CLONING AND CHARACTERIZATION OF A REGION OF THE ENTEROCOCCUS-FAECALISCONJUGATIVE PLASMID, PCF10, ENCODING A SEX PHEROMONE-BINDING FUNCTION

In order to investigate the mechanism by which peptide sex pheromones induce expression of the conjugation functions of certain Enterococcus faecalis plasmids, a biological assay was developed to measure the ab ility of cells carrying the conjugative plasmid pCF10 to bind the sex pheromone cCF10. The data indicated that pCF10 endows its host E.faeca lis cell with the ability to specifically remove (apparently by irreve rsible binding) cCF10 activity from culture medium. The pCF10 DNA enco ding this ability was localized to a 3.4-kb segment within a region in volved in negative control of expression of conjugal transfer function s. This segment also encoded ability to bind the pheromone inhibitor p eptide iCF10. DNA sequencing revealed three open reading frames, which have been denoted prgW (pheromone responsive gene W), prgZ, and prgY. The deduced product of prgW resembled regulatory proteins from other bacteria and eucaryotes, with a very high degree of identity within a putative DNA-binding domain. The prgY gene actually extended into an a djacent region of pCF10 and could encode a protein with significant si milarity to a protein called TraB, believed to be involved in shutdown of pheromone cAD1 production by cells carrying the pheromone-inducibl e hemolysin plasmid pAD1, according to F. Y. An and D. B. Clewell (Abs tr. Gen. Meet. Am. Soc. Microbiol. 1992, H70, 1992). The prgZ gene pro duct showed significant relatedness to binding proteins encoded by oli gopeptide permease (opp) operons in gram-positive and gram-negative ba cteria and is highly similar to a pAD1-encoded protein, TraC, which is believed to mediate sex pheromone cAD1 binding (K. Tanimoto, F. Y. An , and D. B. Clewell, submitted for publication). A Tn5 insertion into prgZ abolished cCF10 binding ability.