Cupriavidus metallidurans CH34 adapted to high Zn concentrations

Cupriavidus metallidurans CH34 is a well-studied metal-resistant beta-proteobacterium and contains a battery of genes involved in the resistance and processing of numerous metals. Here, we performed a directed evolution experiment to obtain derivatives adapted to grow in the presence of zinc at concentrations above the minimal inhibitory concentration for the parental strain. Genetic characterization of one derivative, CH34ZnR, which was also more resistant to cadmium, revealed seven mutations, including inactivation of glpR coding for a DeoR-type transcriptional repressor by insertion of IS1088. The latter resulted in the constitutive expression of the neighboring ABC-type transporter. GlpR and the adjacent ABC transporter are highly similar to the glycerol operon regulator and ATP-driven glycerol importer of Rhizobium leguminosarum bv. viciae VF39, respectively. Deletion of glpR or the ABC transporter and complementation of CH34ZnR with the parental glpR gene further demonstrated that loss of GlpR function, with concomitant induction of the adjacent ABC transporter, is pivotal in the observed phenotype. Moreover, addition of glycerol, presumably by glycerol-mediated reduced GlpR activity, also promoted increased zinc and cadmium resistance. Thus, the ABC-type transporter is proposed to be a new adaptation route during exposure to toxic zinc concentrations.

耐金属贪铜菌CH34（Cupriavidus metallidurans CH34）是一种研究较为深入的金属抗性β-变形菌（beta-proteobacterium），其携带一套参与多种金属抗性与代谢过程的基因。本研究通过定向进化实验，获得了能在高于亲本菌株最低抑菌浓度（minimal inhibitory concentration, MIC）的锌离子浓度下生长的衍生菌株。对其中一株同时对镉具有更高抗性的衍生菌株CH34ZnR进行遗传表征后发现，其携带7处突变，其中包括通过插入IS1088使编码DeoR型转录阻遏蛋白（DeoR-type transcriptional repressor）的glpR基因失活。该突变导致邻近的ABC型转运蛋白（ABC-type transporter）发生组成型表达。GlpR与该相邻ABC型转运蛋白，分别与豌豆根瘤菌bv. viciae株VF39（Rhizobium leguminosarum bv. viciae VF39）的甘油操纵子调节蛋白及ATP驱动的甘油输入蛋白高度同源。敲除glpR或该ABC型转运蛋白，以及用亲本型glpR基因对CH34ZnR进行基因互补实验，进一步证实：GlpR功能丧失并伴随该邻近ABC型转运蛋白的诱导表达，是本次观测到的抗性表型形成的关键因素。此外，添加甘油后，推测通过甘油介导的GlpR活性降低途径，同样可提升菌株对锌与镉的抗性。综上，本研究提出该ABC型转运蛋白是菌株暴露于毒性锌浓度环境时的一种新型适应路径。