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.     

Carbohydrate Catabolism of Selected Strains in the Genus Agrobacterium

Radiorespirometric and enzyme analyses were used to reveal the glucose-catabolizing mechanisms functioning in single strains of seven presumed Agrobacterium species. The Entner-Doudoroff and pentose cycle pathways functioned in A. radiobacter, A. tumefaciens, A. rubi, and A. rhizogenes. Whereas both catabolic pathways were utilized to an almost equal degree in the A. radiobacter and A. tumefaciens strains, use of the Entner-Doudoroff pathway predominated in the A. rubi and A. rhizogenes strains. A stellulatum catabolized glucose almost solely through the Entner-Doudoroff pathway. In A. pseudotsugae and A. gypsophilae, glucose was metabolized mainly through the Emden-Meyerhof-Parnas pathway; the pentose phosphate pathway was also utilized.     

Carbon Metabolism Enzymes of Rhizobium meliloti Cultures and Bacteroids and Their Distribution within Alfalfa Nodules

Several carbon metabolism enzymes were measured in cultured cells and bacteroids of Rhizobium meliloti 102F51 and in alfalfa root nodule cytosol. The enzyme activity levels of the pentose phosphate pathway were much higher than those of the Embden-Meyerhof-Parnas or Entner-Doudoroff pathways in extracts of cultured cells. The pattern of enzyme activities in the bacteroids was different from that of cultured cells.     

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     

Gluconate catabolism in cowpea rhizobia: evidence for a ketogluconate pathway

Enzymatic and radiorespirometric analysis of several strains of cowpea rhizobia revealed the presence of key enzymes of the Entner-Doudoroff (ED) pathway with the operation of the hexose cycle for the dissimilation of gluconate. These bacteria lack the oxidative pentose phosphate (PP) pathway when grown on gluconate. Gluconate-grown cells possessed an operational tricarboxylic acid (TAC) cycle. Enzymes of an ancillary pathway, the ketogluconate (KG) pathway for gluconate catabolism were detected. The presence of this pathway was confirmed by techniques of thin-layer chromatography and radiorespirometry.Abbreviations ED Entner Doudoroff - PP pentose phosphate - EMP Embden-Meyerhof-Parnas - KG ketogluconate - TCA tricarboxylic acid - DKG diketogluconate - PFK phosphofructokinase     

重组运动发酵单胞菌的构建及木糖利用特性研究

将大肠杆菌(Escherichia coli)木糖代谢的关键酶基因.引入到运动发酵单胞菌中,获得能利用木糖发酵生产乙醇的重组工程菌株PZM.混合糖发酵过程中,重组菌利用葡萄糖和木糖生成乙醇的效率分别达到理论值的81.2%和63.1%.     

Glucose Metabolism in Neisseria gonorrhoeae

The metabolism of glucose was examined in several clinical isolates of Neisseria gonorrhoeae. Radiorespirometric studies revealed that growing cells metabolized glucose by a combination on the Entner-Doudoroff and pentose phosphate pathways. A portion of the glyceraldehyde-3-phosphate formed via the Entner-Doudoroff pathway was recycled by conversion to glucose-6-phosphate. Subsequent catabolism of this glucose-6-phosphate by either the Entner-Doudoroff or pentose phosphate pathways yielded CO(2) from the original C6 of glucose. Enzyme analyses confirmed the presence of all enzymes of the Entner-Doudoroff, pentose phosphate, and Embden-Meyerhof-Parnas pathways. There was always a high specific activity of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) relative to that of 6-phosphogluconate dehydrogenase (EC 1.1.1.44). The glucose-6-phosphate dehydrogenase utilized either nicotinamide adenine dinucleotide phosphate or nicotinamide adenine dinucleotide as electron acceptor. Acetate was the only detectable nongaseous end product of glucose metabolism. Following the disappearance of glucose, acetate was metabolized by the tricarboxylic acid cycle as evidenced by the preferential oxidation of [1-(14)C]acetate over that of [2-(14)C]acetate. When an aerobically grown log-phase culture was subjected to anaerobic conditions, lactate and acetate were formed from glucose. Radiorespirometric studies showed that under these conditions, glucose was dissimilated entirely by the Entner-Doudoroff pathway. Further studies determined that this anaerobic dissimilation of glucose was not growth dependent.     

Glucose Catabolism in Strains of Acidophilic, Heterotrophic Bacteria

Pathways of glucose catabolism, potentially operational in six strains of obligately aerobic, acidophilic bacteria, including Acidiphilium cryptum strain Lhet2, were investigated by short-term radiorespirometry and enzyme assays. Short-term radiorespirometry was conducted at pH 3.0 with specifically labeled [¹⁴C]glucose. The high rate and yield of C-1 oxidized to CO₂ indicated that the Entner-Doudoroff, pentose phosphate, or both pathways were operational in all strains. Apparent nonequivalent yields of CO₂ from C-1 and estimated CO₂ from C-4 (C-1 > C-4) were suggestive of simultaneous glucose catabolism by both pathways in all strains tested. Variation in the relative contribution of the two pathways of glucose catabolism appears to account for observed strain differences. Calculation of the actual percent pathway participation was not feasible. Enzyme assays were completed with crude extracts of glucose-grown cells to substantiate the results obtained by radiorespirometry. The key enzymes of the pentose phosphate pathway (6-phosphogluconate dehydrogenase) and the Entner-Doudoroff pathway (2-keto-3-deoxy-6-phosphogluconate aldolase and 6-phosphogluconate dehydrase) were present in all strains examined (PW2, Lhet2, KLB, OP, and QBP). However, none of the strains exhibited detectable levels of the key enzyme of the Embden-Meyerhof-Parnas pathway, 6-phosphofructokinase. All strains contained glucose-6-phosphate dehydrogenase and fructose bisphosphate aldolase. The results of the enzyme study supported the contention that the pentose phosphate and Entner-Doudoroff pathways are operational for glucose catabolism in the acidophilic heterotrophs, and that the Embden-Meyerhof-Parnas pathway is apparently absent.     

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     

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     

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.     

Radiorespirometric Studies of Leucothrix mucor

Catabolic capabilities of Leucothrix mucor were studied by radiorespirometric technique with a liquid scintillation spectrometer. Through measurement of relative rates and total percentages of ¹⁴CO₂ produced, together with the determination of the cellular incorporation of ¹⁴C from different carbon atoms of various labeled substrates, such as simple carbohydrates, intermediary metabolites, and amino acids, the essential characteristics of the primary and secondary pathways operative in this organism were examined. These substrates appeared to be degraded mainly via enzymes of the Entner-Doudoroff pathway in conjunction with the tricarboxylic acid cycle. Estimation of concurrent participation of pathways indicated that 20 to 25% of the administered carbohydrate was catabolized via the pentose phosphate pathway. There was no evidence of the Embden-Meyerhof-Parnas pathway operating in this organism.     

Nonstatistical 13C Distribution during Carbon Transfer from Glucose to Ethanol during Fermentation Is Determined by the Catabolic Pathway Exploited

During the anaerobic fermentation of glucose to ethanol, the three micro-organisms Saccharomyces cerevisiae, Zymomonas mobilis, and Leuconostoc mesenteroides exploit, respectively, the Embden-Meyerhof-Parnas, the Entner-Doudoroff, and the reductive pentose phosphate pathways. Thus, the atoms incorporated into ethanol do not have the same affiliation to the atomic positions in glucose. The isotopic fractionation occurring in each pathway at both the methylene and methyl positions of ethanol has been investigated by isotopic quantitative ¹³C NMR spectrometry with the aim of observing whether an isotope redistribution characteristic of the enzymes active in each pathway can be measured. First, it is found that each pathway has a unique isotope redistribution signature. Second, for the methylene group, a significant apparent kinetic isotope effect is only found in the reductive pentose phosphate pathway. Third, the apparent kinetic isotope effects related to the methyl group are more pronounced than for the methylene group. These findings can (i) be related to known kinetic isotope effects of some of the enzymes concerned and (ii) give indicators as to which steps in the pathways are likely to be influencing the final isotopic composition in the ethanol.     

The genome of Xanthomonas campestris pv. campestris B100 and its use for the reconstruction of metabolic pathways involved in xanthan biosynthesis

The complete genome sequence of the Xanthomonas campestris pv. campestris strain B100 was established. It consisted of a chromosome of 5,079,003bp, with 4471 protein-coding genes and 62 RNA genes. Comparative genomics showed that the genes required for the synthesis of xanthan and xanthan precursors were highly conserved among three sequenced X. campestris pv. campestris genomes, but differed noticeably when compared to the remaining four Xanthomonas genomes available. For the xanthan biosynthesis genes gumB and gumK earlier translational starts were proposed, while gumI and gumL turned out to be unique with no homologues beyond the Xanthomonas genomes sequenced. From the genomic data the biosynthesis pathways for the production of the exopolysaccharide xanthan could be elucidated. The first step of this process is the uptake of sugars serving as carbon and energy sources wherefore genes for 15 carbohydrate import systems could be identified. Metabolic pathways playing a role for xanthan biosynthesis could be deduced from the annotated genome. These reconstructed pathways concerned the storage and metabolization of the imported sugars. The recognized sugar utilization pathways included the Entner-Doudoroff and the pentose phosphate pathway as well as the Embden-Meyerhof pathway (glycolysis). The reconstruction indicated that the nucleotide sugar precursors for xanthan can be converted from intermediates of the pentose phosphate pathway, some of which are also intermediates of glycolysis or the Entner-Doudoroff pathway. Xanthan biosynthesis requires in particular the nucleotide sugars UDP-glucose, UDP-glucuronate, and GDP-mannose, from which xanthan repeat units are built under the control of the gum genes. The updated genome annotation data allowed reconsidering and refining the mechanistic model for xanthan biosynthesis.     

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     

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.     

Mutational Analyses of Glucose Dehydrogenase and Glucose-6-Phosphate Dehydrogenase Genes in Pseudomonas fluorescens Reveal Their Effects on Growth and Alginate Production

The biosynthesis of alginate has been studied extensively due to the importance of this polymer in medicine and industry. Alginate is synthesized from fructose-6-phosphate and thus competes with the central carbon metabolism for this metabolite. The alginate-producing bacterium Pseudomonas fluorescens relies on the Entner-Doudoroff and pentose phosphate pathways for glucose metabolism, and these pathways are also important for the metabolism of fructose and glycerol. In the present study, the impact of key carbohydrate metabolism enzymes on growth and alginate synthesis was investigated in P. fluorescens. Mutants defective in glucose-6-phosphate dehydrogenase isoenzymes (Zwf-1 and Zwf-2) or glucose dehydrogenase (Gcd) were evaluated using media containing glucose, fructose, or glycerol. Zwf-1 was shown to be the most important glucose-6-phosphate dehydrogenase for catabolism. Both Zwf enzymes preferred NADP as a coenzyme, although NAD was also accepted. Only Zwf-2 was active in the presence of 3 mM ATP, and then only with NADP as a coenzyme, indicating an anabolic role for this isoenzyme. Disruption of zwf-1 resulted in increased alginate production when glycerol was used as the carbon source, possibly due to decreased flux through the Entner-Doudoroff pathway rendering more fructose-6-phosphate available for alginate biosynthesis. In alginate-producing cells grown on glucose, disruption of gcd increased both cell numbers and alginate production levels, while this mutation had no positive effect on growth in a non-alginate-producing strain. A possible explanation is that alginate synthesis might function as a sink for surplus hexose phosphates that could otherwise be detrimental to the cell.     

Enzymes of carbohydrate metabolism in fast-growing Rhizobium grown on hexoses or succinate

Enzymatic evidence supports that succinate mediates repression of hexose-catabolising enzymes in fast-growing Rhizobium sp. (Cicer arietinum). Enzymes of the Embden-Myerhof-Parnas, Entner-Doudoroff and pentose phosphate pathways were found present in hexose-grown cells but not in succinate-grown cells. These however could be induced by the presence of hexoses.     

Metabolic Network Analysis of Streptomyces tenebrarius, a Streptomyces Species with an Active Entner-Doudoroff Pathway

Streptomyces tenebrarius is an industrially important microorganism, producing an antibiotic complex that mainly consists of the aminoglycosides apramycin, tobramycin carbamate, and kanamycin B carbamate. When S. tenebrarius is used for industrial tobramycin production, kanamycin B carbamate is an unwanted by-product. The two compounds differ only by one hydroxyl group, which is present in kanamycin carbamate but is reduced during biosynthesis of tobramycin. ¹³C metabolic flux analysis was used for elucidating connections between the primary carbon metabolism and the composition of the antibiotic complex. Metabolic flux maps were constructed for the cells grown on minimal medium with glucose or with a glucose-glycerol mixture as the carbon source. The addition of glycerol, which is more reduced than glucose, led to a three-times-greater reduction of the kanamycin portion of the antibiotic complex. The labeling indicated an active Entner-Doudoroff (ED) pathway, which was previously considered to be nonfunctional in Streptomyces. The activity of the pentose phosphate (PP) pathway was low (10 to 20% of the glucose uptake rate). The fluxes through Embden-Meyerhof-Parnas (EMP) and ED pathways were almost evenly distributed during the exponential growth on glucose. During the transition from growth phase to production phase, a metabolic shift was observed, characterized by a decreased flux through the ED pathway and increased fluxes through the EMP and PP pathways. Higher specific NADH and NADPH production rates were calculated in the cultivation on glucose-glycerol, which was associated with a lower percentage of nonreduced antibiotic kanamycin B carbamate.     

Carbon Metabolism Enzymes of Rhizobium tropici Cultures and Bacteroids

We determined the activities of selected enzymes involved in carbon metabolism in free-living cells of Rhizobium tropici CFN299 grown in minimal medium with different carbon sources and in bacteroids of the same strain. The set of enzymatic activities in sucrose-grown cells suggests that the pentose phosphate pathway, with the participation of the Entner-Doudoroff pathway, is probably the primary route for sugar catabolism. In glutamate- and malate-grown cells, high activities of the gluconeogenic enzymes (phosphoenolpyruvate carboxykinase, fructose-6-phosphate aldolase, and fructose bisphosphatase) were detected. In bacteroids, isolated in Percoll gradients, the levels of activity for many of the enzymes measured were similar to those of malate-grown cells, except that higher activities of glucokinase, glucose-6-phosphate dehydrogenase, and NAD-dependent phosphogluconate dehydrogenase were detected. Phosphoglucomutase and UDP glucose pyrophosphorylase showed high and constant levels under all growth conditions and in bacteroids.