The role of cysteine residues in tellurite resistance mediated by the TehAB determinant

TehATehB is a tellurite (TeO32-) resistance determinant found on the Escher ichia coli chromosome. Normally silent, it specifies a minimal inhibitory c oncentration (MIC) of 2 mug K2TeO3/ml unless upregulated or present on a mu lticopy plasmid which results in an MIC of 128 mug/ml. Both TehA and TehB h ave three cysteine residues. Oligonucleotide site-directed mutagenesis was carried out to systematically replace all six cysteine residues by alaninie s, The results showed that cysteine residues in both TehA and TehB play a r ole in tellurite resistance: A single cysteine change had no effect, howeve r increasing combinations of two or three cysteine substitutions demonstrat ed strong phenotypic effects with minimal inhibitory concentrations ranging from 16-64 mug K2TeO3/ml. A cysteine-free mutant in which all six cysteine residues were replaced by alanines maintained a MIC of 16 mug/ml. Further investigations on the role of cysteines in resistance were studied using th iol reactive reagents on the soluble subunit TehB, These studies confirmed that TehB is a dimer and undergoes a conformational change with tellurite a nd 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. (C) 2000 Academic Press.