Mechanisms of peptide sex pheromone regulation of conjugation in Enterococcus faecalis.

In many gram positive bacteria, horizontal transfer and virulence are regulated by peptide-mediated cell-cell signaling. The heptapeptide cCF10 (C) activates conjugative transfer of the Enterococcus faecalis plasmid pCF10, whereas the iCF10 (I) peptide inhibits transfer. Both peptides bind to the same domain of the master transcription regulator PrgX, a repressor of transcription of the prgQ operon encoding conjugation genes. We show that repression of prgQ by PrgX tetramers requires formation of a pCF10 DNA loop where each of two PrgX DNA-binding sites is occupied by a dimer. I binding to PrgX enhances prgQ repression, while C binding has the opposite effect. Previous models suggested that differential effects of these two peptides on the PrgX oligomerization state accounted for their distinct functions. Our new results demonstrate that both peptides have similar, high-binding affinity for PrgX, and that both peptides actually promote formation of PrgX tetramers with higher DNA-binding affinity than Apo-PrgX. We propose that differences in repression ability of PrgX/peptide complexes result from subtle differences in the structures of DNA-bound PrgX/peptide complexes. Changes in the induction state of a donor cell likely results from replacement of one type of DNA-bound peptide/PrgX tetramer with the other.

In vivo and in vitro analyses of regulation of the pheromone-responsive prgQ promoter by the PrgX pheromone receptor protein.

Expression of conjugative transfer and virulence functions of the Enterococcus faecalis antibiotic resistance plasmid pCF10 is regulated by the interaction of the pheromone receptor protein PrgX with two DNA binding operator sites (XBS1 and XBS2) upstream from the transcription start site of the prgQ operon (encoding the pCF10 transfer machinery) and by posttranscriptional mechanisms. Occupancy of both binding sites by PrgX dimers results in repression of the prgQ promoter. Structural and genetic studies suggest that the peptide pheromone cCF10 functions by binding to PrgX and altering its oligomerization state, resulting in reduced occupancy of XBSs and increased prgQ transcription. The DNA binding activity of PrgX has additional indirect regulatory effects on prgQ transcript levels related to the position of the convergently transcribed prgX operon. This has complicated interpretation of previous analyses of the control of prgQ expression by PrgX. We report here the results of in vivo and in vitro experiments examining the direct effects of PrgX on transcription from the prgQ promoter, as well as quantitative correlation between the concentrations of XBSs, PrgX protein, and prgQ promoter activity in vivo. The results of electrophoretic mobility shift assays and quantitative analysis of prgQ transcription in vitro and in vivo support the predicted roles of the PrgX DNA binding sites in prgQ transcription regulation. The results also suggest the existence of other factors that impede PrgX repression or enhance its antagonism by cCF10 in vivo.

Evidence that highly conserved residues of transmembrane segment 6 of Escherichia coli MntH are important for transport activity.

Nramp (natural resistance-associated macrophage protein) family members have been characterized in mammals, yeast, and bacteria as divalent metal ion/H(+) symporters. In previous work, a bioinformatic approach was used for the identification of residues that are conserved within the Nramp family [Haemig, H. A., and Brooker, R. J. (2004) J. Membr. Biol. 201 (2), 97-107]. On the basis of site-directed mutagenesis of highly conserved negatively charged residues, a model was proposed for the metal binding site of the Escherichia coli homologue, MntH. In this study, we have focused on the highly conserved residues, including two histidines, of transmembrane segment 6 (TMS-6). Multiple mutants were made at the eight conserved sites (i.e., Gly-205, Ala-206, Met-209, Pro-210, His-211, Leu-215, His-216, and Ser-217) in TMS-6 of E. coli MntH. Double mutants involving His-211 and His-216 were also created. The results indicate the side chain volume of these residues is critically important for function. In most cases, only substitutions that are closest in side chain volume still permit transport. In addition, the K(m) for metal binding is largely unaffected by mutations in TMS-6, whereas V(max) values were decreased in all mutants characterized kinetically. Thus, these residues do not appear to play a role in metal binding. Instead, they may comprise an important face on TMS-6 that is critical for protein conformational changes during transport. Also, in contrast to other studies, our data do not strongly indicate that the conserved histidine residues play a role in the pH regulation of metal transport.

Direct evidence for control of the pheromone-inducible prgQ operon of Enterococcus faecalis plasmid pCF10 by a countertranscript-driven attenuation mechanism.

The mating response of Enterococcus faecalis cells carrying the conjugative plasmid pCF10 is controlled by multiple regulatory circuits. Initiation of transcription of the prgQ conjugation operon is controlled by the peptide receptor protein PrgX; binding of the pheromone peptide cCF10 to PrgX abolishes PrgX repression, while binding of the inhibitor peptide iCF10 enhances repression. The results of molecular analysis of prgQ transcripts and genetic studies suggested that the elongation of prgQ transcripts past a putative terminator (IRS1) may be controlled by the interaction of nascent prgQ mRNAs with a small antisense RNA (Anti-Q) encoded within prgQ. Direct evidence for interaction of these RNAs, as well as the resulting effects on readthrough of prgQ transcription, has been limited. Here we report the results of experiments that (i) determine the inherent termination properties of prgQ transcripts in the absence of Anti-Q; (ii) determine the direct effects of the interaction of Anti-Q with nascent prgQ transcripts in the absence of complicating effects of the PrgX protein; and (iii) begin to dissect the structural components involved in these interactions. The results confirm the existence of alternative terminating and antiterminating forms of nascent prgQ transcripts in vivo and demonstrate that the interaction of Anti-Q with these transcripts leads to termination via inhibition of antiterminator formation. In vitro transcription assays support the major results of the in vivo studies. The data support a model for Anti-Q function suggested from recent studies of these RNAs and their interactions in vitro (S. Shokeen, C. M. Johnson, T. J. Greenfield, D. A. Manias, G. M. Dunny, and K. E. Weaver, submitted for publication).