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  • 1
    Electronic Resource
    Electronic Resource
    Springer
    Archives of microbiology 112 (1977), S. 239-246 
    ISSN: 1432-072X
    Keywords: Hydrogen bacteria ; Alcaligenes eutrophus H 16 ; Leucine biosynthesis ; α-Isopropylmalate synthase ; Temperature anomaly ; Cold lability
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract α-Isopropylmalate (IPM) synthase, the first enzyme in the biosynthesis of l-leucine, was purified to a specific activity of 12 μmole/min x mg protein from the valine-isoleucine double auxotrophic mutant A-81 of the hydrogen bacterium Alcaligenes eutrophus H 16. The activity in crude extracts of derepressed cells was 0.106 μmoles of isopropylmalate formed per min and per mg protein. Gel electrophoresis and regel electrophoresis of the isolated main band resulted in several distinct bands, which were not altered by the additions of substrate α-ketoisovalerate, feedback inhibitor leucine or other effectors. The isoelectric points of the enzyme protein was between 3.9 and 4.0. The molecular weight was 114500 daltons and 100000 respectively in the absence and presence of the feedback inhibitor leucine. The enzyme activity depended strongly on the pH, the optimum is at pH 8.2. The enzyme was could labile and exhibits temperature anomalies.
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  • 2
    ISSN: 1432-072X
    Keywords: R-Bodies ; Kappa particles ; Free-living hydrogen bacteria ; Induction ; Electron microscopy ; Chemical composition ; Defective prophages ; Plasmids
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract R-Bodies have been found in a recently isolated pseudomonas-like free-living hydrogen oxidizing bacterium. Their isolation, fine structure and chemical composition are described and compared with the R-bodies from the kappa particles (Caedobacter), obligate endosymbionts of Paramecium aurelia. The 2K 1 R-bodies exhibited essential characteristics of the kappa R-bodies; however, their size and some other structural aspects proved that they represent a new type of R-bodies. The presence of phage tail-like particles in cells induced with Mitomycin C is in favour of the hypothesis that the R-bodies might be coded by defective prophages, or by extrachromosomal elements.
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  • 3
    ISSN: 1432-072X
    Keywords: β-Ketothiolase ; Clostridium pasteurianum ; Alcaligenes eutrophus H 16 ; Isoenzymes ; Inhibition/Inactivation by Coenzyme A
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract 1. Ketothiolase of Clostridium pasteurianum was purified 130-fold by ammonium sulphate fractionation and by column chromatography using DEAE-Sephadex A-50 and hydroxylapatite. Subjected to gel electrophoresis β-ketothiolase revealed two distinct bands; by isoelectric focusing two enzymes with isoelectric points at pH 4.5 and 7.6 were separated. As established by sucrose density gradient centrifugation the molecular weight of both enzymes was found to be 158000. 2. The condensation reaction was measured by a coupled optical test using β-hydroxybutyryl-CoA dehydrogenase as auxiliary enzyme and either acetyl-CoA or free coenzyme A plus acetyl-phosphate and phosphotransacetylase (regenerating system) or acetyl-CoA plus regenerating system as substrates. β-Ketothiolase from C. pasteurianum used only 20% of the chemically synthesized acetyl-CoA; the enzyme from Alcaligenes eutrophus H 16 used 25%. When the regenerating system was added the condensation reaction continued. The enzyme from C. pasteurianum was inactivated by free coenzyme A, while the enzyme from A. eutrophus was inhibited. When acetyl-CoA was added as the substrate the initial velocity determination was impeded by the lack of linearity. With acetyl-CoA as the substrate the K m -value was found to be 2.5 mM acetyl-CoA. If free CoASH (or acetyl-CoA) plus regenerating system was added the K m was 0.44 mM (0.42 mM) acetyl-CoA. 3. The β-ketothiolase activity was measured in the direction of acetoacetyl-CoA cleavage by an optical assay following the decrease of the enol and chelate form of acetoacetyl-CoA by absorption measurement at 305 nm. The activity was maximal at 24 mM MgCl2. The apparent K m values for acetoacetyl-CoA were 0.133 mM and 0.105 mM with 0.065 and 0.016 mM CoASH, respectively. The K m -values as calculated for only the keto form of acetoacetyl-CoA were 0.0471 and 0.0372 mM, respectively. The cleavage reaction was inhibited by high acetoacetyl-CoA concentrations; the inhibition was partially relieved by CoASH. In the range of low concentrations of acetoacetyl-CoA only a slight inhibition by CoASH was observed. The K m for CoASH was found to be 0.0288 and 0.0189 mM with 0.09 and 0.045 mM acetoacetyl-CoA, respectively. High concentrations of CoASH exerted an inhibitory effect on the cleavage reaction. With respect to enzyme kinetics and sensitivity to inhibitors and metabolites the β-ketothiolases of C. pasteurianum and A. eutrophus were rather similar.
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  • 4
    ISSN: 1432-072X
    Keywords: Pyruvate Kinase ; Allosteric Regulation ; Regulation in vivo ; Alcaligenes eutrophus H 16
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The biosynthesis of the enzyme pyruvate kinase (E.C. 2.7.1.40) of Alcaligenes eutrophus (Hydrogenomonas eutropha) H 16 was influenced by the carbon and energy source. After growth on gluconate the specific enzyme activity was high while acetate grown cells exhibited lower activities (340 and 55 μmoles/min·g protein, respectively). The pyruvate kinase from autotrophically grown cells was purified 110-fold. The enzyme was characterized by homotropic cooperative interactions with the substrate phosphoenolpyruvate, the activators AMP, ribose-5-phosphate, glucose-6-phosphate and the inhibitor ortho-phosphate. In addition to phosphate ATP caused inhibition but in this case non-sigmoidal kinetics was obtained. The half maximal substrate saturation constant S0.5 for phosphoenolpyruvate in the absence of any effectors was 0.12 mM, in the presence of 1 mM ribose-5-phosphate 0.07 mM, and with 9 mM phosphate 0.67 mM. The corresponding Hill values were 0.96, 1.1 and 2.75. The ADP saturation curve was hyperbolic even in the presence of the effectors, the K m value was 0.14 mM ADP. When the known intracellular metabolite concentrations in A. eutrophus H 16 were compared with the regulatory sensitivity of the enzyme, it appeared that under the conditions in vivo the inhibition by ATP was more important than the regulation by the allosteric effectors.
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  • 5
    ISSN: 1432-072X
    Keywords: Alcaligenes eutrophus H 16 ; Anthranilate synthase ; Aromatic amino acid biosynthesis ; regulation of
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Properties and regulation of anthranilate synthase from Alcaligenes eutrophus H 16 were investigated. Anthranilate synthase was partially purified from crude extracts by affinity chromatography on tryptophan-substituted Sepharose, and was used for kinetic measurements. During the purification procedure the enzyme was stabilized by 50 mM l-glutamine or during chromatography on DEAE-cellulose and Sephadex G-200 with 30% glycerol, respectively. The glutamine dependent activity of anthranilate synthase was examined; it showed little change between pH 8.4 and pH 9.1. The Arrhenius plot was broken and the activation energy, δH, calculated therefrom amounted to 8.9 kcal/mole up to 30°C and 5.5 kcal/mole at higher temperatures. The molecular weight determined by gelfiltration on Sephadex G-200 and by sucrose density gradient centrifugation resulted in 158000 and 126000, respectively. The K m -values for the two substrates chorismate and glutamine were found to be 5 μM and 560 μM, respectively. Anthranilate synthase was strongly inhibited by l-tryptophan; the only amino acid that affected enzyme activity. Homotropic interactions for chorismate (Hill coefficient n=1.4) were obtained in the presence of l-tryptophan. 50% inhibition were caused by 10 μM l-tryptophan at 100 μM chorismate. The inhibition with respect to l-glutamine was noncompetitive. Anthranilate synthase was not associated to phosphoribosyl transferase and easily separable from the latter by different chromatographic methods.
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  • 6
    Electronic Resource
    Electronic Resource
    Springer
    Archives of microbiology 112 (1977), S. 247-254 
    ISSN: 1432-072X
    Keywords: Hydrogen bacteria ; Alcaligenes eutrophus H 16 ; Leucine biosynthesis ; α-Isopropylmalate synthase ; Cooperativity changes ; Product inhibition ; Substrate specificity
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The purified isopropylmalate synthase of Alcaligenes eutrophus H 16 reacted with the following α-keto acids and acyl-coenzyme A derivatives (in the sequence of decreasing affinities): α-ketoisovalerate, α-keto-n-valerate, α-ketobutyrate and pyruvate; acetyl-CoA, propionyl-CoA, butyryl-CoA. malonyl-CoA, valeryl-CoA, and crotonyl-CoA. α-Ketoisocaproate, however, is a strong inhibitor of the enzyme. All reactions catalyzed by isopropylmalate synthase were inhibited to the same extent by the endproduct l-leucine. the substrate saturation curves of α-ketoisovalerate or other α-keto acids and of acetyl-coenzyme A or other acyl-CoA derivatives had intermediary plateau regions; the Hill coefficient alternated between n H -values higher and lower than 1.0, indicating changes from positive to negative and from negative to positive cooperativity for the substrates. The products, isopropylmalate and free coenzyme A, showed competitive inhibition patterns against both substrates (α-ketoisovalerate and acetyl-CoA). Free coenzyme A (1 μM) inactivated the enzyme irreversibly. The 3′-phosphate of coenzyme A and the free carboxyl group of α-ketoisovalerate were involved in optimal binding of these substrates, but 3′-dephospho-acetyl-coenzyme A and the methylester of α-ketoisovalerate were also converted by this enzyme. A CH3−CH2-grouping of the α-keto acids seemed to be necessary for binding this substrate.
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  • 7
    ISSN: 1432-072X
    Keywords: Ultrastructure ; Micromorphology ; Gram-negative hydrogen bacteria ; Flagellation ; Flagellar fine structure ; Pili
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The cell morphology, the arrangement and fine structure of flagella and the piliation of the following Gram-negative aerobic hydrogen bacteria have been studied: Alcaligenes eutrophus, Alcaligenes paradoxus, Alcaligenes ruhlandii, Pseudomonas flava, Pseudomonas pseudoflava, Pseudomonas palleronii, Pseudomonas facilis, Aquaspirillum autotrophicum, Paracoccus denitrificans, Corynebacterium autotrophicum, and strains MA 2 and SA 35. The identity of the bacteria was examined by their substrate spectra and type of flagellation. Three types of flagellar fine structure were differentiated. The presence of pili was noted in strains of Alcaligenes paradoxus, Pseudomonas flava, P. pseudoflava, P. palleronii, and P. facilis.
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  • 8
    Electronic Resource
    Electronic Resource
    Springer
    Archives of microbiology 114 (1977), S. 203-210 
    ISSN: 1432-072X
    Keywords: Hydrogen bacteria ; Alcaligenes eutrophus H 16 ; Leucine biosynthesis ; α-isopropylmalate synthase ; Regulation ; Feedback inhibition ; Relief of inhibition by valine and isoleucine ; Inhibition by α-ketoisocaproate ; Temperature anomaly
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The α-isopropylmalate synthase (EC 4.1.3.12) from Alcaligenes eutrophus H 16 was inhibited by l-leucine and α-ketoisocaproate. The extent of inhibition was influenced by substrate- and inhibitor concentrations as well as by the pH. Intermediary plateaus, which always appeared in the inhibition curves, suggested cooperative effects. The maximal Hill coefficient was found to be two. At low concentrations of leucine the inhibition mechanism was of the competitive type with respect to substrate acetyl coenzyme A and of the noncompetitive type with respect to substrate α-ketoisovalerate. The inhibition was specifically relieved by the addition of valine or isoleucine. The anomalous effect of temperature on enzyme activity was diminished by leucine. The Arrhenius energy of the reaction increased from about 11 kcal/mole in the absence of leucine to about 18 kcal/mole in the presence of leucine. The further addition of valine reversed this effect. The physiological relevance of the α-ketoisocaproate-mediated inhibition is discussed.
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  • 9
    ISSN: 1432-072X
    Keywords: Ultrastructure ; Micromorphology ; Gram-negative ; Hydrogen bacteria ; Cell envelope ; Cytoplasmic inclusions ; Membranes ; Mesosomes ; Glycogen ; Poly-β-hydroxybutyrate ; Cell wall types
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The fine structure of the cell envelope, of membrane systems and of cytoplasmic inclusions of Gram-negative aerobic hydrogen bacteria has been studied. The results have been tabulated, and three main groups could be recognized: Group 1: Alcaligenes eutrophus, A. paradoxus, A. ruhlandii, Pseudomonas facilis, P. flava, P. pseudoflava, P. palleronii, and Aquaspirillum autotrophicum; Group 2: “Corynebacterium” autotrophicum and strains MA 2 and SA 35; Group 3: Paracoccus denitrificans. Special structures related to the chemoautotrophic way of life of the hydrogen bacteria were not observed.
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  • 10
    ISSN: 1432-072X
    Keywords: Aquaspirillum autotrophicum ; Hydrogen bacterium ; Growth ; Chemolithoautotrophy ; Particulate hydrogenase ; Induction ; Repression ; Natural habitats
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Aquaspirillum autrotrophicum, an aerobic hydrogen bacterium recently isolated from an eutrophic freshwater lake, was characterized physiologically. It grew autotrophically in a fermenter with a doubling time of 4 h. Heterotrophic growth was faster. pH-Optimum ranged from 5.0–7.5, temperature optimum was about 28° C. During autotrophic growth about 10 moles hydrogen were consumed per 1 mole carbon dioxide fixed. Hydrogenase activity is inducible. CO2 did not enhance the oxy-hydrogen reaction by intact cells. The hydrogenase activity was localized in the particulate fraction. The hydrogenase reduced methylene blue and phenazine methosulfate; pyridine nucleotides were not reduced. In cell-free extracts, hydrogenase was sensitive to oxygen. Ribulosebisphosphate carboxylase was present in autotrophically-grown cells and absent from heterotrophically grown cells. Hydrogenase induction in heterotrophically-grown cells followed parabolic kinetics. Oxygen and D-gluconate repressed hydrogenase synthesis, whereas citrate, DL-lactate and pyruvate stimulated its formation. The repressive effect was delayed. The results suggest that the control of hydrogenase synthesis occurred at the transcriptional level, and that mRNA coding for the hydrogenase had a relatively long life span. D-Gluconate was degraded via the Entner-Doudoroff pathway, the enzymes of which were constitutively formed. Enzymes of the pentosephosphate and Embden-Meyerhof pathways (except phosphofructokinase) were present, too. Hydrogen did not inhibit heterotrophic growth. The possible competitive advantage of the physiological properties described with regard to the natural habitat was discussed.
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