Mol Gen Genet (1992) 233:157-160 © Springer-Verlag 1992

Identification of new sex pheromone plasmids in Enterococcus faecalis Reinhard Wirth 1, Anita Friesenegger 1, and Thea Horaud 2 1 Lehrstuhl fiir Mikrobiologie der Universit~it Mfinchen, Maria-Ward-Strasse la, W 8 0 0 0 Miinchen 19, F R G 2 Laboratoire des Staphylocoques et des Streptocoques, Institut Pasteur, F-75724 Paris Cedex 15, France Received October 3, 1991

Summary. We describe the identification of the following new sex pheromone plasmids in Enterococcusfaecalis: a haemolysin-bacteriocin plasmid, piP964; three R plasmids, piP1017, piP1438 and piP1440; and two cryptic conjugative plasmids, piP1141 and pMV120. The identification was based on the formation of cell aggregates on filter membranes during conjugation, on efficient transfer in broth matings, and on a positive clumping reaction of cells carrying these plasmids. In addition these plasmids hybridized with D N A probes specific for sex pheromone-induced structural genes encoding surface proteins required for conjugative transfer of the plasmids. Key words: Sex pheromone plasmids -Enterococcusfaecalis - Sex pheromones - Aggregation substance

Not just 1, but 11 different sex pheromone plasmids have been described so far in E. faecalis. A first hint suggesting why E. faecalis has developed a highly efficient mechanism to collect these plasmids (for which only a few phenotypic traits are known) comes from experiments that indicate that aggregation substance could also function as an adhesin for eucaryotic cells (Kreft et al. 1992). The possible function of the adhesin as a virulence factor for the commensal but perhaps opportunistically pathogenic bacterium E. faeealis might explain why the sex pheromone system is restricted to this species. The study described here had two aims: firstly we wished to screen for new sex pheromone plasmids and characterize them. Secondly we wanted to demonstrate a possible correlation between the formation of mating aggregates on filter membranes by some E. faecalis strains carrying plasmids and the transfer mechanism mediated by sex pheromones.

Introduction The sex pheromone system of Enterococcusfaecalis, first identified by D.B. Clewell and coworkes (Dunny et al. 1978), may be described as a highly efficient plasmid collection mechanism unique to this species. A model of its function (Dunny et al. 1979) states that small linear peptides, which act as sex pheromones, are excreted by recipient strains of E. faecalis not carrying a corresponding plasmid. In the presence of the corresponding sex pheromone the plasmid-bearing donor strain will synthesize an adhesin, called aggregation substance, which mediates, in broth matings, the cell-cell contact required for conjugative transfer of the plasmid between the nonmotile partners. The adhesin is proteinaceous in nature (Yagi et al. 1983) and appears as hair-like structures on the cell surface (Galli et al. 1989; Wanner et al. 1989). Detailed reviews of the E. faecalis sex pheromone system have appeared recently (Clewell and Weaver 1989; Dunny 1990). Correspondence to: R. Wirth

Materials and methods Plasmids. The plasmids used in this study are described in Table 1 (the last four plasmids were used as controls). Clumping assay. The procedure of Dunny et al. (1978) was used with the following slight modifications: the culture supernatant of E. faecalis strain JH2S-S (Tomich et al. 1980) was sterilized by autoclaving and used at a 1:10 dilution. Molecular biological methods. Growth of bacteria, isolation of plasmids, digestion with restriction enzymes and D N A hybridization studies were as described (Galli and Wirth 1991). D N A probes. Four different D N A probes specific for structural genes for sex pheromone-induced surface proteins were used (see Fig. 1). These are: a 0.96 and a 1.2 kb DraI fragment of pAD 1 covering the N- and C-terminal

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portions of the corresponding surface exclusion protein (Weidlich et al. 1992); an oligonucleotide specific for the N-terminus of the aggregation substance gene of all known sex pheromone plasmids (R. Wirth et al., unpublished); a PstI fragment of pAD1 specific for the third quarter of the structural gene for aggregation substance (probe 3 of Fig. 2 in Galli and Wirth 1991).

Results and discussion

The results obtained with the newly identified sex pheromone plasmids are presented in Table 1.

Plasmid size The plasmids have the size (50 to 90 kb) characteristic of sex pheromone plasmids.

Clumping reactions It should be noted that the original transconjugant host strain harbouring the plasmids studied here was E. faecalis JH2-2 (Jacob and Hobbs 1974); with that strain the clumping reaction was barely visible (Ike and Clewell 1984). Therefore, the plasmids were transferred to derivatives of E. faecalis OG1 (Oliver et al. 1977); the clumping reaction was much more pronounced in this genetic background. After induction of the plasmid-bearing strains with culture supernatant from the plasmid-free strain JH2S-S no clumping reaction was seen in the case of piP1017; a weak clumping reaction was observed for piP964, piP1440 and for the controls pPD1 and pAM373. These results suggest that the concentration of sex pheromone in the culture supernatant used might have been limiting in all these cases. However, all the new plasmids described here are transfered in broth mating, and all, except piP964 and its derivative piP1077, induce cell aggregation during conjugative transfer on filter membranes (see Fig. 5 in Pepper et al. 1987).

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The different probes used reflect the genetic organization of sex pheromone-induced genes (see Fig. 1). The results obtained can be summarized as follows: for all plasmids (including the controls pAD1, pPD1, and pJH2) the presence of genes homologous to seal (the structural gene for surface exclusion protein; see Dunny et al. 1985) and to asal (the structural gene for aggregation substance; see Galli et al. 1990) of pAD1 can clearly be observed. The only exception is pAM373; this plasmid is also exceptionally small (36 kb; Clewell et al. 1985). For pPD 1, no signal was observed using the probe specific for the C-terminal region of seal," it remains to be checked whether pPD1 contains a gene for surface exclusion protein that shows only weak homology to the pAD 1-encoded seal gene in the C-terminal region. Our data also indicate that the genetic organization of the plasmids is conserved with the surface exclusion protein gene being located upstream of that for aggregation substance in all lases. This inference stems from the observation that the patterns of hybridizing EcoRI fragments are identical when probes specific for either the C-terminal part of the seal gene or the N-terminal part of the asal gene are used (not shown). Probes specific for the N-terminal part of the seal and the C-terminal part of the asal gene hybridized with different DNA fragments in the various plasmids (not shown). Concluding remarks With the present study the number of identified E. faecalis sex pheromone plasmids in increased to 17. With the exception of piP964 (which seems to be closely related to pJH2) the restriction fragment pattern of the plasmids described here differs from that of the 11 sex pheromone plasmids described earlier; we therefore regard them as "new" sex pheromone plasmids and not secondary isolates of sex pheromone plasmids isolated earlier. The large number of sex pheromone plasmids described raises the question of whether E. faecalis produces different sex pheromones for all these plasmids to induce genes needed for conjugative transfer. Subgroups of sex pheromone plasmids seem to exist, which are defined by their corresponding sex pheromones: pAD1, pAMy1,

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Fig. 1. Genetic organization of structural genes for sex pheromone induced surface proteins of sex pheromone plasmid pAD1 and location of DNA probes used in hybridization studies (see Table 1). The solid line represents plasmid pAD1 ; boxes indicate the structural genes for surface exclusion protein (seal) and aggregation substance (asal) and for open reading frames of unknown function

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pJH2, pBEM10, and piP964 all respond to the same sex pheromone cAD1 (Clewell and Weaver 1989, and this study). Moreover, pAD1, pAMT1, pJH2, and piP964 belong to the same imcompatibility group (Colmar and Horaud 1987; pBEM 10 was not included in that study). Up to now four different sex pheromone have been identified and characterized by chemical methods. From these studies it is clear that more than four different sex pheromones should exist, because some sex pheromone plasmids do not respond to any of them. We suggest that sex pheromone plasmids be defined by their reaction to different sex pheromones in addition to the observed differences in D N A restriction pattern. Acknowledgements. We want to thank those who contributed to this study by providing strains. This work was supported by grant B8 in Sonderforschungsbereich 145 by the Deutsche Forschungsgerneinschaft to R.W.

References Borderon E, Bieth G, Horodniceanu T (1982) Genetic and physical studies of Streptococcus faecalis hemolysin plasmids. FEMS Microbiol Lett 14:51-55 Burdett V (1980) Identification of tetracycline resistance R plasmids in Streptococcus agalactiae (group B). Antirnicrob Agents Chemother 18:753-760 Clewell DB, Weaver KE (1989) Sex pheromones and plasrnid transfer in Enterococcusfaecalis. Plasrnid 21:175-184 Clewell DB, An FY, White BA, Gawron-Burke C (1985) Streptococcus faecalis sex pheromone (cAM373) also produced by Staphylococcus aureus and identification of a conjugative transposon (Tn918). J Bacteriol 162:1212-1220 Colmar I, Horaud T (1987) Enteroeoccus faecalis hernolysinbacteriocin plasmids belong to the same incompatibility group. Appl Environ Microbiol 53:567-570 Dunny GM (1990) Genetic functions and cell-cell interactions in the pheromone-inducible plasmid transfer system of Enterococcus faecalis. Mol Microbiol 4: 689-696 Dunny GM, Brown BL, Clewell DB (1978) Induced cell aggregation and mating in Streptococcusfaecalis: Evidence for a bacterial sex pheromone. Proc Natl Acad Sci USA 75:3479-3483 Dunny GM, Craig RA, Carton RL, Clewell DB (1979) Plasmid transfer in Streptococcus faecalis: Production of multiple sex pheromones by recipients. Plasmid 2:454-465 Dunny GM, Zimmerrnan DL, TortoreUo ML (1985) Induction of surface exclusion (entry exclusion) by Streptococcusfaecalis sex pheromones: Use of monoclonal antibodies to identify an inducible surface antigen involved in the exclusion process. Proc Natl Acad Sci USA 82:8582-8586 Galli D, Wirth R (1991) Comparative analysis of Enterococcus faecalis sex pheromone plasrnids identifies a single homologous DNA region which codes for aggregation substance. J Bacteriol 173:3029-3033 Galli D, Wirth R, Wanner G (1989) Identification of aggregation substances of Enterococcusfaecalis after induction by sex pheromones. Arch Microbiol 151 : 486--490

Note added in proof. We recently identified a plasmid of Enterococcusfaecalis X98 to be a new sex pheromone plasrnid by all the criteria outlined in Table 1. Therefore, the number of sex pheromone plasrnids described is increased to 18. Furthermore, we could show recently that all these plasmids - with the exception of pAM373 - code for a surface protein which is immunologically closely related to pAD 1 encoded aggregation substance (H. Hirt et al., unpublished).

Galli D, Lottspeich F, Wirth R (1990) Sequence analysis of Entero~ eoecusfaecalis aggregation substance encoded by the sex pheromone plasmid pAD1. Mol Microbiol 4:895-904 Horaud T, Delbos F, deCespedes G (1990a) Tn3702, a conjugative transposon in Enteroeoccus faecal&. FEMS Microbiol Lett 72:189-194 Horaud T, Delbos F, Pepper K (1990b) Does a tetracycline resistance determinant of class N exist? Antimicrob Agents Chemother 34:1447-1449 Horodniceanu T, Buu-Hoi A, LeBouguenec C, Bieth G (1982) Narrow host range of some streptococcal R plasmids. Plasmid 8: 199-206 Ike Y, Clewell DB (1984) Genetic analysis of the pAD 1 pheromone response in Streptococcusfaecalis, using transposon Tn917 as an insertional mutagen. J Bacteriol 158: 777-783 Jacob A, Douglas GI, Hobbs SJ (1975) Self-transferable plasmids determining the hemolysin and bacteriocin of Streptococcus faecal& var. zymogenes. J Bacteriol 121 : 863-872 Jacob AE, Hobbs SJ (1974) Conjugal transfer of plasrnid borne multiple antibiotic resistance in Streptococcus faecalis var. zymogenes. J Bacteriol 117:360-372 Kreft B, Marre R, Schramm U, Wirth R (1992) Aggregation substance of Enterococeus faecalis mediates adhesion to cultured renal tubular cells. Infect Imrnun, 60:25-30 LeBouguenec C, Horaud T, Bieth G, Colimon R, Dauguet C (1984) Translocation of antibiotic resistance markers of a plasmid-free Steptococeuspyogenes (group A) strain into different streptococcal hemolysin plasmids. Mol Gen Genet 194:377-387 LeBouguenec C, deCespedes G, Horaud T (1988) Molecular analysis of a composite chromosomal conjugative element (Tn3701) in Streptococcus pyogenes. J Bacteriol 170:3930-3936 Oliver DA, Brown BL, Clewell DB (1977) Analysis of deoxyribonucleic acid in a cariogenic strain of Streptococcus faecal&: an approach to identifying genetic determinants on cryptic plasrnids. J Bacteriol 130:759-765 Pepper K, Horaud T, LeBouguenec C, deCespedes G (1987) Location of antibiotic resistance markers in clinical isolates of Enterococcusfaecalis with similar antibiotypes. Antirnicrob Agents Chernother 31:1394-1402 Tornich PK, An FY, Darnle SP, Clewell DB (1979) Plasmid related transmissibility and multiple drug resistance in Streptococcus faecalis subspecies zymogenes strain DS16. Antimicrob Agents Chemother 15:828-830 Tornich PK, An FY, Clewell DB (1980) Properties of erythromycininducible transposon Tn917 in Streptococcus faecalis. J Bacteriol 141 : 1366-1374 Wanner G, Formanek H, Galli D, Wirth R (1989) Localization of aggregation substances of Enterococcusfaecalis after induction by sex pheromones. Arch Microbiol 151:49t-497 Weidlich G, Wirth R, Galli D (1992) Sex pheromone plasmid pADl-encoded surface exclusion protein of Enterococcusfaecalis. Mol Gen Genet : Yagi Y, Kessler RE, Shaw JH, Lopatin DE, An F, Clewell DB (1983) Plasmid content of Streptococcusfaecalis strain 39-5 and identification of a pheromone (cPD1)-induced surface antigen. J Gen Microbiol 129:1207-1215

Communicated by H. Hennecke