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

TehATehB is a tellurite (TeO32-) resistance determinant found on the Escherichia coli chromosome. Normally silent, it specifies a minimal inhibitory concentration (MIC) of 2 mug K2TeO3/ml unless upregulated or present on a multicopy plasmid which results in an MIC of 128 mug/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 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 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. (C) 2000 Academic Press.