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1.
The oxidation of organic compounds with elemental sulfur or thiosulfate as electron acceptor was studied in the anaerobic hyperthermophilic archaea Thermoproteus tenax and Pyrobaculum islandicum. T. tenax was grown on either glucose or casamino acids and sulfur; P. islandicum on peptone and either elemental sulfur or thiosulfate as electron acceptor. During exponential growth only CO2 and H2S rather than acetate, alanine, lactate, and succinate were detected as fermentation products of both organisms; the ratio of CO2/H2S formed was 1:2 with elemental sulfur and 1:1 with thiosulfate as electron acceptor. Cell extracts of T. tenax and P. islandicum contained all enzymes of the citric acid cycle in catabolic activities: citrate synthase, aconitase, isocitrate dehydrogenase (NADP+-reducing), oxoglutarate: benzylviologen oxidoreductase, succinyl-CoA synthetase, succinate dehydrogenase, fumarase and malate dehydrogenase (NAD+-reducing). Carbon monoxide dehydrogenase activity was not detected. We conclude that in T. tenax and P. islandicum organic compounds are completely oxidized to CO2 with sulfur or thiosulfate as electron acceptor and that acetyl-CoA oxidation to CO2 proceeds via the citric acid cycle.  相似文献   

2.
RNA synthesis during morphogenesis of the fungusMucor racemosus   总被引:6,自引:0,他引:6  
Bacteroides succinogenes produces acetate and succinate as major products of carbohydrate fermentation. An investigation of the enzymes involved indicated that pyruvate is oxidized by a flavin-dependent pyruvate cleavage enzyme to acetyl-CoA and CO2. Active CO2 exchange is associated with the pyruvate oxidation system. Reduction of flavin nucleotides is CoASH-dependent and does not require ferredoxin. Acetyl-CoA is further metabolized via acetyl phosphate to acetate and ATP. Reduced flavin nucleotide is used to reduce fumarate to succinate by a particulate flavin-specific fumarate reductase reaction which may involve cytochrome b. Phosphoenolpyruvate (PEP) is carboxylated to oxalacetate by a GDP-specific PEP carboxykinase. Oxalacetate, in turn, is converted to malate by a pyridine nucleotide-dependent malate dehydrogenase. The organism has a NAD-dependent glyceraldehyde-3-phosphate dehydrogenase. The data suggest that reduced pyridine nucleotides generated during glycolysis are oxidized in malate formation and that the electrons generated during pyruvate oxidation are used to reduce fumarate to succinate.  相似文献   

3.
Enzyme activities conceivably involved in the activation of sulfate were studied with Desulfotomaculum ruminis, D. acetoxidans, D. nigrificans, D. orientis, and Desulfovibrio vulgaris. Cell lysates of these species revealed activities of at least 8 nkat/mg protein (i.e., 480 nmol per min and mg protein) of ATP sulfurylase, acetate kinase, phosphotransacetylase and adenylate kinase. ADP sulfurylase was not detected. Pyrophosphatase activity was high (73 to 97 nkat/mg protein) in Desulfotomaculum orientis and Desulfovibrio vulgaris. In these strains pyrophosphatase was activated by addition of a reductant (dithionite). In Desulfotomaculum ruminis, D. acetoxidans, and D. nigrificans, only low pyrophosphatase activity (2.5 to 6.3 nkat/mg protein) was measured, which was not reductant-activated. Some hints indicated a membrane association of the pyrophosphatase in D. ruminis, and possibly also in D. acetoxidans and D. nigrificans. Activities of a pyrophosphate-dependent acetate kinase (PPi:acetate kinase), a PPi:AMP kinase or a polyphosphate:AMP kinase were not detected or negligible. The results are not in favour of the assumption that pyrophosphate formed by ATP sulfurylase during sulfate activation might be utilized to form acetyl phosphate in Desulfotomaculum species. Contrary results of other authors were shown to be artefacts caused by chemical hydrolysis of acetyl phosphate in the molybdate-sulfuric acid reagent used for phosphate determination.Abbreviations Pi orthophosphate - PPi pyrophosphate - APS adenosine phosphosulfate - AP5A, P1 P5-di(adenosine-5-)pentaphosphate - CTAB cetyltrimethylammonium bromide - MOPS 3-(N-morpholino)propanesulfonic acid - HEPES N(-2-hydroxyethyl)piperazine-N-2-ethanesulfonic acid  相似文献   

4.
The hyperthermophilic anaerobic eubacterium Thermotoga maritima was grown on glucose as carbon and energy source. During growth 1 mol glucose was fermented to 2 mol acetate, 2 mol CO2 and 4 mol H2. The molar growth yicld on glucose (Yglucose) was about 45 g cell dry mass/mol glucose. In the presence of elemental sulfur growing cultures of T. maritima converted 1 mol glucose to 2 mol acetate, 2 mol CO2 about 0.5 mol H2 and about 3.5 mol H2S. Yglucose was about 45 g/mol. Cell extracts contained all enzymes of the Embden-Meyerhof pathway: hexokinase (0.29 U/mg, 50°C), glucose-6-phosphate isomerase (0.56 U/mg, 50°C), phosphofructokinase (0.19 U/mg, 50° C), fructose-1,6-bisphosphate aldolase (0.033 U/mg, 50°C), triosephosphate isomerase (6.3 U/mg, 50°C), glyceraldehyde-3-phosphate dehydrogenase (NAD+ reducing: 0.63 U/mg, 50°C), phosphoglycerate kinase (3.7 U/mg, 50°C), phosphoglycerate mutase (0.4 U/mg, 50°C); enolase (4 U/mg, 80°C), pyruvate kinase (0.05 U/mg, 50°C). Furthermore, cell extracts contained pyruvate: ferredoxin oxidoreductasee (0.43 U/mg, 60°C); NADH: ferredoxin oxidoreductase (benzylviologen reduction: 0.46 U/mg, 80°C); hydrogenase (benzylviologen reduction: 15 U/mg, 80°C), phosphate acetyltransferase (0.13 U/mg, 80°C), acetate kinase (1.2 U/mg, 55°C), lactate dehydrogenase (0.16 U/mg, 80°C) and pyruvate carboxylase (0.02 U/mg, 50°C). The findings indicate that the hyperthermophilic eubacterium T. maritima ferments sugars (glucose) to acetate, CO2 and H2 involving the Embden-Meyerhof pathway, phosphate acetyltransferase and acetate kinase. Thus, the organism differs from the hyperthermophilic archaeon Pyrococcus furiosus which ferments sugars to acetate, CO2 and H2 involving a modified non-phosphorylated Entner-Doudoroff pathway and acetyl-CoA synthetase (ADP forming).  相似文献   

5.
Desulfotomaculum acetoxidans oxidizes acetate to CO2 with sulfate. This organism metabolizes acetate via a pathway in which C1 units rather than tri- and dicarboxylic acids are intermediates. We report here that cell extracts of D. acetoxidans catalyzed an exchange between CO2 and the carboxyl group of acetate at a rate of 90 nmol · min-1 · mg-1 protein which is sufficient to account for the in vivo acetate oxidation rate of 250 nmol · min-1 · mg-1 protein. The reaction was strictly dependent on both ATP and coenzyme A. The extracts contain high activities of acetate kinase (6.3 U · mg-1 protein) and phosphotransacetylase (60 U · mg-1 protein). These findings indicate that acetyl-CoA rather than acetyl-phosphate or acetate is the substrate of the carbon-carbon cleavage activity. Exchange was only observed in the presence of strong reducing agents such as Ti3+. Interestingly, the cell extracts also catalyzed the reduction of CO2 to CO with Ti3+ as electron donor (120 nmol · min-1 · mg-1 protein). Carbon monoxide dehydrogenase and other oxidoreductases involved in acetate oxidation were found to be partially associated with the membrane fraction suggesting a membrane localization of these enzymes.Abbreviations MOPS Morpholinopropane sulfonic acid - Tricine N-tris(hydroxymethyl)-methylglycine - DTT d,l-1,4-Dithiothreitol - DMN 2,3-Dimethyl-1,4-naphthoquinone - MVOX Methyl viologen, oxidized - APS Adenosinephosphosulfate - SRB Sulfate reducing bacteria - U mol product formed per min  相似文献   

6.
Methanosarcina acetivorans, a member of the methanogenic archaea, can grow with carbon monoxide (CO) as the sole energy source and generates, unlike other methanogens, substantial amounts of acetate and formate in addition to methane. Phenotypic analyses of mutant strains lacking the cooS1F operon and the cooS2 gene suggest that the monofunctional carbon monoxide dehydrogenase (CODH) system contributes to, but is not required for, carboxidotrophic growth of M. acetivorans. Further, qualitative proteomic analyses confirm a recent report (Lessner et al., Proc Natl Acad Sci USA, 103:17921–17926, 2006) in showing that the bifunctional CODH/acetyl-CoA synthase (ACS) system, two enzymes involved in CO2-reduction, and a peculiar protein homologous to both corrinoid proteins and methyltransferases are synthesized at elevated levels in response to CO; however, the finding that the latter protein is also abundant when trimethylamine serves as growth substrate questions its proposed involvement in the reduction of methyl-groups to methane. Potential catabolic mechanisms and metabolic adaptations employed by M. acetivorans to effectively utilize CO are discussed.  相似文献   

7.
The marine green sulfur bacterium Chlorobium vibrioforme strain 1930 produced H2 and elemental sulfur from sulfide or thiosulfate under N limitation in the light. H2 production depended on nitrogenase and occurred only in the absence of ammonia. Methionine sulfoximine, an inhibitor of glutamine synthetase, prevented the switch-off by ammonia. In defined syntrophic cocultures of the acetate-oxidizing, sulfur-reducing bacterium Desulfuromonas acetoxidans with green sulfur bacteria, H2 was produced from acetate via a light-driven sulfur cycle. The sulfur-reducing bacterium could not be replaced by sulfate-reducing bacteria in these experiments. In a coculture of the marine Chlorobium vibrioforme strain 1930 and the sulfur-reducing bacterium Desulfuromonas acetoxidans strain 5071, optimum long-term H2 production from acetate was obtained with molecular nitrogen as N source, at low light intensity (110 mol · m-2 · s-1), in sulfide-reduced mineral medium (2 mM Na2S) at pH 6.8. Traces of sulfide (10 M) were sufficient to keep the sulfur cycle running. The coculture formed no poly--hydroxyalkanoates (PHA), but 20%–40% polysaccharide per cell dry mass. Per mol acetate added, the coculture formed 3.1 mol of H2 (78% of the theoretical maximum). Only 8% of the reducing equivalents was incorporated into biomass. The maximum rate of H2 production was 1300 ml H2 per day and g cell dry mass.Non-standard abbrevations MOPS 2-(N-morpholino) propane sulfonic acid - MSX Methionine sulfoximine - PHA poly--hydroxyalkanoates  相似文献   

8.
Chloroflexus aurantiacus OK-70 fl was grown photoautotrophically with hydrogen as electron source. The cultures were subjected to long term labelling experments with 13C-labelled acetate or alanine in the presence of sodium fluoroacetate. The presence of fluoroacetate caused the cells to accumulate large amounts of polyglucose which was hydrolysed and analysed by NMR. The labelling patterns of glucose were symmetric and in agreement with carbohydrate synthesis from acetate and CO2 via pyruvate synthase. The content of carbon derived from added acetate was highest in C2 and C5 of glucose, at least 20% higher than in C1 and C6. About one third of the glucose carbon was derived from added acetate, the rest being from CO2. Contrary to expectations, in glucose formed in the presence of C1-labelled acetate C1 and C6 contained more label than C2 and C5, and with C2-labelled acetate as the tracer glucose was mainly labelled in C2 and C5. Labelled CO2 was formed from acetate labelled at either position. The labelling data indicate a new metabolic pathway in C. aurantiacus. It is suggested that the cells form C1-labelled acetyl-CoA from C2-labelled acetyl-CoA and vice versa by a cyclic mechanism involving concomitant CO2 fixation and that this cycle is the part of the autotrophic CO2 fixation pathways in C. aurantiacus in which acetyl-CoA is formed from CO2.The polyglucose of C. aurantiacus appears to have predominantly (1–4)-linked structure with about 10% (1–6)-linkages as revealed by 13C-NMR.  相似文献   

9.
The strict anaerobe Desulfuromonas acetoxidans can oxidize acetate to CO2 with elemental sulfur as electron acceptor. 14C-labelling experiments and enzyme studies are described revealing that acetate oxidation proceeds via the citric acid cycle with the synthesis of oxaloacetate from acetate and 2 CO2 via pyruvate as anaplerotic reaction. An oxidation of acetate via one carbon unit intermediates as proposed for anaerobic bacteria fermenting acetate to 2 CO2 and 4 H2 was excluded.Dedicated to Professor Dr. Gerhart Drews on the occasion of his 60th birthday  相似文献   

10.
A new moderately thermophilic sulfur-reducing eubacterium was isolated from bottom deposits of Green Lake (Raoul Island, Kermadec archipelago, New Zealand). Cells are short rods, 1.5–1.8 by 0.5–0.7 m, single or in pairs, motile with one polar flagellum, gram-negative with S-layer of subunit structure. Growth occurred between 42 and 77°C with the optimum at 58–60°C and at pH from 6.0 to 7.2 with the optimum at 6.4–6.8. The bacterium was obligately anaerobic and obligately sulfur-respiring, and capable of lithoautotrophic growth on a mineral medium with S° and H2/CO2 gas phase. In addition to molecular hydrogen, a wide range of substrates can be utilized as energy source in the presence of elemental sulfur: pyruvate, acetate, butyrate, pentadecanate, palmitate, stearate. Products are CO2 and H2S. The G+C content of DNA is 33.5 mol%. DNA-DNA homology with the type species of the genus Desulfurella — Desulfurella acetivorans — is 69±2%. A new species, Desulfurella multipotens sp. nov., with the type strain RH-8 is described.  相似文献   

11.
The succinate dehydrogenase from the thermohalophilic bacterium Rhodothermus marinus is a member of the succinate:menaquinone oxidoreductases family. It is constituted by three subunits with apparent molecular masses of 70, 32, and 18 kDa. The optimum temperature for succinate dehydrogenase activity is 80°C, higher than the optimum growth temperature of R. marinus, 65°C. The enzyme shows a high affinity for both succinate (K m = 0.165 mM) and fumarate (K m = 0.10 mM). It contains the canonical iron–sulfur centers S1, S2, and S3, as well as two B-type hemes. In contrast to other succinate dehydrogenases, the S3 center has an unusually high reduction potential of +130 mV and is present in two different conformations, one of which presents an unusual EPR signal with g values at 2.035, 2.009, and 2.001. The apparent midpoint reduction potentials of the hemes, +75 and –65 mV at pH 7.5, are also higher than those reported for other enzymes. The heme with the lower potential (heme bL) presents a considerable dependence of the reduction potential with pH (redox–Bohr effect), having a pK a OX = 6.5 and a pK a red = 8.7. This behavior is consistent with the proposal that in these enzymes menaquinone reduction occurs close to heme bL, near to the periplasmic side of the membrane, and involving dissipation of the proton transmembrane gradient.  相似文献   

12.
A novel type of purple sulfur bacterium was isolated from a hypersaline sulfur spring on the shore of the Dead Sea. The cells of the isolate are irregularly rod-shaped or curved, and motile by means of a tuft of polar flagella. The photosynthetic system, containing bacteriochlorophyll a and carotenoids of the spirilloxanthin series, is located on stacks of lamellar membranes in the cell cytoplasm. The organism can grow either photoautotrophically with sulfide as electron donor, which is oxidized via extracellular sulfur to sulfate, or photoheterotrophically, using acetate, succinate, fumarate, malate or pyruvate as carbon sources. The bacterium is obligately anaerobic, and requires a source of reduced sulfur for growth. The isolate is moderately halophilic, and grows optimally at NaCl concentrations between 3 and 8%, temperatures between 30 and 45°C, and neutral pH. 16S ribosomal RNA oligonucleotide cataloging suggests a close relationship to purple sulfur bacteria of the genus Ectothiorhodospira. As the isolate differs greatly from the described members of the genus Ectothiorhodospira, we describe the isolate as a new species, and propose the name Ectothiorhodospira marismortui sp. nov.  相似文献   

13.
Cell extracts (100,000×g) of acetate grown Methanosarcina barkeri (strain MS) catalyzed CH4 and CO2 formation from acetyl-CoA with specific activities of 50 nmol·min-1·mg protein-1. CH4 formation was found to be dependent on tetrahydromethanopterin (H4MPT) (apparent K M=4 μM), coenzyme M (H-S-CoM), and 7-mercaptoheptanoylthreonine phosphate (H-S-HTP=component B) rather than on methanofuran (MFR) and coenzyme F420 (F420). Methyl-H4MPT was identified as an intermediate. This compound accumulated when H-S-CoM and H-S-HTP were omitted from the assays. These and previous results indicate that methanogenesis from acetate proceeds via acetyl phosphate, acetyl-CoA, methyl-H4MPT, and CH3-S-CoM as intermediates. The disproportionation of formaldehyde to CO2 and CH4 was also studied. This reaction was shown to be dependent on H4MPT, MFR, F420, H-S-CoM, and H-S-HTP.  相似文献   

14.
Growth of Thermoproteus neutrophilus at 85°C was studied using an improved mineral medium with CO2, CO2 plus acetate, CO2 plus propionate, or CO2 plus succinate as carbon sources; sulfur reduction with H2 to H2S was the sole source of energy. None of the carbon compounds added was oxidized to CO2. The organism grew autotrophically with a generation time of 9–14 h, up to a cell density of 0.5 g dry weight per liter (2×109 cells/ml). Propionate did not stimulate, succinate slightly stimulated the growth rate. Acetate, even at low concentrations (0.5 mM), stimulated the growth rate, the generation time being shortened to 3–4 h. Acetate provided 70% of the cell carbon, which shows that Thermoproteus neutrophilus is a facultative autotroph. The path of these carbon precursors into cell compounds was studied by 14C long-term labelling and investigation of enzyme activities. Propionate could not be used as a major carbon source and was incorporated only into isoleucine, probably via the citramalate pathway. Acetate was a preferred carbon source which suppressed autotrophic CO2 fixation: acetate grown cells exhibited an incomplete citric acid cycle in which 2-oxoglutarate dehydrogenase was present, but fumarate reductase was repressed. The succinate incorporation pattern and enzyme pattern indicated that autotrophic CO2 fixation proceeded via a yet to be defined reductive citric acid cycle.  相似文献   

15.
Five strains of obligate anaerobic sulfur-reducing eubacteria that exclusively use acetate as energy and carbon source have been enriched and isolated from anoxic sulfide-containing freshwater mud. The strains were unable to grow in the presence of 2% NaCl. Morphologically the strains were not uniform, cells were either rod-shaped or elongated ovoid. All strains were flagellated with a single polar to subpolar flagellum. They stained gram-negative. Two of the strains were studied in detail. Malate or fumarate was used alternatively to elemental sulfur as electron acceptor. The capacity to grow on acetate as sole organic substrate and to reduce elemental sulfur or polysulfide to sulfide are traits in common with the genus Desulfuromonas. The strains differ from Desulfuromonas acetoxidans by their freshwater origin, morphology, metabolic specialization and their DNA base ratio. Therefore we consider the new isolates as a new species for which the name Desulfuromonas acetexigens is proposed.Friedhelm Bak deceased December 1992  相似文献   

16.
Anaerobically prepared cell extracts of Clostridium kluyveri grown on succinate plus ethanol contained high amounts of 4-hydroxybutyryl-CoA dehydratase, which catalyzes the reversible dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA. The enzyme was purified 12-fold under strictly anaerobic conditions to over 95% homogeneity and had a specific activity of 123 nkat mg-1. The finding of this dehydratase means that all of the enzymes necessary for fermentation of succinate plus ethanol by C. kluyveri have now been demonstrated to exist in this organism and confirms the proposed pathway involving a reduction of succinate via 4-hydroxybutyrate to butyrate. Interestingly, the enzyme is almost identical to the previously isolated 4-hydroxybutyryl-CoA dehydratase from Clostridium aminobutyricum. The dehydratase was revealed as being a homotetramer (m=59 kDa/subunit), containing 2±0.2 mol FAD, 13.6±0.8 mol Fe and 10.8±1.2 mol inorganic sulfur. The enzyme was irreversibly inactivated after exposure to air. Reduction by sodium dithionite also yielded an inactive enzyme which could be reactivated, however, up to 84% by oxidation with potassium hexacyanoferrate(III). The enzyme possesses an intrinsic vinylacetyl-CoA isomerase activity which was also found in 4-hydroxybutyryl-CoA dehydratase from C. aminobutyricum. Moreover, the N-terminal sequences of the dehydratases from both organisms were found to be 63% identical.  相似文献   

17.
Halophilic archaea activate acetate via an (acetate)-inducible AMP-forming acetyl-CoA synthetase (ACS), (Acetate + ATP + CoA Acetyl-CoA + AMP + PPi). The enzyme from Haloarcula marismortui was purified to homogeneity. It constitutes a 72-kDa monomer and exhibited a temperature optimum of 41°C and a pH optimum of 7.5. For optimal activity, concentrations between 1 M and 1.5 M KCl were required, whereas NaCl had no effect. The enzyme was specific for acetate (100%) additionally accepting only propionate (30%) as substrate. The kinetic constants were determined in both directions of the reaction at 37°C. Using the N-terminal amino acid sequence an open reading frame — coding for a 74 kDa protein — was identified in the partially sequenced genome of H. marismortui. The function of the ORF as acs gene was proven by functional overexpression in Escherichia coli. The recombinant enzyme was reactivated from inclusion bodies, following solubilization in urea and refolding in the presence of salts, reduced and oxidized glutathione and substrates. Refolding was dependent on salt concentrations of at least 2 M KCl. The recombinant enzyme showed almost identical molecular and catalytic properties as the native enzyme. Sequence comparison of the Haloarcula ACS indicate high similarity to characterized ACSs from bacteria and eukarya and the archaeon Methanosaeta. Phylogenetic analysis of ACS sequences from all three domains revealed a distinct archaeal cluster suggesting monophyletic origin of archaeal ACS.  相似文献   

18.
19.
A new type of thermophilic cyanobacterial mat, rich in elemental sulfur and containing large numbers of sulfur-reducing bacteria able to utilize different growth substrates at 55° C, was found in the Uzon caldere (Kamchatka). One of the largest groups among these organisms were acetate-oxidizing sulfur-reducing bacteria, numbering 106 cells · cm–3 of mat. The pure culture of a sulfur-reducing eubacterium growing on acetate was isolated. Cells of the new isolate are Gram-negative short rods, often in pairs, motile, with a single polar flagellum. The optimal temperature for growth is 52 to 57° C, with no growth observed at 42 or 73° C. The pH optimum is 6.8 to 7.0. The new isolate is demonstrated to be a true dissimilatory sulfur reducer: it is an obligate anaerobe, it is unable to ferment organic substrates and it can use no electron acceptors other than elemental sulfur. Acetate is the only energy and carbon source, and H2S and CO2 are growth products. No cytochromes were detected. The G+C content of DNA is rather low, only 31.4 mol%. Thus, morphological and physiological features of the new isolate are quite close to those of Desulfuromonas. But on the grounds of a significant difference in the G+C content of DNA, the absence of cytochromes and because of its thermophilic nature, a new genus Desulfurella is proposed with the type species Desulfurella acetivorans.  相似文献   

20.
Whole cells of Desulfobulbus propionicus fermented [1-13C]ethanol to [2-13C] and [3-13C]propionate and [1-13C]-acetate, which indicates the involvement of a randomizing pathway in the formation of propionate. Cell-free extracts prepared from cells grown on lactate (without sulfate) contained high activities of methylmalonyl-CoA: pyruvate transacetylase, acetase kinase and reasonably high activities of NAD(P)-independent L(+)-lactate dehydrogenase NAD(P)-independent pyruvate dehydrogenase, phosphotransacetylase, acetate kinase and reasonably high activity of NAD(P)-independent L(+)-lactate dehydrogenase, fumarate reductase and succinate dehydrogenase. Cell-free extracts catalyzed the conversion of succinate to propionate in the presence of pyruvate, CoA and ATP and the oxaloacetate-dependent conversion of propionate to succinate. After growth on lactate or propionate in the presence of sulfate similar enzyme levels were found except for fumarate reductase which was considerably lower. Fermentative growth on lactate led to higher cytochrome b contents than growth with sulfate as electron acceptor.The labeling studies and the enzyme measurements demonstrate that in Desulfobulbus propionate is formed via a succinate pathway involving a transcarboxylase like in Propionibacterium. The same pathway may be used for the degradation of propionate to acetate in the presence of sulfate.Abbreviations DCPIP 2,6-dichlorophenolindophenol - PEP phosphoenolpyruvate  相似文献   

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