Regulation of the pAD1 sex pheromone response in Enterococcus faecalis: effects of host strain and traA, traB, and C region mutants on expression of an E region pheromone-inducible lacZ fusion

Abstract

Pheromone-induced conjugal transfer of the hemolysin-bacteriocin plasmid pADl of Enterococcusfaecalis is regulated by a cluster of determinants designated traA, traB, and regions C and E. The E region is believed to include a positive regulator that controls many structural genes related to conjugation. The pheromoneinducible Tn917-lac fusion NR5, located in the E region, is regulated by the products of traA, traB, and the C region. To more closely examine the effects of these genes on the induction of E region products, inserts in each of these genes were combined with the NR5 fusion in a novel approach involving triparental matings with a pADi miniplasmid and recombinational mutagenesis. Results indicate that (i) the traA gene product is a key repressor of the pheromone response; (ii) the traB gene product, in cooperation with a gene within or regulated by the E region, controls pheromone shutdown; (iii) a primary function of the C region gene product is in pheromone sensing, with secondary functions in pheromone shutdown and negative regulation; and (iv) the host in which the plasmid resides has a dramatic effect on the regulation of the NR5 fusion in traB and C region mutants. Numerous parallels were observed between the regulation of the NR5 fusion and the regulation of the aggregation and transfer response. These parallels aided in further defining the functions of particular regulatory determinants as well as further establishing the link between the regulation of the E region and the regulation of the aggregation and transfer response.The Enterococcus faecalis plasmid pAD1 (59.6 kilobases) encodes a conjugative transfer system that is induced specifically by a small peptide pheromone, cAD1, produced by potential recipients. When exposed to pheromone, plasmidcontaining cells respond by producing: (i) an aggregation substance which facilitates the formation of mating or selfaggregates, (ii) surface proteins that have been correlated with aggregation substance, and (iii) other functions believed to be essential for the actual physical transfer of the plasmid DNA. Once a recipient acquires pAD1, cAD1 activity is shut down, while the activity of pheromones specific for other plasmids is unaffected. In addition, plasmid-containing cells produce a specific peptide, iAD1, which specifically and competitively inhibits cAD1 activity (for a recent review, see reference 2).The pAD1 pheromone response was previously shown to be regulated by a cluster of genes located in the 15-kilobase EcoRI B restriction fragment (4, 7, 13). Miniplasmids consisting solely of this fragment and containing pheromoneinducible lacZ fusions were found to regulate the fusions in a manner identical to that observed when the same fusions were present on the intact plasmid (14). These results indicated that the regulatory genes on the pAD1 EcoRI B fragment were not only essential but also sufficient for directing a normal pheromone response. Mutational analyses with Tn917 and transcriptional analyses with Tn917-lac have identified genetic det...

Characterization of the traC determinant of the Enterococcus faecalis hemolysin-bacteriocin plasmid pAD1: binding of sex pheromone

“…When pADi is acquired by the recipient, it shuts down production of cAD1; however, pheromones specific for different plasmid systems continue to be secreted. Genes involved in the regulation of the pheromone response have been identified and include determinants for positive (TraEl) and negative (TraA) regulatory proteins (22,29,31). TraA negatively regulates expression of traEl and is also sensitive, at least indirectly, to the mating signal.…”

“…TraA negatively regulates expression of traEl and is also sensitive, at least indirectly, to the mating signal. TraEl is believed to positively regulate in trans all or most of the structural genes involved in the conjugative process (10,29,31). The nature of the regulation of traEl via control of read-through of transcriptional terminators located upstream has recently been reported (12,22,27).…”

“…It is presumed that there is some recognition of the pheromone on the cell surface, and there is genetic evidence for a pAD1 determinant encoding the ability to bind to cAD1 (31). Transposon inserts in what was designated the C region, henceforth designated traC, have been shown to cause the loss of sensitivity to pheromone in E. faecalis OG1X (31). These mutants all exhibit a four-to eightfold increase in iAD1 detectable in culture supernatants, a phenomenon attributed to less binding of the inhibitor to the cell surface.…”

“…1) represents a fragment containing traC subcloned from the miniplasmid pAM2011K (30), which contained a Tn9171ac insertion (NR11), into the shuttle plasmid pAM401 (33). This plasmid and derivatives of it, pAM2601 and pAM2602, each containing a TnS insertion within traC (generated in Escherichia coli by a previously described procedure [27,28]), were tested for their ability to 128 64 a OG1X cells carrying the specified plasmid were grown to mid-exponential phase after which 1 ml of the culture was mixed with 1 ml of synthetic pheromone in N2GT broth (31). After 1 h, the cells were pelleted, the supernatant was boiled for 10 min, and the titer was determined with a microtiter serial twofold dilution assay (9).…”

“…b The number in parentheses corresponds to 40 ng of synthetic pheromone (prepared by the University of Michigan core peptide facility) per ml in N2GT broth (31). Fig.…”