The bacterium is capable of reducing toxic gold(I/III)-complexes and biomineralizing them into metallic gold (Au) nanoparticles, thereby mediating the (trans)formation of Au nuggets. In Au-rich soils, most transition metals do not interfere with the resistance of this bacterium to toxic mobile Au-complexes and can be removed from the cell by plasmid-encoded metal efflux systems. Copper is a noticeable exception: the presence of Au-complexes and Cu-ions results in synergistic toxicity, which is accompanied by an increased cytoplasmic Cu content and formation of Au nanoparticles in the periplasm. The periplasmic Cu-oxidase CopA was not essential for formation of the periplasmic Au nanoparticles. As shown with the purified and reconstituted Cu efflux system CupA, Au-complexes block Cu-dependent release of phosphate from ATP by CupA, indicating inhibition of Cu transport. Moreover, Cu resistance of Au-inhibited cells was similar to that of mutants carrying deletions in the genes for the Cu-exporting P-type ATPases. Consequently, Au-complexes inhibit export of cytoplasmic Cu-ions, leading to an increased cellular Cu-content and decreased Cu/Au resistance. Uncovering the biochemical mechanisms of synergistic Au/Cu-toxicity in explains the issues this bacterium has to face in auriferous environments, where it is as an important contributor to the environmental Au cycle. lives in metal-rich environments, including auriferous soils that contain a mixture of toxic transition metal cations. We demonstrate here that copper ions and gold complexes exert synergistic toxicity because gold ions inhibit the copper-exporting P-type ATPase CupA, which is central to copper resistance in this bacterium. Such a situation should occur in soils overlying Au deposits, in which Cu:Au ratios usually are >> 1. Appreciating how solves the problem of living in environments that contain both Au and Cu is a pre-requisite to understand the molecular mechanisms underlying gold cycling in the environment, and the significance and opportunities of microbiota for specific targeting to Au in mineral exploration and ore processing.

The bacterium Cupriavidus metallidurans is capable of reducing toxic Au(i/iii)-complexes into metallic gold (Au) nano-particles, thereby mediating the (trans)formation of Au nuggets in Earth surface environments. In this study we describe a novel detoxification pathway, which prevents synergistic copper (Cu)/Au-toxicity. Gold-complexes and Cu-ions exert cooperative toxicity, because cellular uptake of Au(i/iii)-complexes blocks Cu(i) export from the cytoplasm by the Cu-efflux pump CupA. Using a combination of micro-analytical and biochemical methods we show that inducible resistance to these Cu/Au mixtures is mediated by the periplasmic Cu(i)-oxidase CopA, which functions as an oxygen-consuming Au(i)-oxidase. With high Au-complex loads the enzymatic activity of CopA detoxifies the reduction pathway of Au(iii)-complexes via Au(i)-intermediates to Au(0) nanoparticles in the periplasm. Thereby the concentration of highly toxic Au(i) in the cytoplasm is diminished, while allowing direct reduction of Au(iii) to Au nanoparticles in the periplasm. This permits C. metallidurans to thrive in Au-rich environments and biomineralise metallic Au.

Zinc is an essential trace element and at the same time it is toxic at high concentrations. In the beta-proteobacterium the highly efficient removal of surplus zinc from the periplasm is responsible for its outstanding metal resistance. Rather than having a typical Zur-dependent, high-affinity ATP-binding cassette transporter of the ABC protein superfamily for zinc uptake at low concentrations, instead has the secondary zinc importer ZupT of the ZRT/IRT (ZIP) family. It is important to understand, therefore, how this zinc-resistant bacterium copes when it is exposed to low zinc concentrations. Members of the Zur regulon in were identified by comparing the transcriptomes of a Δ mutant and its parent strain. The consensus sequence of the Zur-binding box was derived for the promoter-regulatory region using a truncation assay. The motif was used to predict possible Zur-boxes upstream of Zur regulon members. Binding of Zur to these boxes was confirmed. Two Zur-boxes upstream of the gene, encoding a putative zinc chaperone, proved to be required for complete repression of and its downstream genes in cells cultivated in mineral salts medium. A Zur box upstream of each of and permitted low-expression level of these genes plus their up-regulation under zinc starvation conditions. This demonstrates a compartmentalization of zinc homeostasis in with the periplasm being responsible for removal of surplus zinc and cytoplasmic components for management of zinc as an essential co-factor, with both compartments connected by ZupT. Elucidating zinc homeostasis is necessary to understand both host-pathogen interactions and performance of free-living bacteria in their natural environment. acquires zinc under low zinc concentrations by the Zur-controlled ZnuABC importer of the ABC superfamily, and this was also the paradigm for other bacteria. In contrast, the heavy metal-resistant bacterium achieves high tolerance to zinc due to sophisticated zinc handling and efflux systems operating on periplasmic zinc ions, so that removal of surplus zinc is a periplasmic feature in this bacterium. It is shown here that this process is augmented by management of zinc through cytoplasmic zinc chaperones, whose syntheses are controlled by the Zur regulator. This demonstrates a new mechanism to organize zinc homeostasis through compartmentalization.

The Zur regulon is central to zinc homeostasis in the zinc-resistant bacterium Cupriavidus metallidurans. It comprises the transcription regulator Zur, the zinc importer ZupT, and three members of the COG0523 family of metal-chaperoning G3Etype GTPases, annotated as CobW1, CobW2, and CobW3. The operon structures of the zur and cobW1 loci were determined. To analyze the interplay between the Zur regulon components and metal resistance, deletion mutants were constructed from the wild-type strain CH34 and various other strains. The Zur regulon components interacted with the plasmid-encoded and chromosomally encoded metal resistance factors to acquire metals from complexes of EDTA and for homeostasis of and resistance to zinc, nickel, cobalt, and cadmium. The three G3E-type GTPases were characterized in more detail. CobW1 bound only 1 Zn atom per mol of protein with a stability constant slightly above that of 2-carboxy-2=-hydroxy-5=-sulfoformazylbenzene (Zincon) and an additional 0.5 Zn with low affinity. The CobW1 system was necessary to obtain metals from EDTA complexes. The GTPase CobW2 is a zinc storage compound and bound 0.5 to 1.5 Zn atoms tightly and up to 6 more with lower affinity. The presence of MgGTP unfolded the protein partially. CobW3 had no GTPase activity and equilibrated metal import by ZupT with that of the other metal transport systems. It sequestered 8 Zn atoms per mol with decreasing affinity. The three CobWs bound to the metal-dependent protein FolE IB2 , which is encoded directly downstream of cobW1. This demonstrated an important contribution of the Zur regulon components to metal homeostasis in C. metallidurans. IMPORTANCE Zinc is an important transition metal cation and is present as an essential component in many enzymes, such as RNA polymerase. As with other transition metals, zinc is also toxic at higher concentrations so that living cells have to maintain strict control of their zinc homeostasis. Members of the COG0523 family of metal-chaperoning GE3-type GTPases exist in archaea, bacteria, and eucaryotes, including humans, and they may be involved in delivery of zinc to thousands of different proteins. We used a combination of molecular, physiological, and biochemical methods to demonstrate the important but diverse functions of COG0523 proteins in C. metallidurans, which are produced as part of the Zur-controlled zinc starvation response in this bacterium.

Artificial laboratory evolution was used to produce mutant strains of Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA) able to survive on antimicrobial metallic copper surfaces. These mutants were 12- and 60-fold less susceptible to the copper-mediated contact-killing process than their respective parent strains. Growth of the mutant and its parent in complex growth medium was similar. Tolerance to copper ions of the mutants was unchanged. The mutant phenotype remained stable over about 250 generations under non-stress conditions. The mutants and their respective parental strains accumulated copper released from the metallic surfaces to a similar extent. Nevertheless, only the parental strains succumbed to copper stress when challenged on metallic copper surfaces, suffering complete destruction of the cell structure. Whole genome sequencing and global transcriptome analysis were used to decipher the genetic alterations in the mutant strains; however, these results did not explain the copper-tolerance phenotypes on the systemic level. Instead, the mutants shared features with those of stressed bacterial sub-populations entering the early or “shallow” persister state. In contrast to the canonical persister state, however, the ability to survive on solid copper surfaces was adopted by the majority of the mutant strain population. This indicated that application of solid copper surfaces in hospitals and elsewhere has to be accompanied by strict cleaning regimens to keep the copper surfaces active and prevent evolution of tolerant mutant strains. Significance Microbes are rapidly killed on solid copper surfaces by contact-killing. Copper surfaces thus have an important role to play in preventing the spread of nosocomial infections. Bacteria adapt to challenging natural and clinical environments through evolutionary processes, for instance by acquisition of beneficial spontaneous mutations. We wishes to address the question whether mutants can be selected that have evolved to survive contact-killing on solid copper surfaces. We isolated such mutants from E. coli and S. aureus MRSA by artificial laboratory evolution. The ability to survive on solid copper surfaces was a stable phenotype of the mutant population and not restricted to a small sub-population. As a consequence, standard operation procedures with strict hygienic measures are extremely important to prevent emergence and spread of copper-surface-tolerant persister-like bacterial strains if copper surfaces are to be sustainably used to limit the spread of pathogenic bacteria e.g., to curb nosocomial infections.