Glutamine synthetase degradation is controlled by oxidative proteolysis in the marine cyanobacterium Prochlorococcus marinus strain PCC 9511

Prochlorococcus is one of the most important primary producers on Earth; its unusual features and ecological importance have made it a model organism, but nutrient assimilation has received little attention. Glutamine synthetase (GS) plays a key role in nitrogen metabolism and its central position justifies the fine regulation of this enzyme. The aim of this work is to demonstrate the involvement of metal-catalyzed oxidation (MCO) in the control of the biological activity and turnover of GS from Prochlorococcus. In order to study the physiological role of MCO, we have first characterized the in vitro biosynthetic inactivation and degradation of GS in the axenic PCC 9511 strain, testing then the effect of several stress conditions, such as the presence of electron transport inhibitors, darkness and aging, on the inactivation and degradation of GS. It is noteworthy that the physiological substrates of GS could protect the enzyme from the oxidative inactivation and ATP partially reverted this inactivation once the enzyme had been oxidized, being this effect higher in the presence of glutamate. We have also found that the GS from aged cultures is degraded to the same smaller size fragments obtained in the in vitro degradation of GS by an oxidative model system (Fe3+/NADH/NADH oxidase/O2). These results suggest the implication of MCO in the age- and oxidative state-dependent degradation of GS from Prochlorococcus.     

Regulation of glutamine synthetase by metal-catalyzed oxidative modification in the marine oxyphotobacterium Prochlorococcus

The inactivation of glutamine synthetase (GS; EC 6.3.1.2) by metal-catalyzed oxidation (MCO) systems was studied in several Prochlorococcus strains, including the axenic PCC 9511. GS was inactivated in the presence of various oxidative systems, either enzymatic (as NAD(P)H+NAD(P)H-oxidase+Fe³⁺+O₂) or non-enzymatic (as ascorbate+Fe³⁺+O₂). This process required the presence of oxygen and a metal cation, and is prevented under anaerobic conditions. Catalase and peroxidase, but not superoxide dismutase, effectively protected the enzyme against inactivation, suggesting that hydrogen peroxide mediates this mechanism, although it is not directly responsible for the reaction. Addition of azide (an inhibitor of both catalase and peroxidase) to the MCO systems enhanced the inactivation. Different thiols induced the inactivation of the enzyme, even in the absence of added metals. However, this inactivation could not be reverted by addition of strong oxidants, as hydrogen peroxide or oxidized glutathione. After studying the effect of addition of the physiological substrates and products of GS on the inactivation mechanism, we could detect a protective effect in the case of inorganic phosphate and glutamine. Immunochemical determinations showed that the concentration of GS protein significantly decreased by effect of the MCO systems, indicating that inactivation precedes the degradation of the enzyme.     

Regulation of glutamine synthetase by metal-catalyzed oxidative modification in the marine oxyphotobacterium Prochlorococcus.

The inactivation of glutamine synthetase (GS; EC 6.3.1.2) by metal-catalyzed oxidation (MCO) systems was studied in several Prochlorococcus strains, including the axenic PCC 9511. GS was inactivated in the presence of various oxidative systems, either enzymatic (as NAD(P)H+NAD(P)H-oxidase+Fe(3+)+O(2)) or non-enzymatic (as ascorbate+Fe(3+)+O(2)). This process required the presence of oxygen and a metal cation, and is prevented under anaerobic conditions. Catalase and peroxidase, but not superoxide dismutase, effectively protected the enzyme against inactivation, suggesting that hydrogen peroxide mediates this mechanism, although it is not directly responsible for the reaction. Addition of azide (an inhibitor of both catalase and peroxidase) to the MCO systems enhanced the inactivation. Different thiols induced the inactivation of the enzyme, even in the absence of added metals. However, this inactivation could not be reverted by addition of strong oxidants, as hydrogen peroxide or oxidized glutathione. After studying the effect of addition of the physiological substrates and products of GS on the inactivation mechanism, we could detect a protective effect in the case of inorganic phosphate and glutamine. Immunochemical determinations showed that the concentration of GS protein significantly decreased by effect of the MCO systems, indicating that inactivation precedes the degradation of the enzyme.     

Glutathione peroxidase 3 of Saccharomyces cerevisiae suppresses non-enzymatic proteolysis of glutamine synthetase in an activity-independent manner

Glutathione peroxidase 3 (Gpx3) is ubiquitously expressed and is important antioxidant enzyme in yeast. It modulates the activities of redox-sensitive thiol proteins, particularly those involved in signal transduction pathway and protein translocation. Through immunoprecipitation/two-dimensional gel electrophoresis (IP-2DE), MALDI-TOF mass spectrometry, and a pull down assay, we found glutamine synthetase (GS; EC 6.3.1.2) as a candidate interacting protein with Gpx3. GS is a key enzyme in nitrogen metabolism and ammonium assimilation. It has been known that GS is non-enzymatically cleaved by ROS generated by MFO (thiol/ Fe(3+)/O(2) mixed-function oxidase) system. In this study, it is demonstrated that GS interacts with Gpx3 through its catalytic domain both in vivo and in vitro regardless of redox state. In addition, Gpx3 helps to protect GS from inactivation and degradation via oxidative stress in an activity-independent manner. Based on the results, it is suggested that Gpx3 protects GS from non-enzymatic proteolysis, thereby contributing to cell homeostasis when cell is exposed to oxidative stress.     

Glutamine Synthetase Sensitivity to Oxidative Modification during Nutrient Starvation in Prochlorococcus marinus PCC 9511

Glutamine synthetase plays a key role in nitrogen metabolism, thus the fine regulation of this enzyme in Prochlorococcus, which is especially important in the oligotrophic oceans where this marine cyanobacterium thrives. In this work, we studied the metal-catalyzed oxidation of glutamine synthetase in cultures of Prochlorococcus marinus strain PCC 9511 subjected to nutrient limitation. Nitrogen deprivation caused glutamine synthetase to be more sensitive to metal-catalyzed oxidation (a 36% increase compared to control, non starved samples). Nutrient starvation induced also a clear increase (three-fold in the case of nitrogen) in the concentration of carbonyl derivatives in cell extracts, which was also higher (22%) upon addition of the inhibitor of electron transport, DCMU, to cultures. Our results indicate that nutrient limitations, representative of the natural conditions in the Prochlorococcus habitat, affect the response of glutamine synthetase to oxidative inactivating systems. Implications of these results on the regulation of glutamine synthetase by oxidative alteration prior to degradation of the enzyme in Prochlorococcus are discussed.     

Physiological Regulation of Isocitrate Dehydrogenase and the Role of 2-Oxoglutarate in Prochlorococcus sp. Strain PCC 9511

The enzyme isocitrate dehydrogenase (ICDH; EC 1.1.1.42) catalyzes the oxidative decarboxylation of isocitrate, to produce 2-oxoglutarate. The incompleteness of the tricarboxylic acids cycle in marine cyanobacteria confers a special importance to isocitrate dehydrogenase in the C/N balance, since 2-oxoglutarate can only be metabolized through the glutamine synthetase/glutamate synthase pathway. The physiological regulation of isocitrate dehydrogenase was studied in cultures of Prochlorococcus sp. strain PCC 9511, by measuring enzyme activity and concentration using the NADPH production assay and Western blotting, respectively. The enzyme activity showed little changes under nitrogen or phosphorus starvation, or upon addition of the inhibitors DCMU, DBMIB and MSX. Azaserine, an inhibitor of glutamate synthase, induced clear increases in the isocitrate dehydrogenase activity and icd gene expression after 24 h, and also in the 2-oxoglutarate concentration. Iron starvation had the most significant effect, inducing a complete loss of isocitrate dehydrogenase activity, possibly mediated by a process of oxidative inactivation, while its concentration was unaffected. Our results suggest that isocitrate dehydrogenase responds to changes in the intracellular concentration of 2-oxoglutarate and to the redox status of the cells in Prochlorococcus.     

Reversible adenylylation of glutamine synthetase is dynamically counterbalanced during steady-state growth of Escherichia coli

Glutamine synthetase (GS) is the central enzyme for nitrogen assimilation in Escherichia coli and is subject to reversible adenylylation (inactivation) by a bifunctional GS adenylyltransferase/adenylyl-removing enzyme (ATase). In vitro, both of the opposing activities of ATase are regulated by small effectors, most notably glutamine and 2-oxoglutarate. In vivo, adenylyltransferase (AT) activity is critical for growth adaptation when cells are shifted from nitrogen-limiting to nitrogen-excess conditions and a rapid decrease of GS activity by adenylylation is needed. Here, we show that the adenylyl-removing (AR) activity of ATase is required to counterbalance its AT activity during steady-state growth under both nitrogen-excess and nitrogen-limiting conditions. This conclusion was established by studying AR⁻/AT⁺ mutants, which surprisingly displayed steady-state growth defects in nitrogen-excess conditions due to excessive GS adenylylation. Moreover, GS was abnormally adenylylated in the AR⁻ mutants even under nitrogen-limiting conditions, whereas there was little GS adenylylation in wild-type strains. Despite the importance of AR activity, we establish that AT activity is significantly regulated in vivo, mainly by the cellular glutamine concentration. There is good general agreement between quantitative estimates of AT regulation in vivo and results derived from previous in vitro studies except at very low AT activities. We propose additional mechanisms for the low AT activities in vivo. The results suggest that dynamic counterbalance by reversible covalent modification may be a general strategy for controlling the activity of enzymes such as GS, whose physiological output allows adaptation to environmental fluctuations.     

Removal of H₂O₂ and generation of superoxide radical: role of cytochrome c and NADH

In cells, mitochondria, endoplasmic reticulum, and peroxisomes are the major sources of reactive oxygen species (ROS) under physiological and pathophysiological conditions. Cytochrome c (cyt c) is known to participate in mitochondrial electron transport and has antioxidant and peroxidase activities. Under oxidative or nitrative stress, the peroxidase activity of Fe³⁺cyt c is increased. The level of NADH is also increased under pathophysiological conditions such as ischemia and diabetes and a concurrent increase in hydrogen peroxide (H₂O₂) production occurs. Studies were performed to understand the related mechanisms of radical generation and NADH oxidation by Fe³⁺cyt c in the presence of H₂O₂. Electron paramagnetic resonance (EPR) spin trapping studies using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) were performed with NADH, Fe³⁺cyt c, and H₂O₂ in the presence of methyl-β-cyclodextrin. An EPR spectrum corresponding to the superoxide radical adduct of DMPO encapsulated in methyl-β-cyclodextrin was obtained. This EPR signal was quenched by the addition of the superoxide scavenging enzyme Cu,Zn-superoxide dismutase (SOD1). The amount of superoxide radical adduct formed from the oxidation of NADH by the peroxidase activity of Fe³⁺cyt c increased with NADH and H₂O₂ concentration. From these results, we propose a mechanism in which the peroxidase activity of Fe³⁺cyt c oxidizes NADH to NAD^•, which in turn donates an electron to O₂, resulting in superoxide radical formation. A UV-visible spectroscopic study shows that Fe³⁺cyt c is reduced in the presence of both NADH and H₂O₂. Our results suggest that Fe³⁺cyt c could have a novel role in the deleterious effects of ischemia/reperfusion and diabetes due to increased production of superoxide radical. In addition, Fe³⁺cyt c may play a key role in the mitochondrial “ROS-induced ROS-release” signaling and in mitochondrial and cellular injury/death. The increased oxidation of NADH and generation of superoxide radical by this mechanism may have implications for the regulation of apoptotic cell death, endothelial dysfunction, and neurological diseases. We also propose an alternative electron transfer pathway, which may protect mitochondria and mitochondrial proteins from oxidative damage.     

Oxidative inactivation of glutamine synthetase from the cyanobacterium Anabaena variabilis.

In crude extracts of the cyanobacterium Anabaena variabilis, glutamine synthetase (GS) could be effectively inactivated by the addition of NADH. GS inactivation was completed within 30 min. Both the inactivated GS and the active enzyme were isolated. No difference between the two enzyme forms was seen in sodium dodecyl sulfate-gels, and only minor differences were detectable by UV spectra, which excludes modification by a nucleotide. Mass spectrometry revealed that the molecular masses of active and inactive GS are equal. While the Km values of the substrates were unchanged, the Vmax values of the inactive GS were lower, reflecting the inactivation factor in the crude extract. This result indicates that the active site was affected. From the crude extract, a fraction mediating GS inactivation could be enriched by ammonium sulfate precipitation and gel filtration. GS inactivation by this fraction required the presence of NAD(P)H, Fe3+, and oxygen. In the absence of the GS-inactivating fraction, GS could be inactivated by Fe2+ and H2O2. The GS-inactivating fraction produced Fe2+ and H2O2, using NADPH, Fe3+, and oxygen. Accordingly, the inactivating fraction was inhibited by catalase and EDTA. This GS-inactivating system of Anabaena is similar to that described for oxidative GS inactivation in Escherichia coli. We conclude that GS inactivation by NAD(P)H is caused by irreversible oxidative damage and is not due to a regulatory mechanism of nitrogen assimilation.     

Stability of nitrate reductase and source of its reductant in Sorghum seedlings

Pre-incubation of nitrate reductase from Sorghum seedlings with NADH increased enzyme activity by 25%. Ferricyanide had no effect. NADH protected the enzyme from inactivation during storage. Malonate inhibited in vivo nitrate reduction in Sorghum leaves by 95%. The inhibitory effect of malonate was reversed by fumarate. Sodium fluoride in the presence of phosphate also inhibited in vivo nitrate reduction by 60%. It is suggested that NADH generated via the citric acid cycle is utilized for nitrate reduction in Sorghum seedlings.     

Reversible inhibition of mammalian glutamine synthetase by tyrosine nitration

The effect of tyrosine nitration on mammalian GS activity and stability was studied in vitro. Peroxynitrite at a concentration of 5 micro mol/l produced tyrosine nitration and inactivation of GS, whereas 50 micro mol/l peroxynitrite additionally increased S-nitrosylation and carbonylation and degradation of GS by the 20S proteasome. (-)Epicatechin completely prevented both, tyrosine nitration and inactivation of GS by peroxynitrite (5 micro mol/l). Further, a putative "denitrase" activity restored the activity of peroxynitrite (5 micro mol/l)-treated GS. The data point to a potential regulation of GS activity by a reversible tyrosine nitration. High levels of oxidative stress may irreversibly damage and predispose the enzyme to proteasomal degradation.     

Ecology of uncultured Prochlorococcus clades revealed through single-cell genomics and biogeographic analysis

Prochlorococcus is the numerically dominant photosynthetic organism throughout much of the world''s oceans, yet little is known about the ecology and genetic diversity of populations inhabiting tropical waters. To help close this gap, we examined natural Prochlorococcus communities in the tropical Pacific Ocean using a single-cell whole-genome amplification and sequencing. Analysis of the gene content of just 10 single cells from these waters added 394 new genes to the Prochlorococcus pan-genome—that is, genes never before seen in a Prochlorococcus cell. Analysis of marker genes, including the ribosomal internal transcribed sequence, from dozens of individual cells revealed several representatives from two uncultivated clades of Prochlorococcus previously identified as HNLC1 and HNLC2. While the HNLC clades can dominate Prochlorococcus communities under certain conditions, their overall geographic distribution was highly restricted compared with other clades of Prochlorococcus. In the Atlantic and Pacific oceans, these clades were only found in warm waters with low Fe and high inorganic P levels. Genomic analysis suggests that at least one of these clades thrives in low Fe environments by scavenging organic-bound Fe, a process previously unknown in Prochlorococcus. Furthermore, the capacity to utilize organic-bound Fe appears to have been acquired horizontally and may be exchanged among other clades of Prochlorococcus. Finally, one of the single Prochlorococcus cells sequenced contained a partial genome of what appears to be a prophage integrated into the genome.     

Ammonium formation and assimilation in P(SARK)∷IPT tobacco transgenic plants under low N

Wild Type (WT) and transgenic tobacco plants expressing isopentenyltransferase (IPT), a gene encoding the enzyme regulating the rate-limiting step in cytokinins (CKs) synthesis, were grown under limited nitrogen (N) conditions. We analyzed nitrogen forms, nitrogen metabolism related-enzymes, amino acids and photorespiration related-enzymes in WT and P_SARK∷IPT tobacco plants. Our results indicate that the WT plants subjected to N deficiency displayed reduced nitrate (NO₃⁻) assimilation. However, an increase in the production of ammonium (NH₄⁺), by the degradation of proteins and photorespiration led to an increase in the glutamine synthetase/glutamate synthase (GS/GOGAT) cycle in WT plants. In these plants, the amounts of amino acids decreased with N deficiency, although the relative amounts of glutamate and glutamine increased with N deficiency. Although the transgenic plants expressing P_SARK∷IPT and growing under suboptimal N conditions displayed a significant decline in the N forms in the leaf, they maintained the GS/GOGAT cycle at control levels. Our results suggest that, under N deficiency, CKs prevented the generation and assimilation of NH₄⁺ by increasing such processes as photorespiration, protein degradation, the GS/GOGAT cycle, and the formation of glutamine.     

Streamlined Regulation and Gene Loss as Adaptive Mechanisms in Prochlorococcus for Optimized Nitrogen Utilization in Oligotrophic Environments

Prochlorococcus is one of the dominant cyanobacteria and a key primary producer in oligotrophic intertropical oceans. Here we present an overview of the pathways of nitrogen assimilation in Prochlorococcus, which have been significantly modified in these microorganisms for adaptation to the natural limitations of their habitats, leading to the appearance of different ecotypes lacking key enzymes, such as nitrate reductase, nitrite reductase, or urease, and to the simplification of the metabolic regulation systems. The only nitrogen source utilizable by all studied isolates is ammonia, which is incorporated into glutamate by glutamine synthetase. However, this enzyme shows unusual regulatory features, although its structural and kinetic features are unchanged. Similarly, urease activities remain fairly constant under different conditions. The signal transduction protein P_II is apparently not phosphorylated in Prochlorococcus, despite its conserved amino acid sequence. The genes amt1 and ntcA (coding for an ammonium transporter and a global nitrogen regulator, respectively) show noncorrelated expression in Prochlorococcus under nitrogen stress; furthermore, high rates of organic nitrogen uptake have been observed. All of these unusual features could provide a physiological basis for the predominance of Prochlorococcus over Synechococcus in oligotrophic oceans.     

Multiple proteins with single activities or a single protein with multiple activities: The conundrum of cell surface NADH oxidoreductases

Reduction of the cell-impermeable tetrazolium salt WST-1 has been used to characterise two plasma membrane NADH oxidoreductase activities in human cells. The trans activity, measured with WST-1 and the intermediate electron acceptor mPMS, utilises reducing equivalents from intracellular sources, while the surface activity, measured with WST-1 and extracellular NADH, is independent of intracellular metabolism. Whether these two activities involve distinct proteins or are inherent to a single protein is unclear. In this work, we have attempted to address this question by examining the relationship between the trans and surface WST-1-reducing activities and a third well-characterised family of cell surface oxidases, the ECTO-NOX proteins. Using blue native-polyacrylamide gel electrophoresis, we have identified a complex in the plasma membranes of human 143B osteosarcoma cells responsible for the NADH-dependent reduction of WST-1. The dye-reducing activity of the 300 kDa complex was attributed to a 70 kDa NADH oxidoreductase activity that cross-reacted with antisera against the ECTO-NOX protein CNOX. Differences in enzyme activities and inhibitor profiles between the WST-1-reducing NADH oxidoreductase enzyme in the presence of NADH or mPMS and the ECTO-NOX family are reconciled in terms of the different purification methods and assay systems used to study these proteins.     

Glutathione reductase fromSaccharomyces cerevisiae undergoes redox interconversionin situ andin vivo

Redox interconversion of glutathione reductase was studiedin situ withS. cerevisiae. The enzyme was more sensitive to redox inactivation in 24 hour-starved cells than in freshly-grown ones. While 5 μM NADPH or 100 μM NADH caused 50% inactivation in normal cells in 30 min, 0.75 μM NADPH or 50 μM NADH promoted a similar effect in starved cells. GSSG reactivated the enzyme previously inactivated by NADPH, ascertaining that the enzyme was subjected to redox interconversion. Low EDTA concentrations fully protected the enzyme from NADPH inactivation, thus confirming the participation of metals in such a process. Extensive inactivation was obtained in permeabilized cells incubated with glucose-6-phosphate or 6-phosphogluconate, in agreement with the very high specific activities of the corresponding dehydrogenases. Some inactivation was also observed with malate, L-lactate, gluconate or isocitrate in the presence of low NADP⁺ concentrations. The inactivation of yeast glutathione reductase has also been studiedin vivo. The activity decreased to 75% after 2 hours of growth with glucono-δ-lactone as carbon source, while NADPH rose to 144% and NADP⁺ fell to 86% of their initial values. Greater changes were observed in the presence of 1.5 μM rotenone: enzymatic activity descended to 23% of the control value, while the NADH/NAD⁺ and NADPH/NADP⁺ ratios rose to 171% and 262% of their initial values, respectively. Such results indicate that the lowered redox potential of the pyridine nucleotide pool existing when glucono-δ-lactone is oxidized promotesin vivo inactivation of glutathione reductase.     

ATP-dependent deoxyribonuclease in Bacillus subtilis and a mutant deficient in this activity

ATP-dependent DNAse activity was measured in rec⁺ and several rec strains of B. subtilis 168. One of the strains (marker recE₅) was found to lack this activity. The enzyme from the wild type was partially purified and some of its properties were determined. The pH optimum is 9.5. Activity is higher at 50° but inactivation occurs on standing at this temperature. The enzyme requires Mg²⁺ (10^?2M) or Mn²⁺ (2·10^?4M). ATP is an absolute requirement and the only other nucleoside triphosphate that can partially replace it is dATP. Lack of activity in the mutant does not seem to be due to the presence of an inhibitor. Results so far do not allow us to conclude as to whether or not the mutant produces an altered enzyme.     

Characterization of the Reversible Inactivation of Ankistrodesmus braunii Nitrate Reductase by Hydroxylamine

The photoreversible nature of the regulation of nitrate reductase is one of the most interesting features of this enzyme. As well as other chemicals, NH₂OH reversibly inactivates the reduced form of nitrate reductase from Ankistrodesmus braunii. From the partial activities of the enzyme, only terminal nitrate reductase is affected by NH₂OH. To demonstrate that the terminal activity was readily inactivted by NH₂OH, the necessary reductants of the terminal part of the enzyme had to be cleared of dithionite since this compound reacts chemically with NH₂OH. Photoreduced flavins and electrochemically reduced methyl viologen sustain very effective inactivation of terminal nitrate reductase activity, even if the enzyme was previously deprived of its NADH-dehydrogenase activity. The early inhibition of nitrate reductase by NH₂OH appears to be competitive versus NO₃⁻. Since NO₃⁻, as well as cyanate, carbamyl phosphate and azide (competitive inhibitors of nitrate reductase versus NO₃⁻), protect the enzyme from NH₂OH inactivation, it is suggested that NH₂OH binds to the nitrate active site. The NH₂OH-inactivated enzyme was photoreactivated in the presence of flavins, although slower than when the enzyme was previously inactivated with CN⁻. NH₂OH and NADH concentrations required for full inactivation of nitrate reductase appear to be low enough to potentially consider this inactivation process of physiological significance.     

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.