| Antibiotic resistance and virulence determinants of the gram-positive bacterium Enterococcus faecalis can be disseminated via pheromone-inducible conjugative plasmids. These plasmids transfer at a high frequency when plasmid-containing donor cells detect recipient-produced pheromone. Donor cells respond to pheromone by expressing plasmid-encoded conjugation functions, including aggregation substance (AS), which mediates plasmid transfer. AS is also a virulence factor, and is expressed in the absence of recipient cells in vivo. Pheromone is processed from a chromosomally encoded lipoprotein, so donor cells could potentially respond to their own endogenous pheromone. To prevent this energetically wasteful process, the pheromone-inducible plasmid pCF10 encodes two endogenous pheromone control elements; iCF10 and PrgY. iCF10 is an inhibitor peptide that neutralizes supernatant pheromone activity, and PrgY is a putative membrane protein. At the initiation of these studies, the function of PrgY was unknown, and the importance of coordinated regulation of endogenous pheromone by PrgY and iCF10 was unexplored.; The results of this work established a more complete understanding of the mechanism and importance of endogenous pheromone control by PrgY and iCF10. PrgY was shown to decrease endogenous pheromone levels and to have separate functional and specificity domains, and share functionally important conserved residues with homologous proteins in a variety of other prokaryotic and eukaryotic species. These studies established the specificity of PrgY for the pheromone sequence within the preprocessed pheromone peptide, and showed that PrgY function is independent of a membrane protein that is involved in pheromone processing, Eep. PrgY control of endogenous pheromone was shown to be physiologically important and most likely the sole function of PrgY. These combined results were used to establish a model for PrgY predicting that PrgY specifically traps or sequesters pheromone that is produced by the cell. In addition, production of iCF10 was shown to be regulated by endogenous pheromone levels, indicating that the ratio of inhibitor:pheromone is carefully maintained in a donor cell. The importance of this ratio was shown in vivo, where this ratio is disrupted to allow induction of AS by endogenous pheromone. |
