Loss of mobile genomic islands in metal resistant, hydrogen-oxidizing Cupriavidus metallidurans

The genome of the metal resistant, hydrogen-oxidizing bacterium Cupriavidus metallidurans strain CH34 contains horizontally acquired plasmids and genomic islands. Metal-resistance determinants on the two plasmids may exert genetic dominance over other related determinants. To investigate whether these recessive determinants can be activated in the absence of the dominant ones, the transcriptome of the highly zinc-sensitive deletion mutant Δe4 (Δ cadA ΔzntA ΔdmeF ΔfieF ) of the plasmid-free parent AE104 was characterized using gene arrays. As a consequence of some unexpected results, close examination by PCR and genomic re-resequencing of strains CH34, AE104, Δe4 and others revealed that the genomic islands CMGIs 2, 3, 4, D, E, but no other islands or recessive determinants, were deleted in some of these strains. Provided CH34 wild type was kept under alternating zinc and nickel selection pressure, no comparable deletions occurred. All current data suggest that genes were actually deleted and were not, as previously surmised, simply absent from the respective strain. As a consequence, a cured database was compiled from the newly generated and previously published gene array data. Analysis of data from this database indicated that some genes of recessive, no longer needed determinants were nevertheless expressed and up-regulated. Their products may interact with those of the dominant determinants to mediate a mosaic phenotype. The ability to contribute to such a mosaic phenotype may prevent deletion of the recessive determinant. The data suggest that the bacterium actively modifies its genome to deal with metal stress and the same time ensures metal homeostasis. Significance In their natural environment, bacteria continually acquire genes by horizontal gene transfer and newly acquired determinants may become dominant over related ones already present in the host genome. When a bacterium is taken into laboratory culture, it is isolated from the horizontal gene transfer network. It can no longer gain genes, but instead may lose them. This was indeed observed in Cupriavidus metallidurans for loss key metal-resistance determinants when no selection pressure was continuously kept. However, some recessive metal-resistance determinants were maintained in the genome. It is proposed that they might contribute some accessory genes to related dominant resistance determinants, for instance periplasmic metal-binding proteins or two-component regulatory systems. Alternatively, they may only remain in the genome because their DNA serves as a scaffold for the nucleoid. Using C. metallidurans as an example, this study sheds light on the fate and function of horizontally acquired genes in bacteria.

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

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.

The multi metal-resistant bacterium Cupriavidus metallidurans CH34 affects growth and metal mobilization in Arabidopsis thaliana plants exposed to copper

Copper (Cu) is important for plant growth, but high concentrations can lead to detrimental effects such as primary root length inhibition, vegetative tissue chlorosis, and even plant death. The interaction between plant-soil microbiota and roots can potentially affect metal mobility and availability, and, therefore, overall plant metal concentration. Cupriavidus metallidurans CH34 is a multi metal-resistant bacterial model that alters metal mobility and bioavailability through ion pumping, metal complexation, and reduction processes. The interactions between strain CH34 and plants may affect the growth, metal uptake, and translocation of Arabidopsis thaliana plants that are exposed to or not exposed to Cu. In this study, we looked also at the specific gene expression changes in C. metallidurans when co-cultured with Cu-exposed A. thaliana. We found that A. thaliana’s rosette area, primary and secondary root growth, and dry weight were affected by strain CH34, and that beneficial or detrimental effects depended on Cu concentration. An increase in some plant growth parameters was observed at copper concentrations lower than 50 µM and significant detrimental effects were found at concentrations higher than 50 µM Cu. We also observed up to a 90% increase and 60% decrease in metal accumulation and mobilization in inoculated A. thaliana. In turn, copper-stressed A. thaliana altered C. metallidurans colonization, and cop genes that encoded copper resistance in strain CH34 were induced by the combination of A. thaliana and Cu. These results reveal the complexity of the plant-bacteria-metal triad and will contribute to our understanding of their applications in plant growth promotion, protection, and phytoremediation strategies.

Behind the shield of Czc: ZntR controls expression of the gene for the zinc-exporting P-type ATPase ZntA in Cupriavidus metallidurans

In the metallophilic beta-proteobacterium Cupriavidus metallidurans, the plasmid-encoded Czc metal homeostasis system adjusts the periplasmic zinc, cobalt and cadmium concentration, which influences subsequent uptake of these metals into the cytoplasm. Behind this shield, the PIB2-type APTase ZntA is responsible for removal of surplus cytoplasmic zinc ions, thereby providing a second level of defense against toxic zinc concentrations. ZntA is the counterpart to the Zur-regulated zinc uptake system ZupT and other import systems; however, the regulator of zntA expression was unknown. The chromid-encoded zntA gene is adjacent to the genes czcI2C2B2’, which are located on the complementary DNA strand and transcribed from a common promoter region. These genes encode homologs of plasmid pMOL30-encoded Czc components. Candidates for possible regulators of zntA were identified and subsequently tested: CzcI, CzcI2, and the MerR-type gene products of the locus tags Rmet_2302, Rmet_0102, Rmet_3456. This led to the identification of Rmet_3456 as ZntR, the main regulator of zntA expression. Moreover, both CzcIs decreased Czc-mediated metal resistance, possibly to avoid “over-excretion” of periplasmic zinc ions, which could result in zinc starvation due to diminished zinc uptake into the cytoplasm. Rmet_2302 was identified as CadR, the regulator of the cadA gene for an important cadmium-exporting PIB2-type ATPase, which provides another system for removal of cytoplasmic zinc and cadmium. Rmet_0102 was not involved in regulation of the metal resistance systems examined here. Thus, ZntR forms a complex regulatory network with CadR, Zur and the CzcIs. Moreover, these discriminating regulatory proteins assign the efflux systems to their particular function. Importance Zinc is an essential metal for numerous organisms from humans to bacteria. The transportome of zinc uptake and efflux systems controls the overall cellular composition and zinc content in a double feed-back loop. Zinc starvation mediates, via the Zur regulator, an up-regulation of the zinc import capacity via the ZIP-type zinc importer ZupT and an amplification of zinc storage capacity, which together raise the cellular zinc content again. On the other hand, an increasing zinc content leads to ZntR-mediated up-regulation of the zinc efflux system ZntA, which decreases the zinc content. Together, the Zur regulon components and ZntR/ZntA balance the cellular zinc content under both high external zinc concentrations and zinc starvation conditions.

Adaptation of Cupriavidus metallidurans CH34 to Toxic Zinc Concentrations Involves an Uncharacterized ABC-Type Transporter

Cupriavidus metallidurans CH34 is a well-studied metal-resistant β-proteobacterium and contains a battery of genes participating in metal metabolism and resistance. Here, we generated a mutant (CH34ZnR) adapted to high zinc concentrations in order to study how CH34 could adaptively further increase its resistance against this metal. Characterization of CH34ZnR revealed that it was also more resistant to cadmium, and that it incurred seven insertion sequence-mediated mutations. Among these, an IS1088 disruption of the glpR gene (encoding a DeoR-type transcriptional repressor) resulted in the constitutive expression of the neighboring ATP-binding cassette (ABC)-type transporter. GlpR and the adjacent ABC transporter are highly similar to the glycerol operon regulator and ATP-driven glycerol importer of Rhizobium leguminosarum bv. viciae VF39, respectively. Deletion of glpR or the ABC transporter and complementation of CH34ZnR with the parental glpR gene further demonstrated that loss of GlpR function and concomitant derepression of the adjacent ABC transporter is pivotal for the observed resistance phenotype. Importantly, addition of glycerol, presumably by glycerol-mediated attenuation of GlpR activity, also promoted increased zinc and cadmium resistance in the parental CH34 strain. Upregulation of this ABC-type transporter is therefore proposed as a new adaptation route towards metal resistance.

Bioremediation by Cupriavidus metallidurans Strain MSR33 of Mercury-Polluted Agricultural Soil in a Rotary Drum Bioreactor and Its Effects on Nitrogen Cycle Microorganisms

Nitrogen cycle microorganisms are essential in agricultural soils and may be affected by mercury pollution. The aims of this study are to evaluate the bioremediation of mercury-polluted agricultural soil using Cupriavidus metallidurans MSR33 in a rotary drum bioreactor (RDB) and to characterize the effects of mercury pollution and bioremediation on nitrogen cycle microorganisms. An agricultural soil was contaminated with mercury (II) (20–30 ppm) and subjected to bioremediation using strain MSR33 in a custom-made RDB. The effects of mercury and bioremediation on nitrogen cycle microorganisms were studied by qPCR. Bioremediation in the RDB removed 82% mercury. MSR33 cell concentrations, thioglycolate, and mercury concentrations influence mercury removal. Mercury pollution strongly decreased nitrogen-fixing and nitrifying bacterial communities in agricultural soils. Notably, after soil bioremediation process nitrogen-fixing and nitrifying bacteria significantly increased. Diverse mercury-tolerant strains were isolated from the bioremediated soil. The isolates Glutamicibacter sp. SB1a, Brevundimonas sp. SB3b, and Ochrobactrum sp. SB4b possessed the merG gene associated with the plasmid pTP6, suggesting the horizontal transfer of this plasmid to native gram-positive and gram-negative bacteria. Bioremediation by strain MSR33 in an RDB is an attractive and innovative technology for the clean-up of mercury-polluted agricultural soils and the recovery of nitrogen cycle microbial communities.

Cupriavidus metallidurans CH34, a historical perspective on its discovery, characterization and metal resistance

Cupriavidus metallidurans, and in particular type strain CH34, became a model bacterium to study bacterial resistance to metals. Although nowadays the routine use of a wide variety of omics and molecular techniques allow refining, deepening and expanding our knowledge on adaptation and resistance to metals, these were not available at the onset of C. metallidurans research starting from its isolation in 1976. This minireview describes the early research and legacy tools used to study its metal resistance determinants, characteristic megaplasmids, ecological niches and environmental applications.

Space station biomining experiment demonstrates rare earth element extraction in microgravity and Mars gravity

Microorganisms are employed to mine economically important elements from rocks, including the rare earth elements (REEs), used in electronic industries and alloy production. We carried out a mining experiment on the International Space Station to test hypotheses on the bioleaching of REEs from basaltic rock in microgravity and simulated Mars and Earth gravities using three microorganisms and a purposely designed biomining reactor. Sphingomonas desiccabilis enhanced mean leached concentrations of REEs compared to non-biological controls in all gravity conditions. No significant difference in final yields was observed between gravity conditions, showing the efficacy of the process under different gravity regimens. Bacillus subtilis exhibited a reduction in bioleaching efficacy and Cupriavidus metallidurans showed no difference compared to non-biological controls, showing the microbial specificity of the process, as on Earth. These data demonstrate the potential for space biomining and the principles of a reactor to advance human industry and mining beyond Earth.