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.     

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     

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.     

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     

Predominance of gluconate formation from glucose during germination of Bacillus megaterium QM B1551 spores.

Metabolic pathways of glucose during germination of Bacillus megaterium QM B1551 spores were studied by using specifically labeled glucose and gluconate. The Embden-Meyerhof pathway, the pentose cycle, and the direct oxidation route of glucose to gluconate (the gluconate pathway) were all operative at this stage; among those, gluconate accumulation was most predominant, especially in the early stage. Potassium fluoride, an enolase inhibitor, abolished the catabolism by the Embden-Meyerhof pathway totally without affecting gluconate accumulation. Under these conditions glucose was exclusively oxidized to gluconate. Gluconate thus accumulated could be metabolized further via phosphorylation by gluconate kinase. Remarkable gluconate accumulation was also demonstrated in several other spores requiring alanine as an effective germinant. NADH formed by the direct glucose oxidation may serve as a initial ATP source to phosphorylate glucose in germinating spores.     

Gluconate metabolism in germinated spores of Bacillus megaterium QM B1551: primary roles of gluconokinase and the pentose cycle

The metabolic pathway of gluconate, a major product of glucose metabolism during spore germination, was investigated in Bacillus megaterium QM B1551. Compared to the parent, mutant spores lacking gluconokinase could not metabolize gluconate, whereas the revertant simultaneously restored the enzyme activity and the ability to metabolize it, indicating that gluconokinase was solely responsible for the onset of gluconate metabolism. To identify a further metabolic route for gluconate, we determined 14C yields in acetate and CO2 formed from [14C]gluconate, and found that experimental ratios of 14CO2/[14C]acetate obtained from [2-14C]gluconate and [3,4-14C]gluconate were not compatible with the ratios predicted from the Entner-Doudoroff pathway. In contrast, when CO2 release caused by recycling (approx. 30%) was corrected, the ratios almost agreed with those from the pentose cycle. Comparison of specific radioactivities in acetate also supported the conclusion that gluconate was metabolized via the pentose cycle, subsequently metabolized via the Embden-Meyerhof pathway, and finally degraded to acetate and CO2 without a contribution by the Krebs cycle.     

Regulation of Glucose Metabolism in Thiobacillus intermedius

Glucose-yeast extract or glucose-casein hydrolysate-grown Thiobacillus intermedius cells, which use glucose for energy generation, possess high specific activities of the Entner-Doudoroff pathway and related enzymes, 6-phosphogluconate dehydrase, 2-keto-3-deoxy-6-phosphogluconate aldolase, glucokinase, and glucose-6-phosphate dehydrogenase, but low activities of enzymes unique to the pentose shunt and Embden-Meyerhof pathways. Although the synthesis of the latter enzymes remains largely unaffected by the growth environment, that of the former is stimulated by glucose. Radiorespirometric measurements demonstrate an early and parallel respiration of glucose carbon atoms one and four in glucose-casein hydrolysate broth. It is concluded that the Entner-Doudoroff pathway performs an energetic role in glucose metabolism by T. intermedius with the pentose shunt and Embden-Meyerhof pathways functioning mainly in biosynthesis. The presence of thiosulfate in the growth medium inhibits the synthesis of the Entner-Doudoroff pathway and related enzymes. In addition, both thiosulfate and glucose inhibit the synthesis of the Krebs cycle enzymes, nicotinamide adenine dinucleotide phosphate-linked isocitrate and alpha-ketoglutarate dehydrogenases. Thus, repression of enzymes is of significance in the adaptation of T. intermedius to its nutritional environment. The activity of glucose-6-phosphate dehydrogenase of T. intermedius is inhibited by adenosine triphosphate. Such a control could afford the organism a mechanism to regulate the flow of glucose into major energetic and biosynthetic routes.     

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     

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.     

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.     

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     

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%.     

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.     

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.     

Metabolism of Carbohydrates by Pasteurella pseudotuberculosis

Cell-free extracts of Pasteurella pseudotuberculosis and P. pestis catalyzed a rapid and reversible exchange of electrons between pyridine nucleotides. Although the extent of this exchange approximated that promoted by the soluble nicotinamide adenine dinucleotide (phosphate) transhydrogenase of Pseudomonas fluorescens, the reaction in the pasteurellae was associated with a particulate fraction and was not influenced by adenosine-2'-monophosphate. The ability of P. pseudotuberculosis to utilize this system for the maintenance of a large pool of nicotinamide adenine dinucleotide phosphate could not be correlated with significant participation of the Entner-Doudoroff path or catabolic use of the hexose-monophosphate path during metabolism of glucose. As judged by the distribution of radioactivity in metabolic pyruvate, glucose and gluconate were fermented via the Embden-Meyerhof and Entner-Doudoroff paths, respectively. With the exception of hexosediphosphatase, all enzymes of the three paths were detected, although little or no gluconokinase or phosphogluconate dehydrase was present unless the organisms were cultivated with gluconate. The significance of these findings is discussed with respect to the regulation of carbohydrate metabolism in the pasteurellae, related enteric bacteria, and P. fluorescens.     

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     

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.     

Different degradation pathways for glucose and fructose in Rhodopseudomonas capsulata

In Rhodopseudomonas capsulata the enzymes of the Entner-Doudoroff pathway and the Embden-Meyerhof pathway have been examined. Fructose-grown cells contained inducible activities of phosphoenolpyruvate-fructosephospho-transferase and 1-phosphofructokinase and only low levels of fructokinase and 6-phosphofructokinase. Although fructose-grown cells contained, in addition, all the enzymes of the Entner-Doudoroff pathway together with fructose-1,6-diphosphatase and phosphoglucose isomerase, the Entner-Doudoroff pathway was not operative in fructose catabolism and served only the degradation of glucose. The functional separation of glucose and fructose catabolism via the Entner-Doudoroff and a modified Embden-Meyerhof pathway, respectively, was confirmed by different approaches: 1. Radiorespirometric experiments with glucose and fructose labelled in positions 1, 2, 3, 3+4 and 6 have been carried out. The pattern of 14CO2-evolution from position-labelled glucose was characteristic for the Entner-Doudoroff pathway, that from position-labelled fructose for the Embden-Meyerhof pathway. 2. In the presence of arsenite up to 50% of glucose- and fructose-carbon was excreted as pyruvate. Using 1-14C-glucose, 86% of the pyruvate was labelled in the carboxyl group, whereas using 1-14C-fructose only 19% of the pyruvate was labelled in the carboxyl group. 3. A glucose-6-phosphate dehydrogenase-deficient mutant was isolated which lacked a functional Entner-Doudoroff pathway but which was unaltered in its ability to grow on fructose.     

Pathways for metabolism of ketoaldonic acids in an Erwinia sp.

The pathways involved in the metabolism of ketoaldonic acids by Erwinia sp. strain ATCC 39140 have been investigated by use of a combination of enzyme assays and isolation of bacterial mutants. The catabolism of 2,5-diketo-D-gluconate (2,5-DKG) to gluconate can proceed by two separate NAD(P)H-dependent pathways. The first pathway involves the direct reduction of 2,5-DKG to 5-keto-D-gluconate, which is then reduced to gluconate. The second pathway involves the consecutive reduction of 2,5-DKG to 2-keto-L-gulonate and L-idonic acid, which is then oxidized to 5-keto-D-gluconate, which is then reduced to gluconate. Gluconate, which can also be produced by the NAD(P)H-dependent reduction of 2-keto-D-gluconate, is phosphorylated to 6-phosphogluconate and further metabolized through the pentose phosphate pathway. No evidence was found for the existence of the Entner-Doudoroff pathway in this strain.     

Biochemistry of nitrate respiration in Pseudomonas stutzeri. I. Aerobic and nitrate respiration routes of carbohydrate catabolism

Spangler, W. J. (Oregon State University, Corvallis), and C. M. Gilmour. Biochemistry of nitrate respiration in Pseudomonas stutzeri. I. Aerobic and nitrate respiration routes of carbohydrate catabolism. J. Bacteriol. 91:245-250. 1966.-The metabolic pathways of glucose catabolism were studied in Pseudomonas stutzeri under aerobic conditions and under conditions of nitrate respiration. Studies on both glucose and gluconate catabolism, by the radiorespirometric method, indicated that these substrates are degraded in the same manner, i.e., the Entner-Doudoroff and pentose phosphate pathways. There appeared to be no major shift in primary metabolic pathways when nitrate was used as the terminal hydrogen acceptor in nitrate respiration as opposed to aerobic respiration with free molecular oxygen. It was shown that glucose is not degraded to any appreciable extent under anaerobic conditions in the absence of nitrate. Tentative evidence suggests that the tricarboxylic acid cycle functions under both conditions of oxygen relationships and that the rate of carbon oxidation via the tricarboxylic acid cycle is slower with nitrate respiration than under aerobic conditions.