Genetic and transcriptional analyses of the pheromone response of the Enterococcus faecalis plasmid pAM373

pAM373 is a conjugative Enterococcus faecalis plasmid that encodes a response to a peptide sex pheromone secreted by plasmid-free enterococci. It was of interest because a related activity was produced by Staphylococcus aureus and Streptococcus gordonii. Because of clinical concern relating to the possible mobilization of vancomycin resistance determinants from enterococci into pathogens such as S. aureus, further characterization of pAM373 was initiated. Mating experiments demonstrated that pAM373 can transfer into S. aureus, and that when fused with an appropriate plasmid, a cointegrate is formed that can establish in this species. Complete nucleotide sequencing showed that pAM373 is 36,679 by in size and contains 48 open reading frames, 22 of which have homologues in the enterococcal plasmids pAD1, pPD1 or the conjugative transposon Tn916. Transposon insertions in a region corresponding to more than half of the plasmid affected the mating response. While homology at the amino acid level is weaker than among equivalent proteins from other pheromone plasmids, the overall organization of the pheromone response region appears conserved. It contains a gene encoding a pheromone-binding surface protein (TraC), and determinants with negative regulatory activity (e.g. traA) that control expression of downstream structural genes and conjugation functions. The precursor peptide of the inhibitor iAM373 was identified; its gene is located upstream of a transcription terminator t1. Unique features of pAM373 include the lack of determinants equivalent to traB (reduces endogenous pheromone) and sea1 (prevents futile conjugation between homologous donors) of pAD1. It was shown that pAM373 does not utilize an entry exclusion system. The gene for aggregation substance (asa373) differs from other aggregation substances, which generally are highly conserved; its predicted protein is smaller and aggregation does not require the presence of phosphate and divalent canons. Transcriptional analyses indicated that pheromone-inducible transcription from the iam373 promoter is crucial to the regulation of the pheromone response. Induction increased the level of transcripts extending up to t1 and initiated expression of downstream determinants. Finally, a counter-transcript (mD) was identified that is highly transcribed in uninduced cells and disappears after induction. It may reduce transcription from the iam373 promoter in the absence of pheromone.