The role of cysteine residues in tellurite resistance mediated by the TehAB determinant.
TehATehB is a tellurite (TeO(2-)(3)) resistance determinant found on the Escherichia coli chromosome. Normally silent, it specifies a minimal inhibitory concentration (MIC) of 2 microg K(2)TeO(3)/ml unless upregulated or present on a multicopy plasmid which results in an MIC of 128 microg/ml. Both TehA and TehB have three cysteine residues. Oligonucleotide site-directed mutagenesis was carried out to systematically replace all six cysteine residues by alaninies. The results showed that cysteine residues in both TehA and TehB play a role in tellurite resistance: A single cysteine change had no effect, however increasing combinations of two or three cysteine substitutions demonstrated strong phenotypic effects with minimal inhibitory concentrations ranging from 16-64 microg K(2)TeO(3)/ml. A cysteine-free mutant in which all six cysteine residues were replaced by alanines maintained a MIC of 16 microg/ml. Further investigations on the role of cysteines in resistance were studied using thiol reactive reagents on the soluble subunit TehB. These studies confirmed that TehB is a dimer and undergoes a conformational change with tellurite and S-adenosyl-l-methionine binding. Studies using native and SDS denaturing PAGE under reducing and oxidizing conditions suggested that a cysteine in TehB is involved in binding tellurite.
Dyllick-Brenzinger
M
M
Structural Biology Research Group, University of Calgary, Calgary, Alberta, T2N 1N4, Canada.
Liu
M
M
Winstone
T L
TL
Taylor
D E
DE
Turner
R J
RJ
D000409:Alanine / Q000737:chemistry; D000477:Alkylating Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry* / Q000502:physiology*; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004351:Drug Resistance; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D005033:Ethylmaleimide / Q000494:pharmacology; D007460:Iodoacetamide / Q000494:pharmacology; D007461:Iodoacetates / Q000494:pharmacology; D008024:Ligands; D008826:Microbial Sensitivity Tests; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D012436:S-Adenosylmethionine / Q000737:chemistry / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013691:Tellurium / Q000494:pharmacology*; D015854:Up-Regulation
D000477:Alkylating Agents; D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D007461:Iodoacetates; D008024:Ligands; D013439:Sulfhydryl Reagents; C416004:TehA protein, E coli; C119920:tellurite resistance protein B, E coli; D012436:S-Adenosylmethionine; D004228:Dithionitrobenzoic Acid; C026660:tellurous acid; D003545:Cysteine; D013691:Tellurium; D005033:Ethylmaleimide; D000409:Alanine; D007460:Iodoacetamide