The tetracycline resistance plasmid pCF10 (58 kilobases [kb]) of Streptococcusfaecalis possesses two separate conjugation systems. A 25-kb region of the plasmid (designated TRA) was shown previously to determine pheromone response and conjugation functions required for transfer of pCF10 between S. faecalis cells (P. J.Christie and G. M. Dunny, Plasmid 15:230-241, 1986 from the chromosome to various sites on the streptococcal plasmid pAD1 (5,14).Recent work in one of our laboratories resulted in the generation of a physical and genetic map of the pheromoneinducible conjugative S. faecalis tetracycline resistance plasmid pCF10 (4). The major features of this plasmid include a 25-kb region (designated TRA) that determines plasmid transfer and pheromone response functions. A distinct 16-kb region containing the tetracycline resistance determinant (Tet9) showed a high degree of sequence-homology with the conjugative transposon Tn916 (14, 15). However, we were unable to ascribe any of the genetic functions that would be predicted for a conjugative transposon to this region of pCF10. Of particular significance is the stability of the Tetr determinant and its continued association with pCF10 through multiple rounds of conjugative transfer. In contrast, when Tn916 and related elements are inserted into conjugative plasmids, they commonly excise and segregate from these plasmids at high frequency (by a proposed zygotic induction mechanism) (16)

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Fragments, generated by restriction enzyme digestion, of the 58-kilobase Enterococcus (Streptococcus) faecalis tetracycline resistance plasmid pCF10 were cloned and introduced into Escherichia coli and E. faecalis to characterize the pheromone-inducible conjugation system encoded by this plasmid. Western blot (immunoblot) analyses revealed that a 130-kilodalton (kDa) antigen, identical to the Tral30 antigen shown previously to be involved in pCF10-mediated pheromone-inducible surface exclusion, was produced by both bacterial hosts carrying the recombinant plasmid pINY1825 (cloned EcoRI C fragment). Both bacterial hosts carrying pINY1825 also produced various amounts of immunologically related 118-to 125-kDa antigens (designated pre-Tral3O) that resembled antigens produced by E. faecalis cells carrying pCF10. An additional 150-kDa antigen, TralS0, probably involved in pheromone-induced cellular aggregation, was produced by Escherichia coli and E. faecalis hosts carrying pINY1801 (cloned EcoRI C and E fragments). The coding sequences for the TralS0 and Tral30 antigens were further localized in the TRA region of pCF10 by transposon insertion mutagenesis. Western blot analyses of the recombinant strains, and of strains carrying derivatives of pCF10 or various recombinant plasmids containing TnS or Tn917 insertions, suggested that the portion of pCF10 comprising the tra3 through -6 segments (previously defined by Tn917 insertional mutagenesis) contained several genes that are involved in regulating the synthesis of Tral30 and Tral50.Pheromones excreted by Enterococcus (Streptococcus) faecalis recipient cells elicit a complex physiological response among donor cells carrying a certain class of conjugative plasmids that results in enhanced aggregation of donors-and recipients and high levels of donor conjugal DNA transfer (for reviews, see references 7 and 11). In response to pheromones, also called clumping-inducing agents (CIAs), donor cells produce several proteinaceous cell surface antigens, of which one or more are believed to promote cellular aggregation (18,23,26). We have identified three pheromone-inducible surface antigens associated with the 58-kilobase (kb) tetracycline resistance plasmid pCF10 (11,23 tural gene encoding Tral30 or differences in the proteolysis of antigens from cells grown in the presence or absence of pheromone. Regardless of the reason for the different molecular weight forms of this antigen, our previous experiments indicated clearly that the 130-kDa Tral30 protein (but not the lower-molecular-weight pre-Tral30 forms) is involved in a pheromone-inducible surface exclusion process that prevents induced donor cells from acting as conjugal recipients for plasmids related to pCF10 (13).Two additional surface antigens of 73 kDa (Tra73) and 150 kDa (Tral50) are detected only on pheromone-induced cells carrying pCF10 (11,22