Funktion des CzcCB2A-Transporters

近4年来的研究结果表明，CzcCBA跨包膜外排络合物与其他金属外排系统密切相互作用，催化金属拉氏菌对重金属的抗性。CzcCBA的阳离子底物Co(II), Zn（II）和Cd（II）可以从细胞质或周质或两个室室接受。该复合物的中心亚基CzcA （RND蛋白家族）的结构以及相关铜转运体CusA的结构将得到解决。两种蛋白的质周底物结合位点将通过定点诱变来确定。至少对于CzcA，细胞质金属结合位点对该蛋白协同转运行为的重要性将得到阐明。类似的实验也将用金属酸霉的两种p型atp酶，ZntA和CadA进行。本文还将分析其他来自金属化弧菌的rnd蛋白对该细菌的金属抗性的参与情况。通过在特定大肠杆菌突变株中的表达，将鉴定金属化弧菌对钴、锌和镉的吸收系统。最后，胞外阳离子的输出作为一种解毒机制只有在外膜的再摄取受到某种程度的限制时才合理。因此，也将对外膜蛋白在金属抗性中的作用进行评价。

The results obtained during the recent four years show that the CzcCBA transenvelope efflux complex, which catalyzes heavy metal resistance in Ralstonia metallidurans, is working in close interplay with other metal efflux systems. The cationic substrates of CzcCBA Co(II), Zn(II) and Cd(II) may be accepted from the cytoplasm or the periplasm or both compartments. The structure of the central subunit CzcA (RND protein family) of the complex will be solved, additionally, the structure of the related copper transporter CusA. The periplasmic substrate binding sites of either protein will be identified by site-directed mutagenesis. At least for CzcA, the importance of the cytoplasmic metal binding site for the cooperative transport behavior of this protein will be elucidated. Similar experiments will also be performed with the two P-type ATPases from R. metallidurans, ZntA and CadA. Involvement of the other RNDproteins from R. metallidurans for metal resistance in this bacterium will be analyzed. The uptake systems for cobalt, zinc and cadmium of R. metallidurans will be identified by expression in specific E. coli mutant strains. Finally, export of periplasmic cations as a mechanism of detoxification is only reasonable if re-uptake across the outer membrane is somehow limited. Therefore, the role of outer membrane proteins in metal resistance of R. metallidurans will also be evaluated.