Metallidurans CH34 Research Articles

CopH from Cupriavidus metallidurans CH34. A Novel Periplasmic Copper-Binding Protein

The copH gene is one of the 19 open reading frames (ORFs) found in the cop cluster borne by the large plasmid pMol30 in Cupriavidus metallidurans CH34. The entire cluster is involved in detoxification of copper from the cytoplasm as well as from the periplasm. The function of the corresponding protein, CopH, is not yet clear, but it seems to be involved in the late response phase. We have cloned copH and overproduced and purified the corresponding protein. CopH is rather unique as only one paralog can be found in the databases. It is a dimeric protein with a molecular mass of 13 200 Da per subunit and located in the periplasm. The metal binding properties of CopH were examined by using a series of techniques such as UV-visible spectroscopy, circular dichroism (CD), electron paramagnetic resonance (EPR), surface plasmon resonance (SPR), mass spectrometry, and nuclear magnetic resonance (NMR). All together, the corresponding data are consistent with a dimeric protein containing one metal-binding site per subunit. These sites have a high affinity for Cu(II) but can also bind zinc or nickel. CopH does not contain any cysteines or methionines but contains two histidines. EPR and UV-visible features are consistent with the presence of Cu(II) type 2 centers in a nitrogen ligand field. SPR data confirm the involvement of the histidine residues in copper binding. CD and NMR data reveal that CopH is partially unfolded.

Zinc Sorption to Three Gram-Negative Bacteria: Combined Titration, Modeling, and EXAFS Study

The acid-base and Zn sorption properties of three bacteria, Cupriavidus metallidurans CH34, Pseudomonas putida ATCC12633, and Escherichia coli K12DH5alpha, were investigated through an original combination of extended X-ray absorption fine structure (EXAFS) spectroscopy and equilibrium titration studies. Acid-base titration curves of the three strains were fitted with a model accounting for three conceptual reactive sites: an acidic (carboxyl and/or phosphodiester), a neutral (phosphomonoester), and a basic (amine and/or hydroxyl) group. Calculated proton and Zn equilibrium constants and site densities compare with literature data. The nature of Zn binding sites was studied by EXAFS spectroscopy. Phosphoester, carboxyl, and unexpectedly sulfhydryl ligands were identified. Their proportions depended on Zn loading and bacterial strain and were consistent with the titration results. These findings were compared to the structure and site density of the major cell wall components. It appeared that the cumulated theoretical site density of these structures (<2 Zn nm(-2)) was much lower than the total site density of the investigated strains (16-56 Zn nm(-2)). These results suggest a dominant role of extracellular polymeric substances in Zn retention processes, although Zn binding to inner cell components cannot be excluded.

Electricity from low-level H2 in still air ? an ultimate test for an oxygen tolerant hydrogenase

We demonstrate an extreme test of O(2) tolerance for a biological hydrogen-cycling catalyst: the generation of electricity from just 3% H(2) released into still, ambient air using an open fuel cell comprising an anode modified with the unusual hydrogenase from Ralstonia metallidurans CH34, that oxidizes trace H(2) in atmospheric O(2), connected via a film of electrolyte to a cathode modified with the fungal O(2) reductase, laccase.

Paralogs of Genes Encoding Metal Resistance Proteins in Cupriavidus metallidurans Strain CH34

Cupriavidus (Wautersia, Ralstonia, Alcaligenes) metallidurans strain CH34is a well-studied example of a metal-resistant proteobacterium. Genome sequence analysis revealed the presence of a variety of paralogs of proteins that were previously shown to be involved in heavy metal resistance. Which advantage has C. metallidurans in maintaining all these paralogs during evolution? Paralogs investigated belong to the families RND (resistance nodulation cell division) or CHR (chromate resistance). The respective genes were localized by PCR either on one of the two native megaplasmids pMOL28 and pMOL30 of strain CH34, or on its chromosomal DNA. Gene expression was studied by real-time reverse transcriptase PCR and by reporter gene constructs. Genes found to be inducible were disrupted and their contribution to metal resistance measured. When two or three highly related genes were present, usually one was inducible by heavy metals while the other one or two were silent or constitutively expressed. This suggests that C. metallidurans CH34 carries a variety of no longer or not yet used genes that might serve as surplus material for further developments, an advantage that may compensate for the costs of maintaining these genes during evolution.

Precipitation of Silver-Thiosulfate Complex and Immobilization of Silver by Cupriavidus metallidurans 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.

Effect of pH on the toxicity of nickel and other divalent metals to Burkholderia cepacia PR1301

Nickel (Ni) is a common cocontaminant at many waste sites where the soils and sediments often are acidic, thereby influencing metal availability. Growth of Burkholderia cepacia PR1(301) was not affected at 3.41 mM Ni at pH 5, but was inhibited by 73.2% at pH 6 and inhibited completely at pH 7 compared to growth without Ni. This pH effect was not observed in the Ni-resistant strains, Ralstonia metallidurans CH34 and 31A. Predicted Ni speciation did not explain the observed toxicity trends. Sorption of Ni to PR1 increased with increasing pH (1.49, 1.12, and 3.88 mg Ni/g dry weight at pH 5, 6, and 7, respectively), but was low at all three pH values, and most likely does not explain the observed pH effect. Growth inhibition of PRI with increasing pH also was observed for other divalent cations, with growth observed at 4.24 mM Co, 2.22 mM Cd, and 3.82 mM Zn at pH 5 and 6, but totally inhibited at pH 7. These studies suggest that, at circumneutral pH, PRI would be considered sensitive to Ni and other divalent cations, in spite of the ability to grow in higher concentrations at lower pH values.

A probable link between the DedA protein and resistance to selenite

To understand the molecular events involved in the reduction of selenite to non-toxic elemental selenium, 4000 clones of Ralstonia metallidurans CH34 were produced by random Tn 5 transposon integration mutagenesis. Eight mutants were able to resist up to 15 mM selenite while the MIC for the wild-type strain was estimated as 4–6 mM selenite. The identification of the disrupted genes was carried out by Southern blot analysis and inverse PCR. The three resistant mutants containing only one insertion were further characterized. Tn 5 disrupted a gene that encoded a protein which was closely related to proteins of the DedA family. This family represents a group of integral membrane proteins with completely unknown functions. Phenotypic characterization of the dedA mutants and selenite consumption experiments strongly suggest that DedA is involved in the uptake of selenite.

Characterization of the MerD protein from Ralstonia metallidurans CH34: a possible role in bacterial mercury resistance by switching off the induction of the mer operon.

MerD and MerR from Tn4378 found in Ralstonia metallidurans CH34 were purified to homogeneity after overexpression in Escherichia coli. Using electrophoretic mobility shift assays and footprinting experiments, we found that MerD cannot bind to DNA. However, in vitro MerD can form a ternary complex in association with merOP and MerR. The presence of MerD in this complex was demonstrated by Western analysis with antibodies to MerD. To our knowledge, this is the first description of such a ternary complex between MerD-MerR and DNA. The formation and stability of this ternary complex are dependent on the relative concentration of the two proteins and modulated by the presence of mercury. We postulate that MerD could displace Hg-bound MerR from the mer operator to allow new synthesis of metal-free MerR able to switch off the induction of the mer genes when the external mercury is exhausted. This could fully explain how MerD can be a co-regulator repressing the induction of the mer operon.

Crystal Structure of the Oxidized Form of the Periplasmic Mercury-binding Protein MerP from Ralstonia metallidurans CH34

In Ralstonia metallidurans CH34, the gene merP encodes for a periplasmic mercury-binding protein which is capable of binding one mercury atom. The metal-binding site of MerP consists of the highly conserved sequence GMTCXXC found in the family that includes metallochaperones and metal-transporting ATPases. We purified MerP from R. metallidurans CH34 and solved its crystal structure under the oxidized form at 2.0 Å resolution. Superposition with structures of other metal-binding proteins shows that the global structure of R. metallidurans CH34 oxidized MerP follows the general topology of the whole family. The largest differences are observed with the NMR structure of oxidized Shigella flexneri MerP. Detailed analysis of the metal-binding site suggests a direct role for Y66 in stabilizing the thiolate group of C17 during the mercury-binding reaction. The metal-binding site of oxidized MerP is also similar to the metal-binding sites of oxidized copper chaperone for superoxide dismutase and Atx1, two copper-binding proteins from Saccharomyces cerevisiae. Finally, the packing of the MerP crystals suggests that F38, a well-conserved residue in the MerP family may be important in mercury binding and transfer. We propose a possible mechanism of mercury transfer between two CXXC motifs based on a transient bi-coordinated mercury intermediate.

Global analysis of the Ralstonia metallidurans proteome: prelude for the large-scale study of heavy metal response.

A proteome map of Ralstonia metallidurans strain CH34 was constructed using two-dimensional (2-D) gel electrophoresis in combination with automated Edman degradation and mass spectrometry (MS). R. metallidurans CH34 is the type-strain of a family of highly related strains characterized by their multiple resistance to millimolar amounts of heavy metals, conferred by two large plasmids. The protein content of this bacterium grown in minimal medium was separated by 2-D gel electrophoresis using various pH gradients. Protein identification was carried out via N-terminal amino acid sequencing, matrix assisted laser desorption/ionisation-time of flight-mass spectrometry (MALDI-TOF-MS) and tandem MS. So far, 224 different proteins were characterized from 352 protein spots. Although the proteome map is still not complete, one could appraise the importance of proteomics for genome analyses through (i). the identification of previously undetected open reading frames, (ii). the identification of proteins not encoded by the already sequenced genome fragments, (iii). the characterization of protein-encoding genes spanning two different contigs, enabling their merging, and (iv). the precise delineation of the N-terminus of several proteins. Finally, this map will prove a useful tool in the identification of proteins differentially expressed in the presence of different heavy metals.

Biophysical characterization of the MerP-like amino-terminal extension of the mercuric reductase from Ralstonia metallidurans CH34.

The purified native mercuric reductase (MerA) from Ralstonia metallidurans CH34 contains an N-terminal sequence of 68 amino acids predicted to be homologous to MerP, the periplasmic mercury-binding protein. This MerP-like protein has now been expressed independently. The protein was named MerAa by homology with Ccc2a, the first soluble domain of the copper-transporting ATPase from yeast. Deltaa has been characterized using a set of biophysical techniques. The binding of mercury was followed using circular dichroism spectroscopy and electrospray mass spectrometry. The two cysteine residues contained in the consensus sequence GMTC XXC are involved in the binding of one mercury atom, with an apparent affinity comparable to that of MerP for the same metal. The metal-binding site is confirmed by NMR chemical shift changes observed between apo- and metal-bound MerAa in solution. NMR shift and NOE data also indicate that only minor structural changes occur upon metal binding. Further NMR investigation of the fold of MerAa using long-range methyl-methyl NOE and backbone residual dipolar coupling data confirm the expected close structural homology with MerP. (15)N relaxation data show that MerAa is a globally rigid molecule. An increased backbone mobility was observed for the loop region connecting the first beta-strand and the first alpha-helix and comprising the metal-binding domain. Although significantly reduced, this loop region keeps some conformational flexibility upon metal binding. Altogether, our data suggest a role of MerAa in mercury trafficking.

The biphenyl- and 4-chlorobiphenyl-catabolic transposon Tn4371, a member of a new family of genomic islands related to IncP and Ti plasmids.

The nucleotide sequence of the biphenyl catabolic transposon Tn4371 has been completed and analyzed. It confirmed that the element has a mosaic structure made of several building blocks. In addition to previously identified genes coding for a tyrosine recombinase related to phage integrases and for biphenyl degradation enzymes very similar to those of Achromobacter georgiopolitanum KKS102, Tn4371 carries many plasmid-related genes involved in replication, partition, and other, as-yet-unknown, plasmid functions. One gene cluster contains most of the genes required to express a type IV secretion-mating pair formation apparatus coupled with a TraG ATPase, all of which are related to those found on IncP and Ti plasmids. Orthologues of all Tn4371 plasmid-related genes and of the tyrosine recombinase gene were found, with a very similar organization, in the chromosome of Ralstonia solanacearum and on the yet-to-be-determined genomic sequences of Erwinia chrysanthemi and Azotobacter vinelandii. In each of these chromosomal segments, conserved segments were separated by different groups of genes, which also differed from the Tn4371 bph genes. The conserved blocks of genes were also identified, in at least two copies, in the chromosome of Ralstonia metallidurans CH34. Tn4371 thus appears to represent a new family of potentially mobile genomic islands with a broad host range since they reside in a wide range of soil proteobacteria, including plant pathogens.

Construction and use of specific luminescent recombinant bacterial sensors for the assessment of bioavailable fraction of cadmium, zinc, mercury and chromium in the soil

Recombinant luminescent bacterial sensors for the detection of zinc [MC1061(pzntRluc)] and chromate [AE104(pchrBluc)] were constructed. The sensors carry firefly luciferase gene as a reporter under the control of zinc-inducible regulatory unit from Zn resistance system in chromosomal DNA of Escherichia coli or chromate-inducible unit from Ralstonia metallidurans CH34 megaplasmid pMOL28. The response of the sensors was calibrated using Zn 2+, Cr 2O 7 2− and CrO 4 2−, respectively. The detection limit of the zinc sensor for zinc was 40 μM (2.6 mg l −1 of Zn) and that of chromate sensor for dichromate 30 nM (1.6 μg l −1 of Cr) and for chromate 50 nM (2.6 μg l −1 of Cr), respectively. The specificity of the two above-mentioned sensors constructed in this study and a mercury-inducible bacterial sensor MC1061(pmerBR BSluc) constructed by us earlier [Anal. Chem. 73 (2001) 5168] was determined by using different heavy metal compounds. The zinc and mercury sensors were not completely specific to the target metals. The zinc sensor was co-inducible with cadmium and mercury and the mercury sensor with cadmium. The chromate sensor was inducible not only by chromate but also with Cr 3+. The bacterial sensors constructed were used for the estimation of bioavailable fraction of heavy metals in soils spiked with different amounts of zinc, cadmium, mercury and chromate. Both soil–water suspensions and soil–water extracts were analyzed. The results showed that the majority of heavy metals remained adsorbed to soil particles: only 0.6% of Cd, 1.3% of Hg, 2% of Zn and 46% of Cr (VI) were available to the bacterial sensors in soil–water extracts. However, when the soil–water suspensions were analyzed, approximately 20 times more cadmium and 30 times more mercury (12 and 40%, respectively) was available for the sensor bacteria whereas the available fractions of zinc and chromate in soil–water extract and suspension were similar. Thus, this study showed that in case of cadmium and mercury (but not zinc and chromium) the particle-bound metals were also partially bioavailable.

The iron-containing superoxide dismutase of Ralstonia metallidurans CH34

The iron-containing superoxide dismutase (Fe-SOD) of Ralstonia metallidurans CH34 was purified and characterised as a homodimer of 2×21 500 Da containing one iron atom per monomer and exhibiting all the characteristics of the prokaryotic Fe-SODs except for a higher isoelectric point. The protein was 2-fold overexpressed in the presence of selenite, zinc or paraquat. R. metallidurans CH34 was suggested to contain a gene encoding for a manganese-containing SOD located in the inducible chromate resistance operon. Whatever the culture conditions used in this study, including the presence of chromate, only a Fe-SOD, genetically distinct from the putative Mn-SOD, was detected. This Fe-SOD seems to be the only active superoxide dismutase expressed in R. metallidurans CH34.

The iron-containing superoxide dismutase of Ralstonia metallidurans CH34.

The iron-containing superoxide dismutase (Fe-SOD) of Ralstonia metallidurans CH34 was purified and characterised as a homodimer of 2 x 21500 Da containing one iron atom per monomer and exhibiting all the characteristics of the prokaryotic Fe-SODs except for a higher isoelectric point. The protein was 2-fold overexpressed in the presence of selenite, zinc or paraquat. R. metallidurans CH34 was suggested to contain a gene encoding for a manganese-containing SOD located in the inducible chromate resistance operon. Whatever the culture conditions used in this study, including the presence of chromate, only a Fe-SOD, genetically distinct from the putative Mn-SOD, was detected. This Fe-SOD seems to be the only active superoxide dismutase expressed in R. metallidurans CH34.

New genes involved in chromate resistance in Ralstonia metallidurans strain CH34.

Chromate resistance in Ralstonia metallidurans CH34 is based on chromate efflux catalyzed by ChrA efflux pumps. The bacterium harbors two chromate resistance determinants, the previously known chr(1) on plasmid pMOL28 (genes chrI, chrB(1), chrA(1), chrC, chrE, chrF(1)) and chr(2) on the chromosome (genes chrB(2), chrA(2), chrF(2)). Deletion of the genes chrI, chrC, chrA(2), chrB(2) and chrF(2) influenced chromate resistance and transcription from a chrBp(1) ::lacZ fusion. Deletion of the plasmid-encoded gene chrB(1) did not change chromate resistance or chrBp(1) regulation. Northern hybridization and primer-extension experiments were used to study transcription of the plasmid-encoded chr(1) determinant. Transcription of chrB(1), chrA(1) and chrC was induced by chromate. The presence of sulfate influenced transcription positively. The chrBp(1), chrAp(1) and chrCppromoters showed some similarity to heat-shock promoters. Transcription of the gene rpoH encoding a putative heat-shock sigma factor was also induced by chromate, but rpoH was not essential for chromate resistance. The ChrC protein was purified as a homotetramer and exerted superoxide dismutase activity. Thus, possible regulators for chromate resistance (ChrI, ChrB(1), ChrB(2), ChrF(1), and ChrF(2)) and an additional detoxification system (ChrC) were newly identified as parts of chromate resistance in R. metallidurans.

Cloning and functional analysis of the pbr lead resistance determinant of Ralstonia metallidurans CH34.

The lead resistance operon, pbr, of Ralstonia metallidurans (formerly Alcaligenes eutrophus) strain CH34 is unique, as it combines functions involved in uptake, efflux, and accumulation of Pb(II). The pbr lead resistance locus contains the following structural resistance genes: (i) pbrT, which encodes a Pb(II) uptake protein; (ii) pbrA, which encodes a P-type Pb(II) efflux ATPase; (iii) pbrB, which encodes a predicted integral membrane protein of unknown function; and (iv) pbrC, which encodes a predicted prolipoprotein signal peptidase. Downstream of pbrC, the pbrD gene, encoding a Pb(II)-binding protein, was identified in a region of DNA, which was essential for functional lead sequestration. Pb(II)-dependent inducible transcription of pbrABCD from the PpbrA promoter is regulated by PbrR, which belongs to the MerR family of metal ion-sensing regulatory proteins. This is the first report of a mechanism for specific lead resistance in any bacterial genus.

Ralstonia metallidurans CH34 RpoN sigma factor and the control of nitrogen metabolism and biphenyl utilization.

Ralstonia metallidurans CH34 can use biphenyl as carbon and energy source when provided with the catabolic transposon Tn4371. Previous results suggested that this property was dependent on the RNA polymerase subunit sigma(54). The authors sequenced the CH34 rpoN gene and flanking DNA and isolated a CH34 rpoN-deficient strain. Analysis of the sequence revealed a set of features conserved in all rpoN genes and flanking DNA regions previously analysed in other bacterial species. Nevertheless, despite this conservation, CH34 differed even from the closely related strain R. eutropha H16 by one particular ORF. The rpoN null mutation did not affect expression of the Tn4371 bph operon although it did alter the ability of the Tn4371 host strain to grow on biphenyl. The CH34 rpoN mutant had lost the capacity for autotrophic growth and for responding to poor nitrogen sources by a decrease in urease and proline oxidase activity. CH34 RNA polymerase sigma(54) thus positively controls autotrophy as well as nitrogen metabolism but only indirectly affects Tn4371-directed biphenyl utilization.