Nucleotide sequence and characterization of the broad-host-range lactococcal plasmid pWVO1

PLASMID

26,55-66

Nucleotide

(199 1)

Sequence

and Characterization of the Broad-Host-Range Lactococcal Piasmid pWVO1

KEES J. LEENHOUTS, BEREND TOLNER, SIERD BRON, JAN KOIC, GERARD VENEMA, AND Jos F. M. L. SEEGERS Department of Genetics, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands Received February

14, 199 1; revised May 3, 199 1

The nucleotide sequence of the Lactococcuslactis broad-host-range plasmid pWV0 1, replicating in both gram-positive and gram-negative bacteria, was determined. This analysis revealed four open reading frames (ORFs). ORF A appeared to encode a tram-acting 26.8~kDa protein (RepA), necessary for replication. The ORF C product was assumed to play a regulatory role in replication. Both RepA and the ORF C product showed substantial sequence similarity witlr the Rep proteins of the streptococcal plasmid pI.S 1. In addition, the plus origin of replication was identified on the basis of strong similarity with the plus origin of pLS 1. Derivatives of pWVO1 produced single-stranded (ss) DNA in Bacillus sub&s and L. Zactis,suggesting that this plasmid uses the rolling-circle mode of replication. In B. subtilis, but not in L. lactis, the addition of rifampicin resulted in increased levels of ssDNA, indicating that in the former organism the host-encoded RNA polymerase is involved in the conversion of the ssDNA to double-stranded plasmid DNA (dsDNA). Apparently, in L. Iactis the conversion of ss to ds pWV0 1 DNA occurs by a mechanism which does not require the host RNA polymerase. Q 1991Academic press, IIIC.

ber of gram-positive bacteria. The mode of replication of most of these plasmids has been shown to involve single-stranded DNA @DNA) intermediates, most likely as a consequence of rolling-circle replication (RCR) (Gros et al., 1987; Gruss and Ehrlich, 1989). These plasmids will be denoted ssDNA plasmids. The rolling-circle mode of replication is analogous to that proposed for the SSDNA bacteriophages of E. coli, such as 9X 174, G4, and M 13. However, these phages differ from each other in the mechanism of conversion of ssDNA to replicative form (RF) DNA (Baas and Jansz, 1988). An essential plasmid-encoded function for RCR is the Rep protein, which introduces a single-strand nick at the plus origin. This results in the displacement of the plus strand and the concomitant synthesis of a new plus strand. The plus origins of the various plasmids from gram-positive bacteria have been classified into three groups on the basis of sequence similarities in the nick site region (Gruss and Ehrlich, 1989). Likewise, the cog-

The 2.2-kb cryptic plasmid pWVO1 was originally isolated from Lactococcus lactis subsp. cremoris Wg2 (Otto et al., 1982). A genetically marked derivative, pGK12 (Kok et al., 1984), was shown to replicate in a wide variety of gram-positive bacteria (such as bacili, lactococci, streptococci, clostridia, and staphylococci) and gram-negative bacteria, including Escherichia coli. At least two other small plasmids isolated from gram-positive bacteria, the pMVl58-derived streptococcal plasmid pLS1 (Lacks et al., 1986; Van der Lelie et al., 1989) and the lactococcal plasmid pSH7 1 (De Vos, 1987), also replicate in gram-positive as well as gram-negative bacteria. In contrast, other plasmids, mainly isolated from Staphylococcus aureus and Bacillus species, like pT 18 1 (Khan and Novick, 1983), pE194 (Horinouchi and Weisblum, 1982), PUB 110 (McKenzie et al.. 1986), pIM13 (Projan et al., 1987), pTA1060 (Bron et al., 1987), and pIJlO1 (Kendall and Cohen, 1988), have a more limited host range and replicate only in a num55

0147-619X/91

$3.00

Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form ressved.

56

LEENHOUTS ET AL.

nate replication initiator proteins have been classified in three families (Gruss and Ehrlich, 1989). All replication initiator proteins studied so far are able to act in tram (Novick et al., 1982; Projan et al., 1987). For several ssDNA plasmids, minus origins (MOs) of replication have been described in addition to plus origins. Three families of MOs have been distinguished (Gruss and Ehrlich, 1989). These nonessential regions contain imperfect palindromic sequences which are used as initiation sites for the conversion of the displaced single strand to double-stranded (ds) plasmid forms. It is generally believed that the conversion is initiated by the host-encoded RNA polymerase (Gruss and Ehrlich, 1989), analogous to the mechanism of conversion of ssDNA to RF DNA of phage M 13. The absence of a functional MO in RCR plasmids results in the accumulation of ssDNA intermediates and frequently in increased levels of plasmid instability (Boe et al., 1989; Bron et al., 199I; Del Solar et al., 1987; Gruss et al., 1987). The ability of pWVO1 to replicate in both gram-positive and gram-negative bacteria and its wide use as a cloning vector prompted us to analyze the plasmid in more detail. The complete nucleotide sequence was determined and compared with known sequences of other plasmids. Analyses showed that pWV0 1 belongs to the family of ssDNA-generating plasmids. The results also suggest that, at least in L. luctis, the priming of the lagging strand synthesis may not require the host RNA polymerase. MATERIALS

AND METHODS

Bacterial Strains, Plasmids, and Growth Conditions

tions of 5 &ml, both for B. subtilis and L. iactis. Ampicillin (Ap), Cm, and Em were used at final concentrations of 100, 10, and 100 pg/ml, respectively, for E. coli.

(Bio)chemicals Chemicals were of analytical grade and were obtained from Merck (Darmstadt, FRG) or BDH (Poole, UK). Restriction enzymes, Klenow DNA polymerase, endonuclease S1, and T4 DNA ligase were used as indicated by the supplier (Boehringer-Mannheim, FRG).

DNA Techniques DNA techniques were as described by Maniatis et al. (1982). Large-scale and minipreparations of plasmid DNA from E. coli and B. subtilis were obtained essentially as described by Ish-Horowitz and Burke ( 1981) and Birnboim and Doly ( 1979), respectively, with minor modifications for L. luctis (Van der Lelie and Venema, 1987).

DNA SequenceAnalysis The complete nucleotide sequence of both strands of pWVO1 was determined by sequencing either single-stranded DNA of M 13-derived subclones, or double-stranded plasmid DNA, using the T7 DNA polymerase sequencing kit (Pharmacia, Uppsala, Sweden) in the dideoxy chain termination method (Sanger et al., 1977). MicroGenie software (Beckman, Palo Alto, CA) was used for computer-assisted sequence analysis.

Transformations

L. luctis cells were transformed by electroporation using a Bio-Rad Gene Pulser (BioThe bacterial strains and plasmids used are Rad Laboratories, Richmond, CA) as delisted in Table 1. E. coli and B. subtilis were scribed before (Leenhouts et al., 1990). Procultured in TY media (Rottlander and toplasts of B. subtilis were prepared and Trautner, 1970). Ml7 medium supple- transformed according to the method demented with 0.5% glucose (GM17) was used scribed by Chang and Cohen ( 1979). Compefor culturing L. luctis (Terzaghi and Sandine, tent cells of B. subtilis were prepared and 1975). Erythromycin (Em) and chloram- transformed as described by Bron and Venphenicol (Cm) were used at final concentra- ema (1972). Transformation of E. coli cells

NUCLEOTIDE

SEQUENCE OF LACTOCOCCAL PLASMID pWVO1

57

TABLE 1

BACTERL-USTRAINSANDPLASMIDS Bacterial strain or plasmid Bacteria E. coli JMlOl BL21 (DE3)

B. subtilis 8G5 L. lactis MG1363 Plasmids pGK1 pBKl1 pT713 pT7 13repA pORI3

Relevant properties

Source or ref.

supE, thi (lac-proAB), [F’, traD36, proAB, Ia#ZAM 151 F ompTr;;m&nt; bacteriophage DE3 lysogen carrying the T7 RNA polymerase gene controlled by the lacUV5 promoter

Yanisch-Perron et al. (1985) Studier and Moffatt (1986)

Rif, trpc2, tyrl, met his, ura, nit, purA, rib

Bron and Venema (1972)

Rif, plasmid free derivative of L. lactis NCDO712

Gasson (1983)

pWV0 1, carrying the Cm’ gene of pC 194 (20) in the Mb01 site Cm’, pGK1 lacking the 43 I-bp ClaI fragment AP’

Kok et al. (1984)

Ap’, pT7 13 with the 989-bp MvnIMb01 fragment of pWV0 1 Cm’, pGK1 lacking the 989-bp MnvIMboI fragment of pWV0 1

Kok et al. (1984) Tabor and Richardson ( 1985) This work This work

mented with lysozyme (2 mg/ml) and mutanolysin ( 150 U/ml) and the mixtures were placed on ice for 10 min and subsequently T7 RNA Polymerase-DirectedExpression in incubated at 37°C for an additional 10 min. Lysis was completed by the addition of 7 ~1 E. coli 30% sarcosyl (Oramix L30; Seppic, Paris, Protein samples of E. coli BL21 (DE3) France) and incubated for 30 mm at 70°C in were obtained as described by Studier and the presence of proteinase K (20 pg/ml). LyMoffatt (1986). SDS-polyacrylamide gel sates were then extracted twice with phenol (12.5%) electrophoresis was as described by and incubated at 37 “C for 15 min in the presLaemmli ( 1970). ence of RNAse at a final concentration of 0.5 mg/ml. Two 30-~1portions were taken from Detection of ssDNA in Whole-CellLysates of each lysate. One of the portions was incuL. lactis and B. subtilis bated with Sl nuclease (3300 U/ml) for 15 For the preparation of whole-cell lysates, min at 37°C and the other was left untreated. cells were grown to an OD600 of 0.6-0.8. After electrophoresis of the samples in 0.8% Cells from a 4.5-ml culture were harvested by agarose/ethidium bromide gels, the DNA centrifugation and washed once with buffer was transferred to GeneScreen Plus mem(0.15 M NaCl, 50 mM EDTA). The cells branes (NEN Research Products, Boston, were resuspendedin 193~1lysis buffer supple- MA), as described by Chomczynski and was performed by the method of Mandel and Higa (1970).

58

LEENHOUTS ET AL.

ings between the putative RBSs and start codons of ORFs A and D are 10 and 7 bp, respectively. The ORF A stopcodon (TGA) and ORF D startcodon (ATG) partially overlap. Only one putative promoter was identified (upstream of the putative RBS of ORF C, from positions 522 to 527 [-35 region, Inhibition of RNA Polymeraseby Rifampicin TTGTTT] and positions 545 to 550 [- 10 reOvernight cultures of B. subtilis and L. lac- gion, TACACT]; Van der Vossen et al., tis were diluted loo-fold and grown to an 1987). No potential transcription/translation OD600 of 0.6-0.8. De novoprotein synthesis signals could be identified upstream of ORF was then prevented by the addition of Em to B which, therefore, is unlikely to encode a a final concentration of 100 pg/ml and RNA functional polypeptide. polymerase activity was blocked by the addition of rifampicin to a final concentration of 100 pg/ml. Subsequently, the cultures were Similarities of the Putative ORF A and C Products with Proteins Specifiedby the incubated for another hour at 37°C and imStreptococcalPlasmid pLSl mediately used for the preparation of wholecell lysates. The putative products of ORFs A and C showed substantial similarity with two proRESULTS teins encoded by the Streptococcusagalactiae plasmid pLS1 (Lacks et al., 1986). The ORF Nucleotide Seguenceof pWVO1 A protein showed 49.4% similarity to the rep The complete nucleotide sequence of lication initiator protein RepB; the protein pWVO1 is shown in Fig. 1 and has a calcu- encoded by ORF C showed 37.7% similarity lated GC content of 33.4%. Computer-as- to the pLS1 repA gene product, which was sisted analysis revealed the presence of four shown to be the repressor of the repB gene open reading frames (ORF A through D), po- (Del Solar et al., 1989). An alignment of the tentially capable of encoding polypeptides A, amino acid sequencesis presented in Fig. 2. B, C, and D, with molecular sizesof 26.8,7.6, The similarity of the ORF A and ORF C prod5.8, and 5.4 kDa, respectively. All ORFs have ucts of pWV0 1 with known pLS l-encoded ATG as the putative startcodon and show the replication proteins suggeststhat the former same orientation. Upstream of ORF C, with proteins are involved in the replication of a spacing of 8 bp, a potential ribosome bind- pwvo1. ing site (RBS) was present. The free energy (AGO) of binding between this RBS @GAG) ORF A Encodes a Trans-acting Replication and the 3’-end of the 16s rRNA of L. lactis Protein (Ludwig et al., 1985) is -9.4 kcal/mol (TinTo obtain further support for the idea that oco et al., 1973). Potential RBSsare also present upstream of ORFs A (AAGG) and D ORF A encodes an essential pWV0 1 replica(GGGGG) (calculated AG” values of -8.4 tion protein, we cloned the 989-bp MvnIand -8.0 kcal/mol, respectively). The spac- Mb01 fragment, containing ORF A, between Qasba (1984). Detection of the DNA was carried out using the ECL gene detection system (Amersham, Buckinghamshire, UK). Probe labeling and blot handling were carried out according to the suppliers’ instructions.

FIG. 1. Complete nucleotide sequenceof pWV0 1. The sequence is numbered starting at the first nucleotide at the cleavage site of the first CIaI restriction site upstream of ORF B. The deduced amino acid sequencesof the potential ORFs are shown. Asterisks and underlined regions indicate potential RBSs and promoters, respectively. The thick arrows indicate inverted repeats (IR) and the thin arrows indicate direct repeats (DR). This nucleotide. sequencehas been submitted to the EMBL Data Library and assigned the accession number X56954.

60

LEENHOUTS ET AL.

Potential SecondaryStructures and Iterated Sequences Since both the plus and the minus origins in RCR plasmids from gram-positive bacteria have the potential to form secondary hairpin structures (Bron et al., 1989; Gros et al., 1987; Gruss and Ehrlich, 1989), we searched in the pWVO1 sequence for such regions of dyad symmetry. Six potential palindromic structures (Fig. 4) could be identified: from B positions 50 to 181 (I, AG” = -45.7); 228 to 250 (II, AGo = -14.8); 334 to 382 (III, AG” I KXBLTITZSE~LSKSMISVALKSTFXCQEK E = -20.8); 1656 to 1697 (IV, AG” = -18.5); 1719 to 1818 (V, AG” = -12.5);and 1929 to FIG. 2. (A) Alignment of the repA protein pWV0 I and the repB protein from pLS 1.The upper line 2007 (VI, AG” = -28.2). The latter stemdisplays the amino acid sequenceofthe repB protein and loop structure can be formed within a 59-bp the lower line displays that of the ORF A protein. Aster- direct repeat, the first 18 nucleotides of which isks indicate sequence identities and two dots indicate are repeated once more immediately followconservative amino acid replacements. (B) Comparison ing the 59-bp direct repeat. Due to the interof the ORF C protein from pWV0 1 and the repA protein from pLS 1. The upper line represents the amino acid nal symmetry, alternative secondary strucsequence of the repA protein and the lower line that of tures are possible within this region. Inverted the ORF C protein. Symbols as in A. repeat (IR) III contains a region that is highly similar to the consensus sequence of the pE194-type plus origin (Gruss and Ehrlich, the SmaI and BumHI sites of the E. coli ex- 1989; boxed regions in IR III of Fig. 4). For pression vector pT7 13, resulting in plasmid pT713repA. This places the expression of ORF A under control of the inducible T7 97RNA polymerase-specific promoter (Tabor 66and Richardson, 1985). After induction of 42the T7 polymerase in strain E. coli BL2 l(DE3) carrying pT713repA, a protein of ap31proximately 29 kDa was produced that was -29 absent in E. coli BL2l(DE3)(pT713) cells that lacked ORF A (Fig. 3). If the ORF A product is the replication initiator protein, it 2lis expected that plasmids lacking ORE A cannot replicate and that the introduction of ORF A in tram will restore the replication ability. This prediction was tested by deleting FIG. 3. Expression of repA in E. coli BL2 1 (DE3). SamORF A from the pWV0 1-derivative pGK1, ples of E. coli BL2 1 (DE3) carrying either pT7 13 (lane 1) resulting in pORI3. As expected, pORI3 or pT7 13repA (lane 2) were subjected to SDS-polyacrylcould only be constructed in E. coli amide (12.5%) gel electrophoresis. Standard molecular BL21(DE3) when the ORF A product was weight markers: phosphorylase b (97,400); bovine serum albumin (66,200); ovalbumin (42,699); carbonic anhyprovided in tram (on plasmid pT713repA). drase (3 1,000); trypsin inhibitor (2 1,500); and lysozyme In the following sections ORF A will be re- (14,400). Molecular sixes (in kilodaltons) are shown on the left. named repA. l **:

1

t:*:

nvrSES~~ISL~D~~~~FS~~V~

l

.t:

l

l **:

::t:*

l

:*

*

ARKESEQKK

*

FIG. 4. Potential secondary structures and iterated sequencesin pWV0 1. The boxed regions in and near the stem-loop structure III represent the similarity with pE 194-type plus origins. IR: inverted repeat; DR: direct repeat. The putative nick site as found for pLS 1 is indicated with an arrow.

62

LEENHOUTS

pLS 1, containing a pE 194-type plus origin, the nick site has been identified (De la Campa et al., 1990; seeFig. 4). This suggests that also in pWVO1 this region contains the plus origin of replication. Upstream and partially overlapping with IR III, at positions 305 to 341, a 17-bp imperfect repeat (three mismatches) is present. In addition, three direct repeats (of 11, 12, and 12 bp) are located downstream of IR III, at positions 456 to 490 (Fig. 4). Whether these regions are part of the plus origin is not clear at present. Minus origins (MOs) are also known to be located within regions (200-300 bp) of dyad symmetry (Bron et al., 1988; Bron et al., 1989; Gruss et al., 1987; Gruss and Ehrlich, 1989; Van der Lelie et al., 1989). However, none of the potential palindromes in pWV0 1 showed extensive sequence similarity with any known MO from other RCR plasmids.

Production of ssDNA Intermediates of p WV01 Derivatives The similarity between the plus origin and the replication protein of pWVO1 and those of the pEl94-type plasmids suggested that pWVO1 is an RCR plasmid. Therefore, and, since the presence of an MO involved in the conversion of ssDNA intermediates could not be inferred from the pWVO1 sequence (seepreceding section), the ability of pWV0 1 to generate ssDNA was examined. In wholecell lysates of B. subtilis containing either pGK1 or pGKl1 (a deletion derivative of pGK1 which lacks the small 43 1-bp Clul fragment and, consequently, the palindromic structures V and VI), significant amounts of ss plasmid DNA were present (about 30% of the total plasmid DNA mass; see Fig. 5A, lanes 1 and 2). Also in L. lactis some ss plasmid DNA was detectable; however, the amounts were low for either plasmid (~5% of the total plasmid DNA mass; Fig. 5A, lanes 3 and 4). These results confirm the idea that pWV0 1 usesthe rolling-circle mode of replication and that the potential stem-loop

ET AL.

structures V and VI do not play a significant role in the conversion of ss to dsDNA.

Rifampicin Does Not Afect the Accumulation of ssDNA Intermediates in L. lactis To examine the possible role of the host RNA polymerase in the conversion of ssDNA, rifampicin was added to cultures of rifampicin sensitive (Rifs) L. lactis and B. subtilis strains carrying either pGK1 or pGKl1. Inhibition of RNA polymerase did not result in increased amounts of ssDNA intermediates of the pWV0 1-derived plasmids pGK1 and pGKl1 in L. luctis (Fig. 5B, lanes 3 and 4). In contrast, with pMVl58, for which functional MOs have been described (Del Solar et al., 1987; Van der Lelie et al., 1989), an increase in the amount of ssDNA intermediates upon the addition of rifampitin was observed (results not shown). These results suggest that for plasmid pWVO1 the host-encoded RNA polymerase plays no significant role in the conversion of ss to dsDNA. In contrast, in B. subtilis the addition of rifampicin to cells harboring the pWVO1 derivatives resulted in a significant increase in the amount of ssDNA intermediates (Fig. 5B, lanes 1 and 2), indicating that in this organism RNA polymerase is involved in the conversion of ss to dsDNA. DISCUSSION

In the studies described here the complete nucleotide sequence of the 2 178-bp L. luctis subsp. cremoris Wg2 plasmid pWVO1 was determined and analyzed. Comparison of the sequence data of pWVO1 with those of pSH7 1, which was isolated from L. lactis subsp. luctis NCD0712, showed that these plasmids were nearly identical (W. M. de Vos, personal communication). The only significant difference was the absence of the 59bp direct repeat in pSH7 1. Four ORFs were present on the plasmid. ORF D was preceded by a putative RBS and is likely to form one transcriptional unit with ORF C and repA. Interestingly, the stopco-

NUCLEOTIDE

SEQUENCE OF LACTOCOCCAL PLASMID pWVO1

63

FIG. 5. (A) SSDNA production of pGK1 and pGKl1 in B. subtilis and L. luctis. (-), not treated with endonuclease Sl; (+), treated with endonuclease Sl. (B) Effect of rifampicin on SSDNA production of pGK1 and pGKl1 in B. subtilis and L. Zuctis.(-), not treated with rifampicin; (+), treated with rifampicin. (A), (B): lane 1, B. subtilis (pGK1); lane 2, B. subtilis (pGKI1); lane 3, L. luctis (pGK1); lane 4, L. lactis (pGK11). OC, open circular DNA, CC, covalently closed circular DNA; SS, single-stranded DNA.

don of the repA gene (TGA) partially overlaps the putative startcodon (ATG) of ORF D. This suggeststhat the expression of ORF D might be translationally coupled to that of ORF C and repA. The putative product of ORF D was not required for replication, since ORF D could be inactivated by the insertion of an antibiotic-resistance marker in the RsuI site (position 1619) of pGKl1 (unpublished results). The similarity of RepA of pWVO1 with the RepB protein of pLS1 and the extensive homology with DNA sequencessurrounding the nick sites of the pE 194-family of plus origins of replication (De la Campa et al., 1990; Gruss and Ehrlich, 1989) suggests that pWV0 1 is a member of the pE 194-family of RCR plasmids. The putative protein C, encoded by the ORF preceding repA, showed substantial amino acid sequence similarity with RepA of pLS 1. The latter protein has the characteristics of a repressor (Del Solar et al., 1989). For pSH71 it was shown that the ORF C product is a repressor at the transcriptional level for the ORF C-repA operon (W. M. de Vos, personal communication). In addition to the similarities on the amino acid and nucleotide level, there is also similarity in the structural organization of the minimal replicon of the pE 194-family of RCR plasmids and that of pWVO1. In pE194, pLS1,

pSH7 1, and pWV0 1 the gene encoding the replication initiator protein is preceded by a gene encoding a regulatory protein in an operon-like structure. In all these plasmids the plus origin is located upstream of this operon (Byeon and Weisblum, 1990; Lacks et al., 1986; W. M. De Vos, personal communication). In close vicinity to the plus origin direct repeats are present in pWVO1. Although such repeats are not present in most of the small RCR-type gram-positive plasmids, direct repeats have been found near the putative nick site of the Luctobucillus plunturum plasmids pC3Oil (Skaugen, 1989) and pLP1 (Bouia et al., 1989) and the broad-host-range plasmid pLS1 (Puyet et al., 1988). In the latter plasmid, three contiguous 11-bp direct repeats are located 73 bp downstream of the hairpin structure containing the nick site. Recently, it was shown that RepB of pLS1 binds to these direct repeats prior to the nicking of the plus origin (De la Campa et al., 1990). In pWV0 1, three direct repeats of 11, 12, and 12 bp (the first has three mismatches compared to the others) are located 75 bp downstream of IR III. The repeats in these two plasmids also show mutual sequencesimilarity; the sequence S-TCGGCGACTTT-3, present in the pLS1 repeats, resembles that in pWV0 1 (S-TCGCCAACGTTT-3’; three

64

LEENHOUTS ET AL.

mismatches). This similarity suggestsan analogous role of these repeats. In ssDNA-generating plasmids described so far, MOs are required for the efficient conversion of circular ssDNA intermediates into ds plasmid DNA (Boe et al., 1989; Bron et al., 1988; Gruss and Ehrlich, 1989; Lacks et al., 1986). Known MOs are part of large imperfect palindromic structures, generally consisting of 200-300 nucleotides. IRS V and VI could be removed from pWVO1 without affecting the production of ssDNA intermediates both in B. subtilis and L. lactis making it unlikely that these palindromic structures function as an MO. IR II and IR IV consist of only 40 nucleotides or less and, most probably, do not function as MOs. Indeed, IR IV is located just downstream of the putative transcriptional unit ORF C-repA-ORF D and has the characteristics of a transcriptional terminator. The remaining stem-loop structure I, shown in detail in Fig. 6, shows some characteristics of an MO. The consensus sequence 5’-TAGCGT-Y, present in the loop of palAtype MOs (Del Solar et al., 1987), is also located in the loop of structure I. In addition, palA-type MOs contain a so-called RS,-site at the 3’-baseof the stem, containing the 18bp consensus sequence 5’-AAGTTTTCTCGGCATAAA-3’ (Gruss et al., 1987; Novick et al., 1984). A similar sequence(72.2% identical to this consensus) is present in the stem of IR I, however, at the 5’-end. At present, it is not clear whether this region constitutes a functional MO for L. lactis and B. subtilis. Although pa&type MOs are usually inefficient in B. subtilis (Gmss et al., 1987; Novick et al., 1985), it has been shown in our group that the palA MO of pLS 1 has activity in this organism (W. Meijer, personal communication). RNA polymerase has generally been considered to initiate lagging strand synthesis at MOs of RCR plasmids (Boe et al., 1989; Gruss and Ehrlich, 1989). This was, indeed, observed with pWVO1 in B. subtilis. However, the conversion of the ssDNA intermediates to ds pWVO1 DNA did not appear to

u174 n CCGTA c* t? GA T G' TC-CT" C-G

T-r T A T-A A-T

23Oti i351

-CC-CAT f::! A-T T-A A-TG A t:

T-A G T C-G T-A T-A T-A !:I ..TT-AT... 50 181

FIG. 6. Comparison of IR I of pWVO1 with complementary strand origin of @X174 and palA-type minus origins. The boxed regions display the similarity between IR I and the complementary strand origin of +X174. The asterisks indicate the similarity between IR I and a consensus sequence present in the loop of palA-type minus origins (see text). The arrow spans a sequence in hairpin I with homology to the consensusRS, sequence (seetext).

require the host-encoded RNA polymerase in L. lactis, since no inhibitory effect of rifampicin was observed. This observation

NUCLEOTIDE

SEQUENCE OF LACTGCGCCAL

strongly suggests that initiation of lagging strand synthesis of pWVO1 in Lactococcus differs in this organism from that of other RCR plasmids in other bacteria. In this context, it is noteworthy that IR I shows some sequence similarity with the complementary strand origin of +X 174 (Fig. 6). This bacteriophage usesthe primosome complex for complementary strand synthesis, which is not inhibited by rifampicin (Baas and Jansz, 1988). Further experiments will be needed to clarify whether pWVO1 is an RCR plasmid with a new type of MO. ACKNOWLEDGMENTS This work was supported by Unilever Research,Vlaardingen, The Netherlands (K. J. Leenhouts) and by the Dutch Program Committee on Industrial Biotechnology (J. F. M. L. Seegers).We thank Willem de Vos for communicating to us the sequenceofplasmid pSH7 1prior to publication. We thank Henk Mulder for preparing the figures.

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PLASMID pWVO1

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