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PubMed2004

Cloning of a gluconate/polyol dehydrogenase gene from Gluconobacter suboxydans IFO 12528, characterisation of the enzyme and its use for the production of 5-ketogluconate in a recombinant Escherichia coli strain.

A 5-ketogluconate (5-KGA)-forming membrane quinoprotein, gluconate dehydrogenase, was isolated from Gluconobacter suboxydans strain IFO 12528 and partially sequenced. Partial sequences of five internal tryptic peptides were elucidated by mass spectrometry and used to isolate the two adjacent genes encoding the enzyme (EBI accession no. AJ577472). These genes share close homology with sorbitol dehydrogenase from another strain of G. suboxydans (IFO 3255). Substrate specificity of gluconate 5-dehydrogenase (GA 5-DH) turned out to be quite broad, covering many polyols, amino derivatives of carbohydrates, and simple secondary alcohols. There is a broad correlation between the substrate specificity of GA 5-DH and the empirical Bertrand-Hudson rule that predicts the specificity of oxidation of polyols by acetic acid bacteria. Escherichia coli transformed with the genes encoding gluconate dehydrogenase were able to convert gluconic acid into 5-KGA at 75% yield. Furthermore, it was found that 5-KGA can be converted into tartaric acid semialdehyde by a transketolase. These results provide a basis for designing a direct fermentation-based process for conversion of glucose into tartaric acid.

Bacterial Proteins↗
PubMed2003

Cloning and expression of pyrroloquinoline quinone (PQQ) genes from a phosphate-solubilizing bacterium Enterobacter intermedium.

A grass rhizosphere bacterium, Enterobacter intermedium (60-2G), has a strong ability to solubilize insoluble phosphate. Certain phosphate-solubilizing bacteria secrete gluconic acid for this process. The gluconic acid is produced by direct extracellular oxidation of glucose by a glucose dehydrogenase equipped with pyrroloquinoline quinone (PQQ) as a cofactor. A pqq gene cluster producing PQQ was detected in E. intermedium and this sequence conferred phosphate-solubilizing activity to Escherichia coli DH5alpha. The 6,783-bp pqq sequence had six open reading frames (pqqA, B, C, D, E, and F) and showed 50-95% homology to pqq genes of other bacteria. E. coli DH5alpha expressing the E. intermedium pqq genes solubilized phosphate from hydroxyapatite after a pH drop to pH 4.0, which paralleled in time the secretion of gluconic acid. We speculate that production of PQQ in E. coli DH5alpha expressing the pqq cluster activates an endogenous glucose dehydrogenase to permit gluconic acid secretion that solubilizes the insoluble phosphate.

Cloning, Molecular↗
PubMed1992

Oral magnesium and the prevention of preterm labor in a high-risk group of patients.

OBJECTIVE: The null hypothesis of this study is that treatment with oral magnesium gluconate 1 gm four times daily will not decrease the rate of preterm labor and delivery in a high-risk group of pregnant women. STUDY DESIGN: Fifty-four women at risk for preterm delivery were selected randomly to receive magnesium gluconate 1 gm orally four times daily or placebo. These women were monitored prospectively for signs and symptoms of preterm labor. A subgroup of 31 women also received a home uterine activity monitor. The serum magnesium level was measured initially and again 2 weeks after study enrollment. The data were analyzed with Fisher's exact test and analysis of variance. RESULTS: Preterm labor developed in 15 women in the placebo group and in 16 women in the magnesium group. There were no differences in birth weight or gestational age at delivery. The mean increase in serum magnesium level while the patients were taking magnesium gluconate was 0.10 mg/dl (p = not significant). Five women did not have an increase in serum magnesium level and preterm labor developed in all of them. CONCLUSION: Magnesium gluconate in a dose of 1 gm four times daily is not effective for preventing preterm delivery in a high-risk group of patients.

Administration, Oral↗
PubMed2000

Bioavailability, biodistribution, and toxicity of BioZn-AAS(1): a new zinc source. comparative studies in rats.

Food fortification with a proper zinc compound is an economic and effective strategy to prevent zinc deficiency. BioZn-AAS, a zinc gluconate stabilized with glycine, was compared with zinc sulfate (reference standard), zinc hydroxide, and zinc gluconate, all of them labeled with (65)Zn. This preclinical study was performed on Sprague-Dawley rats of both sexes, and the administered dose was 85 microg/kg of zinc. Bioavailability studies showed that absorption of BioZn-AAS was not statistically different than absorption from other sources in female rats (25.65% +/- 2.20% for BioZn-AAS, 28.24% +/- 4. 60% for ZnSO(4), 24.91% +/- 4.02% for Zn[OH](2), and 25.51% +/- 2. 70% for Zn-gluconate). In the case of the male rats, absorption of BioZn-AAS (27.97% +/- 4.20%) was higher (P<0.05) than that from the other compounds (23.15% +/- 2.90% for ZnSO(4), 22.62% +/- 3.90% for Zn[OH](2), and 22.30% +/- 3.90% for Zn-gluconate). Biodistribution studies demonstrated that the zinc from BioZn-AAS followed the same metabolic pathway as zinc from the other sources. Toxicity studies were performed with 50 female and 50 male rats. The value of oral lethal dose 50 (LD(50)) was 2000 mg/kg for female rats and 1900 mg/kg for male rats. Therefore, we conclude that BioZn-AAS has adequate properties to be considered a proper zinc compound for food fortification or dietary supplementation.

Absorption↗
PubMed2003

Polyhydroxyalkanoate (PHA) biosynthesis in Thermus thermophilus: purification and biochemical properties of PHA synthase.

The biosynthesis of polyhydroxyalkanoates (PHAs) was studied, for the first time, in the thermophilic bacterium Thermus thermophilus. Using sodium gluconate (1.5% w/v) or sodium octanoate (10 mM) as sole carbon sources, PHAs were accumulated to approximately 35 or 40% of the cellular dry weight, respectively. Gas chromatographic analysis of PHA isolated from gluconate-grown cells showed that the polyester (Mw: 480,000 g mol(-1)) was mainly composed of 3-hydroxydecanoate (3HD) with a molar fraction of 64%. In addition, 3-hydroxyoctanoate (3HO), 3-hydroxyvalerate (3HV) and 3-hydroxybutyrate (3HB) occurred as constituents. In contrast, the polyester (Mw: 391,000 g mol(-1)) from octanoate-grown cells was composed of 24.5 mol% 3HB, 5.4 mol% 3HO, 12.3 mol% 3-hydroxynonanoate (3HN), 14.6 mol% 3HD, 35.4 mol% 3-hydroxyundecanoate (3HUD) and 7.8 mol% 3-hydroxydodecanoate (3HDD). Activities of PHA synthase, a beta-ketothiolase and an NADPH-dependent reductase were detected in the soluble cytosolic fraction obtained from gluconate-grown cells of T. thermophilus. The soluble PHA synthase was purified 4271-fold with 8.5% recovery from gluconate-grown cells, presenting a Km of 0.25 mM for 3HB-CoA. The optimal temperature of PHA synthase activity was about 70 degrees C and acts optimally at pH near 7.3. PHA synthase activity was inhibited 50% with 25 microM CoA and lost all of its activity when it was treated with alkaline phosphatase. T. thermophilus PHA synthase, in contrary to other reported PHA synthases did not exhibit a lag phase on its kinetics, when low concentration of the enzyme was used. Incubation of PHA synthase with 1 mM N-ethyl-maleimide inhibits the enzyme 56%, indicating that cysteine might be involved in the catalytic site of the enzyme. Acetyl phosphate (10 mM) activated both the native and the dephosphorylated enzyme. A major protein (55 kDa) was detected by SDS-PAGE. When a partially purified preparation was analyzed on native PAGE the major band exhibiting PHA synthase activity was eluted from the gel and analyzed further on SDS-PAGE, presenting the first purification of a PHA synthase from a thermophilic microorganism.

3-Hydroxybutyric Acid↗
PubMed1993

Solubility and intestinal transit time limit calcium absorption in rats.

Six-week-old male rats were placed on two high calcium regimens: one with calcium carbonate and monobasic calcium phosphate, with calcium content increased via calcium carbonate; and another with calcium phosphate and calcium gluconate, with calcium gluconate the source of increased calcium. Animals fed the gluconate-containing diets absorbed 29% of the ingested calcium over the entire calcium intake range, whereas those fed the calcium carbonate diets absorbed 25% over an intake range of 225 to 450 mg Ca/d, but at calcium intakes above 450 mg Ca/d their absorption reached a plateau at approximately 109 mg/d. Active calcium transport decreased with increased calcium intake in both the calcium carbonate- and calcium gluconate-fed groups. Nonsaturable transport was unchanged as a result of increasing calcium intake and did not differ among the diet groups. Because the absorptive processes were unaffected by the calcium source, events in the lumen must have been responsible for the observed differences. Because phosphate is nearly 18 times more soluble than carbonate, very little calcium of calcium carbonate origin can have been solubilized in the presence of phosphate and this, we conclude, accounts for the limit on calcium absorption observed in diets high in calcium carbonate. Moreover, when intake is expressed as soluble calcium, absorption approaches 50%, the value expected when intestinal transit time (approximately 3 h) is multiplied by 16%/h, the experimental value of nonsaturable absorption.

Animals↗
PubMed1988

Fitness effects of a deletion mutation increasing transcription of the 6-phosphogluconate dehydrogenase gene in Escherichia coli.

Directed evolution in microbial organisms provides an experimental approach to molecular evolution in which selective forces can be controlled and favorable mutations analyzed at the molecular level. Here we present an analysis of a mutation selected in Escherichia coli in response to growth in a chemostat in which the limiting nutrient was gluconate. The selectively favored mutation, designated gnd+ (862), occurred in the gene gnd coding for 6-phosphogluconate dehydrogenase, used in gluconate metabolism. Although the allele is strongly favored in chemostats in which the limiting nutrient is gluconate, the selective effects of gnd+ (862) are highly dependent on growth conditions. In chemostats in which growth is limited by a mixture of gluconate and either ribose, glucose, or succinate, the gnd+ (862) allele is favored, disfavored, or neutral according to the relative concentrations of the substrates. The gnd+ (862) allele results from a deletion of 385 nucleotide pairs in the region 5' to the promoter of gnd, and one endpoint of the deletion is contiguous with the terminus of an IS5 insertion sequence located near gnd in E. coli K12. The gnd+ (862) allele shows a marked increase in transcription that accounts for most or all of the increased enzyme activity.

Alleles↗
PubMed1991

Effect of intravenous calcium infusions on serum chemistries in neonates.

In 36 newborn infants admitted to the Children's Hospital of Oklahoma, we studied the hypotheses that intravenous bolus infusions of Ca as calcium gluconate over 10 min may (a) result in acute metabolic acidosis, (b) increase serum osmolality, (c) increase serum free bilirubin, and (d) decrease serum phosphorus concentrations. All infants received 18 mg/kg of elemental calcium, as either a 5 or 10% solution of calcium gluconate. Blood ionized calcium (iCa) rose significantly with i.v. bolus Ca infusion (p less than 0.005) and blood pH and serum P declined significantly (p less than 0.05) with i.v. bolus Ca infusion in infants. Serum free bilirubin was not significantly altered. Serum osmolality rose significantly from baseline with bolus infusion of Ca as a 10% calcium gluconate solution and did not change significantly with bolus infusion of a diluted 5% solution. In neonates, intravenous bolus calcium infusion (a) decreases blood pH, (b) infusion of 10% but not 5% calcium gluconate increases serum osmolality, (c) serum free bilirubin concentrations were not altered, and (d) serum phosphorus concentrations were decreased.

Acid-Base Equilibrium↗
PubMed1983

Treatment of severe hydrofluoric acid exposures.

Previous publications on treatment of hydrofluoric acid exposures have given insufficient emphasis to immediate skin cleansing and prompt restitution of both serum and tissue calcium ion levels. Use of calcium gluconate topically as a 2.5% gel, in eyes as a 1% solution, and by nebulizer as a 2.5% or 3% solution has improved treatment results. Ten percent calcium gluconate solution may be given intravenously as indicated for hypocalcemia. Proper application of the calcium gluconate gel immediately after thorough washing of localized skin burns will produce relief of pain similar to that achieved by subcutaneous infiltration of 5% calcium gluconate solution. This may be used as an end-point for adequacy of treatment.

Accidents, Occupational↗
PubMed1996

Topical treatments for hydrofluoric acid dermal burns. Further assessment of efficacy using an experimental piq model.

Several topical treatments for hydrofluoric acid dermal burns (Zephiran, calcium acetate and magnesium hydroxide antacid soaks, and calcium gluconate gel) were assessed for efficacy in a pig model. Gross appearance and histopathology of treated and untreated burn sites were evaluated. For superficial burns, Zephiran was most effective; calcium acetate, magnesium hydroxide antacid, and calcium gluconate gel were less effective. For deep burns, gross observations showed that calcium acetate and Zephiran were most efficacious, whereas histopathology indicated comparable efficacy of Zephiran, calcium acetate, and calcium gluconate gel for all skin layers. Magnesium hydroxide antacid demonstrated efficacy only for the subdermis. The clinically beneficial effects of both Zephiran and calcium gluconate gel were affirmed. Although results suggest that calcium acetate and magnesium-containing antacids may be beneficial for human hydrofluoric acid dermal burns, these are not established clinical treatments.

Acetates↗
PubMed2003

Impairment of transendothelial leukocyte migration by iron complexes.

Although iron sucrose and iron gluconate are generally well tolerated in patients who are treated for renal anemia, recent clinical studies and cell culture experiments suggested significant toxicity and long-term side effects arising from the use of these iron complexes. Because of the possible role of iron in infection or cardiovascular disease, it was theorized that parenteral iron compounds influence endothelial and PMN interaction in vitro. A well-established double-chamber method was used to assess the effect of different concentrations of iron sucrose and iron gluconate (1, 25, 50, and 100 micro g/ml) on the transendothelial migration of PMN. Preincubation of PMN and endothelial cells as well as preincubation of PMN alone with 25, 50, or 100 micro g/ml iron resulted in a significant decrease in PMN migration. In contrast, after incubation of the endothelial cells alone with iron, no reduction in the transendothelial migration of PMN was observed. Preincubation of PMN and/or endothelial cells with 1 micro g/ml iron did not lead to any decrease in the rate of migrated PMN. The only significant change in experiments with 1 micro g/ml was an increase in PMN migration after preincubation of endothelial cells and PMN with iron gluconate. A four-way ANOVA showed a significant effect of the iron concentration (P < 0.000001), of type of iron complex (P < 0.005), of the preincubation of endothelial cell (P < 0.001), and of the preincubation of PMN with iron (P < 0.000001) on PMN diapedesis. It is concluded that iron sucrose and iron gluconate cause a significant inhibition of transendothelial migration of PMN.

Cell Movement↗
PubMed1993

Nicotinic agonists antagonize quantal size increases and evoked release at frog neuromuscular junction.

1. Previous studies at the frog neuromuscular junction showed that quantal size can be increased two- to fourfold by a variety of treatments, including prior exposure to hypertonic solution (which activates protein kinase A) and insulin (which acts via an unknown pathway). Size increases largely because quanta contain more acetylcholine (ACh). 2. Now the effects of cholinergic agonists on the increases in quantal size have been studied. One muscle from a frog was kept for 2 h in hypertonic sodium gluconate solution. The miniature endplate potential (MEPP) sizes were measured in saline: they had increased about fourfold. The paired muscle went through the same experimental sequence, except that an agonist was added to the hypertonic gluconate solution. Again MEPP sizes were measured in saline. The increase in quantal size was significantly depressed by 0.2 microM 1,1-dimethyl-4-phenyl- piperazinium (DMPP). The sequence of effectiveness of agonists was: DMPP > carbachol > or = ACh = cytisine > oxytremorine. This sequence suggests that the receptor belongs in the nicotinic class. 3. Quantal size is doubled after 1 h in insulin. One micromolar carbachol largely blocked the size increase. 4. The effects of cholinergic antagonists were tested by keeping the experimental muscle in hypertonic gluconate solution containing 1 microM carbachol plus an antagonist. The controls were paired muscles kept in hypertonic gluconate solution (without carbachol or antagonist). MEPP sizes were measured in saline. The depressing action of carbachol on the increase in MEPP size was blocked by 0.2 microM neuronal-bungarotoxin (nBTX). The sequence of effectiveness of antagonists was: nBTX > trimethaphan > d-tubocurarine (dTC). Ten micromolar atropine (without carbachol) depressed the increase in quantal size. Therefore, the antagonist potency of atropine could not be adequately tested. Carbachol action was not blocked by 10 microM hexamethonium or 10 microM mecamylamine. 5. Once quanta are made large they can be converted back to normal size by cholinergic agonists. Muscles in which quantal size had been enlarged were exposed to hypertonic solutions containing the agonist. Quantal size was reduced to a fraction of its former value when the hypertonic solution contained 1 microM carbachol- or 1 microM DMPP. One micromolar oxytremorine had no effect. Carbachol still reduced quantal size when applied in low-Ca2+ solutions, so it does not appear to act by elevating intracellular [Ca2+]. 6. Previous work suggested that the treatments produce a subpopulation of large quanta that are positioned for release.(ABSTRACT TRUNCATED AT 400 WORDS)

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed2002

Engineering of a Bacillus subtilis strain with adjustable levels of intracellular biotin for secretory production of functional streptavidin.

Streptavidin is a biotin-binding protein which has been widely used in many in vitro and in vivo applications. Because of the ease of protein recovery and availability of protease-deficient strains, the Bacillus subtilis expression-secretion system is an attractive system for streptavidin production. However, attempts to produce streptavidin using B. subtilis face the problem that cells overproducing large amounts of streptavidin suffer poor growth, presumably because of biotin deficiency. This problem cannot be solved by supplementing biotin to the culture medium, as this will saturate the biotin binding sites in streptavidin. We addressed this dilemma by engineering a B. subtilis strain (WB800BIO) which overproduces intracellular biotin. The strategy involves replacing the natural regulatory region of the B. subtilis chromosomal biotin biosynthetic operon (bioWAFDBIorf2) with an engineered one consisting of the B. subtilis groE promoter and gluconate operator. Biotin production in WB800BIO is induced by gluconate, and the level of biotin produced can be adjusted by varying the gluconate dosage. A level of gluconate was selected to allow enhanced intracellular production of biotin without getting it released into the culture medium. WB800BIO, when used as a host for streptavidin production, grows healthily in a biotin-limited medium and produces large amounts (35 to 50 mg/liter) of streptavidin, with over 80% of its biotin binding sites available for future applications.

Bacillus subtilis↗
PubMed2002

Intracellular carbon fluxes in riboflavin-producing Bacillus subtilis during growth on two-carbon substrate mixtures.

Metabolic responses to cofeeding of different carbon substrates in carbon-limited chemostat cultures were investigated with riboflavin-producing Bacillus subtilis. Relative to the carbon content (or energy content) of the substrates, the biomass yield was lower in all cofeeding experiments than with glucose alone. The riboflavin yield, in contrast, was significantly increased in the acetoin- and gluconate-cofed cultures. In these two scenarios, unusually high intracellular ATP-to-ADP ratios correlated with improved riboflavin yields. Nuclear magnetic resonance spectra recorded with amino acids obtained from biosynthetically directed fractional (13)C labeling experiments were used in an isotope isomer balancing framework to estimate intracellular carbon fluxes. The glycolysis-to-pentose phosphate (PP) pathway split ratio was almost invariant at about 80% in all experiments, a result that was particularly surprising for the cosubstrate gluconate, which feeds directly into the PP pathway. The in vivo activities of the tricarboxylic acid cycle, in contrast, varied more than twofold. The malic enzyme was active with acetate, gluconate, or acetoin cofeeding but not with citrate cofeeding or with glucose alone. The in vivo activity of the gluconeogenic phosphoenolpyruvate carboxykinase was found to be relatively high in all experiments, with the sole exception of the gluconate-cofed culture.

Bacillus subtilis↗
PubMed1973

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.

Alanine↗
PubMed1993

Genetic definition of the substrate selectivity of outer membrane porin protein OprD of Pseudomonas aeruginosa.

Earlier studies proved that Pseudomonas aeruginosa OprD is a specific porin for basic amino acids and imipenem. It was also considered to function as a nonspecific porin that allowed the size-dependent uptake of monosaccharides and facilitation of the uptake of quinolone and other antibiotics. In the present study, we utilized P. aeruginosa strains with genetically defined levels of OprD to characterize the in vivo substrate selectivity of this porin. An oprD::omega interposon mutant was constructed by gene replacement utilizing an in vitro mutagenized cloned oprD gene. In addition, OprD was overexpressed from the lac promoter by cloning the oprD gene into the broad-host-range plasmid pUCP19. To test the substrate selectivity, strains were grown in minimal medium with limiting concentrations of the carbon sources glucose, gluconate, or pyruvate. In minimal medium with 0.5 mM gluconate, the growth rates of the parent strain H103 and its oprD::omega mutant H729 were only 60 and 20%, respectively, of that of the OprD-overexpressing strain H103(pXH2). In contrast, no significant differences were observed in the growth rates of these three strains on glucose or pyruvate, indicating that OprD selectively facilitated the transport of gluconate. To determine the role of OprD in antibiotic uptake, nine strains representing different levels of OprD and OprF were used to determine the MICs of different antibiotics. The results clearly demonstrated that OprD could be utilized by imipenem and meropenem but that, even when substantially overexpressed, it could not be significantly utilized by other beta-lactams, quinolones, or aminoglycosides. In addition, competition experiments confirmed that imipenem had common binding sites with basic amino acids in the OprD channel, but not with gluconate or glucose.

Amino Acids, Diamino↗
PubMed1994

Loss of protein kinase-catalyzed phosphorylation of HPr, a phosphocarrier protein of the phosphotransferase system, by mutation of the ptsH gene confers catabolite repression resistance to several catabolic genes of Bacillus subtilis.

In gram-positive bacteria, HPr, a phosphocarrier protein of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), is phosphorylated by an ATP-dependent, metabolite-activated protein kinase on seryl residue 46. In a Bacillus subtilis mutant strain in which Ser-46 of HPr was replaced with a nonphosphorylatable alanyl residue (ptsH1 mutation), synthesis of gluconate kinase, glucitol dehydrogenase, mannitol-1-P dehydrogenase and the mannitol-specific PTS permease was completely relieved from repression by glucose, fructose, or mannitol, whereas synthesis of inositol dehydrogenase was partially relieved from catabolite repression and synthesis of alpha-glucosidase and glycerol kinase was still subject to catabolite repression. When the S46A mutation in HPr was reverted to give S46 wild-type HPr, expression of gluconate kinase and glucitol dehydrogenase regained full sensitivity to repression by PTS sugars. These results suggest that phosphorylation of HPr at Ser-46 is directly or indirectly involved in catabolite repression. A strain deleted for the ptsGHI genes was transformed with plasmids expressing either the wild-type ptsH gene or various S46 mutant ptsH genes (S46A or S46D). Expression of the gene encoding S46D HPr, having a structure similar to that of P-ser-HPr according to nuclear magnetic resonance data, caused significant reduction of gluconate kinase activity, whereas expression of the genes encoding wild-type or S46A HPr had no effect on this enzyme activity. When the promoterless lacZ gene was put under the control of the gnt promoter and was subsequently incorporated into the amyE gene on the B. subtilis chromosome, expression of beta-galactosidase was inducible by gluconate and repressed by glucose. However, we observed no repression of beta-galactosidase activity in a strain carrying the ptsH1 mutation. Additionally, we investigated a ccpA mutant strain and observed that all of the enzymes which we found to be relieved from carbon catabolite repression in the ptsH1 mutant strain were also insensitive to catabolite repression in the ccpA mutant. Enzymes that were repressed in the ptsH1 mutant were also repressed in the ccpA mutant.

Bacillus subtilis↗
PubMed2000

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.

Chromogenic Compounds↗