title: Analysis of diphtheria toxin repressor-operator interactions and characterization of a mutant repressor with decreased binding activity for divalent metals.
abstract: The diphtheria toxin repressor (DtxR) is an Fe(2+)-activated protein with sequence-specific DNA-binding activity for the diphtheria toxin (tox) operator. Under high-iron conditions in Corynebacterium diphtheriae, DtxR represses toxin and siderophore biosynthesis as well as iron uptake. DtxR and a mutant repressor with His-47 substituted for Arg-47, designated DtxR-R47H, were purified and compared. Six different divalent cations (Cd2+, Co2+, Fe2+, Mn2+, Ni2+, and Zn2+) activated the sequence-specific DNA-binding activity of DtxR and enabled it to protect the tox operator from DNase I digestion, but Cu2+ failed to activate DtxR. Hydroxyl radical footprinting experiments indicated that DtxR binds symmetrically about the dyad axis of the tox operator. Methylation protection experiments demonstrated that DtxR binding alters the susceptibility to methylation of three G residues within the AT-rich tox operator. These findings suggest that two or more monomers of DtxR are involved in binding to the tox operator, with symmetrical DNA-protein interactions occurring at each end of the palindromic operator. In this regard, DtxR resembles several other well-characterized prokaryotic repressor proteins but differs dramatically from the Fe(2+)-activated ferric uptake repressor protein (Fur) of Escherichia coli. The concentration of Co2+ required to activate DtxR-R47H was at least 10-fold greater than that needed to activate DtxR, but the sequence-specific DNA binding of activated DtxR-R47H was indistinguishable from that of wild-type DtxR. The markedly deficient repressor activity of DtxR-R47H is consistent with a significant decrease in its binding activity for divalent cations.
authors.affiliation: Department of Microbiology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814.
authors.forename: M P
authors.initials: MP
authors.lastname: Schmitt
authors.forename: R K
authors.initials: RK
authors.lastname: Holmes
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002412:Cations / Q000378:metabolism*; D003353:Corynebacterium diphtheriae / Q000235:genetics* / Q000378:metabolism; D004269:DNA, Bacterial / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004167:Diphtheria Toxin / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D007501:Iron / Q000494:pharmacology; D008745:Methylation; D008969:Molecular Sequence Data; D009875:Operator Regions, Genetic; D011485:Protein Binding; D012097:Repressor Proteins / Q000378:metabolism; D013045:Species Specificity
chemical_list: D001426:Bacterial Proteins; D002412:Cations; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D004167:Diphtheria Toxin; C075586:DtxR protein, Corynebacterium diphtheriae; D012097:Repressor Proteins; C068477:ferric uptake regulating proteins, bacterial; D007501:Iron