Short Communication: Lipopolysaccharide composition of Desulfovibrio cell wall

The lipopolysaccharide of the sulphate-reducing bacterium Desulfovibrio desulfuricans was analysed by GC, combined either with flame-ionization detection or with MS, and by standard chemical tests. The major sugar of the polysaccharide portion of the macromolecule was glucose (56%). Low amounts of mannose, galactose, rhamnose and amino sugar were also found. Six fatty acids were identified in the lipid A fraction: 9-octadecenoic, tetracosenoic, heptadecenoic, 10-octadecenoic, eicosenoic and 8-octadecynoic.     

A novel isolate of Desulfovibrio sp. with enhanced ability to reduce Cr(VI)

Kinetic analysis of the reduction of Cr(VI) by resting cell suspensions of Desulfovibrio vulgaris ATCC 29579 and a new isolate, Desulfovibrio sp. (`Oz7') was studied using lactate as the electron donor at 30 °C. The apparent K <sub>m</sub> (K <sub>m app</sub>) and V <sub>max</sub> with respect to Cr(VI) reduction was compared for both strains. Desulfovibio sp. `Oz7' had a K _{m app} of 90 M (threefold lower than that of D. vulgaris ATCC 29579) and a V _max of 120 nmol h^–1 mg^–1 biomass dry wt (approx. 30% lower than for the reference strain). The potential of the new isolate for bioremediation of Cr(VI) wastewaters is discussed.     

Reduction of Cr(VI) by Desulfovibrio vulgaris and Microbacterium sp.

Many industrial wastes contain Cr(VI), a carcinogen and mutagen, the toxicity of which can be ameliorated by reduction to Cr(III). Microbacterium sp. NCIMB 13776 andDesulfovibrio vulgaris NCIMB 8303 reduced Cr(VI) to Cr(III) anoxically using 25 mM sodium citrate buffer (pH 7), with 25 mM sodium acetate and 25 mM sodium formate as electron donors at 30 °C, under which conditions the rates of reduction of 500 M sodium chromate were 77 and 6 nmol h^–1 mg dry cell wt for D. vulgaris and Microbacterium sp., respectively, these being increased to 127 and 17 nmol h^–1 mg dry cell wt in the presence of 20 mM MOPS/NaOH buffer.     

Evaluation of arbitrarily primed PCR for typing of Desulfovibrio desulfuricans strains

Arbitrarily primed polymerase chain reaction (AP-PCR) method was applied to the differentiation of 15 (soil and intestinal) Desulfovibrio desulfuricans strains. The primer M 13, which is a core sequence of phage M 13, was found to be appropriate for the differentiation of isolates of this species. The analysis revealed characteristic band patterns for all of the examined strains of which two soil strains (DV-7 and DV-8) showed identical DNA fingerprints. According to Jaccard's coefficient, the soil bacterial group as well as intestinal bacterial group formed two different clusters. Furthermore, the soil strains showed greater variability than the intestinal isolates. Based on the AP-PCR fingerprints D. desulfuricans strains were differentiated depending on their origin. This study demonstrates that the typing method AP-PCR can be useful in epidemiologic investigations as a rapid and valuable tool for differentiation of the strains of D. desulfuricans species.     

Desulfovibrio alcoholovorans sp. nov., a sulfate-reducing bacterium able to grow on glycerol, 1,2- and 1,3-propanediol

Desulfovibrio strain SPSN was isolated from an anaerobic industrial fermenter fed with waste water from the alcohol industry. The isolate was a gram-negative, non-spore-forming, curved organism, the motility of which is provided by a single polar flagellum. The oxidation of substrates was incomplete and included glycerol and 1,3-propanediol. Sulfate, sulfite, thiosulfate, and sulfur were utilized as electron acceptors. Pyruvate, fumarate and malate could be fermented. The DNA base composition was 64.5±0.3% G+C. Cytochrome c ₃ and desulfoviridin were present. On the basis of these characteristics and because strain SPSN could not be ascribed to any of the existing species, the isolate is established as a new species of the genus Desulfovibrio, and the name Desulfovibrio alcoholovorans is proposed.     

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     

Ferric iron reduction by Desulfovibrio vulgaris Hildenborough wild type and energy metabolism mutants

Desulfovibrio vulgaris Hildenborough wild type and its hyn1, hyd and hmc mutants, lacking genes for periplasmic [NiFe] hydrogenase-1, periplasmic [FeFe] hydrogenase or the transmembrane high molecular weight cytochrome (Hmc) complex, respectively, were able to reduce Fe(III) chelated with nitrilotriacetic acid (NTA), but not insoluble ferric oxide, with lactate as the electron donor. The rate and extent of Fe(III)-NTA reduction followed the order hyn = WT > hmc >> hyd, suggesting that reduction of soluble Fe(III) is a periplasmic process that requires the presence of periplasmic [FeFe] hydrogenase. Reduction of Fe(III)-NTA was not coupled to cell growth. In fact cell concentrations declined when D. vulgaris was incubated with Fe(III)-NTA as the only electron acceptor. Wild type and mutant cells reducing a limiting concentration of sulfate (2 mM), reduced Fe(III)-NTA with similar rates. However, these were similarly incapable of catalyzing subsequent lactate-dependent reduction of Fe(III)-NTA to completion. Periplasmic reduction of Fe(III)-NTA appeared to inhibit the productive, sulfate-reducing metabolism of D. vulgaris, possibly because it prevents the cycling of reducing equivalents needed to achieve a net bioenergetic benefit.     

Metabolism of l-alanine in Desulfotomaculum ruminis and two marine Desulfovibrio strains

The degradation of l-alanine by three strains of sulfate-reducing bacteria that can grow with l-alanine as an energy source was investigated. In Desulfotomaculum ruminis and most likely also in two marine Desulfovibrio strains alanine is converted to pyruvate via an NAD-dependent alanine dehydrogenase. D. ruminis contained high activities of soluble NADH and NADPH dehydrogenases. In the marine strains the activities were much lower and the NADH dehydrogenase was partly associated with the membrane fraction.     

Characterization of Desulfovibrio fructosovorans sp. nov.

Desulfovibrio strain JJ isolated from estuarine sediment differed from all other described Desulfovibrio species by the ability to degrade fructose. The oxidation was incomplete, leading to acetate production. Fructose, malate and fumarate were fermented mainly to succinate and acetate in the absence of an external electron acceptor. The pH and temperature optima for growth were 7.0 and 35° C respectively. Strain JJ was motile by means of a single polar flagellum. The DNA base composition was 64.13% G+C. Cytochrome c ₃ and desulfoviridin were present. These characteristics established the isolate as a new species of the genus Desulfovibrio, and the name Desulfovibrio fructosovorans is proposed.     

Localization of hydrogenase in Desulfovibrio gigas cells

The localization of hydrogenase protein in Desulfovibrio gigas cells grown either in lactate-sulfate or hydrogen-sulfate media, has been investigated by subcellular fractionation with immunoblotting and by electron microscopic immunocytochemistry. Subcellular fractionation experiments suggest that no integral membrane-bound hydrogenase is present in D. gigas. About 40% of the hydrogenase activity could be extracted by treatment of D. gigas cells with Tris-EDTA buffer. The rest of the soluble hydrogenase activity (50%) was found in the soluble fraction which was obtained after disruption of Tris-EDTA extracted cells and high speed centrifugation. Both soluble hydrogenase fractions purified to homogeneity showed identical molecular properties including the N-terminal aminoacid sequences of their large and small subunits. Polyacrylamide gel electrophoresis of the proteins of the subcellular fractions revealed a single band of hydrogenase activity exhibiting the same mobility as purified D. gigas hydrogenase. Western blotting carried out on these subcellular fractions revealed crossreactivity with the antibodies raised against (NiFe) hydrogenase. The lack of crossreactivity with antibodies against (FE) or (NiFeSe) hydrogenases, indicated that only (NiFe) type hydrogenase is present in D. gigas.Immunocytolocalization in ultrathin frozen sections of D. gigas cells grown either in lactate-sulfate, pyruvate-sulfate or hydrogen-sulfate media showed only a (NiFe) hydrogenase located in the periplasmic space. The bioenergetics of D. gigas are discussed in the light of these findings.     

Antigenic diversity of cytochromes c3 from the anaerobic,sulfate-reducing bacteria,Desulfovibrio

An indirect enzyme-linked immunoadsorption assay (ELISA) was developed for cytochrome c₃ using antisera to the cytochromes fromDesulfovibrio africanus Benghazi, Desulfovibrio vulgaris Hildenborough andDesulfovibrio salexigens British Guiana. The ELISA system was used to test for cross-reactions between these antisera and the heterologous antigens. In contrast to previous experiments using the Ouchterlony technique, all of the cytochromes c₃ tested exhibited some degree of cross-reaction. Considerable variation was seen in cross-reactions for cytochromes c₃ from differing strains ofD. desulfuricans. This observation raises questions about the taxonomic relatedness of these strains. No cross-reaction was seen with eukaryotic cytochrome c or withD. vulgaris cytochrome c₅₅₃. The data demonstrate that cytochrome c₃ is capable of undergoing nonprecipitating cross-reactions, and thus may not be as immunologically unique as was once thought.Abbreviations ELISA Enzyme-linked immunoadsorption assay     

Overexpression, purification and immunodetection of DsrD from Desulfovibrio vulgaris Hildenborough

Dissimilatory sulfite reductase (DsrAB) of the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough is an ₂₂ tetramer of 180 kDa, encoded by the dsr operon. In addition to the dsrA and dsrB genes, this operon contains a gene (dsrD) encoding a protein of only 78 amino acids. Although, the function of DsrD is currently unknown, the presence of a dsrD gene has been demonstrated in a variety of sulfate-reducing bacteria and archaea. DsrD was expressed in Escherichia coli at a very high level and purified to homogeneity. Protein blotting experiments, using antisera raised against purified DsrD, demonstrated that it is expressed constitutively in D. vulgaris and does not copurify with DsrAB. Spectroscopic analysis of DsrD indicated that it does not bind either sulfite or sulfide, the substrate and product, respectively of the reaction catalyzed by DsrAB. Thus, although the conservation of this protein and its demonstrated presence in D. vulgaris, suggest an essential function in dissimilatory sulfite reduction, this function remains to be elucidated.     

The dcr gene family of Desulfovibrio: Implications from the sequence of dcrH and phylogenetic comparison with other mcp genes

Desulfovibrio vulgaris Hildenborough contains a family of genes for methyl-accepting chemotaxis proteins (MCPs). Here we report the complete sequence of the gene for Desulfovibrio chemoreceptor H (dcrH). The deduced amino acid sequence of DcrH protein, which has an enlarged N-terminal, ligand binding domain, indicates a structure similar to that of other MCPs. Comparison of the sequences for DcrA, determined earlier, and DcrH indicated that similarity is essentially limited to the C-terminal excitation region. The dcr gene family differs, in this respect, from mcp gene families in other eubacteria (e.g. Escherichia coli and Bacillus subtilis), where MCPs share significant homology throughout their C-terminal signal transduction domains. This may point to an ancient evolutionary origin of the dcr gene family, which is widely distributed throughout the genus Desulfovibrio. The evolutionary origin of mcp genes was traced by comparing nucleotide sequences for the excitation region that is common to all MCPs. Phylogenetic analysis of sequences for thirty mcp genes from nine eubacterial and one archaebacterial species suggested that multiplication of mcp genes has occurred at least twice since the eubacteria diverged from the archaebacteria.Nucleotide accession number: The nucleotide sequence reported in this paper has been entered into GenBank under accession number U30319. Phone: 403-220-6388. Fax: 403-289-9311. Electronic mail address: voordouw@acs.ucalgary.ca.     

Copper resistance in Desulfovibrio strain R2

A sulfate-reducing bacterium, designated as strain R2, was isolated from wastewater of a ball-bearing manufacturing facility in Tomsk, Western Siberia. This isolate was resistant up to 800 mg Cu/l in the growth medium. By comparison, Cu-resistance of reference cultures of sulfate-reducing bacteria ranged from 50 to 75 mg Cu/l. Growth experiments with strain R2 showed that Cu was an essential trace element and, on one hand, enhanced growth at concentrations up to 10 mg/l but, on the other hand, the growth rate decreased and lag-period extended at copper concentrations of >50 mg/l. Phenotypic characteristics and a 1078 bp nucleotide sequence of the 16S rDNA placed strain R2 within the genus Desulfovibrio. Desulfovibrio R2 carried at least one plasmid of approximately of 23.1 kbp. A 636 bp fragment ot the pcoR gene of the pco operon that encodes Cu resistance was amplified by PCR from plasmid DNA of strain R2. The pco genes are involved in Cu-resistance in some enteric and aerobic soil bacteria. Desulfovibrio R2 is a prospective strain for bioremediation purposes and for developing a homologous system for transformation of Cu-resistance in sulfate-reducing bacteria. This revised version was published online in June 2006 with corrections to the Cover Date.     

Glycerol and dihydroxyacetone dissimilation in Desulfovibrio strains

During growth on glycerol two marine Desulfovibrio strains that can grow on an unusually broad range of substrates contained high activities of glycerol kinase, NAD(P)-independent glycerol 3-phosphate dehydrogenase and the other enzymes necessary for the conversion of dihydroxyacetone phosphate to pyruvate. Glycerol dehydrogenase and a specific dihydroxyacetone kinase were absent. During growth on dihydroxyacetone, glycerol kinase is involved in the initial conversion of this compound to dihydroxyacetone phosphate which is then further metabolized. Some kinetic properties of the partially purified glycerol kinase were determined. The role of NAD as electron carrier in the energy metabolism during growth of these strains on glycerol and dihydroxyacetone is discussed.Glycerol also supported growth of three out of four classical Desulfovibrio strains tested. D. vulgaris strain Hildenborough grew slowly on glycerol and contained glycerol kinase, glycerol 3-phosphate dehydrogenase and enzymes for the dissimilation of dihydroxyacetone phosphate. In D. gigas which did not grow on glycerol the enzymes glycerol kinase and glycerol 3-phosphate dehydrogenase were absent in lactate-grown cells.Abbreviations DHA dihydroxyacetone - DHAP dihydroxyacetone phosphate - G3P glycerol 3-phosphate - GAP glyceraldehyde 3-phosphate - 3-PGA 3-phosphoglycerate - 2-PGA 2-phosphoglycerate - 2,3-DPGA 2,3-diphosphoglycerate - PEP phosphoenolpyruvate - DH dehydrogenase - GK glycerol kinase - DHAK dihydroxyacetone kinase - TIM triosephosphate isomerase - PGK 3-phosphoglycerate kinase - PK pyruvate kinase - LDH lactate dehydrogenase - DTT dithiotreitol - HEPES 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid - PIPES piperazine-1,1-bis(2-ethane sulfonic acid) - BV²⁺/BV⁺ oxidized/reduced benzylviologen - PMS phenazine methosulfate - DCPIP 2,6-dichlorophenolindophenol - MTT 3-(4,5-dimethylthiazol-2-yl)-2,4-diphenyltetrazolium bromide     

The anaerobe Desulfovibrio desulfuricans ATCC 27774 grows at nearly atmospheric oxygen levels

Sulfate reducing bacteria of the Desulfovibrio genus are considered anaerobes, in spite of the fact that they are frequently isolated close to oxic habitats. However, until now, growth in the presence of high concentrations of oxygen was not reported for members of this genus. This work shows for the first time that the sulfate reducing bacterium Desulfovibrio desulfuricans ATCC 27774 is able to grow in the presence of nearly atmospheric oxygen levels. In addition, the activity and expression profile of several key enzymes was analyzed under different oxygen concentrations.     

Anerobic degradation of 1,2-propanediol by a new Desulfovibrio strain and D. alcoholovorans

A sulfate-reducing bacterium, strain HDv, was isolated from the anoxic soil of a ricefield using lactate as electron donor. Cells were gram-negative, motile, nonsporulating curved rods, with single polar flagella. Substrates were incompletely oxidized to acetate and included glycerol, 1,2-and 1,3-propanediol. Sulfate, sulfite, thiosulfate, elemental sulfur, fumarate, maleate, and malate were utilized as electron acceptors. Pyruvate, fumarate, maleate, malate and dihydroxyacetone were fermented. Desulfoviridin and c-type cytochromes were present. The DNA base composition was 66.6 ± 0.3 mol% G+C. The isolate was identified as a Desulfovibrio sp.; its metabolic properties were somewhat different from those of previously described Desulfovibrio species. Comparative biochemical study of 1,2-propanediol dissimilation by the new isolate and Desulfovibrio alcoholovorans showed that NAD-dependent dehydrogenases play a key role in the catabolism of this substrate. The hypothetical pathways of 1,2-propanediol degradation by Desulfovibrio spp. are presented.     

Ethanol dissimilation in Desulfovibrio

During growth of ethanol plus sulfate Desulfovibrio gigas and three other Desulfovibrio strains tested contained high NAD-dependent alcohol dehydrogenase activities and dye-linked aldehyde dehydrogenase activities. In lactate-grown cells these activities were lower or absent. In D. gigas an NADH dehydrogenase activity was found which was higher during growth on ethanol than during growth on lactate. The NADH dehydrogenase activity appeared to consist of at least three different soluble enzymes. The aldehyde dehydrogenase activity in D. gigas was highest with benzylviologen as an acceptor and was strongly stimulated by potassium ions. Coenzyme A or phosphate dependency could not be shown, indicating that acetyl-CoA or acetyl phosphate are not intermediates in the conversion of acetaldehyde to acetate.In the absence of sulfate D. gigas was able to convert ethanol to acetate by means of interspecies hydrogen transfer to a methanogen. This conversion, however, did not lead to growth of the Desulfovibrio.Abbreviations DH dehydrogenase - BV²⁺/BV⁺ oxidized/reduced benzylviologen - DCPIP 2,6-dichlorophenolindophenol - MTT 3-(4,5-dimethylthiazol-2-yl)-2,4-diphenyltetrazolium bromide - MV²⁺/MV⁺ oxidized/reduced methylviologen - PMS phenazine methosulfate     

Transformation of selenate and selenite to elemental selenium byDesulfovibrio desulfuricans

Summary Desulfovibrio desulfuricans (DSM 1924) can be adapted to grow in the presence of 10 mM selenate or 0.1 mM selenite. This growth occurred in media containing formate as the electron donor and either fumarate or sulfate as the electron acceptor. As determined by electron microscopy with energy-dispersive X-ray analysis, selenate and selenite were reduced to elemental selenium which accumulated inside the cells. Selenium granules resulting from selenite metabolism were cytoplasmic while granules of selenium resulting from selenate reduction appeared to be in the periplasmic region. The accumulation of red elemental selenium in the media following stationary phase resulted from cell lysis with the liberation of selenium granules. Growth did not occur with either selenate or selenite as the electron acceptor and¹³C nuclear magnetic resonance indicated that neither selenium oxyanion interfered with fumarate respiration. At 1 M selenate and 100 M selenite, reduction byD. desulfuricans was 95% and 97%, respectively. The high level of total selenate and selenite reduced indicated the suitability ofD. desulfuricans for selenium detoxification.     

Characterization of Desulfomicrobium escambium sp. nov. and proposal to assign Desulfovibrio desulfuricans strain Norway 4 to the genus Desulfomicrobium

A sulfate-reducing bacterium, designated strain ESC1, was isolated and found to be a new species. Strain ESC1 is a strictly anaerobic, gram-negative, non-sporeforming, motile, short, round-ended rod often occurring in pairs. Of 31 fermentative substrates tested, only pyruvate was utilized. Sulfate enhanced growth with pyruvate and allowed growth with ethanol, lactate, formate and hydrogen. Both sulfate and thiosulfate were reduced. Lactate was incompletely oxidized to acetate and CO₂. The strain was desulfoviridin negative. The G+C content is 59.9%. These data suggested placement of strain ESC1 in the genus Desulfomicrobium. Comparative 16S rRNA analysis showed that strain ESC1 shares 98% rRNA sequence similarity with Desulfomicrobium baculatum and Desulfovibrio desulfuricans strain Norway 4. The latter two strains shared greater than 99% 16S rRNA sequence similarity. Strain ESC1 has been designated as the new species Desulfomicrobium escambium. We also recommend that D. desulfuricans strain Norway 4 be considered for reclassification as a Desulfomicrobium species.