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.     

Genome streamlining results in loss of robustness of the circadian clock in the marine cyanobacterium Prochlorococcus marinus PCC 9511

The core oscillator of the circadian clock in cyanobacteria consists of 3 proteins, KaiA, KaiB, and KaiC. All 3 have previously been shown to be essential for clock function. Accordingly, most cyanobacteria possess at least 1 copy of each kai gene. One exception is the marine genus Prochlorococcus, which we suggest here has suffered a stepwise deletion of the kaiA gene, together with significant genome streamlining. Nevertheless, natural Prochlorococcus populations and laboratory cultures are strongly synchronized by the alternation of day and night, displaying 24-h rhythms in DNA replication, with a temporal succession of G1, S, and G2-like cell cycle phases. Using quantitative real-time PCR, we show here that in Prochlorococcus marinus PCC 9511, the mRNA levels of the clock genes kaiB and kaiC, as well as a few other selected genes including psbA, also displayed marked diel variations when cultures were kept under a light-dark rhythm. However, both cell cycle and psbA gene expression rhythms damped very rapidly under continuous light. In the closely related Synechococcus sp. WH8102, which possesses all 3 kai genes, cell cycle rhythms persisted over several days, in agreement with established cyanobacterial models. These data indicate a correlation between the loss of kaiA and a loss of robustness in the endogenous oscillator of Prochlorococcus and raise questions about how a basic KaiBC system may function and through which mechanism the daily "lights-on" and "lights-off" signal could be mediated.     

Oxidative Modification of Glutamine Synthetase by Amyloid Beta Peptide

β-Amyloid peptide (Aβ), the main constituent of senile plaques and diffuse amyloid deposits in Alzheimer's diseased brain, was shown to initiate the development of oxidative stress in neuronal cell cultures. Toxic lots of Aβ form free radical species in aqueous solution. It was proposed that Aβ-derived free radicals can directly damage cell proteins via oxidative modification. Recently we reported that synthetic Aβ can interact with glutamine synthetase (GS) and induce inactivation of this enzyme. In the present study we present the evidence that toxic Aβ(25-35) induces the oxidation of pure GS in vitro. It was found that inactivation of GS by Aβ, as well as the oxidation of GS by metal-catalyzed oxidation system, is accompanied by an increase of protein carbonyl content. As it was reported previously by our laboratory, radicalization of Aβ is not iron or peroxide-dependent. Our present observations consistently show that toxic Aβ does not need iron or peroxide to oxidize GS. However, treatment of GS with the peptide, iron and peroxide together significantly stimulates the protein carbonyl formation. Here we report also that Aβ(25-35) induces carbonyl formation in BSA. Our results demonstrate that P-peptide, as well as other free radical generators, induces carbonyl formation when brought into contact with different proteins.     

Nitrogen Starvation and the Regulation of Glutamine Synthetase in Agmenellum quadruplicatum

The level of glutamine synthetase activity in Agmenellum quadruplicatum strain PR-6 was dependent on the nitrogen source used for growth and on the nutritional status of the cells. During exponential growth, glutamine synthetase activity was low in cells grown on ammonia, urea, or nitrate. During the transition from nitrogen replete to nitrogen starved growth, glutamine synthetase activity began to rise. With ammonia as a nitrogen source, glutamine synthetase activity as determined in whole cells increased from 1 nanomole per minute per milliliter during exponential growth to 22 nanomoles per minute per milliliter during severe nitrogen starvation. In cells grown on nitrate the increase was from 5 to 39 nanomoles per minute per milliliter, and in cells grown on urea the increase was from 4 to 31 nanomoles per minute per milliliter.     

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.     

Carbon use efficiencies and allocation strategies in <Emphasis Type="Italic">Prochlorococcus marinus</Emphasis> strain PCC 9511 during nitrogen-limited growth

We studied cell properties including carbon allocation dynamics in the globally abundant and important cyanobacterium Prochlorococcus marinus strain PCC 9511 grown at three different growth rates in nitrogen-limited continuous cultures. With increasing nitrogen limitation, cellular divinyl chlorophyll a and the functional absorption cross section of Photosystem II decreased, although maximal photosynthetic efficiency of PSII remained unaltered across all N-limited growth rates. Chl-specific gross and net carbon primary production were also invariant with nutrient-limited growth rate, but only 20% of Chl-specific gross carbon primary production was retained in the biomass across all growth rates. In nitrogen-replete cells, 60% of the assimilated carbon was incorporated into the protein pool while only 30% was incorporated into carbohydrates. As N limitation increased, new carbon became evenly distributed between these two pools. While many of these physiological traits are similar to those measured in other algae, there are also distinct differences, particularly the lower overall efficiency of carbon utilization. The latter provides new information needed for understanding and estimating primary production, particularly in the nutrient-limited tropical oceans where P. marinus dominates phytoplankton community composition.     

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.     

In vivo regulation of glutamine synthetase activity in the marine chlorophyll b-containing cyanobacterium Prochlorococcus sp. strain PCC 9511 (oxyphotobacteria)

The physiological regulation of glutamine synthetase (GS; EC 6.3.1.2) in the axenic Prochlorococcus sp. strain PCC 9511 was studied. GS activity and antigen concentration were measured using the transferase and biosynthetic assays and the electroimmunoassay, respectively. GS activity decreased when cells were subjected to nitrogen starvation or cultured with oxidized nitrogen sources, which proved to be nonusable for Prochlorococcus growth. The GS activity in cultures subjected to long-term phosphorus starvation was lower than that in equivalent nitrogen-starved cultures. Azaserine, an inhibitor of glutamate synthase, provoked an increase in enzymatic activity, suggesting that glutamine is not involved in GS regulation. Darkness did not affect GS activity significantly, while the addition of diuron provoked GS inactivation. GS protein determination showed that azaserine induces an increase in the concentration of the enzyme. The unusual responses to darkness and nitrogen starvation could reflect adaptation mechanisms of Prochlorococcus for coping with a light- and nutrient-limited environment.     

Glutamine Synthetase in Muscle Is Required for Glutamine Production during Fasting and Extrahepatic Ammonia Detoxification

The main endogenous source of glutamine is de novo synthesis in striated muscle via the enzyme glutamine synthetase (GS). The mice in which GS is selectively but completely eliminated from striated muscle with the Cre-loxP strategy (GS-KO/M mice) are, nevertheless, healthy and fertile. Compared with controls, the circulating concentration and net production of glutamine across the hindquarter were not different in fed GS-KO/M mice. Only a ∼3-fold higher escape of ammonia revealed the absence of GS in muscle. However, after 20 h of fasting, GS-KO/M mice were not able to mount the ∼4-fold increase in glutamine production across the hindquarter that was observed in control mice. Instead, muscle ammonia production was ∼5-fold higher than in control mice. The fasting-induced metabolic changes were transient and had returned to fed levels at 36 h of fasting. Glucose consumption and lactate and ketone-body production were similar in GS-KO/M and control mice. Challenging GS-KO/M and control mice with intravenous ammonia in stepwise increments revealed that normal muscle can detoxify ∼2.5 μmol ammonia/g muscle·h in a muscle GS-dependent manner, with simultaneous accumulation of urea, whereas GS-KO/M mice responded with accumulation of glutamine and other amino acids but not urea. These findings demonstrate that GS in muscle is dispensable in fed mice but plays a key role in mounting the adaptive response to fasting by transiently facilitating the production of glutamine. Furthermore, muscle GS contributes to ammonia detoxification and urea synthesis. These functions are apparently not vital as long as other organs function normally.     

Electron Transport Controls Glutamine Synthetase Activity in the Facultative Heterotrophic Cyanobacterium Synechocystis sp. PCC 6803

Glutamine synthetase (GS) from Synechocystis sp. PCC 6803 was inactivated in vivo by transferring cells from light to darkness or by incubation with the photosynthetic inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea but not with 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone. Addition of glucose prevented both dark and 3-(3,4-dichlorophenyl)-1,1-dimethylurea GS inactivation. In a Synechocystis psbE-psbF mutant (T1297) lacking photosystem II, glucose was required to maintain active GS, even in the light. However, in nitrogen-starved T1297 cells the removal of glucose did not affect GS activity. The fact that dark-inactivated GS was reactivated in vitro by the same treatments that reactivate the ammonium-inactivated GS points out that both nitrogen metabolism and redox state of the cells lead to the same molecular regulatory mechanism in the control of GS activity. Using GS antibodies we detected that dark-inactivated GS displayed a different electrophoretic migration with respect to the active form in nondenaturing polyacrylamide gel electrophoresis but not in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The possible pathway to modulate GS activity by the electron transport flow in Synechocystis cells is discussed.     

Regulation of Glutamate Dehydrogenase and Glutamine Synthetase in Avocado Fruit during Development and Ripening

The activity, protein, and isoenzymic profiles of glutamate de-hydrogenase (GDH) and glutamine synthetase (GS) were studied during development and ripening of avocado (Percea americana Mill. cv Hass) fruit. During fruit development, the activity and protein content of both GDH and GS remained relatively constant. In contrast, considerable changes in these enzymes were observed during ripening of avocado fruit. The specific activity of GDH increased about 4-fold, coincident with a similar increase in GDH protein content and mRNA levels. On the other hand, GS specific activity showed a decline at the end of the ripening process. On the isoenzymic profile of GDH, changes in the prevalence of the seven isoenzymes were found, with a predominance of the more cathodal isoenzymes in the unripe and of the most anodal isoenzymes in the ripe fruit. Two-dimensional electrophoresis revealed that avocado fruit GDH consists of two subunits whose association gives rise to seven isoenzymes. The results support the view that the predominance of the more anodal isoenzymes in the overripe fruit was due to the accumulation of the [alpha]-polypeptide.     

Regulation of Glutamine Synthetase by Light and during Nitrogen Deficiency in Synchronous Chlorella sorokiniana

A method is described to achieve density labeling of proteins in unicellular algae by using ¹³CO₂. This is a satisfactory procedure especially for work on nitrogen metabolism. The increase in activity of glutamine synthetase (EC 6.3.1.2.) and glutamate synthase (EC 1.4.7.1.) in Chlorella sorokiniana mediated by a dark/light shift and by nitrogen starvation were investigated. Using the method of density labeling and isopycnic centrifugation, we demonstrated that the increase in enzyme activity after a dark/light shift is based on activation rather than de novo synthesis. The increase in enzyme activity after transfer to nitrogen-deficient medium is based both on activation and de novo synthesis.     

Expression of Two Different Glutamine Synthetase Polypeptides during Root Development in Phaseolus vulgaris L

Immunoprecipitation and two-dimensional gel electrophoresis analysis of the glutamine synthetase (GS) polypeptides (α and β) during Phaseolus vulgaris root development shows that the α polypeptide is the main component of the enzyme in the embryo and in up to 5 day old roots. From 5 days on, the β polypeptide becomes the root predominant GS monomer. The α/β ratio of the in vitro translated GS polypeptides from the total polysomal RNA isolated at different root ages correlates with the α/β ratio observed in the root extracts. These results suggest that the two root GS polypeptides are encoded by different mRNA species in Phaseolus vulgaris.     

Photophysiological and Photosynthetic Complex Changes during Iron Starvation in Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942

Iron is an essential component in many protein complexes involved in photosynthesis, but environmental iron availability is often low as oxidized forms of iron are insoluble in water. To adjust to low environmental iron levels, cyanobacteria undergo numerous changes to balance their iron budget and mitigate the physiological effects of iron depletion. We investigated changes in key protein abundances and photophysiological parameters in the model cyanobacteria Synechococcus PCC 7942 and Synechocystis PCC 6803 over a 120 hour time course of iron deprivation. The iron stress induced protein (IsiA) accumulated to high levels within 48 h of the onset of iron deprivation, reaching a molar ratio of ∼42 IsiA : Photosystem I in Synechococcus PCC 7942 and ∼12 IsiA : Photosystem I in Synechocystis PCC 6803. Concomitantly the iron-rich complexes Cytochrome b₆f and Photosystem I declined in abundance, leading to a decrease in the Photosystem I : Photosystem II ratio. Chlorophyll fluorescence analyses showed a drop in electron transport per Photosystem II in Synechococcus, but not in Synechocystis after iron depletion. We found no evidence that the accumulated IsiA contributes to light capture by Photosystem II complexes.     

Rapid Decrease in the Glutamine Synthetase Activity during Imbibition of Thermodormant New York Lettuce Seeds

A high glutamine synthetase (GS) activity was detected in dryseeds of New York lettuce but it decreased rapidly during imbibitionat 35°C. The decrease in GS activity was remarkable at 35°C,but not so at 45°C or at 25°C. The activity of extractedGS was relatively constant even at 35°C. The decrease inGS activity stopped immediately after the addition of cycloheximide(CH). This suggests the presence of a GSinactivating systemin the seeds. The amount of ammonia increased abruptly duringthe early stage of imbibition at 35°C, suggesting the blockageof ammonia-assimilation at high temperature. The GS activitythat decreased to a low level during imbibition at 35°Cfor 12 h increased again during the subsequent imbibition atlow temperature (15CC) before the break of thermodormancy. Ahigher GS activity was found in the embryonic axes than in thecotyledons. Partial purification of GS showed that lettuce seedGS was eluted as a single peak on Biogel A1.5m or DEAE-Sephacel(mol wt: 4.4 x 10⁵). Thus the thermodormancy of New York lettuce seeds may be relatedto inactivation of GS during imbibition at high temperatures,and the activity of GS in the embryonic axes may control thegermination of New York lettuce seeds through the regulationof glutamine formation. (Received May 11, 1983; Accepted September 13, 1983)