Physical mapping of the conjugative plasmid pmb1 1 of Enterococcus faecalis

Transcription

1 Letters in Applied Microbiology 1998, 27, Physical mapping of the conjugative plasmid pmb1 1 of Enterococcus faecalis M. Martínez-Bueno 1, E. Valdivia 1, I. Martín 1, R. Quirantes 1 and M. Maqueda 2 1 Departamento de Microbiología, Facultad Ciencias, and 2 Instituto de Biotecnología, University of Granada, Granada, Spain 1826/98: received 9 June 1998 and accepted 15 June 1998 M. MARTÍNEZ-BUENO, E. VALDIVIA, I. MARTÍN, R. QUIRANTES AND M. MAQUEDA The sexpheromone system of Enterococcus faecalis is a form of bacterial conjugation that plays an important role in the horizontal dissemination of genes. The ecological significance of sexual plasmids is to permit a rapid mobilization of genes of interest for the species (e.g. those encoding haemolysins, bacteriocins or antibiotic resistance). The physical mapping of pmb1 1, a conjugative plasmid of Ent. faecalis that responds to ccf10 pheromone, has been undertaken. By means of hybridization with conserved sequences of pcf10 plasmid, the regions harbouring the genes responsible for the pheromone inhibitor and the aggregation and exclusion proteins of this plasmid have been identified. The results demonstrated that plasmids pmb1 1 and pcf10 only show homology in the region involved in the conjugative response, suggesting that this region may be transferred in an independent way to that of the rest of the plasmid. INTRODUCTION One particular class of plasmids commonly found in Enterococcus faecalis is unique in that a mating response is triggered by recipient-produced sex pheromones. These plasmids have the capability to elicit a complex response to small peptides called sex pheromones, resulting in the formation of cell aggregates that facilitate conjugal transfer of the pheromoneresponding plasmids involved. Consequently, conjugation between Gram-positive bacteria is a process initiated by this chemical signal the sex-pheromone and includes several stages of interaction between the donor and recipient cells (Dunny 1990). Most work in this area has been focused on three well known plasmids: the haemolysin-bacteriocin encoding pad1 (Tomich et al. 1979); the Tet r (Tn125) plasmid pcf10 (Christie et al. 1987); and the bacteriocinogenic ppd1 (Yagi et al. 1983). These plasmids are highly homologous in the region which encodes the surface proteins involved in aggregation and exclusion substances (Galli and Wirth 1991; Maqueda et al. 1997). The T1 4 strain of Ent. faecalis harbours the conjugative plasmid pmb1 1 required for the production of, and immunity to, bacteriocin Bc-48 (Lopez-Lara et al. 1991; Martínez- Correspondence to: Mercedes Maqueda, Departamento de Microbiología, Facultad de Ciencias, C/Fuentenueva s/n, Granada, Spain ( mmaqueda@goliat.ugr.es). Bueno et al. 1992). The specific pheromone of plasmids pmb1 1 and pcf10 has been identified (Quirantes et al. 1994) and the existence of homology between the surface proteins encoded by both plasmids has been shown to be similar to regions in previously described plasmids (Quirantes et al. 1995). It has been speculated that the high level of similarity in the region involved in the synthesis of these surface proteins might provide information for localizing this genetic region in pmb1 1. Accordingly, in the present study, the genetic region harbouring the genes responsible for the aggregational response in pmb1 1 has been identified and a physical map of pmb1 1 plasmid has been produced. MATERIALS AND METHODS Bacterial strains and plasmids Strain T1 4 of Ent. faecalis is a transconjugant from a mating between the Bc-48 producer strain A and the plasmidfree OG1X strain (Lopez-Lara et al. 1991; Martínez-Bueno et al. 1992). OG1RF strain harbours the conjugative plasmid pam211 (76 4-kb; pad1::tn916) and OG1SSp strain harbours the pcf10 plasmid (65-kb Tet r plasmid; Nakayama et al. 1994). Enterococcal cells were grown overnight without aeration 1998 The Society for Applied Microbiology

2 126 M. MARTÍNEZ-BUENO ET AL. at 37 C in Brain Heart Infusion broth (BHI-B) or Todd Hewitt broth (TH-B). Escherichia coli DH5a, used for cloning experiments, was grown with shaking in Luria broth (LB) at 37 C. Plasmid DNA isolation, DNA sequencing and sequence analysis Enterococcus faecalis plasmid DNA was extracted according to the Anderson and McKay method (1983). Plasmid from E. coli strains was isolated by alkaline lysis (Maniatis et al. 1989) Standard procedures for molecular cloning, DNA ligation, bacterial transformation, agarose gel electrophoresis and Southern blotting were carried out by standard techniques described by Maniatis et al. (1989). Restriction enzymes, T4 DNA ligase, calf intestine phosphatase and other DNA modifying enzymes were bought from Gibco/BRL and Boehringer and used as recommended by the manufacturers. Hybridization experiments were carried out with digoxigenin-labelled probes according to the instructions of the manufacturers (Boehringer). High- and low-stringency hybridization were performed at 68 C and 45 C, respectively. DNA was sequenced using the dideoxy chain termination method (Sanger et al. 1977). Universal and reverse sequencing primers were obtained from Pharmacia-Biotech. Subsequent sequencing was made by a primer-walking strategy. The nucleotide sequences were aligned and analysed using the Genetics Computer Group (GCG) program package (version 9 1) on a Vax computer (BESTFIT and FASTA). The database used in homology searches was GenBank (Release 97 0). Amplification by the polymerase chain reaction (PCR) PCR mixtures contained 1 mmol l 1 each primer, 200 mmol l 1 deoxynucleoside triphosphate, 5 mmol l 1 MgCl 2, 2 5 U of Taq DNA polymerase (Pharmacia), and 1 reaction buffer (Pharmacia). DNA was amplified with 30 cycles (denaturation, 95 C, 1 min; annealing 48 C, 1 min; extension, 72 C, 1 min kb 1 ) followed by a final extension step at 72 C for 5 min. RESULTS AND DISCUSSION Homology analysis by Southern blotting of pmb1 1 and known plasmid systems To confirm the existence of homology in the genetic region involved in the sexual conjugation of plasmids, a Southern blot analysis was carried out with an EcoRI-digestion of pam211 and pcf10 using pmb1 1 as probe. Plasmid pmb1 1 gave a strong label with several EcoRI-fragments involved in the conjugative region of these plasmids (fig. 1; A, B, E and I fragments of pam211 and B, C, E, F, G and H of pcf10). To localize the homologous regions in pmb1 1, the EcoRIdigestion of pmb1 1 was hybridized with appropriate oligonucleotide sequences from pcf10 (Table 1). For this purpose, the plasmid of strain T1 4 was purified, enzymatically digested and separated in agarose gel. The results showed that EcoRI has multiple cut sites in pmb1 1, yielding at least 11 fragments (A, B, C, D, D?, E, F, G, H, I and J) with sizes of 24, 6, 5 7, 3 7, 3 7, 3 3, 2 1, 1 5, 0 72, 0 66, 0 6 kb, respectively. AS1 and AS2 probes from fragment E of pcf10/ecori mapped inside the structural gene prgb were selected because they encoded the RGDV and LPKTGE sequences that are highly conserved in all conjugative systems described (Kao et al. 1991). Similarly, oligonucleotides ES1 and ES2 of the prga gene (fragment C/EcoRI in pcf10) (Chung et al. 1995) and is (initial sequence of prgq localized in C fragment of pcf10/ecori) (Nakayama et al. 1994) were synthesized. Results of hybridizations obtained for the pmb1 1 plasmid are shown in Fig. 2(b). Probes AS1 and AS2 gave clear labels with G and F/EcoRI fragments; ES1 and is hybridized with E fragment of pmb1 1, while ES2 showed homology with a 3 7 fragment (D?). In all cases, controls gave the hybridization expected. Physical mapping and organization of the conjugative region of pmb1 1 The physical map of pmb1 1 was established by means of simple and double digestions with EcoRI (11 fragments), SacI (linearized pmb1 1) and BglII (10 fragments) (Fig. 3). In all cases, the sum of these fragments was approximately 52 Kb, in agreement with the size previously proposed for this plasmid (Martínez-Bueno et al. 1992). The order and orientation of the EcoRI fragments was determined by Southern blotting with the fragments obtained by BglII digestion. Finally, the organization of the conjugative region was obtained by a PCR strategy, using pmb1 1 as template and the oligonucleotides AS1-AS2, ES1-ES2, is-( ES1) and ES1-AS2 as primers. The PCR products had the expected size and EcoRI sites. In all cases, these products gave a positive hybridization when used as probes (data not shown). According to these results, we propose the following organization for asmb1 (aggregation substance pmb1 1), esmb1 (exclusion substance of pmb1 1) and imb1 (inhibitor of pmb1 1) genes in the 10 6 kb fragment of pmb1 1 (Fig. 3). To determine the degree of homology between these regions a partial sequencing of fragments F, G and E (previously cloned in the puc18 vector of E. coli) was carried out. After alignment of the sequences obtained, homologies

3 PLASMID pmb1-1 OF ENT. FAECALIS 127 Table 1 Oligonucleotides used in this work. Sequences were deduced from conserved regions of the pcf10 plasmid, localized in accession number M64978 of GeneBank Probe Sequence Position Gene AS1 5?-GCTCGTGGTGATGTGTTCT-3? prgb AS2 5?-TCGCCTGTTTTCGGCAA-3? prgb ES1 5?-AGCGAAAAATGCGGAAGC-3? prga -ES1 5?-CGCTTCCGCATTTTTCGC-3? ES2 5?-TGCAACGCTTCTTGTCGG-3? prga is 5?-ATAGGAGGGGTGTAAATG-3? prgq A B Fig. 1 Southern blotting of EcoRI-digested pam211 and pcf10 plasmids using pmb1 1 as probe. Plasmid pmb1 1 was labelled with random primers using digoxigenin-11-dutp. (a) EcoRIrestriction profile of pam211 (2) and pcf10 (3). (b) Hybridization of panel (a) with pmb1 1. (Lane 1) Lambda/HindIII molecular weight markers were found of 95 27% for the G fragment and 94 6% for the F fragment, being greater than 99% for the inhibitory region (results not shown). In summary, comparison of the results obtained with plas- mids pmb1 1 and pcf10 demonstrated a high homology, almost identity, in the regions involved in the aggregational response. The remainder of the plasmid sequences, approximately Kb and encoding, for example, the Bc-48 and Tet r determinants, were completely different. These results suggest that the gene clusters involved in pheromone response (from 25 and 30 Kb) may transfer at a high frequency and independently of the plasmid (usually larger than 56 Kb). This hypothesis can be supported by the existence of alternative origins of replication described in large plasmids (Martínez-Bueno et al. 1992). Additionally, in all conjugative regions analysed, an ori site and repb and repa genes, responsible for copy number and regulation of plasmid replication, respectively, have been described (Chung et al. 1995). Moreover, intraspecific transfer of these gene clusters would allow a rapid mobilization of other genetic determinants which code for phenotypic characters of ecological advantage, such as antibiotic resistance or the production of haemolysins or antimicrobial substances (e.g. bacteriocins or peptide antibiotics). However, the existence of independent genetic units in these plasmids would implicate fusions between them, in a similar way to those effected by the Preproteins of Staphylococcus (Genaro et al. 1987). It is tempting to suggest therefore that this additional function is mediated by the Tra/Mob-proteins (coded by plasmids present in the strains of enterococci) which until now have only been implicated in the transfer/mobilization of plasmids (Guzman and Espinosa 1997). ACKNOWLEDGEMENTS This research was supported by a grant from the Comisión Interministerial de Ciencia y Tecnología (PB ) of the Spanish Ministry of Education and Science. M. M-B received a contract from the University of Granada-CICYT. The authors are grateful to Dr M. Statz for the gift of strain OG1RF (pam211) and Dr Nakayama for sending the strain OG1SSp (pcf10).

4 128 M. MARTÍNEZ-BUENO ET AL. (I) (II) (III) A B A B A B Fig. 2 Southern hybridization of EcoRI-digested pmb1 1, pam211 and pcf10 plasmids with different oligonucleotides labelled with digoxigenin-11-ddutp using the terminal transferase method. (a) EcoRI-restriction profiles of plasmids pam211(2), pcf10(3) and pmb1 1(4). (1) Lambda/HindIII. (b) Hybridization with different probes: (I) AS1, (II) ES2 and (III) is Fig. 3 Restriction map and organization of a 10 6 kb fragment of pmb1 1 involved in aggregation, exclusion and pheromone inhibition compared with the same region in pcf10. The arrow shows the direction of transcription of the genes. The order of these genes was confirmed by PCR using pmb1 1 as template and the oligonucleotides AS1-AS2, ES1-ES2, is-( ES1) and ES1-AS2 as primers. (E: EcoRI, B: BglII, S: SacI) (only some BglII cuts are shown)

5 PLASMID pmb1-1 OF ENT. FAECALIS 129 REFERENCES Anderson, D.G. and McKay, L.L. (1983) A simple and rapid method for isolating large plasmid DNA from lactic streptococci. Applied and Environmental Microbiology 46, Christie, P.J., Korman, R., Zahler, R.Z., Adsit, J.C. and Dunny, G.M. (1987) Two conjugation systems associated with Streptococcus faecalis plasmid pcf10: of a conjugative transposon that transfers between S. faecalis and Bacillus subtilis. Journal of Bacteriology 169, Chung, J.W., Bensing, B.A. and Dunny, G.M. (1995) Genetic analysis of the Enterococcus faecalis plasmid pcf10 involved in positive regulation of conjugative transfer functions. Journal of Bacteriology 177, Dunny, G.M. (1990) Genetic functions and cell cell interactions in the pheromone-inducible plasmid transfer system of Enterococcus faecalis. Molecular Microbiology 4, Galli, D. and Wirth, R. (1991) Comparative analysis of Enterococcus faecalis sex pheromone plasmid identifies a single homologous DNA region which codes for aggregation substance. Journal of Bacteriology 173, Gennaro, M.L., Kornblum, J. and Novick, R.P. (1987) A site specific recombination function in Staphylococcus aureus plasmids. Journal of Bacteriology 169, Guzman, L.M. and Espinosa, M. (1997) The mobilization protein, MobM, of the streptococcal plasmid pmv158 specifically cleaves supercoiled DNA at the plasmid orit. Journal of Molecular Biology 266, Kao, S., Olmsted, S.B., Viksnins, A.S., Gallo, J.C. and Dunny, G.M. (1991) Molecular and genetic analysis of a region of plasmid pcf10 containing positive control genes and structural genes encoding surface proteins involved in pheromone-inducible conjugation in Enterococcus faecalis. Journal of Bacteriology 173, Lopez-Lara, I., Gálvez, A., Martínez-Bueno, M., Maqueda, M. and Valdivia, E. (1991) Purification, characterization and biological effect of a second bacteriocin-like inhibitor from E. faecalis ssp. liquefaciens and its mutant strain B Canadian Journal of Microbiology 37, Maniatis, T., Fritsch, E.F. and Sambrook, J. (1989) Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press. Maqueda, M., Quirantes, R., Martín, I., Gálvez, A., Martínez- Bueno, M. and Valdivia, E. (1997) Chemical signals in Grampositive bacteria: the sex-pheromone system in Enterococcus faecalis. Microbiología Española 13, Martínez-Bueno, M., Valdivia, E., Gálvez, A. and Maqueda, M. (1992) Transfer of a plasmid determining bacteriocin Bc-48 production and immunity, and response to sexual pheromone in Enterococcus faecalis S-48. Plasmid 28, 1 8. Nakayama, J., Ruhfel, R.E., Dunny, G.M., Isogal, Y. and Suzuki, A. (1994) The prgq gene of the Enterococcus faecalis tetracycline resistance plasmid pcf10 encodes a peptide inhibitor, icf10. Journal of Bacteriology 176, Quirantes, R., Gálvez, A., Valvidia, E. et al. (1995) Purification of sex pheromones specific for pmb1 and pmb2 plasmids of Enterococcus faecalis S-48. Canadian Journal of Microbiology 41, Quirantes, R., Martín, I., Valdivia, E., Gálvez, A., Martínez-Bueno, M. and Maqueda, M. (1994) Bacteriocin plasmid pmb1 of Enterococcus faecalis: identification of the cell aggregation substance after induction by sex pheromone. Canadian Journal of Microbiology 40, Sanger, F., Nicklen, S. and Coulson, R.A. (1977) DNA sequencing with chain terminating inhibitors. Proceedings of the National Academy of Sciences USA 74, Tomich, P.K., An, F.Y., Damle, S.P. and Clewell, D.B. (1979) Plasmid related transmissibility and multiple drug resistance in Streptococcus faecalis subspecies zymogenes strain DS16. Antimicrobial Agents and Chemotherapy 15, Yagi, Y., Kessler, R.E., Shaw, J.H., Lopatin, D.E., An, F.Y. and Clewell, D.B. (1983) Plasmid content of Streptococcus faecalis strain 39 5 and identification of a pheromone (cpd1)-induced surface antigen. Journal of General Microbiology 129,