Toxicity of Al to Desulfovibrio desulfuricans

The toxicity of Al to Desulfovibrio desulfuricans G20 was assessed over a period of 8 weeks in a modified lactate C medium buffered at four initial pHs (5.0, 6.5, 7.2, and 8.3) and treated with five levels of added Al (0, 0.01, 0.1, 1.0, and 10 mM). At pH 5, cell population densities decreased significantly and any effect of Al was negligible compared to that of the pH. At pHs 6.5 and 7.2, the cell population densities increased by 30-fold during the first few days and then remained stable for soluble-Al concentrations of <5 x 10(-5) M. In treatments having total-Al concentrations of > or =1 mM, soluble-Al concentrations exceeded 5 x 10(-5) M and limited cell population growth substantially and proportionally. At pH 8.3, soluble-Al concentrations were below the 5 x 10(-5) M toxicity threshold and cell population density increases of 20- to 40-fold were observed. An apparent cell population response to added Al at pH 8.3 was attributed to the presence of large, spirilloidal bacteria (accounting for as much as 80% of the cells at the 10 mM added Al level). Calculations of soluble-Al speciation for the pH 6.5 and 7.2 treatments that showed Al toxicity suggested the possible presence of the Al(13)O(4)(OH)(24)(H(2)O)(12)(7+) "tridecamer" cation and an inverse correlation of the tridecamer concentration and the cell population density. Analysis by (27)Al nuclear magnetic resonance spectroscopy, however, yielded no evidence of this species in freshly prepared samples or those taken 800 days after inoculation. Exclusion of the tridecamer species from the aqueous speciation calculations at pHs 6.5 and 7.2 yielded inverse correlations of the neutral Al(OH)(3) and anionic Al(OH)(4)(-) monomeric species with cell population density, suggesting that one or both of these ions bear primary responsibility for the toxicity observed.     

Serine Biosynthesis in Desulfovibrio desulfuricans

Cell-free extracts of Desulfovibrio desulfuricans possess enzymes which catalyze the synthesis of serine from 3-phosphoglycerate via the intermediates phosphohydroxypyruvate and phosphoserine.     

Characterization of sulfate transport in Desulfovibrio desulfuricans

Uptake of ³⁵S-labelled sulfate was studied with a new isolate of Desulfovibrio desulfuricans, strain CSN. Micromolar additions of sulfate (1–10 M or nmol/mg protein) to cell suspensions incubated in 150 mM KCl at-1°C were almost completely taken up and accumulated about 5,000-fold. Accumulation was not influenced by incubation in NaCl instead of KCl, by acidic pH (5.5) or by incubation under air for 10 min. In alkaline milieu (pH 8.5), after prolonged contact with air (2 h), or after growth with excess sulfate or thiosulfate as electron acceptor, the amount taken up was diminished approximately by half. Pasteurization inhibited sulfate uptake completely. With increasing concentrations of added sulfate (0.1 to 2.5 mM) the intracellular concentration increased only slowly up to 25 mM, and the accumulation factor decreased down to 8. Sulfate transport was reversible. Accumulated sulfate was rapidly lost from the cells after addition of excess non-labelled sulfate or after addition of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP). The ATPase inhibitor dicyclohexylcarbodiimide (DCCD) specifically inhibited sulfate reduction but had no immediate influence on sulfate accumulation. Addition of the phosphate analogue arsenate (5 mM) was without effect. These results were not in favour of an ATP-dependent transport system. The K⁺-H⁺-antiporter nigericin (in 150 mM KCl) and the Na⁺-H⁺-antiporter monensin (in 150 mM NaCl) caused partial inhibition of sulfate accumulation, whereas the K⁺-transporter valinomycin (in 150 mM KCl) and the Na⁺-H⁺ exchange inhibitor amiloride (2 mM) were without effect. The permeant thiocyanate anion (150 mM) inhibited sulfate uptake by 60% at pH 7, and completely at pH 8.5. Although the effects of the different ionophores on the chemiosmotic gradients have not been studied so far, the results indicated that probably both, pH and drive sulfate accumulation and that sulfate is taken up electrogenically in symport with more than 2 protons. The structural sulfate analogues tungstate and molybdate (0.1 mM, each) did not affect sulfate accumulation, although molybdate inhibited sulfate reduction. Chromate completely blocked both of these activities. Sulfite and selenite caused little or no decrease of sulfate accumulation, whereas with thiosulfate and selenate significant inhibition was observed.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - DCCD dicyclohexylcarbodiimide     

Reduction of uranium by Desulfovibrio desulfuricans.

The possibility that sulfate-reducing microorganisms contribute to U(VI) reduction in sedimentary environments was investigated. U(VI) was reduced to U(IV) when washed cells of sulfate-grown Desulfovibrio desulfuricans were suspended in a bicarbonate buffer with lactate or H2 as the electron donor. There was no U(VI) reduction in the absence of an electron donor or when the cells were killed by heat prior to the incubation. The rates of U(VI) reduction were comparable to those in respiratory Fe(III)-reducing microorganisms. Azide or prior exposure of the cells to air did not affect the ability of D. desulfuricans to reduce U(VI). Attempts to grow D. desulfuricans with U(VI) as the electron acceptor were unsuccessful. U(VI) reduction resulted in the extracellular precipitation of the U(IV) mineral uraninite. The presence of sulfate had no effect on the rate of U(VI) reduction. Sulfate and U(VI) were reduced simultaneously. Enzymatic reduction of U(VI) by D. desulfuricans was much faster than nonenzymatic reduction of U(VI) by sulfide, even when cells of D. desulfuricans were added to provide a potential catalytic surface for the nonenzymatic reaction. The results indicate that enzymatic U(VI) reduction by sulfate-reducing microorganisms may be responsible for the accumulation of U(IV) in sulfidogenic environments. Furthermore, since the reduction of U(VI) to U(IV) precipitates uranium from solution, D. desulfuricans might be a useful organism for recovering uranium from contaminated waters and waste streams.     

Reduction of uranium by Desulfovibrio desulfuricans.

The possibility that sulfate-reducing microorganisms contribute to U(VI) reduction in sedimentary environments was investigated. U(VI) was reduced to U(IV) when washed cells of sulfate-grown Desulfovibrio desulfuricans were suspended in a bicarbonate buffer with lactate or H2 as the electron donor. There was no U(VI) reduction in the absence of an electron donor or when the cells were killed by heat prior to the incubation. The rates of U(VI) reduction were comparable to those in respiratory Fe(III)-reducing microorganisms. Azide or prior exposure of the cells to air did not affect the ability of D. desulfuricans to reduce U(VI). Attempts to grow D. desulfuricans with U(VI) as the electron acceptor were unsuccessful. U(VI) reduction resulted in the extracellular precipitation of the U(IV) mineral uraninite. The presence of sulfate had no effect on the rate of U(VI) reduction. Sulfate and U(VI) were reduced simultaneously. Enzymatic reduction of U(VI) by D. desulfuricans was much faster than nonenzymatic reduction of U(VI) by sulfide, even when cells of D. desulfuricans were added to provide a potential catalytic surface for the nonenzymatic reaction. The results indicate that enzymatic U(VI) reduction by sulfate-reducing microorganisms may be responsible for the accumulation of U(IV) in sulfidogenic environments. Furthermore, since the reduction of U(VI) to U(IV) precipitates uranium from solution, D. desulfuricans might be a useful organism for recovering uranium from contaminated waters and waste streams.     

Cytochrome c3 Mutants of Desulfovibrio desulfuricans

To explore the physiological role of tetraheme cytochrome c₃ 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 c₃ in the electron transport pathway from pyruvate to sulfate or sulfite in D. desulfuricans G20.     

Pyruvate-carbon dioxide exchange reaction of Desulfovibrio desulfuricans

Suh, Byungse (University of Kansas, Lawrence), and J. M. Akagi. Pyruvate-carbon dioxide exchange reaction of Desulfovibrio desulfuricans. J. Bacteriol. 91:2281-2285. 1966.-The pyruvate-CO(2) exchange reaction, catalyzed by Desulfovibrio desulfuricans, required the presence of phosphate and coenzyme A. However, the requirement for phosphate disappeared when the concentration of coenzyme A was increased to a level of 3.8 x 10(-3)m. Passing crude extracts through a diethylaminoethyl-cellulose column and an Amberlite CG-50 ion-exchange column, to remove ferredoxin and cytochrome c(3), resulted in a marked decrease in exchange activity; full activity was restored by the addition of ferredoxin or cytochrome c(3). Fe(++) or Co(++) stimulated the exchange of CO(2) into pyruvate.     

Plasmid transfer by conjugation in Desulfovibrio desulfuricans

Conjugational transfer of several IncQ plasmids from Escherichia coli to the strictly anaerobic, sulfate-reducing bacterium Desulfovibrio desulfuricans strain G100A was demonstrated. Plasmid DNA from exconjugants was visualized on agarose gels and was used to transform E. coli to the appropriate antibiotic resistances. Neither transfer of IncW and IncP plasmids to strain G100A, nor transfer of any plasmid to D. desulfuricans strain ATCC 27774 was observed. Conjugation of suicide plasmids containing either Tn5 or Tn9 into D. desulfuricans did not result in detectable transposition. Optimal conditions for conjugational transfer and antibiotic resistance levels of strain G100A were examined.     

Sulfide Production from Cysteine by Desulfovibrio desulfuricans

Two rumen nitrate-reducing isolates of Desulfovibrio desulfuricans were found to hydrolyze cysteine with the production of sulfide and pyruvate. When cultured on agar medium containing yeast extract with nitrate as the primary electron acceptor and ferrous chloride as the indicator, blackening of colonies occurred. The blackening of colonies appeared sooner and was more intense when either cysteine or sulfate was added to the culture medium with nitrate present.     

Metabolic Pathways Leading to Mercury Methylation in Desulfovibrio desulfuricans LS

The synthesis of methylmercury by Desulfovibrio desulfuricans LS was investigated on the basis of ¹⁴C incorporation from precursors and the measurement of relevant enzyme activities in cell extracts. The previously observed incorporation of C-3 from serine into methylmercury was confirmed by measurement of relatively high activities of serine hydroxymethyltransferase and other enzymes of this pathway. High rates of label incorporation into methylmercury from H¹⁴COO^- and H¹⁴CO₃^- prompted the assay of enzymes of the acetyl coenzyme A (CoA) synthase pathway. These enzymes were found to be present but at activity levels much lower than those reported for acetogens. Propyl iodide inhibited methylmercury and acetyl-CoA syntheses to similar extents, and methylmercury synthesis was found to compete with acetyl-CoA synthesis for methyl groups. On the basis of these findings, we propose that in methylmercury synthesis by D. desulfuricans LS the methyl group is transferred from CH₃-tetrahydrofolate via methylcobalamin. The methyl group may originate from C-3 of serine or from formate via the acetyl-CoA synthase pathway. These pathways are not unique to D. desulfuricans LS, and thus the ability of this bacterium to methylate mercury is most likely associated with the substrate specificity of its enzymes.     

The preferred electron acceptor of Desulfovibrio desulfuricans CSN

Abstract: The sulfate-reducing bacterium Desulfovibrio desulfuricans strain CSN (DSM 104) oxidized H₂ with thiosulfate, sulfate, sulfite, nitrite, nitrate and oxygen with rates increasing (in the order listed) from 20 to 525 nmol H₂ min⁻¹ mg⁻¹ protein. Nitrate reduction was induced by nitrate or limiting concentrations of sulfate during growth, while all other activities were constitutive. Oxygen prevented reduction of all other electron acceptors, while nitrate and nitrite blocked the reduction of the sulfur compounds. In the presence of H₂ and reduced sulfur compounds, H₂ was the preferred electron donor. The cells oxidized thiosulfate or sulfite coupled to the reduction of nitrate to ammonia. This represents a novel type of metabolism connecting the sulfur and nitrogen cycles. It is concluded that oxygen is the preferred electron acceptor of D. desulfuricans . Sulfate reduction in oxic environments must be due to different organisms or mechanisms.     

Adhesion of Desulfovibrio desulfuricans and Pseudomonas fluorescens to mild steel surfaces

The adhesion of micro-organisms to metal surfaces has been shown to be important in the corrosion process, but the cell surface structures participating in this adhesion have not previously been identified. Evidence is presented that a bacterial substance taking part in the initial adhesion of Pseudomonas fluorescens and Desulfovibrio desulfuricans (New Jersey) to mild steel is polysaccharide in nature. It is likely that this is present in the outer membrane of the bacterial cells as lipopolysaccharide.     

Methylmercury Resistance in Desulfovibrio desulfuricans Strains in Relation to Methylmercury Degradation

Two strains of Desulfovibrio desulfuricans, one known to synthesize monomethylmercury from ionic mercury, were grown to determine methylmercury toxicity and for comparison with an anaerobic strain of Clostridium pasteurianum, a H₂ producer, and with the broad-spectrum mercury-resistant Pseudomonas putida strain FB-1, capable of degrading 1 μg of methylmercury to methane and elemental mercury in 2 h. The CH₃HgCl resistance of D. desulfuricans strains was 10 times that of P. putida FB-1 and 100 times that of C. pasteurianum. The methylmercury resistance of D. desulfuricans was related to the disappearance of methylmercury from cultures by transformation to dimethylmercury, metacinnabar, methane, and traces of ionic mercury. During a 15-day experiment the kinetics of the two volatile compounds dimethylmercury [(CH₃)₂Hg] and methane were monitored in the liquid by a specific new technique with purge-and-trap gas chromatography in line with Fourier transform infrared spectroscopy and in the headspace by gas chromatography with flame ionization detection. Insoluble metacinnabar (cubic HgS) of biological origin was detected by X-ray diffractometry in the gray precipitate from the insoluble residue of the pellet of a 1-liter culture spiked with 100 mg of CH₃HgCl. This was compared with a 1-liter culture of D. desulfuricans LS spiked with 100 mg of HgCl₂. In a further experiment, it was demonstrated that insoluble, decomposable, white dimethylmercury sulfide [(CH₃Hg)₂S] formed instantly in the reaction of methylmercury with hydrogen sulfide. This organomercurial was extracted with chloroform and identified by gas chromatography in line with mass spectrometry. The D. desulfuricans strains were resistant to high concentrations of methylmercury because they produced insoluble dimethylmercury sulfide, which slowly decomposed under anaerobic conditions to metacinnabar and volatilized to dimethylmercury and methane between pHs 6.2 and 6.5 for high (4.5-g · liter^-1) or low (0.09-g · liter^-1) sulfate contents. Methane was produced from CH₃HgCl at a lower rate than by the broad-spectrum Hg-resistant P. putida strain FB-1.     

Cobalamin-mediated mercury methylation by Desulfovibrio desulfuricans LS.

The prominence of sulfate reducers in mercury biomethylation prompted the examination of the methyl carrier and mercury methylation activity of Desulfovibrio desulfuricans LS. There was a low degree of mercury tolerance and a high degree of methylation during fermentative growth; the opposite was true during sulfate reduction. During 2 days of fermentative growth, up to 37% of HgCl2 was methylated at 0.1 micrograms/ml, but only 1.5% was methylated at 10.0 micrograms/ml. Less than 1% of the added HgCl2 was methylated under sulfate-reducing conditions. D. desulfuricans LS radioimmunoassay results were positive for cobalamin. The addition of CoCl2 and benzimidazole to fermentative cultures increased methylation activity. From D. desulfuricans LS grown in the presence of (57)CoCl2, a corrinoid was extracted and purified. High-performance liquid chromatography analysis of the purified extract yielded a single peak with the retention time of cobalamin, and 97% of the (57)Co radioactivity was associated with this peak. Fast atom bombardment and UV and visible spectra of the isolated corrinoid matched those of cobalamin. When methylated with (14)CH3I, the isolated corrinoid methylated Hg(2+) with a 93.9% preservation of (14)C specific activity. We conclude that D. desulfuricans LS methylates mercury via cobalamin (vitamin B12). Under physiological conditions, the enzymatic catalysis of this reaction is likely.     

Carbon Flow in Mercury Biomethylation by Desulfovibrio desulfuricans

Radiocarbon incorporation from pyruvate and serine into monomethylmercury by Desulfovibrio desulfuricans was consistent with the proposal that the methyl group originates from C-3 of serine. Immunodiagnostic assays measured 4 to 35 μg of tetrahydrofolate and 58 to 161 ng of cobalamin or a closely related cobalt porphyrin per g of cell protein in D. desulfuricans. The light-reversible inhibition of mercury methylation by propyl iodide in D. desulfuricans indicates methyl transfer by a cobalt porphyrin.