Activity of the enzymes of carbon metabolism in Sulfobacillus sibiricus under various conditions of cultivation

The thermoacidophilic iron-oxidizing chemolithotroph Sulfobacillus sibiricus N1T is characterized by steady growth and amplified cell yield when grown in vigorously aerated medium containing Fe2+, glucose, and yeast extract as energy sources. In this case, carbon dioxide, glucose, and yeast extract are used as carbon sources. Glucose is assimilated through the fructose-bisphosphate pathway and the pentose-phosphate pathway. Glyoxylate bypass does not function in S. sibiricus, and the tricarboxylic acid cycle is disrupted at the level of 2-oxoglutarate dehydrogenase. The presence of ribulose-bisphosphate carboxylase indicates that carbon dioxide fixation proceeds through the Calvin cycle. The activity of ribulose-bisphosphate carboxylase is highest in autotrophically grown cells. The cells also contain pyruvate carboxylase, phosphoenolpyruvate carboxylase, phosphoenolpyruvate carboxykinase, and phosphoenolpyruvate carboxytransphosphorylase.     

The Enzymes of Carbon Metabolism in the Thermotolerant Bacillar Strain K1

To determine enzymatic activities in the thermotolerant strain K1 (formerly Sulfobacillus thermosulfidooxidans subsp. thermotolerans), it was grown in a mineral medium with (1) thiosulfate and Fe²⁺ or pyrite (autotrophic conditions), (2) Fe²⁺, thiosulfate, and yeast extract or glucose (mixotrophic conditions), and (3) yeast extract (heterotrophic conditions). Cells grown mixo-, hetero-, and autotrophically were found to contain enzymes of the tricarboxylic acid (TCA) cycle, as well as malate synthase, an enzyme of the glyoxylate cycle. Cells grown organotrophically in a medium with yeast extract exhibited the activity of the key enzymes of the Embden–Meyerhof–Parnas and Entner–Doudoroff pathways. The increased content of carbon dioxide (up to 5 vol %) in the auto- and mixotrophic media enhanced the activity of the enzymes involved in the terminal reactions of the TCA cycle and the enzymes of the pentose phosphate pathway. Carbon dioxide is fixed in the Calvin cycle. The highest activity of ribulose bisphosphate carboxylase was detected in cells grown autotrophically at the atmospheric content of CO₂ in the air used for aeration of the growth medium. The activities of pyruvate carboxylase, phosphoenolpyruvate carboxylase, phosphoenolpyruvate carboxykinase, and phospho-enolpyruvate carboxytransphosphorylase decreased with increasing content of CO₂ in the medium.     

The enzyme of carbon metabolism in the thermotolerant sulfobacillus strain K1

To determine enzymatic activities in the thermotolerant strain K1 (formerly "Sulfobacillus thermosulfidooxidans subsp. thermotolerans"), it was grown in a mineral medium with (1) thiosulfate and Fe2+ or pyrite (autotrophic conditions), (2) Fe2+, thiosulfate, and yeast extract or glucose (mixotrophic conditions), and (3) yeast extract (heterotrophic conditions). Cells grown mixo-, hetero-, and autotrophically were found to contain enzymes of the tricarboxylic acid (TCA) cycle, as well as malate synthase, an enzyme of the glyoxylate cycle. Cells grown organotrophically in a medium with yeast extract exhibited the activity of the key enzymes of the Embden-Meyerhof-Parnas and Entner-Doudoroff pathways. An increased content of carbon dioxide (up to 5 vol%) in the auto- and mixotrophic media enhanced the activity of the enzymes involved in the terminal reactions of the TCA cycle and the enzymes of the pentose phosphate pathway. Carbon dioxide was fixed in the Calvin cycle. The highest activity of ribulose bisphosphate carboxylase was detected in cells grown autotrophically at the atmospheric content of CO2 in the air used for aeration of the growth medium. The activities of pyruvate carboxylase, phosphoenolpyruvate carboxylase, phosphoenolpyruvate carboxykinase, and phosphoenolpyruvate carboxytransphosphorylase decreased with the increasing content of CO2 in the medium.     

Global metabolic response of Escherichia coli to gnd or zwf gene-knockout, based on 13C-labeling experiments and the measurement of enzyme activities

An integrated study on cell growth, enzyme activities and carbon flux redistribution was made to investigate how the central metabolism of Escherichia coli changes with the knockout of genes in the oxidative pentose phosphate pathway (PPP). Mutants deficient in glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were constructed by disrupting the zwf and gnd genes and were grown in minimal media with two different carbon sources, such as glucose or pyruvate. It was shown that the knockout of either gnd or zwf gene did not affect the cell growth rate significantly, but the cellular metabolism was changed. While the specific substrate uptake rate and the specific carbon dioxide evolution rate for either mutant grown on glucose were higher than those obtained for the parent strain, these two rates were markedly decreased in mutants grown on pyruvate. The measurement of enzyme activities implied a significant change in metabolism, when alternative pathways such as the Entner–Doudoroff pathway (EDP) and the malic enzyme pathway were activated in the gnd mutant grown on glucose. As compared with the parent strain, the activities of phosphoglucose isomerase were increased in mutants grown on glucose but decreased in mutants grown on pyruvate. The metabolic flux redistribution obtained based on ¹³C-labeling experiments further indicated that the direction of the flux through the non-oxidative PPP was reversed in response to the gene knockout. Moreover, the knockout of genes caused an increased flux through the tricarboxlic acid cycle in mutants grown on glucose but caused a decrease in the case of using pyruvate. There was also a negative correlation between the fluxes through malic enzyme and isocitrate dehydrogenase in the mutants; and a positive correlation was found between the fluxes through malic enzyme and phosphoenolpyruvate carboxylase.Electronic Supplementary Material Supplementary material is available in the online version of this article at     

Carbon assimilation by different developmental stages of Laminaria saccharina

Various stages of the life cycle of the marine brown alga Laminaria saccharina (L.) Lamour. (Laminariales, Phaeophyta) including male and female gametophytes, female gametes, zygotes and young sporophytes of different age were investigated for their potentials of carbon dioxide (¹⁴CO₂) fixation. Rates of photosynthesis attain the same order of magnitude in all stages. Photosynthetic ¹⁴CO₂-fixation is accompanied by a substantial light independent carbon assimilation. This is confirmed by rate determinations of the equivalent carboxylating enzymes present in the plants, ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) and phosphoenolpyruvate carboxokinase (EC 4.1.1.32) as well as by chromatographic analyses of the appropriate [¹⁴C]-assimilate patterns.Abbreviations RuBP-C ribulose-1,5-bisphosphate carboxylase - PEP-CK phosphoenolpyruvate carboxykinase - PEP phosphoenolpyruvate - PS photosynthesis - DF dark fixation     

Regulation of metabolic pathways in sulfobacilli under different aeration regimes

A comparative study of the activities of the enzymes of carbon metabolism from the cells of moderately thermophilic chemolithotrophic bacteria Sulfobacillus sibiricus (strains N1 and SSO) and Sulfobacillus thermosulfidooxidans subsp. asporogenes (strain 41) was carried out grown in a high layer of medium without forced aeration and cells grown with intense aeration. Limited air access to the growing S. sibiricus N1 cells resulted in switching from the pentose phosphate pathway of glucose metabolism to the Entner-Doudoroff pathway while the Embden-Meyerhof-Parnas pathway persisted. Irrespective of the level of the aeration, in the cells of S. sibiricus SSO and S. thermosulfidooxidans subsp. asporogenes 41, degradation of the glucose occurred via the Entner-Doudoroff and pentose phosphate metabolic pathways, respectively, as well as via the Embden-Meyerhof-Parnas pathway. Prolonged growth of S. sibiricus, strains N1 and SSO, in a high layer of the medium without forced aeration led to the repression of synthesis of most of the tricarboxylic acid cycle (TCA cycle) enzymes, in particular dehydrogenases, as well as of some carboxylases including RuBisCO. The traits of carbon metabolism in various strains of Sulfobacillus under conditions of oxygen deficiency are discussed.     

Autotrophic CO2 fixation in Acetobacterium woodii

Earlier labeling experiments have shown that autotrophically grown Acetobacterium woodii assimilates cell carbon via direct acetyl CoA formation from 2 CO₂, rather than via the Calvin cycle. Cell extracts contained the enzymes required for biosynthesis starting from acetyl CoA and CO₂. Notably, pyruvate synthase, pyruvate phosphate dikinase, and phosphoenolpyruvate carboxytransphosphorylase were present in sufficiently high activities. Ribulose-1,5-bisphosphate carboxylase activity could not be detected. The observed enzyme pattern was consistent with the postulated biosynthetic pathway as deduced from ¹⁴C-labeling experiments.     

Current views on the regulation of autotrophic carbon dioxide fixation via the Calvin cycle in bacteria

The Calvin cycle of carbon dioxide fixation constitutes a biosynthetic pathway for the generation of (multi-carbon) intermediates of central metabolism from the one-carbon compound carbon dioxide. The product of this cycle can be used as a precursor for the synthesis of all components of cell material. Autotrophic carbon dioxide fixation is energetically expensive and it is therefore not surprising that in the various groups of autotrophic bacteria the operation of the cycle is under strict metabolic control. Synthesis of phosphoribulokinase and ribulose-1,5-bisphosphate carboxylase, the two enzymes specifically involved in the Calvin cycle, is regulated via end-product repression. In this control phosphoenolpyruvate most likely has an alarmone function. Studies of the enzymes isolated from various sources have indicated that phosphoribulokinase is the target enzyme for the control of the rate of carbon dioxide fixation via the Calvin cycle through modulation of existing enzyme activity. In general, this enzyme is strongly activated by NADH, whereas AMP and phosphoenol-pyruvate are effective inhibitors. Recent studies of phosphoribulokinase inAlcaligenes eutrophus suggest that this enzyme may also be regulated via covalent modification.     

Inactivation by glucose of phosphoenolpyruvate carboxykinase from Saccharomyces cerevisiae

Phosphoenolpyruvate carboxykinase showed high activity in Saccharomyces cerevisiae grown on gluconeogenic carbon sources. Addition of glucose to such cultures caused a rapid loss of the phosphoenolpyruvate carboxykinase activity. Fructose or mannose had the same effect as glucose, while 2-deoxyglucose or galactose were without effect. The inactivation was an irreversible process, since the regain of the activity was dependent of de novo protein synthesis. Cycloheximide did not prevent inactivation. All strains of the genus Saccharomyces tested showed inactivation of their phosphoenolpyruvate carboxykinase upon addition of glucose; this behaviour was not restricted to this genus.Non-Standard Abbreviations FbPase fructose bisphosphatase [EC 3.1.3.11 fructose-1,6-bisphosphate hydrolase] - PEPCK phosphoenolpyruvate carboxykinase [EC 4.1.49 ATP: oxalacetate carboxylase (transphosphorylating)] - YPE yeast-peptone-ethanol A preliminary account of these results was presented at the Fourth International Symposium on Yeasts, Vienna, Austria, July 1974     

Effect of the growth rate on the enzymatic activities of L-lysine-producing cells of Corynebacterium glutamicum

The activity of 6-phosphogluconate dehydrogenase, aspartate kinase and phosphoenolpyruvate carboxylase has been studied at different dilution rates in aerobic continuous culture of Corynebacterium glutamicum. 6-Phosphogluconate dehydrogenase and aspartate kinase reached their maximum values at the lower dilution rates (0.02–0.06 h^–1), when L-lysine was produced. The phosphoenolpyruvate carboxylase activity seemed to be independent of metabolite synthesis. The production of L-lysine was also studied in non-growing cells in batch cultures. In these conditions, statistical analysis revealed significant differences in L-lysine titres when glucose or gluconic acid were used as carbon sources. Higher L-lysine concentration obtained with gluconic acid was found to be associated with a high 6-phosphogluconate dehydrogenase activity.     

The role of malate in ammonia assimilation in cotyledons of radish (Raphanus sativus L.)

The relationships between the metabolism of malate, nitrogen assimilation and biosynthesis of amino acids in response to different nitrogen sources (nitrate and ammonium) have been examined in cotyledons of radish (Raphanus sativus L.). Measurements of the activities of some key enzymes and pulse-chase experiments with [¹⁴C]malate indicate the operation of an anaplerotic pathway for malate, which is involved in the synthesis of glutamine during increased ammonia assimilation. It is most likely that the tricarboxylicacid cycle is supplied with carbon through entry of malate, formed via the phosphoenolpyruvate (PEP)-carboxylation pathway, when 2-oxoglutarate leaves the cycle to serve as precursor for an increased synthesis of glutamine via glutamate. This might occur predominantly in the cytosol via the activity of the glutamine synthetase/glutamate synthase (GS/GOGAT) cycle, the NADH-dependent GOGAT being the rate-limiting activity.Abbreviations DTT dithiothreitol - EDTA ethylenediamine-tetraacetic acid - GDH glutamate dehydrogenase - GOGAT glutamate synthase (glutamine: 2-oxoglutarate aminotransferase) - GOT aspartate aminotransferase (glutamate: oxaloacetate transaminase) - GS glutamine synthetase - HPLC high-performance liquid chromatography - MCF extraction medium of methanol: chloroform: 7M formic acid, 12:5:3, by vol. - MDH malate dehydrogenase - MSO L-methionine, sulfoximine - PEPCase phosphoenolpyruvate carboxylase - TLC thin-layer chromatography     

Effects of Cytokinin Preparations on the Stability of the Photosynthetic Apparatus of Two Wheat Cultivars Experiencing Water Deficiency

Carboxylase activity of the key enzyme of carbon metabolism, ribulose-bisphosphate carboxylase/oxygenase (RuBisCO; EC 4.1.1.39), and phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31), as well as the intensity of carbon dioxide photosynthetic assimilation in young seedlings and adult leaves of the wheat Triticum aestivum L. cultivars Mironovskaya 808 (a more tolerant) and Lyutestsens758 (a less tolerant), were compared under conditions of progressive water deficiency. The water stress had more pronounced negative effects on all the studied characteristics of the photosynthetic apparatus of the cultivar Lyutestsens758. Its seedlings were more sensitive to water stress. Compounds with a cytokinin activity (6-benzylaminopurine, thidiazuron, kartolin 2, and kartolin 4) played a protective role, increasing the stability of the photosynthetic apparatus under conditions of water deficiency. Preparations of kartolins displayed the maximum protective effect.     

Growth yields and the efficiency of oxidative phosphorylation of Paracoccus denitrificans during two- (carbon) substrate-limited growth

Paracoccus denitrificans was grown aerobically during two-(carbon)substrate-limitation on mannitol and methanol in chemostat cultures. Theoretical growth parameters were calculated based on the presence of 2 or 3 sites in the electron-transport chain of Paracoccus denitrificans. Experimental growth parameters determined during two-(carbon)substrate growth were conform to the presence of 3 sites of oxidative phosphorylation, while cells grown only on mannitol possessed 2 sites. The maximum growth yield on adenosine triphosphate (ATP), corrected for maintenance requirements, determined in chemostat experiments in which the methanol concentration is less than 2.11 times the mannitol concentration was 8.6 g of biomass. When the methanol concentration was more than 2.11 times the mannitol concentration the maximum growth yield on adenosine triphosphate decreased due to the more energy consuming process of CO₂-assimilation. Cells use methanol only as energy source to increase the amount of mannitol used for assimilation purposes. When the methanol concentration in chemostat experiments was more than 2.11 times the mannitol concentration, all mannitol was used for assimilation and excess energy derived from methanol was used for CO₂-assimilation via the ribulose-bisphosphate cycle. The synthesis of ribulosebisphosphate carboxylase was repressed when the methanol concentration in chemostat experiments was less than 2.11 times the mannitol concentration or when Paracoccus denitrificans was grown in batch culture on both methanol and mannitol. When in chemostat experiments the methanol concentration was more than 2.11 times the mannitol concentration ribulose-bisphosphate carboxylase activity could be demonstrated and CO₂-assimilation will occur. It is proposed that energy produced in excess activates or derepresses the synthesis of the necessary enzymes of the ribulose-bisphosphate cycle in Paracoccus denitrificans. Consequently growth on any substrate will be carbonas well as energy-limited. When methanol is present in the nutrient cells of Paracoccus denitrificans synthesize a CO-binding type of cytochrome c, which is essential for methanol oxidase activity.The reason for the increase in efficiency of oxidative phosphorylation from 2 to 3 sites is most probably the occurrence of this CO-binding type of cytochrome c in which presence electrons preferentially pass through the a-type cytochrome region of the electron-transport chain.Non Standard Abbreviations X prosthetic group of methanol dehydrogenase - q _substrate specific rate of consumption of substrate (mol/g biomass. h.) - Y _substrate, Y _substrate ^MAX are respectively the growth yield and the maximum growth yield corrected for maintenance requirements (g biomass/mol) - m _substrate maintenance requirement (mol substrate/g biomass) - specific growth rate (h^-1) - M [methanol]/[mannitol] ratio in the nutrient - N part of mannitol that is assimilated when M=o - R _m amount of methanol-equivalents that has the same energy content as 1 mannitol-equivalent - P/O _N , P/O _F , P/O _X is the amount of ATP produced during electron-transport of two electrons from respectively NADH+H⁺, FADH₂ and XH₂ to oxygen     

Cloning of the gene for phosphoenolpyruvate carboxylase from Azotobacter chroococcum, an enzyme important in aerobic nitrogen fixation

Summary Azotobacter chroococcum Fos 189 is a Tn1-induced mutant which, unlike the parent strain MCD1, does not fix nitrogen in air when provided with glucose or pyruvate as sole carbon sources. Fos 189 showed 5% of parental activity for phosphoenolpyruvate carboxylase though PEP synthetase activity was normal. The A. chroococcum phosphoenolpyruvate carboxylase (ppc) gene was isolated after complementation of an appropriate Escherichia coli mutant using a broad host range gene bank prepared from A. chroococcum genomic DNA. The gene was localised by transposon mutagenesis and subcloning on a minimum DNA fragment of 6.6 kb. Broad host range plasmids containing the A. chroococcum ppc gene complemented the mutation in Fos 189 thereby restoring aerotolerant nitrogen fixation.     

Autotrophic growth of four Sulfolobus strains on tetrathionate and the effect of organic nutrients

Autotrophic growth yields of four strains of Sulfolobus using tetrathionate as sole energy substrate fell in the range 6.2–7.8 g dry weight (mol tetrathionate oxidized)^-1. Autotrophic organisms lacked ribulose 1,5-bis-phosphate carboxylase, but contained pyruvate and phosphoenolpyruvate carboxylases. S. brierleyi and strains B6-2 and LM exhibited mixotrophic growth, with tetrathionate oxidation, CO₂-fixation and organic substrate assimilation occurring concurrently, using media containing glucose or acetate. Yeast extract or succinate supported heterotrophic growth and showed strain-dependent repression of one or both of tetrathionate oxidation and CO₂-fixation resulting in biphasic growth. All four carbon atoms of succinate were assimilated to cell-carbon during growth. Acetate was the major source of cell-carbon during mixotrophic growth. These observations are not inconsistent with the possibility of a reductive carboxylic acid cycle in these organisms. Radiorespirometric analysis of glucose oxidation indicated CO₂ release to occur by means of an Entner-Doudoroff pathway (followed by pyruvate decarboxylation) and oxidative pentose phosphate pathway reactions. There was little evidence from the glucose radiorespirometry of the large-scale use of an oxidative tricarboxylic acid cycle for terminal oxidation of acetate derived from pyruvate. These results demonstrate the considerable metabolic versatility of Sulfolobus strains and show that there is significant variation among them.Abbreviations PIPES Piperazine-N,N-bis (2-ethane sulphonic acid)     

Enzyme patterns during substrate shifts between phenol,acetate and glucose in continuous culture of Trichosporon cutaneum

Summary The soil yeast Trichosporon cutaneum was grown in continuous culture on phenol, acetate or glucose as sole carbon source. The activities of enzymes participating in the tricarboxylic acid cycle, glyoxylate cycle, 3-oxoadipate pathway, pentose phosphate pathway and glycolysis were determined in situ during shifts of carbon sources. Cells grown on phenol or glucose contained basal activity of the glyoxylate-cycle-specific isocitrate lyase. The derepression of the glyoxylate cycle enzymes was partly hindered in the presence of phenol but not in the presence of low levels of glucose. Phenol and glucose caused repression of isocitrate lyase. In the presence of either phenol or glucose, acetate accumulation in the medium increased. However, part of the supplied acetate was utilized simultaneously with phenol or glucose, the utilization rate of either carbon source being reduced in the presence of the other carbon source. Acetate caused repression but not inactivation of the phenol-degrading enzymes, phenol hydroxylase and catechol 1,2-dioxygenase. The simultaneous utilization of phenol and other carbon sources in continuous culture as well as the observed repression-derepression patterns of the involved enzymes reveal T. cutaneum to be an organism of interest for possible use in decontamination processes. Offprint requests to: H. Y. Neujahr Offprint requests to: H. Y. Neujahr     

Mass-spectrometric evidence for the double-carboxylation pathway of malate synthesis by Crassulacean acid metabolism plants in light

Phyllodia of the Crassulacean acid metabolism (CAM) plant Kalanchoë tubiflora were allowed to fix ¹³CO₂ in light and darkness during phase IV of the diurnal CAM cycle, and during prolongation of the regular light period. After ¹³CO₂ fixation in darkness, only singly labelled [¹³C]malate molecules were found. Fixation of ¹³CO₂ under illumination, however, produced singly labelled malate as well as malate molecules which carried label in two, three or four carbon atoms. When the irradiance during ¹³CO₂ fixation was increased, the proportion of singly labelled malate decreased in favour of plurally labelled malate. The irradiance, however, did not change either the ratio of labelled to unlabelled malate molecules found in the tissue after the ¹³CO₂ application, or the magnitude of malate accumulation during the treatment with label. The ability of the tissue to store malate and the labelling pattern changed throughout the duration of the prolonged light period. The results indicate that malate synthesis by CAM plants in light can proceed via a pathway containing two carboxylation steps, namely ribulose-1,5-bisphosphate-carboxylase/oxygenase (EC 4.1.1.39) and phosphoenolpyruvate carboxylase (EC 4.1.1.31) which operate in series and share common intermediates. It can be concluded that, in light, phosphoenolpyruvate carboxylase can also synthesize malate independently of the proceeding carboxylation step by ribulose-1,5-bisphosphate carboxylase/oxygenase.Abbreviations CAM Crassulacean acid metabolism - PEP phosphoenolpyruvate - PEPCase phosphoenolpyruvate carboxylase (EC 4.1.1.31) - RuBPCase ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) - TMS trimethylsilyl     

Phenotypic properties of <Emphasis Type="Italic">Sulfobacillus thermotolerans</Emphasis>: Comparative aspects

The phenotypic characteristics of the species Sulfobacillus thermotolerans Kr1^T, as dependent on the cultivation conditions, are described in detail. High growth rates (0.22–0.30 h^?1) and high oxidative activity were recorded under optimum mixotrophic conditions at 40 °C on medium with inorganic (Fe(II), S⁰, or pyrite-arsenopyrite concentrate) and organic (glucose and/or yeast extract) substrates. In cells grown under optimum conditions on medium with iron, hemes a, b, and, most probably, c were present, indicating the presence of the corresponding cytochromes. Peculiar extended structures in the form of cylindrical cords, never observed previously, were revealed; a mucous matrix, likely of polysaccharide nature, occurred around the cells. In the cells of sulfobacilli grown litho-, organo-, and mixotrophically at 40 °C, the enzymes of the three main pathways of carbon utilization and some enzymes of the TCA cycle were revealed. The enzyme activity was maximum under mixotrophic growth conditions. The growth rate in the regions of limiting temperatures (55 °C and 12–14 °C) decreased two-and tenfold, respectively; no activity of 6-phosphogluconate dehydrogenase, one of the key enzymes of the oxidative pentose phosphate pathway, could be revealed; and a decrease in the activity of almost all enzymes of glucose metabolism and of the TCA cycle was observed. The rate of ¹⁴CO₂ fixation by cells under auto-, mixo-, and heterotrophic conditions constituted 31.8, 23.3, and 10.3 nmol/(h mg protein), respectively. The activities of RuBP carboxylase (it peaked during lithotrophic growth) and of carboxylases of heterotrophic carbon dioxide fixation were recorded. The physiological and biochemical peculiarities of the thermotolerant bacillus are compared versus moderately thermophilic sulfobacilli.     

An analysis of the growth of Gluconobacter oxydans in chemostat cultures

Gluconobacter oxydans was grown successively in glucose and nitrogen-limited chemostat cultures. Construction of mass balances of organisms growing at increasing dilution rates in glucose-limited cultures, at pH 5.5, revealed a major shift from extensive glucose metabolism via the pentose phosphate pathway to the direct pathway of glucose oxidation yielding gluconic acid. Thus, whereas carbon dioxide production from glucose accounted for 49.4% of the carbon input at a dilution rate (D)=0.05 h^-1, it accounted for only 1.3% at D=0.26 h^-1. This decline in pentose phosphate pathway activity resulted in decreasing molar growth yields on glucose. At dilution rates of 0.05 h^-1 and 0.26 h^-1 molar growth yields of 19.5 g/mol and 3.2 g/mol, respectively, were obtained. Increase of the steady state glucose concentration in nitrogen-limited chemostat cultures maintained at a constant dilution rate also resulted in a decreased flow of carbon through the pentose phosphate pathway. Above a threshold value of 15–20 mM glucose in the culture, pentose phosphate pathway activity almost completely inhibited. In G. oxydans the coupling between energy generation and growth was very inefficient; yield values obtained at various dilution rates varied between 0.8–3.4 g/cells synthesized per 0.5 mol of oxygen consumed.     

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