Characterization of mutations that inactivate the diphtheria toxin repressor gene (dtxR).
The diphtheria toxin repressor (DtxR) is an iron-dependent regulator of diphtheria toxin production and iron uptake in Corynebacterium diphtheriae. It is activated in vitro by divalent metal ions including Fe2+, Cd2+, Co2+, Mn2+, Ni2+, and Zn2+. We characterized 20 different mutations in dtxR induced by bisulfite mutagenesis, 18 of which caused single-amino-acid substitutions in DtxR and two of which were chain-terminating mutations. Six of the amino acid replacements were clustered between residues 39 and 52 in a predicted helix-turn-helix motif that exhibits homology with several other repressors and is identified as the putative DNA-binding domain of DtxR. Three substitutions occurred within a predicted alpha-helical region with the sequence His-98-X3-Cys-102-X3-His-106 that resembles metal-binding motifs in several other proteins and is identified as the putative metal-binding site of DtxR. Several purified variants of DtxR with decreased repressor activity failed to bind in gel retardation assays to DNA fragments that contained the tox operator. A quantitative assay for binding of DtxR to 63Ni2+ was also developed. Scatchard analysis revealed that DtxR has a single class of high-affinity 63Ni(2+)-binding sites with a Kd of 2.11 x 10(-6) M and a maximum binding capacity of approximately 1.2 atoms of Ni2+ per DtxR monomer. The P39L, T40I, T44I, and R47H variants of DtxR exhibited normal to slightly decreased 63Ni(2+)-binding activity, but H106Y, which has an amino acid substitution in the presumed metal-binding domain, exhibited markedly decreased 63Ni(2+)-binding activity.
Wang
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Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814.
Schmitt
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Holmes
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D000483:Alleles; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D015153:Blotting, Western; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D009532:Nickel / Q000378:metabolism
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C075586:DtxR protein, Corynebacterium diphtheriae; D009532:Nickel; D004247:DNA