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The sex pheromone plasmids in Enterococcus faecalis are one of the most efficient conjugative plasmid transfer systems known in bacteria. Plasmid transfer rates can reach or exceed 10 ؊1 transconjugants per donor in vivo and under laboratory conditions. We report the completion of the DNA sequence of plasmid pCF10 and the analysis of the transcription profile of plasmid genes, relative to conjugative transfer ability following pheromone induction. These experiments employed a mini-microarray containing all 57 open reading frames of pCF10 and a set of selected chromosomal genes. A clear peak of transcription activity was observed 30 to 60 min after pheromone addition, with transcription subsiding 2 h after pheromone induction. The transcript activity correlated with the ability of donor cells to transfer pCF10 to recipient cells. Remarkably, aggregation substance (Asc10, encoded by the prgB gene) was present on the cell surface for a long period of time after pheromone-induced transcription of prgB and plasmid transfer ability had ceased. This observation could have relevance for the virulence of E. faecalis.The advent of microarray technology allows for a comprehensive analysis of gene expression patterns associated with various biological processes, providing insights into complex regulatory networks. One of the most complex processes is the transfer of large portions of genetic material from a donor cell into a recipient cell by means of conjugation. The plasmids of the sex pheromone family in Enterococcus faecalis are among the most efficient bacterial conjugation systems known (16). The family consists of over 20 plasmids and shows extensive sequence homologies (28). E. faecalis strains can host several of these plasmids. This is exemplified by strain V583, the first vancomycin-resistant isolate in the United States (45), chosen for genome sequencing by The Institute for Genomic Research (TIGR; www.tigr.org). V583 contains two sex pheromone plasmids with homology to the well-characterized pAD1 (pTEF1) and pCF10 (pTEF2) plasmids, respectively. The complete sequences for the pheromone plasmids pAD1 and pAM373 became available recently (14,19). Analysis of the sequences of this group of plasmids allows comparisons and insights into the evolution of these elements.Although the sex pheromone plasmids can be disseminated among enterococcal populations very efficiently, plasmid transfer is highly regulated and only induced by recipient cells in close proximity to plasmid donors. The recipient cells secret 7-to 8-amino-acid-long hydrophobic sex pheromones that are bound by a plasmid-encoded binding protein (44, 51). The pheromone is then taken up into the cell (32) and releases a transcriptional block of the PrgX/TraA family of repressors (5). One of the early transcripts after induction encodes for the surface protein aggregation substance (AS) (9). Expression of AS results in tight physical contact between donor and recipient, allows for plasmid transfer rates of up to 10 Ϫ1 transconjugants/donor (16), and is nece...

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Conjugative transfer of Enterococcus faecalis plasmid pCF10 is induced by the heptapeptide pheromone cCF10. cCF10 produced by plasmid-free recipient cells is detected by pCF10-containing donor cells, which respond by induction of plasmid-encoded transfer functions. The pCF10-encoded membrane protein PrgY is essential to prevent donor cells from responding to endogenously produced pheromone while maintaining the ability to respond to pheromone from an exogenous source; this function has not been identified in any nonenterococcal prokaryotic signaling system. PrgY specifically inhibited endogenous cCF10 and cPD1 (a pheromone that induces transfer of closely related plasmid pPD1) but not cAD1 (which is specific for less-related plasmid pAD1). Ectopic expression of PrgY in plasmid-free recipient cells reduced pheromone activity in culture supernatants and reduced the ability of these cells to acquire pCF10 by conjugation but did not have any effect on the interaction of these cells with exogenously supplied cCF10. The cloned prgY gene could complement a pCF10 prgY null mutation, and complementation was used to identify point mutations impairing PrgY function. Such mutations also abolished the inhibitory effect of PrgY expression in recipients on pheromone production and on acquisition of pCF10. Most randomly generated point mutations identified in the genetic screen mapped to a predicted extracellular domain in the N terminus of PrgY that is conserved in a newly identified family of related proteins from disparate species including Borrelia burgdorferi, Archaeoglobus fulgidus, Arabidopsis thaliana, and Homo sapiens. The combined genetic and physiological data suggest that PrgY may sequester or inactivate cCF10 as it is released from the membrane.