A gold speciation that adds a second layer to synergistic gold-copper toxicity in Cupriavidus metallidurans.

The metal-resistant bacterium Cupriavidus metallidurans occurs in metal-rich environments. In auriferous soils, the bacterium is challenged by a mixture of copper ions and gold complexes, which exert synergistic toxicity. The previously used, self-made Au(III) solution caused a synergistic toxicity of copper and gold that was based on the inhibition of the CupA-mediated efflux of cytoplasmic Cu(I) by Au(I) in this cellular compartment. In this publication, the response of the bacterium to gold and copper was investigated by using a commercially available Au(III) solution instead of the self-made solution. The new solution was five times more toxic than the previously used one. Increased toxicity was accompanied by greater accumulation of gold atoms by the cells. The contribution of copper resistance determinants to the commercially available Au(III) solution and synergistic gold-copper toxicity was studied using single- and multiple-deletion mutants. The commercially available Au(III) solution inhibited periplasmic Cu(I) homeostasis, which is required for the allocation of copper ions to copper-dependent proteins in this compartment. The presence of the gene for the periplasmic Cu(I) and Au(I) oxidase, CopA, decreased the cellular copper and gold content. Transcriptional reporter gene fusions showed that up-regulation of gig, encoding a minor contributor to copper resistance, was strictly glutathione dependent. Glutathione was also required to resist synergistic gold-copper toxicity. The new data indicated a second layer of synergistic copper-gold toxicity caused by the commercial Au(III) solution, inhibition of the periplasmic copper homeostasis in addition to the cytoplasmic one.IMPORTANCEWhen living in auriferous soils, Cupriavidus metallidurans is not only confronted with synergistic toxicity of copper ions and gold complexes but also by different gold species. A previously used gold solution made by using aqua regia resulted in the formation of periplasmic gold nanoparticles, and the cells were protected against gold toxicity by the periplasmic Cu(I) and Au(I) oxidase CopA. To understand the role of different gold species in the environment, another Au(III) solution was commercially acquired. This compound was more toxic due to a higher accumulation of gold atoms by the cells and inhibition of periplasmic Cu(I) homeostasis. Thus, the geo-biochemical conditions might influence Au(III) speciation. The resulting Au(III) species may subsequently interact in different ways with C. metallidurans and its copper homeostasis system in the cytoplasm and periplasm. This study reveals that the geochemical conditions may decide whether bacteria are able to form gold nanoparticles or not.

Full Copper Resistance in Cupriavidus metallidurans Requires the Interplay of Many Resistance Systems.

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. IMPORTANCE The ability of bacteria to maintain homeostasis of the essential-but-toxic "Janus"-faced element copper is important for their survival in many natural environments but also in case of pathogenic bacteria in their respective host. The most important contributors to copper homeostasis have been identified in the last decades and comprise PIB1-type ATPases, periplasmic copper- and oxygen-dependent copper oxidases, transenvelope efflux systems, and glutathione; however, it is not known how all these players interact. This publication investigates this interplay and describes copper homeostasis as a trait emerging from a network of interacting resistance systems.