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Phenotypic and Genetic Characterization of Temperature-Induced Mutagenesis and Mortality in Cupriavidus metallidurans.

ABSTRACT: Cupriavidus metallidurans strains display a decreased viability when incubated in rich medium at a temperature of 37°C compared to their normal growth temperature of 30°C, a phenomenon coined "temperature-induced mortality and mutagenesis" (TIMM). To scrutinize this aberrant phenotype further, the contributions of specific inducers and protective agents were determined. Different growth media, including lysogeny broth (LB) and Schatz, and components, including casamino acids, in particular amino acids (proline, cysteine, glycine, glutamine, leucine, histidine and phenylalanine) and ammonium, were found to induce TIMM at 37°C. Sorbitol was found to counteract TIMM. Furthermore, although TIMM is well conserved within the C. metallidurans species, multiple and strain-specific TIMM inducers exist. Twenty-nine percent of the TIMM survivors inherited resistance to TIMM. Whole-genome sequencing of two resistant derivatives revealed an important role of an uncharacterized oxidoreductase, indicating putative metabolic poisoning when grown in high-concentration nitrogen-containing media at 37°C.

SUBMITTER: Van Houdt R

PROVIDER: S-EPMC8299993 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Central to the ability of Cupriavidus metallidurans to maintain its metal homoeostasis is the metal transportome, composed of uptake and efflux systems. Seven secondary metal import systems, ZupT, PitA, CorA1, CorA2, CorA3, ZntB, and HoxN, interact and are at the core of the metal uptake transportome. The 7-fold deletion mutant ?7 (?zupT ?pitA ?corA1?corA2?corA3?zntB ?hoxN) of parent strain AE104 is still able to maintain its cellular metal content, although at the cost of reduced fitness (M. Herzberg, L. Bauer, A. Kirsten, and D. H. Nies, Metallomics, in press, http://dx.doi.org/10.1039/C5MT00295H). Strain ?7 does not express genes for backup importers, and so ?7 should use metal uptake systems also produced in the AE104 parent cells. These systems should be activated in ?7 by posttranscriptional regulatory processes. The decreased fitness of ?7 correlated with a zinc-dependent downregulation of the overall metabolic backbone of the cells even at nontoxic external zinc concentrations. Responsible for this decreased fitness of ?7 was a negative interference of the activity of two P-type ATPases, MgtA and MgtB, which, on the other hand, kept ?7 at a fitness level higher than that of the ?9 (?7 ?mgtA::kan ?mgtB) mutant strain. This revealed a complicated interplay of the metal uptake transportome of C. metallidurans, which is composed of the seven secondary uptake systems, MgtA, MgtB, and yet-unknown components, with cytoplasmic transition metal pools and posttranscriptional regulatory processes. IMPORTANCE Bacteria, including pathogenic strains, need to make use of the metal composition and speciation of their environment to fulfill the requirement of the cytoplasmic metal content and composition. This task is performed by the bacterial metal transportome, composed of uptake and efflux systems. Seven interacting secondary metal uptake systems are at the core of the metal transportome in C. metallidurans. This publication verifies that posttranscriptional events are responsible for activation of even more, yet-unknown, metal import systems in the 7-fold deletion mutant ?7. Two P-type ATPases were identified as new members of the metal uptake transportome. This publication demonstrates the complexity of the metal transportome and the regulatory processes involved.

Project description:The bacterium Cupriavidus metallidurans can reduce toxic gold(I/III) complexes and biomineralize 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 PIB1-type ATPases. Consequently, Au complexes inhibit export of cytoplasmic Cu ions, leading to an increased cellular Cu content and decreased Cu and Au resistance. Uncovering the biochemical mechanisms of synergistic Au and Cu toxicity in C. metallidurans explains the issues this bacterium has to face in auriferous environments, where it is an important contributor to the environmental Au cycle.IMPORTANCE C. metallidurans 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 C. metallidurans solves the problem of living in environments that contain both Au and Cu is a prerequisite 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.

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Phenotypic and Genetic Characterization of Temperature-Induced Mutagenesis and Mortality in Cupriavidus metallidurans.

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