Characterization of the inducible nickel and cobalt resistance determinant cnr from pMOL28 of Alcaligenes eutrophus CH34.

From pMOL28, one of the two heavy metal resistance plasmids of Alcaligenes eutrophus strain CH34, we cloned an EcoRI-PstI fragment into plasmid pVDZ'2. This hybrid plasmid conferred inducible nickel and cobalt resistance (cnr) in two distinct plasmid-free A. eutrophus hosts, strains AE104 and H16. Resistances were not expressed in Escherichia coli. The nucleotide sequence of the 8.5-kb EcoRI-PstI fragment (8,528 bp) revealed seven open reading frames; two of these, cnrB and cnrA, were assigned with respect to size and location to polypeptides expressed in E. coli under the control of the bacteriophage T7 promoter. The genes cnrC (44 kDa), cnrB (40 kDa), and cnrA (115.5 kDa) are probably structural genes; the gene loci cnrH (11.6 kDa), cnrR (tentatively assigned to open reading frame 1 [ORF]; 15.5 kDa), and cnrY (tentatively assigned to ORF0ab; ORF0a, 11.0 kDa; ORF0b, 10.3 kDa) are probably involved in the regulation of expression. ORF0ab and ORF1 exhibit a codon usage that is not typical for A. eutrophus. The 8.5-kb EcoRI-PstI fragment was mapped by Tn5 transposon insertion mutagenesis. Among 72 insertion mutants, the majority were nickel sensitive. The mutations located upstream of cnrC resulted in various phenotypic changes: (i) each mutation in one of the gene loci cnrYRH caused constitutivity, (ii) a mutation in cnrH resulted in different expression of cobalt and nickel resistance in the hosts H16 and AE104, and (iii) mutations in cnrY resulted in two- to fivefold-increased nickel resistance in both hosts. These genes are considered to be involved in the regulation of cnr. Comparison of cnr of pMOL28 with czc of pMOL30, the other large plasmid of CH34, revealed that the structural genes are arranged in the same order and determine proteins of similar molecular weights. The largest protein CnrA shares 46% amino acid similarity with CzcA (the largest protein of the czc operon). The other putative gene products, CnrB and CnrC, share 28 and 30% similarity, respectively, with the corresponding proteins of czc.     

A new type of Alcaligenes eutrophus CH34 zinc resistance generated by mutations affecting regulation of the cnr cobalt-nickel resistance system.

Spontaneous mutants that were resistant to zinc were isolated from Alcaligenes eutrophus CH34 containing either the native plasmid pMOL28 or a derivative derepressed for its self-transfer, pMOL50. With the cured plasmid-free derivative of CH34, strain AE104, such mutants were not detected. The mutations, which were shown to be located in the plasmid, increased the level of the nickel and cobalt resistance determined by the cnr locus. The chromate resistance closely linked to the cnr locus was not affected by these mutations. In the Znr mutants, the resistance to zinc and nickel was constitutively expressed. Uptake studies showed that the zinc resistance in a Znr mutant resulted from reduced accumulation of zinc ions in comparison with that in the plasmid-free strain. Reduced accumulation of zinc was also observed to a lesser degree in the parental strain induced with nickel, suggesting that zinc interferes with the Ni2+ and Co2+ efflux system. A 12.2-kb EcoRI-XbaI restriction endonuclease fragment containing the cnr locus was cloned from plasmid pMOL28 harboring the mutation and shortened to an 8.5-kb EcoRI-PstI-PstI fragment conferring resistance to zinc, nickel, and cobalt. The 12.2-kb EcoRI-XbaI fragment was also reduced to a 9.7-kb BamHI fragment still encoding weak resistance to nickel and cobalt but not to zinc. Complementation studies demonstrated the recessivity of the cnr mutations with a Znr phenotype. Such mutations thus allow positive selection of mutants affected in the expression of the cnr operon.     

Heavy metal-resistant bacteria as extremophiles: molecular physiology and biotechnological use of Ralstonia sp. CH34

In contrast to thermophilic or psychrophilic organisms, heavy metal-resistant bacteria do not supply enzymes that are active under harsh conditions, but are themselves tools for the evaluation and remediation of heavy metal-contaminated environments. Ralstonia sp. CH34 is a gram-negative bacterium with a remarkable set of resistance determinants, allowing this bacterium to live in extreme environments that are heavily contaminated with toxic metal ions. These heavy metal ions are mostly detoxified by inducible ion efflux systems that reduce the intracellular concentration of a given ion by active export. Because all metal resistance determinants in this bacterium are inducible, their regulatory systems can be used to develop biosensors that measure the biologically important concentrations of heavy metals in an environment. Resistance based on metal ion efflux detoxifies only the cytoplasm of the respective cell. Therefore, this resistance mechanism cannot be used directly to develop biotechnological procedures; however, metal ion efflux can protect a cell in a metal-contaminated environment. Thus, the cell can be enabled to mediate biochemical reactions such as precipitation of heavy metals with the carbon dioxide produced during growth or degradation of xenobiotics. Received: July 11, 1999 / Accepted: December 27, 1999     

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.     

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.     

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.     

Cloning of plasmid genes encoding resistance to cadmium, zinc, and cobalt in Alcaligenes eutrophus CH34.

A 238-kilobase-pair plasmid, pMOL30, confers resistance to cadmium, zinc, and cobalt salts in Alcaligenes eutrophus CH34. After Tn5 mutagenesis, restriction nuclease analysis, and Southern DNA-DNA hybridization, a 9.1-kilobase-pair EcoRI fragment was found to harbor all of these resistance properties and was cloned into the broad-host-range hybrid plasmid pRK290. When transferred to a plasmid-free derivative of CH34, the hybrid plasmid conferred the same degree of resistance as the parent plasmid pMOL30. In two other Alcaligenes strains, the hybrid plasmid was expressed, but to a lower degree than in CH34 derivatives.     

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 2.0A 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.     

Cloning, nucleotide sequence and primary biochemical characterization of esterase EstA from Ralstonia eutropha CH34

A genomic library of Ralstonia eutropha CH34 was screened in Escherichia coli S17-1 for esterase activity by using -naphthyl acetate and Fast Blue RR. A 1,711 bp DNA fragment was subcloned from an esterase-positive clone and sequenced. Esterase EstA was encoded by a 825-bp open reading frame and exhibited significant amino acid similarities with the enzymes involved in the meta-cleavage pathway. EstA is composed of 275 amino aicds with a predicted molecular mass of 30785 Da. The optimal pH for EstA was 7.0, and the enzyme retained more than 65% activity when incubated in buffers with pH 3.8–9.2 for 2 h. EstA was active at temperatures up to 80 °C and retained more than 77% activity after exposure to temperatures below 60 °C for 2 h.     

Cometabolic degradation of dibenzofuran by biphenyl-cultivated Ralstonia sp. strain SBUG 290

Cells of the gram-negative bacterium Ralstonia sp. strain SBUG 290 grown in the presence of biphenyl are able to cooxidize dibenzofuran which has been 1,2-hydroxylated. Meta cleavage of the 1, 2-dihydroxydibenzofuran between carbon atoms 1 and 9b produced 2-hydroxy-4-(3'-oxo-3'H-benzofuran-2'-yliden)but-2-enoic acid, which was degraded completely via salicylic acid. The presence of these intermediates indicates a degradation mechanism for dibenzofuran via lateral dioxygenation by Ralstonia sp. strain SBUG 290.     

The production of phenazine antibiotics by the Pseudomonas aureofaciens strain with plasmid-controlled resistance to cobalt and nickel

Plasmid pBS501 responsible for the resistance of the wild-type Pseudomonas sp. BS501 (pBS501) to cobalt and nickel ions was conjugatively transferred to the rhizosphere Pseudomonas aureofaciens strain BS1393, which is able to synthesize phenazine antibiotics and to suppress a wide range of phytopathogenic microorganisms. The transconjugant P. aureofaciens BS1393 (pBS501) turned out to be resistant to cobalt and nickel with an MIC of 8 mM. When grown in a synthetic medium with 0.25 mM cobalt, the transconjugant accumulated 6 times more cobalt than the wild-type strain BS501 (pBS501) (1.2 and 0.2 microgram Co/mg protein). Electron microscopic studies showed that cobalt accumulates on the surface of transconjugant cells in the form of electron-opaque granules. In a culture medium with 2 mM cobalt or nickel, strain BS1393 produced phenazine-1-carboxylic acid in trace amounts. The transconjugant P. aureofaciens BS1393 (pBS501) produced this antibiotic in still smaller amounts. Unlike the parent strain BS1393, the transconjugant P. aureofaciens BS1393 (pBS501) was able to suppress in vitro the growth of the phytopathogenic fungus Gaeumannomyces graminis var. tritici 1818 in a medium containing 0.5 mM cobalt or nickel.     

Ethanedisulfonate is degraded via sulfoacetaldehyde in Ralstonia sp. strain EDS1

Aerobic enrichment cultures (11) yielded three cultures able to utilise ethane-1,2-disulfonate as sole source of carbon and energy in salts medium. Two pure cultures were obtained and we worked with strain EDS1, which was assigned to the genus Ralstonia on the basis of its 16S rDNA sequence and simple taxonomic tests. Strain EDS1 utilised at least seven alkane(di)sulfonates, ethane-1,2-disulfonate, taurine, isethionate, sulfoacetate, sulfoacetaldehyde and propane-1,3-disulfonate, as well as methanesulfonate and formate. Growth with ethanedisulfonate was concomitant with substrate disappearance and the formation of 2 mol sulfate per mol substrate. The growth yield, 7 g protein (mol C)(-1), indicated quantitative utilisation of the substrate. Ethanedisulfonate-dependent oxygen uptake of whole cells during growth rose to a maximum before the end of growth and then sank rapidly; this was interpreted as evidence for an inducible desulfonative oxygenase that was not active in cell extracts. Inducible sulfoacetaldehyde sulfo-lyase was detected at high activity. Inducible degradation of taurine or isethionate or sulfoacetate via sulfoacetaldehyde sulfo-lyase is interpreted from the data.     

Biodegradation of vinyl chloride and cis-dichloroethene by a Ralstonia sp. strain TRW-1

An aerobic bacterium, Ralstonia sp. strain TRW-1, that assimilates vinyl chloride (VC) or ethene (ETH) as the sole carbon source was isolated from a chloroethene-degrading enrichment culture. Phylogenetic analysis of 16S rDNA sequence of the isolate revealed almost 99% sequence similarity to Ralstonia pickettii. To our knowledge, this is the first report describing the isolation of a member of Ralstonia that can degrade VC as the growth substrate. The measured growth yield values for VC and ETH were 11.27 and 18.90 g protein/mole, respectively. The estimated half-velocity constant K _m values for VC and ETH were 9.09±2.97 and 5.73±2.96 μM, respectively. These values are almost three- to tenfold higher than for other VC-assimilating Mycobacterium sp. The strain also degrades cis-dichloroethene (cis-DCE) in mineral salts medium containing yeast-extract, beef-extract, casamino acids, or peptone. This ability of the strain TRW-1 to degrade cis-DCE in the presence of a nontoxic, water-soluble substrate is relevant to in-situ remediation of cis-DCE-contaminated aquifers.     

Complete biodegradation of 4-fluorocinnamic acid by a consortium comprising Arthrobacter sp. strain G1 and Ralstonia sp. strain H1

A consortium of the newly isolated bacterial strains Arthrobacter sp. strain G1 and Ralstonia sp. strain H1 utilized 4-fluorocinnamic acid for growth under aerobic conditions. Strain G1 converted 4-fluorocinnamic acid into 4-fluorobenzoic acid and used the two-carbon side chain for growth, with some formation of 4-fluoroacetophenone as a dead-end side product. In the presence of strain H1, complete mineralization of 4-fluorocinnamic acid and release of fluoride were obtained. Degradation of 4-fluorocinnamic acid by strain G1 occurred through a β-oxidation mechanism and started with the formation of 4-fluorocinnamoyl-coenzyme A (CoA), as indicated by the presence of 4-fluorocinnamoyl-CoA ligase. Enzymes for further transformation were detected in cell extract, i.e., 4-fluorocinnamoyl-CoA hydratase, 4-fluorophenyl-β-hydroxy propionyl-CoA dehydrogenase, and 4-fluorophenyl-β-keto propionyl-CoA thiolase. Degradation of 4-fluorobenzoic acid by strain H1 proceeded via 4-fluorocatechol, which was converted by an ortho-cleavage pathway.