INTRODUCTION

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Lactococci are known to harbor conjugative plasmids that are used for industrial strain improvement since they encode important metabolic traits such as lactose fermentation, protease activity, bacteriophage resistance, or production of exopolysaccharide (EPS). Therefore, these plasmids are studied for their functional properties as well as for their mode of replication and transfer capacities. Two different mechanisms of replication are known to operate in Lactococcus lactis: rolling circle and theta replication. Rolling circle replication seems to be restricted to relatively small lactococcal plasmids with cryptic functions (19). Two of these, the related promiscuous plasmids pWV01 and pSH71, have been developed into widely used cloning and expression vectors (6). The replication regions of several theta replicating lactococcal plasmids that encode metabolic functions have been analyzed, and all are members of a family of highly related, compatible theta replicons as first identified for plasmid pCI305 (17, 35). They all contain a homologous repB gene encoding the replication protein. The conserved region upstream of repB is likely to include the origin of replication and also contains 22-bp repeats which have a replicon-specific regulatory role in plasmid replication and an inverted repeat overlapping the repB promoter which is a RepB binding site (7).

The capacity for conjugal transfer is an important characteristic of some lactococcal plasmids. Self-transmissible conjugative plasmids have the ability to form effective cell-to-cell contact, while mobilization plasmids are able only to prepare their DNA for transfer (36). The conjugation process in gram-negative bacteria is initiated at the origin of transfer (oriT) by the formation of a relaxosome, usually containing a relaxase and accessory DNA binding proteins. The relaxase catalyzes the cleavage of a specific phosphodiester bond at the nic site in the oriT, after which it is covalently linked to the 5' end of the cleaved strand through a tyrosyl residue. Single-stranded DNA is transferred to the recipient cell and subsequently ligated through the cleaving-joining activity of the relaxase, resembling the process of leading-strand replication by rolling circle replication (20). To date, very little is known about genes required for conjugation in lactococci and other gram-positive bacteria (12). The chromosomally encoded sex factor and the homologous conjugative element pRS01 of L. lactis 712 and ML3, respectively, can mediate a high-frequency transfer of nonconjugative lactose plasmids and confer a cell aggregation (Clu) phenotype (1, 10). The sex factor cluA gene encodes a protein that is involved in cell aggregation during conjugation (14). On the bacteriophage resistance plasmid pCI528, a 2-kb region involved in conjugative mobilization has been identified. It contains a putative oriT and a mobA gene which is predicted to encode a protein involved in mobilization (24).

While EPS production by lactococci has long been known to be a plasmid-encoded trait, it was only recently established that structural genes involved in EPS biosynthesis are located on these plasmids (37, 38). The best-characterized EPS plasmid to date is the 40-kb pNZ4000 from L. lactis NIZO B40, which contains a 12-kb gene cluster encoding EPS biosynthesis (37). Furthermore, it contains multiple replicons, since we were able to separate pNZ4000 in two XhoI-SphI fragments that upon labeling with an erythromycin resistance (Ery^r) marker could each replicate in L. lactis (37). In this study, we report the identification and characterization of the regions involved in plasmid replication and mobilization of this EPS plasmid. Plasmid pNZ4000 contains four functional replicons and two regions involved in mobilization; one codes for an active trans-acting mobilization protein, and both contain a cis-acting oriT region.

	MATERIALS AND METHODS

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Bacterial strains, plasmids, and media. The bacterial strains and plasmids used in this study are listed in Table 1. Escherichia coli was grown in L-broth-based medium at 37°C (33). L. lactis was grown at 30°C in M17 broth (Difco Laboratories) supplemented with 0.5% glucose (GM17). If appropriate, the media contained chloramphenicol (10 µg/ml), erythromycin (10 µg/ml for L. lactis and 150 µg/ml for E. coli), rifampin (50 µg/ml), streptomycin (100 µg/ml), tetracycline (25 µg/ml), or ampicillin (100 µg/ml).

DNA isolation, manipulation, and transfer. Isolation of E. coli plasmid DNA and standard recombinant DNA techniques were performed as described by Sambrook et al. (33). Large-scale isolation of E. coli plasmid DNA for nucleotide sequence analysis was performed with Qiagen columns as instructed by the manufacturer. Isolation and transformation of L. lactis plasmid DNA were performed as previously described (5). For whole-cell lysates of L. lactis, 1.5 ml of a late-log-phase culture was harvested and suspended in 100 µl of a buffer containing 30 mM Tris-HCl (pH 8.0), 3 mM MgCl₂, 25% sucrose, 10 µg of lysozyme ml¹, and 0.1 mg of RNase ml¹. This suspension was incubated at 37°C for 30 min. Lysis was achieved by addition of 100 µl of 2% sodium dodecyl sulfate and vortexing at top speed for 1 min, after which the lysate was treated with 20 µg of proteinase K ml¹ at 37°C for 30 min. Conjugation was performed by filter matings as described before (37). The ratio of donor and recipient was 2:1.

Nucleotide sequence analysis. Automatic double-stranded DNA sequence analysis was performed on both strands with an ALF DNA sequencer (Pharmacia Biotech). Sequencing reactions, performed with an AutoRead sequencing kit, were initiated by using fluorescein-labeled universal and reverse primers and continued with synthetic primers in combination with fluorescein-15-dATP, following the instructions of the manufacturer (Pharmacia Biotech). Sequence data were assembled and analyzed using the PC/GENE program (version 6.70; IntelliGenetics). The GenBank database (February 1998 release) was screened for homologies by using TFASTA.

Construction of plasmids. For replicon screening and plasmid integration, the E. coli plasmid pUC19Ery or pUC18Ery, carrying the Ery^r gene, or pCI182, carrying the tetracycline resistance (Tet^r) gene, was used. For plasmids pNZ4001, pNZ4002, and pNZ4004, a 3.3-kb EcoRI-XbaI fragment, a 3.4-kb EcoRI fragment, and a 2.9-kb Sau3AI fragment of pNZ4000 were cloned into pUC19Ery digested with EcoRI-XbaI, EcoRI, and BamHI, respectively. For plasmid pNZ4003, a 3.7-kb XhoI-HincII fragment of pNZ4000 was cloned into SalI-SmaI-digested pUC18Ery. To construct plasmid pNZ4025, a 3.3-kb EcoRI-XbaI fragment of pNZ4000 was cloned in pUC18 (41) digested with EcoRI-XbaI, and subsequently the pCI182 tetM gene was cloned on a 4.2-kb HincII fragment in the pUC18 HincII site. For plasmids pNZ4026 and pNZ4027, a 3.4-kb EcoRI and a 2.9-kb Sau3AI fragment of pNZ4000 were cloned in pCI182 digested with EcoRI or BglII, respectively.

Nucleotide sequence accession numbers. The complete nucleotide sequences of the replication and mobilization regions are available under GenBank accession no. AF03685, AF03686, and AF03687.

	RESULTS AND DISCUSSION

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The EPS plasmid pNZ4000 contains four functional replicons. The 40-kb plasmid pNZ4000 is essential for EPS production in strain NIZO B40 and includes the 12-kb eps gene cluster involved in EPS biosynthesis (37). The nucleotide sequence of the EPS plasmid was determined, and analysis of the data revealed the unusual presence of four highly homologous replication regions that belong to a family of lactococcal theta replicons (35) which are located outside the eps gene cluster (Fig. 1). DNA fragments carrying these putative replicons were cloned into pUC19Ery or pUC18Ery, which can be used as replicon screening vectors in L. lactis. The resulting plasmids (pNZ4001, pNZ4002, pNZ4003, and pNZ4004) were transformed to L. lactis MG1363, and in all cases Ery^r transformants that harbored plasmids with the expected configuration were obtained (results not shown). These results indicate that all four replicons are functional in L. lactis. Since plasmid replication requires only one of these replicons, pNZ4000 must have derived fragments of several plasmids, which might have formed cointegrates during conjugation processes. This conclusion is corroborated by the presence of complete and truncated copies of ISSI-like elements (Fig. 1), since it is known that ISSI mediates cointegration of the L. lactis ML3 lactose plasmid pSK08 with the conjugal plasmid pRS01 (29). In addition, a complete copy of an IS982-like element is present on pNZ4000 (Fig. 1).

View larger version (18K): [in this window] [in a new window]   FIG. 1.   (A) Physical and genetic map of plasmid pNZ4000. The eps gene cluster is located between IS982 and orfY. (B) Physical and genetic maps of the replication and mobilization regions of pNZ4000. The fragments used for functional analysis are depicted below. A, AccI; C, ClaI; E, EcoRI; H, HincII; N, NcoI; O, XhoI; P, SphI; S, Sau3AI; V, NspV; X, XbaI. For AccI, ClaI, HincII, NcoI, NspV, and Sau3AI, only sites relevant for subcloning are included. Sequences are available under GenBank accession no. AF036485, AF03686, and AF03687.

All four replicons are compatible but show differences in organization. The minimal replicons for repB1, repB2, and repB4 were labeled with the tetM gene to generate pNZ4025, pNZ4026, and pNZ4027, respectively. These plasmids were combined with either pNZ4001, pNZ4002, pNZ4003, or pNZ4004 and transformed to MG1363 to make six strains including all combinations of different replicons carrying a set of Ery^r and Tet^r genes (Table 2). All plasmids had comparable copy numbers, as judged from the intensity of ethidium bromide-stained plasmid DNA separated by agarose gel electrophoresis (results not shown). Stable transformants were obtained for all heteroplasmid combinations following selection for Ery^r and Tet^r, indicating that these replicons are compatible. The compatibility of the plasmids carrying different replicons was confirmed by determining the segregational stability after growth for 35 generations in medium containing no antibiotics (Table 2). Plasmids carrying the replicons with repB1, repB2, and repB4 formed highly stable heteroplasmid combinations. In contrast, the segregational stability of the repB3-containing replicon was significantly lower than that of the others. This was also observed when this replicon was present as a single replicon in MG1363. After 20 generations without selection pressure, 61% of the population was plasmid containing; after 40 generations 16%, and after 60 generations only 3%, of the population contained plasmids. The reason for the difference in stability between the repB3-containing replicon and the other three highly homologous replicons is unclear. It seems that there is interference with the maintenance functions of the repB3-containing replicon which are not directly involved in replication. The orfC genes located downstream of and partly overlapping the repB genes (Fig. 1) are not likely to be involved in this process. The predicted OrfC proteins are homologous to RepB287 (45 and 43% identity for OrfC1 and OrfC2, respectively) encoded by the Tetragenococcus halophilus theta-replicating plasmid pUCL287. RepB287 is not essential for replication, as is OrfC, but its presence reduces the copy number and the segregational stability (2). The N-terminal parts of the OrfC proteins are highly conserved and contain a helix-turn-helix motif which is probably involved in DNA binding. While repB1 and repB2 are followed by almost complete orfC genes, orfC3 and orfC4 encode only the N-terminal parts of OrfC-like proteins. If the role of the lactococcal OrfC was similar to that of RepB287, we would expect the stability of the replicons containing repB1 and repB2 to be lower than that of the replicons containing repB3 and repB4, which is not as we observed.

The EPS plasmid pNZ4000 is a mobilization plasmid. We have previously shown that plasmid pNZ4000 can be conjugally transferred together with the lactose plasmid from the L. lactis NIZO B40 to the recipient strain MG1614 (37). To study the intraspecific conjugative transfer of pNZ4000 in more detail, the Ery^r derivatives pNZ4010 and pNZ4017 were used. These plasmids were transformed to the plasmid-free strain MG1363, and the resulting strains were used as donors in filter matings with strain MG1614. Conjugative transfer of either of these plasmids between these isogenic L. lactis subsp. cremoris strains occurred at a frequency of 10⁶ per donor. Plasmid pNZ4017 was also transformed to the plasmid-free L. lactis subsp. lactis strain IL1403, from which it could be transferred to L. lactis subsp. cremoris MG1614 at a frequency of 10⁸ per donor. These results demonstrate that pNZ4000 can be mobilized from strains MG1363 and IL1403. It is likely that differences in chromosomal conjugation functions account for the differences in transfer efficiency of the pNZ4000 derivatives from both L. lactis subspecies, which are known to share approximately 70 to 80% sequence identity in characterized genes and differ by the presence of a large chromosomal inversion of about half of the genome (13, 21). Furthermore, MG1363 harbors the sex factor that encodes conjugative functions (12), which may play a role in mobilization of pNZ4000.

pNZ4000 contains two functional oriT sites. Mobilization involves a cis-acting oriT region and a trans-acting gene encoding a relaxase (20). Nucleotide sequence analysis of pNZ4000 revealed the presence of a region upstream of repB3 (Fig. 1), which is almost identical (98.3% identity) to a 2.0-kb fragment involved in mobilization of the lactococcal plasmid pCI528 (24). It contains a mobA gene encoding a putative mobilization protein. The upstream region of the mobA gene contains three inverted repeats and a direct repeat and has been postulated to be the oriT region (24). We tested the functionality of the putative oriT sequence (oriT1) by cloning it in the nonconjugative plasmid pNZ124 and transforming the resulting plasmid pNZ4021 to strain MG1363. This strain was mated with MG1614 and chloramphenicol-resistant transconjugants were selected (Table 3). The 1.8-kb region containing the oriT1 sequence was sufficient to achieve conjugal transfer of the nonconjugative plasmid pNZ124, showing that the cloned fragment contains a functional oriT.

View larger version (11K): [in this window] [in a new window]   FIG. 2.   DNA sequence alignment of the IR3 segments of the oriT regions found on pNZ4000 (oriT1 and oriT2) and the sequence of the IncI1 plasmid R64 oriT. For R64 oriT, the inverted repeat is underlined, the NikA binding site is indicated in boldface, and the nic site is indicated with an arrowhead (9).

mobA encodes a product trans-acting on oriT-carrying plasmids. The involvement of mobA in mobilization was studied by comparing the transfer frequencies of plasmids carrying only oriT sequences or carrying oriT and mobA either in cis or in trans (Table 3). When mobA was provided in trans on pNZ4017, the transfer frequencies of pNZ4021 and pNZ4022 increased significantly. The same effect was achieved by when mobA was present in cis as on plasmid pNZ4023, containing oriT1 and mobA. These results indicate that mobA encodes a trans-acting element involved in mobilization.

View larger version (29K): [in this window] [in a new window]   FIG. 3.   Amino acid sequence comparison of the three conserved regions involved in relaxase activity as determined for TraI (27) for the relaxases MobA (Mob), Rlx, and Orf1 from S. aureus plasmids pC221, pS194, and pC223, respectively, MobA (MobA) from pNZ4000, NikB from E. coli plasmid R64 (8), and TraI from the E. coli plasmid RP4. The tyrosine (motif I) and histidine (motif III) residues involved in cleaving-joining reaction, and the serine residue (motif II) involved in DNA binding, are indicated in boldface.