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The gene cluster pbrTRABCD from Cupriavidus metallidurans CH34 is thought to encode a unique, specific resistance mechanism for lead. However, the exact functions of these genes are unknown. In this study we examine the metal specificity and functions of pbrABCD by expressing these genes in different combinations and comparing their ability to restore Pb(2+), Zn(2+) and Cd(2+) resistance in a metal-sensitive C. metallidurans strain DN440. We show that lead resistance in C. metallidurans is achieved through the cooperation of the Zn/Cd/Pb-translocating ATPase PbrA and the undecaprenyl pyrophosphate phosphatase PbrB. While PbrA non-specifically exported Pb(2+), Zn(2+) and Cd(2+), a specific increase in lead resistance was observed when PbrA and PbrB were coexpressed. As a model of action for PbrA and PbrB we propose a mechanism where Pb(2+) is exported from the cytoplasm by PbrA and then sequestered as a phosphate salt with the inorganic phosphate produced by PbrB. Similar operons containing genes for heavy metal translocating ATPases and phosphatases were found in several different bacterial species, suggesting that lead detoxification through active efflux and sequestration is a common lead-resistance mechanism.

Cupriavidus metallidurans CH34 possesses a multitude of metal efflux systems. Here, the function of the novel P(IB4)-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 P(IB2)-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 P(IB2)-type ATPases. Metal transport kinetics performed with inside-out vesicles identified the main substrates for these four exporters, the K(m) values and apparent turn-over numbers. In combination with the mutant data, transport kinetics indicated that CzcP functions as 'resistance enhancer': this P(IB4)-type ATPase exports transition metals Zn(2+), Cd(2+) and Co(2+) much more rapidly than the three P(IB2)-type proteins. However, a basic resistance level has to be provided by the P(IB2)-type efflux pumps because CzcP may not be able to reach all different speciations of these metals in the cytoplasm.