Gluconate Catabolism in Rhizobium japonicum

Gluconate catabolism in Rhizobium japonicum ATCC 10324 was investigated by the radiorespirometric method and by assaying for key enzymes of the major energy-yielding pathways. Specifically labeled gluconate gave the following results for growing cells, with values expressed as per cent (14)CO(2) evolution: C-1 = 93%, C-2 = 57%, C-3 = 30%, C-4 = 70%, C-6 = 39%. The preferential release of (14)CO(2) from C-1 and C-4 indicate that gluconate is degraded primarily by the Entner-Doudoroff pathway but the inequalities between C-1 and C-4 and between C-3 and C-6 indicate that another pathway(s) also participates. The presence of gluconokinase and a system for converting 6-phosphogluconate to pyruvate also indicate a role for the Entner-Doudoroff pathway. The extraordinarily high yield of (14)CO(2) from C-1 labeled gluconate suggests that the other participating pathway is a C-1 decarboxylative pathway. The key enzyme of the pentose phosphate pathway, 6-phosphogluconate dehydrogenase, could not be demonstrated. Specifically labeled 2-ketogluconate and 2,5-diketogluconate were oxidized by gluconate grown cells and gave ratios of C-1 to C-6 of 2.73 and 2.61, respectively. These compare with a ratio of 2.39 obtained with specifically labeled gluconate. Gluconate dehydrogenase, the first enzyme in the ketogluconate pathway found in acetic acid bacteria, was found. Oxidation of specifically labeled pyruvate, acetate, succinate, and glutamate by gluconate-grown cells yielded the preferential rates of (14)CO(2) evolution expected from the operation of the tricarboxylic acid cycle. These data are consistent with the operation of the Entner-Doudoroff pathway and tricarboxylic acid cycle as the primary pathways of gluconate oxidation in R. japonicum. An ancillary pathway for the initial breakdown of gluconate would appear to be the ketogluconate pathway which enters the tricarboxylic acid cycle at alpha-ketoglutarate.     

Chemostat culture of Bacillus caldotenax at 65°C: Activity and regulation of the main metabolic pathways

Bacillus caldotenax was cultivated in chemostat experiments at 65°C with a chemically defined minimal medium. Glycolysis, tricarboxylic acid cycle, pentose phosphate pathway and the respiratory chain were active as demonstrated by measuring the corresponding enzymes. No enzyme activity of the Entner-Doudoroff pathway could be detected. The specific activities of the citrate cycle enzymes were up to 10 times higher as compared to the enzymes of glycolysis. At dilution rates between 0.3 and 2.2 h^-1 none of the main metabolic pathways was regulated. In contrast the isocitrate lyase was regulated (drop of activity with increasing growth rates). As a result of a batch culture with glucose and acetate as carbon sources a regulation model was proposed: glucose, or a metabolite of glucose, represses the isocitrate lyase; in the absence of glucose acetate acts as an inducer.Abbreviations DCIP dichlorphenol indophenol - ED Entner-Doudoroff pathway - EMP Emden-Meyerhof-Parnas pathway - ICL isocitrate lyase - PP pentose phosphate pathway - TCC tricarbonic acid cycle     

Pathways of glucose catabolism during germination of Streptomyces spores

Abstract The participation of the different glucose-catabolic pathways during germination of Streptomyces antibioticus spores was studied. In dormant spores, glucose is catabolized through the pentose phosphate (PP) and the Embden-Meyerhof-Parnas (EMP) pathways, with an active tricarboxylic acid cycle. The relative participation of each catabolic pathway is regulated by germinative or non-germinative conditions. During spore germination, the pentose phosphate pathway continuously increased in its participation in the glucose catabolism and it was the major glucose-catabolic pathway in the exponential phase of growth. In addition, it showed the existence of an active tricarboxylic acid cycle in dormant spores, which was being drained for biosynthetic purposes.     

Enzymes of gluconate metabolism and glycolysis inPenicillium notatum

In addition to the ability of Penicillium notatum to grow on sucrose, glucose, fructose and gluconate, substantial growth occurred on 2-ketogluconate and 5-ketogluconate thereby indicating a diverse sugar metabolism. Cell-free extracts contained all the enzymes of the Embden-Meyerhof-Parnas pathway and for both oxidative and non-oxidative pentose phosphate metabolism. Despite inconsistencies in results between different assay methods for the conventional Entner-Doudoroff (ED) enzymes, the data indicated the route was enzymatically possible. Demonstrations of the activities of the enzymes of the non-phosphorylative equivalent of the ED pathway were achieved. No evidence was found of a phosphorylative linking enzyme between the two pathways. Both 2- and 5-ketogluconate reductases were detected along with gluconate dehydrogenase which suggested interconvertibility between the ketogluconates and gluconate. However, ketogluconokinase, responsible for the conversion of ketogluconate to 2-keto-6-phosphogluconate, was not detected. A scheme for the inter-relationships of routes of gluconate metabolism is discussed.     

Pathways of Carbohydrate Metabolism in Microcyclus Species

Radiorespirometric and enzymatic studies were conducted to determine primary and secondary pathways of carbohydrate catabolism in Microcyclus aquaticus and M. flavus. M. aquaticus catabolizes both glucose and gluconate mainly via the Entner-Doudoroff and pentose phosphate pathways with some concurrent participation of the Embden-Meyerhof pathway. M. flavus, however, oxidizes glucose mainly via the Embden-Meyerhof pathway and gluconate via the Entner-Doudoroff pathway with some simultaneous operation of the pentose phosphate pathway. Both of the organisms showed evidence of the tricarboxylic acid cycle as a secondary pathway for the oxidation of carbohydrates.     

Gluconate accumulation and enzyme activities with extremely nitrogen-limited surface cultures of Aspergillus niger

Batch cultures of Aspergillus niger grown from conidia on a medium with high C/N ratio accumulated gluconate from glucose with a yield of 57%. During almost the whole time of accumulation there was no net synthesis of total protein in the mycelium but the activity per flask and the specific activity of glucose oxidase (EC 1.1.3.4) in mycelial extracts increased whereas both values decreased for glucose dehydrogenase (EC 1.1.99.10) gluconate 6-phosphatase (cf. EC 3.1.3.1, 3.1.3.2), gluconokinase (EC 2.7.1.12), glucose 6-phosphate and phosphogluconate dehydrogenases (EC 1.1.1.49, EC 1.1.1.44), phosphoglucomutase (EC 2.7.5.1), and most enzymes of the Embden-Meyerhof pathway and the tricarboxylic acid cycle. Gluconate dehydratase (EC 4.2.1.39), gluconate dehydrogenase (EC 1.1.99.3) and enzymes of the Entner-Doudoroff pathway could not be detected. By cycloheximide the increase of glucose oxidase activity was inhibited. It is concluded that the high yield of gluconate was due mainly to the net (de novo) synthesis of glucose oxidase which occurred during protein turnover after the exhaustion of the nitrogen source, and which was not accompanied by a net synthesis of the other enzymes investigated. Some gluconate may also have been formed by hydrolytic cleavage of gluconate 6-phosphate.Abbreviations GOD glucose oxidase - GD glucose dehydrogenase - PP pentose phosphate - EM Embden-Meyerhof - TCA tricarboxylic acid     

Metabolism of glucose and gluconate in fast- and slow-growing rhizobia

Enzymatic evidence was sought for the operation of pathways involved in glucose and gluconate catabolisms in fast- and slow-growing Rhizobium species including members of the cowpea group. Enzymes of the Entner-Doudoroff pathway, pentose phosphate pathway and tricarboxylic acid cycle were detected in fast-growing rhizobia but the pentose phosphate pathway was absent in slow-growers, regardless of the carbon source used. When analysed for enzymes of the Embden-Meyerhof-Parnas and Entner-Doudoroff pathways in glucose-grown cells, the pathways were found to operate simultaneously in rhizobia.     

Intermediary carbon metabolism in Frankia

Frankia isolate NPI 0136010 was able to use only propionate and acetate as sole carbon sources and was unable to use hexoses, pentoses, disaccharides, and trisaccharides. Cell free extracts were surveyed for key enzymes of intermediary carbon metabolism. Enzymes of the Embden-Meyerhof-Parnas (EMP) pathway, the tricarboxylic acid (TCA) cycle and glyoxylate shunt were detected while enzymes of the pentose phosphate (PP) and Entner-Doudoroff (ED) pathways were absent. Malic enzyme was present allowing for the conversion of malate to pyruvate and gluconeogenesis. Radiorespirometric analysis confirmed the operation of the TCA cycle and established the methylmalonyl pathway as the route of propionate metabolism. The uptake of propionate was active and mediated by sulfhydryl groups.     

Contributing carbohydrate catabolic pathways in Cyclobacterium marinus.

The primary and secondary pathways of carbohydrate metabolism were determined in a nonfermentative gram-negative ring-forming marine bacterium, Cyclobacterium marinus, by radiorespirometric studies. Whereas glucose is oxidized mainly via the Embden-Meyerhof pathway, gluconate is catabolized mainly via the Entner-Doudoroff pathway, both in conjunction with the tricarboxylic acid cycle as a secondary pathway and with some participation of the pentose phosphate pathway. The operation of these contributing catabolic pathways in this unique marine bacterium was substantiated by assaying the activities of the key enzymes specific to each pathway.     

Glucose catabolism by Thiobacillus A2 grown in chemostat culture under carbon or nitrogen limitation

Thiobacillus A2 was grown in glucose- or ammonium-limited chemostats and relative contributions of the Embden-Meyerhof (EM), Entner-Doudoroff (ED) and pentose phosphate (PP) pathways to glucose catabolism estimated by ¹⁴C-glucose radiorespirometry. In fast growing strain GFI, the EM pathway predominated (41–79%) under all growth conditions with the PP pathway contributing 18–30%. The ED pathway was apparently absent under some conditions of glucose limitation. In contrast, wild type Thiobacillus A2 exhibited predominance of the EM pathway (43–48%) under ammonium-limitation but apparent predominance of the PP pathway (43–55%) under glucose-limitation, although all three pathways were calculated to operate. Under some conditions of glucose limitation the EM pathway was possibly considerably depressed. No clear pattern of response of the three pathways to altered environmental conditions could be deduced, although marked change in pathway activities were obviously induced. Growth yield was apparently unaffected by variation in pathways. The problems of interpreting such complex radiorespirometric data are discussed.Abbreviations EM Embden-Meyerhof - ED Entner-Doudoroff - KDPG 2-keto-3-deoxy-6-phosphogluconate - 6-PG 6-phosphogluconate - PK phosphoketolase - PP pentose phosphate     

Simultaneous operation of three catabolic pathways in the metabolism of glucose by Thiobacillus A2

Enzymes essential to the operation of the Embden-Meyerhof glycolytic pathway, the Entner-Doudoroff pathway and oxidative pentose phosphate pathway were present in Thiobacillus A2 grown on glucose and other sugars. Radiorespirometry under various conditions with Thiobacillus A2 oxidising glucose specifically labelled with ¹⁴C in carbon atoms 1, 2, 3, 3+4, 6 or universally labelled demonstrated the simultaneous operation of the Embden-Meyerhof (48%), Entner-Doudoroff (28%), and pentose phosphate (24%) pathways in release of carbon dioxide from glucose. Growth on succinate, or autotrophically on formate or thiosulphate resulted in repression of most enzymes of the pathways, but high aldolase levels were retained indicating its role in gluconeogenesis and the Calvin cycle. Different fructose diphosphatase activities were found in succinate- and thiosulphate-grown organisms. The results indicate that all three major catabolic pathways for glucose function in Thiobacillus A2 grown on sugars. Thiobacillus acidophilus showed a different radiorespirometric pattern and apparently used the Entner-Doudoroff (64.5%) and pentose phosphate (35.5%) pathways, but showed unusually high release of carbon atom 6, as was also found for T. ferrooxidans.Abbreviations EM Embden-Meyerhof - ED Entner-Doudoroff - EDTA ethylene diamine tetra-acetic acid, disodium salt - FDP fructose 1,6-diphosphate - KDPG 2-keto-3-deoxy-6-phosphogluconate - 6-PG 6-phosphogluconate - Pa Pascal (10⁵ Pa=1 bar) - PP pentose phosphate - POPOP 1,4-di[2-(5-phenyloxazolyl)] benzene - PPO 2,5-diphenyloxazole     

Analysis of growth of Gluconobacter oxydans in glucose containing media

Gluconobacter oxydans oxidizes glucose via alternative pathways: one involves the non-phosphorylative, direct oxidation route to gluconic acid and ketogluconic acids, and the second requires an initial phosphorylation and then oxidation via the pentose phosphate pathway enzymes. During growth of G. oxydans in glucose-containing media, the activity of this pathway is strongly influenced by (1) the pH value of the environment and (2) the actual concentration of glucose present in the culture. At pH values below 3.5 the activity of the pentose phosphate pathway was completely inhibited resulting in an increased requirement of the organism for nutrient substances, and a poor cell yield. At pH 5.5 a triphasic growth response was observed when G. oxydans was grown in a defined medium. Above a threshold value of 5–15 mM glucose, oxidation of both glucose and gluconate by the pentose phosphate pathway enzymes was repressed, causing a rapid accumulation of gluconic acid in the culture medium. When growing under these conditions, a low affinity for the oxidation of glucose was found (K _s=13 mM). Below this threshold glucose concentration, pentose phosphate pathway enzymes were synthesized and glucose was actively assimilated via this pathway. It was shown that de novo enzyme synthesis was necessary for increased pentose phosphate pathway activity and that assimilation of gluconate by washed cell suspensions was inhibited by glucose.     

Regulation of hexose phosphate metabolism in Acetobacter xylinum

The metabolism of glucose and fructose was studied in resting succinate-grown cells of Acetobacter xylinum. From fructose only cellulose and CO(2) were formed by the cells, whereas from glucose, gluconate was formed much more rapidly than these two products. The molar ratio of sugar converted into cellulose to sugar converted into CO(2) was significantly greater than unity for both hexoses. The pattern of label retention in the cellulose formed by the cells from specifically (14)C-labelled glucose, fructose or gluconate corresponded to that of hexose phosphate in a pentose cycle. On the other hand, the isotopic configuration of cellulose arising from variously singly (14)C-labelled pyruvate did not agree with the operation of a pentose cycle on gluconeogenic hexose phosphate. Readily oxidizable tricarboxylic acid-cycle intermediates such as acetate, pyruvate or succinate promoted cellulose synthesis from fructose and gluconate although retarding their oxidation to CO(2). The incorporation into cellulose of C-1 of fructose was greatly increased in the presence of these non-sugar substrates, although its oxidation to CO(2) was greatly diminished. It is suggested that the flow of hexose phosphate carbon towards cellulose or through the pentose cycle in A. xylinum is regulated by an energy-linked control mechanism.     

Analysis of metabolic flux in <Emphasis Type="Italic">Escherichia coli</Emphasis> expressing human-like collagen in fed-batch culture

Metabolic flux distributions of recombinant Escherichia coli BL21 expressing human-like collagen were determined by means of a stoichiometric network and metabolic balancing. At the batch growth stage, the fluxes of the pentose phosphate pathway were higher than the fluxes of the fed-batch growth phase and the production stage. After the temperature was increased, there was a substantially elevated energy demand for synthesizing human-like collagen and heat-shock proteins, which resulted in changes in metabolic fluxes. The activities of the Embden-Meyerhof-Parnas pathway and the tricarboxylic acid cycle were significantly enhanced, leading to a reduction in the fluxes of the pentose phosphate pathway and other anabolic pathways. The temperature upshift also caused an increase in NADPH production by isocitrate dehydrogenase in the tricarboxylic acid cycle. The metabolic model predicted the involvement of a transhydrogenase that generates additional NADH from NADPH, thereby increasing ATP regeneration in the respiratory chain. These data indicated that the maintenance energy for cellular activity increased with the increase in biomass in fed-batch culture, and that cell growth and synthesis of human-like collagen could clearly represent the changes in metabolic fluxes. At the production stage, more NADPH was used to synthesize human-like collagen than for maintaining cellular activity, cell growth, and cell propagation. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Physiology of Sporeforming Bacteria Associated with Insects: Metabolism of Bacillus popilliae Grown in Third-Instar Popillia japonica Newman Larvae

The timing and relative participation of concurrent pathways of carbohydrate metabolism as well as the extent of terminal respiratory activity were determined by radiorespirometry with 14-C substrates and by enzyme assays for vegetative and sporulating cells of the bacterium Bacillus popilliae cultured in whole, intact Popillia japonica (Japanese beetle) larvae. During vegetative proliferation, the pentose phosphate pathway predominates in the bacterial cells with minor involvement of the Embden-Meyerhof-Parnas pathway. As the cells proceed through sporulation, pentose phosphate and Embden-Meyerhof-Parnas activity remains constant. No tricarboxylic cycle activity is evident during growth and sporulation of B. popilliae. The results demonstrate (i) predominantly aerobic metabolism for carbohydrate assimilation within in vivo sporulating cells, (ii) a major contrast to the metabolism of other aerobic sporeforming bacteria that exhibit derepression of tricarboxylic acid cycle enzymatic activity at the onset of sporulation, and (iii) no causal necessity of the cycle to B. popilliae sporogeny.     

Triple catabolic pathways for glucose in a fast-growing strain ofThiobacillus A2

Glucose catabolism was evaluated radiorespirometrically in a fast-growing strain (GFI) ofThiobacillus A2, using glucose specifically labelled ith¹⁴C in carbon atoms 1, 2, 3, 3 + 4, 6 and universally labelled. Three simultaneously functional catabolic pathways were found to operate: Embden Meyerhof (54.6%), Entner-Doudoroff (34.4%) and pentose phosphate (11%).Abbreviations EM Embden-Meyerhof - ED Enter-Doudoroff - PP pentose phosphate     

Calculation of the pentose phosphate and Embden-Myerhoff pathways from a single incubation with [U-14C]- and [5-3H]glucose

A method that simultaneously determines Embden-Myerhoff pathway and pentose phosphate pathway (PPP) activities from an incubation with [U-14C]- and [5-3H]glucose is presented. The method relies on the use of unlabeled pyruvate and lactate to dilute out radiolabel entering the tricarboxylic acid cycle. Gluconeogenesis from pyruvate is prevented by the use of an incubation chamber that maintains a CO2 (and bicarbonate) free environment. The method, which includes the contribution by the recycling steps of the PPP, is especially useful when biological material is limited or developmental timing is critical.     

Pathways of carbohydrate metabolism in peripheral nervous tissue. I. The contribution of alternative routes of glucose utilization in peripheral nerve and brain

The activities of enzymes of the glycolytic route, the pentose phosphate pathway, the tricarboxylic acid cycle and lipogenesis have been measured in rat sciatic nerve and brain. Parallel studies have been made of the utilization of 14 C-labelled glucose and pyruvate in these two tissues. Comparison of the enzyme profiles and flux through alternative routes was based on activity relative to the rate of glucose phosphorylation as measured by the rate of formation of 3H2O from [2-3H]glucose. The contributions of the pentose phosphate pathway and lipogenesis to glucose utilization were substantially higher in sciatic nerve than brain. The relatively high activity of transketolase (EC 2.2.1.1) and transaldolase (EC 2.2.1.2) suggested a special role for these enzymes in sciatic nerve.     

Intracellular carbon fluxes in riboflavin-producing Bacillus subtilis during growth on two-carbon substrate mixtures

Metabolic responses to cofeeding of different carbon substrates in carbon-limited chemostat cultures were investigated with riboflavin-producing Bacillus subtilis. Relative to the carbon content (or energy content) of the substrates, the biomass yield was lower in all cofeeding experiments than with glucose alone. The riboflavin yield, in contrast, was significantly increased in the acetoin- and gluconate-cofed cultures. In these two scenarios, unusually high intracellular ATP-to-ADP ratios correlated with improved riboflavin yields. Nuclear magnetic resonance spectra recorded with amino acids obtained from biosynthetically directed fractional (13)C labeling experiments were used in an isotope isomer balancing framework to estimate intracellular carbon fluxes. The glycolysis-to-pentose phosphate (PP) pathway split ratio was almost invariant at about 80% in all experiments, a result that was particularly surprising for the cosubstrate gluconate, which feeds directly into the PP pathway. The in vivo activities of the tricarboxylic acid cycle, in contrast, varied more than twofold. The malic enzyme was active with acetate, gluconate, or acetoin cofeeding but not with citrate cofeeding or with glucose alone. The in vivo activity of the gluconeogenic phosphoenolpyruvate carboxykinase was found to be relatively high in all experiments, with the sole exception of the gluconate-cofed culture.     

The pathways of amino acid oxidation during germination of mustard seeds (Sinapis alba)

During the germination of Sinapis alba seed, alanine and some other amino acids were oxidized via the tricarboxylic acid cycle (TCA), although the pentose phosphate pathway (PPP) was also operative and may account for ca 50% of glucose oxidation. The relative operation of the PPP and the TCA cycle was influenced by changes in the concentrations of glutamic acid and glycine.