[Routes of incorporation of C14 carbon dioxide into dicarboxylic amino acids during chemosynthesis of Hydrogenomonas eutropha Z-1].
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Carbon Dioxide↗
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[Biosynthesis of phosphoenolpyruvate from pyruvate by extracts from Hydrogenomonas eutropha strain H 16].
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Adenosine Monophosphate↗
[Comparative characteristics of the amino acid composition of the protein fractions of the hydrogen bacteria Hydrogenomonas eutropha in meat and wheat].
An attempt was made to compare the biological value of the biological mass of the hydrogen bacteria Tydrogenomas eutropha, of meat and wheat on the ground of the fractional and amino acids composition of their proteins. Substantial differences in the distribution of proteins and amino acids in all of the three objects examined were revealed. It is shown that more than one half of the entire protein contained in the biological mass of the hydrogen bacteria is made up of poorly soluble structural proteins difficultly amenable to the action of digestive enzymes. It is this fraction where the bulk of essential amino acids is concentrated. The data obtained imply that the biological value of the biological mass of hydrogen bacteria is higher than in wheat, but lower than in meat.
Amino Acids↗
[Autotrophic growth of Hydrogenomonas eutropha during optimal gas supply].
Growth of hydrogen bacteria was studied in conditions of continuous cultivation at concentrations of 4 and 8 gram/litre. Growth characteristics were plotted as a function of cell concentration and oxygen partial pressure in the gas phase. The cells were also cultivated in periodic conditions to a concentration of 51 gram/litre at a productivity of 2.0--4.5 gram/litre per hours.
Carbon Dioxide↗
Current research on regenerative systems.
Multiple studies directed toward the development of a regenerative life support system have shown that easily synthesized organic compounds and microbiological materials are potentially capable of being used as foods for long-duration space missions. Animal feeding studies have supported these views. The organic compounds presently believed to offer the greatest potential are glycerol, simple glycerol derivatives such as triacetin, and formose sugars. Laboratory studies indicate that glycerol can be synthesized from formaldehyde which in turn is obtained by the direct catalytic oxidation of methane, a by-product of the Sabatier reaction used in spacecraft atmosphere control system. Formose sugars are derived from the self-condensation of formaldehyde. Mixtures of glycerol and triacetin have been shown to be suitable as a major component of diets fed to weanling rats for prolonged periods. These compounds do not exist as stereoisomers and therefore offer advantages over the formose sugars. Hydrogenomonas eutropha is the microbiological system under investigation. An automated system for the continuous autotrophic production of Hydrogenomonas bacteria is in operation, and the nutritional requirements for growth in the system using urea as a nitrogen source are being studied. Nutritional evaluation of Hydrogenomonas bacteria has shown they are capable of supplying the total protein requirement of growing rats for prolonged periods. The potential and problems of these regenerative systems and the prospects for the accomplishment of a totally regenerative food system will be discussed.
Animals↗
Regulation of the pyruvate kinase from Alcaligenes eutrophus H 16 in vitro and in vivo.
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 mumoles/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 nonsigmoidal 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 Km 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.
Acetates↗
The regulation of poly-beta-hydroxybutyrate metabolism in Azotobacter beijerinckii.
1. The enzymes beta-ketothiolase, acetoacetyl-CoA reductase, acetoacetate-succinate CoA-transferase (;thiophorase') and d(-)-3-hydroxybutyrate dehydrogenase have been partially purified from crude extracts of glucose-grown nitrogen-fixing batch cultures of Azotobacter beijerinckii. The condensation of acetyl-CoA to acetoacetyl-CoA catalysed by beta-ketothiolase is inhibited by CoASH, and the reverse reaction is inhibited by acetoacetyl-CoA. Acetoacetyl-CoA reductase has K(m) for acetoacetyl-CoA of 1.8mum and is inhibited by acetoacetyl-CoA above 10mum. The enzyme utilizes either NADH or NADPH as electron donor. The second enzyme of poly-beta-hydroxybutyrate degradation, d(-)-3-hydroxybutyrate dehydrogenase, is NAD(+)-specific and is inhibited by NADH, pyruvate and alpha-oxoglutarate. CoA transferase is inhibited by acetoacetate, the product of hydroxybutyrate oxidation. In continuous cultures poly-beta-hydroxybutyrate biosynthesis ceased on relaxation of oxygen-limitation and the rates in situ of oxygen consumption and carbon dioxide evolution of such cultures increased without a concomitant increase in glucose uptake. 2. On the basis of these and other findings a cyclic mechanism for the biosynthesis and degradation of poly-beta-hydroxybutyrate is proposed, together with a regulatory scheme suggesting that poly-beta-hydroxybutyrate metabolism is controlled by the redox state of the cell and the availability of CoASH, pyruvate and alpha-oxoglutarate. beta-Ketothiolase plays a key role in the regulatory process. Similarities to the pathways of poly-beta-hydroxybutyrate biosynthesis and degradation in Hydrogenomonas are discussed.
Acetoacetates↗
The facile isolation of a structural phospholipoprotein from hydrogenomon as facilis and Neurospora crassa.
The paper describes a new and gentle procedure for isolating "structural" phospholipoproteins from organisms and the first such isolation from a procaryotic microbe, Hydrogenomonas facilis. The amino acid composition of its protein moiety resembles that of "structural protein" from other sources. Although disc gel electrophoresis has shown the protein to be heterogeneous, this is attributed to aggregation.
Amino Acids↗
Carbon isotope fractionation by ribulose-1,5-bisophosphate carboxylase from various organisms.
Carbon isotope fractionation by structurally and catalytically distinct ribulose-1,5-bisphosphate carboxylases from one eucaryotic and four procaryotic organisms has been measured under nitrogen. The average fractionation for 40 experiments was -34.1 per thousand with respect to the delta(13)C of the dissolved CO(2) used, although average fractionations for each enzyme varied slightly: spinach carboxylase, -36.5 per thousand; Hydrogenomonas eutropha, -38.7 per thousand; Agmenellum quadruplicatum, -32.2 per thousand; Rhodospirillum rubrum, -32.1 per thousand; Rhodopseudomonas sphaeroides peak I carboxylase, -31.4 per thousand; and R. sphaeroides peak II carboxylase, -28.3 per thousand. The carbon isotope fractionation value was largely independent of method of enzyme preparation, purity, or reaction temperature, but in the case of spinach ribulose-1,5-bisphosphate carboxylase fractionation, changing the metal cofactor used for enzyme activation had a distinct effect on the fractionation value. The fractionation value of -36.5 per thousand with Mg(2+) as activator shifted to -29.9 per thousand with Ni(2+) as activator and to -41.7 per thousand with Mn(2+) as activator. These dramatic metal effects on carbon isotope fractionation may be useful in examining the catalytic site of the enzyme.
Journal Article↗
Bacterial growth kinetics on diphenylmethane and naphthalene-heptamethylnonane mixtures.
Experiments were carried out to determine if diphenylmethane is utilized by a species of Pseudomonas (Hydrogenomonas) in the dissolved state regardless of the physical state (liquid or solid) of the undissolved diphenylmethane suspended in the medium. Bacterial growth rates in the presence of various amounts of solid or liquid diphenylmethane indicate that liquid diphenylmethane is utilized at the aqueous-diphenylmethane interface but that solid diphenylmethane is not. A Pseudomonas sp. that was isolated on naphthalene (solid), but could not utilize heptamethylnonane, was grown in the presence of various amounts of a naphthalene-heptamethylnonane mixture (liquid). The growth rates indicate that the bacterium could utilize naphthalene at the aqueous-hydrocarbon interface, which is not the case in the absence of the heptamethylnonane.
Journal Article↗
Bacterial degradation of diphenylmethane, a DDT model substrate.
A strain of Hydrogenomonas was isolated by elective culture in a solution with diphenylmethane, an analogue of DDT, as the sole carbon source. Constitutive enzymes effected the oxidation and fission of one of the benzene rings of diphenylmethane, and phenylacetic acid was found as a major degradation product. Small amounts of phenylglyoxylic and benzoic acids were also generated from diphenylmethane by the bacterium. Phenylacetic acid, which contains the second benzene ring of diphenylmethane, was metabolized by inducible enzymes.
Benzene Derivatives↗
Energy generation and utilization in hydrogen bacteria.
Studies on the relationship between cell synthesis and energy utilization in Hydrogenomonas eutropha have shown that the amount of oxidative energy required for synthetic reactions depends on the conditions of growth. The energy of hydrogen oxidation was most efficiently used when growth conditions were optimal (continuous culture, cells in exponential growth phase) and when the rate of growth was limited by H(2) or O(2) supply. Under these conditions, 2 to 2.5 atoms of oxygen were consumed by the oxyhydrogen reaction for the concomitant conversion of 1 mole of CO(2) to cell matter. This conversion efficiency, expressed as the O/C energyyield value, was observed with continuous cultures. A less efficient conversion was found with batch cultures. With limiting concentrations of CO(2) the rate of hydrogen oxidation was relatively high, and the O/C value was dependent on the growth rate. With nonlimiting concentrations of CO(2), the rate of hydrogen oxidation was strictly proportional to the rate of CO(2) fixation, and the O/C value was independent of growth rate. This proportionality between the rate of H(2) oxidation and the rate of CO(2) fixation suggested that energy supply regulates the (maximum) rate of growth. From the energy-yield measurements, we concluded that the oxidation of 1 mole of H(2) yields the equivalent of 2 moles of adenosine triphosphate for H. eutropha, and that at least 5 moles of this high-energy phosphate is required for the conversion of 1 mole of CO(2) into cellular constituents.
Carbon Dioxide↗
Autolytic activity associated with competent group H streptococci.
Competent cells of group H streptococci strains Wicky and Challis autolyzed markedly when placed at 37 C in 0.05 m tris(hydroxymethyl)methyl-amino-propane sulfonic acid buffer (pH 9.0 to 9.1) containing 0.02 m 2-mercaptoethanol, whereas noncompetent cells autolyzed slightly. Autolysis of competent Wicky cells did not occur at 0 C or after the cells were heated at 100 C for 5 min. Culture fluids derived from strain Challis that contained competence factor (CF) activity did not contain lytic activity. Addition of native deoxyribonucleic acid (DNA) to competent Wicky cells caused a retardation in the rate of autolysis; ribonucleic acid and alkali-denatured DNA had less of an effect. Supernantant fluids derived from competent cell lysates lysed noncompetent Wicky cells but were inactive against cells of Hydrogenomonas eutropha, a group A Streptococcus, and against a commercial lysozyme substrate (Micrococcus lysodeikticus). This lytic activity was inactivated by heat (5 min at 100 C). Electron microscopic observations of autolyzed cells showed that autolysis occurs only at the site of cross-wall formation. A close relationship between the development of competence and autolysis is suggested by the fact that certain conditions that prevent the establishment of the competent state in Wicky populations (such as no CF, addition of CF simultaneously with chloramphenicol, and addition of trypsin-inactivated CF) also prevent autolysis. This observation emphasizes the indirect or inductive nature of CF on these processes.
Autolysis↗
Ribulose diphosphate carboxylase from autotrophic microorganisms.
Thiobacillus denitrificans was grown anaerobically with nitrate as an acceptor in both sterile and nonsterile media. Ribulose diphosphate carboxylase was stable throughout the exponential growth phase and declined slowly only after cells reached the stationary phase. Reversible inactivation of the carboxylase occurred in extracts as a result of bicarbonate omission. The enzyme was purified 32-fold with excellent recovery of a preparation which was 50 to 60% pure by the criterion of polyacrylamide gel electrophoresis. This purified preparation catalyzed the fixation of 1.25 mumoles of CO(2) per min per mg of protein at pH 8.1 and 30 C, and the molecular weight of ribulose diphosphate carboxylase was approximately 350,000 daltons. A striking biphasic time course of CO(2) fixation that was independent of protein and ribulose diphosphate concentration was observed. The optimal pH of the enzyme assay was fairly broad, ranging from 7 to 8.2. Kinetic dependence upon bicarbonate, ribulose diphosphate, and Mg(2+) was characterized and indicated that bicarbonate and Mg(2+) must combine with enzyme prior to addition of ribulose diphosphate. Antiserum to ribulose diphosphate carboxylase from Hydrogenomonas eutropha was only slightly inhibitory when added to the enzyme from T. denitrificans, and the mixture did not precipitate. Cyanide (4 x 10(-5)m) gave 61% inhibition of the enzyme from T. denitrificans. Ribulose diphosphate carboxylase in extracts of H. eutropha, H. facilis, Chromatium D, Rhodospirillum rubrum, and Chlorella pyrenoidosa were also inhibited to varying extents by cyanide and antiserum to the H. eutropha enzyme.
Anaerobiosis↗
Immunologically cross-reacting proteins in cell walls of many bacteria.
Antibodies to a protein present in the cell envelope of Hydrogenomonas facilis agglutinate many gram-negative bacteria and a few gram-positive bacteria. Immunodiffusion studies of extracts from the various organisms tested indicate the presence of components sharing antigenic determinants with the cell envelope protein in all bacteria which can be agglutinated and a few that cannot. Cross-reacting components were not detected in extracts of Mycoplasma laidlawii, Anacystis nidulans, and several eukaryotic organisms.
Agglutination Tests↗
Distribution of the isopropylmalate pathway to leucine among diverse bacteria.
alpha-Isopropylmalate synthase and beta-isopropylmalate dehydrogenase activities were detected in extracts of the following organisms: Chromatium D, Rhodopseudomonas spheroides, Hydrogenomonas H16, Pseudomonas aeruginosa, Pseudomonas fluorescens, Vibrio extorquens, Rhizobium japonicum, Alcaligenes viscolactis, Escherichia coli B, Proteus vulgaris, Aerobacter aerogenes, Salmonella typhimurium, Micrococcus sp., Micrococcus lysodeikticus, Bacillus polymyxa, Bacillus subtilis, and Nocardia opaca. The alpha-isopropylmalate synthase activity in these extracts was inhibited by low concentrations of l-leucine. Taken together with other data, these results suggest that the isopropylmalate pathway is widespread among organisms that can synthesize leucine.
Alcohol Oxidoreductases↗
Phosphorylation in hydrogen bacteria.
The electron-transport system of cell-free extracts obtained from Hydrogenomonas H-20 has been studied with particular reference to phosphorylation associated with the oxyhydrogen reaction. Cell-free preparations of this organism exhibit oxidative phosphorylation with hydrogen and succinate as electron donors. This activity could be uncoupled with a number of agents. Ratios of phosphorylative activity to oxidative activity observed varied from 0.2 to 0.7. Factors affecting the efficiency of phosphorylation were examined. Inhibitor and spectrophotometric studies indicated that phosphorylation with hydrogen as electron donor occurs exclusively at a site in an abbreviated electron transport chain between H(2) and cytochrome b. The possible occurrence of a cytochrome b oxidase and the requirement for a quinone are discussed, as well as the correlation between the abbreviated pathway and the energy generation by the cell. Evidence is presented which indicates that nicotinamide adenine dinucleotide does not participate in the hydrogen oxidation path which is coupled to adenosine triphosphate formation.
Antimycin A↗
Presence of rhapidosomes in various species of bacteria and their morphological characteristics.
Rod-shaped structures have been observed in cells of Pseudomonas, Photobacterium, Proteus, and Saprospira by use of the negative-contrast stain. These structures, referred to as rhapidosomes, appear to be normal components of these cells. Other bacteria including Escherichia, Salmonella, Shigella, Klebsiella, Micrococcus, Bacillus, Mycobacterium, Rhodospirillum, and Hydrogenomonas genera failed to reveal these structures. The rhapidosomes of Saprospira were found to consist of two rods, one encasing a narrower, longer structure. In contrast, the rhapidosomes of Pseudomonas, Proteus, and Photobacterium were without the rigid inner structure, but were occasionally seen filled with a homogeneous material as observed by the negative stain. Ultrathin sections of Pseudomonas cells indicate that these rhapidosomes are embedded within or are in close association with the nucleoplasm.
Alcaligenes↗