Effect of temperature on the activity and synthesis of glucose-catabolizing enzymes in Pseudomonas fluorescens.

The activity of the enzymes of the oxidative non-phosphorylated pathway, glucose and gluconate dehydrogenases, were not significantly affected by changes in the assay temperature. Both enzymes demonstrated only a threefold difference in activity when compared at assay temperatures of 30 degrees C and 5 degrees C. In contrast, the enzymes involved in the direct phosphorylation and catabolism of glucose or its oxidation products, gluconate and 2-ketogluconate, exhibited a more pronounced response to decreasing assay temperatures. At least one enzyme in each pathway, involved in the direct phosphorylation and catabolism of glucose or 2-ketogluconate (2KG), demonstrated an eightfold decrease in activity with a decrease in assay temperature from 30 degrees C to 5 degrees C. A similar decrease in assay temperature resulted in a fivefold decrease in activity of the enzymes involved in the direct phosphorylation and catabolism of gluconate. The observed differential effect of temperature on the activity of the enzymes of glucose catabolism and on the accumulation of direct oxidation products during growth with glucose in P. fluorescens E-20 is discussed. Growth with glucose at 5 or 20 degrees C resulted in high induced levels of all glucose-catabolizing enzymes examined when compared with the levels of these same enzymes in pyruvate-grown cells. However, only low levels of glucose dehydrogenase were detected during growth at 30 degrees C with glucose, gluconate, or 2-KG. Similarly, only low levels of gluconate dehydrogenase were detected during growth with glucose at 30 degrees C, although a weak induction was observed during growth with gluconate or 2-KG at 30 degrees C. The levels of 2-KG kinase plus KPG reductase during growth at 30 degrees C were undetectable with glucose, weakly induced with gluconate, and fully induced with 2-KG. High induced levels of glucose dehydrogenase, gluconate dehydrogenase, and 2-KG kinase plus KPG reductase were present during growth at 20 degrees C with glucose or 2-KG. The low levels of glucose and gluconate dehydrogenases present at a growth temperature of 30 degrees C was not due to heat lability of the enzymes at this temperature. The low amounts of these two enzymes during growth with glucose at 30 degrees C probably prevented sufficient inducer(s) formation from glucose to allow induction of enzymes of 2-KG catabolism. The results demonstrated that temperature may regulate the pathways of glucose dissimilation by regulating, either directly or indirectly, the activity and synthesis of the enzymes involved in these pathways.     

The influence of the culture pH value on the direct glucose oxidative pathway in Klebsiella pneumoniae NCTC 418

Klebsiella pneumoniae NCTC 418 was cultured aerobically in chemostat cultures (D=0.3 h^-1; 35°C) under respectively carbon-, phosphate-, potassium-, sulphate-, and ammonia-limited conditions with glucose as the sole carbon and energy source. The effect of the external pH value on glucose metabolism and on the enzymes of the direct glucose oxidative pathway was examined. The pH value of the medium had a profound influence on both the activity and the synthesis of the glucose dehydrogenase and the gluconate dehydrogenase. At pH values ranging from pH 5.5 to pH 6.0 maximal activity and synthesis of these enzymes resulted in a more than 80% conversion of the glucose consumed into gluconate and 2-ketogluconate under potassium-or phosphate-limited conditions. On the other hand, no gluconate and/or 2-ketogluconate production could be detected when K. pneumoniae was cultured at pH 8.0. Whereas the synthesis of gluconate dehydrogenase seemingly was completely repressed, still some glucose dehydrogenase was present. The lack of glucose dehydrogenase activity at pH 8.0 was shown not to be due to the dissociation of the cofactor PQQ from the enzyme.Abbreviations DCIP dichlorophenol indophenol - PQQ pyrroloquinoline quinone [2,7,9-tricarboxy-1H-pyrrolo (2,3-f) quinoline-4,5-dione] - WB Wurster's Blue [1,4-bis-(dimethylamino)-benzene perchlorate]     

Glucolysis in Pseudomonas putida: Physiological Role of Alternative Routes from the Analysis of Defective Mutants

A number of mutants in which glucolysis is impaired have been isolated from Pseudomonas putida. The study of their behavior shows that this organism possesses a single glucolytic pathway with physiological significance. The first step of the pathway consists in the oxidation of glucose into gluconate. Two proteins with glucose dehydrogenase activity appear to exist in P. putida but the reasons for this duplicity are not clear. The process continues with the formation of 2-ketogluconate which is in turn converted into gluconate-6-phosphate. This is proved by the fact that mutants unable to form gluconate-6-phosphate from 2-ketogluconate show extremely slow growth on glucose or gluconate (generation times are increased more than 100 times). Other possible routes for the conversion of glucose into gluconate-6-phosphate, the glucose-6-phosphate pathway, or the direct phosphorylation of the gluconate formed by glucose oxidation are only minor shunts in P. putida. The Entner-Doudoroff enzymes, which catalyze the conversion of gluconate-6-phosphate into pyruvate and triosephosphate, appear to be essential to grow on glucose and also on gluconate and 2-ketogluconate. A significative role of the pentose route in the catabolism of these substrates is not apparent from this study. In contrast, P. putida strains showing no activity of the Entner-Doudoroff enzymes grow readily on fructose, although there is evidence that this hexose is at least partially catabolized via gluconate-6-phosphate.     

Participation of the Entner-Doudoroff pathway in Escherichia coli strains with an inactive phosphotransferase system (PTS- Glc+) in gluconate and glucose batch cultures

The activity of the enzymes of the central metabolic pathways has been the subject of intensive analysis; however, the Entner-Doudoroff (ED) pathway has only recently begun to attract attention. The metabolic response to edd gene knockout in Escherichia coli JM101 and PTS- Glc+ was investigated in gluconate and glucose batch cultures and compared with other pyruvate kinase and PTS mutants previously constructed. Even though the specific growth rates between the strain carrying the edd gene knockout and its parent JM101 and PTS- Glc+ edd and its parent PTS- Glc+ were very similar, reproducible changes in the specific consumption rates and biomass yields were obtained when grown on glucose. These results support the participation of the ED pathway not only on gluconate metabolism but on other metabolic and biochemical processes in E. coli. Despite that gluconate is a non-PTS carbohydrate, the PTS- Glc+ and derived strains showed important reductions in the specific growth and gluconate consumption rates. Moreover, the overall activity of the ED pathway on gluconate resulted in important increments in PTS- Glc+ and PTS- Glc+ pykF mutants. Additional results obtained with the pykA pykF mutant indicate the important contribution of the pyruvate kinase enzymes to pyruvate synthesis and energy production in both carbon sources.     

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 uptake of 2-ketogluconate by Pseudomonas putida

The uptake of 2-ketogluconate is inducible in Pseudomonas putida: 2-ketogluconate, glucose, gluconate, glycerol and glycerate were each good nutritional inducers of this ability. 2-Ketogluconate uptake obeyed saturation kinetics (apparent K _min 2-ketogluconate-grown cells was 0.4 mM). 2-Ketogluconate was transported against a concentration gradient, apparently in an unchanged state, and the process required metabolic energy, all of which indicate an active transport system.A number of independently isolated mutants with deranged activity of a common glucose-gluconate uptake system were found to be also defective in 2-ketogluconate transport. Strains unable to transport 2-ketogluconate which grew readily on glucose and gluconate were also isolated. These results suggest that 2-ketogluconate transport is governed by at least two genetic elements: one which is also required to take up glucose and gluconate and another which appears to be specific for 2-ketogluconate transport. Similarly glucose and gluconate transport appears to require at least one factor which is not necessary for 2-ketogluconate transport, as suggested by the lack of induction of the common glucose-gluconate uptake system by glycerol and glycerate, substrates which are good inducers of 2-ketogluconate uptake.Abbreviations CCCP carbonyl-cyanide-m-chlorophenyl-hydrazone - cpm radioactivity counts per minute - GGU glucose-gluconate uptake - PFU plaque forming units - U.V. ultraviolet Dedicated to Prof. Roger Y. Stainer on the occasion of his 60th birthday     

6-Phosphogluconate dehydratase deficiency in pleiotropic carbohydrate-negative mutant strains of Pseudomonas aeruginosa.

Mutants of Pseudomonas aeruginosa, strain PAO, have been isolated that are unable to grow on mannitol, glucose, gluconate, or 2-ketogluconate, cut that exhibit wild-type growth on pyruvate, lactate, citrate, succinate, or acetate. Although some of these mutants were also unable to grow on glycerol, the mutations formed a single linkage group by quantitative transductional analysis with phage F116 on glucose minimal agar medium. Cell extracts of all mutant strains were either lacking or severely deficient in 6-phosphogluconate dehydratase activity. Glu+ transductants derived from mutant strains that retained the wild-type ability for growth at the expense of glycerol also regained the ability to grow on all C-6 compounds. Although a role for the pentose phosphate pathway in the catabolism of C6 substrates was not found, a functional Entner-Doudoroff pathway appears to be essential for the catabolism of mannitol, glucose, gluconate, and 2-ketogluconate.     

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.     

Role of the pentose phosphate pathway and the Entner–Doudoroff pathway in glucose metabolism of Gluconobacter oxydans 621H

Glucose catabolism by the obligatory aerobic acetic acid bacterium Gluconobacter oxydans 621H proceeds in two phases comprising rapid periplasmic oxidation of glucose to gluconate (phase I) and oxidation of gluconate to 2-ketogluconate or 5-ketogluconate (phase II). Only a small amount of glucose and part of the gluconate is taken up into the cells. To determine the roles of the pentose phosphate pathway (PPP) and the Entner–Doudoroff pathway (EDP) for intracellular glucose and gluconate catabolism, mutants defective in either the PPP (Δgnd, Δgnd zwf*) or the EDP (Δedd–eda) were characterized under defined conditions of pH 6 and 15 % dissolved oxygen. In the presence of yeast extract, neither of the two pathways was essential for growth with glucose. However, the PPP mutants showed a reduced growth rate in phase I and completely lacked growth in phase II. In contrast, the EDP mutant showed the same growth behavior as the reference strain. These results demonstrate that the PPP is of major importance for cytoplasmic glucose and gluconate catabolism, whereas the EDP is dispensable. Reasons for this difference are discussed.     

Genetic and biochemical characterization of the pathway in Pantoea citrea leading to pink disease of pineapple

Pink disease of pineapple, caused by Pantoea citrea, is characterized by a dark coloration on fruit slices after autoclaving. This coloration is initiated by the oxidation of glucose to gluconate, which is followed by further oxidation of gluconate to as yet unknown chromogenic compounds. To elucidate the biochemical pathway leading to pink disease, we generated six coloration-defective mutants of P. citrea that were still able to oxidize glucose into gluconate. Three mutants were found to be affected in genes involved in the biogenesis of c-type cytochromes, which are known for their role as specific electron acceptors linked to dehydrogenase activities. Three additional mutants were affected in different genes within an operon that probably encodes a 2-ketogluconate dehydrogenase protein. These six mutants were found to be unable to oxidize gluconate or 2-ketogluconate, resulting in an inability to produce the compound 2,5-diketogluconate (2,5-DKG). Thus, the production of 2,5-DKG by P. citrea appears to be responsible for the dark color characteristic of the pink disease of pineapple.     

Effect of dissolved oxygen concentration on the metabolism of glucose inPseudomonas putida BM014

The effect of dissolved oxygen concentration on the metabolism of glucose inPseudomonas putida BM014 was investigated. Glucose was completely converted to 2-ketogluconatevia extracellular oxdative pathway and then taken up for cell growth under the condition of sufficient dissolved oxygen concentration. On the other hand, oxygen limitation below dissolved oxygen tension (DOT) value of 20% of air saturation caused the shift of glucose metabolism from the extracellular oxidative pathway to the intracellular phosphorylative pathway. Specific activities of hexokinase and gluconate kinase in intracellular phosphorylation pathway decreased as the DOT increased, while 2-ketogluconokinase activity in extracellular oxidative pathway increased under the same condition. This result can be usefully applied to microbial transformation of glucose to 2-ketogluconate, the synthetic precursor for iso-vitamine C, with almost 100% yieldvia extracellular oxidation by simple DOT control.     

Characterization of enzymes involved in the central metabolism of Gluconobacter oxydans

Gluconobacter oxydans is an industrially important bacterium that lacks a complete Embden–Meyerhof pathway (glycolysis). The organism instead uses the pentose phosphate pathway to oxidize sugars and their phosphorylated intermediates. However, the lack of glycolysis limits the amount of NADH as electron donor for electron transport phosphorylation. It has been suggested that the pentose phosphate pathway contributes to NADH production. Six enzymes predicted to play central roles in intracellular glucose and gluconate flux were heterologously overproduced in Escherichia coli and characterized to investigate the intracellular flow of glucose and gluconates into the pentose phosphate pathway and to explore the contribution of the pentose phosphate pathway to NADH generation. The key pentose phosphate enzymes glucose 6-phosphate dehydrogenase (Gox0145) and 6-phosphogluconate dehydrogenase (Gox1705) had dual cofactor specificities but were physiologically NADP- and NAD-dependent, respectively. Putative glucose dehydrogenase (Gox2015) was NADP-dependent and exhibited a preference for mannose over glucose, whereas a 2-ketogluconate reductase (Gox0417) displayed dual cofactor specificity for NAD(P)H. Furthermore, a putative gluconokinase and a putative glucokinase were identified. The gluconokinase displayed high activities with gluconate and is thought to shuttle intracellular gluconate into the pentose phosphate pathway. A model for the trafficking of glucose and gluconates into the pentose phosphate pathway and its role in NADH generation is presented. The role of NADPH in chemiosmotic energy conservation is also discussed.     

Regulation of the glucolytic enzymes inPseudomonas putida

Summary The synthesis of glucose catabolizing enzymes is under inductive control inPseudomonas putida. Glucose, gluconate and 2-ketogluconate are the best nutritional inducers of these enzymes. Mutants unable to catabolize gluconate or 2-ketogluconate synthesized relatively high levels of glucose dehydrogenase and gluconate-6P dehydrase activities when grown in the presence of these substrates. This identifies both compounds as true inducers of these enzymes. KDGP aldolase is induced by its substrate, as evidenced by the inability of mutant cells unable to form KDGP to produce this enzyme at levels above the basal one. A 3-carbon compound appears to be the inducer of glyceraldehyde-3P dehydrogenase. This pattern of regulation suggests that there is a low degree of coordinate control in the synthesis of the glucolytic enzymes byP. putida. This is also supported by the lack of proportionality found in the levels of two enzymes governed by the same inducers, glucose dehydrogenase and gluconate-6P dehydrase, in cells grown on different conditions.Abbrevitions P phosphate - KDGP 2-Keto-3-deoxygluconate-6-phosphate - GDH glucose dehydrogenase - GNDH gluconate dehydrogenase - GK glucokinase - GNK gluconokinase - KGK ketogluconokinase - KGR 2-Ketogluconate-6-phosphate reductase - GPDH glucose-6-phosphate dehydrogenase - GNPD gluconate-6-phosphate dehydrase - KDGPA 2-Keto-3-deoxygluconate-6-phosphate aldolase - GAPDH glyceraldehyde-3-phosphate dehydrogenase     

Biochemical characterization and sequence analysis of the gluconate:NADP 5-oxidoreductase gene from Gluconobacter oxydans.

Gluconate:NADP 5-oxidoreductase (GNO) from the acetic acid bacterium Gluconobacter oxydans subsp. oxydans DSM3503 was purified to homogeneity. This enzyme is involved in the nonphosphorylative, ketogenic oxidation of glucose and oxidizes gluconate to 5-ketogluconate. GNO was localized in the cytoplasm, had an isoelectric point of 4.3, and showed an apparent molecular weight of 75,000. In sodium dodecyl sulfate gel electrophoresis, a single band appeared corresponding to a molecular weight of 33,000, which indicated that the enzyme was composed of two identical subunits. The pH optimum of gluconate oxidation was pH 10, and apparent Km values were 20.6 mM for the substrate gluconate and 73 microM for the cosubstrate NADP. The enzyme was almost inactive with NAD as a cofactor and was very specific for the substrates gluconate and 5-ketogluconate. D-Glucose, D-sorbitol, and D-mannitol were not oxidized, and 2-ketogluconate and L-sorbose were not reduced. Only D-fructose was accepted, with a rate that was 10% of the rate of 5-ketogluconate reduction. The gno gene encoding GNO was identified by hybridization with a gene probe complementary to the DNA sequence encoding the first 20 N-terminal amino acids of the enzyme. The gno gene was cloned on a 3.4-kb DNA fragment and expressed in Escherichia coli. Sequencing of the gene revealed an open reading frame of 771 bp, encoding a protein of 257 amino acids with a predicted relative molecular mass of 27.3 kDa. Plasmid-encoded gno was functionally expressed, with 6.04 U/mg of cell-free protein in E. coli and with 6.80 U/mg of cell-free protein in G. oxydans, which corresponded to 85-fold overexpression of the G. oxydans wild-type GNO activity. Multiple sequence alignments showed that GNO was affiliated with the group II alcohol dehydrogenases, or short-chain dehydrogenases, which display a typical pattern of six strictly conserved amino acid residues.     

Glucose and Gluconate Metabolism in a Mutant of Escherichia coli Lacking Gluconate-6-phosphate Dehydrase

A mutant lacking gluconate-6-phosphate dehydrase (the first enzyme of the Entner-Doudoroff pathway) was isolated after ethyl methane sulfonate mutagenesis of Escherichia coli. Other enzymes of gluconate metabolism (gluconokinase, gluconate-6-phosphate dehydrogenase, and 2-keto-3-deoxygluconate-6-phosphate aldolase) were present in the mutant. When the mutant was grown on gluconate-1-(14)C, alanine isolated from protein was unlabeled, showing that the dehydrase was absent in vivo and that the sole pathway of gluconate metabolism in the mutant was the hexose monophosphate shunt. The mutant grew on gluconate with a doubling time of 155 min, compared with the parent strain's 56 min. On glucose and fructose it grew with normal doubling times. Thus, in E. coli, the Entner-Doudoroff pathway is used for gluconate metabolism but not for glucose metabolism.     

Effect of temperature on diauxic growth with glucose and organic acids in Pseudomonas fluorescens

Growth of Pseudomonas fluorescens in batch culture with glucose and organic acids resulted in typical diauxic responses at 30° C but no detectable diauxic lag at 5° C.At 30° C, organic acids were preferentially utilized during the first growth phase. Glucose utilization was delayed unitl onset of the second growth phase. Systems involved in direct uptake and catabolism of glucose responded in a manner compatible with respression by malate and/or its metabolites and induction by glucose and/or its metabolites. The oxidative non-phosphorylated pathway, through gluconate and 2-ketogluconate (2-KG) as intermediates, was not induced during either growth phase.At 5° C, growth with glucose and organic acids was biphasic but without diauxic lag. Organic acids were preferentially utilized during the first growth phase. Although carbon from glucose was not fully catabolized until onset of the second growth phase, glucose was oxidized to and accumulated extracellularly as gluconate and 2-KG during the first growth phase. No significant repression of glucose-catabolizing enzymes was observed during growth with organic acids in the presence of glucose. However, uptake activities for gluconate and 2-KG did not increase significantly until onset of the second growth phase.Thus, at low temperatures, psychrotrophic P. fluorescens oxidized glucose to extracellular 2-KG, while growing on preferred carbon sources. The 2-KG was then catabolized after depletion of the organic acid.