Biomedical subjects

G Gottschalk

Publications and source records attributed to G Gottschalk.

At least 55 records · Page 3Linked to original sources

PubMed1993

Purification and characterization of a coenzyme-A-dependent succinate-semialdehyde dehydrogenase from Clostridium kluyveri.

Cell extracts of Clostridium kluyveri, grown on ethanol plus succinate contained a succinyl-CoA:CoA transferase (0.28 U/mg), a coenzyme-A-dependent succinate-semialdehyde dehydrogenase (0.73 U/mg) and a NAD(+)-dependent 4-hydroxybutyrate dehydrogenase (0.25 U/mg). The semialdehyde dehydrogenase, which catalyzed the NADPH-dependent reduction of succinyl-CoA to succinate semialdehyde, was purified 59-fold to homogeneity. A molecular mass of 115000 Da was determined for the native enzyme; SDS/PAGE revealed one protein band at 55,000, indicating that the active form is a dimer. The enzyme was highly specific for succinyl-CoA and succinate semialdehyde. The pH optimum was 7.0 for the reduction of succinyl-CoA, and 8.5 for the reverse reaction. Km values were determined for both the forward and reverse directions. The kinetic data suggest a ping-pong mechanism.

Acyl Coenzyme A↗

PubMed1992

Growth temperature-dependent activity of glycerol dehydratase in Escherichia coli expressing the Citrobacter freundii dha regulon.

Using the cosmid pWE15, a genomic library of Citrobacter freundii DNA in Escherichia coli ECL707 was prepared and screened for glycerol utilization. Six out of approximately 3000 clones were positive. One clone, harboring the recombinant cosmid pRD1, expressed glycerol dehydratase in high activity when grown at 28 degrees C but not at 37 degrees C. The growth temperature had little effect on the activity of the other enzymes encoded by the dha regulon. When the glycerol-containing medium was supplemented with corrinoids, the recombinant E. coli strain produced 1,3-propanediol in high amounts at 28 degrees C.

Citrobacter freundii↗

PubMed1992

Presence of a sodium-translocating ATPase in membrane vesicles of the homoacetogenic bacterium Acetobacterium woodii.

Inverted membrane vesicles of the homoacetogenic bacterium Acetobacterium woodii catalyzed the hydrolysis of ATP with a rate of 100-150 nmol.min-1.mg protein-1. The ATPase was stimulated 1.4-1.6-fold by NaCl and inhibited by N,N'-dicyclohexylcarbodiimide tributyltin or azide. The degree of inhibition caused by F0-directed but not F1-directed inhibitors was affected by the Na+ concentration in the medium. These experiments indicated the presence of a sodium-translocating ATPase. This was verified by transport studies. Upon addition of ATP to inverted vesicles, 22Na+ was actively transported into the intravesicular space up to a 24-fold accumulation. Na+ transport was inhibited by the sodium ionophore N,N,N',N',-tetracyclohexyl-1,2-phenyl-enedioxydiacetamide but stimulated by valinomycin with potassium whereas the protonophore 3,5,-di-tert-butyl-4-hydroxybenzylidenemalonitrile was without effect. N,N'-dicyclohexylcarbodiimide and tributyltin inhibited 22Na+ transport. These experiments are in accordance with a primary electrogenic Na+ transport as catalyzed by a F1F0-ATPase.

Adenosine Triphosphatases↗

PubMed1992

Purification and characterization of F420H2-dehydrogenase from Methanolobus tindarius.

The oxidation of F420H2 (reduced coenzyme F420) is a key reaction in the final step of methanogenesis. This step is catalyzed in Methanolobus tindarius by the membrane-bound F420H2-dehydrogenase which was purified 31-fold to apparent homogeneity. The apparent molecular mass of the native enzyme was 120 kDa. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed the presence of five different subunits of apparent molecular masses of 45 kDa, 40 kDa, 22 kDa, 18 kDa and 17 kDa. The purified F420H2-dehydrogenase, which was yellowish, contained 16 +/- 2 mol iron and 16 +/- 3 mol acid-labile sulfur/mol enzyme. No flavin could be detected. The oxygen-stable enzyme catalyzed the oxidation of F420H2 (apparent Km = 5.4 microM) with methylviologen and metronidazole as electron acceptors at a specific rate of 13 mumol.min-1.mg-1 (kcat = 25.5 s-1). The isoelectric point was at pH 5.0. The temperature optimum was at 37 degrees C and the pH optimum at 6.8.

Catalysis↗

PubMed1992

Isopropylbenzene (cumene)--a new substrate for the isolation of trichloroethene-degrading bacteria.

Various bacterial isolates from enrichments with isopropylbenzene (cumene), toluene or phenol as carbon and energy sources were tested as to their potential to oxidize trichloroethene (TCE). In contrast to toluene and phenol, all isolates enriched on isopropylbenzene were able to oxidize TCE. Two isolates, strain JR1 and strain BD1, were identified as Pseudomonas spec. and as Rhodococcus erythropolis, respectively. TCE oxidation was accompanied by the liberation of stoichiometric amounts of chloride. Initial TCE oxidation rate increased proportional to the substrate concentration from 25 to 200 microM TCE. Maximal initial TCE-degradation rates found here were 4 to 5 nmol.min-1.mg protein-1. The TCE degradation rate decreased with time. The two isolates showed a temperature optimum for TCE degradation between 10 and 20 degrees C. In addition to TCE, R. erythropolis BD1 degraded only cis- and trans-dichloroethene whereas Pseudomonas spec. JR1 was able to oxidize also 1,1-dichloroethene, vinyl chloride, trichloroethane, and 1,2-dichloroethane.

Benzene Derivatives↗

PubMed1992

Purification and properties of a F420-nonreactive, membrane-bound hydrogenase from Methanosarcina strain Gö1.

The distribution of the F420-reactive and F420-nonreactive hydrogenases from the methylotrophic Methanosarcina strain Gö1 indicated a membrane association of the F420-nonreactive enzyme. The membrane-bound F420-nonreactive hydrogenase was purified 42-fold to electrophoretic homogeneity with a yield of 26.7%. The enzyme had a specific activity of 359 mumol H2 oxidized.min-1.mg protein-1. The purification procedure involved dispersion of the membrane fraction with the detergent Chaps followed by anion exchange, hydrophobic and hydroxylapatite chromatography. The aerobically prepared enzyme had to be reactivated anaerobically. Maximal activity was observed at 80 degrees C. The molecular mass as determined by native gel electrophoresis and gel filtration was 77,000 and 79,000, respectively. SDS gel electrophoresis revealed two polypeptides with molecular masses of 60,000 and 40,000 indicating a 1:1 stoichiometry. The purified enzyme contained 13.3 mol S2-, 15.1 mol Fe and 0.8 mol Ni/mol enzyme. Flavins were not detected. The amino acid sequence of the N-termini of the subunits showed a higher degree of homology to eubacterial uptake-hydrogenases than to F420-dependent hydrogenases from other methanogenic bacteria. The physiological function of the F420-nonreactive hydrogenase from Methanosarcina strain Göl is discussed.

Amino Acid Sequence↗

PubMed1992

Energetics of methanogenesis studied in vesicular systems.

Methanogenesis is restricted to a group of prokaryotic microorganisms which thrive in strictly anaerobic habitats where they play an indispensable role in the anaerobic food chain. Methanogenic bacteria possess a number of unique cofactors and coenzymes that play an important role in their specialized metabolism. Methanogenesis from a number of simple substrates such as H2 + CO2, formate, methanol, methylamines, and acetate is associated with the generation of transmembrane electrochemical gradients of protons and sodium ions which serve as driving force for a number of processes such as the synthesis of ATP via an ATP synthase, reverse electron transfer, and solute uptake. Several unique reactions of the methanogenic pathways have been identified that are involved in energy transduction. Their role and importance for the methanogenic metabolism are described.

Adenosine Triphosphate↗

PubMed1992

Physiological Events in Clostridium acetobutylicum during the Shift from Acidogenesis to Solventogenesis in Continuous Culture and Presentation of a Model for Shift Induction.

The pH of continuous cultures of Clostridium acetobutylicum growing at pH 5.6 was allowed to decrease to 4.3 after acid production and thereby to shift the cultures from acetate and butyrate to acetone and butanol formation. Several parameters were determined during the shift. An increase in the intracellular acid concentration to 440 mM was recorded. An excess of undissociated butyric acid but not of acetic acid just before the shift to solventogenesis was followed by a decline in acid production and subsequently by the uptake of acids. The intracellular ATP concentration reached a minimum before the onset of solventogenesis; this presumably reflects the ATP-consuming proton extrusion connected with the increase in the DeltapH from 0.7 to 1.4 units. The pool of NADH plus NADPH exhibited a drastic increase until solventogenesis was induced. The changes in the ATP and ADP and NADH plus NADPH pools during these pH shift experiments were the beginning of a stable metabolic oscillation which could also be recorded as an oscillation of the culture redox potential under steady-state solventogenic conditions. Similar changes were observed when the shift was induced by the addition of butyrate and acetate (50 mM each) to the continuous culture. However, when methyl viologen was added, important differences were found: ATP levels did not reach a minimum, acetoacetate decarboxylase activity could not be measured, and butanol but not acetone was produced. A model for the shift is proposed; it assumes the generation of two signals, one by the changed ATP and ADP levels and the other by the increased NAD(P)H level.

Journal Article↗

PubMed1992

Parameters Affecting Solvent Production by Clostridium pasteurianum.

The effect of pH, growth rate, phosphate and iron limitation, carbon monoxide, and carbon source on product formation by Clostridium pasteurianum was determined. Under phosphate limitation, glucose was fermented almost exclusively to acetate and butyrate independently of the pH and growth rate. Iron limitation caused lactate production (38 mol/100 mol) from glucose in batch and continuous culture. At 15% (vol/vol) carbon monoxide in the atmosphere, glucose was fermented to ethanol (24 mol/100 mol), lactate (32 mol/100 mol), and butanol (36 mol/100 mol) in addition to the usual products, acetate (38 mol/100 mol) and butyrate (17 mol/100 mol). During glycerol fermentation, a completely different product pattern was found. In continuous culture under phosphate limitation, acetate and butyrate were produced only in trace amounts, whereas ethanol (30 mol/100 mol), butanol (18 mol/100 mol), and 1,3-propanediol (18 mol/100 mol) were the major products. Under iron limitation, the ratio of these products could be changed in favor of 1,3-propanediol (34 mol/100 mol). In addition, lactate was produced in significant amounts (25 mol/100 mol). The tolerance of C. pasteurianum to glycerol was remarkably high; growth was not inhibited by glycerol concentrations up to 17% (wt/vol). Increasing glycerol concentrations favored the production of 1,3-propanediol.

Journal Article↗

PubMed1992

N5-methyl-tetrahydromethanopterin:coenzyme M methyltransferase of Methanosarcina strain Gö1 is an Na(+)-translocating membrane protein.

To determine the cellular localization of components of the methyltransferase system, we separated cell extracts of Methanosarcina strain Gö1 into cytoplasmic and inverted-vesicle fractions. Measurements demonstrated that 83% of the methylene-tetrahydromethanopterin reductase activity resided in the cytoplasm whereas 88% of the methyl-tetrahydromethanopterin:coenzyme M methyltransferase (methyltransferase) was associated with the vesicles. The activity of the methyltransferase was stimulated 4.6-fold by ATP and 10-fold by ATP plus a reducing agent [e.g., Ti(III)]. In addition, methyltransferase activity depended on the presence of Na+ (apparent Km = 0.7 mM) and Na+ was pumped into the lumen of the vesicles in the course of methyl transfer from methyl-tetrahydromethanopterin not only to coenzyme M but also to hydroxycobalamin. Both methyl transfer reactions were inhibited by 1-iodopropane and reconstituted by illumination. A model for the methyl transfer reactions is presented.

Carrier Proteins↗

PubMed1991

A sodium-stimulated ATP synthase in the acetogenic bacterium Acetobacterium woodii.

Experiments with resting cells of Acetobacterium woodii were performed to elucidate the coupling ion used by the ATP synthase. A. woodii synthesized ATP in response to an artificial delta pH, indicating the presence of a proton-translocating ATPase. On the other hand, a delta pNa, as well as a proton diffusion potential, could serve as a driving force for ATP synthesis with the latter strictly dependent on Na+. These results are indicative for the presence of a Na(+)-translocating ATP synthase in A. woodii.

Gram-Positive Rods↗

PubMed1990

Reduced coenzyme F420: heterodisulfide oxidoreductase, a proton- translocating redox system in methanogenic bacteria.

Washed everted vesicles of the methanogenic bacterium strain Go1 were found to couple the F420H2-dependent heterodisulfide reduction with the transfer of protons across the membrane into the lumen of the everted vesicles. The transmembrane electrochemical potential of protons thereby generated was shown to be competent in driving ATP synthesis from ADP + Pi, exhibiting a stoichiometry of 2 H+ translocated or 0.4 ATP synthesized per F420H2 oxidized. This enzyme system exhibits the phenomenon of coupling and uncoupling and represents a different kind of electron transport chain with the heterodisulfide of 2-mercaptoethanesulfonate and 7-mercaptoheptanoylthreonine phosphate as terminal electron acceptor. The heterodisulfide and methane are formed in the methyl coenzyme M reductase reaction. The reducing equivalents are derived from reduced coenzyme F420, which represents an analogue of NADH + H+ in other respiratory chains. It is assumed that the proton-translocating oxidoreductase discovered in strain Go1 is of principal importance to all methanogenic bacteria not utilizing H2.

Journal Article↗

PubMed1990

Transfer and expression of the tetracycline resistance transposon Tn925 in Acetobacterium woodii.

The Enterococcus faecalis conjugative plasmid pCF10 was used to introduce Tn925 into Acetobacterium woodii by filter mating. Tetracycline resistance was transferred at frequencies of about 10(-6) per donor, but no plasmid DNA was found in the transconjugants. DNA hybridization analyses of HindIII-digested chromosomal DNA demonstrated the insertion of Tn925 at a variety of locations, whereas wild type DNA showed no hybridization at all. The transconjugants were used as donor in mating experiments with tetracycline-sensitive Bacillus subtilis. Transfer of tetracycline resistance was observed at frequencies of 10(-8) per recipient.

Bacillus subtilis↗

PubMed1989

ATP synthesis coupled to methane formation from methyl-CoM and H2 catalyzed by vesicles of the methanogenic bacterial strain Gö1.

Methanogenesis from methyl-CoM and H2, as catalyzed by inside-out vesicle preparations of the methanogenenic bacterium strain Gö1, was associated with ATP synthesis. That this ATP synthesis proceeded via an uncoupler-sensitive transmembrane proton gradient was concluded from the following results: 1. Various inhibitors that affected methane formation (e.g. 2-bromomethanesulfonate) also prevented ATP synthesis. 2. The protonophore 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile, in combination with the K+ ionophore valinomycin, inhibited ATP synthesis completely without affecting methanogenesis. 3. The ATP synthase inhibitor diethylstilbestrol inhibited ATP synthesis. 4. Addition of the detergent sulfobetaine inhibited both methane formation and ATP synthesis; the former but not the latter could be restored by adding titanium(III) citrate as electron donor. In addition it was shown that ATP synthesis could also be driven by transmembrane proton gradients artificially imposed on the vesicles. Furthermore net methanogenesis-dependent ATP formation was shown by measuring [32P]phosphate incorporation.

ATP Synthetase Complexes↗

PubMed1989

Dependence on membrane components of methanogenesis from methyl-CoM with formaldehyde or molecular hydrogen as electron donors.

Methane formation from 2-(methylthio)-ethanesulfonate (methyl-CoM) and H2 by the soluble fraction from the methanogenic bacterium strain Gö1 was stimulated up to tenfold by the addition of the membrane fraction. This stimulation was observed with membranes from various methanogenic species belonging to different phylogenetic families, but not with membranes from Escherichia coli or Acetobacterium woodii. Treatment of the membranes with strong oxidants, i.e. O2 and K3[Fe(CN)6], or with SH reagents, i.e. Ag+, p-chloromercuribenzoate or iodoacetamide, caused an irreversible decrease or loss in stimulatory activity, as did heat treatment at temperatures above 78 degrees C. Methanogenesis from methyl-CoM with formaldehyde instead of H2 as electron donor depended similarly on the membrane fraction. With membranes, 1 mol HCHO was oxidized to 1 mol CO2 and allowed the formation of 2 mol CH4 from 2 mol CH3-CoM. Without membranes, per mol of HCHO oxidized 1 mol H2 was formed and 1 mol CH4 was produced from CH3-CoM; the rate was 10-20% of that in the presence of membranes. When methyl-CoM was replaced by an artificial electron acceptor system consisting of methylviologen and metronidazole, the formaldehyde-oxidizing activity was no longer stimulated by the membrane fraction. These results demonstrate for the first time an essential function of membrane components in methanogenic electron transfer.

Cell Wall↗

PubMed1989

Purification and characterization of the pyruvate-ferredoxin oxidoreductase from Clostridium acetobutylicum.

The pyruvate-ferredoxin oxidoreductase from Clostridium acetobutylicum was purified to homogeneity and partially characterized. A 9.2-fold purification was achieved in a three step purification procedure: ammonium sulfate fractionation, chromatography on Phenyl Sepharose and on Procion Blue H-EGN12. The pure enzyme exhibited a specific activity of 25 U/mg of protein. Homogeneity of the pyruvate-ferredoxin oxidoreductase was confirmed by native polyacrylamide gel electrophoresis and sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis. The molecular weight was determined to be 123,000/monomer. The subunit composition of the native enzyme could not be determined because of the instability of the pure enzyme. The pyruvate-ferredoxin oxidoreductase is sensitive to oxygen and dilution during purification. The dilution inactivation could be partially overcome by the addition of 300 microM coenzyme A or 50% ethyleneglycol. A thiamine pyrophosphate content of 0.39 mol per mol of enzyme monomer was found, the iron and sulfur content was 4.23 and 0.91, respectively. The pH-optimum was at pH 7.5 and the temperature optimum was at 60 degrees C. Kinetic constants were measured in the forward reaction. The apparent Km for pyruvate and coenzyme A were 322 microM and 3.7 microM, respectively. With 2-ketobutyrate the pyruvate-ferredoxin oxidoreductase showed 12.5% of the activity compared to pyruvate. No activity was found with 2-ketoglutarate. Ferredoxin from Clostridium pasteurianum could be used as physiological electron acceptor.

Chromatography, Ion Exchange↗

PubMed1989

Production and Utilization of Ethanol by the Homoacetogen Acetobacterium woodii.

Acetobacterium woodii formed ethanol as a fermentation product in addition to acetate when the phosphate concentration of the medium was between 0.2 and 8.4 mM. Considerable amounts of alanine were also found (2 to 11 mM). Supplementation with phosphate caused a shift to acetate as the only end product. Ethanol could also serve as a substrate for A. woodii. The fermentation yielded predominantly acetate and was strictly dependent on high bicarbonate concentrations. 1-Propanol, 1-butanol, and 1-pentanol were converted to the corresponding fatty acids but allowed only marginal growth. A. wieringae and A. carbinolicum grown under identical conditions were also able to form ethanol, and A. wieringae could use ethanol as a substrate, too. Alcohol dehydrogenase and acetaldehyde dehydrogenase activities were determined in A. woodii. Activity stains of polyacrylamide gels with crude extracts allowed the detection of acetaldehyde dehydrogenase but not of alcohol dehydrogenase. Trace amounts of methane were detected during growth of A. woodii on glucose and ethanol.

Journal Article↗

PubMed1989

Sodium dependence of acetate formation by the acetogenic bacterium Acetobacterium woodii.

Growth of Acetobacterium woodii on fructose was stimulated by Na+; this stimulation was paralleled by a shift of the acetate-fructose ratio from 2.1 to 2.7. Growth on H2-CO2 or on methanol plus CO2 was strictly dependent on the presence of sodium ions in the medium. Acetate formation from formaldehyde plus H2-CO by resting cells required Na+, but from methanol plus H2-CO did not. This is analogous to H2-CO2 reduction to methane by Methanosarcina barkeri, which involves a sodium pump (V. Müller, C. Winner, and G. Gottschalk, Eur. J. Biochem. 178:519-525, 1988). This suggests that the reduction of methylenetetrahydrofolate to methyltetrahydrofolate is the Na+-requiring reaction. A sodium gradient (Na+ out/Na+ in = 32, delta pNa = -91 mV) was built up when resting cells of A. woodii were incubated under H2-CO2. Acetogenesis was inhibited when the delta pNa was dissipated by monensin.

Acetates↗