The adaptive genome of Desulfovibrio vulgaris Hildenborough

Peculiar attributes revealed by sequencing the genome of Desulfovibrio vulgaris Hildenborough are analyzed, particularly in relation to the presence of a phosphotransferase system (PTS). The PTS is a typical bacterial carbohydrate transport system functioning via group translocation. Novel avenues for investigations are proposed emphasizing the metabolic diversity of D. vulgaris Hildenborough, especially the likely utilization of mannose-type sugars. Comparative analysis with PTS from other Gram-negative and Gram-positive bacteria indicates regulatory functions for the PTS of D. vulgaris Hildenborough, including catabolite repression and inducer exclusion. Chemotaxis towards PTS substrates is considered. Evidence suggests that this organism may not be a strict anaerobic sulfate reducer typical of the ocean, but a versatile organism capable of bidirectional transmigration and adaptation to both water and terrestrial environments.     

Carbon monoxide cycling by Desulfovibrio vulgaris Hildenborough

Sulfate-reducing bacteria, like Desulfovibrio vulgaris Hildenborough, use the reduction of sulfate as a sink for electrons liberated in oxidation reactions of organic substrates. The rate of the latter exceeds that of sulfate reduction at the onset of growth, causing a temporary accumulation of hydrogen and other fermentation products (the hydrogen or fermentation burst). In addition to hydrogen, D. vulgaris was found to produce significant amounts of carbon monoxide during the fermentation burst. With excess sulfate, the hyd mutant (lacking periplasmic Fe-only hydrogenase) and hmc mutant (lacking the membrane-bound, electron-transporting Hmc complex) strains produced increased amounts of hydrogen from lactate and formate compared to wild-type D. vulgaris during the fermentation burst. Both hydrogen and CO were produced from pyruvate, with the hyd mutant producing the largest transient amounts of CO. When grown with lactate and excess sulfate, the hyd mutant also exhibited a temporary pause in sulfate reduction at the start of stationary phase, resulting in production of 600 ppm of headspace hydrogen and 6,000 ppm of CO, which disappeared when sulfate reduction resumed. Cultures with an excess of the organic electron donor showed production of large amounts of hydrogen, but no CO, from lactate. Pyruvate fermentation was diverse, with the hmc mutant producing 75,000 ppm of hydrogen, the hyd mutant producing 4,000 ppm of CO, and the wild-type strain producing no significant amount of either as a fermentation end product. The wild type was most active in transient production of an organic acid intermediate, tentatively identified as fumarate, indicating increased formation of organic fermentation end products in the wild-type strain. These results suggest that alternative routes for pyruvate fermentation resulting in production of hydrogen or CO exist in D. vulgaris. The CO produced can be reoxidized through a CO dehydrogenase, the presence of which is indicated in the genome sequence.     

Purification and characterization of the formate dehydrogenase from Desulfovibrio vulgaris Hildenborough

Abstract Formate dehydrogenase from Desulfovibrio vulgaris Hildenborough, a sulfate-reducing bacterium, has been isolated and characterized. The enzyme is composed of three subunits. A high molecular mass subunit (83 500 Da) is proposed to contain a molybdenum cofactor, a 27 000 Da subunit is found to be similar to the Fe-S subunit of the formate dehydrogenase from Escherichia coli and a low molecular mass subunit (14000 Da) holds a c -type heme. The presence of heme c in formate dehydrogenase is reported for the first time and is correlated to the peculiar low oxidoreduction potential of the metabolism of these strictly anaerobic bacteria. In vitro measurements have shown that a monoheme cytochrome probably acts as a physiological partner of the enzyme in the periplasm.     

Function of periplasmic hydrogenases in the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough

The sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough possesses four periplasmic hydrogenases to facilitate the oxidation of molecular hydrogen. These include an [Fe] hydrogenase, an [NiFeSe] hydrogenase, and two [NiFe] hydrogenases encoded by the hyd, hys, hyn1, and hyn2 genes, respectively. In order to understand their cellular functions, we have compared the growth rates of existing (hyd and hyn1) and newly constructed (hys and hyn-1 hyd) mutants to those of the wild type in defined media in which lactate or hydrogen at either 5 or 50% (vol/vol) was used as the sole electron donor for sulfate reduction. Only strains missing the [Fe] hydrogenase were significantly affected during growth with lactate or with 50% (vol/vol) hydrogen as the sole electron donor. When the cells were grown at low (5% [vol/vol]) hydrogen concentrations, those missing the [NiFeSe] hydrogenase suffered the greatest impairment. The growth rate data correlated strongly with gene expression results obtained from microarray hybridizations and real-time PCR using mRNA extracted from cells grown under the three conditions. Expression of the hys genes followed the order 5% hydrogen>50% hydrogen>lactate, whereas expression of the hyd genes followed the reverse order. These results suggest that growth with lactate and 50% hydrogen is associated with high intracellular hydrogen concentrations, which are best captured by the higher activity, lower affinity [Fe] hydrogenase. In contrast, growth with 5% hydrogen is associated with a low intracellular hydrogen concentration, requiring the lower activity, higher affinity [NiFeSe] hydrogenase.     

Extraction of periplasmic hydrogenase from Desulfovibrio vulgaris (Hildenborough)

Abstract Periplasmic hydrogenase from Desulfovibrio vulgaris (Hildenborough) was extracted according to the method of van der Westen [8] and the effect of trace minerals on the extractability of this enzyme was investigated. The final growth yields in the presence or absence of trace minerals were the same; however, the growth was much faster and the amount of periplasmic hydrogenase extracted was significantly lower in the presence of trace minerals. Polyacrylamide gel electrophoresis showed the presence of 2 hydrogenases in D. vulgaris , one soluble and the other possibly membrane-bound.     

Desulfovibrio vulgaris Hildenborough prefers lactate over hydrogen as electron donor

Desulfovibrio vulgaris can use lactate as an electron donor and accumulate hydrogen. Hydrogen can also be consumed as an electron donor when lactate is depleted or absent. The aim of this study was to determine whether D. vulgaris has an electron donor preference system between lactate and hydrogen and how this system is regulated. In order to be sure that D. vulgaris was grown under the same conditions except for electron donors, continuous growth mode was conducted and the optical density (600 nm) was kept constant. When 20 mmol/l lactate was the sole electron donor, it was depleted after 9 h of incubation while hydrogen was accumulated to 1,500 ppm. After that, the hydrogen level was decreased to and maintained at 400 ppm. When 1,200 ppm hydrogen was provided as the electron donor, the culture reached an OD of 0.2 after 24 h incubation and hydrogen was consumed to 600 ppm. When 1,200 ppm hydrogen and 20 mmol/l lactate were both present, the lactate was consumed during the first 9 h incubation and hydrogen was accumulated to 1,800 ppm. D. vulgaris used hydrogen as an electron donor after the lactate was depleted and the hydrogen level was decreased to 600 ppm. D. vulgaris has both pathways to utilize lactate and hydrogen as electron donors. It prefers lactate over hydrogen and the system is regulated by lactate starvation.     

Cloning and sequencing of the gene encoding flavodoxin from Desulfovibrio vulgaris Hildenborough

Abstract The gene encoding flavodoxin from Desulfovibrio vulgaris Hildenborough (148 amino acid residues), the first flavoprotein for which a three-dimensional structure has been determined, was cloned with the use of two synthetic oligonucleotides, designed to recognize the coding sequence for amino acid residues 11–19 and 98–103, respectively. The two oligonucleotides were used to screen a library of 900 λ-clones of the D. vulgaris chromosome. A single clone, λFL1, reacting with both probes was isolated. The entire structural gene for flavodoxin is contained in the 15 kb insert of λFL1 as found by nucleic acid sequencing. The codon usage in the flavodoxin gene is strongly biased towards G or C in the third codon position. A table in which codon usage information from all genes of D. vulgaris sequenced to date is combined is presented and should facilitate further gene cloning with oligonucleotide probes.     

Identification of a membrane-bound hydrogenase of Desulfovibrio vulgaris (Hildenborough)

Two hydrogenase activities from Desulfovibrio vulgaris (Hildenborough) could be distinguished immunologically and biochemically. The first activity, described as hydrogenase I, corresponded to the soluble enzyme located in the periplasmic space of D. vulgaris. Hydrogenase I had a high specific activity and was sensitive to inhibition by CO. The second activity, hydrogenase II, was located in the membrane fraction, had a lower specific activity and was not affected by CO. The enzymes exhibited different electrophoretic mobilities in polyacrylamide gels, and reacted differently when exposed to proteases. Antibodies raised against purified periplasmic hydrogenase of D. vulgaris reacted with hydrogenase I, but not with hydrogenase II.     

The genome sequence of the anaerobic, sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough

Desulfovibrio vulgaris Hildenborough is a model organism for studying the energy metabolism of sulfate-reducing bacteria (SRB) and for understanding the economic impacts of SRB, including biocorrosion of metal infrastructure and bioremediation of toxic metal ions. The 3,570,858 base pair (bp) genome sequence reveals a network of novel c-type cytochromes, connecting multiple periplasmic hydrogenases and formate dehydrogenases, as a key feature of its energy metabolism. The relative arrangement of genes encoding enzymes for energy transduction, together with inferred cellular location of the enzymes, provides a basis for proposing an expansion to the 'hydrogen-cycling' model for increasing energy efficiency in this bacterium. Plasmid-encoded functions include modification of cell surface components, nitrogen fixation and a type-III protein secretion system. This genome sequence represents a substantial step toward the elucidation of pathways for reduction (and bioremediation) of pollutants such as uranium and chromium and offers a new starting point for defining this organism's complex anaerobic respiration.     

Kinetic properties of hydrogenase isolated from Desulfovibrio vulgaris (Hildenborough)

Hydrogenase of Desulfovibrio vulgaris shows nonlinear kinetics in hydrogen production with both the natural electron carrier, cytochrome c3, and the artificial donor, methyl viologen semiquinone. Increasing concentrations of salt progressively inhibit the hydrogen production, as do increasing amounts of dimethylsulfoxide (Me2SO). Hydrogen consumption activity does not change up to 30% (v/v) of Me2SO. Preincubation in Me2SO up to 55% (v/v) does not affect the hydrogen uptake or production. The production activity of the enzyme shows an optimum around pH 6. When plotted as a function of redox potential the activity can be fitted to a Nernst equation with n = 1. Midpoint potentials calculated at various values follow approximately the hydrogen electrode to pH 6. Thereafter, there is a shift of about 40 mV to higher redox potentials.     

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.     

Biofilm formation in Desulfovibrio vulgaris Hildenborough is dependent upon protein filaments

Desulfovibrio vulgaris Hildenborough is a Gram-negative sulfate-reducing bacterium (SRB), and the physiology of SRBs can impact many anaerobic environments including radionuclide waste sites, oil reservoirs and metal pipelines. In an attempt to understand D. vulgaris as a population that can adhere to surfaces, D. vulgaris cultures were grown in a defined medium and analysed for carbohydrate production, motility and biofilm formation. Desulfovibrio vulgaris wild-type cells had increasing amounts of carbohydrate into stationary phase and approximately half of the carbohydrate remained internal. In comparison, a mutant that lacked the 200 kb megaplasmid, strain DeltaMP, produced less carbohydrate and the majority of carbohydrate remained internal of the cell proper. To assess the possibility of carbohydrate re-allocation, biofilm formation was investigated. Wild-type cells produced approximately threefold more biofilm on glass slides compared with DeltaMP; however, wild-type biofilm did not contain significant levels of exopolysaccharide. In addition, stains specific for extracellular carbohydrate did not reveal polysaccharide material within the biofilm. Desulfovibrio vulgaris wild-type biofilms contained long filaments as observed with scanning electron microscopy (SEM), and the biofilm-deficient DeltaMP strain was also deficient in motility. Biofilms grown directly on silica oxide transmission electron microscopy (TEM) grids did not contain significant levels of an exopolysaccharide matrix when viewed with TEM and SEM, and samples stained with ammonium molybdate also showed long filaments that resembled flagella. Biofilms subjected to protease treatments were degraded, and different proteases that were added at the time of inoculation inhibited biofilm formation. The data indicated that D. vulgaris did not produce an extensive exopolysaccharide matrix, used protein filaments to form biofilm between cells and silica oxide surfaces, and the filaments appeared to be flagella. It is likely that D. vulgaris used flagella for more than a means of locomotion to a surface, but also used flagella, or modified flagella, to establish and/or maintain biofilm structure.