Energy coupling to nitrite respiration in the sulfate-reducing bacterium Desulfovibrio gigas.

By use of a membrane fraction prepared from Desulfovibrio gigas grown in a lactate-sulfate medium, synthesis of ATP was demonstrated to be coupled to the oxidation of molecular hydrogen and reduction of either nitrite or hydroxylamine. This phosphorylation was uncoupled from electron transport by pentachlorophenol, methyl viologen, and gramicidin, but not by oligomycin. The extrusion of protons from the cells was shown to be coupled to the hydrogen-nitrite respiratory system, and, assuming the localization of nitrite reductase on the outer side of the plasma membrane, H+/2e- values of 2.0 +/- 0.3 were obtained. Energy coupling observed with this system appears to be due to electron transfer-coupled proton translocation rather than vectorial electron transfer associated with hydrogen oxidation.     

Pathways for utilization of carbon reserves in Desulfovibrio gigas under fermentative and respiratory conditions.

The sulfate-reducing bacterium Desulfovibrio gigas accumulates large amounts of polyglucose as an endogenous carbon and energy reserve. In the absence of exogenous substrates, the intracellular polysaccharide was utilized, and energy was conserved in the process (H. Santos, P. Fareleira, A. V. Xavier, L. Chen, M.-Y. Liu, and J. LeGall, Biochem. Biophys. Res. Commun. 195:551-557, 1993). When an external electron acceptor was not provided, degradation of polyglucose by cell suspensions of D. gigas yielded acetate, glycerol, hydrogen, and ethanol. A detailed investigation of the metabolic pathways involved in the formation of these end products was carried out, based on measurements of the activities of glycolytic enzymes in cell extracts, by either spectrophotometric or nuclear magnetic resonance (NMR) assays. All of the enzyme activities associated with the glycogen cleavage and the Embden-Meyerhof pathway were determined as well as those involved in the formation of glycerol from dihydroxyacetone phosphate (glycerol-3-phosphate dehydrogenase and glycerol phosphatase) and the enzymes that catalyze the reactions leading to the production of ethanol (pyruvate decarboxylase and ethanol dehydrogenase). The key enzymes of the Entner-Doudoroff pathway were not detected. The methylglyoxal bypass was identified as a second glycolytic branch operating simultaneously with the Embden-Meyerhof pathway. The relative contribution of these two pathways for polyglucose degradation was 2:3. 13C-labeling experiments with cell extracts using isotopically enriched glucose and 13C-NMR analysis supported the proposed pathways. The information on the metabolic pathways involved in polyglucose catabolism combined with analyses of the end products formed from polyglucose under fermentative conditions provided some insight into the role of NADH in D. gigas. In the presence of electron acceptors, NADH resulting from polyglucose degradation was utilized for the reduction of sulfate, thiosulfate, or nitrite, leading to the formation of acetate as the only carbon end product besides CO2. Evidence supporting the role of NADH as a source of reducing equivalents for the production of hydrogen is also presented.     

Purification and properties of protoporphyrinogen oxidase from an anaerobic bacterium, Desulfovibrio gigas.

Protoporphyrinogen oxidase has been solubilized from plasma membranes of Desulfovibrio gigas. The enzyme was purified to apparent homogeneity with single silver-stained protein bands on isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gels. This protoporphyrinogen oxidase has a molecular weight (Mr) of 148,000 and is composed of three dissimilar subunits of Mrs 12,000, 18,500, and 57,000, which are held together by sulfhydryl bonds. Unlike other protoporphyrinogen oxidases, which use molecular oxygen as an electron acceptor, this enzyme does not couple to oxygen. The protoporphyrinogen oxidase donates electrons to 2,6-dichlorophenol-indophenol but not to NAD+, NADP+, flavin adenine dinucleotide, or flavin mononucleotide. The natural physiological electron acceptor of the protoporphyrinogen oxidase from D. gigas is unknown. By using 2,6-dichlorophenol-indophenol as the electron acceptor, the Km and Vmax values for oxidation of protoporphyrinogen were determined to be 21 microM and 8.38 nmol/min per 70 micrograms of protein, respectively. The catalytic rate constant, Kcat, was calculated to be 17.7 mol of protoporphyrin formed per mole of enzyme per min of incubation, and the Kcat/Km was 0.84. Energies of activation were calculated from Arrhenius plots with 7,429 cal (ca. 31,080 J)/mol per degree below 10 degrees C and 1,455 cal (ca. 6,088, J)/mol per degree above 10 degrees C. Optimum enzyme activity was at 23 degrees C, and inhibition was observed with both N-ethylmaleimide and iodoacetamide.     

A genomic island of the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough promotes survival under stress conditions while decreasing the efficiency of anaerobic growth

A 47 kb genomic island (GEI) bracketed by 50 bp direct repeats, containing 52 annotated genes, was found to delete spontaneously from the genome of Desulfovibrio vulgaris Hildenborough. The island contains genes for site-specific recombinases and transposases, rubredoxin:oxygen oxidoreductase-1 (Roo1) and hybrid cluster protein-1 (Hcp1), which promote survival in air and nitrite stress. The numbering distinguishes these from the Roo2 and Hcp2 homologues for which the genes are located elsewhere in the genome. Cells with and without the island (GEI⁺ and GEI^- cells respectively) were obtained by colony purification. GEI^- cells arise in anaerobic cultures of colony-purified GEI⁺ cells, indicating that the site-specific recombinases encoded by the island actively delete this region. GEI⁺ cells survive better in microaerophilic conditions due to the presence of Roo1, whereas the Hcps appear to prevent inhibition by sulfur and polysulfide, which are formed by chemical reaction of sulfide and nitrite. Hence, the island confers resistance to oxygen and nitrite stress. However, GEI^- cells have a higher growth rate in anaerobic media. Microarrays and enzyme activity stains indicated that the GEI^- cells have increased expression of genes, which promote anaerobic energy conservation, explaining the higher growth rate. Hence, while lowering the efficiency of anaerobic metabolism, the GEI increases the fitness of D. vulgaris under stress conditions, a feature reminiscent of pathogenicity islands which allow more effective colonization of environments provided by the targeted hosts.     

Localization of dehydrogenases, reductases, and electron transfer components in the sulfate-reducing bacterium Desulfovibrio gigas.

Various dehydrogenases, reductases, and electron transfer proteins involved in respiratory sulfate reduction by Desulfovibrio gigas have been localized with respect to the periplasmic space, membrane, and cytoplasm. This species was grown on a lactate-sulfate medium, and the distribution of enzyme activities and concentrations of electron transfer components were determined in intact cells, cell fractions prepared with a French press, and lysozyme spheroplasts. A significant fraction of formate dehydrogenase was demonstrated to be localized in the periplasmic space in addition to hydrogenase and some c-type cytochrome. Cytochrome b, menaquinone, fumarate reductase, and nitrite reductase were largely localized on the cytoplasmic membrane. Fumarate reductase was situated on the inner aspect on the membrane, and the nitrite reductase appeared to be transmembraneous. Adenylylsulfate reductase, bisulfite reductase (desulfoviridin), pyruvate dehydrogenase, and succinate dehydrogenase activities were localized in the cytoplasm. Significant amounts of hydrogenase and c-type cytochromes were also detected in the cytoplasm. Growth of D. gigas on a formate-sulfate medium containing acetate resulted in a 10-fold increase in membrane-bound formate dehydrogenase and a doubling of c-type cytochromes. Growth on fumarate with formate resulted in an additional increase in b-type cytochrome compared with lactate-sulfate-grown cells.     

Transition from anaerobic to aerobic growth conditions for the sulfate-reducing bacterium Desulfovibrio oxyclinae results in flocculation

A chemostat culture of the sulfate-reducing bacterium Desulfovibrio oxyclinae isolated from the oxic layer of a hypersaline cyanobacterial mat was grown anaerobically and then subjected to gassing with 1% oxygen, both at a dilution rate of 0.05 h(-1). The sulfate reduction rate under anaerobic conditions was 370 nmol of SO(4)(2-) mg of protein(-1) min(-1). At the onset of aerobic gassing, sulfate reduction decreased by 40%, although viable cell numbers did not decrease. After 42 h, the sulfate reduction rate returned to the level observed in the anaerobic culture. At this stage the growth yield increased by 180% compared to the anaerobic culture to 4.4 g of protein per mol of sulfate reduced. Protein content per cell increased at the same time by 40%. The oxygen consumption rate per milligram of protein measured in washed cell suspensions increased by 80%, and the thiosulfate reduction rate of the same samples increased by 29% with lactate as the electron donor. These findings indicated possible oxygen-dependent enhancement of growth. After 140 h of growth under oxygen flux, formation of cell aggregates 0.1 to 3 mm in diameter was observed. Micrometer-sized aggregates were found to form earlier, during the first hours of exposure to oxygen. The respiration rate of D. oxyclinae was sufficient to create anoxia inside clumps larger than 3 microm, while the levels of dissolved oxygen in the growth vessel were 0.7 +/- 0.5 microM. Aggregation of sulfate-reducing bacteria was observed within a Microcoleus chthonoplastes-dominated layer of a cyanobacterial mat under daily exposure to oxygen concentrations of up to 900 microM. Desulfonema-like sulfate-reducing bacteria were also common in this environment along with other nonaggregated sulfate-reducing bacteria. Two-dimensional mapping of sulfate reduction showed heterogeneity of sulfate reduction activity in this oxic zone.     

Zinc-, cobalt- and iron-chelated forms of adenylate kinase from the Gram-negative bacterium Desulfovibrio gigas

Adenylate kinase (AK) from the sulphate-reducing bacterium Desulfovibrio gigas (AK) has been characterized earlier as a Co²⁺/Zn²⁺-containing enzyme, which is an unusual characteristic for adenylate kinases from Gram-negative bacteria, in which these enzymes are normally devoid of metal ions. AK was overexpressed in E. coli and homogeneous Co²⁺-, Zn²⁺- and Fe²⁺-forms of the enzyme were obtained under in vivo conditions. Their structural stability and spectroscopic and kinetic properties were compared. The thermal denaturation of Co²⁺- and Zn²⁺-forms of AK was studied as a cooperative two-state process, sufficiently reversible at pH 10, which can be correctly interpreted in terms of a simple two-state thermodynamic model. In contrast, the thermally induced denaturation of Fe²⁺-AK is irreversible and strongly dependent upon the scan rate, suggesting that this process is under kinetic control. Practically identical contents of secondary-structure elements were found for all the metal-chelated-forms of AK upon analysis of circular dichroism data, while their tertiary structures were significantly different. The peculiar tertiary structure of Fe²⁺-AK, in contrast to Co²⁺- and Zn²⁺-AK, and the consequent changes in the physico-chemical and enzymatic properties of the enzyme are discussed.     

Variations in the spectrum of desulfoviridin from Desulfovibrio gigas.

Desulfoviridin preparations from D. gigas showed variations in the position of the absorption maximum the beta-peak) in the 580-nm region of the specturm. On treatment with Na2S2O4 a preparation with a beta-peak at 585 nm was affected rapidly, the 585-nm peak shifting to the 596-nm region; this was partially reversed by K3Fe(CN)6. Treatment of the original preparation with K3Fe(CN)6 resulted in a shift of the beta-peak to 582-583 nm. Desulfoviridins with beta-peaks from 580 to 583 nm were not rapidly affected by Na2S2O4. The spectrum of the chromophore of desulfoviridin way also affected by Na2S2O4 with the peak at 587 nm shifting to 597 nm; this effect was completely reversed by oxygen. There was no evidence to show that spectral variations in desulfoviridin preparations were due to the loss or acquisition of metal ions during growth or to the selection of mutants containing spectrally different desulfoviridins. It is suggested that during biosynethesis oal detachment of the chromophore, thus causing a change towards the spectral properites of the detached chromophore.     

Oxidation of protoporphyrinogen in the obligate anaerobe Desulfovibrio gigas.

The anaerobic oxidation of protoporphyrinogen to protoporphyrin was demonstrated in extracts of Desulfovibrio gigas. Protoporphyrin formation occurred in the presence of nitrite, hydroxylamine, sulfite, thiosulfate, ATP plus sulfate, NAD+, NADP+, flavin adenine dinucleotide, flavin mononucleotide, fumarate, 2,6-dichlorophenol-indophenol, methyl viologen, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. With dialyzed cell extracts, highest activities were observed with sulfite, NAD+, and NADP+ as electron acceptors. The enzyme for protoporphyrinogen oxidation was localized in the membrane of D. gigas and displayed optimal activity at pH 7.3 and 28 degrees C.     

Direct electrochemistry of the Desulfovibrio gigas aldehyde oxidoreductase.

This work reports on the direct electrochemistry of the Desulfovibrio gigas aldehyde oxidoreductase (DgAOR), a molybdenum enzyme of the xanthine oxidase family that contains three redox-active cofactors: two [2Fe-2S] centers and a molybdopterin cytosine dinucleotide cofactor. The voltammetric behavior of the enzyme was analyzed at gold and carbon (pyrolytic graphite and glassy carbon) electrodes. Two different strategies were used: one with the molecules confined to the electrode surface and a second with DgAOR in solution. In all of the cases studied, electron transfer took place, although different redox reactions were responsible for the voltammetric signal. From a thorough analysis of the voltammetric responses and the structural properties of the molecular surface of DgAOR, the redox reaction at the carbon electrodes could be assigned to the reduction of the more exposed iron cluster, [2Fe-2S] II, whereas reduction of the molybdopterin cofactor occurs at the gold electrode. Voltammetric results in the presence of aldehydes are also reported and discussed.     

Characterization of the periplasmic hydrogenase from Desulfovibrio gigas.

The hydrogenase of the sulfate-reducer $\text{Desulfovibrio gigas}$ has been purified to homogeneity. The pure enzyme shows a specific activity of 90 μmoles H₂ evolved/min./mg protein. Its molecular weight is 89,500 and its is composed of two different subunits (mol. wt. : 62,000 and 26,000) which are not covalently bound. The absorption spectrum of the enzyme is characteristic of an iron-sulfur protein. The millimolar extinction coefficients of the hydrogenase are 46.5 and 170 respectively at 400 and 280 nm. It contains about 12 iron atoms and 12 acid-labile sulfur groups per molecule and the quantitative extrusion of the Fe-S centers of the hydrogenase indicates the presence of 3 Fe₄S₄ clusters. This hydrogenase has 21 half-cystine residues per molecule and a preponderance of aromatic amino-acids.     

Effect of enzymic assay conditions on sulfite reduction catalysed by desulfoviridin from Desulfovibrio gigas.

The type and the amount of end products resulting from sulfite reduction catalysed by a single partially purified desulfoviridin preparation from Desulfovibrio gigas were shown to depend upon the enzymic assay conditions employed. Both manometric and spectrophotometric assays were used, with reduced methyl viologen serving as the electron donor in each system. Trithionate, thiosulfate, tetrathionate and sulfide were identified as possible end products. In the manometric assays, sulfide production was favoured by high reduced methyl viologen concentrations, low sulfite concentrations and a pH value of 7.0 as opposed to 6.0. In the spectrophotometric assays, results approaching the stoichiometric conversion of sulfite to sulfide were obtained only at high initial reduced methyl viologen concentrations.     

Calcium is required for the reduction of sulfite from hydrogen in a reconstituted electron transfer chain from the sulfate reducing bacterium, Desulfovibrio gigas

Calcium is found a strong stimulator of sulfite reduction from hydrogen. A coupling protein of molecular weight 65,000 can be isolated from Desulfovibrio gigas. It functions in a reconstituted electron transfer chain between hydrogenase and sulfite reductase. Its N-terminal sequence shows high homologies with calcium or magnesium binding sites from other calcium-binding proteins.     

Crystal structure of the 16 heme cytochrome from Desulfovibrio gigas: a glycosylated protein in a sulphate-reducing bacterium

Sulphate-reducing bacteria have a wide variety of periplasmic cytochromes involved in electron transfer from the periplasm to the cytoplasm. HmcA is a high molecular mass cytochrome of 550 amino acid residues that harbours 16 c-type heme groups. We report the crystal structure of HmcA isolated from the periplasm of Desulfovibrio gigas. Crystals were grown using polyethylene glycol 8K and zinc acetate, and diffracted beyond 2.1 A resolution. A multiple-wavelength anomalous dispersion experiment at the iron absorption edge enabled us to obtain good-quality phases for structure solution and model building. DgHmcA has a V-shape architecture, already observed in HmcA isolated from Desulfovibrio vulgaris Hildenborough. The presence of an oligosaccharide molecule covalently bound to an Asn residue was observed in the electron density maps of DgHmcA and confirmed by mass spectrometry. Three modified monosaccharides appear at the highly hydrophobic vertex, possibly acting as an anchor of the protein to the cytoplasmic membrane.     

Purification and characterization of an iron superoxide dismutase and a catalase from the sulfate-reducing bacterium Desulfovibrio gigas

The iron-containing superoxide dismutase (FeSOD; EC 1.15.1.1) and catalase (EC 1.11.1.6) enzymes constitutively expressed by the strictly anaerobic bacterium Desulfovibrio gigas were purified and characterized. The FeSOD, isolated as a homodimer of 22-kDa subunits, has a specific activity of 1,900 U/mg and exhibits an electron paramagnetic resonance (EPR) spectrum characteristic of high-spin ferric iron in a rhombically distorted ligand field. Like other FeSODs from different organisms, D. gigas FeSOD is sensitive to H(2)O(2) and azide but not to cyanide. The N-terminal amino acid sequence shows a high degree of homology with other SODs from different sources. On the other hand, D. gigas catalase has an estimated molecular mass of 186 +/- 8 kDa, consisting of three subunits of 61 kDa, and shows no peroxidase activity. This enzyme is very sensitive to H(2)O(2) and cyanide and only slightly sensitive to sulfide. The native enzyme contains one heme per molecule and exhibits a characteristic high-spin ferric-heme EPR spectrum (g(y,x) = 6.4, 5.4); it has a specific activity of 4,200 U/mg, which is unusually low for this class of enzyme. The importance of these two enzymes in the context of oxygen utilization by this anaerobic organism is discussed.     

Gene expression by the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough grown on an iron electrode under cathodic protection conditions

The genome sequence of the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough was reanalyzed to design unique 70-mer oligonucleotide probes against 2,824 probable protein-coding regions. These included three genes not previously annotated, including one that encodes a c-type cytochrome. Using microarrays printed with these 70-mer probes, we analyzed the gene expression profile of wild-type D. vulgaris grown on cathodic hydrogen, generated at an iron electrode surface with an imposed negative potential of -1.1 V (cathodic protection conditions). The gene expression profile of cells grown on cathodic hydrogen was compared to that of cells grown with gaseous hydrogen bubbling through the culture. Relative to the latter, the electrode-grown cells overexpressed two hydrogenases, the hyn-1 genes for [NiFe] hydrogenase 1 and the hyd genes, encoding [Fe] hydrogenase. The hmc genes for the high-molecular-weight cytochrome complex, which allows electron flow from the hydrogenases across the cytoplasmic membrane, were also overexpressed. In contrast, cells grown on gaseous hydrogen overexpressed the hys genes for [NiFeSe] hydrogenase. Cells growing on the electrode also overexpressed genes encoding proteins which promote biofilm formation. Although the gene expression profiles for these two modes of growth were distinct, they were more closely related to each other than to that for cells grown in a lactate- and sulfate-containing medium. Electrochemically measured corrosion rates were lower for iron electrodes covered with hyn-1, hyd, and hmc mutant biofilms than for wild-type biofilms. This confirms the importance, suggested by the gene expression studies, of the corresponding gene products in D. vulgaris-mediated iron corrosion.