Hydrogen metabolism in Desulfovibrio desulfuricans strain New Jersey (NCIMB 8313)--comparative study with D. vulgaris and D. gigas species

This article aims to study hydrogen production/consumption in Desulfovibrio (D.) desulfuricans strain New Jersey, a sulfate reducer isolated from a medium undergoing active biocorrosion and to compare its hydrogen metabolism with two other Desulfovibrio species, D. gigas and D. vulgaris Hildenborough. Hydrogen production was followed during the growth of these three bacterial species under different growth conditions: no limitation of sulfate and lactate, sulfate limitation, lactate limitation, pyruvate/sulfate medium and in the presence of molybdate. Hydrogen production/consumption by D. desulfuricans shows a behavior similar to that of D. gigas but a different one from that of D. vulgaris, which produces higher quantities of hydrogen on lactate/sulfate medium. The three species are able to increase the hydrogen production when the sulfate became limiting. Moreover, in a pyruvate/sulfate medium hydrogen production was lower than on lactate/sulfate medium. Hydrogen production by D. desulfuricans in presence of molybdate is extremely high. Hydrogenases are key enzymes on production/consumption of hydrogen in sulfate reducing organisms. The specific activity, number and cellular localization of hydrogenases vary within the three Desulfovibrio species used in this work, which could explain the differences observed on hydrogen utilization.     

Sulfate Reduction by a Desulfovibrio Species Isolated from Sheep Rumen

Several dissimilatory, sulfate-reducing bacteria were isolated from the rumen fluid of sheep fed purified diets containing sulfate. One isolate, strain D, was selected for characterization. This organism is a nonsporeforming, obligately anaerobic, mesophilic, nonmotile, gram-negative, straight rod. Cell-free extracts show absorption maxima for cytochrome c(3) and desulfoviridin, characteristic of Desulfovibrio. Carbohydrates, as a sole carbon source, will support growth. Lactate supports growth in the presence of sulfate, not in its absence, whereas glucose or pyruvate support growth either in the presence or absence of sulfate. The isolate has a deoxyribonucleic acid base composition of 61.2% guanine plus cytosine, which is similar to that of several other species of Desulfovibrio; however, it differs from previously described species in morphology, motility, and carbon source utilization. Cell-free extracts of this bacterium exhibit adenosine 5'-triphosphate-sulfurylase, adenosine-5'-phosphosulfate-reductase, and hydrogenase activity. After incubation of cell-free extracts with adenine 5'-triphosphate and (35)SO(4) (2-), adenosine-5'-phosphosulfate rather than 3'-phosphoadenosine-5'-phosphosulfate was shown to be labeled, indicating that the pathway of sulfate reduction in this organism is similar to that of other dissimilatory sulfate reducers. This is the first report of a Desulfovibrio sp. isolated from the rumen.     

Immunocytochemical localization of APS reductase and bisulfite reductase in three Desulfovibrio species

The localization of APS reductase and bisulfite reductase in Desulfovibrio gigas, D. vulgaris Hildenborough and D. thermophilus was studied by immunoelectron microscopy. Polyclonal antibodies were raised against the purified enzymes from each strain. Cells fixed with formaldehyde/glutaraldehyde were embedded and ultrathin sections were incubated with antibodies and subsequently labeled with protein A-gold. The bisulfite reductase in all three strains and APS reductase in d. gigas and D. vulgaris were found in the cytoplasm. The labeling of d. thermophilus with APS reductase antibodies resulted in a distribution of gold particles over the cytoplasmic membrane region. The localization of the two enzymes is discussed with respect to the mechanism and energetics of dissimilatory sulfate reduction.     

The 'strict' anaerobe Desulfovibrio gigas contains a membrane-bound oxygen-reducing respiratory chain

Sulfate-reducing bacteria are considered as strict anaerobic microorganisms, in spite of the fact that some strains have been shown to tolerate the transient presence of dioxygen. This report shows that membranes from Desulfovibrio gigas grown in fumarate/sulfate contain a respiratory chain fully competent to reduce dioxygen to water. In particular, a membrane-bound terminal oxygen reductase, of the cytochrome bd family, was isolated, characterized, and shown to completely reduce oxygen to water. This oxidase has two subunits with apparent molecular masses of 40 and 29 kDa. Using NADH or succinate as electron donors, the oxygen respiratory rates of D. gigas membranes are comparable to those of aerobic organisms (3.2 and 29 nmol O(2) min(-1) mg protein(-1), respectively). This 'strict anaerobic' bacterium contains all the necessary enzymatic complexes to live aerobically, showing that the relationships between oxygen and anaerobes are much more complex than originally thought.     

Characterization and complete amino acid sequence of ferredoxin II from Desulfovibrio vulgaris Miyazaki

One of 2 ferredoxins, Fd II, purified from Desulfovibrio vulgaris Miyazaki (DvM) has been characterized and its complete amino acid sequence established. Fd II is composed of 63 amino acid residues and contains 7 cysteinyl residues but has only 4 iron atoms in an iron-sulfur cluster of a standard redox potential of -405 mV. The arrangement of cysteinyl residues in the protein suggests that some cysteinyl residues are not directly involved in ligation to the iron-sulfur cluster. Homology is recognized among Fd II (DvM), Fd I (D. desulfuricans Norway), Fd I (D. africanus Benghazi), and Fd (D. gigas). Although Fd I and Fd II in DvM are poorly homologous, the C-terminal half of Fd I is fairly homologous to the N-terminal half of Fd II. Fd II is 40% as effective as Fd I as an electron carrier for pyruvate dehydrogenase coupled with hydrogenase and cytochrome c3.     

Genome sequence of the mercury-methylating and pleomorphic Desulfovibrio africanus Strain Walvis Bay

Desulfovibrio africanus strain Walvis Bay is an anaerobic sulfate-reducing bacterium capable of producing methylmercury (MeHg), a potent human neurotoxin. The mechanism of methylation by this and other organisms is unknown. We present the 4.2-Mb genome sequence to provide further insight into microbial mercury methylation and sulfate-reducing bacteria.     

Whole-cell antigens of members of the sulfate-reducing genus Desulfovibrio

Antisera have been developed against the wholecell antigens of Desulfovibrio africanus Benghazi and Walvis Bay, D. vulgaris Hildenborough, D. salexigens British Guiana, D. gigas, and D. desulfuricans Essex 6. An enzymelinked immunoadsorption assay (ELISA) was developed to measure the reaction of these antisera with the homologous and heterologous antigens. The ELISA method demonstrated a reaction between pre-immune sera and cells of D. africanus, D. gigas and D. desulfuricans, suggesting the presence of a lectin-like substance on these cell surfaces. Extensive cross-reactions were seen between the antisera and heterologous cells, suggesting the sharing of a number of surface antigens amongst the Desulfovibrio. However, the pattern of these cross-reactions was different from that observed for an ELISA reaction developed for the cytochrome c₃ from various Desulfovibrio.Abbreviation ELISA enzyme-linked immunoadsorption assay     

Diversity of dissimilatory bisulfite reductase genes of bacteria associated with the deep-sea hydrothermal vent polychaete annelid Alvinella pompejana

A unique community of bacteria colonizes the dorsal integument of the polychaete annelid Alvinella pompejana, which inhabits the high-temperature environments of active deep-sea hydrothermal vents along the East Pacific Rise. The composition of this bacterial community was characterized in previous studies by using a 16S rRNA gene clone library and in situ hybridization with oligonucleotide probes. In the present study, a pair of PCR primers (P94-F and P93-R) were used to amplify a segment of the dissimilatory bisulfite reductase gene from DNA isolated from the community of bacteria associated with A. pompejana. The goal was to assess the presence and diversity of bacteria with the capacity to use sulfate as a terminal electron acceptor. A clone library of bisulfite reductase gene PCR products was constructed and characterized by restriction fragment and sequence analysis. Eleven clone families were identified. Two of the 11 clone families, SR1 and SR6, contained 82% of the clones. DNA sequence analysis of a clone from each family indicated that they are dissimilatory bisulfite reductase genes most similar to the dissimilatory bisulfite reductase genes of Desulfovibrio vulgaris, Desulfovibrio gigas, Desulfobacterium autotrophicum, and Desulfobacter latus. Similarities to the dissimilatory bisulfite reductases of Thermodesulfovibrio yellowstonii, the sulfide oxidizer Chromatium vinosum, the sulfur reducer Pyrobaculum islandicum, and the archaeal sulfate reducer Archaeoglobus fulgidus were lower. Phylogenetic analysis separated the clone families into groups that probably represent two genera of previously uncharacterized sulfate-reducing bacteria. The presence of dissimilatory bisulfite reductase genes is consistent with recent temperature and chemical measurements that documented a lack of dissolved oxygen in dwelling tubes of the worm. The diversity of dissimilatory bisulfite reductase genes in the bacterial community on the back of the worm suggests a prominent role for anaerobic sulfate-reducing bacteria in the ecology of A. pompejana.     

Purification and characterization of cytochrome c3, ferredoxin, and rubredoxin isolated from Desulfovibrio desulfuricans Norway.

Different electron carriers of the non-desulfoviridin-containing, sulfate-reducing bacterium Desulfovibrio desulfuricans (Norway strain) have been studied. Two nonheme iron proteins, ferredoxin and rubredoxin, have been purified. This ferredoxin contains four atoms of non-heme iron and acid-labile sulfur and six residues of cysteine per molecule. Its amino acid composition suggests that it is homologous with the other Desulfovibrio ferredoxins. The rubredoxin is also an acidic protein of 6,000 molecular weight and contains one atom of iron and four cysteine residues per molecule. The amino acid composition and molecular weight of the cytochrome c3 from D. desulfuricans (strain Norway 4) are reported. Its spectral properties are very similar to those of the other cytochromes c3 (molecular weight, 13,000) of Desulfovibrio and show that it contains four hemes per molecule. This cytochrome has a very low redox potential and acts as a carrier in the coupling of hydrogenase and thiosulfate reductase in extracts of Desulfovibrio gigas and Desulfovibrio desulfuricans (Norway strain) in contrast to D. gigas cytochrome c3 (molecular weight, 13,000). A comparison of the activities of the cytochrome c3 (molecular weight, 13,000) of D. gigas and that of D. desulfuricans in this reaction suggests that these homologous proteins can have different specificity in the electron transfer chain of these bacteria.     

Spectroscopic studies on APS reductase isolated from the hyperthermophilic sulfate-reducing archaebacterium Archaeglobus fulgidus.

Adenylyl sulfate (APS) reductase, the key enzyme of the dissimilatory sulfate respiration, catalyzes the reduction of APS (the activated form of sulfate) to sulfite with release of AMP. A spectroscopic study was carried out with the APS reductase purified from the extremely thermophilic sulfate-reducing archaebacterium Archaeoglobus fulgidus DSM 4304. Combined ultraviolet/visible spectroscopy and low temperature electron paramagnetic resonance (EPR) studies were used in order to characterize the active centers and the reactivity towards AMP and sulfite of this enzyme. The A. fulgidus APS reductase is an iron-sulfur flavoprotein containing two distinct [4Fe-4S] clusters (Centers I and II) very similar to the homologous enzyme from Desulfovibrio gigas. Center I, which has a high redox potential, is reduced by AMP and sulfite, and Center II has a very negative redox potential.     

Extractable and lipopolysaccharide fatty acid and hydroxy acid profiles from Desulfovibrio species

An analysis of the phospholipid ester-linked and the lipopolysaccharide (LPS) fatty acids and hydroxy fatty acids of six lactate-utilizing Desulfovibrio-type sulfate-reducing bacteria (SRB) has been performed using capillary gas-liquid chromatography-mass spectrometry (GLC-MS). The concentrations of normal fatty acids were essentially similar, with the possible exception of a high content of normal fatty acids in the LPS of Desulfovibrio gigas. Determination of monounsaturated acid double bond configuration was performed by GLC-MS analysis of the derivatized fatty acids. A total of nine branched chain and eight straight chain monounsaturated fatty acids was detected in the Desulfovibrio species analyzed. The major component detected in five Desulfovibrio was the 17-carbon iso-branched monoenoic acid which showed cis unsaturation [i17:1(n-7)c] seven carbons from the terminal methyl group of the fatty acid chain. D. gigas, in contrast, contained almost no unsaturated fatty acids and was greatly enriched in iso-branched 15:0. Major differences between strains were found in the phospholipid and LPS hydroxy fatty acids. These components, in addition to the i17:1(n-7)c and other characteristic branched chain unsaturated acids, can possibly be utilized as signatures of the lactate-utilizing SRB.     

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.     

Microcalorimetric studies of the growth of sulfate-reducing bacteria: comparison of the growth parameters of some Desulfovibrio species.

We performed a comparative study of the growth energetics of some species of Desulfovibrio by measuring microcalorimetric and molar growth yield values. Lactate and pyruvate were used as energy sources for sulfate reduction. On lactate-sulfate media Desulfovibrio desulfuricans Norway, Desulfovibrio gigas, and Desulfovibrio africanus exhibited molar growth yields of 4.1 +/- 0.6, 3.7 +/- 1.7, and 1.8 +/- 0.1 g/mol, respectively, whereas on pyruvate-sulfate media the molar growth yields were higher (8.5 +/- 0.8, 7.7 +/- 1.6, and 3.5 +/- 0.5 g/mol, respectively). Thus, we found that D. africanus was the least efficient species in converting energy into cell material. The uncoupling of energy in this strain was obvious since its catabolic activities were high compared with those of the two other strains. The enthalpy changes associated with lactate and pyruvate metabolism were -49 +/- 0.7 and -70.2 +/- 6.0 jK/mol, respectively, for D. desulfuricans, -76.6 +/- 1.8 and -91.2 +/- 1.1 kJ/mol, respectively, for D. gigas, and -78.8 +/- 7.2 and -88.0 +/- 6.2 kJ/mol, respectively, for D. africanus. D. gigas and D. africanus produced only acetate, CO2 and hydrogen sulfide as metabolic end products. In addition to these normal end products, D. desulfuricans Norway produced a small amount of butanol. This butanol production was interpreted as reflecting a regulatory system of electron flow during the catabolism of both substrates. Such metabolism was comparable to that reported for D. vulgaris, which lost part of the reducing power of its energy sources through hydrogen evolution.     

Localized intracellular polyphosphate formation by Desulfovibrio gigas.

The dissimilatory sulphate-reducing bacterium Desulfovibrio gigas, frequently sub-cultured, often contained spherical granules which stained metachromatically with some basic dyes. The granules were examined in situ by transmission electron microscopy of whole organisms and thin sections. The granules were isolated from broken bacteria as a water-insoluble, non-crystalline, white material containing magnesium, phosphorus and organic carbon, but devoid of sulphur and nitrogen. The molar ratio of phosphorus to magnesium (1 to 17) was close to the proportions in magnesium tripolyphosphate. Infrared absorption spectra for the white material and magnesium tripolyphosphate were similar.     

Purification and characterization of a tungsten-containing formate dehydrogenase from Desulfovibrio gigas

An air-stable formate dehydrogenase (FDH), an enzyme that catalyzes the oxidation of formate to carbon dioxide, was purified from the sulfate reducing organism Desulfovibrio gigas (D. gigas) NCIB 9332. D. gigas FDH is a heterodimeric protein [alpha (92 kDa) and beta (29 kDa) subunits] and contains 7 +/- 1 Fe/protein and 0.9 +/- 0.1 W/protein. Selenium was not detected. The UV/visible absorption spectrum of D. gigas FDH is typical of an iron-sulfur protein. Analysis of pterin nucleotides yielded a content of 1.3 +/- 0.1 guanine monophosphate/mol of enzyme, which suggests a tungsten coordination with two molybdopterin guanine dinucleotide cofactors. Both M?ssbauer spectroscopy performed on D. gigas FDH grown in a medium enriched with (57)Fe and EPR studies performed in the native and fully reduced state of the protein confirmed the presence of two [4Fe-4S] clusters. Variable-temperature EPR studies showed the presence of two signals compatible with an atom in a d(1) configuration albeit with an unusual relaxation behavior as compared to the one generally observed for W(V) ions.     

Cloning, sequencing and expression of the tetraheme cytochrome c(3) from Desulfovibrio gigas

The gene encoding the tetraheme cytochrome c(3) from Desulfovibrio gigas was cloned and sequenced from a 2.7-kb EcoRI-PstI insert of D. gigas DNA. The derived amino acid sequence showed that the D. gigas cytochrome c(3) is synthesized as a precursor protein with an N-terminal signal peptide sequence of 25 residues and allowed the correction of the previous reported amino acid sequence (Matias et al. Protein Science 5 (1996) 1342-1354). Expression in D. vulgaris (Hildenborough) was possible by conjugal transfer of a recombinant broad-host-range vector pSUP104 containing a SmaI fragment of the D. gigas cytochrome c(3) gene. Biochemical, immunological and spectroscopic analysis of the purified protein showed that the recombinant cytochrome is identical to that isolated from D. gigas.     

The third subunit of desulfoviridin-type dissimilatory sulfite reductases.

In addition to the 50-kDa (alpha) and 40-kDa (beta) subunits, an 11-kDa polypeptide has been discovered in highly purified Desulfovibrio vulgaris (Hildenborough) dissimilatory sulfite reductase. This is in contrast with the hitherto generally accepted alpha 2 beta 2 tetrameric subunit composition. Purification, high-ionic-strength gel-filtration, native electrophoresis and isoelectric focussing do not result in dissociation of the 11-kDa polypeptide from the complex. Densitometric scanning of SDS gels and denaturing gel-filtration indicate a stoichiometric occurrence. A similar 11-kDa polypeptide is present in the desulfoviridin of D. vulgaris oxamicus (Monticello), D. gigas and D. desulfuricans ATCC 27774. We attribute an alpha 2 beta 2 gamma 2 subunit structure to desulfoviridin-type sulfite reductases. N-terminal sequences of the alpha, beta and gamma subunits are reported.     

DsrC is involved in fermentative growth and interacts directly with the FlxABCD–HdrABC complex in Desulfovibrio vulgaris Hildenborough

DsrC is a key protein in dissimilatory sulfur metabolism, where it works as co-substrate of the dissimilatory sulfite reductase DsrAB. DsrC has two conserved cysteines in a C-terminal arm that are converted to a trisulfide upon reduction of sulfite. In sulfate-reducing bacteria, DsrC is essential and previous works suggested additional functions beyond sulfite reduction. Here, we studied whether DsrC also plays a role during fermentative growth of Desulfovibrio vulgaris Hildenborough, by studying two strains where the functionality of DsrC is impaired by a lower level of expression (IPFG07) and additionally by the absence of one conserved Cys (IPFG09). Growth studies coupled with metabolite and proteomic analyses reveal that fermentation leads to lower levels of DsrC, but impairment of its function results in reduced growth by fermentation and a shift towards more fermentative metabolism during sulfate respiration. In both respiratory and fermentative conditions, there is increased abundance of the FlxABCD–HdrABC complex and Adh alcohol dehydrogenase in IPFG09 versus the wild type, which is reflected in higher production of ethanol. Pull-down experiments confirmed a direct interaction between DsrC and the FlxABCD–HdrABC complex, through the HdrB subunit. Dissimilatory sulfur metabolism, where sulfur compounds are used for energy generation, is a key process in the ecology of anoxic environments, and is more widespread among bacteria than previously believed. Two central proteins for this type of metabolism are DsrAB dissimilatory sulfite reductase and its co-substrate DsrC. Using physiological, proteomic and biochemical studies of Desulfovibrio vulgaris Hildenborough and mutants affected in DsrC functionality, we show that DsrC is also relevant for fermentative growth of this model organism and that it interacts directly with the soluble FlxABCD-HdrABC complex that links the NAD(H) pool with dissimilatory sulfite reduction.     

Characterization of the Desulfovibrio desulfuricans ATCC 27774 DsrMKJOP complex--a membrane-bound redox complex involved in the sulfate respiratory pathway

Sulfate-reducing organisms use sulfate as an electron acceptor in an anaerobic respiratory process. Despite their ubiquitous occurrence, sulfate respiration is still poorly characterized. Genome analysis of sulfate-reducing organisms sequenced to date permitted the identification of only two strictly conserved membrane complexes. We report here the purification and characterization of one of these complexes, DsrMKJOP, from Desulfovibrio desulfuricans ATCC 27774. The complex has hemes of the c and b types and several iron-sulfur centers. The corresponding genes in the genome of Desulfovibrio vulgaris were analyzed. dsrM encodes an integral membrane cytochrome b; dsrK encodes a protein homologous to the HdrD subunit of heterodisulfide reductase; dsrJ encodes a triheme periplasmic cytochrome c; dsrO encodes a periplasmic FeS protein; and dsrM encodes another integral membrane protein. Sequence analysis and EPR studies indicate that DsrJ belongs to a novel family of multiheme cytochromes c and that its three hemes have different types of coordination, one bis-His, one His/Met, and the third a very unusual His/Cys coordination. The His/Cys-coordinated heme is only partially reduced by dithionite. About 40% of the hemes are reduced by menadiol, but no reduction is observed upon treatment with H2 and hydrogenase, irrespective of the presence of cytochrome c3. The aerobically isolated Dsr complex displays an EPR signal with similar characteristics to the catalytic [4Fe-4S]3+ species observed in heterodisulfide reductases. Further five different [4Fe-4S](2+/1+) centers are observed during a redox titration followed by EPR. The role of the DsrMKJOP complex in the sulfate respiratory chain of Desulfovibrio spp. is discussed.