Biomedical subjects

B Schink

Publications and source records attributed to B Schink.

At least 55 records · Page 3Linked to original sources

PubMed1997

Desulfovibrio inopinatus, sp. nov., a new sulfate-reducing bacterium that degrades hydroxyhydroquinone.

A new sulfate-reducing bacterium was isolated from marine sediment with hydroxyhydroquinone (1,2,4-trihydroxybenzene) as the sole electron and carbon source. Strain HHQ 20 grew slowly with doubling times of > 20 h and oxidized hydroxyhydroquinone, lactate, pyruvate, ethanol, fructose, and ribose incompletely to acetate and carbon dioxide, with concomitant reduction of sulfate to sulfide. Cells were large, vibrio-shaped, and gram-negative with a G+C content of 49.7 mol%, and contained desulfoviridin. Based on analysis of the 16S rRNA sequence, strain HHQ 20 was found to be related to the genus Desulfovibrio but formed a separate line, thus justifying the establishment of a new species within this genus. Hydroxyhydroquinone was the only aromatic compound utilized among numerous hydroxybenzoates, hydroxybenzenes, methoxybenzoates, and methoxybenzenes tested, suggesting that phloroglucinol and resorcinol are not degradation intermediates. Cell-free extracts of strain HHQ 20 did not contain pyrogallol-phloroglucinol transhydroxylase activity. First experiments indicated that this strain uses a new reductive pathway for anaerobic hydroxyhydroquinone degradation.

Acetates↗

PubMed1997

Energetics of syntrophic cooperation in methanogenic degradation.

Fatty acids and alcohols are key intermediates in the methanogenic degradation of organic matter, e.g., in anaerobic sewage sludge digestors or freshwater lake sediments. They are produced by classical fermenting bacteria for disposal of electrons derived in simultaneous substrate oxidations. Methanogenic bacteria can degrade primarily only one-carbon compounds. Therefore, acetate, propionate, ethanol, and their higher homologs have to be fermented further to one-carbon compounds. These fermentations are called secondary or syntrophic fermentations. They are endergonic processes under standard conditions and depend on intimate coupling with methanogenesis. The energetic situation of the prokaryotes cooperating in these processes is problematic: the free energy available in the reactions for total conversion of substrate to methane attributes to each partner amounts of energy in the range of the minimum biochemically convertible energy, i.e., 20 to 25 kJ per mol per reaction. This amount corresponds to one-third of an ATP unit and is equivalent to the energy required for a monovalent ion to cross the charged cytoplasmic membrane. Recent studies have revealed that syntrophically fermenting bacteria synthesize ATP by substrate-level phosphorylation and reinvest part of the ATP-bound energy into reversed electron transport processes, to release the electrons at a redox level accessible by the partner bacteria and to balance their energy budget. These findings allow us to understand the energy economy of these bacteria on the basis of concepts derived from the bioenergetics of other microorganisms.

Alcohols↗

PubMed1996

One molecule of molybdopterin guanine dinucleotide is associated with each subunit of the heterodimeric Mo-Fe-S protein transhydroxylase of Pelobacter acidigallici as determined by SDS/PAGE and mass spectrometry.

The molybdenum-containing iron-sulfur protein 1,2,3,5-tetrahydroxybenzene: 1,2,3-trihydroxybenzene hydroxyltransferase (transhydroxylase) of Pelobacter acidigallici was investigated by various techniques including mass spectrometry and electron paramagnetic resonance. Mass spectrometry confirmed that the 133-kDa protein is a heterodimer consisting of an alpha subunit (100.4 kDa) and a beta subunit (31.3 kDa). The presence of a molybdenum cofactor was documented by fluorimetric analysis of the oxidized form A of molybdopterin. The enzyme contained 1.55 +/- 0.14 mol pterin and 0.92 +/- 0.25 mol molybdenum/mol enzyme (133 kDa). Alkylation of the molybdenum cofactor with iodoacetamide formed di(carboxamidomethyl)-molybdopterin. Upon acid hydrolysis, 1.4 mol 5'GMP/mol enzyme (133 kDa) was released indicating that molybdenum is bound by a molybdopterin guanine dinucleotide. The alpha and beta subunits were separated by preparative gel electrophoresis. Both subunit fractions were free of molybdenum but contained equal amounts of a fluorescent form of the molybdenum cofactors. Mass spectrometry at various pH values revealed that an acid-labile cofactor was released from the large subunit and also from the small subunit. At X-band, 5-25 K, transhydroxylase (as isolated) showed minor EPR resonances with apparent g values around 4.3, 2.03 and, depending on the preparation, a further signal at g of approximately 1.98. This signal was still detectable above 70 K and was attributed to a Mo(V) center. Upon addition of dithionite, a complex set of intense resonances appeared in the region g 2.08-1.88. From their temperature dependence, three distinct sites could be identified: the Fe-S center I with gx,y,z at approximately 1.875, 1.942 and 2.087 (gav 1.968, detectable < 20 K); the Fe-S center II with gx,y,z at approximately 1.872, 1.955 and 2.051 (gav 1.959, detectable > 20 K); and the Mo(V) center consisting of a multiple signal around g 1.98 (detectable > 70 K).

Amino Acid Sequence↗

PubMed1996

Sodium-dependent succinate decarboxylation by a new anaerobic bacterium belonging to the genus Peptostreptococcus.

An anaerobic bacterium was isolated from a polluted sediment, with succinate and yeast extract as carbon and energy sources. The new strain was Gram-positive, the cells were coccal shaped, the mol% G+G content of the genomic DNA was 29, and the peptidoglycan was of the L-ornithine-D-glutamic acid type. Comparative sequence analysis of the 16S rRNA gene showed the new strain to belong to the genus Peptostreptococcus. Succinate, fumarate, pyruvate, 3-hydroxybutyrate and lysine supported growth. Succinate was degraded to propionate and presumably CO2, with a stoichiometric cell yield. Key enzymes of the methylmalonyl-CoA decarboxylase pathway were present. The methylmalonyl-CoA decarboxylase activity was avidin-sensitive and sodium dependent, and about 5 mM Na+ was required for maximal activity. Whole cells, however, required at least 50 mM sodium for maximal succinate decarboxylation activity and to support the maximum growth rate. Sodium-dependent energy conservation coupled to succinate decarboxylation is shown for the first time to occur in a bacterium belonging to the group of Gram-positive bacteria containing the peptostreptococci and their relatives.

Bacteria, Anaerobic↗

PubMed1996

Complete assimilation of cysteine by a newly isolated non-sulfur purple bacterium resembling Rhodovulum sulfidophilum (Rhodobacter sulfidophilus).

A rod-shaped, motile, phototrophic bacterium, strain SiCys, was enriched and isolated from a marine microbial mat, with cysteine as sole substrate. During phototrophic anaerobic growth with cysteine, sulfide was produced as an intermediate, which was subsequently oxidized to sulfate. The molar growth yield with cysteine was 103 g mol-1, in accordance with complete assimilation of electrons from the carbon and the sulfur moiety into cell material. Growth yields with alanine and serine were proportionally lower. Thiosulfate, sulfide, hydrogen, and several organic compounds were used as electron donors in the light, whereas cystine, sulfite, or elemental sulfur did not support phototrophic anaerobic growth. Aerobic growth in the dark was possible with fructose as substrate. Cultures of strain SiCys were yellowish-brown in color and contained bacteriochlorophyll a, spheroidene, spheroidenone, and OH-spheroidene as major photosynthetic pigments. Taking the morphology, photosynthetic pigments, aerobic growth in the dark, and utilization of sulfide for phototrophic growth into account, strain SiCys was assigned to the genus Rhodovulum (formerly Rhodobacter) and tentatively classified as a strain of R. sulfidophilum. In cell-free extracts in the presence of pyridoxal phosphate, cysteine was converted to pyruvate and sulfide, which is characteristic for cysteine desulfhydrase activity (l-cystathionine gamma-lyase, EC 4.4. 1.1).

Cystathionine gamma-Lyase↗

PubMed1996

Phylogenetic positions of Desulfofustis glycolicus gen. nov., sp. nov., and Syntrophobotulus glycolicus gen. nov., sp. nov., two new strict anaerobes growing with glycolic acid.

The glycolate-oxidizing, sulfate-reducing bacterium strain PerGlyS and the syntrophically glycolate-oxidizing bacterium strain FlGlyR were studied with respect to their phylogenetic relationships on the basis of in vitro amplification and direct sequencing of 16S rRNA-encoding DNA. Strain PerGlyS clustered with representatives of the delta subclass of the class Proteobacteria, close to "Desulforhopalus vacuolatus" but sufficiently distinct to preclude its assignment to this genus. These organisms, together with Desulfobulbus propionicus, represent a phylogenetic subgroup among members of the delta subclass of Proteobacteria. Strain FlGlyR was found to cluster with the gram-positive bacteria with low-G + C DNA, and Desulfitobacterium dehalogenans and Desulfotomaculum orientis are its closest relatives. Other species of the genus Desulfotomaculum are phylogenetically only moderately closely related to these organisms. These results necessitate the establishment of new genera and species for these two strains. Strain PerGlyS was designated the type strain of Desulfofustis glycolicus gen. nov., sp. nov., and strain FlGlyR was designated the type strain of Syntrophobotulus glycolicus gen. nov., sp. nov.

Bacteria, Anaerobic↗

PubMed1996

Clostridium ultunense sp. nov., a mesophilic bacterium oxidizing acetate in syntrophic association with a hydrogenotrophic methanogenic bacterium.

A syntrophic acetate-oxidizing bacterium, strain BST (T = type strain), was isolated from a previously described mesophilic triculture that was able to syntrophically oxidize acetate and form methane in stoichiometric amounts. Strain BST was isolated with substrates typically utilized by homoacetogenic bacteria. Strain BST was a spore-forming, gram-positive, rod-shaped organism which utilized formate, glucose, ethylene glycol, cysteine, betaine, and pyruvate. Acetate and sometimes formate were the main fermentation products. Small amounts of alanine were also produced from glucose, betaine, and cysteine. Strain BST grew optimally at 37 degrees C and pH 7. The G+C content of the DNA of strain BST was 32 mol%. A 16S rRNA sequence analysis revealed that strain BST was a member of a new species of the genus Clostridium. We propose the name Clostridium ultunense for this organism; strain BS is the type strain of C. ultunense.

Acetates↗

PubMed1996

Anaerobic, nitrate-dependent microbial oxidation of ferrous iron.

Enrichment and pure cultures of nitrate-reducing bacteria were shown to grow anaerobically with ferrous iron as the only electron donor or as the additional electron donor in the presence of acetate. The newly observed bacterial process may significantly contribute to ferric iron formation in the suboxic zone of aquatic sediments.

Journal Article↗

PubMed1995

14CO2 exchange with acetoacetate catalyzed by dialyzed cell-free extracts of the bacterial strain BunN grown with acetone and nitrate.

The nitrate-reducing bacterial strain BunN is able to grow with acetone and nitrate under anoxic conditions. Dialyzed crude cell-free extracts of acetone-plus-nitrate-grown cells of strain BunN catalyzed the exchange of 14CO2 into acetoacetate in an ADP-dependent reaction. The rates of exchange catalyzed by extracts of acetate-grown or 3-hydroxybutyrate-grown cells were only 13% of that catalyzed by extracts of acetone-grown cells. The activity was enzymic since it was destroyed by boiling and was proportional to the amount of added extract. The optimal acetoacetate concentration was 100 mM and the apparent Km was 11.1 mM. The pH optimum was 6.5, the exchange was not dependent on the addition of biotin, and the activity was not inhibited by avidin. The exchange activity was not stimulated (less than two fold) by a variety of metal ions or by a range of possible cofactors. Under optimal conditions (100 mM acetoacetate, 5 mM ADP, 10 mM NaHCO3, pH 6.5, under N2), the exchange activity was 2.7 nmol.min-1.mg protein-1; 2% of the in vivo carboxylation activity of acetone-plus-nitrate-grown cultures. It is suggested that the exchange reaction is a partial reaction catalyzed by the enzyme (or enzyme complex) that carboxylates acetone, and that the methods developed in this study provide a means with which to investigate this reaction further.

Acetoacetates↗

PubMed1995

Metabolic pathways and energetics of the acetone-oxidizing, sulfate-reducing bacterium, Desulfobacterium cetonicum.

Acetone degradation by cell suspensions of Desulfobacterium cetonicum was CO2-dependent, indicating initiation by a carboxylation reaction. Degradation of butyrate was not CO2-dependent, and acetate accumulated at a ratio of 1 mol acetate per mol butyrate degraded. In cultures grown on acetone, no CoA transfer apparently occurred, and no acetate accumulated in the medium. No CoA-ligase activities were detected in cell-free crude extracts. This suggested that the carboxylation of acetone to acetoacetate, and its activation to acetoacetyl-CoA may occur without the formation of free acetoacetate. Acetoacetyl-CoA was thiolytically cleaved to two acetyl-CoA, which were oxidized to CO2 via the acetyl-CoA/carbon monoxide dehydrogenase pathway. The measured intracellular acyl-CoA ester concentrations allowed the calculation of the free energy changes involved in the conversion of acetone to acetyl-CoA. At in vivo concentrations of reactants and products, the initial steps (carboxylation and activation) must be energy-driven, either by direct coupling to ATP, or coupling to transmembrane gradients. The delta G' of acetone conversion to two acetyl-CoA at the expense of the energetic equivalent of one ATP was calculated to lie very close to 0 kJ (mol acetone)-1. Assimilatory metabolism was by an incomplete citric acid cycle, lacking an activity oxidatively decarboxylating 2-oxoglutarate. The low specific activities of this cycle suggested its probable function in anabolic metabolism. Succinate and glyoxylate were formed from isocitrate by isocitrate lyase. Glyoxylate thus formed was condensed with acetyl-CoA to form malate, functioning as an anaplerotic sequence. A glyoxylate cycle thus operates in this strictly anaerobic bacterium. Phosphoenolpyruvate (PEP) carboxykinase formed PEP from oxaloacetate.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetone↗

PubMed1995

Electron transport phosphorylation driven by glyoxylate respiration with hydrogen as electron donor in membrane vesicles of a glyoxylate-fermenting bacterium.

The syntrophically glycolate-fermenting bacterium in the methanogenic binary coculture FlGlyM was isolated in pure culture (strain FlGlyR) with glyoxylate as sole substrate. This strain disproportionated 12 glyoxylate to 7 glycolate, 10 CO2, and 3 hydrogen. Glyoxylate was oxidized via the malyl-CoA pathway. All enzymes of this pathway, i.e. malyl-CoA lyase/malate: CoA ligase, malic enzyme, and pyruvate synthase, were demonstrated in cell-free extracts. Glycolate dehydrogenase, hydrogenase, and ATPase, as well as menaquinones as potential electron carriers, were present in the membranes. Everted membrane vesicles catalyzed hydrogen-dependent glyoxylate reduction to glycolate [86-207 nmol min-1 (mg protein)-1] coupled to ATP synthesis from ADP and Pi [38-82 nmol min-1 (mg protein)-1)]. ATP synthesis was abolished entirely by protonophores or ATPase inhibitors (up to 98 and 94% inhibition, respectively) indicating the involvement of proton-motive force in an electron transport phosphorylation driven by a new glyoxylate respiration with hydrogen as electron donor. Measured reaction rates in vesicle preparations revealed a stoichiometry of ATP formation of 0.2-0.5 ATP per glyoxylate reduced.

Adenosine Triphosphate↗

PubMed1995

Lactosphaera gen. nov., a new genus of lactic acid bacteria, and transfer of Ruminococcus pasteurii Schink 1984 to Lactosphaera pasteurii comb. nov.

The phylogenetic position and physiology of strain KoTa2T (T = type strain), which was previously classified as a Ruminococcus pasteurii strain, were studied. A determination of the 16S ribosomal DNA sequence of this taxon revealed its position within the radiation of the gram-positive lactic acid bacteria having low DNA G+C contents and that it is closely related to the genus Carnobacterium. L-Lactic acid was produced from glucose by a fructose-1,6-bisphosphate-activated lactate dehydrogenase, and oxygen tolerance was observed, characteristics which are consistent with assignment to this group. On the basis of its phenotypic characteristics and unique signature nucleotides, we propose that strain KoTa2 (= DSM 2381 = ATCC 35945) should be transferred to a new genus, Lactosphaera gen. nov., as the type strain of the species Lactosphaera pasteurii comb. nov.

Anaerobiosis↗

PubMed1995

Pathway of butyrate catabolism by Desulfobacterium cetonicum.

Desulfobacterium cetonicum 480 oxidized butyrate to 1 mol of acetate and 2 mol of CO2; this reaction was coupled to reduction of sulfate to sulfide. Butyrate was activated by coenzyme A (CoA) transfer from acetyl-CoA, and butyryl-CoA was oxidized to acetyl-CoA by a classical beta-oxidation pathway. Acetyl-CoA was oxidized through the acetyl-CoA/carbon monoxide dehydrogenase pathway. There was a rapid exchange of 14CO2 into the intermediate CoA esters and into acetate and butyrate, showing that all of the steps involved in the oxidation of butyrate to acetyl-CoA are reversible.

Acyl Coenzyme A↗

PubMed1995

Purification and characterization of acetylene hydratase of Pelobacter acetylenicus, a tungsten iron-sulfur protein.

Acetylene hydratase of the mesophilic fermenting bacterium Pelobacter acetylenicus catalyzes the hydration of acetylene to acetaldehyde. Growth of P. acetylenicus with acetylene and specific acetylene hydratase activity depended on tungstate or, to a lower degree, molybdate supply in the medium. The specific enzyme activity in cell extract was highest after growth in the presence of tungstate. Enzyme activity was stable even after prolonged storage of the cell extract or of the purified protein under air. However, enzyme activity could be measured only in the presence of a strong reducing agent such as titanium(III) citrate or dithionite. The enzyme was purified 240-fold by ammonium sulfate precipitation, anion-exchange chromatography, size exclusion chromatography, and a second anion-exchange chromatography step, with a yield of 36%. The protein was a monomer with an apparent molecular mass of 73 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point was at pH 4.2. Per mol of enzyme, 4.8 mol of iron, 3.9 mol of acid-labile sulfur, and 0.4 mol of tungsten, but no molybdenum, were detected. The Km for acetylene as assayed in a coupled photometric test with yeast alcohol dehydrogenase and NADH was 14 microM, and the Vmax was 69 mumol.min-1.mg of protein-1. The optimum temperature for activity was 50 degrees C, and the apparent pH optimum was 6.0 to 6.5. The N-terminal amino acid sequence gave no indication of resemblance to any enzyme protein described so far.

Acetaldehyde↗

PubMed1994

O-demethylation by the homoacetogenic anaerobe Holophaga foetida studied by a new photometric methylation assay using electrochemically produced cob(I)alamin.

The previously studied complete methyl transfer sequence of tetrahydrofolate-dependent O-demethylation catalyzed by Holophaga foetida strain TMBS4 extracts was separated into two steps using cobalamins as non-physiological substrates: electrochemically produced cob(I) alamin served as methyl acceptor for phenyl methyl ether demethylation, yielding methylcob(III)alamin (reaction I), and methylcob(III)alamin served as donor for tetrahydrofolate methylation, yielding 5-methyl tetrahydrofolate (reaction II). Both reactions were measured with a new and direct photometric assay of cob(I)alamin methylation (or the reverse reaction) at 540 nm, the isobestic wavelength of the cob(II)alamin/cob(I)alamin redox couple (delta epsilon 540 = 4.40 nM-1.cm-1. The rates of reactions I and II were proportional to protein concentration, unlike the complete reaction sequence. Small components of cell extract did not affect activity of reactions I and II. Isovanillate demethylation by extracts of synringate-grown cells (reaction I) required reductive activation by cob(I)alamin and was inhibited and inactivated by cob(II)alamin, indicating that the reaction mechanism was a nucleophilic attack of an enzyme-bound corrinoid in the reduced Co(I) state on the methyl carbon of the ether, rather than a radical attack. Only phenyl methyl ethers were demethylated; demethylation rates were enhanced by ortho-hydroxyl or para-carboxyl groups, but reduced by additional meta substituents. The rate of isovanillate demethylation was 81 nmol.min-1.(mg protein)-1 [0.76 mM cob(I)alamin] and apparent kinetic constants for cob(I)alamin were: Km = 1.2 mM, Vmax = 220 nmol min-1.(mg protein)-1, and Vmax/Km = 180 nmol.min-1.(mg protein) 1.mM-1 3,5-Dihydroxyanisole demethylation by extracts of 3,5-dihydroxyanisole-grown cells (also reaction I) was much slower. Reaction II did not require activation; specific activity and the specificity constant for methylcob(III)alamin were much lower.

Anisoles↗

PubMed1994

Fermentative degradation of acetone by an enrichment culture in membrane-separated culture devices and in cell suspensions.

A mixed culture, WoAct, growing on acetone, consisted of two dominant morphotypes: a rod-shaped acetone-fermenting bacterium producing acetate, and an acetate-utilizing Methanosaeta species. Dense cell suspensions, largely free of the aceticlastic methanogen and supplemented with bromoethanesulfonate, were able to degrade acetone and grow in small volumes in membrane-separated culture devices in which the acetate produced could diffuse into a large volume of medium. Acetone degradation and growth halted when the acetate concentration reached about 10 to 12 mM. Cell suspensions were able to degrade acetone in the absence of active methanogenesis, but the addition of 10 mM acetate inhibited acetone metabolism. Addition of an active culture of Methanosaeta sp. greatly stimulated the rate of acetone degradation. The results show that acetate removal in the mixed culture is not a prerequisite for growth and acetone degradation by the acetone-fermenting bacterium.

Acetates↗

PubMed1994

Transhydroxylase of Pelobacter acidigallici: a molybdoenzyme catalyzing the conversion of pyrogallol to phloroglucinol.

Trihydroxybenzenes are degraded anaerobically through the phloroglucinol pathway. In Pelobacter acidigallici as well as in Pelobacter massiliensis, pyrogallol is converted to phloroglucinol in the presence of 1,2,3,5-tetrahydroxybenzene by intermolecular hydroxyl transfer. The enzyme catalyzing this reaction was purified to chromatographic and electrophoretic homogeneity. Gel filtration and electrophoresis revealed a heterodimer structure with an apparent molecular mass of 127 kDa for the native enzyme and 86 kDa and 38 kDa, respectively, for the subunits. The enzyme was not sensitive to oxygen. HgCl2, p-chloromercuribenzoic acid, and CuCl2 inhibited strongly the reaction indicating an essential function of SH-groups. Transhydroxylase had a pH-optimum of 7.0 and a pI of 4.1. The apparent temperature optimum was in the range of 53 degrees C to 58 degrees C. The activation energy for the conversion of pyrogallol and 1,2,3,5-tetrahydroxybenzene to phloroglucinol and tetrahydroxybenzene was 31.4 kJ per mol. Purified enzyme exhibited a specific activity of 3.1 mol min-1 mg-1 protein and an apparent Km for pyrogallol and 1,2,3,5-tetrahydroxybenzene of 0.70 mM and 0.71 mM, respectively. The enzyme was found to contain per mol heterodimer 1.1 mol molybdenum, 12.1 mol iron and 14.5 mol acid-labile sulfur. Requirement for molybdenum for transhydroxylating enzyme activity was proven also by cultivation experiments. No hints for the presence of flavins were obtained. The results presented here support the hypothesis that a redox reaction is involved in this intermolecular hydroxyl transfer.

Bacteria, Anaerobic↗

PubMed1994

Fermentative degradation of triethanolamine by a homoacetogenic bacterium.

With triethanolamine as sole source of energy and organic carbon, a strictly anaerobic, gram-positive, rod-shaped bacterium, strain LuTria 3, was isolated from sewage sludge and was assigned to the genus Acetobacterium on the basis of morphological and physiological properties. The G+C content of the DNA was 34.9 +/- 1.0 mol %. The new isolate fermented triethanolamine to acetate and ammonia. In cell-free extracts, a triethanolamine-degrading enzyme activity was detected that formed acetaldehyde as reaction product. Triethanolamine cleavage was stimulated 30-fold by added adenosylcobalamin (co-enzyme B12) and inhibited by cyanocobalamin or hydroxocobalamin. Ethanolamine ammonia lyase, acetaldehyde:acceptor oxidoreductase, phosphate acetyltransferase, acetate kinase, and carbon monoxide dehydrogenase were measured in cell-free extracts of this strain. Our results establish that triethanolamine is degraded by a corrinoid-dependent shifting of the terminal hydroxyl group to the subterminal carbon atom, analogous to a diol dehydratase reaction, to form an unstable intermediate that releases acetaldehyde. No anaerobic degradation of triethylamine was observed in similar enrichment assays.

Biodegradation, Environmental↗