A new circadian class 2 gene, opcA, whose product is important for reductant production at night in Synechococcus elongatus PCC 7942

Gene expression in the cyanobacterium Synechococcus elongatus PCC 7942 is under the control of a circadian oscillator, such that peaks and troughs of expression recur with a periodicity of about 24 h in the absence of environmental cues. This can be monitored easily as light production from luciferase gene fusions to S. elongatus promoters. All promoters seem to exhibit circadian oscillation of expression, but the phasing of peak and trough times differs among different genes. The majority of genes are designated class 1, with expression peaks near dusk or subjective dusk (the time corresponding to dusk in the absence of a diurnal cycle). A minority, of which purF is an example, have expression peaks approximately 12 h out of phase with class 1 genes. A screen of Tn5 mutants for those in which purF phasing is altered revealed a mutant that carries an insertion in the opcA gene, previously identified as essential for glucose-6-phosphate dehydrogenase function. However, a different enzymatic reporter and in vitro luciferase assays revealed that the expression pattern of the purF promoter is not altered by opcA inactivation, but rather the reduced flavin mononucleotide substrate of luciferase is limiting at the time of the natural circadian peak. The results suggest that OpcA is involved in temporally separated reductant-generating pathways in S. elongatus and that it has a role outside of its function in activating glucose-6-phosphate dehydrogenase. The opcA gene, expected to be cotranscribed with fbp and zwf, was shown to have its own class 2 promoter, whereas the fbp promoter was determined to be in class 1. Thus, opcA expression is likely to be constitutive by virtue of the activity of two promoters in nearly opposite circadian phases.     

Adaptations of Pseudomonas aeruginosa to the cystic fibrosis lung environment can include deregulation of zwf, encoding glucose-6-phosphate dehydrogenase

Cystic fibrosis (CF) patients are highly susceptible to chronic pulmonary disease caused by mucoid Pseudomonas aeruginosa strains that overproduce the exopolysaccharide alginate. We showed here that a mutation in zwf, encoding glucose-6-phosphate dehydrogenase (G6PDH), leads to a approximately 90% reduction in alginate production in the mucoid, CF isolate, P. aeruginosa FRD1. The main regulator of alginate, sigma-22 encoded by algT (algU), plays a small but demonstrable role in the induction of zwf expression in P. aeruginosa. However, G6PDH activity and zwf expression were higher in FRD1 strains than in PAO1 strains. In PAO1, zwf expression and G6PDH activity are known to be subject to catabolite repression by succinate. In contrast, FRD1 zwf expression and G6PDH activity were shown to be refractory to such catabolite repression. This was apparently not due to a defect in the catabolite repression control (Crc) protein. Such relaxed control of zwf was found to be common among several examined CF isolates but was not seen in other strains of clinical and environmental origin. Two sets of clonal isolates from individual CF patient indicated that the resident P. aeruginosa strain underwent an adaptive change that deregulated zwf expression. We hypothesized that high-level, unregulated G6PDH activity provided a survival advantage to P. aeruginosa within the lung environment. Interestingly, zwf expression in P. aeruginosa was shown to be required for its resistance to human sputum. This study illustrates that adaptation to the CF pulmonary environment by P. aeruginosa can include altered regulation of basic metabolic activities, including carbon catabolism.     

Resistance of Deinococcus radiodurans to Mutagenesis Is Facilitated by Pentose Phosphate Pathway in the mutS1 Mutant Background

MutS1 is a key protein involved in mismatch repair system for ensuring fidelity of replication and recombination in Deinococcus radiodurans. The zwf gene encodes glucose-6-phosphate dehydrogenase (G6PD) in the pentose phosphate (PP) pathway, which provides adequate metabolites as precursors of DNA repair. In this study, mutS1 and zwf were disrupted by homologous recombination. The zwf mutant (Deltazwf) and the zwf/mutS1 double mutant (Deltazwf/mutS1) were sensitive to ultraviolet (UV) light, H(2)O(2), and DNA cross-linking agent mitomycin C (MMC), whereas the mutS1 mutant (DeltamutS1) showed resistance to UV light, H(2)O(2) and MMC as the wild-type strain. Inactivation of mutS1 resulted in a 3.3-fold increase in frequency of spontaneous rifampicin-resistant mutagenesis and a 4.9-fold increment in integration efficiency of a donor point-mutation marker during bacterial transformation. Although inactivation of zwf had no obvious effect compared with the wild-type strain, dual disruption of zwf and mutS1 resulted in a 4.7-fold increase in mutation frequency and a 7.4-fold increase in integration efficiency. These results suggest that inactivation of the PP pathway decreases the resistance of D. radiodurans cells to DNA damaging agents and increases mutation frequency and integration efficiency in the mutS1 mutant background.     

The opcA and ΨopcB regions in Neisseria: genes, pseudogenes, deletions, insertion elements and DNA islands

Previous data have indicated that the opc gene encoding an immunogenic invasin is specific to Neisseria meningitidis (Nm) and is lacking in Neisseria gonorrhoeae (Ng). The data presented here show that Nm and Ng both contain two paralogous opc-like genes, opcA, corresponding to the former opc gene, and (psi)opcB, a pseudogene. The predicted OpcA and OpcB proteins possess transmembrane regions with conserved non-polar faces but differ extensively in four of the five surface-exposed loops. Gonococcal OpcA was expressed weakly under in vitro conditions, and it is unknown whether these bacteria can express this protein at high levels. Analysis of the sequences flanking opcA and (psi)opcB revealed a framework of conserved housekeeping genes interspersed with DNA islands. These regions also contained several pseudogenes, deletions and IS elements, attesting to considerable genome plasticity. Both opcA and (psi)opcB are located on DNA islands that have probably been imported from unrelated bacteria. A third island encodes the dcmD/dcrD R/M genes in Ng versus a small open reading frame in most strains of Nm. Rare strains of Nm were identified in which the R/M island has been imported. DNA islands in Nm and Ng seem to have been acquired by recombination via conserved flanking housekeeping genes rather than by insertion of mobile genetic elements.     

A study of the role of the hexose monophosphate pathway with respect to fatty acid biosynthesis in sporulation of Saccharomyces cerevisiae

13C NMR was used to study the pattern of label incorporation from [2-13C]acetate into trehalose during sporulation in Saccharomyces cerevisiae. A wild-type strain and a strain homozygous for the zwf1 mutation (which affects glucose-6-phosphate dehydrogenase) were used. In the wild-type it was possible to deduce the cycling of glucose 6-phosphate around the hexose monophosphate pathway whilst in the mutant strain this did not occur. The requirement of the hexose monophosphate pathway for providing NADPH for fatty acid biosynthesis was examined using 13C NMR and GC/MS. The wild-type strain produced a typical profile of fatty acids with palmitoleic acid being the most abundant whereas the mutant contained only one-quarter the amount of total fatty acid. As zwf1 homozygous diploids are able to sporulate this indicates that the large amount of fatty acid biosynthesis observed in sporulation of wild-type strains is not essential to the process.     

Functional expression of human glucose-6-phosphate dehydrogenase in Escherichia coli

The complete coding sequence for human glucose-6-phosphate-dehydrogenase (G6PD) was inserted downstream from the tac promoter of a plasmid, pJF118EH, which also carries the lacIq repressor gene. When Escherichia coli strains (that are unable to grow on glucose due to the absence of functional zwf (G6PD-) and pgi genes) were transformed with this plasmid (pAC1), they were able to grow on glucose as sole carbon source. The rate of growth on glucose was faster in the presence of the inducer of the tac promoter, isopropyl-beta-D-thiogalactopyranoside (IPTG). Extracts of the transformed cells contained a G6PD activity that was not detectable in the parental strains and that was inducible by IPTG. The G6PD activities from normal E. coli and from pAC1-transformed cells comigrated with human G6PD when subjected to electrophoresis on agarose gels. However, when denatured, the G6PD produced by pAC1 was, like the human enzyme, distinguishable from the E. coli-encoded enzyme on the basis of its immunoreactivity with antibody specific for human G6PD. Therefore, human G6PD can be expressed in E. coli and can function to complement the bacterial enzyme deficiency.     

Genetic regulation of glucose 6-phosphate dehydrogenase activity in the inbred mouse

The erythrocyte glucose 6-phosphate dehydrogenase activity characteristic of each of 16 inbred mouse strains falls into one of three distinct classes. Strains C57L/J and C57BR/cdJ represent the low activity class: strains A/J and A/HeJ represent the high activity class; other strains have intermediate activities. There is no evidence that structural variation is responsible for the variation in G6PD activity, since partially purified enzyme from each class has the same thermal stability, pH-activity profile, Michaelis constants for G6P and NADP, electrophoretic mobility, and activity using 2-deoxy d-glucose 6-phosphate as substrate. The activities of 6-phosphogluconate dehydrogenase and glucose phosphate isomerase do not differ in erythrocytes of the three G6PD activity classes. Young red cells have higher G6PD activities than old red cells and there is evidence that the intracellular stability of the enzyme is reduced in red cells of strain C57L/J. G6PD activities in kidney and skeletal and cardiac muscle from animals with low red cell G6PD are slightly lower than the activities in kidney and muscle from animals with high red cell G6PD activity. The quantitative differences in red cell G6PD activity are not regulated by X-linked genes, but by alleles at two or more autosomal loci. A simple genetic model is proposed in which alleles at two unlinked, autosomal loci, called Gdr-1 and Gdr-2 regulate G6PD activity in the mouse erythrocyte.     

NADPH production by the pentose phosphate pathway in the zona fasciculata of rat adrenal gland.

Biosynthesis of steroid hormones in the cortex of the adrenal gland takes place in smooth endoplasmic reticulum and mitochondria and requires NADPH. Four enzymes produce NADPH: glucose-6-phosphate dehydrogenase (G6PD), the key regulatory enzyme of the pentose phosphate pathway, phosphogluconate dehydrogenase (PGD), the third enzyme of that pathway, malate dehydrogenase (MDH), and isocitrate dehydrogenase (ICDH). However, the contribution of each enzyme to NADPH production in the cortex of adrenal gland has not been established. Therefore, activity of G6PD, PGD, MDH, and ICDH was localized and quantified in rat adrenocortical tissue using metabolic mapping, image analysis, and electron microscopy. The four enzymes have similar localization patterns in adrenal gland with highest activities in the zona fasciculata of the cortex. G6PD activity was strongest, PGD, MDH, and ICDH activity was approximately 60%, 15%, and 7% of G6PD activity, respectively. The K(m) value of G6PD for glucose-6-phosphate was two times higher than the K(m) value of PGD for phosphogluconate. As a consequence, virtual flux rates through G6PD and PGD are largely similar. It is concluded that G6PD and PGD provide the major part of NADPH in adrenocortical cells. Their activity is localized in the cytoplasm associated with free ribosomes and membranes of the smooth endoplasmic reticulum, indicating that NADPH-demanding processes related to biosynthesis of steroid hormones take place at these sites. Complete inhibition of G6PD by androsterones suggests that there is feedback regulation of steroid hormone biosynthesis via G6PD.     

A comparison of gene organization in the zwf region of the genomes of the cyanobacteria Synechococcus sp. PCC 7942 and Anabaena sp. PCC 7120

Abstract The region of the genome encoding the glucose-6-phosphate dehydrogenase gene zwf was analysed in a unicellular cyanobacterium, Synechococcus sp. PCC 7942, and a filamentous, heterocystous cyanobacterium, Anabaena sp. PCC 7120. Comparison of cyanobacterial zwf sequences revealed the presence of two absolutely conserved cysteine residues which may be implicated in the light/dark control of enzyme activity. The presence in both strains of a gene fbp , encoding fructose-1,6-bisphosphatase, upstream from zwf strongly suggests that the oxidative pentose phosphate pathway in these organisms may function to completely oxidize glucose 6-phosphate to CO₂. The amino acid sequence of fructose-1,6-bisphosphatase does not support the idea of its light activation by a thiol/disulfide exchange mechanism. In the case of Anabaena sp. PCC 7120, the tal gene, encoding transaldolase, lies between zwf and fbp .     

Glucose-6-phosphate dehydrogenase modulates vascular endothelial growth factor-mediated angiogenesis

Glucose-6-phosphate dehydrogenase (G6PD), the first enzyme of the pentose phosphate pathway, is the principal intracellular source of NADPH. NADPH is utilized as a cofactor by vascular endothelial cell nitric-oxide synthase (eNOS) to generate nitric oxide (NO*). To determine whether G6PD modulates NO*-mediated angiogenesis, we decreased G6PD expression in bovine aortic endothelial cells using an antisense oligodeoxynucleotide to G6PD or increased G6PD expression by adenoviral gene transfer, and we examined vascular endothelial growth factor (VEGF)-stimulated endothelial cell proliferation, migration, and capillary-like tube formation. Deficient G6PD activity was associated with a significant decrease in endothelial cell proliferation, migration, and tube formation, whereas increased G6PD activity promoted these processes. VEGF-stimulated eNOS activity and NO* production were decreased significantly in endothelial cells with deficient G6PD activity and enhanced in G6PD-overexpressing cells. In addition, G6PD-deficient cells demonstrated decreased tyrosine phosphorylation of the VEGF receptor Flk-1/KDR, Akt, and eNOS compared with cells with normal G6PD activity, whereas overexpression of G6PD enhanced phosphorylation of Flk-1/KDR, Akt, and eNOS. In the Pretsch mouse, a murine model of G6PD deficiency, vessel outgrowth from thoracic aorta segments was impaired compared with C3H wild-type mice. In an in vivo Matrigel angiogenesis assay, cell migration into the plugs was inhibited significantly in G6PD-deficient mice compared with wild-type mice, and gene transfer of G6PD restored the wild-type phenotype in G6PD-deficient mice. These findings demonstrate that G6PD modulates angiogenesis and may represent a novel angiogenic regulator.     

Efficient production of 2-deoxy-scyllo-inosose from d-glucose by metabolically engineered recombinant Escherichia coli

2-Deoxy-scyllo-inosose (DOI) is a six-membered carbocycle formed from d-glucose-6-phosphate catalyzed by 2-deoxy-scyllo-inosose synthase (DOIS), a key enzyme in the biosynthesis of 2-deoxystreptamine-containing aminocyclitol antibiotics. DOI is valuable as a starting material for the benzene-free synthesis of catechol and other benzenoids. We constructed a series of metabolically engineered Escherichia coli strains by introducing a DOIS gene (btrC) from Bacillus circulans and disrupting genes for phosphoglucose isomerase, d-glucose-6-phosphate dehydrogenase, and phosphoglucomutase (pgi, zwf and pgm, respectively). It was found that deletion of the pgi gene, pgi and zwf genes, pgi and pgm genes, or all pgi, zwf and pgm genes significantly improved DOI production by recombinant E. coli in 2YTG medium (3% glucose) up to 7.4, 6.1, 11.6, and 8.4 g l(-1), respectively, compared with that achieved by wild-type recombinant E. coli (1.5 g l(-1)). Moreover, E. coli mutants with disrupted pgi, zwf and pgm genes showed strongly enhanced DOI productivity of up to 29.5 g l(-1) (99% yield) in the presence of mannitol as a supplemental carbon source. These results demonstrated that DOI production by metabolically engineered recombinant E. coli may provide a novel, efficient approach to the production of benzenoids from renewable d-glucose.     

Glucose-6-phosphate dehydrogenase and the oxidative pentose phosphate cycle protect cells against apoptosis induced by low doses of ionizing radiation

The initial and rate-limiting enzyme of the oxidative pentose phosphate shunt, glucose-6-phosphate dehydrogenase (G6PD), is inhibited by NADPH and stimulated by NADP(+). Hence, under normal growth conditions, where NADPH levels exceed NADP(+) levels by as much as 100-fold, the activity of the pentose phosphate cycle is extremely low. However, during oxidant stress, pentose phosphate cycle activity can increase by as much as 200-fold over basal levels, to maintain the cytosolic reducing environment. G6PD-deficient (G6PD(-)) cell lines are sensitive to toxicity induced by chemical oxidants and ionizing radiation. Compared to wild-type CHO cells, enhanced sensitivity to ionizing radiation was observed for G6PD(-) cells exposed to single-dose or fractionated radiation. Fitting the single-dose radiation response data to the linear-quadratic model of radiation-induced cytotoxicity, we found that the G6PD(-) cells exhibited a significant enhancement in the alpha component of radiation-induced cell killing, while the values obtained for the beta component were similar in both the G6PD(-) and wild-type CHO cell lines. Here we report that the enhanced alpha component of radiation-induced cell killing is associated with a significant increase in the incidence of ionizing radiation-induced apoptosis in the G6PD(-) cells. These data suggest that G6PD and the oxidative pentose phosphate shunt protect cells from ionizing radiation-induced cell killing by limiting the incidence of radiation-induced apoptosis. The sensitivity to radiation-induced apoptosis was lost when the cDNA for wild-type G6PD was transfected into the G6PD(-) cell lines. Depleting GSH with l-BSO enhanced apoptosis of K1 cells while having no effect in the G6PD(-) cell line     

Use of cellular glucose-6-phosphate dehydrogenase for cell quantitation: applications in cytotoxicity and apoptosis assays

A fluorescence-based microplate assay was developed to quantify cell death based upon the measurement of glucose-6-phosphate dehydrogenase (G6PD) activity. G6PD is a cytosolic enzyme and leaks from cells when plasma membrane integrity is compromised. In this assay, cell death is measured by correlating the activity of extracellular G6PD to the reduction of resazurin to the fluorescent product, resorufin, via a coupled-enzyme reaction. The coupled-enzyme reaction permits rapid signal amplification from small amounts of G6PD, an advantage over assays based on resazurin alone. This assay is rapid, nontoxic, and amenable to high-throughput screening. The assay has a Z' factor of 0.78.