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

Abstract

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 ␣-amylase reporter gene, amyL, by use of a novel promoter probe vector, pAMYem1. For pheromone-regulated ␣-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 ␣-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 pheromonedependent functions are unaffected. The discrepancy between the new data and those of former studies defining TraE1 as a trans-acting positive regulator is discussed.

Isolation of VanB-Type Enterococcus faecalis Strains from Nosocomial Infections: First Report of the Isolation and Identification of the Pheromone-Responsive Plasmids pMG2200, Encoding VanB-Type Vancomycin Resistance and a Bac41-Type Bacteriocin, and pMG2201, Encoding Erythromycin Resistance and Cytolysin (Hly/Bac)

“…There are reports that traE1 acts in trans (8,45). Another report shows that traE1 acts as a cis element in gene regulation (46). All of the analyses were performed under artificial conditions with cloned elements of the regions on multicopy vector plasmids; thus, these activities might differ from the activity in the wild-type plasmid.…”

Characterization of the Pheromone Response of theEnterococcus faecalisConjugative Plasmid pCF10: Complete Sequence and Comparative Analysis of the Transcriptional and Phenotypic Responses of pCF10-Containing Cells to Pheromone Induction

“…Studies of the transcriptional response of the plasmid-encoded genes on sex pheromone plasmids have focused on the regions upstream of the respective AS genes. Regulation in the pAD1 system shows some differences, most notably the presence of an apparent in trans regulatory protein (TraE1) that is absent in the pCF10 system (34,37). In pCF10, the transcriptional start site for the prgB transcript is 5 kb upstream of the gene start (9), in the prgQ locus, which encodes the iCF10 inhibitor peptide and several RNAs involved in regulation of expression of downstream genes.…”

DNA-Binding Proteins Regulating pIP501 Transfer and Replication

“…Additionally, in case of TraJ from plasmid R1 and F-related plasmids, regulation of the transfer operon via a sense/antisense RNA system has been shown (Koraimann et al, 1991 , 1996 ; Mark Glover et al, 2015 ). For Gram-positive bacteria, the sex-pheromone-responsive enterococcal plasmids, particularly pCF10, are those with the best studied regulatory processes controlling conjugative transfer (Tanimoto et al, 1996 ; Muscholl-Silberhorn, 2000 ; Dunny, 2007 , 2013 ; Folli et al, 2008 ).…”