Structural organization of pLP1, a cryptic plasmid from Lactobacillus plantarum CCM 1904

PLASMID

22, 185-192 (1989)

Structural from

Organization

of pLP1, a Cryptic

Lactobacillus

plantarum

CCM

Plasmid 1904

ABDELHAK BOUIA, FRANCOISE BRINGEL, LLJCIE FREY, BENO~T KAMMERER, ABDELKADER BELARBI, ARMEL GUYONVARCH, AND JEAN-CLAUDE HUBERT’ Laboratoire de microbiologic, Universite’ Louis-Pusteur, Institut de Biologic A4oltkdaire et Cellulaire du CNRS, 4, rue Bluise-Puscal, 67070 Strasbourg Cedex. France Received June 2 I. 1989: revised November

13, 1989

To construct shuttle vectors based on an endogenous replicon, we isolated a small cryptic plasmid (pLP1) from Lactobacillus plantarum CCM 1904. The nucleotide sequence (2093 bp, 38.25 GC mol%) revealed one major open reading frame encoding for a 3 17 amino acid protein (Rep). Comparisons with proteins encoded by other Gram-positive bacteria plasmids strongly suggest that the protein encoded by pLPl has a replicative role. The presence of a consensus sequence including a tyrosine residue known to be the replication protein binding site to the DNA (in phage #X 174) strengthens this hypothesis. The DNA sequence contains also a sequence similar to the pC194 origin nick sequence, which initiates the plasmid replication at the plus origin, characteristic of plasmids which replicate following a rolling circle mechanism via single-stranded DNA intermediates. A set of 13 direct rcpcats of 17 bp could be involved ia the expression of the incompatibility or in the copy number control as in the other plasmids. A promoter sequence located at the rep 5’ region has been identified and is functional in Buci1fu.c .suhtilis. D ~~~~caderns

Plasmids have been found in different lactobacilli where they can be involved in the glucidic metabolism (Liu Ed al., 198X), amino acid metabolism (Shay et al., 1988), and drug resistance (Vescovo et al., 1982; Axelsson et al., 1988). A LactobacilIus reuteri plasmid favors the growth rate ability of the bacteria in the cecum of gnotobiotic mice (San-a et al., 1989). But usually, no physiological role has been assigned to the studied plasmids which therefore were designated as cryptic plasmids. Few lactobacilli plasmid studies at a DNA level have been undertaken: a L. acidophilus promoter sequence from a cryptic plasmid has been described by Damiani et al. (1987) and the L. hilgardii cryptic plasmid pLAB 1000 has been analyzed by Josson et al. (1989). L. plantarum is a species of considerable industrial and medical interest. The study of L. ylantarurn gene structure and expression will be possible when efficient DNA recombination techniques are available. To this end, an autonomous replicating vector based on a ’ To whom correspondence should bc addressed.

selected endogenous replicon was constructed. In this paper, we report the cloning and sequence analysis of the pLP1 plasmid present in L. plantarum CCM 1904. MATERIALS

AND

METHODS

Bacterial Strains and Growth Media L. plantarvm CCM 1904 (ATCC 80 14) has been obtained from the Czechoslovak Collection of Microorganisms. Cultures were incubated at 30°C on MRS (de Man et al., 1960) solid or liquid media without agitation. M13mp18 or M13mp19 (Norrander et al., 1983) phage propagation and Escherichia coli pUC19 plasmid (Norrander et aZ., 1983) or E. coli-LactobaciAus hybrid plasmid amplification were done in E. coli JM103 (Messing et al., 198 1). Cultures were propagated at 37°C on solid or liquid LB media (Maniatis et al., 1982) with 50 pg ampicillin/ml as required. Bacilius subtilis MI 112 (Tanaka and Sakaguchi, 1978) was transformed with the promoter probe vector pGR71 (Goldfarb et al., 198 1) and was cultivated on the LB plate at

186

BOUIA

ET AL.

extraction of pLP1 from L. plantarum yields about 20 pg/liter of culture. To obtain sufficient quantities of the plasmid for structural studies, a set of pUC 19-pLP 1 hybrid plasmids DNA Techniques (PULP) was constructed. The resulting plasChromosomal or plasmid DNA from L. mids, PULP 1, pULP2, pULP2 1, and pULP22 pluntarum CCM 1904 was extracted following described by Bringel et al. (accompanying paKlaenhammer’s protocol (1984) with two per), can be amplified and maintained in E. modifications: Mutanolysine at 75 kg/ml was coli. By recutting with the appropriate restricused at 37°C instead of lysozyme at 0°C (lytion enzyme, pLP1 fragments were easily obsozyme at 0°C has no effect on our strains at tained and recloned into the M 13mpl8 or 19 low concentration). Plasmid DNA extraction phagespolylinker for sequencing. from E. coli and from B. subtilis was achieved by the alkaline lysis method (Maniatis et al., pLPl Structure and DNA SequenceAnalysis 1982). Electrophoresis was performed in 0.8% agaFigure 1 shows a pLP 1 restriction map with rose gels (ultrapure, low melting point, from the critical restriction endonuclease sites utiBethesdaResearchlaboratories) in Tris-borate lized to subclone fragments into the M 13mp18 buffer, pH 8.2 (Meyers et al., 1976). or 19 phages for sequencing. The nucleotide DNA-restricted fragments were directly ex- sequence (Fig. 2) of the 2093 bp was detertracted from gel (Hubert et al., 1980) and li- mined for both strands. Each base pair was gated with the appropriate linearized vectors. counted from the first AT pair of the far E. coli transformation by DNA ligation Hind111 site upstream from the major open mixtures was performed following the CaC& reading frame ATG initiation codon (Fig. 1). method (Mandel and Higa, 1970) and B. subThe GC percentage found for this plasmid tilis transformation following the Contente is 38.25 mol% which is lower than the value and Dubnau method (1979). (44-46 mol%) for L. plantarum (Kandler and The DNA sequences were determined Weiss, 1986). Becauseof the broad host range by the dideoxynucleotide chain-terminating of this plasmid among lactic acid bacteria, as method @anger et al., 1977) after subcloning was shown by Bringel et al. (accompanying the different DNA restriction fragments in the paper), no real significance must be given to M 13mp 18 or 19 phages.The universal primer this absence of correlation between the two (Biolabs M 13 sequencing primer, 17-mer) was GC mole percents.The pLP1 plasmid was also used as initiation primer. In order to read the found in Leuconostoc mesenteroides(GC 37sequenceentirely in both orientations, six oli- 40 mol%; Garvie, 1986), in Lactobaciffus curgonucleotides had to be synthesized to serve vatus and Lactobacillus sake (GC 42-44 as primers. mol%; Kandler and Weiss, 1986). GC mole The polyacrylamide-urea gel electropho- percent of ssDNA plasmids or ssDNA phages resis method @anger and Coulson, 1978) was is usually lower than that of the host. usedto generatethe sequencing ladders. DNA A major reading frame (Fig. 1) which exsequenceswere analyzed with the University tends over 95 1 nucleotides shows identifiable of Wisconsin Genetics Computer Group se- ribosome binding sites at the consensus disquence analysis software package (Devereux tance from the translational start reported for et al., 1984). genesfrom Gram-positive bacteria (Moran et al., 1982). RESULTS pLP 1 contains a set of 13 contiguous direct repeats of 17 bp (5’~CATTATCATGTAGTL. pluntarum CCM 1904 harbors at least GCG-3’) located outside of the major ORF* three plasmids. The smallest of theseplasmids, a 2.1-kb plasmid designatedpLP 1, was chosen * Abbreviation used: ORF, open reading frame. to be investigated at the molecular level. Direct 37°C supplemented with 20 pg kanamycin/ ml or 5 pg chloramphenicol/ml.

STRUCTURE OF PLASMID pLPl AhdUAMlJ

187

HindUI

\ PWII

FIG. 1. pLPl plasmid simplified restriction map: only the restriction sites used for pLP1 subcloning into M 13mpl8 or mpl9 phagesare shown. For each subclone thin arrows indicate the sequencingdirection and the determined sequencelength. The positions of oligonucleotides synthesized for sequencing (1168-I 183, 5’-GTTGTCGCAAAATGGG-3’; 13IO- 1326,5’-CCAATTTATTTGGAGTG-3’; 1568- 1585, S-CATGAAGTTGTGTTGTGC-3’; 1602- 1586,5’-AGGTTTTGATGGGTTTA-3’; 1330- 1311, S-CTTACACTCCAAATAAATTG-3’; 1037-1021,5’-CGTGTTCCAGCTAAGGC-3’ ) are indicated by small squares.Grey arrow, position and direction of the major ORF encoding the replication protein (Rep). Hatched line, set of 13 17bp direct repeats.

between position 1335 and position 1554 as shown in Fig. 2. The related RSFl 0 10 (Persson and Nordstrom, 1986) and R1162 (Lin et al., 1987) broad-host-range plasmids carry a set of three and one-half perfectly conserved 20-bp directly repeated sequencesessential for their incompatibility expression. In the caseof the plasmid RI 162, these direct repeats are also involved in the copy number control and the plasmid in vitro replication. The role of pLP1 direct repeats is yet not known, but further experiments consisting of partial or total deletions of the direct repeats are under investigation and will show if the pLP1 repeats are also involved in plasmid incompatibility expression. At position 2051 begins a sequence (5’TTCTTATCTTGATA-3’) which is identical to the known plus origin of replication sequencesofpC194, pUBll0 (Gros et al., 1987) pBAA1 (Devine et al., 1989), and pCBl0 1

(Minton and Oultram, 1988) and is highly similar to that of the phage 4X 174 (5’-CCCCCAACTTGATA-3’; Langeveld et al., 1978). Therefore, this sequencemay be the pLP1 plus origin of replication which includes the nick site. ORF SequenceAnalysis The major ORF extends from nucleotide 262 to nucleotide 1212 over 95 1 bp. The 5’ upstream region contains a 5’-GGAGG-3’ Shine and Dalgarno sequence( 1974) boxed in Fig. 2 at positions -13 to -9 from the ATG codon. Nothing is known about the L. plantarum 3’ end 16 S rRNA sequencebut the sequence found here also exhibits complementarity with the B. subtilis 3’ end 16 S rRNA sequence (Moran et al., 1982). Potential promoter regions following consensus sequencespointed

BOUIA

ET AL.

FIG.2. pLPl plasmid nucleotide sequence and predicted Rep protein amino acid sequence. Boxed, Shine and Dalgamo sequence, initiation codon, and putative plus origin of replication; the vertical arrow, position of the possible nick site; horizontal black arrows, 17-bp direct repeats; horizontal hatched arrows, inverted repeats. This nucleotide sequence has been submitted to Gen Bank and assigned the accession number M31223.

189

STRUCTURE OF PLASMID pLPl

A 240

U A A

G G

U-A ,G-C LA-U C-G/ u - rlG 250

230

C

Shine & Dalgamo sequence

U- A C-G G-C A-U U Q codon

215

270

... FIG. 3. Possible hairpin loop containing the putative Shine and Dalgamo sequenceand the pLP 1 main ORF initiation codon. This loop can play a role in the mRNA translation regulation hence in the replication regulation. The free energy of formation (as defined by Freier et al., 1986) of this loop is - 14.5 kcal/mol.

out by Graves and Rabinowitz (1986) were found. The most plausible area for transcriptional initiation seemsto be the TATTCT sequence (positions 169- 174) for the - 10 region and the sequence TTGATT (positions 143148) for the -35 region. Another possible Pribnow box could be the TATAA sequence located at positions 202-206. The comparison of pLP 1 sequencewith the L. acidophilus cryptic plasmid pPV741 promoter region described by Damiani et al. (1987) revealed no similarity. The presenceof a promoter activity in this region was confirmed by the cloning of the HindIII-Hind111 pLP1 fragment (positions 1 to 206) into the Hind111 site of the promoter probe vector pGR71 (Goldfarb et al., 1981). The recombinant plasmid with the HindIII-Hind111 fragment in the same orientation relative to pLP 1, designatedpFB 1, was usedto transform the B. subtilis MI1 12 strain. Cells harboring the pFB 1 plasmid exhibited chloramphenicol resistance(minimal inhibitory concentrations, 2 pg/ml for MI 112 [pGR7 11; 5 pg/ml for MI 112 [pF’Bl]) indicating that the HindIIIHind111 fragment contains a sequence active

on the pGR7 1 CAT gene transcription initiation in B. subtilis. S1 nuclease mapping currently in progresswill reveal the precise transcription initiation site. Northern hybridization between total RNA and pLP1 DNA corresponding to the ORF revealed a unique 1.3-kb transcript (data not shown). One inverted repeat in the 5’ region (Figs. 2 and 3) could form a hairpin structure which occludes the ribosome binding sequence,suggesting that this region could play a regulatory role in protein translation and plasmid replication modulation. The putative protein of 3 17 amino acids encoded by the ORF was compared to the NBRF protein sequencedata bank using the methods of Wilbur and Lipman (1983) and Lipman and Pearson (1985). Significant similarities (Fig. 4) were observed among proteins involved in Gram-positive bacteria plasmids replication such as PUB 110 and pC194 (Staphylococcus aureus), pFTB 14 (B. amyloliquefaciens), pBAA 1 (B. subtilis), pIJ 101 (Streptomyces lividans), and pCB 101 (Clostridium butyricum) and also with the phage 4X 174 replication protein. This result strongly

190

BOUIA ET AL. PC194 PFTBl4 pBAA1 pm1 10 PLPl

...........................

MCYNMEKYTEKKQRNQVFQKFIKRHIGE ..... MYSSENDYSILE~ATGKKRDWRGKKRRANLMAEHYEALKKRIGAPY ............ ............................................ MANHYEALNSKIGAPY NGVSFNIMCPNSSIYSDEKSRVLVDKTKSGKVRPWREKKIANVDYFELLHILE ....... .................. MSEIFKDKTENGKVRPWRERKIENVRYAEYLAILE .......

q

PC194 pFTf314 pBAA1 pm110 PLPl

PC194 pFw14 pEAA pLlB110 PLPl

QVQKIRQNN DIRRVKGRV

PC194 PFrBl4 pEAA pUBi PLPl

DIRRVKGKA

PC194 pFm14 peAA pm1 10 PLPl

229 PC1 94 PFTBl4 pBAA1 puEl110 PLPl FIG. 4. Similarities among replication proteins of Gram-positive bacteria plasmids (pC194 (Horinouchi and Weisblum, 1982) and pUBll0 (McKenzie et al.. 1986, 1987) from Staphylococcus aureus, pFTB14 from Bacillus amyloliquefaciens (Murai et al., 1987),pBAA 1 from B. subtilis (Devine et al., 1989), pCBl0 1 from Clostridium butyricum (Minton and Oultram, 1988) and pIJ 101 from Streptomyces lividans (Kendall and Cohen, 1988)and 6x174 bacteriophage (Sangeret al., 1978)).Boldface, amino acids common to pLPl protein and other proteins; boxed, amino acids common to all replication proteins. Only the replication protein region around the tyrosine (Y) residue implicated in the phage CpX174 protein A enzymatic site is shown for pCBlO1 and pIJ 101 (these short sequencesimilarities have already been described by Gruss and Ehrlich ( 1989)).

suggeststhat the ORF-encoded protein (Rep) is implicated in pLP1 replication. As in the other replication protein sequences cited, a tyrosine residue is present at position 229 (Fig. 4). This amino acid is described as the 4X 174 protein A linkage site to the DNA when nicking occurs at the plus origin (Gros et al., i987; van Mansfeld et al., 1986). DISCUSSION

pLP 1, the first plasmid from a L. plantarwn strain to be sequenced,shows an organization

similar to that of many other plasmids from Gram-positive bacteria. Its unique open reading frame encodes for a replication protein (Rep). The strong similarity of the Rep proteins argues for a common rep gene origin. Thus, the pLP1 plasmid seemsto belong to a large plasmid family. Its simple structure supports the choice of pLP1 as a model for the genetic study of this plasmid family in lactic acid bacteria. The similarity between pLP1 and Grampositive bacteria plasmids or the E. coii phage $X 174 strongly suggeststhat pLP 1 also adopts

STRUCTURE OF PLASMID pLPl

a rolling-circle mechanism involving singlestranded DNA intermediates for its replication (te Riele et al., 1986a,b; Gros et al., 1987; reviewed by Gruss and Ehrlich, 1989). Thus, the use of pLP1 replicon as the base for cloning vector constructions is not recommended since Ballester et al. (1989) and Peeterset al. ( 1988) demonstrated that single-stranded DNA intermediates stimulate illegitimate recombinations which lead to high structural plasmid instability or deletions. On the other hand, the presence of pLP1 in many strains and its incurability by treatment with chemical agents(novobiocine, a&dine) or after growth at sublethal temperatures indicate that pLP1 has a broad host range and is stably inherited. Different shuttle vectors have been constructed and are currently used to transform L. plantarum or other lactobacilli. The first results are discussed by B&gel et al. (see accompanying paper). ACKNOWLEDGMENTS This work was supported by the CNRS (ATP Biotechnologies 95 l-20 140)and by a grant from the Minis&e de la Recherche et de la Technologie 87T0173.

REFERENCES AXELSSON,L. T., AHRN~, S. E. I., ANDERSSON,M. C., AND STAHL,S. R. (1988). Identification and cloning of a plasmid-encodederythromycin resistancedeterminant from Lactdbacillus reuteri. Plasmid 20, 17l- 174. BALLESTER,S., LOPEZ,P., ESPINOSA,M., ALONSO,J. C., AND LACKS,S. A. (1989). Plasmid structural instability associatedwith pC194 replication functions.J. Bacterial. 171,2271-2277.

BRINGEL,F., FREY,L., AND HUBERT,J.-C. (1989). Characterization, cloning, curing, and distribution in lactic acid bacteria of pLP1, a plasmid from Lactobacillus plantarum CCM 1904 and its use in shuttle vector construction. Plasrnid 22, 193-202. CONTENTE,S., AND DUBNAU,D. ( 1979).Characterization of plasmid transformation in Bacillus subtilis: Kinetic properties and the effect of DNA conformation. Mol. Gen. Genet. 167,25 l-258. DAMIANI, G., ROMAGNOLI,S., FERRETTI,L., MORELLI, L., Bo’rr~zr, V., AND SGARAMELLA,V. (1987). Sequence and functional analysis of a divergent promoter from a cryptic plasmid of Lactobacillus acidophilus 168 S. Plasmid 17, 69-72.

DE MAN, J. C., ROGOSA,M., AND SHARPE,M. E. (1960). A medium for the cultivation of lactobacilli. J. Appl. Bacterial. 23, 130- 135.

191

DEVEREUX,J., HAEBERLI,P., AND SMITHIES,0. (1984). A comprehensive set of sequenceanalysis programs for the VAX. Nucleic Acids Res. 12, 387-395. DEVINE, K. M., HOGAN, S. T., HIGGINS, D. G., AND MCCONNELL,D. J. (1989). Replication and segregational stability of Bacillus plasmid pBAA 1. J. Bacterial. 171, 1166-l 172. FREIER,S. M., KIERZEK, R., JAEGER,J. A., SUGIMOTO, N., CARUTHERS,M. H., NEILSON,T., AND TURNER, D. H. (1986). Improved free-energyparameters for predictions of RNA duplex stability. Proc. Natl. Acad. Sci. USA 83,9373-9377.

GARVIE, E. I. (1986). Genus Leuconostoc van Tieghem 1878.In “Bergey’s Manual of SystematicBacteriologyVol. 2” (P. H. A. Sneath, N. S. Mair, M. E. Sharpe, and J. G. Holt, Eds.), pp. 1071-1075. Williams & Wilkins, Baltimore. GOLDFARB,D. S., DOI, R. H., AND RODRIGUEZ,R. L. (1981). Expression of TnY-derived chloramphenicol resistancein Bacillus subtilis. Nature (London) 293,30931 1. GRAVES,M. C., AND RABINOWITZ,J. C. (1986). In vivo and in vitro transcription of the Clostridium pasteurianum ferredoxin gene. Evidence for “extended” promoter elements in Gram-positive organisms. J. Biol. Chem. 261, 11,409-l 1,415. GROS,M. F., TE RIELE,H., AND EHRLICH,S. D. (1987). Rolling circle replication of single-stranded DNA plasmid pC194. EMBO J. 6,3863-3869. GRUSS,A., AND EHRLICH,S. D. (1989). The family of highly interrelated single-strandeddeoxyribonucleic acid plasmids. Microbial. Rev. 53,23 1-24 1. HORINOUCHI,S., AND WEISBLUM,B. (1982). Nucleotide sequenceand functional map of pCl94, a plasmid that specifiesinducible chloramphenicol resistance.J. Bacteriol. 150, 8 15-825.

HUBERT,J.-C., BACH,M.-L., AND LACROUTE,F. (1980). Measureof asymmetrical transcription of the yeastOMP decarboxylasegeneexpressedin yeast or in E. coli. Curr. Genet. 2, 103-107. JOSSON, K., SCHEIRLINCK, T., MICHIELS,F., PLATTEEUW, C., STANSSENS, P., Joos, H., DHAESE,P., ZABEAU,M., AND MAHILLON,J. (1989). Characterization of a grampositive broad-host-range plasmid isolated from Lactobacillus hilgardii. Plasmid 21, 9-20.

KANDLER,O., AND WEISS,N. (1986). Genus Lactobacillus Beijerinck 1901. In “Bergey’s Manual of Systematic Bacteriology-Vol. 2” (P. H. A. Sneath, N. S. Mair, M. E. Sharpe, and J. G. Holt, Eds.), pp. 1209-1234. Williams & Wilkins, Baltimore. KENDALL, K. J., AND COHEN,S. N. (1988). Complete nucleotide sequenceof the Streptomyces lividans plasmid pIJ 101 and correlation of the sequencewith genetic properties. J. Bacterial. 170, 4634-465 1. KLAENHAMMER,T. R. (1984). A general method for plasmid isolation in lactobacilli. Curr. Microbial. 10, 2328.

LANGEVELD,S. A., VAN MANSFELD,A. D. M., BAAS, P. D., JANSZ,H. S., VANARKEL,G. A., AND WEISBEEK, P. J. (1978). Nucleotide sequenceof the origin of rep-

192

BOUIA

lication in bacteriophage @X174 RF DNA. Nature (London) 271,4 17-420. LIN, L.-S., KIM, Y.-J., AND MEYER,R. J. (1987). The 20 bp, directly repeated DNA sequenceof broad host range plasmid Rl162 exerts incompatibility in vivo and inhibits R 1162DNA replication in vitro. Mol. Gen. Gene/.

ET AL.

PERSSON,C., AND NORDSTROM,K. (1986). Control of replication of the broad host range plasmid RSFlOlO: The incompatibility determinant consistsof directly repeated DNA sequences.Mol. Gen. Genet. 203, 189192. SANGER,F., AND COULSON,A. R. (I 978). The useof thin 208,390-397. acrylamide gels for DNA sequencing. FEBS Len. 87, LIPMAN, D. J., AND PEARSON,W. R. (1985). Rapid and 107-l 10. sensitive protein similarity searches.Science227, 1435- SANGER,F., COULSON,A. R., FRIEDMANN,T., AIR, 1441. G. M., BARRELL,B. G., BROWN,N. L., FIDDES,J. C., LIU, M.-L., KONDO,J. K., BARNES,M. B., AND BARTHOHUTCHISON,C. A., III, SLOCOMBE, P. M., AND SMITH, LOMEW,D. T. (1988).Plasmid-linked maltose utilization M. ( 1978). The nucleotide sequence of bacteriophage in Lactobacillus ssp. Biochimie 70, 35 l-355. $X 174. J. Mol. Biol. 125, 225-246. MANDEL, M., AND HIGA, A. (1970). Calcium-dependent SANGER,F., NICKLEN, S., AND COULSON,A. R. (1977). bacteriophage DNA infection. J. Mol. Biol. 53, 159DNA sequencing with chain-terminating inhibitors. 162. Proc. Natl. Acad. Sci. USA 74, 5463-5467. MANIATIS,T., FRITSCH,E. F., AND SAMBROOK,J. ( 1982). SARRA,P. G., VESCOVO,M., AND BOTTAZZI,V. (1989). “Molecular Cloning: A Laboratory Manual.” Cold Antagonism and adhesion among isogenic strains of Spring Harbor Laboratory, Cold Spring Harbor, NY. Lactobacillus reuteri in the caecum of gnotobiotic mice. MCKENZIE,T., HOSHINO,T., TANAKA, T., AND SUEOKA, Microbiologica 12, 69-74. N. (1986). The nucleotide sequenceof pUBll0: Some SHAY, B. J., EGAN, A. F., WRIGHT, M., AND ROGERS, salient features in relation to replication and its reguP. J. (1988). Cysteine metabolism in an isolate of Laclation. Plasmid 15, 93-103. tobacillus sake: Plasmid composition and cysteine transport. FEMS Microbial. Lett. 56, 183- 188. MCKENZIE,T., HOSHINO,T., TANAKA, T., AND SUEOKA, N. (1987). A revision of the nucleotide sequence and SHINE,J., AND DALGARNO,L. (1974). The 3’-terminal sequence of Escherichia coli 16s ribosomal RNA: functional map of pUBll0. Plasmid 17, 83-85. Complementarity to nonsense triplets and ribosome MESSING,J., CREA, R., AND SEEBURG,P. H. (1981). A binding sites.Proc. Natl. Acad. Sci. USA 71, 1342- 1346. systemfor shotgun DNA sequencing.Nucleic Acids Rex TANAKA, T., AND SAKAGUCHI,K. (1978). Construction 9, 309-32 I. of a recombinant plasmid composed of B. subtilis leuMEYERS,J. A., SANCHEZ,D., ELWELL,L. P., AND FALtine genes and a B. subtilis (natto) plasmid: Its use as KOW, S. (1976). Simple agarose gel electrophoretic cloning vehicle in B. subtilis 168.Mol. Gen. Genet. 165, method for the identification and characterization of 269-276. plasmid deoxyribonucleic acid. J Bacterial. 127, 1529TE RIELE, H., MICHEL, B., AND EHRLICH,S. D. (1986a). 1537. Single-stranded plasmid DNA in Bacillus subtilis and MINTON, N. P., AND OULTRAM,J. D. (1988). Host:vector Staphylococcusaureus. Proc. Natl. Acad. Sci. USA 83, systemsfor genecloning in Clostridium. Microbial. Sci. 2541-2545. 5,310-315. MORAN,C. P., JR., LANG, N., LEGRICE,S. F. J., LEE,G., TE RIELE,H., MICHEL, B., AND EHRLICH,S. D. (1986b). Are single-stranded circles intermediates in plasmid STEPHENS, M., SONENSHEIN, A. L., PERO,J., AND LoDNA replication? EMBO J. 5, 63 l-637. SICK, R. (1982). Nucleotide sequencesthat signal the initiation of transcription and translation in Bacillus VAN MANSFELD,A. D. M., VAN TEEFFELEN,H. A. A. M., BAAS,P. D., AND JANSZ,H. S. (1986). Two juxtaposed subtilis. Mol. Gen. Genet. 186, 339-346. tyrosyl-OH groups participate in 4X 174geneA protein MURAI, M., MIYASHITA, H., ARAKI, H., SEKI, T., AND catalysed cleavage and ligation of DNA. Nucleic Acids OSHIMA, Y. (1987). Molecular structure of the repliRex 14,4229-4238. cation origin of a Bacillus amyloliquefaciens plasmid VESCOVO,M., MORELLI, L., AND BOTTAZZI,V. (1982). pFTBl4. Mol. Gen. Genet. 210,92-100. Drug resistance plasmids in Lactobacillus acidophilus NORRANDER,J., KEMPE, T., AND MESSING,J. (1983). and Lactobacillus reuteri. Appl. Environ. Microbial. 43, Construction of improved M 13 vectors using oligode50-56. oxynucleotidedirected mutagenesis.Gene26, 101- 106. WILBUR,W. J., ANDLIPMAN,D. J. (1983).Rapid similarity PEETERS,B. P. H., DE BOER,J. H., BRON,S., AND VEsearchesof nucleic acid and protein data banks. Proc. NEMA,G. (1988). Structural plasmid instability in BaNatl. Acad. Sci. VSA 80, 726-730. cillus subtilis: Effect ofdirect and inverted repeats.Mol. Communicated by S. Dusk0 Ehrlich Gen. Genet. 212,450-458.