Differential expression of Desulfovibrio vulgaris genes in response to Cu(II) and Hg(II) toxicity

The response of Desulfovibrio vulgaris to Cu(II) and Hg(II) was characterized. Both metals increased the lag phase, and Cu(II) reduced cell yield at concentrations as low as 50 microM. mRNA expression was analyzed using random arbitrarily primed PCR, differential display, and quantitative PCR. Both Cu(II) and Hg(II) (50 micro M) caused upregulation of mRNA expression for an ATP binding protein (ORF2004) and an ATPase (ORF856) with four- to sixfold increases for Hg(II) and 1.4- to 3-fold increases with Cu(II). These results suggest that D. vulgaris uses an ATP-dependent mechanism for adapting to toxic metals in the environment.     

Differential Expression of Desulfovibrio vulgaris Genes in Response to Cu(II) and Hg(II) Toxicity

The response of Desulfovibrio vulgaris to Cu(II) and Hg(II) was characterized. Both metals increased the lag phase, and Cu(II) reduced cell yield at concentrations as low as 50 μM. mRNA expression was analyzed using random arbitrarily primed PCR, differential display, and quantitative PCR. Both Cu(II) and Hg(II) (50 μM) caused upregulation of mRNA expression for an ATP binding protein (ORF2004) and an ATPase (ORF856) with four- to sixfold increases for Hg(II) and 1.4- to 3-fold increases with Cu(II). These results suggest that D. vulgaris uses an ATP-dependent mechanism for adapting to toxic metals in the environment.     

Cr(VI) detoxification by Desulfovibrio vulgaris strain Hildenborough: microbe–metal interactions studies

Toxic heavy metals constitute a worldwide environmental pollution problem. Bioremediation technologies represent efficient alternatives to the classic cleaning-up of contaminated soil and ground water. Most toxic heavy metals such as chromium are less soluble and toxic when reduced than when oxidized. Sulfate-reducing bacteria (SRB) are able to reduce heavy metals by a chemical reduction via the production of H₂S and by a direct enzymatic process involving hydrogenases and c₃ cytochromes. We have previously reported the effects of chromate [Cr(VI)] on SRB bioenergetic metabolism and the molecular mechanism of the metal reduction by polyhemic cytochromes. In the current work, we pinpoint the bacteria–metal interactions using Desulfovibrio vulgaris strain Hildenborough as a model. The bacteria were grown in the presence of high Cr(VI) concentration, where they accumulated precipitates of a reduced form of chromium, trivalent chromium [Cr(III)], on their cell surfaces. Moreover, the inner and outer membranes exhibited precipitates that shared the spectroscopic signature of trivalent chromium. This subcellular localization is consistent with enzymatic metal reduction by cytochromes and hydrogenases. Regarding environmental significance, our findings point out the Cr(VI) immobilization mechanisms of SRB; suggesting that SRB are highly important in metal biogeochemistry.     

Orange protein from <Emphasis Type="Italic">Desulfovibrio alaskensis</Emphasis> G20: insights into the Mo–Cu cluster protein-assisted synthesis

A novel metalloprotein containing a unique [S₂MoS₂CuS₂MoS₂]^3? cluster, designated as Orange Protein (ORP), was isolated for the first time from Desulfovibrio gigas, a sulphate reducer. The orp operon is conserved in almost all sequenced Desulfovibrio genomes and in other anaerobic bacteria, however, so far D. gigas ORP had been the only ORP characterized in the literature. In this work, the purification of another ORP isolated form Desulfovibrio alaskensis G20 is reported. The native protein is monomeric (12443.8 ± 0.1 Da by ESI–MS) and contains also a MoCu cluster with characteristic absorption bands at 337 and 480 nm, assigned to S–Mo charge transfer bands. Desulfovibrio alaskensis G20 recombinant protein was obtained in the apo-form from E. coli. Cluster reconstitution studies and UV–visible titrations with tetrathiomolybdate of the apo-ORP incubated with Cu ions indicate that the cluster is incorporated in a protein metal-assisted synthetic mode and the protein favors the 2Mo:1Cu stoichiometry. In Desulfovibrio alaskensis G20, the orp genes are encoded by a polycistronic unit composed of six genes whereas in Desulfovibrio vulgaris Hildenborough the same genes are organized into two divergent operons, although the composition in genes is similar. The gene expression of ORP (Dde_3198) increased 6.6 ± 0.5 times when molybdate was added to the growth medium but was not affected by Cu(II) addition, suggesting an involvement in molybdenum metabolism directly or indirectly in these anaerobic bacteria.     

The bacterial metallome: composition and stability with specific reference to the anaerobic bacterium Desulfovibrio desulfuricans

In bacteria, the intracellular metal content or metallome reflects the metabolic requirements of the cell. When comparing the composition of metals in phytoplankton and bacteria that make up the macronutrients and the trace elements, we have determined that the content of trace elements in both of these microorganisms is markedly similar. The trace metals consisting of transition metals plus zinc are present in a stoichometric molar formula that we have calculated to be as follows: Fe₁Mn_0.3Zn_0.26Cu_0.03Co_0.03Mo_0.03. Under conditions of routine cultivation, trace metal homeostasis may be maintained by a series of transporter systems that are energized by the cell. In specific environments where heavy metals are present at toxic levels, some bacteria have developed a detoxification strategy where the metallic ion is reduced outside of the cell. The result of this extracellular metabolism is that the bacterial metallome specific for trace metals is not disrupted. One of the microorganisms that reduces toxic metals outside of the cell is the sulfate-reducing bacterium Desulfovibrio desulfuricans. While D. desulfuricans reduces metals by enzymatic processes involving polyhemic cytochromes c ₃ and hydrogenases, which are all present inside the cell; we report the presence of chain B cytochrome c nitrite reductase, NrfA, in the outer membrane fraction of D. desulfuricans ATCC 27774 and discuss its activity as a metal reductase.     

Identification of genes that confer sediment fitness to Desulfovibrio desulfuricans G20

Signature-tagged mutants of Desulfovibrio desulfuricans G20 were screened, and 97 genes crucial for sediment fitness were identified. These genes belong to functional categories including signal transduction, binding and transport, insertion elements, and others. Mutants with mutations in genes encoding proteins involved in amino acid biosynthesis, hydrogenase activity, and DNA repair were further characterized.     

Functional expression of Desulfovibrio vulgaris Hildenborough cytochrome c3 in Desulfovibrio desulfuricans G200 after conjugational gene transfer from Escherichia coli.

Plasmid pJRDC800-1, containing the cyc gene encoding cytochrome c3 from Desulfovibrio vulgaris subsp. vulgaris Hildenborough, was transferred by conjugation from Escherichia coli DH5 alpha to Desulfovibrio desulfuricans G200. The G200 strain produced an acidic cytochrome c3 (pI = 5.8), which could be readily separated from the Hildenborough cytochrome c3 (pI = 10.5). The latter was indistinguishable from cytochrome c3 produced by D. vulgaris subsp. vulgaris Hildenborough with respect to a number of chemical and physical criteria.     

Identification of Genes That Confer Sediment Fitness to Desulfovibrio desulfuricans G20

Signature-tagged mutants of Desulfovibrio desulfuricans G20 were screened, and 97 genes crucial for sediment fitness were identified. These genes belong to functional categories including signal transduction, binding and transport, insertion elements, and others. Mutants with mutations in genes encoding proteins involved in amino acid biosynthesis, hydrogenase activity, and DNA repair were further characterized.     

Cytochrome c(3) mutants of Desulfovibrio desulfuricans

To explore the physiological role of tetraheme cytochrome c(3) in the sulfate-reducing bacterium Desulfovibrio desulfuricans G20, the gene encoding the preapoprotein was cloned, sequenced, and mutated by plasmid insertion. The physical analysis of the DNA from the strain carrying the integrated plasmid showed that the insertion was successful. The growth rate of the mutant on lactate with sulfate was comparable to that of the wild type; however, mutant cultures did not achieve the same cell densities. Pyruvate, the oxidation product of lactate, served as a poor electron source for the mutant. Unexpectedly, the mutant was able to grow on hydrogen-sulfate medium. These data support a role for tetraheme cytochrome c(3) in the electron transport pathway from pyruvate to sulfate or sulfite in D. desulfuricans G20.     

Improving sample treatment for in-solution protein identification by peptide mass fingerprint using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Three ultrasonic energy sources were studied to speed up the sample treatment for in-solution protein identification by peptide mass fingerprint using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Protein reduction, alkylation, and enzymatic digestion steps were done in 15 min. Nine proteins, including zinc resistance-associated protein precursor from Desulfovibrio desulfuricans strain G20 and split-soret cytochrome c from D. desulfuricans ATCC27774 were successfully identified with the new protocol.     

Oxidation of benzaldehydes to benzoic acid derivatives by three Desulfovibrio strains

Desulfovibrio vulgaris Marburg, "Desulfovibrio simplex" XVI, and Desulfovibrio sp. strain MP47 used benzaldehydes such as vanillin, 3,4,5-trimethoxybenzaldehyde, protocatechualdehyde, syringaldehyde, p-anisaldehyde, p-hydroxybenzaldehyde, and 2-methoxybenzaldehyde as electron donors for sulfate reduction and carbon dioxide and/or components of yeast extract as carbon sources for cell synthesis. The aldehydes were oxidized to their corresponding benzoic acids. The three sulfate reducers oxidized up to 7 mM vanillin and up to 4 mM p-anisaldehyde. Higher concentrations of vanillin or p-anisaldehyde were toxic. In addition, pyridoxal hydrochloride and o-vanillin served as electron donors for sulfate reduction. Salicylaldehyde, pyridine-2-aldehyde, pyridine-4-aldehyde, and 4-hydroxy-3-methoxybenzylalcohol were not oxidized. No molecular hydrogen was detected in the gas phase. The oxidized aldehydes were not further degraded.     

Oxidation of benzaldehydes to benzoic acid derivatives by three Desulfovibrio strains.

Desulfovibrio vulgaris Marburg, "Desulfovibrio simplex" XVI, and Desulfovibrio sp. strain MP47 used benzaldehydes such as vanillin, 3,4,5-trimethoxybenzaldehyde, protocatechualdehyde, syringaldehyde, p-anisaldehyde, p-hydroxybenzaldehyde, and 2-methoxybenzaldehyde as electron donors for sulfate reduction and carbon dioxide and/or components of yeast extract as carbon sources for cell synthesis. The aldehydes were oxidized to their corresponding benzoic acids. The three sulfate reducers oxidized up to 7 mM vanillin and up to 4 mM p-anisaldehyde. Higher concentrations of vanillin or p-anisaldehyde were toxic. In addition, pyridoxal hydrochloride and o-vanillin served as electron donors for sulfate reduction. Salicylaldehyde, pyridine-2-aldehyde, pyridine-4-aldehyde, and 4-hydroxy-3-methoxybenzylalcohol were not oxidized. No molecular hydrogen was detected in the gas phase. The oxidized aldehydes were not further degraded.     

Desulfovibrio longreachii sp. nov., a sulfate-reducing bacterium isolated from the Great Artesian Basin of Australia

Abstract A new mesophilic, thermotolerant sulfate-reducing bacterium, was isolated from the flowing bore waters of a deep aquifer, the Great Artesian Basin, Australia. The strain, designated isolate AB16910a, is a curved rod and resembled members of the genus Desulfovibrio . However, the isolate can be differentiated from other members of the Desulfovibrio species because of the high G+C content of 69±0.25% the 16S rRNA sequence data and other physiological characteristics. The name Desulfovibrio longreachii is proposed for the new isolate.     

Proteome analysis of Desulfovibrio desulfuricans G20 mutants using the accurate mass and time (AMT) tag approach

Abundance values obtained from direct LC-MS analyses were used to compare the proteomes of six transposon-insertion mutants of Desulfovibrio desulfuricans G20, the lab strain (G20lab) and a sediment-adapted strain (G20sediment). Three mutations were in signal transduction histidine kinases, and three mutations were in other regulatory proteins. The high-throughput accurate mass and time (AMT) tag proteomic approach was utilized to analyze the proteomes. A total of 1318 proteins was identified with high confidence, approximately 35% of all predicted proteins in the D. desulfuricans G20 genome. Proteins from all functional categories were identified. Significant differences in the abundance of 30 proteins were detected between the G20lab strain and the G20sediment strain. Abundances of proteins for energy metabolism, ribosomal synthesis, membrane biosynthesis, transport, and flagellar synthesis were affected in the mutants. Specific examples of proteins down-regulated in mutants include a putative tungstate transport system substrate-binding protein and several proteins related to energy production, for example, 2-oxoacid:acceptor oxidoreductase, cytochrome c-553, and formate acetyltransferase. In addition, several signal transduction mechanism proteins were regulated in one mutant, and the abundances of ferritin and hybrid cluster protein were reduced in another mutant. However, the similar abundance of universal stress proteins, heat shock proteins, and chemotaxis proteins in the mutants revealed that regulation of chemotactic behavior and stress regulation might not be observed under our growth conditions. This study provides the first proteomic overview of several sediment fitness mutants of G20, and evidence for the difference between lab strains and sediment-adapted strains at the protein level.     

Immunological cross-reactivities of adenosine-5'-phosphosulfate reductases from sulfate-reducing and sulfide-oxidizing bacteria

Crude extracts from 14 species of sulfate-reducing bacteria comprising the genera Desulfovibrio, Desulfotomaculum, Desulfobulbus, and Desulfosarcina and from three species of sulfide-oxidizing bacteria were tested in an enzyme-linked immunosorbent assay with polyclonal antisera to adenosine 5'-phosphosulfate reductase from Desulfovibrio desulfuricans G100A. The results showed that extracts from Desulfovibrio species were all highly cross-reactive, whereas extracts from the other sulfate-reducing genera showed significantly less cross-reaction. An exception was Desulfotomaculum orientis, which responded more like Desulfovibrio species than the other Desulfotomaculum strains tested. Extracts from colorless or photosynthetic sulfur bacteria were either unreactive or exhibited very low levels of reactivity with the antibodies to the enzyme from sulfate reducers. These results were confirmed by using partially purified enzymes from sulfate reducers and the most cross-reactive sulfide oxidizer, Thiobacillus denitrificans. Two types of monoclonal antibodies to adenosine 5'-phosphosulfate reductase were also isolated. One type reacted more variably with the enzymes of the sulfate reducers and poorly with the Thiobacillus enzyme, whereas the second reacted strongly with Desulfovibrio, Desulfotomaculum orientis, and Thiobacillus enzymes.     

EPR and redox properties of Desulfovibrio vulgaris Miyazaki hydrogenase: comparison with the Ni-Fe enzyme from Desulfovibrio gigas.

We have carried out a detailed redox titration monitored by EPR on the hydrogenase from Desulfovibrio vulgaris Miyazaki. Typical 3Fe and nickel signals have been observed, which are very similar to those given by Desulfovibrio gigas hydrogenase in all the characteristic redox states of the enzyme. This confirms that D. vulgaris Miyazaki hydrogenase is a Ni-Fe enzyme closely related to that from D. gigas, as was recently proposed on the basis of sequence comparisons (Deckers, H.M., Wilson, F.R. and Voordouw, G. (1990) J. Gen. Microb. 136, 2021-2028).     

Tolerance and stress response of sclerotiogenic <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> G15 to copper and lead

Aspergillus oryzae G15 was cultured on Czapek yeast extract agar medium containing different concentrations of copper and lead to investigate the mechanisms sustaining metal tolerance. The effects of heavy metals on biomass, metal accumulation, metallothionein (MT), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) were evaluated. Cu and Pb treatment remarkably delayed sclerotial maturation and inhibited mycelial growth, indicating the toxic effects of the metals. Cu decreased sclerotial biomass, whereas Pb led to an increase in sclerotial biomass. G15 bioadsorbed most Cu and Pb ions on the cell surface, revealing the involvement of the extracellular mechanism. Cu treatment significantly elevated MT level in mycelia, and Pb treatment at concentrations of 50–100 mg/L also caused an increase in MT content in mycelia. Both metals significantly increased MDA level in sclerotia. The variations in MT and MDA levels revealed the appearance of heavy metal-induced oxidative stress. The activities of SOD, CAT, and POD varied with heavy metal concentrations, which demonstrated that tolerance of G15 to Cu and Pb was associated with an efficient antioxidant defense system. In sum, the santioxidative detoxification system allowed the strain to survive in high concentrations of Cu and Pb. G15 depended mostly on sclerotial differentiation to defend against Pb stress.     

Uranium reduction by Desulfovibrio desulfuricans strain G20 and a cytochrome c3 mutant

Previous in vitro experiments with Desulfovibrio vulgaris strain Hildenborough demonstrated that extracts containing hydrogenase and cytochrome c3 could reduce uranium(VI) to uranium(IV) with hydrogen as the electron donor. To test the involvement of these proteins in vivo, a cytochrome c3 mutant of D. desulfuricans strain G20 was assayed and found to be able to reduce U(VI) with lactate or pyruvate as the electron donor at rates about one-half of those of the wild type. With electrons from hydrogen, the rate was more severely impaired. Cytochrome c3 appears to be a part of the in vivo electron pathway to U(VI), but additional pathways from organic donors can apparently bypass this protein.     

Adhesion of biodegradative anaerobic bacteria to solid surfaces.

In order to exploit the ability of anaerobic bacteria to degrade certain contaminants for bioremediation of polluted subsurface environments, we need to understand the mechanisms by which such bacteria partition between aqueous and solid phases, as well as the environmental conditions that influence partitioning. We studied four strictly anaerobic bacteria, Desulfomonile tiedjei, Syntrophomonas wolfei, Syntrophobacter wolinii, and Desulfovibrio sp. strain G11, which theoretically together can constitute a tetrachloroethylene- and trichloroethylene-dechlorinating consortium. Adhesion of these organisms was evaluated by microscopic determination of the numbers of cells that attached to glass coverslips exposed to cell suspensions under anaerobic conditions. We studied the effects of the growth phase of the organisms on adhesion, as well as the influence of electrostatic and hydrophobic properties of the substratum. Results indicate that S. wolfei adheres in considerably higher numbers to glass surfaces than the other three organisms. Starvation greatly decreases adhesion of S. wolfei and Desulfovibrio sp. strain G11 but seems to have less of an effect on the adhesion of the other bacteria. The presence of Fe(3+) on the substratum, which would be electropositive, significantly increased the adhesion of S. wolfei, whereas the presence of silicon hydrophobic groups decreased the numbers of attached cells of all species. Measurements of transport of cells through hydrophobic-interaction and electrostatic-interaction columns indicated that all four species had negatively charged cell surfaces and that D. tiedjei and Desulfovibrio sp. strain G11 possessed some hydrophobic cell surface properties. These findings are an early step toward understanding the dynamic attachment of anaerobic bacteria in anoxic environments.     

Regulation of arsenate resistance in Desulfovibrio desulfuricans G20 by an arsRBCC operon and an arsC gene

Desulfovibrio desulfuricans G20 grows and reduces 20 mM arsenate to arsenite in lactate-sulfate media. Sequence analysis and experimental data show that D. desulfuricans G20 has one copy of arsC and a complete arsRBCC operon in different locations within the genome. Two mutants of strain G20 with defects in arsenate resistance were generated by nitrosoguanidine mutagenesis. The arsRBCC operons were intact in both mutant strains, but each mutant had one point mutation in the single arsC gene. Mutants transformed with either the arsC1 gene or the arsRBCC operon displayed wild-type arsenate resistance, indicating that the two arsC genes were equivalently functional in the sulfate reducer. The arsC1 gene and arsRBCC operon were also cloned into Escherichia coli DH5alpha independently, with either DNA fragment conferring increased arsenate resistance. The recombinant arsRBCC operon allowed growth at up to 50 mM arsenate in LB broth. Quantitative PCR analysis of mRNA products showed that the single arsC1 was constitutively expressed, whereas the operon was under the control of the arsR repressor protein. We suggest a model for arsenate detoxification in which the product of the single arsC1 is first used to reduce arsenate. The arsenite formed is then available to induce the arsRBCC operon for more rapid arsenate detoxification.