Tungstate can substitute for molybdate in sustaining growth of Methanobacterium thermoautotrophicum

Methanobacterium thermoautotrophicum (strain Marburg) was found to grow on media supplemented with tungstate rather than with molybdate. The Archaeon then synthesized a tungsten iron-sulfur isoenzyme of formylmethanofuran dehydrogenase. The isoenzyme was purified to apparent homogeneity and shown to be composed of four different subunits of apparent molecular masses 65 kDa, 53 kDa, 31 kDa, and 15 kDa and to contain per mol 0.4 mol tungsten, <0.05 mol molybdenum, 8 mol non-heme iron, 8 mol acid-labile sulfur and molybdopterin guanine dinucleotide. Its molecular and catalytic properties were significantly different from those of the molybdenum isoenzyme characterized previously. The two isoenzymes also differed in their metal specificity: the active molybdenum isoenzyme was only synthesized when molybdenum was available during growth whereas the active tungsten isoenzyme was also generated during growth of the cells on molybdate medium. Under the latter conditions the tungsten isoenzyme was synthesized containing molybdenum rather than tungsten.Abbreviations MFR methanofuran - CHO-MFR N-formylmethanofuran - MGD molybdopterin guanine dinucleotide - MAD molybdopterin adenine dinucleotide - MHD molybdopterin hypoxanthine dinucleotide - FPLC fast protein liquid chromatography - SDS/PAGE sodium dodecylsulfate/polyacrylamide gel electrophoresis - ICP-MS inductively coupled plasma mass spectrometry     

Tungstate does not support synthesis of active formylmethanofuran dehydrogenase in Methanosarcina barkeri

Cell extracts of Methanosarcina barkeri grown on methanol in media supplemented with molybdate exhibited a specific activity of formylmethanofuran dehydrogenase of approximately 1 U (1 mol/min)/mg protein. When the growth medium was supplemented with tungstate rather than with molybdate, the specific activity was only 0.04 U/mg. Despite this reduction in specific activity growth on methanol was not inhibited. An inhibition of both growth and synthesis of active formylmethanofuran dehydrogenase was observed, however, when H₂ and CO₂ were the energy substrates. The results indicate that, in contrast to Methanobacterium wolfei and Methanobacterium thermoautotrophicum, M. barkeri possesses only a molybdenum containing formylmethanofuran dehydrogenase and not in addition a tungsten isoenzyme.     

A molybdenum and a tungsten isoenzyme of formylmethanofuran dehydrogenase in the thermophilic archaeon Methanobacterium wolfei.

We have recently reported that the thermophilic archaeon Methanobacterium wolfei contains two formylmethanofuran dehydrogenases, I and II. Formylmethanofuran dehydrogenase II, which is preferentially expressed in tungsten-grown cells, has been purified and shown to be a tungsten-iron-sulfur protein. We have now purified and characterized formylmethanofuran dehydrogenase I from molybdenum-grown cells and shown that it is a molybdenum-iron-sulfur protein. The purified enzyme, with a specific activity of 27 U/mg protein, was found to be composed of three subunits of apparent molecular mass 64 kDa, 51 kDa, and 31 kDa and to contain per mol 146-kDa molecule approximately 0.23 mol molybdenum, 0.46 mol molybdopterin guanine dinucleotide, and 6.6 mol non-heme iron but no tungsten (< 0.01 mol). The molybdenum enzyme differed from the tungsten enzyme (8 U/mg) in that it catalyzed the oxidation of N-furfurylformamide and formate and was inactivated by cyanide. The two enzymes also differed significantly in the pH optimum, in the apparent Km for the electron acceptor, and in the chromatographic behaviour. The molybdenum enzyme and the tungsten enzyme were similar, however, in that the N-terminal amino acid sequences determined for the alpha and beta subunits were identical up to residue 23, indicating that the two proteins are isoenzymes. The molybdenum enzyme, as isolated, was found to display an EPR signal derived from molybdenum as evidenced by isotope substitution.     

Evidence for the occurrence of autolytic enzymes in Methanobacterium wolfei

Cultures of the pseudomurein-containing archaebacterium Methanobacterium wolfei regularly lysed a short while after the energy source H₂ was exhausted, or when H₂ in growing cultures was replaced by N₂. During lysis of cells, the DNA was released into the culture medium.No intact cell wall sacculi of lysed cells could be detected, but a soluble fragment of the pseudomurein was isolated and characterized.The lysate of Methanobacterium wolfei was used to lyse other species of the genus Methanobacterium. Since no phages were detected, autolytic enzymes probably are responsible for cell lysis.     

Formate utilization by members of the genus Methanobacterium

Washed cell suspensions of Methanobacterium formicicum MF, Methanobacterium bryantii M.o.H.G. and Methanobacterium strain FR-2 but not Methanobacterium bryantii M.o.H., were shown to produce hydrogen and methane from formate. Levels of dissolved gases (H₂ and CH₄) were continuously and simultaneously monitored within a closed reaction vessel using membrane inlet mass spectrometry. Growth on formate (0–50mM), measured by methane production and increase in absorbance, was observed for both M. formicicum MF and M. bryantii M.o.H.G. but not with Methanobacterium strain FR-2 or M. bryantii M.o.H.     

Properties of the tungsten-substituted molybdenum formylmethanofuran dehydrogenase from Methanobacterium wolfei.

In Methanobacterium wolfei two formylmethanofuran dehydrogenases are present, one of which is a molybdenum- and the other a tungsten enzyme. We report here that also the 'molybdenum' enzyme contained tungsten when the archaeon was grown on molybdenum-deprived medium supplemented with tungstate (1 microM). Unexpectedly the tungsten-substituted molybdenum enzyme was catalytically active and displayed a rhombic EPR signal which was attributed to tungsten by the characteristic 183W splitting.     

Effect of tungsten and molybdenum on growth of a syntrophic coculture of <Emphasis Type="Italic">Syntrophobacter fumaroxidans</Emphasis> and <Emphasis Type="Italic">Methanospirillum hungatei</Emphasis>

The effect of tungsten (W) and molybdenum (Mo) on the growth of Syntrophobacter fumaroxidans and Methanospirillum hungatei was studied in syntrophic cultures and the pure cultures of both the organisms. Cells that were grown syntropically were separated by Percoll density centrifugation. Measurement of hydrogenase and formate dehydrogenase levels in cell extracts of syntrophically grown cells correlated with the methane formation rates in the co-cultures. The effect of W and Mo on the activity of formate dehydrogenase was considerable in both the organisms, whereas hydrogenase activity remained relatively constant. Depletion of tungsten and/or molybdenum, however, did not affect the growth of the pure culture of S. fumaroxidans on propionate plus fumarate significantly, although the specific activities of hydrogenase and especially formate dehydrogenase were influenced by the absence of Mo and W. This indicates that the organism has a low W or Mo requirement under these conditions. Growth of M. hungatei on either formate or H₂/CO₂ required tungsten, and molybdenum could replace tungsten to some extent. Our results suggest a more prominent role for H₂ as electron carrier in the syntrophic conversion of propionate, when the essential trace metals W and Mo for the functioning of formate dehydrogenase are depleted.     

The stability of the molybdenum-azotochelin complex and its effect on siderophore production in Azotobacter vinelandii

 Azotobacter vinelandii produces five siderophores with different metal binding properties, depending on the concentrations of Fe(III) and molybdate in the growth medium. The three lower protonation constants of the unusual bis(catecholamide) siderophore azotochelin (L) were determined by a simultaneous spectrophotometric and potentiometric titration as log K ₅=3.65(5), log K ₄=7.41(3) and log K ₃=8.54(4). The metal-ligand equilibrium constant for [MoO₂(L)]^3– was obtained from analysis of the absorbance concentration data: at 20  °C and pH 6.6, log K _eq=4(1). Based on an average log K _a value of 12.1 for the two basic phenolic oxygens of azotochelin, the equilibrium formation constant was converted into the conventional formation constant K _f(MoL) = [MoO₂L^{3 –}]/[MoO₂ ²⁺][L^{5 –}] = 10³⁵ M^–1. To assess the influence of molybdenum-siderophore interactions on metal uptake in A. vinelandii, the dose-response effect of molybdate in the growth medium on siderophore biosynthesis was followed by UV-vis spectroscopy and HPLC. It could be shown that the formation of molybdenum siderophore complexes clearly reduces the concentration of free siderophores available for iron solubilization. Furthermore, in media with initial molybdate concentrations up to 100 μM, the molybdenum azotochelin complex is the predominant molybdenum species, suggesting that azotochelin might also possess sequestering activity towards molybdenum. Even higher molybdate levels result in a complete repression of the synthesis of the tetradentate siderophore azotochelin, while they initiate the alternative release of the more efficient iron chelator, the hexadentate siderophore protochelin. Received: 20 April 1998 / Accepted: 29 June 1998     

Photoreactivation in Methanobacterium thermoautotrophicum

The sensitivity of three methanogenic bacteria towards ultraviolet irradiation was similar to the UV-sensitivity of Escherichia coli. The lethal effects of UV-irradiation in Methanobacterium thermoautotrophicum Marburg and in Methanobacterium thermoautotrophicum H but not in Methanococcus vannielii were reversed by exposure to visible light. In cell suspensions of Methanobacterium thermoautotrophicum that had been irradiated to 0.1% survival, 90% of the UV-caused damage was photorepairable. The in vivo action spectrum for photoreactivation suggests that in this organism a deazaflavin, probably F₄₂₀, functions as the chromophore of the photoreactivating enzyme.     

Exposure to sodium molybdate results in mild oxidative stress in Drosophila melanogaster

Objectives: The study was conducted to assess the redox status of Drosophila flies upon oral intake of insulin-mimetic salt, sodium molybdate (Na₂MoO₄).

Methods: Oxidative stress parameters and activities of antioxidant and associated enzymes were analyzed in two-day-old D. melanogaster insects after exposure of larvae and newly eclosed adults to three molybdate levels (0.025, 0.5, or 10 mM) in the food.

Results: Molybdate increased content of low molecular mass thiols and activities of catalase, superoxide dismutase, glutathione-S-transferase, and glucose-6-phosphate dehydrogenase in males. The activities of these enzymes were not affected in females. Males exposed to molybdate demonstrated lower carbonyl protein levels than the control cohort, whereas females at the same conditions had higher carbonyl protein content and catalase activity than ones in the control cohort. The exposure to 10 mM sodium molybdate decreased the content of protein thiols in adult flies of both sexes. Sodium molybdate did not affect the activities of NADP-dependent malate dehydrogenase and thioredoxin reductase in males or NADP-dependent isocitrate dehydrogenase in either sex at any concentration.

Discussion: Enhanced antioxidant capacity in upon Drosophila flies low molybdate levels in the food suggests that molybdate can be potentially useful for the treatment of certain pathologies associated with oxidative stress.     

Distorted octahedral coordination of tungstate in a subfamily of specific binding proteins

Bacteria and archaea import molybdenum and tungsten from the environment in the form of the oxyanions molybdate (MoO₄ ²⁻) and tungstate (WO₄ ²⁻). These substrates are captured by an external, high-affinity binding protein, and delivered to ATP binding cassette transporters, which move them across the cell membrane. We have recently reported a crystal structure of the molybdate/tungstate binding protein ModA/WtpA from Archaeoglobus fulgidus, which revealed an octahedrally coordinated central metal atom. By contrast, the previously determined structures of three bacterial homologs showed tetracoordinate molybdenum and tungsten atoms in their binding pockets. Until then, coordination numbers above four had only been found for molybdenum/tungsten in metalloenzymes where these metal atoms are part of the catalytic cofactors and coordinated by mostly non-oxygen ligands. We now report a high-resolution structure of A. fulgidus ModA/WtpA, as well as crystal structures of four additional homologs, all bound to tungstate. These crystal structures match X-ray absorption spectroscopy measurements from soluble, tungstate-bound protein, and reveal the details of the distorted octahedral coordination. Our results demonstrate that the distorted octahedral geometry is not an exclusive feature of the A. fulgidus protein, and suggest distinct binding modes of the binding proteins from archaea and bacteria. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. K. Hollenstein and M. Comellas-Bigler contributed equally to this work.     

The Effect of the Simultaneous Addition of Molybdenum and Tungsten to the Culture Medium on the Formate Dehydrogenase Activity from Methylobacterium sp. RXM

Shake flask cultivation of the facultative methylotroph Methylobacterium sp. RXM was carried out by using a statistical experimental design to investigate the role of metal association on the formate dehydrogenase (FDH) levels. The maximal values of FDH activity were obtained for tungsten concentration up to 0.6 μM and for molybdenum concentration between 0.6 and 0.9 μM. The negative polynomial parameter (β₂) for tungsten compared with the positive polynomial parameter (β₁) for molybdenum on the FDH activity suggested that the latter metal exerts a stronger influence on the enzyme stimulation than the tungsten metal. A negative interaction between both metals was found, suggesting that tungsten and molybdenum shared an antagonistic effect on the enzyme activity. Received: 21 October 1997 / Accepted: 8 December 1997     

Nitrate as a sole nitrogen source forMethanococcus thermolithotrophicus and its effect on growth of several methanogenic bacteria

Methanococcus (Mc.) thermolithotrophicus can use nitrate as the sole source of nitrogen, but four other species of methanogens cannot. The growth rate was similar on both nitrate and ammonium, but yields were 20–25% lower on nitrate.Mc. thermolithotrophicus, Methanobacterium thermoautotrophicum, andMethanobrevibacterium smithii were not inhibited by 20 mM nitrate, butMethanospirillum hungatei was inhibited 35%, andMethanosarcina barkeri was completely inhibited by 20 mM nitrate. WhenMc. thermolithotrophicus was growing with nitrate as the sole source of nitrogen, growth was dependent on either molybdenum or tungsten, and the presence of both gave the best growth response; vanadium or chromium did not replace the requirement for these metals. Growth on ammonium could not be strictly demonstrated to require either of these metals, but both molybdenum and tungsten stimulated growth.     

Function of methylcobalamin: coenzyme M methyltransferase isoenzyme II in Methanosarcina barkeri

Methanosarcina barkeri was recently shown to contain two cytoplasmic isoenzymes of methylcobalamin: coenzyme M methyltransferase (methyltransferase 2). Isoenzyme I predominated in methanol-grown cells and isoenzyme II in acetate-grown cells. It was therefore suggested that isoenzyme I functions in methanogenesis from methanol and isoenzyme II in methanogenesis from acetate. We report here that cells of M. barkeri grown on trimethylamine, H₂/CO₂, or acetate contain mainly isoenzyme II. These cells were found to have in common that they can catalyze the formation of methane from trimethylamine and H₂, whereas only acetate-grown cells can mediate the formation of methane from acetate. Methanol-grown cells, which contained only low concentrations of isoenzyme II, were unable to mediate the formation of methane from both trimethylamine and acetate. These and other results suggest that isoenzyme II (i) is employed for methane formation from trimethylamine rather than from acetate, (ii) is constitutively expressed rather than trimethylamine-induced, and (iii) is repressed by methanol. The constitutive expression of isoenzyme II in acetate-grown M. barkeri can explain its presence in these cells. The N-terminal amino acid sequences of isoenzyme I and isoenzyme II were analyzed and found to be only 55% similar.Abbreviations H-S-CoM coenzyme M or 2-mercaptoethane-sulfonate - CH₃-S-CoM methyl-coenzyme M or 2(methylthio)-ethanesulfonate - [Co] cobalamin - CH₃-[Co] methylcobalamin - H₄MPT tetrahydromethanopterin - CH₃-H₄MPT N ⁵-methyltetrahydromethanopterin - MT₁ methyltransferase 1 or methanol: 5-hydroxybenzimidazolyl cobamide methyltransferase - MT₂ methyltransferase 2 or methylcobalamin: coenzyme M methyltransferase - Mops morpholinopropanesulfonate - 1 U = 1 mol/min     

Isolation and Characterization of Methanobacterium thermoautotrophicum ΔH Mutants Unable To Grow under Hydrogen-Deprived Conditions

By using random mutagenesis and enrichment by chemostat culturing, we have developed mutants of Methanobacterium thermoautotrophicum that were unable to grow under hydrogen-deprived conditions. Physiological characterization showed that these mutants had poorer growth rates and growth yields than the wild-type strain. The mRNA levels of several key enzymes were lower than those in the wild-type strain. A fed-batch study showed that the expression levels were related to the hydrogen supply. In one mutant strain, expression of both methyl coenzyme M reductase isoenzyme I and coenzyme F₄₂₀-dependent 5,10-methylenetetrahydromethanopterin dehydrogenase was impaired. The strain was also unable to form factor F₃₉₀, lending support to the hypothesis that the factor functions in regulation of methanogenesis in response to changes in the availability of hydrogen.     

Sulfate inhibition of molybdate assimilation by planktonic algae and bacteria: some implications for the aquatic nitrogen cycle

Molybdenum is required for both dinitrogen fixation and nitrate assimilation. In oxic waters the primary form of molybdenum is the molybdate anion. Using radioactive [⁹⁹Mol Na₂MoO₄, we have shown that the transport of molybdate by a natural assemblage of freshwater phytoplankton is light-dependent and follows typical saturation kinetics. The molybdate anion is strikingly similar to sulfate and we present data to show that sulfate is a competitive inhibitor of molybdate assimilation by planktonic algae and bacteria. The ability of freshwater phytoplankton to transport molybdate is inhibited at sulfate concentrations as low as 5% of those in seawater and at sulfate: molybdate ratios as low as 50 to 100 times lower than those found in seawater, Similarly, the growth of both a freshwater bacterium and a saltwater diatom was inhibited at sulfate: molybdate ratios lower than those in seawater.The ratio of sulfate to molybdate is 10 to 100 times greater in seawater than in fresh water. This unfavorable sulfate: molybdate ratio may make molybdate less biologically available in the sea. The sulfate: molybdate ratio may explain, in part, the low rates of nitrogen fixation in N-limited salt waters.     

The anaerobic degradation of l-serine and l-threonine in enterobacteria: networks of pathways and regulatory signals

The mechanisms controlling the biosynthesis and degradation of l-serine and l-threonine are remarkably complex. Their metabolism forms a network of pathways linking several amino acids, central primary metabolites such as pyruvate, oxaloacetate and 3-phosphoglycerate, and C₁ metabolism. Studies on the degradation of these amino acids in Escherichia coli have revealed the involvement of fascinating enzymes that utilise quite diverse catalytic mechanisms. Moreover, it is emerging that both environmental and metabolic signals have a major impact in controlling enzyme synthesis. This is exemplified by the anaerobically regulated tdc operon, which encodes a metabolic pathway for the degradation of serine and threonine. Studies on this pathway are beginning to provide insights into how an organism adapts its genetic makeup to meet the physiological demands of the cell. Received: 30 August 1998 / Accepted: 9 October 1998     

Sodium dependence of growth and methane formation in Methanobacterium thermoautotrophicum

Methanobacterium thermoautotrophicum was found to require sodium for growth and for CO₂ reduction to methane. The dependence of the rate of growth and methane formation on the sodium concentration was hyperbolic with an apparent K _s for sodium of approximately 1 mM. The findings indicate that sodium has a specific function in the energy metabolism of this bacterium.