Pheromone-regulated expression of sex pheromone plasmid pAD1-encoded aggregation substance depends on at least six upstream genes and a cis-acting, orientation-dependent factor.
Conjugative transfer of Enterococcus faecalis-specific sex pheromone plasmids relies on an adhesin, called aggregation substance, to confer a tight cell-to-cell contact between the mating partners. To analyze the dependence of pAD1-encoded aggregation substance, Asa1, on pheromone induction, a variety of upstream fragments were fused to an alpha-amylase reporter gene, amyL, by use of a novel promoter probe vector, pAMY-em1. For pheromone-regulated alpha-amylase activity, a total of at least six genes, traB, traC, traA, traE1, orfY, and orf1, are required: TraB efficiently represses asa1 (by a mechanism unrelated to its presumptive function in pheromone shutdown, since a complete shutdown is observed exclusively in the presence of traC); only traC can relieve traB-mediated repression in a pheromone-dependent manner. In addition to traB, traA is required but not sufficient for negative control. Mutational inactivation of traE1, orfY, or orf1, respectively, results in a total loss of alpha-amylase activity for constructs normally mediating constitutive expression. Inversion of a fragment covering traA, P(0), and traE1 without disrupting any gene or control element switches off amyL or asa1 expression, indicating the involvement of a cis-acting, orientation-dependent factor (as had been shown for plasmid pCF10). Unexpectedly, pAD1 represses all pAMY-em1 derivatives in trans, while its own pheromone-dependent functions are unaffected. The discrepancy between the new data and those of former studies defining TraE1 as a trans-acting positive regulator is discussed.
Muscholl-Silberhorn
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AB
Universität Regensburg, NWFIII-Mikrobiologie, D-93053 Regensburg, Germany. albrecht.muscholl@biologie.uni-regensburg.de
D018829:Adhesins, Bacterial / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D013293:Enterococcus faecalis / Q000235:genetics*; D036781:Fimbriae Proteins; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005822:Genetic Vectors; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010675:Pheromones / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D000516:alpha-Amylases / Q000235:genetics / Q000378:metabolism
D018829:Adhesins, Bacterial; D001426:Bacterial Proteins; D008565:Membrane Proteins; D010675:Pheromones; C065937:aggregation substance, Enterococcus faecalis; C082576:traB protein, Bacteria; C074790:traC protein, Plasmid F; D036781:Fimbriae Proteins; D000516:alpha-Amylases