The pheromone-responding conjugative bacteriocin plasmid pPD1 (59 kb) of Enterococcus faecalis was mapped physically by using a relational clone approach, and transposon analysis with Tn917 (Em r ) or Tn916 (Tc r ) facilitated the location of the bacteriocin-related genes in a segment of about 6.7 kb. Tn917 insertions within a 3-kb region resulted in constitutive clumping. The nucleotide sequence of the region that included the insertions giving rise to constitutive clumping was determined. The region of pPD1 spanned about 8 kb and was found to contain a number of open reading frames, some of which were named on the basis of homologies with two other pheromone-responding plasmids, pAD1 and pCF10. The genes were arranged in the sequence repB-repA-traC-traB-traA-ipd-traE-traF-orfY-sea-1 with all but repB and traA oriented in the same (left-toright) direction. traC and traB corresponded, respectively, to traC and traB of pAD1 and to prgY and prgZ of pCF10.Certain conjugative plasmids in Enterococcus faecalis transfer at a high frequency in broth mating, a phenomenon relating to a response to specific peptide sex pheromones secreted by potential recipients. The sex pheromone induces the synthesis of a surface aggregation substance that facilitates the formation of a mating aggregate (5-7, 14, 15, 18, 48). When a given plasmid is acquired, secretion of the related pheromone is prevented; however, unrelated pheromones continue to be produced. In addition, the plasmid itself determines the production of a peptide that acts as a competitive inhibitor of the corresponding pheromone (8,10,11,27,32).Several pheromone-responding plasmids have been reported, and the plasmids in E. faecalis have been found to encode such traits as hemolysin-bacteriocin production, UV resistance, and drug resistance (5-7). Of these plasmids, the pheromone-related conjugation systems best studied are pAD1 (60 kb) (6,7,9,12,23,33,35,36,40,41,44,45) and pCF10 (54 kb) (4, 30, 37), which confer responses to sex pheromones cAD1 and cCF10, respectively. pAD1 encodes a hemolysin-bacteriocin mediated by the same genetic determinant (2, 3, 5, 26) and belongs to incompatibility group incHly (13,25). Most (over 90%) of the Hly-Bac plasmids identified in clinical isolates are identical and exhibit extensive homology to pAD1, respond to cAD1, and represent incompatibility group incHly (24,25,29). pAD1 is a representative of this group. pCF10 carries a tetracycline resistance determinant located on transposon Tn925. Genes involved in regulation of the pheromone response have been identified and are clustered in a 7-kb region on each of the plasmids. In pAD1, these genes are arranged in the order traE1-iad-traA-traC-traB (6). The traE1 product is a key positive regulator for expression of downstream structural genes, including determinant asa-1 for the aggregation substance and other conjugation-related genes.The traA product has been shown to represent a key negative regulator of traE1 expression. The traB product is involved in shutdown of endogenous cAD1 p...

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