Selection of Escherichia coli mutants lacking glucose-6-phosphate dehydrogenase or gluconate-6-phosphate dehydrogenase

Glucose is metabolized in Escherichia coli chiefly via the phosphoglucose isomerase reaction; mutants lacking that enzyme grow slowly on glucose by using the hexose monophosphate shunt. When such a strain is further mutated so as to yield strains unable to grow at all on glucose or on glucose-6-phosphate, the secondary strains are found to lack also activity of glucose-6-phosphate dehydrogenase. The double mutants can be transduced back to glucose positivity; one class of transductants has normal phosphoglucose isomerase activity but no glucose-6-phosphate dehydrogenase. An analogous scheme has been used to select mutants lacking gluconate-6-phosphate dehydrogenase. Here the primary mutant lacks gluconate-6-phosphate dehydrase (an enzyme of the Enter-Doudoroff pathway) and grows slowly on gluconate; gluconate-negative mutants are selected from it. These mutants, lacking the nicotinamide dinucleotide phosphate-linked glucose-6-phosphate dehydrogenase or gluconate-6-phosphate dehydrogenase, grow on glucose at rates similar to the wild type. Thus, these enzymes are not essential for glucose metabolism in E. coli.     

Phenotypic suppression of phosphofructokinase mutations in Escherichia coli by constitutive expression of the glyoxylate shunt

Fructose-6-phosphate kinase (pfkA) mutants have impaired growth on carbon sources which enter glycolysis at or above the level of fructose-6-phosphate, but the degree of impairment depends on the carbon source (e.g., growth on glucose is very much slower than growth on glucose-6-phosphate). The present report contains considerable data on this complicated growth phenotype and derives mainly from the finding of a class of partial revertants which grow as fast on glucose as on glucose-6-phosphate; the reversion mutation is shown to be constitutivity of the glyoxylate shunt (iclR(c)). iclR(c) does not increase the fructose-6-phosphate kinase level in the mutants, and the exact mechanism of the partial phenotypic suppression is not understood. However, iclR(c) was already known to suppress some mutations which affected phosphoenolpyruvate levels, and H. L. Kornberg and J. Smith have suggested (1970) that the growth phenotype of pfkA mutants might be related to pathways of phosphoenolpyruvate formation. Surprisingly, the hexose-monophosphate shunt is not necessary for the suppression, which therefore must act to restore metabolism via the residual phosphofructokinase activity present in all pfkA mutants. A mutant totally lacking phosphofructokinase activity was not suppressed.     

Control of amino sugar metabolism in Escherichia coli and isolation of mutants unable to degrade amino sugars

1. Growth of Escherichia coli on glucosamine results in an induction of glucosamine 6-phosphate deaminase [2-amino-2-deoxy-d-glucose 6-phosphate ketol-isomerase (deaminating), EC 5.3.1.10] and a repression of glucosamine 6-phosphate synthetase (l-glutamine-d-fructose 6-phosphate aminotransferase, EC 2.6.1.16); glucose abolishes these control effects. 2. Growth of E. coli on N-acetylglucosamine results in an induction of N-acetylglucosamine 6-phosphate deacetylase and glucosamine 6-phosphate deaminase, and in a repression of glucosamine 6-phosphate synthetase; glucose diminishes these control effects. 3. The synthesis of amino sugar kinases (EC 2.7.1.8 and 2.7.1.9) is unaffected by growth on amino sugars. 4. Glucosamine 6-phosphate synthetase is inhibited by glucosamine 6-phosphate. 5. Mutants of E. coli that are unable to grow on N-acetylglucosamine have been isolated, and lack either N-acetylglucosamine 6-phosphate deacetylase (deacetylaseless) or glucosamine 6-phosphate deaminase (deaminaseless). Deacetylaseless mutants can grow on glucosamine but deaminaseless mutants cannot. 6. After growth on glucose, deacetylaseless mutants have a repressed glucosamine 6-phosphate synthetase and a super-induced glucosamine 6-phosphate deaminase; this may be related to an intracellular accumulation of acetylamino sugar that also occurs under these conditions. In one mutant the acetylamino sugar was shown to be partly as N-acetylglucosamine 6-phosphate. Deaminaseless mutants have no abnormal control effects after growth on glucose. 7. Addition of N-acetylglucosamine or glucosamine to cultures of a deaminaseless mutant caused inhibition of growth. Addition of N-acetylglucosamine to cultures of a deacetylaseless mutant caused lysis, and secondary mutants were isolated that did not lyse; most of these secondary mutants had lost glucosamine 6-phosphate deaminase and an uptake mechanism for N-acetylglucosamine. 8. Similar amounts of (14)C were incorporated from [1-(14)C]-glucosamine by cells of mutants and wild-type growing on broth. Cells of wild-type and a deaminaseless mutant incorporated (14)C from N-acetyl[1-(14)C]glucosamine more efficiently than from N[1-(14)C]-acetylglucosamine, incorporation from the latter being further decreased by acetate; cells of a deacetylaseless mutant showed a poor incorporation of both types of labelled N-acetylglucosamine.     

Glucose-6-phosphate dehydrogenase deficiency in pleiotropic carbohydrate-negative mutant strains of Rhizobium meliloti

Several mutant strains of Rhizobium meliloti isolated after nitrosoguanidine mutagenesis were selected as unable to grow on mannose. Some of them also failed to grow on glucose, fructose, ribose, and xylose but grew on L-arabinose, galactose, and many other carbon sources. Biochemical analysis demonstrated that the mutants lacked NAD- and NADP-linked glucose-6-phosphate dehydrogenase activities that reside on a single enzyme species. One such mutant was found to accumulate glucose-6-phosphate, and this could partially explain the inhibition of growth observed on mixtures of permissive and nonpermissive carbon sources. Symbiotic properties remained unaffected in all these mutants.     

Utilization of gluconate by Escherichia coli. Induction of gluconate kinase and 6-phosphogluconate dehydratase activities

1. A mutant of Escherichia coli, devoid of phosphopyruvate synthetase, glucosephosphate isomerase and 6-phosphogluconate dehydrogenase activities, grew readily on gluconate and inducibly formed an uptake system for gluconate, gluconate kinase and 6-phosphogluconate dehydratase while doing so. 2. This mutant also grew on glucose 6-phosphate and inducibly formed 6-phosphogluconate dehydratase; however, the formation of the gluconate uptake system and gluconate kinase was not induced under these conditions. 3. The use of the Entner–Doudoroff pathway for the dissimilation of 6-phosphogluconate, derived from either gluconate or glucose 6-phosphate, by this mutant was also demonstrated by the accumulation of 2-keto-3-deoxy-6-phosphogluconate (3-deoxy-6-phospho-l-glycero-2-hexulosonate) from both these substrates in a similar mutant that also lacked phospho-2-keto-3-deoxygluconate aldolase activity. 4. Glucose 6-phosphate inhibits the continued utilization of fructose by cultures of the mutants growing on fructose, as it does in wild-type E. coli. 5. The mutants do not use glucose for growth. This is shown to be due to insufficiency of phosphopyruvate, which is required for glucose uptake.     

Effects of edd and pgi disruptions on inosine accumulation in Escherichia coli

Using an inosine-producing mutant of Escherichia coli, the contributions of the central carbon metabolism for overproducing inosine were investigated. Sodium gluconate instead of glucose was tested as a carbon source to increase the supply of ribose-5-phosphate through the oxidative pentose phosphate pathway. The edd (6-phosphogluconate dehydrase gene)-disrupted mutant accumulated 2.5 g/l of inosine from 48 g/l of sodium gluconate, compared with 1.4 g/l of inosine in the edd wild strain. The rpe (ribulose phosphate 3-epimerase gene)-disrupted mutant resulted in low cell growth and low inosine production on glucose and on gluconate. The disruption of pgi (glucose-6-phosphate isomerase gene) was effective for increasing the accumulation of inosine from glucose but resulted in low cell growth. The pgi-disrupted mutant accumulated 3.7 g/l of inosine from 40 g/l of glucose when 8 g/l of yeast extract was added to the medium. Furthermore, to improve effective utilization of adenine, the yicP (adenine deaminase gene)-disrupted mutant was evaluated. It showed higher inosine accumulation, of 3.7 g/l, than that of 2.8 g/l in the yicP wild strain when 4 g/l of yeast extract was added to the medium.     

Glucose and Gluconate Metabolism in an Escherichia coli Mutant Lacking Phosphoglucose Isomerase

A single gene mutant lacking phosphoglucose isomerase (pgi) was selected after ethyl methane sulfonate mutagenesis of Escherichia coli strain K-10. Enzyme assays revealed no pgi activity in the mutant, whereas levels of glucokinase, glucose-6-phosphate dehydrogenase, and gluconate-6-phosphate dehydrogenase were similar in parent and mutant. The amount of glucose released by acid hydrolysis of the mutant cells after growth on gluconate was less than 2% that released from parent cells; when grown in the presence of glucose, mutant and parent cells contained the same amount of glucose residues. The mutant grew on glucose one-third as fast as the parent; it also grew much slower than the parent on galactose, maltose, and lactose. On fructose, gluconate, and other carbon sources, growth was almost normal. In both parent and mutant, gluconokinase and gluconate-6-phosphate dehydrase were present during growth on gluconate but not during growth on glucose. Assay and degradation of alanine from protein hydrolysates after growth on glucose-1-(14)C and gluconate-1-(14)C showed that in the parent strain glucose was metabolized by the glycolytic path and the hexose monophosphate shunt. Gluconate was metabolized by the Entner-Doudoroff path and the hexose monophosphate shunt. The mutant used glucose chiefly by the shunt, but may also have used the Entner-Doudoroff path to a limited extent.     

Transient repression of beta-galactosidase synthesis by glucose-6-phosphate in a mutant of Escherichia coli lacking enzyme II specific for glucose in the phosphoenolpyruvate-sugar phosphotransferase system.

The effects of glucose and glucose-6-phosphate in initiating the repression of beta-galactosidase synthesis were studied using a mutant of Escherichia coli K12 which lacks glucose-specific enzyme II of the phosphoenolpyruvate-sugar phosphotransferase system. It was found that glucose-6-phosphate causes transient repression of beta-galactosidase synthesis but glucose does not cause transient repression in this mutant. Evidence was obtained that both the presence of an active transport system for glucose-6-phosphate in the cells and glucose-6-phosphate in the medium are necessary for the initiation of transient repression. No metabolism of glucose-6-phosphate is required. Upon depletion of glucose-6-phosphate in the medium the transient repression was reversed. After the reversal the rate of enzyme synthesis was high in the cells which had been exposed to a high concentration of glucose-6-phosphate. It was concluded that the translocation of glucose-6-phosphate across the membranes is the primary event which affects both the initiation of and the recovery from the transient repression. During the transient repression the cellular content of cyclic adenosine 3',5'-monophosphate decreased significantly.     

Isolation of a Regulatory Mutant of Fructose-1, 6-diphosphatase in Saccharomyces carlsbergensis

By selecting for growth on glycerol, but absence of growth on glucose, a mutant of Saccharomyces carlsbergensis was isolated which does not grow on glucose, fructose, mannose, or sucrose, which shows long-term adaptation to maltose, but which can grow normally on galactose, ethanol, or glycerol. In the mutant, fructose diphosphatase is not inactivated after the addition of glucose, fructose or mannose to the medium, resulting in the simultaneous presence of fructose diphosphatase and phosphofructokinase activity. Under these conditions, a cycle is probably catalyzed between fructose-6-phosphate and fructose-1,6-diphosphate, resulting in the net consumption of adenosine triphosphate and an immediate stop of protein synthesis.     

Uncoupling of the glucose growth defect and the deregulation of glycolysis in Saccharomyces cerevisiae Tps1 mutants expressing trehalose-6-phosphate-insensitive hexokinase from Schizosaccharomyces pombe

In the yeast Saccharomyces cerevisiae inactivation of trehalose-6-phosphate (Tre6P) synthase (Tps1) encoded by the TPS1 gene causes a specific growth defect in the presence of glucose in the medium. The growth inhibition is associated with deregulation of the initial part of glycolysis. Sugar phosphates, especially fructose-1,6-bisphosphate (Fru1,6bisP), hyperaccumulate while the levels of ATP, Pi and downstream metabolites are rapidly depleted. This was suggested to be due to the absence of Tre6P inhibition on hexokinase. Here we show that overexpression of Tre6P (as well as glucose-6-phosphate (Glu6P))-insensitive hexokinase from Schizosaccharomyces pombe in a wild-type strain does not affect growth on glucose but still transiently enhances initial sugar phosphate accumulation. We have in addition replaced the three endogenous glucose kinases of S. cerevisiae by the Tre6P-insensitive hexokinase from S. pombe. High hexokinase activity was measured in cell extracts and growth on glucose was somewhat reduced compared to an S. cerevisiae wild-type strain but expression of the Tre6P-insensitive S. pombe hexokinase never caused the typical tps1Delta phenotype. Moreover, deletion of TPS1 in this strain expressing only the Tre6P-insensitive S. pombe hexokinase still resulted in a severe drop in growth capacity on glucose as well as sensitivity to millimolar glucose levels in the presence of excess galactose. In this case, poor growth on glucose was associated with reduced rather than enhanced glucose influx into glycolysis. Initial glucose transport was not affected. Apparently, deletion of TPS1 causes reduced activity of the S. pombe hexokinase in vivo. Our results show that Tre6P inhibition of hexokinase is not the major mechanism by which Tps1 controls the influx of glucose into glycolysis or the capacity to grow on glucose. In addition, they show that a Tre6P-insensitive hexokinase can still be controlled by Tps1 in vivo.     

Regulation of alternate peripheral pathways of glucose catabolism during aerobic and anaerobic growth of Pseudomonas aeruginosa.

Glucose may be converted to 6-phosphogluconate by alternate pathways in Pseudomonas aeruginosa. Glucose is phosphorylated to glucose-6-phosphate, which is oxidized to 6-phosphogluconate during anaerobic growth when nitrate is used as respiratory electron acceptor. Mutant cells lacking glucose-6-phosphate dehydrogenase are unable to catabolize glucose under these conditions. The mutant cells utilize glucose as effectively as do wild-type cells in the presence of oxygen; under these conditions, glucose is utilized via direct oxidation to gluconate, which is converted to 6-phosphogluconate. The membrane-associated glucose dehydrogenase activity was not formed during anaerobic growth with glucose. Gluconate, the product of the enzyme, appeared to be the inducer of the gluconate transport system, gluconokinase, and membrane-associated gluconate dehydrogenase. 6-Phosphogluconate is probably the physiological inducer of glucokinase, glucose-6-phosphate dehydrogenase, and the dehydratase and aldolase of the Entner-Doudoroff pathway. Nitrate-linked respiration is required for the anaerobic uptake of glucose and gluconate by independently regulated transport systems in cells grown under denitrifying conditions.     

6-phosphogluconolactonase mutants of Escherichia coli and a maltose blue gene

Mutants lacking an enzyme of the oxidative branch of the hexose monophosphate shunt, 6-phosphogluconolactonase (pgl), have been selected as a new class of glucose-negative derivatives of a phosphoglucose isomerase (pgi) mutant. Glucose negativity is not as complete as in mutants lacking phosphoglucose isomerase and glucose-6-phosphate dehydrogenase. Pgi(+), pgl(-) strains have been constructed by transduction and grow almost normally on glucose. Genetic mapping shows that pgl lies between chlD and att-lambda, in the same position as and identical with a blu gene described by Adhya and Schwartz. These blu mutants grown on maltose were recognized by their property to turn blue after treatment with iodine. It is not known how phosphogluconolactonase deficiency causes this reaction; it might be related to accumulation of 6-phosphogluconolactone.     

A temperature-sensitive mutant of Sacchromyces cerevisiae defective in the specific phosphatase of trehalose biosynthesis

Abstract A temperature-sensitive mutant of Saccharomyces cerevisiae has been isolated which accumulates a large pool of trehalose-6-phosphate when shifted to temperatures above 34°C nonpermissive for growth. This indicates that its defect is in the second enzyme of trehalose biosynthesis, the hydrolase that converts trehalose-6-phosphate to trehalose. Trehalose is made continouosly when yeast is growing on high glucose or when it is starved for a nitrogen source, and accumulates as cells enter the stationary phase. Revertants of the mutant able to grow at 37°C arise spontaneously and no longer accumulate trehalose-6-phosphate at this temperature. Also the kinetics of trehalose-6-phosphate accumulation in the mutant following a 25–37°C shift resemble the kinetics of inhibition of RNA and protein synthesis. It is probable therefore that accumulation of high levels of this metabolic intermediate is inhibitory to growth.     

Effects of edd and pgi Disruptions on Inosine Accumulation in Escherichia coli

Using an inosine-producing mutant of Escherichia coli, the contributions of the central carbon metabolism for overproducing inosine were investigated. Sodium gluconate instead of glucose was tested as a carbon source to increase the supply of ribose-5-phosphate through the oxidative pentose phosphate pathway. The edd (6-phosphogluconate dehydrase gene)-disrupted mutant accumulated 2.5 g/l of inosine from 48 g/l of sodium gluconate, compared with 1.4 g/l of inosine in the edd wild strain. The rpe (ribulose phosphate 3-epimerase gene)-disrupted mutant resulted in low cell growth and low inosine production on glucose and on gluconate. The disruption of pgi (glucose-6-phosphate isomerase gene) was effective for increasing the accumulation of inosine from glucose but resulted in low cell growth. The pgi-disrupted mutant accumulated 3.7 g/l of inosine from 40 g/l of glucose when 8 g/l of yeast extract was added to the medium. Furthermore, to improve effective utilization of adenine, the yicP (adenine deaminase gene)-disrupted mutant was evaluated. It showed higher inosine accumulation, of 3.7 g/l, than that of 2.8 g/l in the yicP wild strain when 4 g/l of yeast extract was added to the medium.     

Evidence for glucose-6-phosphate transport in rat liver microsomes

The existence of glucose-6-phosphate transport across the liver microsomal membrane is still controversial. In this paper, we show that S3483, a chlorogenic acid derivative known to inhibit glucose-6-phosphatase in intact microsomes, caused the intravesicular accumulation of glucose-6-phosphate when the latter was produced by glucose-6-phosphatase from glucose and carbamoyl-phosphate. S3483 also inhibited the conversion of glucose-6-phosphate to 6-phosphogluconate occurring inside microsomes in the presence of electron acceptors (NADP or metyrapone). These data indicate that liver microsomal membranes contain a reversible glucose-6-phosphate transporter, which furnishes substrate not only to glucose-6-phosphatase, but also to hexose-6-phosphate dehydrogenase.     

Glucose dehydrogenase, glucose-6-phosphate dehydrogenase and hexokinase in liver of rainbow trout (Salmo gairdneri). Effects of starvation and temperature variations.

1. Activities of trout liver glucose dehydrogenase (GDH, EC 1.1.1.47) and glucose-6-phosphate dehydrogenase (G6PD, EC 1.1.1.49) were increased after a sudden drop in water temperature, but not in long-time cold acclimated as compared with warm acclimated trout. 2. Possibly, the activities of GDH and G6PD were temporarily increased in connection with metabolic adaptation to the lower temperature. 3. The activities of GDH and G6PD were not changed by the stress of handling. 4. Partially purified trout liver GDH has a lower activation energy with glucose than with glucose-6-phosphate as substrate, and the Km (glucose) decreases with decreasing assay temperature. 5. At low temperatures, the activity of trout liver GDH with glucose as substrate may be comparable to that of glucose-6-phosphate. 6. Partially purified beef liver GDH has a high activation energy with glucose as substrate, and the Km (glucose) does not change with the assay temperature. 7. Hexokinase (HK, EC 2.7.1.1) and GDH activities were unchanged when trout were deprived of food for 4 weeks. Apparently, the trout liver glucose utilization did not adapt to the starvation.     

Utilization of Glucose and the Effect of Organic Compounds on the Chemolithotroph Thiobacillus ferrooxidans

The utilization of glucose by the chemolithotroph Thiobacillus ferrooxidans results in a repression of the ability to oxidize iron, the substrate for autotrophic growth. An assay with resting cells was used to measure iron oxidation rates. Concomitant with the decreased iron oxidation rates, the enzyme responsible for carbon dioxide fixation, ribulose diphosphate (RuDP) carboxylase, was also repressed. Maximum iron oxidation rates precede peak RuDP carboxylase levels, consistent with the role of these processes in autotrophic metabolism in nonrepressed cells. The degree of iron oxidation repression depends on the organic substrate supplied, as does the level of RuDP carboxylase. The uptake of glucose parallels an increase in synthesis of glucose-6-phosphate dehydrogenase and the accumulation in cells of poly-beta-hydroxybutyrate. The organism is also capable of growing on glucose and other organic supplements in the absence of its inorganic energy source; growth rates depend on the organic substrate supplied.     

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.