<Emphasis Type="Italic">Cupriavidus metallidurans</Emphasis>: evolution of a metal-resistant bacterium

Cupriavidus metallidurans CH34 has gained increasing interest as a model organism for heavy metal detoxification and for biotechnological purposes. Resistance of this bacterium to transition metal cations is predominantly based on metal resistance determinants that contain genes for RND (resistance, nodulation, and cell division protein family) proteins. These are part of transenvelope protein complexes, which seem to detoxify the periplasm by export of toxic metal cations from the periplasm to the outside. Strain CH34 contains 12 predicted RND proteins belonging to a protein family of heavy metal exporters. Together with many efflux systems that detoxify the cytoplasm, regulators and possible metal-binding proteins, RND proteins mediate an efficient defense against transition metal cations. To shed some light into the origin of genes encoding these proteins, the genomes of C. metallidurans CH34 and six related proteobacteria were investigated for occurrence of orthologous and paralogous proteins involved in metal resistance. Strain CH34 was not much different from the other six bacteria when the total content of transport proteins was compared but CH34 had significantly more putative transition metal transport systems than the other bacteria. The genes for these systems are located on its chromosome 2 but especially on plasmids pMOL28 and pMOL30. Cobalt–nickel and chromate resistance determinants located on plasmid pMOL28 evolved by gene duplication and horizontal gene transfer events, leading to a better adaptation of strain CH34 to serpentine-like soils. The czc cobalt–zinc–cadmium resistance determinant, located on plasmid pMOL30 in addition copper, lead and mercury resistance determinants, arose by duplication of a czcICAB core determinant on chromosome 2, plus addition of the czcN gene upstream and the genes czcD, czcRS, czcE downstream of czcICBA. C. metallidurans apparently evolved metal resistance by horizontal acquisition and by duplication of genes for transition metal efflux, mostly on the two plasmids, and decreased the number of uptake systems for those metals. This paper is dedicated to Dr. Max Mergeay for a long time of cooperation, constructive competition and friendship.     

CPU-GPU hybrid accelerating the Zuker algorithm for RNA secondary structure prediction applications

Background

Different Cupriavidus metallidurans strains isolated from metal-contaminated and other anthropogenic environments were genotypically and phenotypically compared with C. metallidurans type strain CH34. The latter is well-studied for its resistance to a wide range of metals, which is carried for a substantial part by its two megaplasmids pMOL28 and pMOL30.

Results

Comparative genomic hybridization (CGH) indicated that the extensive arsenal of determinants involved in metal resistance was well conserved among the different C. metallidurans strains. Contrary, the mobile genetic elements identified in type strain CH34 were not present in all strains but clearly showed a pattern, although, not directly related to a particular biotope nor location (geographical). One group of strains carried almost all mobile genetic elements, while these were much less abundant in the second group. This occurrence was also reflected in their ability to degrade toluene and grow autotrophically on hydrogen gas and carbon dioxide, which are two traits linked to separate genomic islands of the Tn4371-family. In addition, the clear pattern of genomic islands distribution allowed to identify new putative genomic islands on chromosome 1 and 2 of C. metallidurans CH34.

Conclusions

Metal resistance determinants are shared by all C. metallidurans strains and their occurrence is apparently irrespective of the strain's isolation type and place. Cupriavidus metallidurans strains do display substantial differences in the diversity and size of their mobile gene pool, which may be extensive in some (including the type strain) while marginal in others.     

New mobile genetic elements in <Emphasis Type="Italic">Cupriavidus metallidurans</Emphasis> CH34, their possible roles and occurrence in other bacteria

Cupriavidus metallidurans strain CH34 is a β-Proteobacterium that thrives in low concentrations of heavy metals. The genetic determinants of resistance to heavy metals are located on its two chromosomes, and are particularly abundant in the two megaplasmids, pMOL28 and pMOL30. We explored the involvement of mobile genetic elements in acquiring these and others traits that might be advantageous in this strain using genome comparison of Cupriavidus/Ralstonia strains and related β-Proteobacteria. At least eleven genomic islands were identified on the main replicon, three on pMOL28 and two on pMOL30. Multiple islands contained genes for heavy metal resistance or other genetic determinants putatively responding to harsh environmental conditions. However, cryptic elements also were noted. New mobile genetic elements (or variations of known ones) were identified through synteny analysis, allowing the detection of mobile genetic elements outside the bias of a selectable marker. Tn4371-like conjugative transposons involved in chemolithotrophy and degradation of aromatic compounds were identified in strain CH34, while similar elements involved in heavy metal resistance were found in Delftia acidovorans SPH-1 and Bordetella petrii DSM12804. We defined new transposons, viz., Tn6048 putatively involved in the response to heavy metals and Tn6050 carrying accessory genes not classically associated with transposons. Syntenic analysis also revealed new transposons carrying metal response genes in Burkholderia xenovorans LB400, and other bacteria. Finally, other putative mobile elements, which were previously unnoticed but apparently common in several bacteria, were also revealed. This was the case for triads of tyrosine-based site-specific recombinases and for an int gene paired with a putative repressor and associated with chromate resistance.     

The <Emphasis Type="Italic">cnr</Emphasis>-like operon in strain <Emphasis Type="Italic">Comamonas</Emphasis> sp. encoding resistance to cobalt and nickel

Plasmid pBS501 was detected in the strain Comamonas sp. BS501. This plasmid specifies high level of induced resistance (5 mM) to cobalt/nickel both in the host strain and in related strains C. testosteroni B-1241 and C. acidovorans B-1251. Hybridization analysis revealed a homology of pBS501 restriction fragments with the only well-characterized operon cnrXYHCBAT that resides in plasmid pMOL28 from Cupriavidus metallidurans CH34. Essential differences in the structural organization of the cobalt/nickel resistance determinant were found between plasmid pBS501 and the cnr operon.     

The response of <Emphasis Type="Italic">Cupriavidus metallidurans</Emphasis> CH34 to spaceflight in the international space station

The survival and behavior of Cupriavidus metallidurans strain CH34 were tested in space. In three spaceflight experiments, during three separate visits to the ‘International Space Station’ (ISS), strain CH34 was grown for 10–12 days at ambient temperature on mineral agar medium. Space- and earth-grown cells were compared post-flight by flow cytometry and using 2D-gel protein analysis. Pre-, in- and post-flight incubation conditions and experiment design had a significant impact on the survival and growth of CH34 in space. In the CH34 cells returning from spaceflight, 16 proteins were identified which were present in higher concentration in cells developed in spaceflight conditions. These proteins were involved in a specific response of CH34 to carbon limitation and oxidative stress, and included an acetone carboxylase subunit, fructose biphosphate aldolase, a DNA protection during starvation protein, chaperone protein, universal stress protein, and alkyl hydroperoxide reductase. The reproducible observation of the over-expression of these same proteins in multiple flight experiments, indicated that the CH34 cells could experience a substrate limitation and oxidative stress in spaceflight where cells and substrates are exposed to lower levels of gravity and higher doses of ionizing radiation. Bacterium C. metallidurans CH34 was able to grow normally under spaceflight conditions with very minor to no effects on cell physiology, but nevertheless specifically altered the expression of a few proteins in response to the environmental changes.     

The ABC-transporter AtmA is involved in nickel and cobalt resistance of <Emphasis Type="Italic">Cupriavidus metallidurans</Emphasis> strain CH34

Cupriavidus metallidurans CH34 genome contains an ortholog of Atm1p named AtmA (Rmet_0391, YP_582546). In Saccharomyces cerevisiae, the ABC-type transport system Atm1p is involved in export of iron–sulfur clusters from mitochondria into the cytoplasm for assembly of cytoplasmic iron–sulfur containing proteins. An ∆atmA mutant of C. metallidurans was sensitive to nickel and cobalt but not iron cations. AtmA increased also resistance to these cations in Escherichia coli strains that carry deletions of the genes for other nickel and cobalt transport systems. In C. metallidurans, atmA expression was not significantly induced by nickel and cobalt, but repressed by zinc. AtmA was purified as a 70 kDa protein after expression in E. coli. ATPase activity of AtmA was stimulated by nickel and cobalt.     

Molecular characterization influencing metal resistance in the Cupriavidus/Ralstonia genomes

Our environment is stressed with a load of heavy and toxic metals. Microbes, abundant in our environment, are found to adapt well to this metal-stressed condition. A comparative study among five Cupriavidus/Ralstonia genomes can offer a better perception of their evolutionary mechanisms to adapt to these conditions. We have studied codon usage among 1051 genes common to all these organisms and identified 15 optimal codons frequently used in highly expressed genes present within 1051 genes. We found the core genes of Cupriavidus metallidurans CH34 have a different optimal codon choice for arginine, glycine and alanine in comparison with the other four bacteria. We also found that the synonymous codon usage bias within these 1051 core genes is highly correlated with their gene expression. This supports that translational selection drives synonymous codon usage in the core genes of these genomes. Synonymous codon usage is highly conserved in the core genes of these five genomes. The only exception among them is C. metallidurans CH34. This genomewide shift in synonymous codon choice in C. metallidurans CH34 may have taken place due to the insertion of new genes in its genomes facilitating them to survive in heavy metal containing environment and the co-evolution of the other genes in its genome to achieve a balance in gene expression. Structural studies indicated the presence of a longer N-terminal region containing a copper-binding domain in the cupC proteins of C. metallidurans CH3 that helps it to attain higher binding efficacy with copper in comparison with its orthologs.     

Chromosome mapping in Alealigenes eutrophus CH34

Mutants and mobilizing plasmids were developed as genetic tools in Alcaligenes eutrophus CH34. In order to map the chromosome, spontaneous and ethyl methane sulphonate (EMS)-induced mutants (mostly auxotrophs) were isolated. Another source of mutants was provided by the phenomenon of temperature-induced mortality and mutagenesis that is observed at 37° C and is characteristic of many metallotolerant strains of A. eutrophus. Plasmid pULB113 (RP4::miniMu) was used to map the available mutations. Twenty-five loci were ordered in a circular map. pMOL50, a rearranged derivative of plasmid pMOL28, which was obtained in a survivor at 37° C and displayed chromosome mobilizing activity (Cma+), was also used to mobilize chromosomal markers: resulting linkages were stronger than with pULB113, allowing confirmation of the circularity of the A. eutrophus CH34 chromosome with a small number of crosses.     

From industrial sites to environmental applications with <Emphasis Type="Italic">Cupriavidus metallidurans</Emphasis>

Cupriavidus metallidurans CH34 and related strains are adapted to metal contaminated environments. A strong resistance to environmental stressors and adaptation make it ideal strains for survival in decreasing biodiversity conditions and for bioaugmentation purposes in environmental applications. The soil bacterium C. metallidurans is able to grow chemolithoautotrophically on hydrogen and carbon dioxide allowing a strong resilience under conditions lacking organic matter. The biofilm growth on soil particles allows coping with starvation or bad conditions of pH, temperature and pollutants. Its genomic capacity of two megaplasmids encoding several heavy metal resistance operons allowed growth in heavy metal contaminated habitats. In addition its specific siderophores seem to play a role in heavy metal sequestration besides their role in the management of bioavailable iron. Efflux ATPases and RND systems pump the metal cations to the membrane surface where polysaccharides serve as heavy metal binding and nucleation sites for crystallisation of metal carbonates. These polysaccharides contribute also to flotation under specific conditions in a soil-heavy metals–bacteria suspension mixture. An inoculated moving bed sand filter was constructed to treat heavy metal contaminated water and to remove the metals in the form of biomass mixed with metal carbonates. A membrane based contactor allowed to use the bacteria as well in a versatile wastewater treatment system and to grow homogeneously formed heavy metal carbonates. Its behaviour toward heavy metal binding and flotation was combined in a biometal sludge reactor to extract and separate heavy metals from metal contaminated soils. Finally its metal-induced heavy metal resistance allowed constructing whole cell heavy metal biosensors which, after contact with contaminated soil, waste, solids, minerals and ashes, were induced in function of the bioavailable concentration (Cd, Zn, Cu, Cr, Co, Ni, Tl, Pb and Hg) in the solids and allowed to investigate the speciation of immobilization of those metals. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

X-ray structure of the metal-sensor CnrX in both the apo- and copper-bound forms

Both the X-ray structures of the apo- and the copper-bound forms of the metal-sensor domain (residues 31-148) of CnrX from Cupriavidus metallidurans CH34 were obtained at 1.74 Å resolution from a selenomethionine derivative. This four-helix hooked-hairpin is the first structure of a metal-sensor in an ECF-type signaling pathway. The copper ion is bound in a type 2-like center with a 3N1O coordination in the equatorial plane and shows an unprecedented remote fifth axial ligand with Met93 contributing a weak S-Cu bond. The signal onset cannot be explained by conformational changes associated with CnrX metallation.     

Specific PCR to identify the heavy-metal-resistant bacterium <Emphasis Type="Italic">Cupriavidus metallidurans</Emphasis>

The aim of this study is to develop a polymerase chain reaction (PCR) assay for rapid detection of Cupriavidus metallidurans. PCR primers targeting the Signal transduction histidine kinase gene were designed and designated Cm-F1/Cm-R1. Strains of C. metallidurans were positively identified. The size of the PCR products was 437 bp, as expected. This PCR method enables monitoring of industrial, environmental and clinical sources for presence of C. metallidurans.     

Characterization of the metabolically modified heavy metal-resistant Cupriavidus metallidurans strain MSR33 generated for mercury bioremediation

Background

Mercury-polluted environments are often contaminated with other heavy metals. Therefore, bacteria with resistance to several heavy metals may be useful for bioremediation. Cupriavidus metallidurans CH34 is a model heavy metal-resistant bacterium, but possesses a low resistance to mercury compounds.

Methodology/Principal Findings

To improve inorganic and organic mercury resistance of strain CH34, the IncP-1β plasmid pTP6 that provides novel merB, merG genes and additional other mer genes was introduced into the bacterium by biparental mating. The transconjugant Cupriavidus metallidurans strain MSR33 was genetically and biochemically characterized. Strain MSR33 maintained stably the plasmid pTP6 over 70 generations under non-selective conditions. The organomercurial lyase protein MerB and the mercuric reductase MerA of strain MSR33 were synthesized in presence of Hg²⁺. The minimum inhibitory concentrations (mM) for strain MSR33 were: Hg²⁺, 0.12 and CH₃Hg⁺, 0.08. The addition of Hg²⁺ (0.04 mM) at exponential phase had not an effect on the growth rate of strain MSR33. In contrast, after Hg²⁺ addition at exponential phase the parental strain CH34 showed an immediate cessation of cell growth. During exposure to Hg²⁺ no effects in the morphology of MSR33 cells were observed, whereas CH34 cells exposed to Hg²⁺ showed a fuzzy outer membrane. Bioremediation with strain MSR33 of two mercury-contaminated aqueous solutions was evaluated. Hg²⁺ (0.10 and 0.15 mM) was completely volatilized by strain MSR33 from the polluted waters in presence of thioglycolate (5 mM) after 2 h.

Conclusions/Significance

A broad-spectrum mercury-resistant strain MSR33 was generated by incorporation of plasmid pTP6 that was directly isolated from the environment into C. metallidurans CH34. Strain MSR33 is capable to remove mercury from polluted waters. This is the first study to use an IncP-1β plasmid directly isolated from the environment, to generate a novel and stable bacterial strain useful for mercury bioremediation.     

CzcE from Cupriavidus metallidurans CH34 is a copper-binding protein

CzcE is encoded by the most distal gene of the czc determinant that allows Cupriavidus metallidurans CH34 to modulate its internal concentrations of cobalt, zinc and cadmium by regulation of the expression of the efflux pump CzcCBA. We have overproduced and purified CzcE. CzcE is a periplasm-located dimeric protein able to bind specifically 4 Cu-equivalent per dimer. Spectrophotometry and EPR are indicative of type II copper with typical d-d transitions. Re-oxidation of fully reduced CzcE led to the formation of an air stable semi-reduced form binding both 2 Cu(I) and 2 Cu(II) ions. The spectroscopic characteristics of the semi-reduced form are different of those of the oxidized one, suggesting a change in the environment of Cu(II).     

Mobilization of Selenite by Ralstonia metallidurans CH34

Ralstonia metallidurans CH34 (formerly Alcaligenes eutrophus CH34) is a soil bacterium characteristic of metal-contaminated biotopes, as it is able to grow in the presence of a variety of heavy metals. R. metallidurans CH34 is reported now to resist up to 6 mM selenite and to reduce selenite to elemental red selenium as shown by extended X-ray absorption fine-structure analysis. Growth kinetics analysis suggests an adaptation of the cells to the selenite stress during the lag-phase period. Depending on the culture conditions, the medium can be completely depleted of selenite. Selenium accumulates essentially in the cytoplasm as judged from electron microscopy and energy-dispersive X-ray analysis. Elemental selenium, highly insoluble, represents a nontoxic storage form for the bacterium. The ability of R. metallidurans CH34 to reduce large amounts of selenite may be of interest for bioremediation processes targeting selenite-polluted sites.