Received 24 April 1989/Accepted 14 September 1989

Transcription

1 JOURNAL OF BACTERIOLOGY, Jan. 1990, p /9O/ $02.00/0 1990, American Society for Microbiology Vol. 172, No.1 Strains of Lactococcus lactis subsp. lactis DANIËL VAN DER LELIE,t HAN A. B. WaSTEN, SIERD BRON,* LINDA OSKAM, AND GERARD VENEMA Department of Genetics, Center of Biological Sciences, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands Received 24 April 1989/Accepted 14 September 1989 pmv1s8, a non-self-transmissible plasmid encoding tetracycline resistance, was conjugaily transferred from Enterococcusfaecalis JH2O3 to Lactococcus lactis subsp, lactis IL14O3. This transfer appeared to be dependent on the cotransfer ofthe conjugative plasmids pampl or pipsol. Intraspecies conjugal transfer ofpmv1s8 also occurred in strain IL14O3. In contrast to the transfer from E. faecalis, transfer in IL14O3 did not require the presence of a conjugative plasmid in the donor strain but, rather, appeared to be dependent on putative chromosomal functions in strain IL14O3. The transfer of pmv1s8 from strain IL14O3 required the presence of an active pmv1s8-encoded protein, which showed homology to the Pre (plasmid recombination enzyme) proteins encoded by several smail plasmids extracted from Staphylococcus aureus, such as pt18l. Lactococci, which comprise the strains Lactococcus lac- resolution and plasmid segregation after transfer, was astis subsp. lactis, Lactococcus lactis subsp. cremoris, and sumed to underlie the mobilization. Whether homologous Lactococcus lactis subsp. diacetylactis, are important in the recombination is a strict requirement for the mobilization is manufacture of dairy products. Owing to concerted efiorts in uncertain, because it has been observed that the sma1l severa1laboratories, this group of organisms recently be- lactococca1 shuttle vector pck1, which has no detectable came accessible to genetic manipulation by recombinant homology with pam131, is mobilizable by pam131 (12). DNA technologies. It is expected that these techniques wil1 A derivative ofpam131, pmv 1l131, has also been shown to be applied in the near future to improve the properties of be capable of mobilizing pmv158 between Enterococcus existing lactococcal strains and to add new properties for faecalis strains (23). pmv158 is a small, broad-host-range specia1 purposes (40). An important requirement for the application of DNA plasmid that was origina1ly isolated from Streptococcus agalactiae and that encodes resistance to tetracycline (4). technologies concerns the development of efficient DNA Derivatives of this plasmid have proved to be useful for the transfer systems. Although in some model strains plasmid cloning of genes in Streptococcus pneumoniae (35). This DNA can be conveniently transferred by transduction (29), prompted us to examine whether pmv158 and its derivatives protoplast fusion (13), protqplast transformation (20), and could be used as mobilizable cloning vectors for lactococci more recently, electroporation (16, 31, 38), severallactococ- and, if so, to examine the mechanism underlying the mobica1 strains have often proven to be refractory to these lization. transfer systems. An alternative way to introduce foreign In the studies presented here we show that the conjugal DNA into lactococcal strains concerns conjugation. Many transfer of pmv158 from Enterococcus faecalis JH203 to L. important lactococcal properties, like lactose and sucrose lactis subsp. lactis IL1403 depends on the cotransfer of metabolism, proteinase production, nisin production and pam131 or pip501. In contrast, the transfer of pmv158 from resistance, bacteriocin production and resistance, and bac- strain IL1403 to L. lactis subsp. lactis MG1363 does not teriophage insensitivity, are encoded by conjuga1ly transfer- require the cotransfer of pam131 or pip501, but appears to able plasmids (for a recent review, see reference 12). be mediated by chromosomally encoded conjugation func- Of specia1 interest for the transfer of recombinant plas- tions. The results further indicate that a pmv158-encoded mids from well-transformable to transformation-refractory protein, with similarity to the Pre (plasmid recombination lactococcal hosts is the observation that nonconjuga1 plas- enzyme) protein of pt181 (15) and related plasmids (37a), is mids can be mobilized for transfer by conjugally active required for the conjuga1 transfer of this plasmid. plasmids. Thus, it has been reported that the' broad-hostrange plasmids pam131 (7), pip501 (17), and the pip501 derivative pv A797 (11) can mobilize certain nonconjugal MATERIALS AND METHODS plasmids (34). Romero et al. (32) were able to extend Bacterial strains, plasmids, and media. The strains and pv A797-mediated mobilization of the non-self-transmissible plasmids pv A838 (24) and psa3 (8) to lactococcal strains plasmids used in this study are listed in Table 1. and to Streptococcus salivarius subsp. thermophilus by TY broth (33) was used to culture Escherichia coli cells. For platings, TY broth was solidified with 1.5% agar (BBL using the transformable strain L. lactis subsp. lactis LM2301 as an intermediate donor. In these cases, cointegrate formation between the conjugative and the nonconjugative plas- Microbiology Systems, Cockeysville, Md.). L. lactis subsp. lactis and Enterococcus faecalis were mids, as a result of homologous recombination followed by cultured and plated on M17 (36) broth and agar supplemented with 0.5% glucose (GM17). Erythromycin was added at final concentrations of 5 ~g/rnl for L. lactis subsp. lactis * Corresponding author. and Enterococcus faecalis; tetracycline was added at final t Present address: Transgene SA, Strasbourg Cedex concentration of 4 ~g/ml for L. lactis subsp. lactis and France. Enterococcus faecalis and 10 ~g/ml for Escherichia coli.

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5 depend directlyon the cotransfer of the conjugative plasmid. The matings with E.faecalis JH203 as the donor might be an example of this situation. In the case of mobilization of pmv158 by the L. lactis subsp. lactis IL1403 conjugation function, we could not select for the possible cotransfer of the putative chromosomally located functions. The mechanism of this transfer is, therefore, totally unclear. Surprisingly, the presence of the Pre protein was also required for this mobilization event. In this context, it may be of interest to compare the transfer of pmv158 with that of the staphylococcal plasmid pc221 (Archer and Projan, Abstr. Annu. Meet. Am. Soc. Microbiol. 1988). This plasmid contains two open reading frames, ORF-A and ORF-B, the first of which is considered to be responsible for the relaxation of covalently closed circular plasmid DNA. These open reading frames have been shown to be necessary for the mobilization of pc221 by the conjugative plasmid pgo1 (37). Although no homology between the products of these two open reading frames and the Pre protein of pmv158 could be detected, the possibility that the Pre protein also acts as a nickase, making pmv158 a target for the conjugation machinery, cannot be dismissed. It is attractive to believe that conjugal transfer systems of non-self-transmissible plasmids similar to the system described here may have a rather wide applicability in grampositive bacteria. This speculation is based on the observation that a considerable number of gram-positive plasmids contain a gene encoding Pre-like proteins (15, 37a), which might be required for mobilization. These plasmids replicate in, for example, Bacillus subtilis, Staphylococcus aureus, streptococci and lactococci. Another requirement for transfer, the presence of conjugation functions, can possibly be fulfilled by several conjugative plasmids. In addition to pam131, pip501, and pgo1, other large gram-positive plasmids may contain such functions. ACKNOWLEDGMENTS This work was supported by the Biotechnology Action Program of the Commission of the European Communities. We thank V. Burdett for the kind gift of E. faecalis JH203(pMV158) and H. Mulder for preparing the figures. LITERA TURE CITED 1. Balganesh, T. S., and S. A. Lacks Plasmid vector for cloning in Streptococcus pneumoniae and strategies for enrichment for recombinant plasmids. Gene 29: Brasch, M. A., and R. J. Meyer Genetic organization of plasmid Rl162 DNA involved in conjugative mobilization. J. Bacteriol. 167: Brasch, M. A., and R. J. Meyer A 38 base-pair segment of DNA is required in cis for conjugative mobilization of broadhost-range plasmid Rl162. J. Mol. Biol. 198: Burdett, V Identification of tetracycline-resistant R- plasmids in Streptococcus agalactiae (group B). Antimicrob. Agents Chemother. 18: Chomczynski, P., and P. K. Qasba Alkaline transfer of DNA to plastic membrane. Biochem. Biophys. Res. Commun. 122: Chopin, A., M..C. Chopin, A. Moillo-Batt, and P. 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6 biol. 54: Romero, D. A., P. Slos, C. Robert, I. Caste"ino, and A. Mercenier Conjugative mobilization as an alternative vector delivery system for lactic streptococci. Appl. Environ. Microbiol. 53: Rottlander, E., and T. A. Trautner Genetic and transfection studies with Bacillus subtilis phage SP50. J. Mol. Biol. 108: Smidt, M. D., and D. B. Clewe" Return of Streptococcus faecalis DNA by cloning in Escherichia coli to its original host via transformation of Streptococcus sanguis fo"owed by conjugative mobilization. J. Bacteriol. 160: Stassi, D. L., P. Lopéz, M. Espinosa, and S. A. Lacks Cloning of chromosomal genes in Streptococcus pneumoniae. Proc. Natl. Acad. Sci. USA 78: Terzaghi, B. E., and W. E. Sandine Improved medium for lactic streptococci and their bacteriophages. Appl. Microbiol. 29: Thomas, w. D., Jr., and G. L. Archer Identification and cloning of the conjugative transfer region of staphylococcal plasmid pgo1. J. Bacteriol. 171: a.van der Lelie, D., S. Bron, G. Venerna, and L. Oskam Similarity of minus origins of replication and flanking open reading frames of plasmids publ10, ptb913, and pmv158. Nucleic Acids Res. 17: van der Lelie, D., J. M. B. M. van der Vossen, and G. Venerna Effect of plasmid incompatibility on DNA transfer to Streptococcus cremoris. Appl. Environ. Microbiol. 54: van der Lelie, D., and G. Venerna Bacillus subtilis generates a major specific deletion in pam131. Appl. Environ. Microbiol. 53: Venerna, G., and J. Kok Improving dairy starter cultures. Tibtech 5: Vieira, J., and J. Messing The puc plasmids, an M13 mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19: