Mixotrophic Growth of Hydrogenomonas eutropha

Mixotrophic growth conditions were established by the addition of lactate to cultures of Hydrogenomonas eutropha growing autotrophically in a gaseous environment of H(2), O(2), and CO(2) (6:2:1). The specific growth rate of mixotrophic cultures was double that of the autotrophic cultures, and lactate disappearance paralleled growth. Growth yields in mixotrophic cultures were significantly greater than those in heterotrophic cultures for equal quantities of lactate consumed. The magnitude of the increase in yield was directly proportional to the absolute growth rate at the time of lactate addition to the starting autotrophic culture and to the time under mixotrophic conditions. The specific activities of hydrogenase and ribulose diphosphate carboxylase decreased during mixotrophic growth; the total activities increased somewhat. The results suggested that the complete autotrophic and heterotrophic physiologies functioned simultaneously under mixotrophic contions.     

Gas Consumption and Growth Rate of Hydrogenomonas eutropha in Continuous Culture

The bacterium Hydrogenomonas eutropha is under consideration for use in a regenerative life-support system for manned space missions of long duration. A 4-liter continuous culture unit containing the organism was operated for a period of 272 days under autotrophic environmental conditions. The best steady-state run achieved with this unit was observed over a 22-day time interval after 181 days of operation. During this time, the culture consumed an average of 22.9 +/- 2.0 ml of carbon dioxide per min, 38.1 +/- 3.3 ml of oxygen per min, and 128.5 +/- 10.6 ml of hydrogen per min. It required 18.7 +/- 1.2 liters of fresh nutrient medium per 24 hr to maintain a constant, preestablished cell population of 1.65 g (dry weight) per liter. The ratio of consumption of carbon dioxide, oxygen, and hydrogen varied from 1:1.2:4.5 to 1:1.9:6.6, with an average of 1:1.7:5.7. Based on these values, approximately 60 liters of the culture would be necessary to balance the gas exchange of one man.     

pH-Conditional, Ammonia Assimilation-Deficient Mutants of Hydrogenomonas eutropha: Isolation and Growth Characteristics

Two mutants of the facultative autotroph Hydrogenomonas eutropha were isolated by using a modified penicillin selection method. The mutation involved was unusual in that its effect on cellular growth was conditional with regard to extracellular pH and the type of substrate employed. Growth of both mutants was abnormal under autotrophic conditions and during heterotrophic cultivation in the presence of organic substrates which lacked an amino group. Abnormal growth was characterized by linear growth rates which were low at pH 6.0 and moderate at pH 7.2. In contrast, growth of the mutants was normal on most amino acids. Those substrates yielding abnormal growth were oxidized at normal rates by the mutants, indicating the mutation did not impair their uptake or metabolism. The data suggest that the mutants are defective in their ability to assimilate inorganic nitrogen into organic forms, and this defect is strongly influenced by pH.     

Purification and Regulatory Properties of Fructose 1,6-Diphosphatase from Hydrogenomonas eutropha

Fructose diphosphatase of Hydrogenomonas eutropha H 16, produced during autotrophic growth, was purified 247-fold from extracts of cells. The molecular weight of the enzyme was estimated to be 170,000. The enzyme showed a pH optimum of 8.5 in both crude extracts and purified preparation. The shape of the pH curve was not changed in the presence of ethylenediaminetetraacetic acid. The enzyme required Mg²⁺ for activity. The MgCl₂ saturation curve was sigmoidal and the degree of positive cooperativity increased at lower fructose diphosphate concentrations. Mn²⁺ can replace Mg²⁺, but maximal activity was lower than that observed with Mg²⁺ and the optimal concentration range was narrow. The fructose diphosphate curve was also sigmoidal. The purified enzyme also hydrolyzed sedoheptulose diphosphate but at a much lower rate than fructose diphosphate. The enzyme was not inhibited by adenosine 5′-monophosphate but was inhibited by ribulose 5-phosphate and adenosine 5′-triphosphate. Adenosine 5′-triphosphate did not affect the degree of cooperativity among the sites for fructose diphosphate. The inhibition by adenosine 5′-triphosphate was mixed and by ribulose 5-phosphate was noncompetitive. An attempt was made to correlate the properties of fructose diphosphatase from H. eutropha with its physiological role during autotrophic growth.     

Purification and regulatory properties of phosphoribulokinase from Hydrogenomonas eutropha H 16

1. Phosphoribulokinase was purified 286-fold from extracts of autotrophically grown cells. 2. The enzyme had a molecular weight of 237000 and showed a pH optimum of 9.0 in both crude extracts and purified preparation. MgCl(2) was required for activity; full activation was obtained at 5mm-MgCl(2) and the K(m) was approx. 0.5mm. 3. The ATP-saturation curve was sigmoidal and the degree of positive co-operativity increased at higher MgCl(2) concentrations. The ATP-binding sites appeared to be non-interacting at low ribulose 5-phosphate concentrations. 4. Lineweaver-Burk plots for ribulose 5-phosphate showed abrupt transitions between apparently linear sections. The apparent K(m) and V(max.) values increased with increasing concentrations of ribulose phosphate. The transitions may be explained by a sequence of negative and positive co-operativity in the catalytic rate constants. 5. Phosphoribulokinase activity was inhibited by AMP and phosphoenolpyruvate and was activated by NADH. The presence of AMP or phosphoenolpyruvate increased s(0.5) (substrate concentration required for half-maximal velocity) for both ribulose 5-phosphate and ATP but V(max.) was not changed. The sigmoidicity of the ATP-saturation curve increased in the presence of AMP but was not affected by phosphoenolpyruvate. The transitions in the ribulose 5-phosphate-saturation curves were more abrupt in the presence of either inhibitor. NADH lowered the s(0.5) for both ribulose 5-phosphate and ATP. The activator did not affect the degree of positive co-operativity between ATP-binding sites, but the ribulose 5-phosphate-binding sites appeared to be non-interacting in its presence. 6. A sequence of positive and negative co-operativity in the interactions of AMP-binding sites was suggested by the Hill plots. In the presence of NADH (and phosphoenolpyruvate) the sensitivity to inhibition by AMP was less below a certain AMP concentration and increased above that concentration. 7. Examination of the interactions between ligands indicated that phosphoribulokinase can be regulated effectively by changes in effector concentrations similar to those reported to occur in vivo.     

Protein Quality of the Bacterium Hydrogenomonas eutropha

Hydrogenomonas eutropha cells harvested from semicontinuous autotrophic culture and washed free of substrate contain about 13% of nitrogen on a dry-solids basis. Biological value and digestibility of the bacterial nitrogen were determined in the rat by use of an abbreviated Mitchell-Thomas nitrogen balance technique and casein as the standard protein. Casein nitrogen was 99% digestible, and that of both whole boiled and sonically ruptured bacterial cells was 93%. Biological value of casein and the bacterial preparations was uniformly 77%. Amino acid composition of the bacteria, as in the case of casein, indicates a first limitation of sulfur-containing amino acids. These compositional features suggest that H. eutropha may be potentially valuable as a protein supplement in animal feeds.     

Utilization of organic nitrogen compounds by Hydrogenomonas eutropha

The chemolithotroph, Hydrogenomonas eutropha, was tested for its ability to utilize a variety of single nitrogen sources during growth in an atmosphere of H₂? O₂? CO₂ The present data show that H. eutropha can utilize the nitrogen from many, but not all, amino acids, several sulfur-containing amino acids, glucosamine, and two aliphatic amides. The nitrogen concentration that supported maximum growth for NH₄Cl, L -glutamate, L -glutamine, urea, and glycine was in the 0.010–0.019M range. H. eutropha failed to remove the nitrogen from primary and secondary amines, eycloleucine, tert-DL -leucine, DL -p-fluorophenylalanine, DL -5-methyltryptophan, creatine, and creatine. This microorganism was able to partially degrade at least six substituted indoles and/or tryptophan catabolites and six substituted imidazoles and/or histidine catabolites. All of a series of 17 dipeptides were able to serve as a nitrogen source for growth in the absence of NH₄Cl. Extracts of H. eutropha were able to catalyze the hydrolysis of 16 α-dipeptides, 2 tripetides, a tetrapeptide, a polypeptide, a β-aspartyl peptide, 2 γ-glutamyl peptides, a N-acetyl amino acid, and 4 amino acid amides. These results emphasize the effectiveness of H. eutropha in utilizing a wide diversity of organic nitrogenous compounds containing amino and amide groups, heterocyclic rings, and peptide bonds.     

Factors Affecting the Synthesis and Degradation of Ribulose-1,5-Diphosphate Carboxylase in Hydrogenomonas facilis and Hydrogenomonas eutropha

Hydrogenomonas facilis and H. eutropha cultured in fructose medium retained high levels of ribulose-1,5-diphosphate carboxylase only when the following conditions were fulfilled: low aeration, FeCl(3) addition to fructose medium, and cell harvest at or prior to mid-exponential phase of growth. Repression of carboxylase synthesis was demonstrated under conditions of high oxygen tension during growth of H. eutropha on fructose. Upon depletion of fructose in the growth medium, carboxylase activity fell abruptly in both organisms. The decline could not be attributed to a repressive mechanism. Rapid inactivation of carboxylase was promoted by transfer of mid-exponential-phase H. eutropha to a basal salts medium lacking fructose. During severe fructose starvation, N(2), H(2), 80% H(2) to 20% air, 2,4-dinitrophenol, actinomycin D, streptomycin, bicarbonate, and magnesium ion deficiency spared carboxylase. Nitrogen starvation or chloramphenicol afforded no protection during severe starvation. In vitro inactivation was also demonstrated in crude cell-free extracts from nonstarved, fructose-grown H. eutropha. Substrate bicarbonate protected against this loss. Inactivation of the carboxylase could not be demonstrated either by starvation of autotrophically grown cells or in autotrophic extracts. Autotrophic extracts mixed with heterotrophic extracts lost their carboxylase activity, but mixing with heterotrophic extracts that had been heated to 50 C resulted in no loss of activity. Mechanisms are proposed to accommodate these observations.     

Quaternary structure and oxygenase activity of D-ribulose-1,5-bisphosphate carboxylase from Hydrogenomonas eutropha.

Electrophoretically homogeneous ribulose-1,5-bisphosphate (RuBP) carboxylase was obtained from autotropically grown Hydrogenomonas eutropha by sedimentation of the 105,000 X g supernatant in a discontinuous sucrose gradient and by ammonium sulfate fractionation followed by another sucrose gradient centrifugation. The molecular weight of the enzyme determined by light scattering was 490,000 +/- 15,000. The enzyme could be dissociated by sodium dodecyl sulfate into three types of subunits, and the molecular weights (+/- 10%) could be measured. There were two species of large subunits, L and L' (molecular weight 56,000 and 52,000, respectively) and one species of small subunits (molecular weight, 15,000). The mole ratio of L to L' was 5:3, and the overall mole ratio of the small to large subunits was 1.08. The simplest quaternary structure of the enzyme is L5L'3S8. The enzyme contained RuBP oxygenase activity as evidenced by the O2-dependent production of phosphoglycolate and 3-phosphoglyceric acid in equimolar quantities from RuBP.     

Autotrophic and Heterotrophic Metabolism of Hydrogenomonas I. Growth Yields and Patterns Under Dual Substrate Conditions

Auxotrophic mutants of Hydrogenomonas eutropha and H. facilis requiring utilizable amino acids were employed to demonstrate the simultaneous utilization of H(2) and an organic substrate for growth. The ratio of the cell yields under dual substrate conditions compared to heterotrophic conditions indicated the relative contributions of the autotrophic and heterotrophic systems to the growth of the organism. Wildtype H. eutropha grown under simultaneous conditions exhibited a dicyclic growth pattern, the first cycle representing either heterotrophic or simultaneous growth and the second cycle representing autotrophic growth. The duration of the changeover period was either very short with no plateau or long with a plateau up to 8 hr, depending upon the organic substrate. The growth rate under simultaneous conditions with some organic substrates was faster than either the autotrophic or heterotrophic rate, but was not the sum of the two rates. The data suggest that, in the presence of both organic and inorganic substrates, heterotrophic metabolism functions normally but autotrophic metabolism is partially repressed.     

Bicarbonate Requirement for Elimination of the Lag Period of Hydrogenomonas eutropha

Carbon dioxide and oxygen concentrations have a profound effect on the lag period of chemoautotrophically grown Hydrogenomonas eutropha. Minimum lag periods and high growth rates were obtained in shaken flask cultures with a prepared gas mixture containing 70% H(2), 20% O(2), and 10% CO(2). However, excessively long lag periods resulted when the same gas mixture was sparged through the culture. The lag period was shortened in sparged cultures by decreasing both the pO(2) and the pCO(2), indicating that gas medium equilibration had not occurred in shaken cultures. The lag period was completely eliminated at certain concentrations of O(2) and CO(2). The optimum pO(2) was 0.05 atm, but the optimum pCO(2) varied according to the pH of the medium and physiological age of the inoculum. At pH 6.4, the pCO(2) required to obtain immediate growth of exponential, postexponential, and stationary phase inocula at equal specific rates was 0.02, 0.05, and 0.16 atm, respectively. With each 0.3-unit increase in the pH of the medium, a 50% decrease in the CO(2) concentration was needed to permit growth to occur at the same rate. The pCO(2) changes required to compensate for the pH changes of the medium had the net effect of maintaining a constant bicarbonate ion concentration. Initial growth of H. eutropha was therefore indirectly related to pCO(2) and directly dependent upon a constant bicarbonate ion concentration.     

Nutritional Value of Lipids in Hydrogenomonas eutropha as Measured in the Rat

Hydrogenomonas eutropha is known to accumulate lipid, comprised largely of polymerized beta-hydroxybutyric acid, when maintained in nitrogen-deficient medium. This lipid was very poorly absorbed by mice from bacteria-containing diets, even though nitrogen absorption was adequate. The monomer, free beta-hydroxybutyric acid, was well absorbed from purified diet. Rats fed the monomer or butyric acid ate less food and grew more slowly than rats fed corn oil.     

pH-conditional, ammonia assimilation-deficient mutants of Hydrogenomonas eutropha: evidence for the nature of the mutation

Two amination-deficient mutants of Hydrogenomonas eutropha, characterized by pH-dependent linear growth on non-amino acid substrates, were investigated to determine the exact nature of the mutation. Glutamate dehydrogenase, the only aminating enzyme found in wild-type cells, was present at similar levels in mutant cells. Phenylalanine and aspartate, which allowed normal growth of the mutants, could transaminate 2-oxoglutarate to glutamate, whereas alanine, which does not support normal growth, could not transfer its amino nitrogen to form glutamate. In H. eutropha, l-alanine is apparently synthesized by beta-decarboxylation of aspartate. Studies with NH(4) (+) ions as the sole nitrogen source demonstrated that growth rates of the mutant strains were dependent on both extracellular pH and NH(4) (+) ion concentration. Comparison of these results revealed that the growth rate of mutant cultures was proportional to the concentration of extracellular NH(3). Wild-type cultures were not dependent on extracellular NH(3) since exponential growth rates did not vary with pH or NH(4) (+) ion concentration. The results suggest that the mutant strains lack an NH(4) (+) ion transport system and consequently are dependent on NH(3) diffusion which does not support optimal amination rates. The significance of the findings for the amino acid metabolism of H. eutropha is discussed.