Analysis of Pheromone Binding and Pheromone Reception by Enterococcus faecalis
Bacteriocin plasmid pPD1 in Enterococcus faecalis encodes a mating response to a small-peptide sex pheromone, cPD1, secreted from recipient cells5. To investigate how a pPD1-harboring donor cell receives the pheromone signal, a tritiated pheromone, [3H]cPD1, was synthesized which was fully active during bioassay. Initially, [3H]cPD1 was incubated with donor cells harboring pAM351 (=pPD1: Tn916) or plasmid-free recipient cells and then the radioactivities of the cells were measured. The activity of the donor cells was about five times higher than that of the recipient cells (Table 1). This result suggests that the donor cells bind or uptake pheromone. The radioactivity of the donor cells reached a maximum in 20 to 30 minutes at both 4°C and 37°C. Lysozyme-treated cells, spheroplasts, harboring pAM351, showed a pheromone-binding activity higher than the intact cells did (Table 1). This indicates that the cell wall is an obstacle for the pheromone to approach the cell membrane. Membranes of donor cells showed no detectable binding activity specific to cPD1 (data not shown). On the other hand, cell-free extract showed high binding activity (Table 1). These results suggest that the pheromone is internalized into cells and bound to some component existing in the cytosol. Furthermore, the binding component was found to have a molecular weight as high as 30,000 Da based on gel permeation chromotography. Scatchard analysis with the donor spheroplast displayed an apparent Kd of 0.45 nM and approximately a hundred binding sites per cell.
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