Characterization of the effects of regulators PrgY and PrgZ on the inducibility of plasmid pCF10 in Enterococcus faecalis

Abstract

Enterococcus faecalis is an increasingly significant pathogenic bacterium in humans, as well as a commensal member of the intestinal flora. Pheromone-responsive plasmid systems play an important role in the dissemination of antibiotic resistance among this and other species. pCF10 is a well-characterized member of this class of plasmids, and allows for transmission of tetracycline resistance in E. faecalis. The experiments described in this thesis were designed to assess the effects of three genes thought to be important for the response of plasmid pCF10 to the peptide mating pheromone cCF10 in E. faecalis: 1) the chromosomal locus responsible for pheromone production- ccfA, 2) the plasmid-encoded negative regulator prgY thought to be responsible for sequestration of endogenous pheromone at the cell surface, and 3) the plasmid-encoded positive regulator prgZ, an oligopeptide permease subunit A homolog responsible for specific import of pheromone. In this study transcription emanating from regulatory regions of this plasmid was characterized over time using reverse transcription quantitative polymerase chain reaction (RT-qPCR). Results from these assays were confirmed using phenotypic analysis and Northern blotting. Expression of ccfA was shown to be important to moderate the pheromone response. This moderation effect is believed to be the result of basal induction of donor cells, resulting in production of the peptide inhibitor iCF10 from the plasmid. As the first study to probe PrgY function at the level of transcription, RNA quantitation revealed that levels of endogenous pheromone cCF10 resulting from processing of the CcfA lipoprotein precursor are insufficient to fully induce a conjugation response in the absence of negative regulator PrgY, suggesting secondary negative regulation. Finally, PrgZ was shown to be important for regulated shutdown of the conjugation response in addition to initiation. This lends evidence to previous research suggesting specific import of inhibitor iCF10 by PrgZ. Probing the effects of these regulators over time in strains that differ in their ability to secrete pheromone allows us to paint a more complete regulatory picture of this complex and dynamic system.