<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.     

Precipitation of Silver-Thiosulfate Complex and Immobilization of Silver by <Emphasis Type="BoldItalic">Cupriavidus metallidurans</Emphasis> CH34

Cupriavidus metallidurans CH34 is a facultative chemolithotrophic bacterium that possesses two megaplasmids (pMOL28 and pMOL30) that confer resistance to eleven metals. The ability of Cupriavidus metallidurans CH34 to resist silver is described here. Electronic microscopy, energy-dispersive X-ray (EDX) and X-ray diffractometry (DRX) observations revealed that C. metallidurans CH34 strongly associated silver with the outer membrane, under chloride chemical form. Using derivate strains of C. metallidurans CH34, which carried only one or no megaplasmid, we show that this resistance seems to be carried by pMOL30.     

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.     

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.     

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.     

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.     

Factors influencing the biosorption of gadolinium by micro-organisms and its mobilisation from sand

The present work was devoted to the study of the biosorption capacities of various microbial species (Bacillus subtilis, Pseudomonas aeruginosa, Ralstonia metallidurans CH34 previously Alcaligenes eutrophus CH34, Mycobacterium smegmatis, Saccharomyces cerevisiae) for ions of the lanthanide gadolinium (Gd³⁺). The uptake by sand of this element was also measured. Saturation curves and Scatchard models were established for all biosorbants used in this work. The results enabled us to determine the binding affinities and the maximum capacities for biosorption of Gd³⁺, which ranged from 350 μmol g⁻¹ for B. subtilis to 5.1 μmol g⁻¹ for S. cerevisiae. This study demonstrated the usefulness of optimisation of experimental conditions in biosorption investigations. Experimental results showed that biosorption could be influenced by the growth stage and by the composition of the growth medium of microbial cells. Finally, particular attention was given to the transfer of gadolinium ions from a loaded sand to a bacterial suspension. Received: 8 November 1999 / Received revision: 3 February 2000 / Accepted: 4 February 2000     

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.     

Protein dynamics during seed germination under copper stress in Arabidopsis over-expressing <Emphasis Type="Italic">Potentilla superoxide dismutase</Emphasis>

Copper (Cu), though an essential micronutrient for plants, poses toxicity at higher concentrations possibly by inducing oxidative stress. With the background that enzyme superoxide dismutase (SOD) ameliorates oxidative stress, the present work focused on understanding physiological and proteomic response of Arabidopsis seeds constitutively over-expressing copper–zinc SOD of Potentilla atrosanguinea (PaSOD) during germination in response to varied concentrations of copper sulphate (Cu stress). Transgenics showed higher germination percentage and required less “mean time to germination” under Cu-stress. In response to Cu stress, 39 differentially expressed protein spots were detected by 2-D electrophoresis in proteins of germinating wild type (WT) and transgenic seeds, of which 14 spots appeared exclusively in transgenics. Among the rest 25 protein spots, 14 showed down-regulation, one showed up-regulation, and 10 spots disappeared. MALDI-TOF and subsequent peptide mass fingerprinting analysis revealed that the down-regulated proteins in transgenics were related to oxidative stress, detoxification, germination, intermediary metabolism and regulatory proteins. Up-regulated proteins in WT and down-regulated proteins in transgenic during Cu stress were the same. Changes in key proteins, vis-à-vis alleviation of oxidative stress in transgenic Arabidopsis over-expressing PaSOD possibly alleviated toxicity of Cu-induced stress during seed germination, resulting in higher germination rate and germination percentage.     

Adaptive stress response to menadione-induced oxidative stress in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> KNU5377

The molecular mechanisms involved in the ability of yeast cells to adapt and respond to oxidative stress are of great interest to the pharmaceutical, medical, food, and fermentation industries. In this study, we investigated the time-dependent, cellular redox homeostasis ability to adapt to menadione-induced oxidative stress, using biochemical and proteomic approaches in Saccharomyces cerevisiae KNU5377. Time-dependent cell viability was inversely proportional to endogenous amounts of ROS measured by a fluorescence assay with 2′,7′-dichlorofluorescin diacetate (DCFHDA), and was hypersensitive when cells were exposed to the compound for 60 min. Morphological changes, protein oxidation and lipid peroxidation were also observed. To overcome the unfavorable conditions due to the presence of menadione, yeast cells activated a variety of cell rescue proteins including antioxidant enzymes, molecular chaperones, energy-generating metabolic enzymes, and antioxidant molecules such as trehalose. Thus, these results show that menadione causes ROS generation and high accumulation of cellular ROS levels, which affects cell viability and cell morphology and there is a correlation between resistance to menadione and the high induction of cell rescue proteins after cells enter into this physiological state, which provides a clue about the complex and dynamic stress response in yeast cells.     

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.     

Solution structure of GSP13 from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> exhibits an S1 domain related to cold shock proteins

GSP13 encoded by gene yugI is a σ^B-dependent general stress protein in Bacillus subtilis, which can be induced by heat shock, salt stress, ethanol stress, glucose starvation, oxidative stress and cold shock. Here we report the solution structure of GSP13 and it is the first structure of S1 domain containing protein in Bacillus subtilis. The structure of GSP13 mainly consists of a typical S1 domain along with a C-terminal 50-residue flexible tail, different from the other known S1 domain containing proteins. Comparison with other S1 domain structures reveals that GSP13 has a conserved RNA binding surface, and it may function similarly to cold shock proteins in response to cold stress.     

Coordinated expression and immunogenicity of an outer membrane protein from Salmonella enterica serovar Typhi under iron limitation, oxidative stress and anaerobic conditions

Summary Successful pathogens overcome the environmental stresses by the coordinated expression of various genes and eventually proteins. Since, the surface of the microbe is likely to come in contact with the host initially, an attempt was made to identify the outer membrane proteins (OMPs), if any, which may get expressed under more than one environmental conditions simulating the in vivo ones. In the present study, Salmonella enterica serovar Typhi was grown under iron-limited, oxidative stress as well as anaerobic conditions and the OMP profiles were compared. A 69 kDa OMP was found to express with enhanced intensity under the selected stress conditions in comparison to normal conditions. The phenotypic similarity among the proteins was assessed on the basis of their molecular weight, cross reactivity and HPLC. The protein expressed under oxidative stress and anaerobic conditions reacted with the antibodies raised against iron-regulated outer membrane protein (IROMP), indicating the sharing of at least some of the epitopes. A single peak observed after subjecting the pooled 69 kDa protein sample and appearance of a single band on SDS-PAGE thereafter, confirmed the purity and phenotypic similarity of the 69 kDa OMP. Reactivity of pooled 69 kDa protein with 85% of sera from typhoid patients revealed the in vivo expression of this protein. The results of this study indicate the coordination of this phenotype under iron stress, oxidative stress and anaerobic conditions. In view of the expression of the 69 kDa protein under the selected stress conditions and their in vivo immunogenicity, these findings may be relevant for the better understanding of the host–microbe interactions and for the further development of diagnostic and preventive strategies.     

Production of pigment-free pullulan by swollen cell in <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis> NG which cell differentiation was affected by pH and nutrition

A black yeast strain “NG” was isolated from strawberry fruit and identified as Aureobasidium pullulans. Strain NG displayed yeast-like cell (YL), swollen cell (SC), septate swollen cell (SSC), meristematic structure (MS), and chlamydospore (CH) morphologies. pH was the key factor regulating cell morphogenesis of strain NG. Differentiation of YL controlled by extracellular pH had no relationship with nutrition level. YL was maintained at pH >6.0, but was transformed into SC at pH ∼4.5. SC, a stable cell type of A. pullulans, could bud, septate, or transform into MS or CH, in response to nutrition level and low pH. SC produced swollen cell blastospores (SCB) at pH 2.1 with abundant nutrition, and could transform into MS at lower pH (1.5). SC was induced to form CH by low level nutrition and pH <3, and this transition was suppressed by adjusting pH to ∼4.5. Crude polysaccharides without pigment (melanin) were produced by SC of strain NG. Pullulan content of the polysaccharides was very high (98.37%). Fourier-transform infrared spectroscopy confirmed that chemical structures of the polysaccharides and standard pullulan were identical. Swollen cells produced 2.08 mg/ml non-pigmented polysaccharides at 96 h in YPD medium. Controlling pH of fermentation is an effective and convenient method to harvest SC for melanin-free pullulan production.     

Oxidative stress by <Emphasis Type="Italic">Helicobacter pylori</Emphasis> causes apoptosis through mitochondrial pathway in gastric epithelial cells

Helicobacter pylori is a gram negative bacterium that infects the human stomach of approximately half of the world’s population. It produces oxidative stress, and mitochondria are one of the possible targets and the major intracellular source of free radicals. The present study was aimed at determining mitochondrial alterations in H. pylori-infected gastric epithelial cells and its relationship with oxidative stress, one of the recognized causes of apoptotic processes. Cells were treated with a strain of H. pylori for 24 h. Cellular oxidative burst, antioxidant defense analysis, mitochondrial alterations and apoptosis-related processes were measured. Our data provide evidence on how superoxide acts on mitochondria to initiate apoptotic pathways, with these changes occurring in the presence of mitochondrial depolarization and other morphological and functional changes. Treatment of infected cells with Vitamin E prevented increases in intracellular ROS and mitochondrial damage consistent with H. pylori inducing a mitochondrial ROS mediated programmed cell death pathway.     

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.