Phenotypic suppression of a fructose-1,6-diphosphate aldolase mutation in Escherichia coli

Strain NP 315 of Escherichia coli possesses a thermolabile fructose-1, 6-diphosphate (FDP) aldolase; its growth on carbohydrate substrates is inhibited probably as a consequence of the accumulation of high intracellular levels of FDP. Studies of one class of phenotypic revertants of strain NP 315 which have regained their ability to grow on C(6) substrates at 40 C showed that in these strains the buildup of the inhibitory FDP pool is prevented by additional mutations in enzymes catalyzing the conversion of the substrate offered in the medium to FDP. For example, mutations affecting 6-phosphogluconate dehydrogenase activity (gnd(-)) may be selected in great number without any mutagenesis and enrichment simply by isolating revertants of strain NP 315 able to grow on gluconate at 40 C. Similarly, an additional mutation in phosphoglucose isomerase (pgi(-)) restores the ability of these fda(-)gnd(-) strains to grow on glucose at 40 C. Glucose metabolism of these fda(-)gnd(-)pgi(-) strains was investigated. The enzymes of the Entner-Doudoroff pathway are induced to an appreciable extent upon growth of these mutants on glucose medium; further evidence for glucose degradation via this route (which normally is induced only in the presence of gluconate) was provided by following the fate of the C1 label of radioactive glucose in l-alanine. Predominant labeling of the carboxyl-carbon of l-alanine was observed, inciating a major contribution of the Entner-Doudoroff path to pyruvate formation from glucose. Chromatographic analysis of the intermediates of glucose metabolism showed further that glucose apparently is at least partly metabolized via a bypass consisting of the accumulation of extracellular gluconic acid which arises by dephosphorylation of 6-phosphogluconolactone and possibly of 6-phosphogluconate. This extracellular gluconate is then taken up and metabolized in the normal manner via the Entner-Doudoroff enzymes.     

Carbohydrate catabolism of Mima polymorpha. II. Abortive catabolism of glucose

Marus, Adrienne (University of Cincinnati, Cincinnati, Ohio), and Emily J. Bell. Carbohydrate catabolism of Mima polymorpha. II. Abortive catabolism of glucose. J. Bacteriol. 91:2229-2236. 1966.-Mima polymorpha, unable to grow in the presence of glucose as a sole carbon and energy source, is able to obtain supplemental, utilizable energy from the partial catabolism of this substrate. Various enzymes of hexose catabolism have been assayed in this organism and in M. polymorpha M, a mutant obtained by ultraviolet irradiation. The parent strain contains a functional glucose dehydrogenase, glucose-6-phosphate dehydrogenase, diphosphofructoaldolase, and a 2-keto-3-deoxy-6-phosphogluconate aldolase, but is lacking in glucokinase, gluconokinase, 2-ketogluconokinase, and 6-phosphogluconate dehydrogenase. The enzymes present indicate partially functioning hexose diphosphate and Entner-Doudoroff pathways. The absence of kinases explains the inability of the strain to grow on glucose and an absence of 6-phosphogluconate dehydrogenase would indicate the absence of the complete pentose pathway. The mutant strain, M. polymorpha M, possesses, in addition to those enzymes produced by the wild type, both gluconokinase and 6-phosphogluconate dehydrogenase. The presence of the former explains the mutant's ability to grow on glucose, and the presence of the latter indicates a more complete pentose shunt. The supplemental energy obtained from partial glucose catabolism (to gluconic acid) may be obtained from a cytochrome-linked reaction of the glucose dehydrogenase.     

Activity of two catabolic enzymes of the phosphogluconate pathway in mesquite roots inoculated with Azospirillum brasilense Cd.

The mesquite amargo (Prosopis articulate), one of the main nurse trees of the Sonoran Desert in Mexico, is responsible for major, natural re-vegetation processes. It exudes gluconic acid in root exudates, a favorite carbon source for the plant growth-promoting bacterium Azospirillum brasilense. Two enzymes, gluconokinase (EC 2.7.1.12) and 6-phosphogluconate dehydrogenase (EC 1.1.1.44), participating in the phosphogluconate pathway, are active in the bacteria. Bacterial 6-phosphogluconate dehydrogenase is a constitutive enzyme, while gluconokinase is induced upon exposure to gluconic acid. Both enzymes are active in young, non-inoculated mesquite seedlings growing under hydroponic conditions. When A. brasilense Cd bacteria are inoculated on the root system, the roots exhibit much higher activity of gluconokinase, but not 6-phosphogluconate dehydrogenase. Mesquite roots exhibit high levels of root colonization by the inoculating bacteria. At the same time, and also for plants growing under sand culture conditions, the seedlings grew taller, greener, had longer leaves, and were heavier.     

Altered growth-rate-dependent regulation of 6-phosphogluconate dehydrogenase level in hisT mutants of Salmonella typhimurium and Escherichia coli.

In Escherichia coli, the level of 6-phosphogluconate dehydrogenase is directly proportional to the cellular growth rate during growth in minimal media. This contrasts with the report by Winkler et al. (M. E. Winkler, J. R. Roth, and P. E. Hartman, J. Bacteriol. 133:830-843, 1978) that the level of the enzyme in Salmonella typhimurium LT-2 strain SB3436 is invariant. The basis for the difference in the growth-rate-dependent regulation between the two genera was investigated. Expression of gnd, which encodes 6-phosphogluconate dehydrogenase, was growth rate uninducible in strain SB3436, as reported previously, but it was 1.4-fold growth rate inducible in other S. typhimurium LT-2 strains, e.g., SA535. Both the SB3436 and SA535 gnd genes were growth rate inducible in E. coli K-12. Moreover, the nucleotide sequences of the regulatory regions of the two S. typhimurium genes were identical. We concluded that a mutation unlinked to gnd is responsible for the altered growth rate inducibility of 6-phosphogluconate dehydrogenase in strain SB3436. Transductional analysis showed that the altered regulation is due to the presence of a mutation in hisT, the gene for the tRNA modification enzyme pseudouridine synthetase I. A complementation test showed that the regulatory defect conferred by the hisT mutation was recessive. In E. coli, hisT mutations reduced the extent of growth rate induction by the same factor as in S. typhimurium. The altered regulation conferred by hisT mutations was not simply due to their general effect of reducing the polypeptide chain elongation rate, because miaA mutants, which lack another tRNA modification and have a similarity reduced chain growth rate, had higher rather than lower 6-phosphogluconate dehydrogenase levels. Studies with genetic fusions suggested that hisT mutations lower the gnd mRNA level. The data also indicated that hisT is involved in translational control of gnd expression, but not the aspect mediated by the internal complementary sequence.     

Relationship between catabolism of glycerol and metabolism of hexosephosphate derivatives by Pseudomonas aeruginosa.

The relationship between catabolism of glycerol and metabolism of hexosephosphate derivatives in Pseudomonas aeruginosa was studied by comparing the growth on glycerol and enzymatic constitution of strain PAO with these characteristics of glucose-catabolic mutants and revertants. Growth of strain PAO on glycerol induced a catabolic oxidized nicotinamide adenine dinucleotide-linked glyceraldehyde-phosphate dehydrogenase and seven glucose-catabolic enzymes. The results indicated that these enzymes were induced by a six-carbon metabolite of glucose. All strains possessed a constitutive anabolic Embden-Meyerhof-Parnas pathway allowing limited conversion of glycerol-derived triosephosphate to hexosephosphate derivatives, which was consistent with induction of these enzymes by glycerol. Phosphogluconate dehydratase-deficient mutants grew on glycerol. However, mutants lacking both phosphogluconate dehydrogenase and phosphogluconate dehydratase were unable to grow on glycerol, although these strains possessed all of the enzymes needed for degradation of glycerol. These mutants apparently were inhibited by hexosephosphate derivatives, which originated from glycerol-derived triosephosphate and could not be dissimilated. This conclusion was supported by the fact that revertants regaining only a limited capacity to degrade 6-phosphogluconate were glycerol positive but remained glucose negative.     

Specific Deletion Occurring in the Directed Evolution of 6-Phosphogluconate Dehydrogenase in ESCHERICHIA COLI

A novel genetic change leading to increased activity of 6-phosphogluconate dehydrogenase (6PGD) in E. coli has been observed. The mutation is a deletion of approximately 0.4 kilobase pairs occurring between the structural gene of 6PGD (gnd) and one copy of an insertion element (IS5) found normally in E. coli K12 a few hundred base pairs upstream (counterclockwise) from gnd at 44 minutes on the conventional genetic map. The deletion is associated with a threefold higher activity of 6PGD and a 57% increase in the maximum growth rate when cells are grown in gluconate.     

Enzymes of glucose metabolism in Frankia sp.

Enzymes of glucose metabolism were assayed in crude cell extracts of Frankia strains HFPArI3 and HFPCcI2 as well as in isolated vesicle clusters from Alnus rubra root nodules. Activities of the Embden-Meyerhof-Parnas pathway enzymes glucokinase, phosphofructokinase, and pyruvate kinase were found in Frankia strain HFPArI3 and glucokinase and pyruvate kinase were found in Frankia strain HFPCcI2 and in the vesicle clusters. An NADP+-linked glucose 6-phosphate dehydrogenase and an NAD-linked 6-phosphogluconate dehydrogenase were found in all of the extracts, although the role of these enzymes is unclear. No NADP+-linked 6-phosphogluconate dehydrogenase was found. Both dehydrogenases were inhibited by adenosine 5-triphosphate, and the apparent Km's for glucose 6-phosphate and 6-phosphogluconate were 6.86 X 10(-4) and 7.0 X 10(-5) M, respectively. In addition to the enzymes mentioned above, an NADP+-linked malic enzyme was detected in the pure cultures but not in the vesicle clusters. In contrast, however, the vesicle clusters had activity of an NAD-linked malic enzyme. The possibility that this enzyme resulted from contamination from plant mitochondria trapped in the vesicle clusters could not be discounted. None of the extracts showed activities of the Entner-Doudoroff enzymes or the gluconate metabolism enzymes gluconate dehydrogenase or gluconokinase. Propionate- versus trehalose-grown cultures of strain HFPArI3 showed similar activities of most enzymes except malic enzyme, which was higher in the cultures grown on the organic acid. Nitrogen-fixing cultures of strain HFPArI3 showed higher specific activities of glucose 6-phosphate and 6-phosphogluconate dehydrogenases and phosphofructokinase than ammonia-grown cultures.     

Growth-phase-dependent induction of 6-phosphogluconate dehydrogenase and glucose 6-phosphate dehydrogenase in the cyanobacterium Synechococcus sp. PCC7942.

In most cyanobacteria, the only known pathway for oxidation of stored carbohydrate in the dark or under energy-limiting conditions is the hexose monophosphate shunt. To determine whether the increased use of the shunt under these conditions derives from an increase in the activity level of the respective enzymes, we measured the effect of growth phase during the growth of batch cultures of Synechococcus sp. strain PCC7942 on the specific activity of 6-phosphogluconate dehydrogenase (6PGD) and glucose 6-phosphate dehydrogenase. The specific activities were constant during the exponential growth phase of the culture, but they increased about fivefold during the transition into stationary phase. As an approach to determining the level of expression at which the growth-phase-dependent regulation of 6PGD level is exerted, we constructed operon and gene fusions between the gnd gene, which encodes 6PGD, and the Escherichia coli lacZ gene, which encodes beta-galactosidase (beta Gal). Strains harboring the fusions integrated into the cyanobacterial chromosome were prepared, and the growth-phase dependence of beta Gal level was determined. The specific activity of beta Gal in cultures of both types of fusion strains increased during the transition into stationary phase, indicating that the growth-phase-dependent regulation is on the gnd mRNA level. Characterization of the growth-phase-dependent induction of 6PGD in strains carrying differing amounts of DNA upstream from the gnd structural gene led to the localization of the promoter and the regulatory site on the restriction map of the gene, whose sequence has previously been determined.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutational Analysis of the Pentose Phosphate and Entner-Doudoroff Pathways in Gluconobacter oxydans Reveals Improved Growth of a {Delta}edd {Delta}eda Mutant on Mannitol

The obligatory aerobic acetic acid bacterium Gluconobacter oxydans 621H oxidizes sugars and sugar alcohols primarily in the periplasm, and only a small fraction is metabolized in the cytoplasm. The latter can occur either via the Entner-Doudoroff pathway (EDP) or via the pentose phosphate pathway (PPP). The Embden-Meyerhof pathway is nonfunctional, and a cyclic operation of the tricarboxylic acid cycle is prevented by the absence of succinate dehydrogenase. In this work, the cytoplasmic catabolism of fructose formed by oxidation of mannitol was analyzed with a Δgnd mutant lacking the oxidative PPP and a Δedd Δeda mutant devoid of the EDP. The growth characteristics of the two mutants under controlled conditions with mannitol as the carbon source and enzyme activities showed that the PPP is the main route for cytoplasmic fructose catabolism, whereas the EDP is dispensable and even unfavorable. The Δedd Δeda mutant (lacking 6-phosphogluconate dehydratase and 2-keto-3-deoxy-6-phosphogluconate aldolase) formed 24% more cell mass than the reference strain. In contrast, deletion of gnd (6-phosphogluconate dehydrogenase) severely inhibited growth and caused a strong selection pressure for secondary mutations inactivating glucose-6-phosphate dehydrogenase, thus preventing fructose catabolism via the EDP also. These Δgnd zwf* mutants (with a mutation in the zwf gene causing inactivation of the glucose-6-phosphate dehydrogenase) were almost totally disabled in fructose catabolism but still produced about 14% of the carbon dioxide of the reference strain, possibly by catabolizing substrates from the yeast extract. Overexpression of gnd in the reference strain improved biomass formation in a similar manner as deletion of edd and eda, further confirming the importance of the PPP for cytoplasmic fructose catabolism.     

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.     

Gluconate Regulation of Glucose Catabolism in Pseudomonas fluorescens

Induction of Entner-Doudoroff pathway enzymes in Pseudomonas fluorescens was investigated to study the role of gluconate as a possible inducer. Glucose oxidase-deficient mutants were isolated and characterized. One of these mutants, gox-7, was deficient in particulate glucose oxidase; another mutant, gox-17, was deficient in particulate glucose and gluconate oxidase activities. Gluconate, but not glucose, induced synthesis of gluconokinase and 6-phosphogluconate dehydratase in both mutants. High constitutive levels of 2-keto-3-deoxy-6-phosphogluconate aldolase were found when both mutants were grown on glucose. Growth of parent and both mutant strains on glycerol also resulted in high levels of Entner-Doudoroff pathway enzymes. It was concluded that glucose cannot serve as an inducer molecule for derepression of Entner-Doudoroff pathway enzymes in P. fluorescens. Evidence presented provides good support for gluconate being the true inducer of this pathway in P. fluorescens. A relationship is presented for explaining distribution of the Entner-Doudoroff pathway in certain groups of bacteria.     

The route of ethanol formation in Zymomonas mobilis

1. Enzymic evidence supporting the operation of the Entner-Doudoroff pathway in the anaerobic conversion of glucose into ethanol and carbon dioxide by Zymomonas mobilis is presented. 2. Cell extracts catalysed the formation of equimolar amounts of pyruvate and glyceraldehyde 3-phosphate from 6-phosphogluconate. Evidence that 3-deoxy-2-oxo-6-phosphogluconate is an intermediate in this conversion was obtained. 3. Cell extracts of the organism contained the following enzymes: glucose 6-phosphate dehydrogenase (active with NAD and NADP), ethanol dehydrogenase (active with NAD), glyceraldehyde 3-phosphate dehydrogenase (active with NAD), hexokinase, gluconokinase, glucose dehydrogenase and pyruvate decarboxylase. Extracts also catalysed the overall conversion of glycerate 3-phosphate into pyruvate in the presence of ADP. 4. Gluconate dehydrogenase, fructose 1,6-diphosphate aldolase and NAD-NADP transhydrogenase were not detected. 5. It is suggested that NAD is the physiological electron carrier in the balanced oxidation-reduction involved in ethanol formation.     

Fructose catabolism in Azospirillum brasilense and Azospirillum lipoferum.

The pathways for catabolism of fructose were investigated in the type strains of Azospirillum lipoferum and Azospirillum brasilense grown aerobically with (NH4)2SO4 as the nitrogen source. When grown on fructose, the former species possessed a complete Entner-Doudoroff pathway, whereas the latter species lacked activity for glucose-6-phosphate dehydrogenase. Both species possessed a complete catabolic Embden-Meyerhof-Parnas pathway. Neither species possessed the key enzyme of the hexose monophosphate pathway, 6-phosphogluconate dehydrogenase. Both species could phosphorylate fructose to fructose-1-phosphate by means of a phosphoenolpyruvate-phosphotransferase system, and high activities of 1-phosphofructokinase occurred. Both species possessed glucokinase activity, but only A. lipoferum had hexokinase activity; moreover, the cells of A. brasilense were nearly impermeable to glucose, accounting for the inability of this species to grow on glucose. Both species possessed pyruvate dehydrogenase, a complete tricarboxylic acid cycle, a glyoxylate shunt, and malic enzyme. Analysis of the acidic end products for both species indicated the formation of only small amounts of various organic acids, and most of the titratable acidity was due to utilization of the ammonium ions of the medium. Gluconic acid was not formed during growth of either species on fructose but was detected during growth of A. lipoferum on glucose; this species also possessed an NADP-linked glucose dehydrogenase and gluconokinase.     

A novel gnd mutation leading to increased L-lysine production in Corynebacterium glutamicum

Toward more efficient L-lysine production, we have been challenging genome-based strain breeding by the approach of assembling only relevant mutations in a single wild-type background. Following the creation of a new L-lysine producer Corynebacterium glutamicum AHP-3 that carried three useful mutations (lysC311, hom59, and pyc458) on the relevant downstream pathways, we shifted our target to the pentose phosphate pathway. Comparative genomic analysis for the pathway between a classically derived L-lysine producer and its parental wild-type identified several mutations. Among these mutations, a Ser-361-->Phe mutation in the 6-phosphogluconate dehydrogenase gene (gnd) was defined as a useful mutation for L-lysine production. Introduction of the gnd mutation into strain AHP-3 by allelic replacement led to approximately 15% increased L-lysine production. Enzymatic analysis revealed that the mutant enzyme was less sensitive than the wild-type enzyme to allosteric inhibition by intracellular metabolites, such as fructose 1,6-bisphosphate, D-glyceraldehyde 3-phosphate, phosphoribosyl pyrophosphate, ATP, and NADPH, which were known to inhibit this enzyme. Isotope-based metabolic flux analysis demonstrated that the gnd mutation resulted in 8% increased carbon flux through the pentose phosphate pathway during L-lysine production. These results indicate that the gnd mutation is responsible for diminished allosteric regulation and contributes to redirection of more carbon to the pentose phosphate pathway that was identified as the primary source for NADPH essential for L-lysine biosynthesis, thereby leading to improved product formation.     

Mutations affecting gluconate catabolism in Escherichia coli. Genetic mapping of the locus for the thermosensitive gluconokinase

An Escherichia coli strain unable to use gluconate was isolated by spontaneous curing of lambda cI857 s7 xis6 b515 b519, lambda cI857 s7 delta(A-att) dargI valS lysogens. Two lesions, linked to asd and pyrB markers, respectively, were necessary to produce this phenotype. The asd-linked mutation gnt-17, of regulatory type, seems to affect the expression of the major system of gluconate utilization (min 75) as well as that of 6-phosphogluconate dehydratase (gene edd, min 41), the first enzyme of the Entner-Doudoroff pathway. A closely linked suppressor of gnt-17 causes constitutivity of these activities; this suppressor resembles gntR, which is also in the asd region. Hence, it is possible that gnt-17 is a super-repressing allele of gntR, rather than a positive controlling element. Lesion gnt-17 alone does not prevent the utilization of gluconate; for this, the mutation gnt-18 at 96.9 min is also necessary. This mutation abolishes the thermosensitive gluconokinase activity and thus eliminates the subsidiary ability to catabolize gluconate. Accordingly, gnt-18 seems to be allelic with gntV, the locus postulated as being in the pyrB region specifying the thermosensitive gluconokinase.     

Genetics and physiology of proline utilization in Saccharomyces cerevisiae: mutation causing constitutive enzyme expression.

A mutation resulting in inducer-independent expression of the proline-degradative enzymes was isolated in the yeast Saccharomyces cerevisiae. Strains carrying the mutation, put3, are partially constitutive for proline oxidase and delta 1-pyrroline-5-carboxylate dehydrogenase when grown on a medium lacking proline and are hyperinducible for both enzyme activities when grown on a proline-containing medium. put3 segregates as a single nuclear gene, is not linked to either of the presumed structural genes for proline oxidase and delta 1-pyrroline-5-carboxylate dehydrogenase, and does not affect proline transport. When heterozygous in diploid strains, put3 behaves neither fully dominant nor fully recessive. Endogenous induction by proline has been eliminated as a cause of the inducer-independent enzyme expression in the put3 mutant and the mutation is believed to be in a regulatory component of the proline-degradative pathway.