Dyllick-Brenzinger M, Liu M, Winstone T L, Taylor D E, Turner R J
Structural Biology Research Group, University of Calgary, Calgary, Alberta, T2N 1N4, Canada.
Biochem Biophys Res Commun. 2000 Oct 22;277(2):394-400. doi: 10.1006/bbrc.2000.3686.
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.
TehATehB是在大肠杆菌染色体上发现的一种亚碲酸盐（TeO(2-)(3)）抗性决定因子。通常处于沉默状态，它指定的最低抑菌浓度（MIC）为2微克K(2)TeO(3)/毫升，除非被上调或存在于多拷贝质粒上，此时MIC为128微克/毫升。TehA和TehB都有三个半胱氨酸残基。进行了寡核苷酸定点诱变，用丙氨酸系统地取代所有六个半胱氨酸残基。结果表明，TehA和TehB中的半胱氨酸残基在亚碲酸盐抗性中起作用：单个半胱氨酸变化没有影响，然而，两个或三个半胱氨酸取代组合的增加显示出强烈的表型效应，最低抑菌浓度范围为16 - 64微克K(2)TeO(3)/毫升。一个无半胱氨酸突变体，其中所有六个半胱氨酸残基都被丙氨酸取代，其MIC维持在16微克/毫升。使用硫醇反应试剂对可溶性亚基TehB进行了关于半胱氨酸在抗性中作用的进一步研究。这些研究证实TehB是一种二聚体，并且在与亚碲酸盐和S-腺苷-L-甲硫氨酸结合时会发生构象变化。在还原和氧化条件下使用天然和SDS变性聚丙烯酰胺凝胶电泳的研究表明，TehB中的一个半胱氨酸参与亚碲酸盐的结合。
