Transfer functions of the Streptococcus faecalis plasmid pAD1: organization of plasmid DNA encoding response to sex pheromone.

The conjugative plasmid pAD1 (59.6 kilobases) of Streptococcus faecalis shows a 10,000-fold increase in transfer frequency following induction by the sex pheromone cAD1. Mutagenesis of the plasmid with transposon Tn917 was undertaken to determine the region(s) of pAD1 required for the mating response. The relevant genetic material was found to be distributed over a 31.2-kilobase contiguous region of the plasmid. Although insertions in two previously identified regions (traA and traB) exhibited increased transfer frequencies, insertions in five new regions (D, E, F, G, and H) decreased the ability of pAD1 to transfer. Insertions in region H allowed the cells to form visible mating aggregates, but the plasmid transfer frequency was decreased to levels below detection during a 1-h broth mating. Mutants with mutations in region G were able to form aggregates; however, insertions in regions D, E, and F prevented aggregate formation. Insertions in region C decreased the sensitivity of the cell to exogenous cAD1 and exhibited increased activity of the pheromone inhibitor iAD1. Surface protein profiles produced by a number of these mutants were examined, and in some cases were found to be different from those of the wild type. A map showing the various regions is presented, and related aspects of the regulation of the pAD1 mating response are discussed.

Identification of pheromone-induced surface proteins in Streptococcus faecalis and evidence of a role for lipoteichoic acid in formation of mating aggregates.

The conjugative transfer of the Streptococcus faecalis plasmid pAD1 is characterized by a 10,000-fold increase in frequency following sex pheromone (cAD1) induction. Before the increase in plasmid transfer, donor cells synthesize a proteinaceous adhesin that facilitates the formation of mating aggregates. Four novel surface proteins appearing after exposure of pAD1-containing cells to sex pheromone have been identified. Thirty minutes after induction, a 130-kilodalton (kDa) protein was detectable by Western blotting. A 74-kDa protein, the major species present, and a pair of bands at 153 and 157 kDa were evident 45 min after induction. Induced cells containing another conjugative S. faecalis plasmid, pPD1, gave rise to three high-molecular-weight proteins of the same size (130, 153, and 157 kDa) as those synthesized by pAD1-containing cells. These proteins cross-reacted with antisera raised against induced cells containing pAD1. However, the major protein species produced by pPD1-containing cells had a molecular weight of 78,000 and did not cross-react significantly with the corresponding band of the pAD1 system. Pheromone-induced transfer of the two plasmids, when both were present in the same cell, was independent; induction was limited to the pheromone-specified plasmid. The possibility that lipoteichoic acid might act as a receptor (binding substance) for the induced adhesin protein was also explored. Free lipoteichoic acid (isolated from S. faecalis) inhibited clumping of induced cells, apparently by acting as a competitive inhibitor of the cellular binding substance.