Cupriavidus Metallidurans CH34 Research Articles

Size-, Composition- and Shape-Dependent Toxicological Impact of Metal Oxide Nanoparticles and Carbon Nanotubes toward Bacteria

Ecotoxicological effects of nanoparticles (NP) are still poorly documented while their commercialization for industrial and household applications increases. The aim of this study was to evaluate the influence of physicochemical characteristics on metal oxide NP and carbon nanotubes toxicological effects toward bacteria. Two strains of bacteria, Cupriavidus metallidurans CH34 and Escherichia coli MG1655 were exposed to TiO(2) or Al(2)O(3) NP or to multiwalled-carbon nanotubes (MWCNT). Particular attention was paid on optimizing NP dispersion to obtain nonagglomerated suspensions. Our results show that NP toxicity depends on their chemical composition, size, surface charge, and shape but not on their crystalline phase. MWCNT toxicity does not depend on their purity. Toxicity also depends on the bacterial strain: E. coli MG1655 is sensitive to NP, whereas C. metallidurans CH34 is not. Interestingly, NP are accumulated in both bacterial strains, and association between NP and bacteria is necessary for bacterial death to occur. NP may then represent a danger for the environment, causing the disappearance of some sensitive bacterial strains such as E. coli MG1655, but also being mobilized by nonsensitive strains such as C. metallidurans CH34 and transported through the whole ecosystem.

CzcP is a novel efflux system contributing to transition metal resistance inCupriavidus metalliduransCH34

Cupriavidus metallidurans CH34 possesses a multitude of metal efflux systems. Here, the function of the novel P(IB4)-type ATPase CzcP is characterized, which belongs to the plasmid pMOL30-mediated cobalt-zinc-cadmium (Czc) resistance system. Contribution of CzcP to transition metal resistance in C. metallidurans was compared with that of three P(IB2)-type ATPases (CadA, ZntA, PrbA) and to other efflux proteins by construction and characterization of multiple deletion mutants. These data also yielded additional evidence for an export of metal cations from the periplasm to the outside of the cell rather than from the cytoplasm to the outside. Moreover, metal-sensitive Escherichia coli strains were functionally substituted in trans with CzcP and the three P(IB2)-type ATPases. Metal transport kinetics performed with inside-out vesicles identified the main substrates for these four exporters, the K(m) values and apparent turn-over numbers. In combination with the mutant data, transport kinetics indicated that CzcP functions as 'resistance enhancer': this P(IB4)-type ATPase exports transition metals Zn(2+), Cd(2+) and Co(2+) much more rapidly than the three P(IB2)-type proteins. However, a basic resistance level has to be provided by the P(IB2)-type efflux pumps because CzcP may not be able to reach all different speciations of these metals in the cytoplasm.

From industrial sites to environmental applications with Cupriavidus metallidurans

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.

Assessment of uranium and selenium speciation in human and bacterial biological models to probe changes in their structural environment

Abstract This study illustrates the potential of physicochemical techniques to speciate uranium (U) and selenium (Se) in biological samples. Speciation, defined here as the study of structural environment, of both toxic elements, was characterized at several levels in biological media and directly in human cells or bacteria once the metal(loid)s were internalized. External speciation that is extracellular speciation in culture media was predicted by thermodynamic equilibrium computer modelling using the JChess software and validated by spectroscopic measurements (XANES and EXAFS). Internal speciation that is intracellular speciation in eukaryotic and prokaryotic cells was studied in vitro with a soil bacterium Cupriavidus metallidurans CH34 and ROS 17/2.8 osteoblasts, human cells responsible for bone formation. XANES, EXAFS, HPLC-ICP-MS and SDS-PAGE coupled to particle induced X-ray emission (PIXE) permitted the identification and quantification of complexes formed with organic or inorganic molecules and/or larger proteins.

Introduction to a special Festschrift issue celebrating the microbiology of Cupriavidus metallidurans strain CH34

Although this series of ten reports focuses on themicrobiology of a single bacterium namely the beta-proteobacterium Cupriavidus metallidurans CH34,this volume is of general interest for several reasons.Firstly, this unusual multiple toxic metal ion-resistantbacterium has been a model for how microbes handlesuch toxic metal (loids) from the time of its firstisolation (Mergeay et al. 1978, 1985). Moreover, itcontinues to be a useful model, as seen in this series ofreportsaswellasinanysearchoftheliterature,usedforan expanding range of interests from metal responsephysiology (Nies et al. 1990, 1989; Nies 2000; Rouxet al. 2001; Sendra et al. 2006) and basic moleculargenetics (Lejeune et al. 1983), to genomics (theorganism is unusual in having two chromosomes plustwo large plasmids; Mergeay et al. 2003), microbialecology (Vander Auwera, this series), to applicationsinenvironmentalbiotechnology(Diels,thisseries)andeven in space research (Leys, this series).As an introductory aside, but a necessary explana-tion here, strain CH34 is complicated in that it has hadat least five official names, starting as a Pseudomonasstrain (Mergeay et al. 1978), then Alcaligeneseutrophus (Lejeune et al. 1983), then Ralstonia eutro-pha, then Ralstonia sp., then Ralstonia metallidurans,to Wautersia metallidurans and finally (we hope)Cupriavidusmetallidurans,including twochangesinasingle year (Vandamme and Coenye 2004), as thetaxonomy of related bacteria evolved. Interestedreaders (if one wants useful and comprehensiveliterature) are advised to also search by ‘‘CH34’’ andnot only by genus and species names. The letters CHrefer to Christian Houba, at that time (in 1976), astudent from the Universite´ of Lie`ge, who first isolatedmetal-resistant bacteria in a region in Belgium wellknown for its metallurgical industry historical past aswell as for metal-accumulating plants.The current reports are from a small FestschriftSymposium on work with strain CH34 that was heldat the SCK CEN (the Belgian Nuclear ResearchCentre of Mol) in April 2008. The science reported,included herein and much more that did not result inmanuscripts, was a wonderful endorsement of theimportance of this fascinating microbe, the benefits ofa research program continuing over decades, and thefellowship and collegiality of the small internationalgroup of CH34 workers. We hope this continues.References

Amine- and Carboxyl- Quantum Dots Affect Membrane Integrity of Bacterium Cupriavidus metallidurans CH34

The present study examines the interaction of amine- and carboxyl- PEG core/shell quantum dots (QDs) with metal resistant bacterium Cupriavidus metallidurans CH34. The evolution of the number of QDs, their hydrodynamic radius, diffusion coefficients, and single particle fluorescence were characterized before and during the contact with bacterium by fluorescence correlation spectroscopy (FCS). The obtained results showed that at nanomolar concentrations the amine- and carboxyl-PEG-QDs with average hydrodynamic radiuses of 16.4 and 13.5 nm, form stable dispersions in the absence and presence of 15 mgC L(-1) HA. The decrease of the number of fluorescent particles in the bacterial medium, determined by FCS, together with the increase of the fluorescence of bacterial cells over the background, found by flow cytometry (FCM), demonstrated the association of QDs to C. metallidurans. Furthermore, QDs enhanced the level of the reactive oxygen species in the bacterial cells and augmented the percentage of the cells with damaged and leaky membranes as probed by FCM in combination with 5-(and-6)-carboxy-27'-dichlorodihydrofluorescein diacetate and propidium iodide stains. No difference in the behavior of amine- and carboxyl-PEG-QDs was found, suggesting that different functional groups in the surface coating have no effect on bacterium-QD interactions under the studied conditions. The presence of HA does not affect the hydrodynamic characteristics of the functionalized QDs, but prevented the damage to the bacterial membrane. The slight decrease in the bacterial growth found after exposure of C. metallidurans to these QDs was attributed to the nanoparticles themselves rather the cadmium, zinc, or selenium ions released from the QDs.

New mobile genetic elements in Cupriavidus metallidurans CH34, their possible roles and occurrence in other bacteria

Cupriavidus metallidurans strain CH34 is a beta-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 beta-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.

ArsR arsenic-resistance regulatory protein from Cupriavidus metallidurans CH34

The Cupriavidus metallidurans CH34 arsR gene, which is part of the arsRIC(2)BC(1)HP operon, and its putative arsenic-resistance regulatory protein were identified and characterized. The arsenic-induced transcriptome of C. metallidurans CH34 showed that the genes most upregulated in the presence of arsenate were all located within the ars operon, with none of the other numerous heavy metal resistance systems present in CH34 being induced. A transcriptional fusion between the luxCDABE operon and the arsR promoter/operator (P/O) region was used to confirm the in vivo induction of the ars operon by arsenite and arsenate. The arsR gene was cloned into expression vectors allowing for the overexpression of the ArsR protein as either his-tagged or untagged protein. The ability of the purified ArsR proteins to bind to the ars P/O region was analyzed in vitro by gel mobility shift assays. ArsR showed an affinity almost exclusively to its own ars P/O region. Dissociation of ArsR and its P/O region was metal dependent, and based on decreasing degrees of dissociation three groups of heavy metals could be distinguished: As(III), Bi(III), Co(II), Cu(II), Ni(II); Cd(II); Pb(II) and Zn(II), while no dissociation was observed in the presence of As(V).

Unprecedented Binding Cooperativity between CuI and CuII in the Copper Resistance Protein CopK from Cupriavidus metallidurans CH34: Implications from Structural Studies by NMR Spectroscopy and X-Ray Crystallography

The bacterium Cupriavidus metallidurans CH34 is resistant to high environmental concentrations of many metal ions, including copper. This ability arises primarily from the presence of a large plasmid pMOL30 which includes a cluster of 19 cop genes that respond to copper. One of the protein products CopK is induced at high levels and is expressed to the periplasm as a small soluble protein (8.3 kDa). Apo-CopK associates in solution to form a dimer (K(D) approximately 10(-5) M) whose structure was defined by NMR and X-ray crystallography. The individual molecules feature two antiparallel beta-sheets arranged in a sandwich-like structure and interact through C-terminal beta-strands. It binds Cu(II) with low affinity (K(D)(Cu(II)) > 10(-6) M) but Cu(I) with high affinity (K(D)(Cu(I)) = 2 x 10(-11) M). Cu(I)-CopK was also a dimer in the solid state and featured a distorted tetrahedral site Cu(I)(S-Met)(3)(NCS). The isothiocyanato ligand originated from the crystallization solution. Binding of Cu(I) or Ag(I), but not of Cu(II), favored the monomeric form in solution. While Ag(I)-CopK was stable as isolated, Cu(I)-CopK was moderately air-sensitive due to a strong binding cooperativity between Cu(I) and Cu(II). This was documented by determination of the Cu(I) and Cu(II) binding affinities in the presence of the other ion: K(D)(Cu(I)) = 2 x 10(-13) M and K(D)(Cu(II)) = 3 x 10(-12) M, that is, binding of Cu(II) increased the affinity for Cu(I) by a factor of approximately 10(2) and binding of Cu(I) increased the affinity for Cu(II) by a factor of at least 10(6). Stable forms of both Cu(I)Cu(II)-CopK and Ag(I)Cu(II)-CopK were isolated readily. Consistent with this unprecedented copper binding chemistry, NMR spectroscopy detected three distinct forms: apo-CopK, Cu(I)-CopK and Cu(I)Cu(II)-CopK that do not exchange on the NMR time scale. This information provides a valuable guide to the role of CopK in copper resistance.

The ABC-transporter AtmA is involved in nickel and cobalt resistance of Cupriavidus metallidurans 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.

Lead(II) resistance in Cupriavidus metallidurans CH34: interplay between plasmid and chromosomally-located functions

Proteome and transcriptome analysis, combined with mutagenesis, were used to better understand the response of Cupriavidus metallidurans CH34 against lead(II). Structural Pb(II)-resistance genes of the pMOL30-encoded pbrUTRABCD operon formed the major line of defense against Pb(II). However, several general stress response mechanisms under the control of alternative sigma factors such as sigma24/rpoK, sigma32/rpoH and sigma28/fliA were also induced. In addition, the expression of the pbrR(2) cadA pbrC(2) operon of the CMGI-1 region and the chromosomally encoded zntA were clearly induced in the presence of Pb(II), although their respective gene products were not detected via proteomics. After inactivation of the pbrA, pbrB or pbrD genes, the expression of the pbrR(2) cadA pbrC(2) operon went up considerably. This points towards synergistic interactions between pbrUTRABCD and pbrR(2) cadA pbrC(2) to maintain a low intracellular Pb(II) concentration, where pbrR(2) cadA pbrC(2) gene functions can complement and compensate for the mutations in the pbrA and pbrD genes. This role of zntA and cadA to complement for the loss of pbrA was further confirmed by mutation analysis. The pbrB:: colonsTn(Km2) mutation resulted in the most significant decrease of Pb(II) resistance, indicating that Pb(II) sequestration, avoiding re-entry of this toxic metal ion, forms a critical step in the pbr-encoded Pb(II) resistance mechanism.

Cupriavidus metallidurans: 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.

Biostructural analysis of the metal-sensor domain of CnrX from Cupriavidus metallidurans CH34

In Cupriavidus metallidurans CH34, the proteins CnrX, CnrY, and CnrH regulate the expression of the cnrCBA operon that codes for a cation-efflux pump involved in cobalt and nickel resistance. The periplasmic part of CnrX can be defined as the metal sensor in the signal transduction complex composed of the membrane-bound anti-sigma factor CnrY and the extra-cytoplasmic function sigma factor CnrH. A soluble form of CnrX was overproduced and purified. This protein behaves as a dimer in solution as judged from gel filtration, sedimentation velocity experiments, and NMR. Native crystals diffracting to 2.3 A using synchrotron radiation were obtained using the hanging-drop vapor-diffusion method. They belong to the primitive monoclinic space group P2(1), with unit cell parameters a = 31.87, b = 74.80, c = 93.67 A, beta = 90.107 degrees. NMR data and secondary structure prediction suggest that this protein is essentially formed by helices.

Molecular Structure and Metal-binding Properties of the Periplasmic CopK Protein Expressed in Cupriavidus metallidurans CH34 During Copper Challenge

The copK gene is localized on the pMOL30 plasmid of Cupriavidus metallidurans CH34 within the complex cop cluster of genes, for which 21 genes have been identified. The expression of the corresponding periplasmic CopK protein is strongly upregulated in the presence of copper, leading to a high periplasmic accumulation. The structure and metal-binding properties of CopK were investigated by NMR and mass spectrometry. The protein is dimeric in the apo state with a dissociation constant in the range of 10 - 5 M estimated from analytical ultracentrifugation. Mass spectrometry revealed that CopK has two high-affinity Cu(I)-binding sites per monomer with different Cu(I) affinities. Binding of Cu(II) was observed but appeared to be non-specific. The solution structure of apo-CopK revealed an all-β fold formed of two β-sheets in perpendicular orientation with an unstructured C-terminal tail. The dimer interface is formed by the surface of the C-terminal β-sheet. Binding of the first Cu(I)-ion induces a major structural modification involving dissociation of the dimeric apo-protein. Backbone chemical shifts determined for the 1Cu(I)-bound form confirm the conservation of the N-terminal β-sheet, while the last strand of the C-terminal sheet appears in slow conformational exchange. We hypothesize that the partial disruption of the C-terminal β-sheet is related to dimer dissociation. NH-exchange data acquired on the apo-protein are consistent with a lower thermodynamic stability of the C-terminal sheet. CopK contains seven methionine residues, five of which appear highly conserved. Chemical shift data suggest implication of two or three methionines (Met54, Met38, Met28) in the first Cu(I) site. Addition of a second Cu(I) ion further increases protein plasticity. Comparison of the structural and metal-binding properties of CopK with other periplasmic copper-binding proteins reveals two conserved features within these functionally related proteins: the all-β fold and the methionine-rich Cu(I)-binding site.

PIXE ANALYSES OF THE SOLUBLE AND MEMBRANE SE-CONTAINING PROTEINS EXTRACTED FROMCUPRIAVIDUS METALLIDURANSCH34 AFTER SELENIUM OXIDES CHALLENGE

The soil bacterium Cupriavidus metallidurans CH34 resist selenite by reducing it into the insoluble and less toxic elemental selenium. Two mechanisms of reduction of selenium oxides in C. metallidurans CH34 were highlighted: assimilation leading to organic species and detoxification leading to precipitation of selenite in nanoparticules of elemental selenium. The alkyl selenide detected as an intermediate product during assimilation of selenite or as the major accumulated chemical form during assimilation of selenate was identified as selenomethionine.Soluble and membrane proteins were extracted from C. metallidurans CH34 submitted to selenium oxides challenge. After separation by SDS-PAGE, µPIXE analyses were used for Se identification and quantification at a micrometer scale. The profiles of Se distribution in the different samples suggest a non-specific incorporation of selenium probably reflecting the incorporation of selenomethionin in place of the naturally occurring methionin.

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).

Overproduction, purification and preliminary X-ray diffraction analysis of CzcE from Cupriavidus metallidurans CH34.

CzcE is encoded by the czc determinant that allows Cupriavidus metallidurans CH34 to modulate its internal concentrations of cobalt, zinc and cadmium. This periplasmic protein was overproduced in its mature form in Escherichia coli and purified in two steps. After preliminary screening of crystallization conditions using a robot, well diffracting crystals were obtained using the hanging-drop vapour-diffusion method. Crystals diffracted to 1.96 A using synchrotron radiation. They belonged to the monoclinic space group C2, with unit-cell parameters a = 105.54, b = 29.68, c = 71.10 A. The asymmetric unit is expected to contain a dimer, in agreement with the quaternary structure deduced from gel-filtration experiments.

Physiological changes induced in bacteria following pH stress as a model for space research

The physiology of the environmental bacterium Cupriavidus metallidurans CH34 (previously Ralstonia metallidurans) is being studied in comparison to the clinical model bacterium Escherichia coli in order to understand its behaviour and resistance under extreme conditions (pH, temperature, etc.). This knowledge is of importance in the light of the potential use and interest of this strain for space biology and bioremediation. Flow cytometry provides powerful means to measure a wide range of cell characteristics in microbiological research. In order to estimate physiological changes associated with pH stress, flow cytometry was employed to estimate the extent of damage on cell size, membrane integrity and potential, and production of superoxides in the two bacterial strains. Suspensions of C. metallidurans and E. coli were submitted to a 1-h pH stress (2 to 12). For flow cytometry, fluorochromes, including propidium iodide, 3, 3 ′ -dihexyloxacarbocyanine iodide and hydroethidine were chosen as analytical parameters for identifying the physiological state and the overall fitness of individual cells. A physiologic state of the bacterial population was assessed with a Coulter EPICS XL analyser based on the differential uptakes of these fluorescent stains. C. metallidurans cells exhibited a different staining intensity than E. coli cells. For both bacterial strains, the physiological status was only slightly affected between pH 6 and 8 in comparison with pH 7 which represents the reference pH. Moderate physiological damage could be observed at pH 4 and 5 as well as at pH 9 in both strains. At pH 2, 10 and 12, membrane permeability and potential and superoxide anion production were increased to high levels showing dramatic physiological changes. It is apparent that a range of significant physiological alterations occurs after pH stress. Fluorescent staining methods coupled with flow cytometry are useful and complementary for monitoring physiological changes induced not only by pH stress but also temperature and oxidative stress, radiation, pressure as well as space stress.

Effect of cultivation and experimental conditions on the surface reactivity of the metal-resistant bacteria Cupriavidus metallidurans CH34 to protons, cadmium and zinc

The surface chemistry of the cell wall of the metal resistant bacterium Cupriavidus metallidurans CH34 was investigated through proton exchange and zinc and cadmium sorption experiments. The effect of organic and mineral nutrients availability, culture age and viability on cell wall reactivity to H +, Zn or Cd was specifically addressed. Parameter sensitivity studies allowed constraining the pH-validity domain of the titration experiments and defining experimental conditions that permit reproducible experiments with this bacterium. The results were satisfactorily fitted with a non-electrostatic model that allowed the determination of the stability constants of three discrete acid–base functional groups differing in acidity at bacterial cell surfaces. These results revealed that C. metallidurans CH34 did not particularly stand out in terms of its surface reactivity as compared to metal-sensitive bacteria. This may confirm a generic global reactivity of all bacteria towards non-redox sensitive metals. The same reactivity to zinc was observed for C. metallidurans CH34 cells grown in LB-rich or TSM-mineral media. Cell surface reactivity was found to be independent of organic substrates availability but strongly dependent on cell growth stage and cell viability. Zinc sorption by C. metallidurans CH34 was only slightly (15% decrease) affected by phosphate availability. This suggests the involvement of phosphorus sites in metal binding. Zn and Cd stability constants compared to those of strong chelating ligands but were higher than those of weak ligands, such as acetic acid or phosphoric acid. This indicates that bacterial cells strongly compete with small dissolved organic components that are potentially less reactive to metals than bacteria. This competition potentially affects metal mobility in soils.

Transcriptomic and proteomic analyses of the pMOL30-encoded copper resistance in Cupriavidus metallidurans strain CH34

The four replicons of Cupriavidus metallidurans CH34 (the genome sequence was provided by the US Department of Energy-University of California Joint Genome Institute) contain two gene clusters putatively encoding periplasmic resistance to copper, with an arrangement of genes resembling that of the copSRABCD locus on the 2.1 Mb megaplasmid (MPL) of Ralstonia solanacearum, a closely related plant pathogen. One of the copSRABCD clusters was located on the 2.6 Mb MPL, while the second was found on the pMOL30 (234 kb) plasmid as part of a larger group of genes involved in copper resistance, spanning 17 857 bp in total. In this region, 19 ORFs (copVTMKNSRABCDIJGFLQHE) were identified based on the sequencing of a fragment cloned in an IncW vector, on the preliminary annotation by the Joint Genome Institute, and by using transcriptomic and proteomic data. When introduced into plasmid-cured derivatives of C. metallidurans CH34, the cop locus was able to restore the wild-type MIC, albeit with a biphasic survival curve, with respect to applied Cu(II) concentration. Quantitative-PCR data showed that the 19 ORFs were induced from 2- to 1159-fold when cells were challenged with elevated Cu(II) concentrations. Microarray data showed that the genes that were most induced after a Cu(II) challenge of 0.1 mM belonged to the pMOL30 cop cluster. Megaplasmidic cop genes were also induced, but at a much lower level, with the exception of the highly expressed MPL copD. Proteomic data allowed direct observation on two-dimensional gel electrophoresis, and via mass spectrometry, of pMOL30 CopK, CopR, CopS, CopA, CopB and CopC proteins. Individual cop gene expression depended on both the Cu(II) concentration and the exposure time, suggesting a sequential scheme in the resistance process, involving genes such as copK and copT in an initial phase, while other genes, such as copH, seem to be involved in a late response phase. A concentration of 0.4 mM Cu(II) was the highest to induce maximal expression of most cop genes.