Vulgaris Strain Hildenborough Research Articles

Oxygen isotope effects during microbial sulfate reduction: applications to sediment cell abundances

The majority of anaerobic biogeochemical cycling occurs within marine sediments. To understand these processes, quantifying the distribution of active cells and gross metabolic activity is essential. We present an isotope model rooted in thermodynamics to draw quantitative links between cell-specific sulfate reduction rates and active sedimentary cell abundances. This model is calibrated using data from a series of continuous culture experiments with two strains of sulfate reducing bacteria (freshwater bacterium Desulfovibrio vulgaris strain Hildenborough, and marine bacterium Desulfovibrio alaskensis strain G-20) grown on lactate across a range of metabolic rates and ambient sulfate concentrations. We use a combination of experimental sulfate oxygen isotope data and nonlinear regression fitting tools to solve for unknown kinetic, step-specific oxygen isotope effects. This approach enables identification of key isotopic reactions within the metabolic pathway, and defines a new, calibrated framework for understanding oxygen isotope variability in sulfate. This approach is then combined with porewater sulfate/sulfide concentration data and diagenetic modeling to reproduce measured 18O/16O in porewater sulfate. From here, we infer cell-specific sulfate reduction rates and predict abundance of active cells of sulfate reducing bacteria, the result of which is consistent with direct biological measurements.

Deconstructing the Dissimilatory Sulfate Reduction Pathway: Isotope Fractionation of a Mutant Unable of Growth on Sulfate.

The sulfur isotope record provides key insight into the history of Earth's redox conditions. A detailed understanding of the metabolisms driving this cycle, and specifically microbial sulfate reduction (MSR), is crucial for accurate paleoenvironmental reconstructions. This includes a precise knowledge of the step-specific sulfur isotope effects during MSR. In this study, we aim at resolving the cellular-level fractionation factor during dissimilatory sulfite reduction to sulfide within MSR, and use this measured isotope effect as a calibration to enhance our understanding of the biochemistry of sulfite reduction. For this, we merge measured isotope effects associated with dissimilatory sulfite reduction with a quantitative model that explicitly links net fractionation, reaction reversibility, and intracellular metabolite levels. The highly targeted experimental aspect of this study was possible by virtue of the availability of a deletion mutant strain of the model sulfate reducer Desulfovibrio vulgaris (strain Hildenborough), in which the sulfite reduction step is isolated from the rest of the metabolic pathway owing to the absence of its QmoABC complex (ΔQmo). This deletion disrupts electron flux and prevents the reduction of adenosine phosphosulfate (APS) to sulfite. When grown in open-system steady-state conditions at 10% maximum growth rate in the presence of sulfite and lactate as electron donor, sulfur isotope fractionation factors averaged −15.9‰ (1 σ = 0.4), which appeared to be statistically indistinguishable from a pure enzyme study with dissimilatory sulfite reductase. We coupled these measurements with an understanding of step-specific equilibrium and kinetic isotope effects, and furthered our mechanistic understanding of the biochemistry of sulfite uptake and ensuing reduction. Our metabolically informed isotope model identifies flavodoxin as the most likely electron carrier performing the transfer of electrons to dissimilatory sulfite reductase. This is in line with previous work on metabolic strategies adopted by sulfate reducers under different energy regimes, and has implications for our understanding of the plasticity of this metabolic pathway at the center of our interpretation of modern and palaeo-environmental records.

CO2 exposure at pressure impacts metabolism and stress responses in the model sulfate-reducing bacterium Desulfovibrio vulgaris strain Hildenborough.

Geologic carbon dioxide (CO2) sequestration drives physical and geochemical changes in deep subsurface environments that impact indigenous microbial activities. The combined effects of pressurized CO2 on a model sulfate-reducing microorganism, Desulfovibrio vulgaris, have been assessed using a suite of genomic and kinetic measurements. Novel high-pressure NMR time-series measurements using 13C-lactate were used to track D. vulgaris metabolism. We identified cessation of respiration at CO2 pressures of 10 bar, 25 bar, 50 bar, and 80 bar. Concurrent experiments using N2 as the pressurizing phase had no negative effect on microbial respiration, as inferred from reduction of sulfate to sulfide. Complementary pressurized batch incubations and fluorescence microscopy measurements supported NMR observations, and indicated that non-respiring cells were mostly viable at 50 bar CO2 for at least 4 h, and at 80 bar CO2 for 2 h. The fraction of dead cells increased rapidly after 4 h at 80 bar CO2. Transcriptomic (RNA-Seq) measurements on mRNA transcripts from CO2-incubated biomass indicated that cells up-regulated the production of certain amino acids (leucine, isoleucine) following CO2 exposure at elevated pressures, likely as part of a general stress response. Evidence for other poorly understood stress responses were also identified within RNA-Seq data, suggesting that while pressurized CO2 severely limits the growth and respiration of D. vulgaris cells, biomass retains intact cell membranes at pressures up to 80 bar CO2. Together, these data show that geologic sequestration of CO2 may have significant impacts on rates of sulfate reduction in many deep subsurface environments where this metabolism is a key respiratory process.

Metallomics of two microorganisms relevant to heavy metal bioremediation reveal fundamental differences in metal assimilation and utilization

Although as many as half of all proteins are thought to require a metal cofactor, the metalloproteomes of microorganisms remain relatively unexplored. Microorganisms from different environments are likely to vary greatly in the metals that they assimilate, not just among the metals with well-characterized roles but also those lacking any known function. Herein we investigated the metal utilization of two microorganisms that were isolated from very similar environments and are of interest because of potential roles in the immobilization of heavy metals, such as uranium and chromium. The metals assimilated and their concentrations in the cytoplasm of Desulfovibrio vulgaris strain Hildenborough (DvH) and Enterobacter cloacae strain Hanford (EcH) varied dramatically, with a larger number of metals present in Enterobacter. For example, a total of 9 and 19 metals were assimilated into their cytoplasmic fractions, respectively, and DvH did not assimilate significant amounts of zinc or copper whereas EcH assimilated both. However, bioinformatic analysis of their genome sequences revealed a comparable number of predicted metalloproteins, 813 in DvH and 953 in EcH. These allowed some rationalization of the types of metal assimilated in some cases (Fe, Cu, Mo, W, V) but not in others (Zn, Nd, Ce, Pr, Dy, Hf and Th). It was also shown that U binds an unknown soluble protein in EcH but this incorporation was the result of extracellular U binding to cytoplasmic components after cell lysis.

Variation among Desulfovibrio Species in Electron Transfer Systems Used for Syntrophic Growth

Mineralization of organic matter in anoxic environments relies on the cooperative activities of hydrogen producers and consumers linked by interspecies electron transfer in syntrophic consortia that may include sulfate-reducing species (e.g., Desulfovibrio). Physiological differences and various gene repertoires implicated in syntrophic metabolism among Desulfovibrio species suggest considerable variation in the biochemical basis of syntrophy. In this study, comparative transcriptional and mutant analyses of Desulfovibrio alaskensis strain G20 and Desulfovibrio vulgaris strain Hildenborough growing syntrophically with Methanococcus maripaludis on lactate were used to develop new and revised models for their alternative electron transfer and energy conservation systems. Lactate oxidation by strain G20 generates a reduced thiol-disulfide redox pair(s) and ferredoxin that are energetically coupled to H(+)/CO(2) reduction by periplasmic formate dehydrogenase and hydrogenase via a flavin-based reverse electron bifurcation process (electron confurcation) and a menaquinone (MQ) redox loop-mediated reverse electron flow involving the membrane-bound Qmo and Qrc complexes. In contrast, strain Hildenborough uses a larger number of cytoplasmic and periplasmic proteins linked in three intertwining pathways to couple H(+) reduction to lactate oxidation. The faster growth of strain G20 in coculture is associated with a kinetic advantage conferred by the Qmo-MQ-Qrc loop as an electron transfer system that permits higher lactate oxidation rates under elevated hydrogen levels (thereby enhancing methanogenic growth) and use of formate as the main electron-exchange mediator (>70% electron flux), as opposed to the primarily hydrogen-based exchange by strain Hildenborough. This study further demonstrates the absence of a conserved gene core in Desulfovibrio that would determine the ability for a syntrophic lifestyle.

Homology modelling and molecular dynamics simulations of wild type and mutated flavodoxins from Desulfovibrio vulgaris (Miyazaki F): insight into FMN–apoprotein interactions

The flavodoxin from Desulfovibrio vulgaris, strain Miyazaki F (FD-DvMF), binds one molecule of flavin mononucleotide (FMN) as a cofactor and is considered to associate with electron transport reactions. However, although the 3D structure of the related FD from D. vulgaris strain Hildenborough has been determined, that for FD-DvMF has not. In this study, we have predicted the protein structures of the wild type and the W59F, Y97F and W59F-Y97F substitutional mutants of FD-DvMF by a homology modelling approach. Subsequently, the dynamic properties of these four FD-DvMF variants were investigated by molecular dynamics simulations. The results revealed that peptide O of Trp59 formed H-bond with Tyr99OH only in Y97F, leading to FMN being buried deeper inside the protein than in the other three variants and reducing the accessibility of water to FMN. The phosphate oxygen atoms formed extensive H-bonds with amino acid residues in the 10-loop region in all variants resulting in the highest degree of stabilisation. The OH groups of the ribityl chain and the isoalloxazine ring formed H-bonds with amino acid residues of the 60- and 90-loop regions, respectively. The decomposition free energy calculations suggest that the greatest contribution come from the 10-loop region, which is compatible with the published data. That the calculated binding and decomposition free energies were both greatest in Y97F is proposed to be due to the H-bond between peptide O of Trp59 and Tyr99OH.

Structural and functional relationships in the hybrid cluster protein family: structure of the anaerobically purified hybrid cluster protein fromDesulfovibrio vulgarisat 1.35 Å resolution

The hybrid cluster protein (HCP) from the sulfate-reducing bacterium Desulfovibrio vulgaris strain Hildenborough has been isolated and crystallized anaerobically. The phase problem was solved for a P2(1)2(1)2(1) crystal form using multiple-wavelength anomalous diffraction data collected in the vicinity of the Fe K absorption edge. Although the overall protein structure is essentially the same as that previously obtained, it shows that the nature of the hybrid cluster has particular differences when isolated and crystallized in the absence of oxygen and this provides insight into the structural features associated with changes in the oxidation state. A comparison between HCPs and carbon monoxide dehydrogenases (CoDs) shows that they possess a similar fold and that the dehydrogenases have a related cluster at the equivalent HCP hybrid cluster position. This helps to understand the nature of the hybrid cluster and to predict a dimeric structure for class 3 HCPs, which lack the N-terminal region.

Cr(VI) detoxification by Desulfovibrio vulgaris strain Hildenborough: microbe–metal interactions studies

Toxic heavy metals constitute a worldwide environmental pollution problem. Bioremediation technologies represent efficient alternatives to the classic cleaning-up of contaminated soil and ground water. Most toxic heavy metals such as chromium are less soluble and toxic when reduced than when oxidized. Sulfate-reducing bacteria (SRB) are able to reduce heavy metals by a chemical reduction via the production of H2S and by a direct enzymatic process involving hydrogenases and c3 cytochromes. We have previously reported the effects of chromate [Cr(VI)] on SRB bioenergetic metabolism and the molecular mechanism of the metal reduction by polyhemic cytochromes. In the current work, we pinpoint the bacteria-metal interactions using Desulfovibrio vulgaris strain Hildenborough as a model. The bacteria were grown in the presence of high Cr(VI) concentration, where they accumulated precipitates of a reduced form of chromium, trivalent chromium [Cr(III)], on their cell surfaces. Moreover, the inner and outer membranes exhibited precipitates that shared the spectroscopic signature of trivalent chromium. This subcellular localization is consistent with enzymatic metal reduction by cytochromes and hydrogenases. Regarding environmental significance, our findings point out the Cr(VI) immobilization mechanisms of SRB; suggesting that SRB are highly important in metal biogeochemistry.

Nitrate reduction by Desulfovibrio desulfuricans: A periplasmic nitrate reductase system that lacks NapB, but includes a unique tetraheme c-type cytochrome, NapM

Many sulphate reducing bacteria can also reduce nitrite, but relatively few isolates are known to reduce nitrate. Although nitrate reductase genes are absent from Desulfovibrio vulgaris strain Hildenborough, for which the complete genome sequence has been reported, a single subunit periplasmic nitrate reductase, NapA, was purified from Desulfovibrio desulfuricans strain 27774, and the structural gene was cloned and sequenced. Chromosome walking methods have now been used to determine the complete sequence of the nap gene cluster from this organism. The data confirm the absence of a napB homologue, but reveal a novel six-gene organisation, napC- napM– napA– napD– napG– napH. The NapC polypeptide is more similar to the NrfH subgroup of tetraheme cytochromes than to NapC from other bacteria. NapM is predicted to be a tetra-heme c-type cytochrome with similarity to the small tetraheme cytochromes from Shewanella oneidensis. The operon is located close to a gene encoding a lysyl-tRNA synthetase that is also found in D. vulgaris. We suggest that electrons might be transferred to NapA either from menaquinol via NapC, or from other electron donors such as formate or hydrogen via the small tetraheme cytochrome, NapM. We also suggest that, despite the absence of a twin-arginine targeting sequence, NapG might be located in the periplasm where it would provide an alternative direct electron donor to NapA.

Hydrogenases in sulfate-reducing bacteria function as chromium reductase.

The ability of sulfate-reducing bacteria (SRB) to reduce chromate VI has been studied for possible application to the decontamination of polluted environments. Metal reduction can be achieved both chemically, by H(2)S produced by the bacteria, and enzymatically, by polyhemic cytochromes c(3). We demonstrate that, in addition to low potential polyheme c-type cytochromes, the ability to reduce chromate is widespread among [Fe], [NiFe], and [NiFeSe] hydrogenases isolated from SRB of the genera Desulfovibrio and Desulfomicrobium. Among them, the [Fe] hydrogenase from Desulfovibrio vulgaris strain Hildenborough reduces Cr(VI) with the highest rate. Both [Fe] and [NiFeSe] enzymes exhibit the same K(m) towards Cr(VI), suggesting that Cr(VI) reduction rates are directly correlated with hydrogen consumption rates. Electron paramagnetic resonance spectroscopy enabled us to probe the oxidation by Cr(VI) of the various metal centers in both [NiFe] and [Fe] hydrogenases. These experiments showed that Cr(VI) is reduced to paramagnetic Cr(III), and revealed inhibition of the enzyme at high Cr(VI) concentrations. The significant decrease of both hydrogenase and Cr(VI)-reductase activities in a mutant lacking [Fe] hydrogenase demonstrated the involvement of this enzyme in Cr(VI) reduction in vivo. Experiments with [3Fe-4S] ferredoxin from Desulfovibrio gigas demonstrated that the low redox [Fe-S] (non-heme iron) clusters are involved in the mechanism of metal reduction by hydrogenases.

Reduced hybrid cluster proteins (HCP) from Desulfovibrio desulfuricans ATCC 27774 and Desulfovibrio vulgaris (Hildenborough): X-ray structures at high resolution using synchrotron radiation.

The hybrid cluster proteins from the sulfate reducing bacteria Desulfovibrio desulfuricans ATCC 27774 ( Dd) and Desulfovibrio vulgaris strain Hildenborough ( Dv) have been isolated and crystallized anaerobically. In each case, the protein has been reduced with dithionite and the crystal structure of the reduced form elucidated using X-ray synchrotron radiation techniques at 1.25 A and 1.55 A resolution for Dd and Dv, respectively. Although the overall structures of the proteins are unchanged upon reduction, there are significant changes at the hybrid cluster centres. These include significant movements in the position of the iron atom linked to the persulfide moiety in the oxidized as-isolated proteins and the sulfur atom of the persulfide itself. The nature of these changes is described and the implications with respect to the function of hybrid cluster proteins are discussed.

Thermodynamics of cleaning-up of waste waters polluted by chromium

The effect of Cr(VI) on Desulfovibrio vulgaris strain Hildenborough bioenergetic metabolism was monitored by microcalorimetry and the concomitant reduction of this metal was studied. Results showed that Cr(VI) is reduced by the bacterium and that the bacterial growth is altered, involving a strong modification of the metabolism of the bacteria. An absence of correlation between Cr(VI) reduction and cell growth is observed, suggesting that Cr(VI) does not yield energy to support anaerobic growth. The analysis of the enzymatic characteristics of Cr(VI) reduction are in progress.

Rubrerythrin and rubredoxin oxidoreductase in Desulfovibrio vulgaris: a novel oxidative stress protection system.

Evidence is presented for an alternative to the superoxide dismutase (SOD)-catalase oxidative stress defense system in Desulfovibrio vulgaris (strain Hildenborough). This alternative system consists of the nonheme iron proteins, rubrerythrin (Rbr) and rubredoxin oxidoreductase (Rbo), the product of the rbo gene (also called desulfoferrodoxin). A Deltarbo strain of D. vulgaris was found to be more sensitive to internal superoxide exposure than was the wild type. Unlike Rbo, expression of plasmid-borne Rbr failed to restore the aerobic growth of a SOD-deficient strain of Escherichia coli. Conversely, plasmid-borne expression of two different Rbrs from D. vulgaris increased the viability of a catalase-deficient strain of E. coli that had been exposed to hydrogen peroxide whereas Rbo actually decreased the viability. A previously undescribed D. vulgaris gene was found to encode a protein having 50% sequence identity to that of E. coli Fe-SOD. This gene also encoded an extended N-terminal sequence with high homologies to export signal peptides of periplasmic redox proteins. The SOD activity of D. vulgaris is not affected by the absence of Rbo and is concentrated in the periplasmic fraction of cell extracts. These results are consistent with a superoxide reductase rather than SOD activity of Rbo and with a peroxidase activity of Rbr. A joint role for Rbo and Rbr as a novel cytoplasmic oxidative stress protection system in D. vulgaris and other anaerobic microorganisms is proposed.

Identification of a putative histidine base and of a non-protein nitrogen ligand in the active site of Fe-hydrogenases by one-dimensional and two-dimensional electron spin-echo envelope-modulation spectroscopy.

The active H-cluster of the Fe-hydrogenases from Megasphaera elsdenii and Desulfovibrio vulgaris (strain Hildenborough) has been investigated with one- and two-dimensional pulsed EPR spectroscopy. In both complexes the coordination of a nitrogen-containing ligand was found. The unusual quadrupole interaction parameters (D. vulgaris: quadrupole coupling constant, K = 1.20 MHz, asymmetry parameter eta = 0.32, M. elsdenii: K = 1.23 MHz, eta = 0.25) indicate a non-protein type of nitrogen and are consistent with cyanide as ligand to the H-cluster. The additional interactions measured on the EPR signal of the inactivated H-cluster in D. vulgaris hydrogenase are consistent with an imidazole interaction similar to that found in Rieske-type iron-sulfur clusters. Since a His residue near the putative H-cluster binding motif of Cys residues, His371, is the only conserved His in Fe-hydrogenases, it is a likely candidate for the base that accepts the proton in the heterolytic cleavage of molecular hydrogen. The inactivation of the enzyme is accompanied by direct binding of the imidazole ring to the H-cluster.

Kinetic Studies on the Electron-Transfer Reaction between Cytochrome c3 and Flavodoxin from Desulfovibrio vulgaris Strain Hildenborough

The kinetic properties of the electron-transfer process between reduced Desulfovibrio vulgaris cytochrome c3 and D. vulgaris flavodoxin have been studied by anaerobic stopped-flow techniques. Anaerobic titrations of reduced cytochrome c3 with oxidized flavodoxin show a stoichiometry of 4 mol of flavodoxin required to oxidize the tetraheme cytochrome. Flavodoxin neutral semiquinone and oxidized cytochrome c3 are the only observable products of the reaction. At pH 7.5, the four-electron-transfer reaction is biphasic. Both the rapid and the slow phases exhibit limiting rates as the flavodoxin concentration is increased with respective rates of 73.4 and 18.5 s-1 and respective Kd values of 65.9 +/- 9.4 microM and 54.5 +/- 13 microM. A biphasic electron-transfer rate is observed when the ionic strength is increased to 100 mM KCl; however, the observed rate is no longer saturable, and relative second-order rate constants of 5.3 x 10(5) and 8.5 x 10(4) M-1 s-1 are calculated. The magnitude of the rapid phase of electron transfer diminishes with the level of heme reduction when varying reduced levels of the cytochrome are mixed with oxidized flavodoxin. No rapid phase is observed when 0.66e(-)-reduced cytochrome c3 reacts with an approximately 25-fold molar excess of flavodoxin. At pH 6.0, the electron-transfer reaction is monophasic with a limiting rate of 42 +/- 1.4 s-1 and a Kd value of approximately 8 microM. Increasing the ionic strength of the pH 6.0 solution to 100 microM KCl results in a biphasic reaction with relative second-order rate constants of 5.3 x 10(5) and 1.1 x 10(4) M-1 s-1. Azotobacter vinelandii flavodoxin reacts with reduced D. vulgaris cytochrome c3 in a slow, monophasic manner with limiting rate of electron transfer of 1.2 +/- 0.06 s-1 and a Kd value of 80.9 +/- 10.7 microM. These results are discussed in terms of two equilibrium conformational states for the cytochrome which are dependent on the pH of the medium and the level of heme reduction [Catarino et al. (1991) Eur. J. Biochem. 207, 1107-1113].

Simulation of the electrochemical behavior of multi-redox systems. Current potential studies on multiheme cytochromes.

The direct unmediated electrochemical response of the tetrahemic cytochrome c3 isolated from sulfate reducers Desulfovibrio baculatus (DSM 1743) and D. vulgaris (strain Hildenborough), was evaluated using different electrode systems [graphite (edge cut), gold, semiconductor (InO2) and mercury)] and different electrochemical methods (cyclic voltammetry and differential pulse voltammetry). A computer program was developed for the theoretical simulation of a complete cyclic voltammetry curve, based on the method proposed by Nicholson and Shain [Nicholson, R.S. & Shain, I. (1964) Anal. Chem. 36, 706-723], using the Gauss-Legendre method for calculation of the integral equations. The experimental data obtained for this multi-redox center protein was deconvoluted in to the four redox components using theoretically generated cyclic voltammetry curves and the four mid-point reduction potentials determined. The pH dependence of the four reduction potentials was evaluated using the deconvolution method described.

Purification and characterization of desulfoferrodoxin. A novel protein from Desulfovibrio desulfuricans (ATCC 27774) and from Desulfovibrio vulgaris (strain Hildenborough) that contains a distorted rubredoxin center and a mononuclear ferrous center.

A new type of non-heme iron protein was purified to homogeneity from extracts of Desulfovibrio desulfuricans (ATCC 27774) and Desulfovibrio vulgaris (strain Hildenborough). This protein is a monomer of 16-kDa containing two iron atoms per molecule. The visible spectrum has maxima at 495, 368, and 279 nm and the EPR spectrum of the native form shows resonances at g = 7.7, 5.7, 4.1 and 1.8 characteristic of a high-spin ferric ion (S = 5/2) with E/D = 0.08. Mössbauer data indicates the presence of two types of iron: an FeS4 site very similar to that found in desulforedoxin from Desulfovibrio gigas and an octahedral coordinated high-spin ferrous site most probably with nitrogen/oxygen-containing ligands. Due to this rather unusual combination of active centers, this novel protein is named desulfoferrodoxin. Based on NH2-terminal amino acid sequence determined so far, the desulfoferrodoxin isolated from D. desulfuricans (ATCC 27774) appears to be a close analogue to a recently discovered gene product from D. vulgaris (Brumlik, M.J., and Voordouw, G. (1989) J. Bacteriol. 171, 49996-50004), which was suggested to be a rubredoxin oxidoreductase. However, reduced pyridine nucleotides failed to reduce the desulforedoxin-like center of this new protein.

Electron transfer between the hydrogenase from Desulfovibrio vulgaris (Hildenborough) and viologens. 2. Investigations by chronoamperometry.

The electron transfer kinetics between the hydrogenase from Desulfovibrio vulgaris (strain Hildenborough) and the mediators methyl viologen, di-(n-aminopropyl) viologen and propyl viologen sulfonate have been investigated by chronoamperometry. Second-order rate constants were calculated on basis of the theory for a simple catalytic mechanism and are compared with the results obtained before by cyclic voltammetry (preceding paper in this journal). From the ionic-strength dependence and the observed differences in the rate constants for the differently charged viologens, the existence of an electrostatic interaction between mediator and a negatively charged part of the protein is confirmed. Chronoamperometry (computer-controlled) was found to possess advantages over cyclic voltammetry in the determination of homogeneous rate constants (faster, more accurate, and better reproducibility).

Electron transfer between the hydrogenase from Desulfovibrio vulgaris (Hildenborough) and viologens. 1. Investigations by cyclic voltammetry.

The electron transfer kinetics between the hydrogenase from Desulvovibrio vulgaris (strain Hildenborough) and three different viologen mediators has been investigated by cyclic voltammetry. The mediators methyl viologen, di(n-aminopropyl) viologen and propyl viologen sulfonate differ in redox potential and in net charge. Dependent on the pH both the one- and two-electron-reduced forms or only the two-electron-reduced form of the viologens are effective in electron exchange with hydrogenase. Calculations of the second-order rate constant k for the reaction between reduced viologen and hydrogenase are based on the theory of the simplest electrocatalytic mechanism. Values for k are in the range of 10(6)-10(7) M-1 s-1 and increase in the direction propyl viologen sulfonate----methyl viologen----di(n-aminopropyl) viologen. An explanation is based on electrostatic interactions. It is proposed that the electron transfer reaction is the rate-determining step in the catalytic mechanism.

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.