Enterococcus faecalis sex pheromone plasmid pAM373: analyses of TraA and evidence for its interaction with RpoB

The Enterococcus faecalis plasmid pAM373 (36.7kb) encodes a mating response to the sex pheromone cAM373 secreted by recipient (plasmid-free) bacteria. Like certain other conjugative enterococcal plasmids, a key regulator of the pheromone response is a negatively acting protein, TraA, which is believed to interact with internalized pheromone to influence expression from a key transcriptional promoter P(0). An earlier report showed that in the case of pAM373 most, but not all, transposon-insertion mutations in traA differed from those in the case of pAD1 and pCF10 in that they did not give rise to the normally characteristic constitutive clumping. We show here that this phenomenon relates to a host effect involving an RpoB-related mutation associated with rifampin resistance. When harboring traA mutants, rifampin-sensitive hosts exhibited constitutive clumping, whereas rifampin-resistant hosts did not-despite the fact that the latter host exhibited a normal pheromone-inducible clumping response when harboring a wild-type plasmid. The data imply that TraA normally remains associated with the transcription complex after induction. In addition the promoter of traA, designated P(a), was shown to be located about 600bp upstream of the translational start site, as clones containing traA required this site to complement traA mutants in trans. Transcription from P(a) also gave rise to a short (130 nt) transcript, mD, expressed at a high level in uninduced cells. An earlier observation suggesting that TraA negatively affected transcriptional readthrough into the 3' end of traA from the t(ac) intrinsic bidirectional terminator between traA and the opposing, adjacent traC was supported by TraA complementation studies. Evidence is also presented suggesting that this regulation at t(ac) also involves an additional, possibly cis-acting, element.