JOURNAL OF BACTERIOLOGY, Jan. 1990, p. 47-52
Vol. 172, No. 1
0021-9193/90/010047-06$02.00/0 Copyright C 1990, American Society for Microbiology
Conjugal Mobilization of Streptococcal Plasmid pMV158 between Strains of Lactococcus lactis subsp. lactis DANIEL VAN DER LELIE, HAN A. B. WOSTEN, 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
pMV158, a non-self-transmissible plasmid encoding tetracycline resistance, was conjugally transferred from Enterococcusfaecalis JH203 to Lactococcus lactis subsp. lactis IL1403. This transfer appeared to be dependent on the cotransfer of the conjugative plasmids pAMj31 or pIP501. Intraspecies conjugal transfer of pMV158 also occurred in strain IL1403. In contrast to the transfer from E. faecalis, transfer in IL1403 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 IL1403. The transfer of pMV158 from strain IL1403 required the presence of an active pMV158-encoded protein, which showed homology to the Pre (plasmid recombination enzyme) proteins encoded by several small plasmids extracted from Staphylococcus aureus, such as pT181.
Lactococci, which comprise the strains Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, and Lactococcus lactis subsp. diacetylactis, are important in the manufacture of dairy products. Owing to concerted efforts in several laboratories, this group of organisms recently became accessible to genetic manipulation by recombinant DNA technologies. It is expected that these techniques will be applied in the near future to improve the properties of existing lactococcal strains and to add new properties for special purposes (40). An important requirement for the application of DNA technologies concerns the development of efficient DNA transfer systems. Although in some model strains plasmid DNA can be conveniently transferred by transduction (29), protoplast fusion (13), protoplast transformation (20), and more recently, electroporation (16, 31, 38), several lactococcal strains have often proven to be refractory to these transfer systems. An alternative way to introduce foreign DNA into lactococcal strains concerns conjugation. Many important lactococcal properties, like lactose and sucrose metabolism, proteinase production, nisin production and resistance, bacteriocin production and resistance, and bacteriophage insensitivity, are encoded by conjugally transferable plasmids (for a recent review, see reference 12). Of special interest for the transfer of recombinant plasmids from well-transformable to transformation-refractory lactococcal hosts is the observation that nonconjugal plasmids can be mobilized for transfer by conjugally active plasmids. Thus, it has been reported that the broad-hostrange plasmids pAM,1 (7), pIP501 (17), and the pIP501 derivative pVA797 (11) can mobilize certain nonconjugal plasmids (34). Romero et al. (32) were able to extend pVA797-mediated mobilization of the non-self-transmissible plasmids pVA838 (24) and pSA3 (8) to lactococcal strains and to Streptococcus salivarius subsp. thermophilus by using the transformable strain L. lactis subsp. lactis LM2301 as an intermediate donor. In these cases, cointegrate formation between the conjugative and the nonconjugative plasmids, as a result of homologous recombination followed by *
resolution and plasmid segregation after transfer, was assumed to underlie the mobilization. Whether homologous recombination is a strict requirement for the mobilization is uncertain, because it has been observed that the small lactococcal shuttle vector pCK1, which has no detectable homology with pAM,1, is mobilizable by pAMP1 (12). A derivative of pAMP1, pMVA&P1, has also been shown to be capable of mobilizing pMV158 between Enterococcus faecalis strains (23). pMV158 is a small, broad-host-range plasmid that was originally isolated from Streptococcus agalactiae and that encodes resistance to tetracycline (4). Derivatives of this plasmid have proved to be useful for the cloning of genes in Streptococcus pneumoniae (35). This prompted us to examine whether pMV158 and its derivatives could be used as mobilizable cloning vectors for lactococci and, if so, to examine the mechanism underlying the mobilization. In the studies presented here we show that the conjugal transfer of pMV158 from Enterococcus faecalis JH203 to L. lactis subsp. lactis IL1403 depends on the cotransfer of pAM31 or pIP501. In contrast, the transfer of pMV158 from strain IL1403 to L. lactis subsp. lactis MG1363 does not require the cotransfer of pAM,1 or pIP501, but appears to be mediated by chromosomally encoded conjugation functions. The results further indicate that a pMV158-encoded protein, with similarity to the Pre (plasmid recombination enzyme) protein of pT181 (15) and related plasmids (37a), is required for the conjugal transfer of this plasmid. MATERIALS AND METHODS Bacterial strains, plasmids, and media. The strains and plasmids used in this study are listed in Table 1. TY broth (33) was used to culture Escherichia coli cells. For platings, TY broth was solidified with 1.5% agar (BBL Microbiology Systems, Cockeysville, Md.). L. lactis subsp. lactis and Enterococcus faecalis were cultured and plated on M17 (36) broth and agar supplemented with 0.5% glucose (GM17). Erythromycin was added at final concentrations of 5 ,ug/ml for L. lactis subsp. lactis and Enterococcus faecalis; tetracycline was added at final concentration of 4 pug/ml for L. lactis subsp. lactis and Enterococcus faecalis and 10 ,g/ml for Escherichia coli.
Corresponding author.
t Present address: Transgene SA, 67082 Strasbourg Cedex, France.
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VAN DER
J. BACTERIOL.
LELIE ET AL.
TABLE 2. Conjugal transfer of pMV158 from E. faecalis JH203 to L. lactis subsp. lactis IL1403 (Rif)
TABLE 1. Bacterial strains and plasmids used in this study Source or Bacterial strain or Genotype or
plasmid
markera
reference
V. Burdett
Enterococcus faecalis JH203
Escherichia coli JM83
Plasmids pIP501
pAMP1 pMV158 pLS1 pLW1 pLW2 pLW5
Plasmid
frequencya pAM,13
pIP501
Mobilization
frequencya Emr + Tcrb
Tcrc
pMV158
Vol. 172, No. 1
0021-9193/90/010047-06$02.00/0 Copyright C 1990, American Society for Microbiology
Conjugal Mobilization of Streptococcal Plasmid pMV158 between Strains of Lactococcus lactis subsp. lactis DANIEL VAN DER LELIE, HAN A. B. WOSTEN, 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
pMV158, a non-self-transmissible plasmid encoding tetracycline resistance, was conjugally transferred from Enterococcusfaecalis JH203 to Lactococcus lactis subsp. lactis IL1403. This transfer appeared to be dependent on the cotransfer of the conjugative plasmids pAMj31 or pIP501. Intraspecies conjugal transfer of pMV158 also occurred in strain IL1403. In contrast to the transfer from E. faecalis, transfer in IL1403 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 IL1403. The transfer of pMV158 from strain IL1403 required the presence of an active pMV158-encoded protein, which showed homology to the Pre (plasmid recombination enzyme) proteins encoded by several small plasmids extracted from Staphylococcus aureus, such as pT181.
Lactococci, which comprise the strains Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, and Lactococcus lactis subsp. diacetylactis, are important in the manufacture of dairy products. Owing to concerted efforts in several laboratories, this group of organisms recently became accessible to genetic manipulation by recombinant DNA technologies. It is expected that these techniques will be applied in the near future to improve the properties of existing lactococcal strains and to add new properties for special purposes (40). An important requirement for the application of DNA technologies concerns the development of efficient DNA transfer systems. Although in some model strains plasmid DNA can be conveniently transferred by transduction (29), protoplast fusion (13), protoplast transformation (20), and more recently, electroporation (16, 31, 38), several lactococcal strains have often proven to be refractory to these transfer systems. An alternative way to introduce foreign DNA into lactococcal strains concerns conjugation. Many important lactococcal properties, like lactose and sucrose metabolism, proteinase production, nisin production and resistance, bacteriocin production and resistance, and bacteriophage insensitivity, are encoded by conjugally transferable plasmids (for a recent review, see reference 12). Of special interest for the transfer of recombinant plasmids from well-transformable to transformation-refractory lactococcal hosts is the observation that nonconjugal plasmids can be mobilized for transfer by conjugally active plasmids. Thus, it has been reported that the broad-hostrange plasmids pAM,1 (7), pIP501 (17), and the pIP501 derivative pVA797 (11) can mobilize certain nonconjugal plasmids (34). Romero et al. (32) were able to extend pVA797-mediated mobilization of the non-self-transmissible plasmids pVA838 (24) and pSA3 (8) to lactococcal strains and to Streptococcus salivarius subsp. thermophilus by using the transformable strain L. lactis subsp. lactis LM2301 as an intermediate donor. In these cases, cointegrate formation between the conjugative and the nonconjugative plasmids, as a result of homologous recombination followed by *
resolution and plasmid segregation after transfer, was assumed to underlie the mobilization. Whether homologous recombination is a strict requirement for the mobilization is uncertain, because it has been observed that the small lactococcal shuttle vector pCK1, which has no detectable homology with pAM,1, is mobilizable by pAMP1 (12). A derivative of pAMP1, pMVA&P1, has also been shown to be capable of mobilizing pMV158 between Enterococcus faecalis strains (23). pMV158 is a small, broad-host-range plasmid that was originally isolated from Streptococcus agalactiae and that encodes resistance to tetracycline (4). Derivatives of this plasmid have proved to be useful for the cloning of genes in Streptococcus pneumoniae (35). This prompted us to examine whether pMV158 and its derivatives could be used as mobilizable cloning vectors for lactococci and, if so, to examine the mechanism underlying the mobilization. In the studies presented here we show that the conjugal transfer of pMV158 from Enterococcus faecalis JH203 to L. lactis subsp. lactis IL1403 depends on the cotransfer of pAM31 or pIP501. In contrast, the transfer of pMV158 from strain IL1403 to L. lactis subsp. lactis MG1363 does not require the cotransfer of pAM,1 or pIP501, but appears to be mediated by chromosomally encoded conjugation functions. The results further indicate that a pMV158-encoded protein, with similarity to the Pre (plasmid recombination enzyme) protein of pT181 (15) and related plasmids (37a), is required for the conjugal transfer of this plasmid. MATERIALS AND METHODS Bacterial strains, plasmids, and media. The strains and plasmids used in this study are listed in Table 1. TY broth (33) was used to culture Escherichia coli cells. For platings, TY broth was solidified with 1.5% agar (BBL Microbiology Systems, Cockeysville, Md.). L. lactis subsp. lactis and Enterococcus faecalis were cultured and plated on M17 (36) broth and agar supplemented with 0.5% glucose (GM17). Erythromycin was added at final concentrations of 5 ,ug/ml for L. lactis subsp. lactis and Enterococcus faecalis; tetracycline was added at final concentration of 4 pug/ml for L. lactis subsp. lactis and Enterococcus faecalis and 10 ,g/ml for Escherichia coli.
Corresponding author.
t Present address: Transgene SA, 67082 Strasbourg Cedex, France.
47
48
VAN DER
J. BACTERIOL.
LELIE ET AL.
TABLE 2. Conjugal transfer of pMV158 from E. faecalis JH203 to L. lactis subsp. lactis IL1403 (Rif)
TABLE 1. Bacterial strains and plasmids used in this study Source or Bacterial strain or Genotype or
plasmid
markera
reference
V. Burdett
Enterococcus faecalis JH203
Escherichia coli JM83
Plasmids pIP501
pAMP1 pMV158 pLS1 pLW1 pLW2 pLW5
Plasmid
frequencya pAM,13
pIP501
Mobilization
frequencya Emr + Tcrb
Tcrc
pMV158
