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     

Reduction of molybdate by sulfate-reducing bacteria

Molybdate is an essential trace element required by biological systems including the anaerobic sulfate-reducing bacteria (SRB); however, detrimental consequences may occur if molybdate is present in high concentrations in the environment. While molybdate is a structural analog of sulfate and inhibits sulfate respiration of SRB, little information is available concerning the effect of molybdate on pure cultures. We followed the growth of Desulfovibrio gigas ATCC 19364, Desulfovibrio vulgaris Hildenborough, Desulfovibrio desulfuricans DSM 642, and D. desulfuricans DSM 27774 in media containing sub-lethal levels of molybdate and observed a red-brown color in the culture fluid. Spectral analysis of the culture fluid revealed absorption peaks at 467, 395 and 314 nm and this color is proposed to be a molybdate–sulfide complex. Reduction of molybdate with the formation of molybdate disulfide occurs in the periplasm D. gigas and D. desulfuricans DSM 642. From these results we suggest that the occurrence of poorly crystalline Mo-sulfides in black shale may be a result from SRB reduction and selective enrichment of Mo in paleo-seawater.     

Purification and characterization of an alcohol dehydrogenase from 1,2-propanediol-grownDesulfovibrio strain HDv

The sulfate-reducing bacterimDesulfovibrio strain HDv (DSM 6830) grew faster on (S)- and on (R, S)-1,2-propanediol (μ_max 0.053 h⁻) than on (R)-propanediol (0.017 h⁻¹) and ethanol (0.027 h⁻¹). From (R, S)-1,2-propanediol-grown cells, an alcohol dehydrogenase was purified. The enzyme was oxygen-labile, NAD-dependent, and decameric; the subunit mol. mass was 48 kDa. The N-terminal amino acid sequence indicated similarity to alcohol dehydrogenases belonging to family III of NAD-dependent alcohol dehydrogenases, the first 21 N-terminal amino acids being identical to those of theDesulfovibrio gigas alcohol dehydrogenase. Best substrates were ethanol and propanol (K _m of 0.48 and 0.33 mM, respectively). (R, S)-1,2-Propanediol was a relatively poor substrate for the enzyme, but activities in cell extracts were high enough to account for the growth rate. The enzyme showed a preference for (S)-1,2-propanediol over (R)-1,2-propanediol. Antibodies raised against the alcohol dehydrogenase ofD. gigas showed cross-reactivity with the alcohol dehydrogenase ofDesulfovibrio strain HDv and with cell extracts of six other ethanol-grown sulfate-reducing bacteria.     

Accumulation and incorporation of 185W-tungsten into proteins of Clostridium acidiurici and Clostridium cylindrosporum

Uptake of tungstate by growing cells was unaffected by the presence of molybdate in Clostridium cylindrosporum, whereas in C. acidiurici the accumulation was decreased by molybdate at 10^-6 mol/l tungstate and higher concentrations. The labelling pattern of soluble proteins by ¹⁸⁵W-tungsten indicated after gel chromatography the presence of three different tungstoproteins in both bacteria. Formate dehydrogenase activity always eluted at a maximum of tungsten labelling. The incorporation of tungsten into formate dehydrogenase containing fractions and a possible tungsten-binding-storage protein was independent of the presence of excess molydate pointing to a genuine role for tungstate in these bacteria.     

Differentiation between Clostridium acidiurici and Clostridium cylindrosporum on the basis of specific metal requirements for formate dehydrogenase formation

The formate dehydrogenases of Clostridium acidiurici and of C. cylindrosporum coupled the oxidation of formate with the reduction of viologen dyes. The basal activity level was about 0.85 moles/min s mg of protein for both species. The level of formate dehydrogenase of C. acidiurici increased 12-fold when 10^-7 M tungstate and selenite were present during growth. Molybdate exerted no effect. On the other hand, molybdate and selenite were required to increase the formate dehydrogenase of C. cylindrosporum, and tungstate exhibited an antagonistic effect in this organism.Growth on hypoxanthine generally depended on the addition of bicarbonate. Supplementation with tungstate and selenite accelerated growth of C. acidiurici and increased again the level of formate dehydrogenase. The addition of both, molybdate and selenite was necessary to initiate growth of C. cylindrosporum and to form an active formate dehydrogenase.The differences in the requirement for metal ion supplementation to form high levels of formate dehydrogenase and their involvement in hypoxanthine degradation can be used to differentiate between C. acidiurici and C. cylindrosporum.Abbreviation FDH formate dehydrogenase     

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.     

Selenium requirement for active xanthine dehydrogenase from Clostridium acidiurici and Clostridium cylindrosporum

The xanthine dehydrogenase of Clostridium acidiurici and C. cylindrosporum was assayed with methyl viologen as acceptor. In C. acidiurici the basal activity level was about 0.3 mol/min x mg of protein. Cells grown on uric acid in the presence of 10^-7 M selenite showed a 14-fold increase in xanthine dehydrogenase activity, which decreased with higher selenite concentrations (10^-5 M). The supplementation with 10^-7 M molybdate or tungstate was without effect. High concentrations of tungstate decreased the xanthine dehydrogenase if selenite was also present. In comparison, high concentrations of molybdate affected only a small decrease in activity level at the optimal concentration for selenite and relieved to some degree the inhibitory effect of 10^-5 M selenite. With hypoxanthine and xanthine as substrates for growth again only the addition of selenite was necessary to show a similar increase in xanthine dehydrogenase activity. C. acidiurici could be grown in a mineral medium. Both xanthine dehydrogenase and formate dehydrogenase exhibited the highest level of activity if selenite and tungstate were present in that medium.In C. cylindrosporum the basal activity level of xanthine dehydrogenase was about 0.95 mol/min x mg of protein. The addition of 10^-7 M selenite to the growth medium increased the activity level about 3-fold, but the highest level (3.7 U/mg) was reached if 10^-7 M molybdate was also added. The presence of tungstate resulted in a decreased enzyme activity.     

The role of tungstate and/or molybdate in the formation of aldehyde oxidoreductase in Clostridium thermoaceticum and other acetogens; immunological distances of such enzymes

Besides Clostridium thermoaceticum and C. formicoaceticum other resting acetogenic clostridia such as C. aceticum and C. thermoautotrophicum and to a lesser extent non-clostridial acetogens such as Butyribacterium methylotrophicum and Eubacterium limosum were able to reduce propionate to propanol at the expense of carbon monoxide or formate. Methylviologen usually increased the reduction rate. Ten M molybdate in the growth medium decreased this capability for C. thermoaceticum but increased it or had no effect for the other organisms. Ten M tungstate in the growth medium increased the aldehyde oxidoreductase activity in all organisms. Crude extracts of C. thermoaceticum cells grown in the presence of 10 M or 1 mM molybdate showed by ELISA the same or even a 4 fold concentration of aldehyde oxidoreductase in the latter case. However, the enzymic activity was very low in both cases. Omission of dithionite in the growth medium diminished the antigen by a factor of about 8. The immunological distance between the enzyme from C. thermoaceticum and C. thermoautotrophicum was rather low but very large to C. formicoaceticum and undeterminably large to the other organisms.Abbreviations Ald-DH aldehyde dehydrogenase - AOR aldehyde oxidoreductase - CO-DH carbon-monoxide dehydrogenase - ELI-SA enzyme-linked immunosorbent assay - FDH formate dehydrogenase - MV methylviologen - V⁺⁺ oxidised - V^+. reduced viologen     

Environmental regulation of alcohol metabolism in thermotolerant methylotrophic Bacillus strains

The thermotolerant methylotroph Bacillus sp. C1 possesses a novel NAD-dependent methanol dehydrogenase (MDH), with distinct structural and mechanistic properties. During growth on methanol and ethanol, MDH was responsible for the oxidation of both these substrates. MDH activity in cells grown on methanol or glucose was inversely related to the growth rate. Highest activity levels were observed in cells grown on the C₁-substrates methanol and formaldehyde. The affinity of MDH for alcohol substrates and NAD, as well as V _max, are strongly increased in the presence of a M _r 50,000 activator protein plus Mg²⁺-ions [Arfman et al. (1991) J Biol Chem 266: 3955–3960]. Under all growth conditions tested the cells contained an approximately 18-fold molar excess of (decameric) MDH over (dimeric) activator protein. Expression of hexulose-6-phosphate synthase (HPS), the key enzyme of the RuMP cycle, was probably induced by the substrate formaldehyde. Cells with high MDH and low HPS activity levels immediately accumulated (toxic) formaldehyde when exposed to a transient increase in methanol concentration. Similarly, cells with high MDH and low CoA-linked NAD-dependent acetaldehyde dehydrogenase activity levels produced acetaldehyde when subjected to a rise in ethanol concentration. Problems frequently observed in establishing cultures of methylotrophic bacilli on methanol- or ethanol-containing media are (in part) assigned to these phenomena.Abbreviations MDH NAD-dependent methanol dehydrogenase - ADH NAD-dependent alcohol dehydrogenase - A1DH CoA-linked NAD-dependent aldehyde dehydrogenase - HPS hexulose-6-phosphate synthase - G6Pdh glucose-6-phosphate dehydrogenase     

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     

Molybdenum cofactor amounts in Chlamydomonas reinhardtii depend on the Nit5 gene function related to molybdate transport

Strain 21gr from Chlamydomonas reinhardtii is a cryptic mutant defective in the Nit5 gene related to the biosynthesis of molybdenum cofactor (MoCo). In spite of this mutation, this strain has active MoCo and can grow on nitrate media. In genetic crosses, the Nit5 mutation cosegregated with a phenotype of resistance to high concentrations of molybdate and tungstate. Molybdate/tungstate toxicity was much higher in nitrate than in ammonium media. Strain 21gr showed lower amounts of MoCo activity than the wild type both when grown in nitrate and after growth in ammonium and nitrate induction. However, nitrate reductase (NR) specific activity was similar in wild type and 21gr cells. Tungstate, either at nanomolar concentrations in nitrate media or at micromolar concentrations during growth in ammonium and nitrate induction, strongly decreased MoCo and NR amounts in wild‐type cells but had a slight effect in 21gr cells. Molybdate uptake activity of ammonium‐grown cells from both the wild‐type and 21gr strains was small and blocked by sulphate 0·3 mM . However, cells from nitrate medium showed a molybdate uptake activity insensitive to sulphate. This uptake activity was much higher and more sensitive to inhibition by tungstate in the wild type than in strain 21gr. These results suggest that strain 21gr has a high affinity and low capacity molybdate transport system able to discriminate efficiently tungstate, and lacks a high capacity molybdate/tungstate transport system, which operates in wild‐type cells upon nitrate induction. This high capacity molybdate transport system would account for both the stimulating effect of molybdate on MoCo amounts and the toxic effects of tungstate and molybdate when present at high concentrations.     

Defluviimonas denitrificans gen. nov., sp. nov., and Pararhodobacter aggregans gen. nov., sp. nov., non-phototrophic Rhodobacteraceae from the biofilter of a marine aquaculture

Three Gram-negative bacterial strains were isolated from the biofilter of a recirculating marine aquaculture. They were non-pigmented rods, mesophiles, moderately halophilic, and showed chemo-organoheterotrophic growth on various sugars, fatty acids, and amino acids, with oxygen as electron acceptor; strains D9-3^T and D11-58 were in addition able to denitrify. Phototrophic or fermentative growth could not be demonstrated. Phylogenetic analysis of the 16S rRNA gene sequences placed D9-3^T and D11-58, and D1-19^T on two distinct branches within the alpha-3 proteobacterial Rhodobacteraceae, affiliated with, but clearly separate from, the genera Rhodobacter, Rhodovulum, and Rhodobaca. Based on morphological, physiological, and 16S rRNA-based phylogenetic characteristics, the isolated strains are proposed as new species of two novel genera, Defluviimonas denitrificans gen. nov., sp. nov. (type strain D9-3^T = DSM 18921^T = ATCC BAA-1447^T; additional strain D11-58 = DSM19039 = ATCC BAA-1448) and Pararhodobacter aggregans gen. nov., sp. nov (type strain D1-19^T = DSM 18938^T = ATCC BAA-1446^T).     

The effect of molybdate and tungstate ions on the metabolic rates and enzyme activities in methanol-grown Methylobacterium sp. RXM

Feed-switching experiments were carried out in steady-state methanol-excess chemostat Methylobacterium sp. RXM cultures at a fixed dilution rate, temperature and pH (0.10 h^–1, 30° C and 6.95, respectively). The removal of molybdate from the nutrient supply led to a metabolic energy deficiency reflected in the molar growth yield and biomass values. High carbon conversion efficiency was linked with high formate dehydrogenase (FDH) activity and observed only when either molybdate or tungstate was added to the feed medium. A constant coenzyme ratio NAD⁺/K-ferri-cyanide linked to FDH activity was found during the enzyme stimulation period following the feed-switching experiment with tungstate addition, which suggests that both activities belong to the same enzyme. Quantitative metabolic responses (carbon conversion efficiency, methanol and O₂ consumption rates, CO₂ production rate and respiratory quotient) were measured in between steady-states just after the shift in the nutrient supply composition. Correspondence to: F. M. Gírio     

Evidence for a tungsten-stimulated aldehyde dehydrogenase activity of Desulfovibrio simplex that oxidizes aliphatic and aromatic aldehydes with flavins as coenzymes

The aldehyde dehydrogenase activity of the sulfate-reducing bacterium Desulfovibrio simplex strain DSM 4141 was characterized in cell-free extracts. Oxygen-sensitive, constitutive aldehyde dehydrogenase activity was found in cells grown on l(+)-lactate, hydrogen, or vanillin with sulfate as the electron acceptor. A 1.83- to 2.6-fold higher specific activity was obtained in cells grown in media supplemented with 1 μM WO₄ ^2–. The aldehyde dehydrogenase in cell-free extracts catalyzed the oxidation of aliphatic (K _m < 20 μM) and aromatic aldehydes (K _m < 0.32 mM) using methyl viologen as the electron acceptor. Flavins (FMN and FAD) were also active and are proposed to be the natural cofactors, while no activity was obtained with NAD⁺ or NADP⁺. ¹⁸⁵WO₄ ^2– was incorporated in vivo into D. simplex; it was found exclusively in the soluble fraction (≥ 98%). Anionic-exchange chromatography demonstrated coelution of ¹⁸⁵W with two distinct peaks, the first one containing hydrogenase and formate dehydrogenase activities, and the second one aldehyde dehydrogenase activity. Received: 7 February 1997 / Accepted: 6 June 1997     

Some properties of formate dehydrogenase,accumulation and incorporation of 185W-tungsten into proteins of Clostridium formicoaceticum

Formate dehydrogenase of Clostridium formicoaceticum used only methyl and benzyl viologen, but not NAD as electron acceptor. The S_0.5 values were 0.9×10^-4 M for formate and 5.8×10^-3 M for methyl viologen. Using potassium phosphate buffer a pH-optimum of 7.9 was observed. The initial velocity of the formate dehydrogenase activity reached a maximum at 70°C, whereas the activity was stable only up to 50°C. The level of formate dehydrogenase in C. formicoaceticum was increased to its maximum when 10^-6 M selenite and 10^-7 M tungstate were added to a synthetic medium. Addition of molybdate instead of tungstate did not increase the level of formate dehydrogenase. ¹⁸⁵W-tungsten was concentrated about 100-fold by C. formicoaceticum; molybdate had no major effect on the uptake of tungsten. ¹⁸⁵W-tungsten was found almost exclusively in the soluble fluid and was predominantly recovered after chromatography in a protein of about 88000 molecular weight. Occasionally a labelled protein of low molecular weight was observed. Again molybdate added even in high molar excess did not influence the labelling pattern. No radioactivity peak could be obtained at the elution peak of formate dehydrogenase activity. The extreme instability of formate dehydrogenase prevented further purification.Abbreviations FDH formate dehydrogenase - DTE dithioerythritol - HEPES hydroxyethylpiperazine N-2-ethane sulconic acid - TEA triethylamine - DCPIP 2,6-dichlorophenolindophenol - PMS phenazine methosulfate - TTC triphenyltetrazolium     

Growth yields of green sulfur bacteria in mixed cultures with sulfur and sulfate reducing bacteria

1. Dry weight yields from mixed cultures ofProsthecochloris aestuarii orChlorobium limicola with the sulfur reducingDesulfuromonas acetoxidans were determined on different growth limiting amounts of acetate, ethanol or propanol. The obtained yields agreed well with values predicted from stoichiometric calculations. 2. From mixed cultures of twoChlorobium limicola strains withDesulfovibrio desulfuricans orD. gigas on ethanol as the growth limiting substrate, dry weight yields were obtained as calculated for the complete utilization of the ethanol by the mixed cultures. 3. Dry weight yield determinations for two pure cultures ofChlorobium limicola with different growth limiting amounts of sulfide in the absence and presence of excess acetate confirmed that acetate is incorporated byChlorobium in a fixed proportion to sulfide; compared to the yield in the absence of acetate the yield is increased two to threefold in the presence of acetate. 4. The lowest possible sulfide concentrations necessary for optimal growth of mixed cultures of eitherProsthecochloris orChlorobium withDesulfuromonas on acetate were 7–8 mg H₂S per liter of medium. 5. Doubling times at the growth rate limiting light intensities of 5, 10, 20, 50, 100 and 200 lux were determined under optimal growth conditions for the following phototrophic bacteria:Prosthecochloris aestuarii, Chlorobium phaeovibriodes, Chromatium vinosum andRhodopseudomonas capsulata. Reasonably good growth was still obtained withProsthecochloris at 10 and 5 lux light intensity at which no growth of the purple bacteria could be observed.     

A comparative study of aldehyde dehydrogenase and alcohol dehydrogenase activities in crucian carp and three other vertebrates: apparent adaptations to ethanol production

Summary In the final step of the pathway producing ethanol in anoxic crucian carp (Carassius carassius L.), acetaldehyde is reduced to ethanol by alcohol dehydrogenase. The presence of aldehyde dehydrogenase in the tissues responsible for ethanol production could cause an undesired oxidation of acetaldehyde to acetate coupled with a reduction of NAD⁺ to NADH. Moreover, acetaldehyde could competitively inhibit the oxidation of reactive biogenic aldehydes. In the present study, the distribution of aldehyde dehydrogenase (measured with a biogenic aldehyde) and alcohol dehydrogenase (measured with acetaldehyde) were studied in organs of crucian carp, common carp (Cyprinus carpio L.), rainbow trout (Salmo gairdneri Richardson), and Norwegian rat (Rattus norvegicus Berkenhout). The results showed that alcohol dehydrogenase and aldehyde dehydrogenase activities were almost completely spatially separated in the crucian carp. These enzymes occurred together in the other three vertebrates. In the crucian carp, alcohol dehydrogenase was only found in red and white skeletal muscle, while these tissues contained exceptionally low aldehyde dehydrogenase activities. Moreover, the low aldehyde dehydrogenase activity found in crucian carp red muscle was about 1000 times less sensitive to inhibition by acetaldehyde than that found in other tissues and other species. The results are interpreted as demonstrating adaptations to avoid a depletion of ethanol production, and possibly inhibition of biogenic aldehyde metabolism.Abbreviations ADH alcohol dehydrogenase - ALDH aldehyde dehydrogenase - DOPAL 3,4-dihydroxyphenylacetaldehyde - MAO monoamine oxidase - PCA perchloric acid     

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     

Molybdate metabolism in Aspergillus nidulans. I. Mutations affecting nitrate reductase and-or xanthine dehydrogenase

Summary Further evidence supports the hypothesis that nitrate reductase and xanthine dehydrogenase are molybdo-enzymes inAspergillus nidulans, probably sharing a molybdenum-containing cofactor. This evidence includes (1) five-fold greater toxicity of tungstate on nitrate and hypoxanthine than on other nitrogen sources, (2) locus-specific molybdate reparability of both nitrate reductase and xanthine dehydrogenase at one (cnxE) of five (cnx) loci where mutation can result in pleiotropic loss of both enzyme activities, and (3) an additional class of mutants (molB) which are both molybdate resistant and partially defective in utilization of nitrate and hypoxanthine as nitrogen sources. Moreover, the phenotypes on molybdate-containing media of various mutants altered in the regulation of nitrate reductase synthesis and the ability of nitrate to protect against molybdate toxicity suggest that incorporation of molybdenum into nitrate reductase or into something having the same control properties as nitrate reductase can detoxify molybdate. However, mutations affecting regulation of xanthine dehydrogenase synthesis do not affect growth responses to molybdate. The properties of another class of molybdate resistance mutations (molA) suggest that there is another nitrate-inducible intracellular molybdate detoxification mechanism in addition to the one having identical control properties to nitrate reductase.