Cadmium and zinc are effluxed from cells of Ralstonia metallidurans by the RND-protein complex CzcCBA, the CDF-protein CzcD and by two CPx-type ATPases, CadA and ZntA. The genes of the czc-determinant (czcCBA, czcD) are located on megaplasmid pMOL30 but the cadA and zntA genes on the chromosome. Expression of zntA from R. metallidurans predominantly mediated resistance to zinc and expression of cadA predominantly mediated resistance to cadmium in the metallsensitive Escherichia coli strain GG48. Both transporters decreased the cellular content of zinc or cadmium in this organism. The purified proteins ZntA or CadA bind to Cd2+, Zn2+ or Cu2+ in vitro. In the plasmid-free R. metallidurans strain AE104, single gene deletions of cadA or zntA only had a moderate effect on cadmium or zinc resistance but zinc resistance decreased 6-fold and cadmium resistance decreased 350-fold in cadA zntA double mutation strains. Neither single nor double gene mutations affected zinc resistance in the presence of the RND-protein complex CzcCBA. In contrast, cadmium resistance of the cadA zntA double mutant could be increased only partially by the presence of CzcCBA. The reporter gene fusions with lacZ indicated that expression of cadA was induced by cadmium but not by zinc in R. metallidurans strain AE104. In the absence of the zntA gene, expression of cadA occurred at lower cadmium concentrations and zinc now served as an inducer. In contrast, expression of zntA was induced by both zinc and cadmium, and the induction pattern did not change in the absence of CadA. However, expression of both genes, zntA and cadA, was diminished in the presence of CzcCBA. This indicated that CzcCBA efficiently decreased cytoplasmic cadmium and zinc concentrations. |