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Summary

Cupriavidus metallidurans CH34 possesses a multitude of metal efflux systems. Here, the function of the novel PIB4-type ATPase CzcP is characterized, which belongs to the plasmid pMOL30-mediated cobalt-zinc-cadmium (Czc) resistance system. Contribution of CzcP to transition metal resistance in C. metallidurans was compared with that of three PIB2-type ATPases (CadA, ZntA, PrbA) and to other efflux proteins by construction and characterization of multiple deletion mutants. These data also yielded additional evidence for an export of metal cations from the periplasm to the outside of the cell rather than from the cytoplasm to the outside. Moreover, metal-sensitive Escherichia coli strains were functionally substituted in trans with CzcP and the three PIB2-type ATPases. Metal transport kinetics performed with inside-out vesicles identified the main substrates for these four exporters, the Km values and apparent turn-over numbers. In combination with the mutant data, transport kinetics indicated that CzcP functions as ‘resistance enhancer’: this PIB4-type ATPase exports transition metals Zn2+, Cd2+ and Co2+ much more rapidly than the three PIB2-type proteins. However, a basic resistance level has to be provided by the PIB2-type efflux pumps because CzcP may not be able to reach all different speciations of these metals in the cytoplasm.