In Vivo and In Vitro Analyses of Regulation of the Pheromone-Responsive prgQ Promoter by the PrgX Pheromone Receptor Protein

Expression of conjugative transfer and virulence functions of theEnterococcus faecalis antibiotic resistance plasmid pCF10 is regulated by the interaction of the pheromone receptor protein PrgX with two DNA binding operator sites (XBS1 and XBS2) upstream from the transcription start site of theprgQ operon (encoding the pCF10 transfer machinery) and by posttranscriptional mechanisms. Occupancy of both binding sites by PrgX dimers results in repression of theprgQ promoter. Structural and genetic studies suggest that the peptide pheromone cCF10 functions by binding to PrgX and altering its oligomerization state, resulting in reduced occupancy of XBSs and increasedprgQ transcription. The DNA binding activity of PrgX has additional indirect regulatory effects onprgQ transcript levels related to the position of the convergently transcribedprgX operon. This has complicated interpretation of previous analyses of the control ofprgQ expression by PrgX. We report here the results ofin vivo andin vitro experiments examining the direct effects of PrgX on transcription from theprgQ promoter, as well as quantitative correlation between the concentrations of XBSs, PrgX protein, andprgQ promoter activityin vivo . The results of electrophoretic mobility shift assays and quantitative analysis ofprgQ transcriptionin vitro andin vivo support the predicted roles of the PrgX DNA binding sites inprgQ transcription regulation. The results also suggest the existence of other factors that impede PrgX repression or enhance its antagonism by cCF10in vivo .