A bioinformatic approach was used for the identification of residues that are conserved within the Nramp family of metal transporters. Site-directed mutagenesis was then carried out to change six conserved acidic residues (i.e., Asp-34, Glu-102, Asp-109, Glu-112, Glu-154, and Asp-238) in the E. coli Nramp homolog mntH. Of these six, five of them, Asp-34, Glu-102, Asp-109, Glu-112, and Asp-238 appear to be important for function since conservative substitutions at these sites result in a substantial loss of transport function. In addition, all of the residues within the signature sequence of the Nramp family, DPGN, were also mutated in this study. Each residue was changed to several different side chains, and of ten site-directed mutations made in this motif, only P35G showed any measurable level of (54)Mn(2+) uptake with a V(max) value of approximately 10% of wild-type and a slightly elevated K(m) value. Overall, the data are consistent with a model where helix breakers in the conserved DPGN motif in TMS-1 provide a binding pocket in which Asp-34, Asn-37, Asp-109, Glu-112 (and possibly other residues) are involved in the coordination of Mn(2+). Other residues such as Glu-102 and Asp238 may play a role in the release of Mn(2+) to the cytoplasm or may be involved in maintaining secondary structure.
E xpression of genes required for conjugative transfer of Enterococcus faecalis plasmid pCF10 is controlled by cell-cell signaling. The plasmid encodes a sensing system to increase expression of the prgQ operon, which encodes the transfer machinery, in response to the peptide mating pheromone cCF10 (LVTLVFV) (16). cCF10 is excreted into the growth medium by plasmid-free E. faecalis cells that can serve as conjugative recipients for pCF10 transfer. Since cCF10 production is chromosomally encoded, pCF10 carries genes whose products block self-induction of donor cells by endogenous pheromone. These include prgY, whose protein product reduces pheromone production by the host cell (7), and prgQ, which encodes a 22-amino-acid polypeptide that is processed to an exported 7-amino-acid inhibitor peptide, iCF10 (AI TLIFI) (25). The iCF10 peptide can be imported into donor cells, where it competitively inhibits binding of cCF10 and serves to neutralize any residual endogenous pheromone that escapes PrgY inhibition. For further review of pheromone peptide signaling in E. faecalis, see references 16 and 13.Recent studies have implicated the prgQ promoter as the regulatory target for pheromone signaling in the pCF10 system, as illustrated in Fig. 1A. In the present model, transcription from the prgQ promoter P Q is repressed by PrgX. PrgX dimers bind specifically to two "operator" DNA binding sites (XBS1 and XBS2) in the intergenic region between prgX and prgQ (3). The lower-affinity XBS2 site overlaps P Q , and PrgX occupancy of XBS2 is proposed to inhibit transcription by steric hindrance of RNA polymerase (RNAP) binding to P Q . While XBS2 can be bound by a PrgX dimer in the absence of XBS1, our data (3) suggest that the affinity of the PrgX-XBS2 binary interaction is so low that it would not allow PrgX to compete effectively with RNA polymerase for binding to this region of pCF10 and repress prgQ transcription initiation. Genetic and structural data (19,20,27) suggest that protein-protein interactions between pairs of PrgX dimers bound to the XBSs and the resulting DNA loop formation may favor a repressing structure (state iv) shown in Fig. 1A. Both iCF10 and cCF10 bind to the same region of PrgX, and binding of either peptide has no direct effects on the structure of the DNA binding domain. Instead, the peptides have different effects on the structure of the C terminus of PrgX that cause opposing changes on the PrgX oligomerization state. It has been suggested that the repressing tetramer structure is enhanced by iCF10 binding, while cCF10 binding is predicted to favor conversion of tetramers to dimers and ultimately reduce P Q repression (20,27).In the absence of pheromone, PrgX repression of prgQ operon transcription is incomplete, allowing for a basal level of expression of an ϳ380-nucleotide (nt) RNA (Qs) whose 5= segment encodes iCF10; induced cells contain increased levels of Qs, and longer transcripts that encode the conjugative transfer machinery (5, 9). The mRNA from which PrgX is translated is transcribed from ...
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.
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