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 activated by cPD1, one of several peptide sex pheromones secreted by plasmid-free recipient cells, and is blocked by a donor-produced peptide inhibitor, iPD1, Using a tritiated pheromone, [H-3]cPD1, we investigated how pPD1-harboring donor cells receive these peptide signals. Donor cells rapidly incorporated [H-3]cPD1, The cell extract but not the membrane fraction of the donor strain exhibited significant [H-3] cPD1-binding activity, On the basis of these data and those of tracer studies, it was demonstrated that cPD1 was internalized, where it bound to a high-molecular-weight compound. The cell extract of a strain carrying the traA-bearing multicopy plasmid (pDLHH21) also exhibited high [H-3] cPD1-binding activity, A recombinant TraA exhibited a dissociation constant of 0.49 +/- 0.08 nM against [H-3]cPD1. iPD1 competitively inhibited [H-3]cPD1 binding to TraA, whereas pheromones and inhibitors relating to other plasmid systems did not. These results show that TraA is a specific intracellular receptor for cPD1 and that iPD1 acts as an antagonist for TraA, A strain carrying the traC-bearing multicopy plasmid (pDLES23) exhibited significant [H-3]cPD1-binding activity. A strain carrying traC-disrupted pPD1 (pAM351CM) exhibited lower [H-3] cPD1-binding activity as well as lower sensitivity to cPD1 than a wild-type donor 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-binding protein, supports donor cells to receive cPD1.