Full copper resistance in Cupriavidus metallidurans requires the interplay of many resistance systems

Abstract

The metal-resistant bacterium Cupriavidus metallidurans uses its copper resistance components to survive the synergistic toxicity of copper ions and gold complexes in auriferous soils. The cup, cop, cus, and gig determinants encode as central component the Cu(I)-exporting PIB1-type ATPase CupA, the periplasmic Cu(I)-oxidase CopA, the transenvelope efflux system CusCBA, and the Gig system with unknown function, respectively. The interplay of these systems with each other and with glutathione (GSH) was analyzed. Copper resistance in single and multiple mutants up to the quintuple mutant was characterized in dose-response curves, Live/Dead-staining, and atomic copper and glutathione content of the cells. The regulation of the cus and gig determinants was studied using reporter gene fusions and in case of gig also RT-PCR studies, which verified the operon structure of gigPABT. All five systems contributed to copper resistance in the order of importance: Cup, Cop, Cus, GSH, and Gig. Only Cup was able to increase copper resistance of the Δcop Δcup Δcus Δgig ΔgshA quintuple mutant but the other systems were required to increase copper resistance of the Δcop Δcus Δgig ΔgshA quadruple mutant to the parent level. Removal of the Cop system resulted in a clear decrease of copper resistance in most strain backgrounds. Cus cooperated with and partially substituted Cop. Gig and GSH cooperated with Cop, Cus, and Cup. Copper resistance is thus the result of an interplay of many systems.