Enterococcus faecalis pheromone binding protein, PrgZ, recruits a chromosomal oligopeptide permease system to import sex pheromone cCF10 for induction of conjugation.

Conjugative transfer of the plasmid pCF10 by Enterococcus faecalis donor cells occurs in response to a peptide sex pheromone, cCF10, secreted by recipients. The plasmid-encoded cCF10 binding protein, PrgZ, is similar in sequence to binding proteins (OppAs) encoded by oligopeptide permease (opp) operons. Mutation of prgZ decreased the sensitivity of donor cells to pheromone, whereas inactivation of the chromosomal E. faecalis opp operon abolished response at physiological concentrations of pheromone. Affinity chromatography experiments demonstrated the interaction of the pheromone with several putative intracellular regulatory molecules, including an RNA molecule required for positive regulation of conjugation functions. These data suggest that processing of the pheromone signal involves recruitment of a chromosomal Opp system by PrgZ and that signaling occurs by direct interaction of internalized pheromone with intracellular effectors.

Identification and characterization of the genes of Enterococcus faecalis plasmid pCF10 involved in replication and in negative control of pheromone-inducible conjugation.

The prgB gene of the Enterococcus faecalis pheromone-inducible conjugative plasmid pCF10 encodes the surface protein Asc10. This protein mediates cell aggregation and its expression results in high-frequency transfer of the plasmid from donor to recipient. To identify the minimum region necessary for negative regulation of prgB expression, target plasmids were constructed containing a recently identified positive control region and a prgB::lacZ transcriptional fusion; expression of prgB in cells carrying these plasmids was thus verified by beta-galactosidase assay. The target plasmids were used in genetic studies with compatible plasmids containing cloned pCF10 genes supplying putative negative control functions to define the minimum region of pCF10 required for shutdown of prgB expression in the absence of exogenous pheromone. The minimum segment required for negative control, as indentified by deletion analysis, was a 6.9-kb region extending from the 5' end of a gene called prgN, through a previously identified gene, prgX. The DNA in this region, which had not been previously characterized (2.85 kb), was sequenced, and several potential regulatory genes and plasmid replication genes were identified. Genetic analysis indicated that the prgN, -Y, and -X genes are involved in negative control; prgW may also play a role in negative control, since it appeared to be required for expression of prgY. prgX, or a closely adjacent DNA sequence, acted in cis. The region of pCF10 containing negative control genes was also shown to function as an autonomous replicon in E. faecalis.