MOLECULAR MECHANISM OF PEPTIDE-SPECIFIC PHEROMONE SIGNALING IN ENTEROCOCCUS-FAECALIS - FUNCTIONS OF PHEROMONE RECEPTOR TRAA AND PHEROMONE-BINDING PROTEIN TRAC ENCODED BY PLASMID PPD1

Conjugative transfer of the Enterococcus faecalis plasmid pPD1 is acti vated by cPD1, one of several peptide sex pheromones secreted by plasm id-free recipient cells, and is blocked by a donor-produced peptide in hibitor, iPD1, Using a tritiated pheromone, [H-3]cPD1, we investigated how pPD1-harboring donor cells receive these peptide signals. Donor c ells rapidly incorporated [H-3]cPD1, The cell extract but not the memb rane fraction of the donor strain exhibited significant [H-3] cPD1-bin ding activity, On the basis of these data and those of tracer studies, it was demonstrated that cPD1 was internalized, where it bound to a h igh-molecular-weight compound. The cell extract of a strain carrying t he traA-bearing multicopy plasmid (pDLHH21) also exhibited high [H-3] cPD1-binding activity, A recombinant TraA exhibited a dissociation con stant of 0.49 +/- 0.08 nM against [H-3]cPD1. iPD1 competitively inhibi ted [H-3]cPD1 binding to TraA, whereas pheromones and inhibitors relat ing to other plasmid systems did not. These results show that TraA is a specific intracellular receptor for cPD1 and that iPD1 acts as an an tagonist for TraA, A strain carrying the traC-bearing multicopy plasmi d (pDLES23) exhibited significant [H-3]cPD1-binding activity. A strain carrying traC-disrupted pPD1 (pAM351CM) exhibited lower [H-3] cPD1-bi nding activity as well as lower sensitivity to cPD1 than a wild-type d onor strain, Some of the other pheromones and inhibitors inhibited [H- 3]cPD1 binding to the traC transformant like cPD1 and iPD1 did, These results show that TraC, as an extracellular less-specific pheromone-bi nding protein, supports donor cells to receive cPD1.