The molecular mechanism and peptide signaling response of PrgX used to control pheromone-induced conjugative transfer ofpCF10

Control of expression of conjugation functions encoded by some Enterococcus faecalis plasmids involves cell-cell communication mediated by oligopeptides. Peptides can function as either positive or negative effectors. In the case of the antibiotic resistance plasmid pCF10, the cCF10 pheromone and iCF10 inhibitor peptides compete as signals for and against conjugative transfer. Two plasmid-encoded negative regulators, PrgX protein and Qa RNA, repress conjugation genes in uninduced donor cells. Induction by pheromone results in expression of conjugation factors, including aggregation substance (Asc10); Asc10 then acts to mediate high-frequency plasmid transfer resulting in dissemination of the plasmid among the population.;PrgX had been shown to play a key role in negative regulation of pCF10 conjugation, and Qa was identified as an active RNA, acting in repression. However, at the initiation of this study it was unknown how PrgX and Qa RNA each specifically contributed to the process of negative regulation. Many functions of PrgX had been discovered, but the structural requirements for the various PrgX functions were unknown. Likewise, the molecular mechanism utilized by PrgX to repress the prgQ promoter (PQ) was a mystery.;In this study, independent roles for PrgX and Qa RNA in the regulation of PQ were established; PrgX was revealed as a peptide signal receptor and Qa was shown to be a pheromone-insensitive transcriptional attenuator. Regulation of PQ was shown to require the formation of a DNA loop. Additionally, the same DNA loop was found to be required for control of PrgX autoregulation and Qa production. Furthermore the nature of the pheromone-PrgX interaction was elucidated and the potential biological significance of various signaling peptides in the context of pCF10 regulation was investigated. The data from a collaborative project to determine the three dimensional crystal structure of PrgX alone and in complex with cCF10 supports the proposed model of PrgX function. The results of these genetic and biochemical studies establish a more refined view of the molecular role played by not only PrgX, but also Qa RNA, cCF10, and iCF10 in the negative regulatory circuit and induction of pCF10 plasmid transfer.