electrophoresed as described in MATERIALS AND METHODS. 1, pCD1 DNA; 2, ,1)3'1' DNA cleaved with EcoRI; 3, pCD1 DNA cleaved with EcoRI; 4, Ad-2 DNA cleaved with EcoRI and BamHI; 5, pMB9 DNA cleaved with EcoRI; 6, pCD1 DNA cleaved with BglII; 7, pMB9 DNA cleaved with BglII; 8, pCD1 DNA cleaved with BamHl; 9, pMB9 DNA cleaved with Ban~HI, 10, pMB9 DNA, 11, pCD1 DNA.

162

this fragment, suggest that it consists of DNA derived from pMB9. Therefore, the thyP3 gene should reside on the smaller EcoRI fragment. Both BglII sites are located on the smaller EcoRI fragment. One BglII site is very close to the EcoRI site furthest from the BamHI site and the small fragment that should be generated by a combined digestion with these two endonucleases could not be observed on agarose gels. To determine the molecular weight of this fragment a second plasmid was derived by using a limit BglII digest of pCD1 to transform C600 Thy- to tetracycline resistance. The plasmid isolated from this Thy- Tet R clone, pCD2, has nuclease patterns consistent with its being the product of in vivo recircularization of the large BglII fragment of pCD1 (see Fig. 3). Comparison of the EcoRI digest and the EcoRI, Bg/II digest of pCD2, indicates a 0.04 md length for the short fragment.

Evidence that pCD1 contains the thyP3 gene Because the pMB9 plasmid was prepared in a Thy ÷ strain of E. coil, it is remotely possible that pCD1 actually contains an E. coil Thy* gene. Although the high efficiency of transformation of B. subtilis by pCD1 would suggest this is not C~e case, nevertheless, additional evidence of the origin of the thy gene cloned tin pCD1 was sought. Recently, Southern described an elegant technique for hybridization of DNA from agarose gels. This technique, as modified by C. Hutchinson, III, provides a means for determining homology between the thyP3 gene of bacteriophage ~3T and the thy gene of pCD1. As shown in Fig. 5, the smaller molecular weight fragment (B) generated by an EcoRI digest of pCD1 DNA hybridized only with pCD1 DNA and ~3T DNA. There is no hybridization of the larger fragment (A), corresponding to pMB9 with &3T DNA. Note also that the larger ~ragment (A) only hybridizes with the larger fragment (A) of pCD1 DNA as expected. Although this hybridization does not rule out the possibility that the thyP3 gene may share homologous sequences with an E. eoli thy gene, it is consistent with the conclusion that pCD1 DNA does contain the thyP3 gene of bacteriophage 4~3T. DISCUSSION

One of the most exciting prospects raised by the new recombinant DNA technology is the possibility Of expressing foreign DNA to make novel proteins in a prokaryotic host. For example, Struhl et ah (1976) have recently reported complementation of auxotrophic mutants of E. coil by yeast genes cloned in bacteriophage k vectors. C~,ang and Cohen (1974)demonstrated that genes responsible for antibiotic resistance functioned in hosts that did not normally exchange genetic material with the donor. The nitrogen fixation genes from Kiebsiella pneumoniae, carried on the recombinant plasmid RP41, have been shown to function in both E. coil (Dixon and Cannon, 1976) and also in the more distantly related organism Azotobacter vinelandii (Cannon and Postgate, 1976). Although the expression of eukaryotic DNA in prokatTotic cells provides the most dramatic use of cloning to cross species barriers, the well

163

Fig.5. Hybridization of EcoRI digested DNA to @3T and pCD1 plasmid DNA. Nonradioactive, undigested @3T DNA and non-radioactive EcoRI digested pCD1 DNA were eluted to a cellulose nitrate (millipore) membrane after agarose gel electrophoresis as described in MATERIALS AND METHODS. Tracing of this DNA was from a photograph of the agarose slab gel. Radioactive pCD1 DNA was digested with EcoRI endonuclease and subjected to agarose gel electrophoresis. The slab gel was divided and each piece was ~luted to the same membrane at right angles to the non-radioactive DNA. The larger fragment (A) and the smaller fragment (B) of the EcoRI digested pCD1 DNA are indicated.

defined genetic systems o f p r o k a r y o t e s such as E. coil, and B. subtilis provide model organisms in which to study replication, regulation and expression of cloned genes. Here we r e p o r t a gene, thyP3, f r o m Bacillus subtilis bacteriophage • 3T, coding for t h y m i d y l a t e synthetase, which functions b o t h in K coil and in B. subtilis to convert t h y m i n e a u x o t r o p h s to p r o t o t r o p h s . When thyP3 is cloned in t h e plasmid pMB9 in E. coli it results in a thymidylate synthetase level nine times as high as in a B. subtilis lysogen o f ~3T. Interestingly, this is approximately the same e n z y m e level as is f o u n d in extracts of B. subtilis during a lytic infection with the clear plaque m u t a n t ~ 3 T C

164

(Tucker, 1969). In both cases there are multiple copies of the thyP3 gene in the cell. Shnflar gene dosage effects have been reported for the tryptophan biosynthetic enzymes when the trp operon is cloned in ColE1 (Hershfield et al., 1974). Hence, it appears that this B. subtilis thy gene is transcribed and translated efficiently in K coli. In addition toprovidingevidence of expression of the thyP3 gene, successful transformati~: of B. subtiiis withithe hybrid DNA has helped to Characterize the process of transformation inB: subtilis.i : ~ t h O u ~ previous work in our laboratory i n d i c a t ~ that classical .... restriCtion,modification mechanisms probably were not the major factors in preventing transformation by foreign DNA (Wilson and Young, 1972, Wilson and Young, 1973) it was not possible to determine what effect replication of transforming DNA in a distantly related organism would have on its subsequent activity. The thyP3 gene apparently transforms/3, subtiUs with the same efficiency regardless of the source of the donor DNA. This conclusion is deduced from results obtained after digestion with EcoRI endonuclease. Based on the difference in the molecular weights of the pCD1 plasmid and ~3T genome, the plasmid borne gene, thyP3, is approximately 30-fold less efficient in transforming B. subtiUs than the same gene when it exists as part of the ~3T DNA (Fig. 1). Digestion of pCD1 with EcoRI results in a further 8-fold decrease (Table I) yielding a final reduction of 240-fold. This final reduction is similar to the 200-fold reduction in transforming activity observed with EcoRI digested ~3T DNA (Graham et al,, 1977). This accords with our previous work showing that heterologous chromosomal DNA is n o t restricted during tran~ formation in B. subtiUs and argues against the existence of a common modification system in the bacillus genospecies that is responsible for the limited genetic exchange among its members. The absence of restriction of this DNA propagated in the host E. coil supports our original contention that the nucleotide sequence homology between the donor and recipient DNA is the major contributor to limited genetic exchange by foreign DNA. In both the E. coli calcium dependent and the B. subtilis transformation systems, the efficiency of transformation is a function of the molecular weight of the donor DNA (Cosloy and Oishi, 1973; Morrison and Guild, 1972). Studies directed toward elucidating the critical size of the donor DNA necessary for maximum efficiency of transformation have been hampered by the fact that the DNA preparations were not very homogeneous. The thyP3 gene of the hybrid plasmid appears to be a model substrate for studies relating the size of the donor fragment to the efficiency of transformation. For example, at non-saturating levels of DNA, Thy ÷ transforming activity of pCD1 was reduced 8-fold by lin:.it digestion with EcoRI. The size of the EcoRI fragment (2.0 rod) may be very near the 'threshold size for efficient transforming DNA and is quite close to the value (1.2 rod) reported by Morrison and Guild (1972) for randomly sheared DNA fragments. ...... The origin of the actual fragment bearing the thyP3 region of the pCDI

165 plasmid is not clear. It is evident that pCD1 was not the result of the simple insertion of an EcoRI fragment of ~3T into the pMB9 EcoRI site because the thyP3 bearing fragment of pCD1 does not comigrate on gels with an EcQRI fragment of ~3T. Likewise the smaller BglH fragment of pCD1 does not comigrate with a fragment from a BglII digest of ~3T DNA, yet Fig. 5 shows that the cloned fragment hybridizes to intact ~3T DNA. One explanation is that a deletion occurred in the EcoRI thyP3 fragment during the cloning procedure. There is considerable interest in developing an in vitro cloning system in B. subtilis. This prokaryote has a high frequency transformation system along with a well developed genetic map. B. subtilis is a non-pathogenic spore forming soil bacterium. Infections with this and related bacilli have only been reported in compromised hosts (Farrar, 1963; Pearson, 1970; Ihde and Armstrong, 1973). In addition, it undergoes a morphogenetic differentiation process resulting in spore formation. Knowledge of the regulation of this sequentially controlled process should be greatly advanced with the possibility of constructing partially diploid strains using cloned genes. At this time, plasmid vectors appear to be the most promising means of cloning genes in B. subtilis. Small plasmids have been identified in several bacillus species (Lovett and Bramucci, 1974, Lovett and Bramucci, 1975; Lovett, et al., 1976) and these molecules can be transformed into strain 168, the best characterized, competent bacillus strain. So far, none of these carry an easily selectable marker. It may be possible to construct a useful vector in vitro by using the purified thyP3 gene inserted into existing plasmids. ACKNOWLEDGEMENTS

We would like to thank Peggy Spear for providing technical assistanceand preparing restrictionendonucleases. Discussions of thiswork with Scott Graham and Len Mayer were greatly appreciated. Construction of hybrid D N A and all operations involvingthe isolationand use of this D N A were performed under conditions which adhered to the recommendations establishedfor recombinant molecule technology developed at the Asilomar Conference. This work was supported by American Cancer Society grant V-27. C H D is a predoctoral student supported by the department of Radiation Biology and Biophysics.

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