The role of polyglucose in oxygen-dependent respiration by a new strain of Desulfovibrio salexigens

Abstract: Desulfovibrio salexigens strain Mastl was isolated from the oxic/anoxic interface of a marine sediment. Growth under sulfate-reducing conditions was accompanied by polyglucose accumulation in the cell with every substrate tested. Highest polyglucose storage was found with glucose (0.8–1.0 g polyglucose (g protein)⁻¹), but the growth rate with this substrate was very low (0.015 h⁻¹). Anaerobically grown cells of strain Mastl exhibited immediate oxygen-dependent respiration. The endogenous oxygen reduction rate was proportional to the polyglucose content. The rate of aerobic respiration of pyruvate was also directly related to the polyglucose content indicating that this organism was only able to respire with oxygen as long as polyglucose was present. Maximum oxygen reduction rates were found at air saturating concentrations and were relatively low (3–50 nmol O₂ min⁻¹ (mg protein)⁻¹). Catalase was constitutively present in anaerobically grown cells. When batch cultures were exposed to oxygen, growth ceased immediately and polyglucose was oxidized to acetate within 40–50 h. Like the oxygen reduction activity, the nitro blue tetrazolium (NBT)-reduction activity in these cells was proportional to the polyglucose content. Under anaerobic starvation conditions there was no correlation between the NBT-reduction activity and polyglucose concentration and polyglucose was degraded slowly within 240 h. The ecological significance of aerobic polyglucose consumption is discussed.     

Synthesis,storage and degradation of polyglucose in chlorobium thiosulfatophilum

Cultures of Chlorobium thiosulfatophilum form polyglucose during growth. The polyglucose is laid down within the cells as rosette-like granules, which are made up from smaller grains. The size of each granule appears to be limited to less than 30 nm, since an increase in polyglucose content leads to more granules being formed rather than an increase in granule size.The polyglucose in washed cells is fermented in the dark to acetate, propionate, caproate and succinate, of which acetate by far comprises the largest fraction (68%). During incubation of washed cells without hydrogen donor, the level of polyglucose decreases regardless of whether the cells are incubated in the dark or in the light. Since the products formed from polyglucose under the two different conditions are not the same, it is suggested that polyglucose in the dark serves as an energy source, whereas when in the light the role of polyglucose is mainly to provide the cell with reducing power.     

Polyglucose particles histochemically synthesized by glycogen synthetase activity in the living chick retina

Summary Electron histochemical techniques for glycogen synthetase has been applied to the living retina of the chick and the polyglucose particles synthesized from UDPG in the paraboloid of the accessory cone were compared with those synthesized by the conventional histochemical techniques.In the retinal incubated in the medium for glycogen synthetase in vivo, synthesized polyglucose particles were located in the cytoplasmic matrices and most of the particles were less than 200 Å in diameter. These particles were rather well stainable with lead citrate and filled the cytoplasmic matrices. However, the tubular structures were not flattened, but slightly dilated. Compared with polyglucose particles synthesized in vitro by glycogen synthetase, those demonstrated by the in vivo histochemical techniques showed closer resemblance to native glycogen particles in size and stainability with lead citrate.The polyglucose particles synthesized from UDPG by glycogen synthetase were apparently different from those synthesized from glucose-1-phosphate by phosphorylase and branching glycosyltransferase.     

Cytochemistry and morphology of synaptic structures in the cerebral cortex of rat

Summary Glycogen synthetase and phosphorylase activities in the paraboloid glycogen of the accessory cone of the chick retina were studied electron histochemically, while the paraboloid glycogen was observed by electron microscopy.Some of the paraboloid of the accessory cone of the chick retina contained abundant glycogen granules, but some showed no glycogen granules. Some inner segments of the accessory cones were occupied by deposition of glycogen granules.Polyglucose particles synthesized by glycogen synthetase activity in the chick paraboloid were demonstrated in fine granular form with diameter from 70 to 130 Å. These particles were less stainable with lead citrate than native glycogen granules. Synthesized polyglucose particles were located in the cytoplasmic matrices and expanded them. Lamellar and membrane structures were not related to synthesized polyglucose.Polyglucose particles synthesized by phosphorylase activity in the chick paraboloid were located in the cytoplasmic matrices and expanded them widely. Tubular structure appeared to be flattened by deposition of synthesized polyglucose particles. These features showed the resemblance to the inner segment of the accessory cone filled with a great amount of glycogen granules. Synthesized polyglucose was demonstrated in macromolecular form with diameter from 200 to 500 Å. There were no relationships between lamellar or membrane structures and polyglucose.The present study suggests that the chick paraboloid not only is a field for active glycogen metabolism, but also becomes a storage of glycogen.     

Pathways for utilization of carbon reserves in Desulfovibrio gigas under fermentative and respiratory conditions.

The sulfate-reducing bacterium Desulfovibrio gigas accumulates large amounts of polyglucose as an endogenous carbon and energy reserve. In the absence of exogenous substrates, the intracellular polysaccharide was utilized, and energy was conserved in the process (H. Santos, P. Fareleira, A. V. Xavier, L. Chen, M.-Y. Liu, and J. LeGall, Biochem. Biophys. Res. Commun. 195:551-557, 1993). When an external electron acceptor was not provided, degradation of polyglucose by cell suspensions of D. gigas yielded acetate, glycerol, hydrogen, and ethanol. A detailed investigation of the metabolic pathways involved in the formation of these end products was carried out, based on measurements of the activities of glycolytic enzymes in cell extracts, by either spectrophotometric or nuclear magnetic resonance (NMR) assays. All of the enzyme activities associated with the glycogen cleavage and the Embden-Meyerhof pathway were determined as well as those involved in the formation of glycerol from dihydroxyacetone phosphate (glycerol-3-phosphate dehydrogenase and glycerol phosphatase) and the enzymes that catalyze the reactions leading to the production of ethanol (pyruvate decarboxylase and ethanol dehydrogenase). The key enzymes of the Entner-Doudoroff pathway were not detected. The methylglyoxal bypass was identified as a second glycolytic branch operating simultaneously with the Embden-Meyerhof pathway. The relative contribution of these two pathways for polyglucose degradation was 2:3. 13C-labeling experiments with cell extracts using isotopically enriched glucose and 13C-NMR analysis supported the proposed pathways. The information on the metabolic pathways involved in polyglucose catabolism combined with analyses of the end products formed from polyglucose under fermentative conditions provided some insight into the role of NADH in D. gigas. In the presence of electron acceptors, NADH resulting from polyglucose degradation was utilized for the reduction of sulfate, thiosulfate, or nitrite, leading to the formation of acetate as the only carbon end product besides CO2. Evidence supporting the role of NADH as a source of reducing equivalents for the production of hydrogen is also presented.     

Kinetics of polyglucose breakdown in Chlorobium

Washed cells of Chlorobium limicola f. thiosulfatophilum photoassimilate CO₂ and acetate into polyglucose, which is laid down within the cells as rosette-like granules. When the cells are incubated in the dark, the polyglucose is broken down. Experiments using electron microscopy and labelling with ¹⁴C-acetate have revealed that degradation of polyglucose occurs in such a way that all the granules are subject to degradation simultaneously and the polyglucose which has been formed most recently in the light, becomes metabolized in the dark first.     

Quantitation of dextran 70 in peritoneal dialysate from patients administered 7.5% polyglucose

A method using gel permeation chromatography was evaluated for the quantitation of dextran 70 in dialysate samples containing polyglucose. Dialysate samples containing dextran 70 and polyglucose were pretreated using the enzyme α-amylase to selectively hydrolyze the α(1–4)-linked polyglucose, while leaving the α(1–6)-linked dextran 70 intact. Following sample deproteinization with trichloroacetic acid, dextran 70 was quantitated using gel permeation chromatography with refractive index detection. This method was evaluated for accuracy, precision, specificity, linearity, range, and analyte stability. Adequate method linearity with a correlation of >0.999 was established over the range of dextran 70 concentration from 1 to 0.025 mg/ml. Method precision was approximately 2% R.S.D. and accuracy (% recovery) was approximately 98–100% in the typical sample concentration range (1–0.5 mg/ml). This method was applied to the determination of intraperitoneal fluid kinetics in continuous ambulatory peritoneal dialysis (CAPD) patients administered daily night-time intraperitoneal exchanges with either 7.5% polyglucose or 4.25% dextrose. Dextran 70 was added to the dialysis solutions to yield an initial concentration of 1 mg/ml. Dialysate samples were collected at various times over a 10-h dwell-time and assayed for dextran 70. Intraperitoneal volume profiles based on dextran 70 concentrations and drain volumes were then calculated for each dialysis solution.     

Macromolecular particles of polyglucose synthesized under histochemical conditions in the endothelial cells of rabbit blood vessels

Synopsis New polyglucose was synthesized artificially under histochemical conditions from glucose 1-phosphate by phosphorylase and branching glycosyltransferase in the endothelial cells of rabbit blood vessels. In electron micrographs it appeared as a large macromolecular structure of spheroidal branching bodies. The polyglucose particles were much larger in size than those observed previously. They were synthesized in intracellular cytoplasmic matrices and also within nuclei.     

Direct determination of polyglucose metabolites in plasma using anion-exchange chromatography with pulsed amperometric detection

High-performance anion-exchange chromatography with pulsed amperometric detection (HPAE–PAD) was evaluated for the quantitation of polyglucose metabolites (DP2–DP7) in human plasma. The method was investigated for accuracy, precision, specificity, linearity, range and analyte stability. Samples were prepared by dilution into the standard range (0.1–10 μg/ml) followed by deproteinization using a 30?000 molecular mass cut-off filtration device. The limit of detection was 0.05 μg/ml for all metabolites. Method precision for DP2–DP7 varied from approximately 2% R.S.D. in the upper range to approximately 15% R.S.D. at the limit of quantitation. Samples were stable following one or two freeze–thaw cycles and, after preparation, they could be refrigerated for up to 72 h. Application of this method to clinical plasma samples from continuous ambulatory peritoneal dialysis (CAPD) patients administered one daily night-time intraperitoneal exchange of 2 l of 7.5% polyglucose solution for four weeks indicated that plasma levels of DP2, DP3 and DP4 increased from baseline levels of <0.01 g/l to steady-state levels of 1.2±0.3, 1.2±0.3 and 0.4±0.1 g/l (mean±S.D.), respectively. These steady state plasma levels for DP2 and DP3 are comparable to previously reported levels in patients administered daily overnight 7.5% polyglucose dialysis solution.     

Regulation of growth and photosynthesis by Oscillatoria agardhii grown with a light/dark cycle

Abstract The cyanobacterium Oscillatoria agardhii was grown in turbidostat cultures with the light energy supply in either the continuous mode or in the pulsed mode (8/16 h light/dark (L/D) cycle). The light irradiance value used was sufficient to allow the maximal growth rate to be attained, when supplied continuously. Adaptation of O. agardhii to the L/D cycle was characterized by an increase in pigment content and photosynthetic performance, accompanied by a decrease in growth rate. This mode of adaptation resembled the adaptation of O. agardhii to continuous low light intensities. It is suggested that in this case the L/D cycle provokes this adaptation in order to allow the cells to accumulate carbohydrate rapidly during the light period. This was attributed to the storage of polyglucose, which served as a carbon and energy source for growth in the dark. The utilization of polyglucose in the dark was able to sustain the synthesis of all other cell components at the same rate as when cells were growing in the light. The growth yield in the dark, whilst metabolizing internally stored polyglucose, was 0.52 g cell C/g polyglucose C, or 0.62 g cell dry weight/g polyglucose. Although in the pulsed mode there is a 66% loss in light irradiance per 24 h when compared with a continuous light regime, the growth rate of the cyanobacteria grown in the pulsed mode was only 35% lower than the growth rate of a culture grown in continuous light. This can be explained by a high growth yield in the dark and by increased CO₂ fixation rates in the light of cells grown in the pulsed mode.     

Carbon dioxide fixation in green sulphur bacteria

1. About one-third of the CO(2) fixed during photosynthesis by washed suspensions of Chlorobium thiosulfatophilum strain 8346 gave rise to alpha-oxoglutarate and branched-chain oxo acids, mainly beta-methyl-alpha-oxovalerate. Another one-third to one-half gave rise to a polyglucose. 2. The fixation of CO(2) was inhibited by fluoroacetate, increasing concentrations up to 1mm stimulating the accumulation of alpha-oxoglutarate and causing a decrease in the formation of the branched-chain oxo acids and polyglucose. 3. Acetate was converted into the same products as was CO(2). 4. Fluoroacetate (1mm) had a negligible effect on the formation of polyglucose from acetate and caused a slight inhibition of the formation of the branched-chain oxo acids and increased accumulation of alpha-oxoglutarate. 5. Iodoacetate (1mm) strongly inhibited polyglucose formation from acetate and caused accumulation of pyruvate. The formation of the branched-chain oxo acids from acetate was only slightly affected by this inhibitor. 6. Pyruvate can be metabolized by this organism in the presence of a suitable electron donor whether CO(2) is present or not. In the absence of CO(2) pyruvate is converted into polyglucose. 7. The accumulation of oxo acids during CO(2) fixation is completely inhibited by NH(4) (+) ions. The formation of the branched-chain oxo acids is considerably decreased by the presence of isoleucine, leucine or valine, or a mixture of these. 8. CO(2) fixation in two other strains of Chlorobium appears to exhibit a similar pattern to that in C. thiosulfatophilum strain 8346. 9. It is concluded that in washed suspensions, CO(2) is fixed mainly by a mechanism involving the reductive carboxylic acid cycle. Acetate, the product of the cycle, is converted into polyglucose via pyruvate synthase and a reversal of glycolysis or into branched-chain oxo acids by an unknown mechanism.     

Occurrence,structure and function of intracellular polyglucose in the obligate chemolithotroph Thiobacillus neapolitanus

Nitrogen-limited cells of the obligate chemolithotroph Thiobacillus neapolitanus formed an intracellular polymer during growth in the chemostat. This polymer was isolated and characterized as a branched polyglucose composed of units joined by -14 and -16 linkages. Polyglucose in T. neapolitanus can be considered a storage compound since formation of this compound took place during excess of energy and CO₂ whilst shortage of CO₂ resulted in rapid breakdown of polyglucose. Moreover the breakdown of polyglucose generated metabolically useful energy as could be demonstrated by polyglucose-dependent protein synthesis. Possession of polyglucose did not influence the viability of T. neapolitanus during prolonged periods of energy starvation. Activities of key enzymes of the oxidative pentose phosphate cycle, glucose-6-phosphatedehydrogenase and 6-phospho-gluconate-dehydrogenase, were demonstrated in cell free extracts of T. neapolitanus and appeared to increase 5- and 3-fold, respectively, during growth on NO ₃ ^- instead of NH ₄ ⁺ as a nitrogen source.     

In vivo 31P and 13C nuclear magnetic resonance studies of acetate metabolism in Chromatium vinosum

31P and 13C nuclear magnetic resonance (NMR) experiments were performed on suspensions of the phototrophic bacterium Chromatium vinosum incubated anaerobically in the dark. 31P NMR spectra revealed that during prolonged dark incubation high ATP levels are maintained. This phenomenon was independent of the presence of the energy reserves polyglucose and polyphosphate. 13C NMR experiments revealed that the amino acids glutamate, aspartate, and alanine are the major products of acetate incorporation in the dark. Apart from these amino acids, poly-beta-hydroxybutyrate was also formed. Acetate metabolism was markedly stimulated by the presence of polyglucose. The specific 13C activity of glutamate C-2 was approximately 50% that of glutamate C-4. The idea is discussed that this difference is the consequence of the maintenance of redox balance during entry of acetate into cell metabolism.     

Oxygen Consumption by Desulfovibrio Strains with and without Polyglucose

The kinetics of oxygen reduction by Desulfovibrio salexigens Mast1 and the role of polyglucose in this activity were examined and compared with those of strains of D. desulfuricans and D. gigas. Oxidation rates were highest at air saturation (up to 40 nmol of O₂ min⁻¹ mg of protein⁻¹) and declined with decreasing oxygen concentrations. Studies with cell extracts (CE) indicated that NADH oxidase was entirely responsible for the oxygen reduction in strain Mast1. In D. desulfuricans CSN, at least three independent systems appeared to reduce oxygen. Two were active at all oxygen concentrations (NADH oxidase and NADPH oxidase), and one was maximally active at less than 10 μM oxygen. In contrast to D. gigas and D. salexigens strains, the D. desulfuricans strains also contained NADH peroxidase and NADPH peroxidase activities and did not accumulate polyglucose under nonlimiting growth conditions. At air saturation, initial activities of the oxidases and peroxidases of cells harvested at the end of the log phase were on the order of 20 to 140 nmol of O₂ min⁻¹ mg of protein⁻¹. In all strains, these enzymes were relatively stable but were susceptible to inactivation as soon as substrates were added to the assay mixture. Under those conditions, all oxidation activity disappeared after ca. 1 h of incubation. The same finding was observed with whole cells of D. desulfuricans CSN and D. desulfuricans ATCC 27774, but inactivation was less pronounced with cells of D. salexigens Mast1. It appeared that the presence of polyglucose in the whole cells retarded the process of inactivation of NADH oxidase, but this property was lost in crude CE. In spite of the effect of polyglucose on the oxidative potential, oxygen-dependent growth of D. salexigens Mast1 could be demonstrated neither in batch nor in continuous culture.     

Microbial community engineering for biopolymer production from glycerol

In this work, the potential of using microbial community engineering for production of polyhydroxyalkanoates (PHA) from glycerol was explored. Crude glycerol is a by-product of the biofuel (biodiesel and bioethanol) industry and potentially a good substrate for bioplastic production. A PHA-producing microbial community was enriched based on cultivation in a feast–famine regime as successfully applied before for fatty acids-based biopolymer production. A glycerol-fed sequencing batch reactor operated at a 2-day liquid and biomass residence time and with feast–famine cycles of 24 h was used to enrich a mixed community of PHA producers. In a subsequent fed-batch PHA production step under growth-limiting conditions, the enriched mixed community produced PHA up to a dry weight content of 80 wt.%. The conversion efficiency of substrate to PHA on electron basis was 53%. Since glycerol is entering the metabolic pathways of the cell in the glycolytic pathway, it was anticipated that besides PHA, polyglucose could be formed as storage polymer as well. Indeed, polyglucose was produced in low amounts (∼10 wt.%). The results indicated that the feast–famine-based enrichment strategy was comparably successful to obtain a microbial community compared to fatty acids-based enrichment described before.     

Application of histochemistry to a living organ

Summary Histochemical application in a living animal was tested on the paraboloid of the accessory cone of the chick retina.After the anterior part of the eye had been cut with a Graefe's knife under ether anesthesia, the posterior part was filled with the medium for phosphorylase under exposure to light. The specimens were embedded for routine electron microscopy and the paraboloid of the accessory cone was observed by electron microscopy.Polyglucose particles were synthesized from glucose-1-phosphate in the paraboloid by the activities of phosphorylase and branching glycosyl transferase and found to be in the cytoplasmic matrices. These particles were larger in size and better stainable with lead citrate than those found in the paraboloid of the retina incubated in the medium in vitro by the conventional histochemical method. Overproduction of polyglucose particles was not found in the paraboloid of the retina incubated in the medium in vivo. These findings suggest that polyglucose particles synthesized in vivo have a close resemblance to native glycogen particles and that glycogen metabolism is regulated by the living cell. Glycolysis may not be related to the membranous structures.Therefore, application of enzyme histochemical techniques to the living organ can demonstrate more accurate morphological aspects of metabolism in the cell.     

Binding of calcium by cellulose membranes and Sephadex

Calcium ions bind to polyglucose matrices (dialysis membranes and Sephadex) with low affinity, but in amounts sufficient to markedly influence the rate of dialysis of Ca⁺⁺. In applications where such interaction is critical (binding studies by flow dialysis or ultrafiltration), Ca⁺⁺ binding can be largely prevented by acetylation of the membrane and/or high salt concentration.     

Polyglucose synthesis in Chloroflexus aurantiacus studied by 13C-NMR

Chloroflexus aurantiacus OK-70 fl was grown photoautotrophically with hydrogen as electron source. The cultures were subjected to long term labelling experments with ¹³C-labelled acetate or alanine in the presence of sodium fluoroacetate. The presence of fluoroacetate caused the cells to accumulate large amounts of polyglucose which was hydrolysed and analysed by NMR. The labelling patterns of glucose were symmetric and in agreement with carbohydrate synthesis from acetate and CO₂ via pyruvate synthase. The content of carbon derived from added acetate was highest in C2 and C5 of glucose, at least 20% higher than in C1 and C6. About one third of the glucose carbon was derived from added acetate, the rest being from CO₂. Contrary to expectations, in glucose formed in the presence of C1-labelled acetate C1 and C6 contained more label than C2 and C5, and with C2-labelled acetate as the tracer glucose was mainly labelled in C2 and C5. Labelled CO₂ was formed from acetate labelled at either position. The labelling data indicate a new metabolic pathway in C. aurantiacus. It is suggested that the cells form C1-labelled acetyl-CoA from C2-labelled acetyl-CoA and vice versa by a cyclic mechanism involving concomitant CO₂ fixation and that this cycle is the part of the autotrophic CO₂ fixation pathways in C. aurantiacus in which acetyl-CoA is formed from CO₂.The polyglucose of C. aurantiacus appears to have predominantly (1–4)-linked structure with about 10% (1–6)-linkages as revealed by ¹³C-NMR.     

Chemical analysis of the outer cyst wall and inclusion material ofBdellovibrio bdellocysts

The outer cyst wall and inclusion material fromBdellovibrio bdellocysts were isolated and their chemical composition was determined. The outer cyst wall is primarily peptidoglycan containing glucosamine, muramic acid, alanine, glutamic acid, and diaminopimelic acid. The cyst walls are resistant to lysozyme, but are rendered sensitive following deacylation and N-acetylation. Isolated inclusions were degraded quantitatively to glucose by HCl and by amyloglucosidase, whereas α-amylase degraded the polymer only partially with the release of reducing groups. The inclusion material is therefore an amylopectin-like polysaccharide, being a polyglucose containing both α-1,4 and α-1,6 linkages.     

Structure of rigid-layer of Rhizobium cell wall. I. Purification on the peptidoglycan from the cellulose microfibrils

The bag shaped peptidoglycan layer of Rhizobium cell wall was isolated from intact cells after treatment with sodium dodecylsulfate and trypsin, chymotrypsin or pepsin digestion. Results of chemical analysis of acid hydrolyzed peptidoglycan revealed beside two amino sugars: glucosamine and muramic acid, three major amino acids; alanine, glutamic acid and 2,6-diaminopimelic acid and also significant amount of glucose. Evidence were provided that the polyglucose found in peptidoglycan preparations of three strains of Rhizobium trifolii, one of Rhizobium leguminosarum and one of Rhizobium meliloti consist of cellulose microfibrils. The content of cellulose present in Rhizobium peptidoglycans ranged from 60 to 80%. Methods of peptidoglycan purification from the cellulose microfibrils are described.