Analysis of expression of prgX, a key negative regulator of the transfer of the Enterococcus faecalis pheromone-inducible plasmid pCF10

Conjugative transfer of pCF10, a plasmid found in Enterococcus faecalis, is induced by the peptide pheromone cCF10 and the donor-recipient aggregation is mediated by PrgB. Expression of prgB in pCF10 is negatively regulated by PrgX. The prgX gene is adjacent to prgQ which provides the promoter for prgB expression. The prgX and prgQ genes are transcribed in opposite directions. A deletion spanning nucleotides + 259 to +398 from the prgQ transcription initiation site abolished the prgX gene products at both RNA and protein levels. An RNA, named Qa, was found to be transcribed in the antisense direction in the prgQ region. The transcription start site and the promoter were found to be almost identical with those of the traD determinant in pAD1, another pheromone-responsive plasmid. The first inverted repeat sequence in prgQ, IRS1, was required for the full activity of the Rn promoter. Although the size of the major Qa RNA detected by Northern blot analysis was too short (ca 120 nt) to be an mRNA for PrgX protein, the transcription from the Qa promoter was shown to proceed through to prgX. Transcriptional fusion ana lyses showed that the transcription of prgX requires one or more trans elements. Moreover, deletion of prgX greatly reduced the level of the Qa RNA and abolished transcription of prgX. Although transcription initiation from the Qa promoter was not increased by PrgX, transcriptional readthrough to prgX was increased by the protein. Based on these data, we conclude that transcription of prgX is initiated from the Qa promoter in prgQ, and PrgX autoregulates its transcription either by mediating transcriptional readthrough or increasing mRNA stability. The PrgX protein, prgX RNA, and the Qa RNA were downregulated by cCF10 induction; however, prgX gene products were still detected for at least 40 minutes after induction. (C) 2000 Academic Press.