Herzberg M, Bauer L, Kirsten A, Nies D H
Molecular Microbiology, Institute for Biology/Microbiology, Martin-Luther-University, Halle-Wittenberg, Germany.
Metallomics. 2016 Mar;8(3):313-26. doi: 10.1039/c5mt00295h.
The beta-proteobacterium Cupriavidus metallidurans is able to grow in metal-contaminated environments due to having sophisticated metal efflux systems. Here, the contribution of all seven known secondary metal uptake systems (ZupT, PitA, CorA1, CorA2, CorA3, ZntB, HoxN) to metal resistance is characterized. In a strategic deletion approach, all ten double deletion mutants, a variety of triple and quadruple mutants, and from the Δ4 mutant (ΔzupT ΔcorA1 ΔcorA2 ΔcorA3) the mutants Δ5 (=Δ4 ΔpitA), Δ6 (=Δ4 ΔpitA ΔzntB), and finally Δ7 (ΔzupT ΔcorA1 ΔcorA2 ΔcorA3 ΔpitA ΔzntB ΔhoxN) were constructed. Metal resistance, metal content, and regulation of expression of these genes were characterized in these mutants. The ΔzupT single deletion strain exhibited an extended lag phase in Tris-buffered liquid mineral salts medium (TMM) compared to its parent strain AE104, indicating a decreased fitness level. Further deletions up to Δ6 did not influence growth in TMM without added metals but fitness of the Δ7 strain dropped to a lower level compared to Δ6, Δ5 and ΔzupT. The cells of the Δ7 multiple deletion strain still contained all essential metals, demonstrating that additional metal import systems must exist in C. metallidurans. PitA was an important contributor of metal:phosphate complexes to C. metallidurans. Up to Δ5 no evidence was found for increased expression of the transporter genes to recruit substitutes for the deleted importers. Only the hoxN-lacZ reporter gene fusion displayed a changed expression pattern in the Δ6 strain, indicating recruitment of HoxN. Metal resistance of the deletion strains decreased along the deletion series although all strains still contained metal efflux systems: up to the Δ6 mutant the overall fitness was kept at the ΔzupT mutant strain level at the cost of a diminished competence to handle μM concentrations of transition metals. Together, these data demonstrated an important contribution of the seven secondary metal import systems to metal homeostasis in this bacterium.
β-变形菌金属抗性贪铜菌（Cupriavidus metallidurans）能够在金属污染环境中生长，这归因于其拥有复杂的金属外排系统。在此，对所有七个已知的二级金属摄取系统（ZupT、PitA、CorA1、CorA2、CorA3、ZntB、HoxN）对金属抗性的贡献进行了表征。采用策略性缺失方法，构建了所有十个双缺失突变体、多种三缺失和四缺失突变体，从Δ4突变体（ΔzupT ΔcorA1 ΔcorA2 ΔcorA3）开始，依次构建了Δ5（=Δ4 ΔpitA）、Δ6（=Δ4 ΔpitA ΔzntB），最后构建了Δ7（ΔzupT ΔcorA1 ΔcorA2 ΔcorA3 ΔpitA ΔzntB ΔhoxN）。对这些突变体的金属抗性、金属含量以及这些基因的表达调控进行了表征。与亲本菌株AE104相比，ΔzupT单缺失菌株在Tris缓冲液体矿物盐培养基（TMM）中表现出延长的延迟期，表明其适应性水平降低。进一步缺失直至Δ6对不添加金属的TMM中的生长没有影响，但与Δ6、Δ5和ΔzupT相比，Δ7菌株的适应性降至更低水平。Δ7多重缺失菌株的细胞仍含有所有必需金属，这表明金属抗性贪铜菌中必定存在其他金属导入系统。PitA是金属抗性贪铜菌中金属 - 磷酸盐复合物的重要贡献者。直至Δ5，未发现转运蛋白基因表达增加以招募缺失导入蛋白替代物的证据。只有hoxN - lacZ报告基因融合在Δ6菌株中显示出变化后的表达模式，表明招募了HoxN。尽管所有菌株仍含有金属外排系统，但缺失菌株的金属抗性沿缺失系列下降：直至Δ6突变体，总体适应性保持在ΔzupT突变体菌株水平，但代价是处理微摩尔浓度过渡金属的能力减弱。总之，这些数据证明了这七个二级金属导入系统对该细菌中金属稳态的重要贡献。
