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| CHARACTERIZATION OF MUTATIONS THAT INACTIVATE THE DIPHTHERIA-TOXIN REPRESSOR GENE (DTXR) |
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| Author(s): WANG ZO, SCHMITT MP, HOLMES RK |
| Source: INFECTION AND IMMUNITY Volume: 62 Issue: 5 Pages: 1600-1608 Published: MAY 1994 |
| Times Cited: 38 References: 43 |
| Abstract: The diphtheria toxin repressor (DtxR) is an iron-dependent regulator of diphtheria toxin production and iron uptake in Corynebacterium diptheriae. It is activated in vitro by divalent metal ions including Fe2+, Cds(2+) 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:ere 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-X(3)-Cys-102-X(3)-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 Ni-63(2+) was also developed. Scatchard analysis revealed that DtxR has a single class of high-affinity Ni-63(2+)-binding sites with a K-d 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 Ni-63(2+)-binding activity, but HI06Y, which has an amino acid substitution in the presumed metal-binding domain, exhibited markedly decreased Ni-63(2+)-binding activity. |
| Document Type: Article |
| Language: English |
Addresses:
1. UNIFORMED SERV UNIV HLTH SCI, DEPT MICROBIOL & IMMUNOL, BETHESDA, MD 20814 USA |
| Publisher: AMER SOC MICROBIOLOGY, 1325 MASSACHUSETTS AVENUE, NW, WASHINGTON, DC 20005-4171 |
| Subject Category: Immunology; Infectious Diseases |
| IDS Number: NH313 |
| ISSN: 0019-9567 |
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