Molec. gen. Genet. 176, 239 245 (1979) © by Springer-Verlag 1979

Construction of a Colony Bank of E. coli Containing Hybrid Plasmids Representative of the Bacillus subtilis 168 Genome Expression of Functions Harbored by the Recombinant Plasmids in B. subtilis Georges Rapoport, Andr6 Klier, Alain Billault, Fran~oise Fargette, and Raymond Dedonder Department of Microbiology, Institut de Recherche en Biologic Mol+culaire, C.N.R.S. et Universit6 Paris VII, 2, place Jussieu, F-75221 Paris Cedex 05 (France)

Summary. A collection of about 2500 clones containing hybrid plasmids representative of nearly the entire genome of B. subtilis 168 was established in E. coIi SK1592 by using the poly(dA)-poly(dT) joining method with randomly sheared DNA fragments and plasmid pHV33, a bifunctional vector which can replicate in both E. coli and B. subtilis. Detection of cloned recombinant DNA molecules was based on the insertional inactivation of the Tc gene occurring at the unique BamHI cleavage site present in the vector plasmid. Thirty individual clones of the collection were shown to hybridize specifically with a B. subtilis rRNA probe. CCC-recombinant plasmids extracted from E. coli were pooled in lots of 100 and used to transform auxotrophic mutants of B. subtilis 168. Complementation of these auxotrophic mutations was observed for several markers such as thr, leuA, hisA, glyB and purB. In several cases, markers carried by the recombinant plasmids were lost from the plasmid and integrated into the chromosomal DNA. Loss of genetic markers from the hybrid plasmids did not occur when a rec- recipient strain of B. subtilis was used.

Introduction The introduction of recombinant DNA technology has markedly improved our ability to study bacterial and eukaryotic genes in greater details (for review see Beers and Bassett, 1977; Boyer and Nicosia, 1978). It is now possible to cut a genome into small fragments, each carried on a hybrid plasmid as a separate Abbreviations: ApR: resistance to ampicillin; TCR: resistance to tetracycline, Cm R: resistance to chloramphenicol; CCC : covalently closed circular duplex; Mdal: megadalton For o fJ))rints contact: G. Rapoport

clone. The construction of such a "colony bank" should facilitate studies on the expression in vivo and in vitro of genes, particularly for determining the nature of the signals involved in the regulation of transcription and translation. In prokaryotes such a collection has already been obtained for E. coli DNA (Clarke and Carbon, 1975, 1976). In the case of B. subtilis, cloning of specific chromosomal fragments in E. coli has been demonstrated by several investigators (Ehrlich et al., 1976; Segall and Losick, 1977; Mahler and Halvorson, 1977; Horinouchi etal., 1977 ; Chi et al., 1978; Nagahari and Sakaguchi, 1978 ; Bonamy et al., 1978), some of the genes studied being expressed in the heterologous host cells (Ehrlich et al., 1976; Mahler and Halvorson, 1977; Chi et al., 1978; Nagahari and Sakaguchi, 1978; Duncan et al., 1978). Subsequently, specific cloning vectors of B. subtilis carrying antibiotic resistance characters have been constructed (Keggins et al., 1978; Gryczan and Dubnau, 1978) which can in some cases replicate and express their functions both in E. coli and B. subtilis (Ehrlich, 1978; Kreft et al., 1978; Chang and Cohen, 1979). Keggins et al. (1978) have reported that DNA from several bacilli can be cloned and expressed in B. subtilis, as detected by complementation of an auxotrophic mutation. The construction of a cloning vehicle in B. subtilis 168 composed of B. subtilis leucine genes and a B. subtilis natto plasmid has also been described (Tanaka and Sakaguchi, 1978). Our aim was to establish a colony bank of E. coli containing plasmids with inserted DNA fragments corresponding to the entire genome of B. subtilis. For that purpose large fragments of DNA obtained by mechanical shearing were used instead of restriction endonuclease cleavage which may cause loss of genetic markers. The cloning vector chosen was pHV33, a plasmid constructed by linking pC194 to pBR322; this new plasmid can replicate in both E. coli and B. subtilis (Ehrlich, 1978, and unpublished

0026-8925/79/0176/0239/$0t.40

G. Rapoport et al. : Bank of B. subtilis DNA

240 results). Using pHV33, hybrid plasmids were constructed in vitro by the poly(dA), poly(dT) joining m e t h o d a n d t h e n c l o n e d i n E. coli. C l o n i n g f i r s t i n E. coli h a s t h e a d v a n t a g e o f a l l o w i n g t h e f o r m a t i o n and amplification of stable CCC-recombinant DNA plasmids which can then subsequently be used to t r a n s f o r m B. subtilis. A colony bank of about 2500 clones was obtained a n d t h e size o f t h e i n s e r t e d D N A f r a g m e n t s o f a b o u t 3 Mdal was estimated to be large enough to include hybrid plasmids representative of nearly the entire B. subtilis g e n o m e . C l o n e s c o n t a i n i n g s p e c i f i c s e g m e n t s of DNA were identified; these include ones which code for rRNA and others which are capable of complementing several auxotrophic mutations i n B. subtilis. W e o b s e r v e d t h a t p l a s m i d s c o n t a i n i n g g e n e t i c m a r k e r s l o s e t h e m a r k e r s i n rec +, b u t n o t i n recs t r a i n s o f B. subtilis.

Materials and Methods

30mM Tris, 0.1mM dithiothreitol, l m M COC12, 0,04raM [3H] dATP (20 gCi), 10 gg of bovine serum albumin, 27 ~tg of DNA and 0.5 gg of terminal deoxynucleotidyl transferase from calf thymus (PL Biochemicals, 14000 units/mg protein). The reaction was allowed to proceed at 37° C for 120 min and the polydeoxyadenylated B. subtilis DNA was repurified by phenol extraction and concentrated by ethanol precipitation. An average of 300 residues of dA were added per end of molecule of DNA, based upon the incorporation of [3H] dAMP. Poly (dT) extensions were added to pHV33 DNA in a reaction mixture similar to that described above, using 0.05mM [3H] TTP in place of [3H] dATP, 9 gg of DNA and 0.25 gg of terminal transferase. The reaction mixture was incubated at 37°C for 45 min. Polythymidylated pHV33 DNA was phenol extracted and ethanol precipitated. Under these conditions approximately 250 residues of dT were added per end of DNA molecule. 1 gg of pHV33-(dT)2~6 DNA and 2 gg of B. subtilis-(dA)~ DNA were mixed at 5 gg/ml in 10 mM TrisHC1 pH 8.0, 250 mM NaC1 and 0.2 mM Na EDTA and allowed to anneal at 42 ° C for 120 min. Annealed hybrid DNA could be stored at 40 C for months without loss of transforming activity. Potential biohazards associated with the experiments described in this publication have been reviewed by the French National Control Committee. The appropriate experiments were performed at PI level containment.

Bacteria

Isolation and Analysis of Plasmid DATA

E. coli strain SK1592 (tonA gal thi sbcB15 endA hsdR4 hsdM +) from S.R. Kushner was used throughout. B. subtilis mutants derived from strain 168 (trpC2) included QB79 (sacT'30), QB666 (hisA1 thr5 leuA8 sacA321) QB934 (tre12 metC3 glyB133 trpC2), QB944 (purA16 cysA14 trpC2), QB3006 (thr5 leuA8 recE4), QB3043 (purB33 glefru man mtl), were prepared in this laboratory. E. coli and B. subtilis were grown in L liquid medium.

Pools of 100 clones of E. coli were grown in a mineral medium containing 1% casein hydrolysate (Oxoid) and 0.5% glucose. At mid-log growth phase 250 gg/ml Cm was added and the cultures were maintained at 37 ° C for 16 h to allow plasmid amplification. CCC-DNA was extracted from small volumes of E. coli and B. subtilis cultures according to a new technique (Birnboim and Doly, manuscript in preparation), which involves selective denaturation of chromosomal DNA, but not CCC-DNA, with NaOH. Plasmid DNA was also purified from large volume (1 to 2 1) on hydroxyapatite according to Colman et al. (1978). DNA fractions were analyzed by electrophoresis on horizontal 0.6% agarose slab gels. Electrophoresis was carried out in 40 mM Tris, 20 mM Na acetate, 2 mM Na EDTA buffer pH 7.8 for 16 h at 3V/cm. Gels were stained with ethidium bromide (1 gg/ml) and photographed under UV irradiation.

Transformation Procedure E. eoli SK1592 was transformed as described (Lederberg and Cohen, 1974). Resistant cells were selected on L-agar plates supplemented with antibiotics [Ap (100 gg/ml) or Tc (15 gg/ml)]. Individual clones were grown in L medium in presence of 100 gg/ml Ap and stored at 20° C in L broth diluted with an equal volume of 85 % glycerol (v/v). Induction of competence and transformation of B. subtilis strains were as described (Niaudet and Ehrlich, 1979). Transformants were selected for Cm R (3 gg/ml Cm) on minimum medium agar plates supplemented with 0.1% glycerol (Dedonder et al., 1977). The appropriate nutritional requirements were added to a final concentration of 20 gg/ml. Individual transformants were grown in L medium in presence of 3 gg/ml Cm and stored at - 70 ° C after addition of glycerol to 15% (v/v).

Preparation of Plasmid Hybrids B. subtilis DNA (75 lag/ml in 0.15 NaC1, 0,015 M Na citrate) isolated from strain QB79 (sacTC30) was sheared at 0 ° C by 6 passages through a hypodermic needle, ethanol precipitated and passed through a Sepharose 2B column to separate fragments of 2-10 Mdal (ascending part of the excluded peak). Plasmid pt-IV33 was cleaved in the Tc gene by the restriction enzyme BamHI under conditions described by the supplier (Boehringer), then phenol extracted and precipitated by ethanol. Extensions of poly (dA) or poly (dT) were added to the 3'hydroxyl termini of the DNAs essentially as described by Lobban and Kaiser (1973). Poly(dA) tails were added to DNA fragments in a reaction mixture of 0.1 ml containing 140 mM cacodylic acid,

DNA-RNA Hybridization rRNA from B. subtilis was labeled with [~:32p]ATP and T4 polynucleotide kinase as described (Maizels, 1977). The specific radioactivity of the probe was 5 x 1 0 6 cpm/btg RNA. E. coli colonies on Millipore filters (0.45 ~t, 47 mm diameter; 50 colonies per filter) were prepared for in situ hybridization according to Grunstein and Hogness (1975). In order to reduce the background E. coli rRNA was included as a competitor in the hybridization reaction. One-half ml of reaction mixture containing 8 × l0 s cpm [3ZP]rRNA, 20 ~g E. colirRNA in 0.3 M NaC1, 0.03 M Na citrate40% formamide was used per filter.

Results Construction o f a Collection o r E . coli Clones Containing p H V 3 3 - B , subtilis D N A Plasmids The cloning vector used pHV33 (molecular weight, 4.7 M d a l ) is a T c R r e v e r t a n t o f t h e h y b r i d p l a s m i d

G. Rapoport et al. : Bankof B. subtilis DNA pHV14 constructed by linking pC194 originally issued from S. aureus (Ehrlich, 1978) to pBR322 derived from E. coli (Boyer et al., 1977) at their unique HindlII site. Plasmid pHV33 can replicate in both E. coli and B. subtilis; it confers Ap R, Cm R and Tc R to E. coli but confers only CmR to B. subtilis. This kind of heterospecific barriers to gene expression has been described previously (Ehrlich, 1978; Kreft et al., 1978). With the highly transformable strain of E. coli SK1592 used in these experiments, the rate of transformation for any of the three antibiotic characters was about 10~ transformants per lag of pure CCC-DNA prepared by the hydrexyapatite chromatography method, whereas with B. subtilis 168 the frequency of transformation for Cm R was approximately 10 6 transformants per lag of DNA. The plasmid vector contains a single BamHI site located in the Tc gene; insertional inactivation of the Tc gene allows identification of inserted recombinant DNA molecules in E. coli host cells. B. subtilis DNA and the linearized plasmid vector were extended by poly(dA) and poly(dT) respectively and the annealed, but unligated hybrid DNA was used to transform E. coli SK1592 as mentioned in Materials and Methods. Transformants were selected for Ape: the efficiency of transformation was about 5 x 104 transformants per gg of annealed total DNA. Over 3000 colonies were picked and transferred to Tc containing plates. About 80% of the Ap n clones were sensitive to Tc, suggesting that B. subtilis DNA has been inserted into the Tc gene of pHV33. Each of the 2500 Ap e clones sensitive to Tc was grown separately in the presence of 100 lag/ml Ap, amplified by treatment with a high concentration of Cm (250 lag/ml) and stored at 20 ° C.

Size o f the Recombinant Plasmids

Hybrid plasmid DNAs were purified from pools of 100 clones by the NaOH treatment method (see Materials and Methods). The 25 extracts obtained corresponding to the entire collection were examined separately by agarose gel electrophoresis: the size of the CCC-DNAs ranged from 6 to 15 Mdal, with a mean value of about 8 Mdal. Considering the molecular weight of the plasmid vector (4.7 Mdal) the average size of the B. subtilis inserts is approximately 3 Mdal. The probability that a given unique DNA sequence is present in the collection was estimated by the equation given by Clarke and Carbon (1976 ; see also Collins, 1977): P = l - ( 1 - g ' ) N with

g'=(1-X/L)g

241 where P is the probability that the required sequence occurs intact; g is the average size of the fragments cloned as a fraction of the total genome; N is the number of clones needed to obtain the degree of certainty P; X is the length of the sequence required intact; L is the length of the fragment cloned. The size of the B. subtilis chromosome was estimated between 2 to 3 x 109 daltons (Hariharan and Hutchinson, 1973; Wake, 1973; Hoch, 1978). If the length of the sequence required for a complete gene is 1 Mdal, and the average size of the fragments cloned 3 Mdal, and assuming a molecular weight of 3 x 109 daltons for the entire genome of B. subtilis (which is the upper limit), the probability of finding any cloned gene system at random was calculated to be about 80% with 2500 clones.

Characterization o f Hybrid Plasmids

A direct method to establish that clones in a colony bank do carry hybrid plasmids is to screen the clones for the presence of heterologous rDNA. This was done by the technique of Grunstein and Hogness (1975) using [32p] labeled rRNA from B. subtilis as a probe. Thirty positive clones were detected in the total collection. This value is in good agreement with the number of ribosomal genes estimated in B. subtilis (about 10 cistrons for 23s and 16s rRNA) (Potter et al., 1977). Recombinant plasmids containing B. subtilis rDNA were extracted from these clones and the size of the plasmids was estimated by agarose gel electrophoresis between 7 and 9 Mdal. Plasmid DNA isolated from five clones selected at random were next used to transform the reference B. subtiIis strain 168 for Cm ~. The frequency of transformation was about 106 transformants per gg of CCC-DNA prepared by the hydroxyapatite separation technique. In each case the size of the recombinant plasmid was found identical to that of the plasmid originally isolated from E. coli. When extracted from B. subtilis and used to transform E. coli SK1592, these plasmids yielded approximately 107 transformants per gg of DNA for the Ap ~ character, similar to the efficiencies of transformation of the original plasmids grown in E. coli. It seems therefore likely that in these cases the transfer of the plasmids from E. coli to B. subtilis did not significantly modify DNA recombinant molecules. The entire collection was then screened for hybrid plasmids capable of complementing auxotrophic mutations in B. subtilis 168. The 25 extracts of 100 clones each were used to transform competent cells of the following auxotrophic strains of B. subtilis: QB666, QB934 and QB3043. Direct selection was applied for

242

simultaneous CmR and for complementation of one nutritional marker. It is important to note that the presence of plasmid DNA in a CCC form is absolutely necessary for transforming B. subtilis. When a mixture of p H V 3 3 - ( d T ) ~ DNA was annealed with B. subtilis-(dA)~-o DNA and used for transformation, no Cm R colonies were detected. A similar result has already been reported with linearized or nicked circular plasmid DNA (Gryczan and Dubnau, 1978; Ehrlich, 1978; Duncan et al., 1978; Contente and Dubnau, 1979; Chang and Cohen, 1979). With CCC-hybrid plasmids the number of Cm R transformants was proportional to the amount of DNA, up to 100 ng per assay. The efficiencies were close to 5 x 104 transformants per gg of DNA, about 20 times lower than the value observed with recombinant plasmids isolated by the hydroxyapatite separation method. Each of the 25 extracts containing 100 ng DNA was used to transform the appropriate B. subtilis auxotrophs. Despite the relatively low yield of Cm r transformants, recombinant DNA plasmids that complemented several mutant alleles were detected: thr5 (about one hundred clones Thr +) leuA8 (3 clones Leu+), hisA1 (1 clone His+), glyB133 (2 clones Gly +) and purB33 (2 clones Ade+). Complementation for each marker was observed with only one particular extract. Expression of the threonine marker is strikingly high compared to the other characters expressed. Unfortunately the nature of the thr mutations is still unknown in B. subtilis (Young and Wilson, 1976). The phenotype of each transformant was verified by replica plating on appropriate media. Phenotypic reversion among the Cm R transformants could reasonably be ruled out on the basis of the reversion rate known for the different auxotrophic mutations studied (less than 10- 6). Furthermore genetic markers were identified in several hybrid plasmids extracted from B. subtilis transformants (see below). The plasmid collection appears therefore to contain recombinant DNA markers widely distributed on the B. subtiffs circular chromosome (Fig. 1). However, not all genetic markers sought were actually detected among the Cm R transformants. So far, attempts to obtain complementation of the following auxotrophic mutations were unsuccessful: trpC2, purA16, metC3, cysA14. Complemented B. subtilis transformants were then screened for their plasmid content. Supercoiled DNA was prepared by the NaOH treatment method. Table 1 summarizes some of their properties. Plasmid bands were readily detected by agarose gel electrophoresis in 8 cases out of 11 examined. The size of the recombinant plasmids ranged from 7 to 15 Mdal, indicating insertion of foreign DNA segments in pHV33 of 2 to 8 Mdal. A more sensitive test to detect

G. Rapoport et al. : Bank of B. subtilis DNA

Fig. 1. Simplified map ofB. subtilis 168 (Lepesant-Kejzlarovfi et al., 1975) indicating position of genetic markers tested in the present study. Complementation of auxotrophic mutations obtained by transformation with recombinant plasmids are underlined

the presence of hybrid plasmids was based on the capacity of the extracts to transform E. coli SK1592 for Ap e. Transformants displaying this phenotype were found in the same 8 cases. The transformation rate of E. coli by pHV33 and by the hybrid plasmids extracted from the transformants by the NaOH method was 10-100 x lower than that obtained with more highly purified plasmid DNAs, depending upon the extract tested. The absence of plasmid DNA was thus confirmed in extracts of clones 2, 3 and 9.. The same DNA preparations were then used to transform B. subtilis and selection was applied either for Cm R or for both Cm R and auxotrophic complementation. In all cases, except the three clones mentioned above in which no plasmid DNA was observed, transformants to Cm R were obtained with a frequency of about 104 transformants per gg of DNA, which is about the same as that observed with recombinant DNA plasmids prepared from E. coli. However, complementation for auxotrophic markers was found in only 2 cases, for clone 1 and for clone 8 (Table 1). Loss of DNA markers suffered by most of the plasmids recovered from the complemented B. subtilis may have been due to in vivo integration of one or two markers into the chromosomal DNA. This hypothesis was tested by using chromosomal DNA extracted from clone 2 to transform strain QB666. Selection was applied for only one character: either Cm k or Thr ÷. Transformants were actually obtained for both markers (for instance 650 Cm~ colonies and 600 Thr-- colonies in one typical experiment performed at DNA saturation). It is therefore likely that integration of genetic markers carried by

243

G. Rapoport et ai. : Bank of B. subtilis D N A Table 1. Properties of the plasmids extracted from complemented transformants of Bacillus subtilis Relevant properties of the extracts

Clones examined 1 Thr +~

2 Thr +

3 Thr +

4 His +

5 Leu +

6 Leu +

7 Leu +

8 Gly +

9 Gly +

10 Ade +

11 Ade +

Detection of plasmid b Estimated size (Mdal)

+ 7

n.d

n.d.

+ 7

+ 9

+ 15

+ I5

+ 7.5

n.d.

+ 7.5

+ 8

Transformation of E. coli for Ap R~

+

-

-

+

+

+

+

+

-

+

+

Transformation of B. subtilis for Cm R

+

-

-

+

+

+

+

+

-

+

+

Complementation of B. subtilis for auxotrophic markers d

+

.

+

-

-

-

.

.

.

.

.

All B. subtilis transformants were initially selected for Cm R. Extracts of plasmid D N A were made by the N a O H treatment method (see Materials and Methods) a Three clones were picked at random among the Thr + transformants b The presence of plasmid was monitored by agarose gel electrophoresis (see Materials and Methods) (n.d. = n o t detected) c Strain SK1592 was transformed with the different extracts and selection was applied for Ap R d Strain QB666, QB934 and QB3043 were transformed for Cm R alone and for both Cm R and auxotrophic complementation. Signs + o r refer to a positive or a negative response concerning the property envisaged

Table 2. Properties of the plasmids isolated from the transformants Cm~Thr + of a B, subtilis rec- strain Relevant properties of the plasmids

Frequency ~

Detection of the plasmid Size (Mdal)

+ 7 _+0.2

100

Transformation of SK1592 for Ap R

+

100

Transformation of QB666 for Cm R

+

100

Transformation of QB666 for Thr +

+

100

QB3006 (thr5 leuA8 recE4) was transformed for both Cm R and Thr + with a recombinant plasmid containing the thr character extracted from E. coli SK1592. Twenty clones CmRThr + were picked at random and their plasmid analyzed as mentioned in Table 1 a Percentage of the clones examined giving a positive response concerning the property envisaged

the recombinant plasmids did occur into the chromosomal DNA. The fate of a particular genetic character (in a recombinant plasmid prepared from E. coli) was followed in a strain of B. subtilis harboring the recE4 marker. The presence of this recombination-deficient mutation prevents the integration of chromosomal D N A fragments into the bacterial chromosome during standard transformation (Dubnau and Cirigliano, 1974). Strain QB3006 (thr5 leuA8 recE4) was

transformed to Cm R with the recombinant plasmid obtained from E. coll. which has been shown to complement strain QB666 for Thr + (see Table 1). All the transformants selected for Cm R were also Thr +, with a frequency of about 103 transformants/gg D N A obtained by the N a O H treatment method. Twenty Thr ÷ isolates were picked at random and analyzed for their plasmid content (Table 2). The following results were obtained: i) all the Thr ÷ transformants contained CCCD N A molecules showing the same electrophoretic mobility, with a size equivalent to that of the plasmid extracted from clone 1 (Table 1). ii) all the plasmids recovered were able to transform E. coli SK1592 for Ap R. iii) all the plasmids recovered were able to transform strain QB666 for Cm R and Thr + at the same frequency (about 2 x 104 transformants/gg DNA). It is concluded from these data that the genetic markers carried by a recombinant plasmid are not integrated into the chromosomal D N A when a strain harboring the mutation recE4 was used as a recipient cell.

Discussion

The results presented demonstrate that the hybrid plasmid pHV33 is potentially useful as a vector for molecular cloning in both E. coli and B. subtilis. The poly(dA)-poly(dT) "tailing" method is a very effec-

244

tive way of recovering E. coli transformants carrying hybrid plasmids in about 80% of the cases. However, this method could not be applied directly to B. subtilis as annealed circular molecules of DNA which are not CCC have no transforming activity in this bacterial species. The rationale used to establish a colony bank of B. subtilis DNA was to use E. coli as a host which can be transformed by these molecules; circularization of the annealed recombinant molecules and their amplification proceeds easily in E. coli. Also E. eoli has the advantage of maintaining stably the recombinant plasmids under selective conditions, which is not the case in B. subtilis rec +. From the results which we have obtained it appears that a colony bank of 2500 clones containing inserted DNA fragments of about 3 Mdal is sufficient to find any segment of B. subtilis DNA (total molecular weight of about 3 x 109 daltons) with a probability level higher than 80%. The collection was easily screened for the presence of hybrid plasmids containing ribosomal DNA sequences of B. subtilis and thirty such clones were identified. Pooled fractions of CCC-plasmids purified from E. coli by the rapid NaOH-treatment method were shown to be capable of complementing auxotrophic requirements of B. subtilis mutants with an acceptable efficiency. Although expression of any cloned marker is unlikely, for instance in the case where several cistrons are under the regulation of one promoter in the same operon, a relatively good proportion of genetic functions have been complemented in mutants of B. subtiIis. These markers are widely distributed along the chromosomal map. Eleven clones of rec + B. subtilis transformed for nutritional markers have been examined for the state of the genes introduced into the cells. Three situations were encountered when the cells were grown in the presence of Cm: i) an apparently intact plasmid can be recovered from the cell and used to transform E. coIi for Ap R and B. subtilis for Cm ~ and for the nutritional marker. ii) a plasmid DNA can be recovered from the cell and used to transform E. coli for Ap R and B. subtilis for Cm R, but not for the nutritional marker. Since the original B. subtilis remains prototrophic for the nutritional marker, it is presumed that the gene has been integrated into the bacterial chromosome. iii) no plasmid DNA can be detected by agarose gel electrophoresis of a cell extract, and the extract does not transform either E. coli or B. subtilis for antibiotic or nutritional markers. However, the original B. subtilis cell remains prototrophic for the nutritional marker and Cm ~.

G. Rapoport et al. : Bank of B. subtilis DNA

In Table 1, clones 1 and 8 fall into the first class, whereas clones 4, 5, 6, 7, 10 and 11 fall into the second class and clones 2, 3 and 9 fall into the third class. The phenomenon of transfer of markers from plasmid to chromosomal DNA appears to be dependent upon the rec gene, since in recE4 cells, similar recombination of genes did not appear to occur (Table 2). Evidence for integration of markers into the host chromosome was provided by the fact that chromosomal DNA isolated from clone 2 readily transforms recipient cells to a Cm R and Thr + phenotype. Integration and expression of a foreign marker, in this case the Cm character originally issued from S. aureus, into the chromosome of B. subtilis is of strong interest. Integration into the B. subtilis chromosome of an entire chimeric plasmid composed of plasmid pMB9 and a DNA fragment carrying the thymidylate synthetase gene from phage /322 has been recently reported (Duncan et al., 1978). It is also worth noting that no case was found in which the Cm character was lost from the plasmid into the chromosome while the complementing marker was maintained into the recombinant plasmid. This is deduced by the fact that, when present, all the plasmids isolated from the transformant clones coded for Cm R in recipient cells. Our experiments indicate that the use of rec- mutants will be of great help for DNA cloning in B. subtilis. Keggins et al. (1978) have already shown that a clone containing a Trp fragment of B. subtilis is stably maintained in a recE4 strain. A recombinant plasmid constructed by linking B. subtilis Leu genes and a B. subtilis natto plasmid has also been recently shown to be stably maintained in a strain of B. subtilis harboring the recE4 marker (Tanaka and Sakaguchi, 1978). We have now shown that is has been possible to clone a thr-complementing DNA fragment carried by a recombinant plasmid extracted from E. coli by using a B. subtilis recE4 recipient. The colony bank in our case is maintained in E. coli where it appears to be stable. It should be useful for isolating other DNA fragments and for constructing merodiploid strains in B. subtilis which will permit genetic complementation analyses and also for a variety of studies on gene expression, transformation and sporulation. Acknowledgments. We are indebded to Dr S.D. Ehrlich for providing us the plasmid pHV33. We gratefully acknowledge Dr H.C. Birnboim for communicating the rapid method of plasmid extraction before publication and for correcting this manuscript. We thank also Mrs J. Walle for technical assistance. This work was supported by grants from the Centre National de la Recherche Scientifique (n ° 3557), from the D616gation G6n~rale ~tla Recherche Scientifique et Technique (n ° 77 7 0293), from the Commissariat ~ l'Energie Atomique, and from the Fondation pour la Recherche M+dicale Frangaise.

G. Rapoport et al. : Bank of B. subtitis DNA

References Beers, R.F., Bassett, E.G.: Recombinant molecules: Impact on science and society, pp. 1 531. New-York: Raven Press 1977 Bonamy, C., Keryer, E., Szulmajster, J. : Cloning of Bacillus subtilis DNA fragments carrying spore genes. In: Spores VII (Chambliss, G. and Vary, J.C., eds), pp. I39 143. Washington: Amer. Soc. Microbiol. 1978 Boyer, H.W., Betlach, M., Bolivar, F., Rodriguez, R.L., Honeycker, H.L., Shine, J., Goodman, H.M. : The construction of molecular cloning vehicles In: Recombinant molecules: Impact on science and society (Beers, R.F. and Bassett, E.G., eds.), pp. 9-20. NewYork: Raven Press 1977 Boyer, H.W., Nicosia, S. : Genetic engineering, pp. 1 298. Amsterdam-NewYork-Oxford: Elsevier/North Holland Biomedical Press 1978 Chang, S., Cohen, S.N. : High frequency transformation of Bacillus subtilis protoplasts by plasmid DNA. Mol. Gen. Genet. 168, II1-115 (1979) Chi, N-Y.W., Ehrlich, S.D., Lederberg, J.: Functional expression of two Bacillus subtilis chromosomal genes in Escherichia coli. J. Bacteriol. 133, 816-821 (1978) Clarke, L., Carbon, J.: Biochemical construction and selection of hybrid plasmids containing specific segments of the Escherichia coli genome. Proc. Natl. Acad. Sci. U.S.A. 72, 4361-4365 (1975) Clarke, L., Carbon, J. : A colony bank containing synthetic ColEI hybrid plasmids representative of the entire E. coli genome. Cell 9, 91-99 (1976) Collins, J. :Gene cloning with small plasmids. Curr. Top. Microbiol. hnmunol. 78, 121 170 (1977) Colman, A., Byers, M.J., Primrose, S.B., Lyons, A. : Rapid purification of plasmid DNAs by hydroxyapatite chromatography. Eur. J. Biochem. 91, 303-310 (1978) Contente, S., Dubnau, D. : Characterization ofplasmid transformation in Bacillus subtilis: Kinetic properties and the effect of DNA conformation. Mol. Gem Genet. 167, 251-258 (1979) Dedonder, R.A., Lepesant, J-A., Lepesant-Kejzlarov~, J., Billault, A., Steinmetz, M., Kunst, F. : Construction of a kit of reference strains for rapid genetic mapping in Bacillus subtilis 168. Appl. Environ. Microbiol. 33, 989-993 (1977) Dubnau, D., Cirigtiano, C. : Fate of transforming deoxyribonucleic acid after uptake by competent Bacillus subtilis: Size and distribution of the integrated donor segments. J. Bacteriol. 117, 488-493 (1974) Duncan, C.H., Wilson, G.A., Young, F.E. : Mechanism of integrating foreign DNA during transformation of Bacillus subtilis. Proc. Natl. Acad. Sci. U.S.A. 75, 3664-3668 (1978) Ehrlich, S.D., Bursztyn-Pcttegrew, H., Stroynowski, 1., Lederberg, J. : Expression of the thymidylate synthetase gene of the Bacillus subtilis bacteriophage Phi-3-T in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 73, 4145-4149 (1976) Ehrlich, S.D. : DNA cloning in Bacillus subtilis. Proc. Natl. Acad. Sci. U.S.A. 75, 1433 1436 (1978) Grunstein, M., Hogness, D.S.: Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gone. Proc. Natl. Acad. Sci. U.S,A. 72, 3961-3965 (1975) Gryczan, T.J., Dubnau, D.: Construction and properties of chimeric plasmids in Bacillus subtilis. Proc. Natl. Acad. Sci. U.S.A. 75, 1428-1432 (1978) Hariharan, P.V., Hutchinson, F.: Neutral sucrose gradient sedimentation of very large DNA from Bacillus subtilis. J. Mol. Biol. 75, 479494 (1973)

245 Hoch, J.A.: Developmental genetics at the beginning of a new era. In: Spores VII (Chambliss, G. and Vary, J.C., eds.), pp. 119 121. Washington: Amer. Soc. Microbiol. 1978 Horinouchi, S., Uozumi, T., Hoshino, T., Ozaki, A., Nakajima, S., Beppu, T., Arima, K. : Molecular cloning and in vitro transcription of Bacillus subtilis plasmid in Escherichia coli. Mol. Gen. Genet. 157, 175-182 (1977) Keggins, K.M., Lovett, P.S., Duvall, E.J.: Molecular cloning of genetically active fragments of Bacillus DNA in Bacillus subtilis and properties of the vector plasmid pUB 110. Proc. Natl. Acad. Sci. U.S.A. 75, 1423-1427 (1978) Kreft, J. Bernhard, K., Goebel, W. : Recombinant plasmids capable of replication in B. subtilis and E. coll. Mol. Gen. Genet. 162, 59-67 (1978) Lederberg, E.M., Cohen, S.N. : Transformation of Sahnonella thyphimurium by plasmid deoxyribonucleic acid. J. Bacteriol. 119, 1072-1074 (t974) Lepesant-Kejzlarov/l, J., Lepesant, J-A., Walle, J., Billault, A., Dedonder, R. : Revision of the linkage map of Bacillus subtilis 168 : Indications for circularity of the chromosome. J. Bacteriol. 121, 823-834 (1975) Lobban, P.E., Kaiser, A.D. : Enzymatic end-to-end joining of DNA molecules. J. Mol. Biol. 78, 453-471 (1973) Mahler, I., Halvorson, H.O.: Transformation of Escherichia coli and Bacillus subtilis with a hybrid plasmid, J. Bacteriol. 131, 374-377 (1977) Maizels, N. : RNA labeling mediated by T4 polynucleotide kinase. In : ICN-UCLA Syrup. on Molecular and Cellular Biology (Wilcox, G., Abelson, J., Fox, C.F., eds.), Vol. 8, pp. 247-251. New York: Academic Press 1977 Nagahari, K., Sakaguchi, K. : Cloning of Bacillus subtilis leucine A, B and C genes with Escherichia coli plasmids and expression of the leuC gene in E. coll. Mol. Gen. Genet. 158, 263-270 (1978) Niaudet, B., Ehrlich, S.D. : In vitro genetic labeling of Bacillus subtilis cryptic plasmid pHV400. Plasmid 2, 48-58 (1979) Potter, S.S., Bott, K.F., Newbold, J.E.: Two-dimensional restriction analysis of the Bacillus subtilis genome: Gone purification and ribosomal ribonucleic acid gone organization. J. Bacteriol. 129, 492-500 (1977) Segall, J., Losick, R. : Cloned Bacillus subtilis DNA containing a g e n e that is activated early during sporulation. Cell 11, 751 761 (1977) Tanaka, T., Sakaguchi, K. : Construction of a recombinant plasmid composed of B. subtilis leucine genes and a B. subtilis (natto) plasmid: its use as cloning vehicle in B. subtilis 168. Mol. Gen. Goner. 165, 269-276 (1978) Wake, R.G. : Circularity of the Bacillus subtilis chromosome and further studies on its bidirectional replication. J. Mol. Biol. 77, 569 575 (1973) Young, F.E., Wilson, G.A. : Revision of the linkage map of Bacillus subtilis. In:Handbook ofbiochem, molec, biol. (Fasman, G.D., ed.), Vol. 2, 3rd ed., pp. 686-703. Cleveland : Chemical Rubber Co. Press 1976

Communicated

by W. Gehring

Received June 2, I979