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.     

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.     

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.     

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.     

Unique organization of the dnaA region from Prochlorococcus marinus CCMP1375, a marine cyanobacterium

In order to study DNA replication control elements in cyanobacteria we cloned and sequenced the dnaA gene from the marine cyanobacterium Prochlorococcus marinus. The dnaA gene is ubiquitous among bacteria and encodes the DNA replication initiation factor DnaA. The deduced amino acid sequence of the P. marinus DnaA protein shows highest similarity to the DnaA protein from the freshwater cyanobacterium Synechocystis sp. PCC6803. Using a solid-phase DNA binding assay we demonstrated that both cyanobacterial DnaA proteins specifically recognize chromosomal origins, oriC, of Escherichia coli and Bacillus subtilis in vitro. The genetic environment of dnaA is not conserved between the two cyanobacteria. Upstream of the P. marinusdnaA gene we identified a gene encoding a putative ATP-binding cassette (ABC) transport protein. The gor gene encoding glutathione reductase lies downstream of dnaA. Comparison of the genetic structure of dnaA regions from 15 representative bacteria shows that the pattern of genes flanking dnaA is not universally conserved among them.     

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.     

FtsZ degradation in the cyanobacterium Anabaena sp. strain PCC 7120

In prokaryotes, cell division is normally achieved by binary fission, and the key player FtsZ is considered essential for the complete process. In cyanobacteria, much remains unknown about several aspects of cell division, including the identity and mechanism of the various components involved in the division process. Here, we report results obtained from a search of the players implicated in cell division, directly associating to FtsZ in the filamentous, heterocyst-forming cyanobacterium Anabaena sp. PCC 7120. Histidine tag pull-downs were used to address this question. However, the main observation was that FtsZ is a target of proteolysis. Experiments using various cell-free extracts, an unrelated protein, and protein blot analyses further supported the idea that FtsZ is proteolytically cleaved in a specific manner. In addition, we show evidence that both FtsZ termini seem to be equally prone to proteolysis. Taken together, our data suggest the presence of an unknown player in cyanobacterial cell division, opening up the possibility to investigate novel mechanisms to control cell division in Anabaena PCC 7120.     

Genome reduction by deletion of paralogs in the marine cyanobacterium Prochlorococcus

Several isolates of the marine cyanobacterial genus Prochlorococcus have smaller genome sizes than those of the closely related genus Synechococcus. In order to test whether loss of protein-coding genes has contributed to genome size reduction in Prochlorococcus, we reconstructed events of gene family evolution over a strongly supported phylogeny of 12 Prochlorococcus genomes and 9 Synechococcus genomes. Significantly, more events both of loss of paralogs within gene families and of loss of entire gene families occurred in Prochlorococcus than in Synechococcus. The number of nonancestral gene families in genomes of both genera was positively correlated with the extent of genomic islands (GIs), consistent with the hypothesis that horizontal gene transfer (HGT) is associated with GIs. However, even when only isolates with comparable extents of GIs were compared, significantly more events of gene family loss and of paralog loss were seen in Prochlorococcus than in Synechococcus, implying that HGT is not the primary reason for the genome size difference between the two genera.     

A quantitative proteomic analysis of light adaptation in a globally significant marine cyanobacterium Prochlorococcus marinus MED4

In this study, we conducted biological and technical replicate proteomic experiments using isobaric tags for relative and absolute quantification (iTRAQ), to elucidate the light adaptation strategies of Prochlorococcus marinus MED4. The MED4 strain is adapted to an oceanic environment characterized by low nutrient levels, and ever-changing light intensities. Approximately 11% of the proteome was identified, with an average coefficient of variation of iTRAQ quantification values of 0.15. Fifteen proteins were deemed to be statistically and significantly differentially expressed in changing light intensities, particularly the down-regulation of photosystem-related proteins, and the up-regulation of the stress-related chaperone GroEL in high light compared to low light.     

ADP-ribosylation of glutamine synthetase in the cyanobacterium Synechocystis sp. strain PCC 6803.

Glutamine synthetase (GS) inactivation was observed in crude cell extracts and in the high-speed supernatant fraction from the cyanobacterium Synechocystis sp. strain PCC 6803 following the addition of ammonium ions, glutamine, or glutamate. Dialysis of the high-speed supernatant resulted in loss of inactivation activity, but this could be restored by the addition of NADH, NADPH, or NADP+ and, to a lesser extent, NAD+, suggesting that inactivation of GS involved ADP-ribosylation. This form of modification was confirmed both by labelling experiments using [32P]NAD+ and by chemical analysis of the hydrolyzed enzyme. Three different forms of GS, exhibiting no activity, biosynthetic activity only, or transferase activity only, could be resolved by chromatography, and the differences in activity were correlated with the extent of the modification. Both biosynthetic and transferase activities were restored to the completely inactive form of GS by treatment with phosphodiesterase.     

Glutamine synthetase from the marine cyanobacteria Prochlorococcus spp: characterization,phylogeny and response to nutrient limitation

The regulation of glutamine synthetase (EC 6.3.1.2) from Prochlorococcus was previously shown to exhibit unusual features: it is not upregulated by nitrogen starvation and it is not inactivated by darkness (El Alaoui et al. (2001) Appl Environ Microbiol 67: 2202-2207). These are probably caused by adaptations to oligotrophic environments, as confirmed in this work by the marked decrease in the enzymatic activity when cultures were subjected to iron or phosphorus starvation. In order to further understand the adaptive features of ammonium assimilation in this cyanobacterium, glutamine synthetase was purified from two Prochlorococcus strains: PCC 9511 (high-light adapted) and SS120 (low-light adapted). We obtained approximately 100-fold purified samples of glutamine synthetase electrophoretically homogeneous, with a yield of approximately 30%. The estimated molecular mass of the subunits was roughly the same for both strains: 48.3 kDa. The apparent Km constants for the biosynthetic activity were 0.30 mM for ammonium, 1.29 mM for glutamate and 1.35 mM for ATP; the optimum pH was 8.0. Optimal temperature was surprisingly high (55 degrees C). Phylogenetic analysis of glnA from three Prochlorococcus strains (MED4, MIT9313 and SS120) showed they group closely with marine Synechococcus isolates, in good agreement with other studies based on 16 S RNA sequences. All of our results suggest that the structure and kinetics of glutamine synthetase in Prochlorococcus have not been significantly modified during the evolution within the cyanobacterial radiation, in sharp contrast with its regulatory properties.     

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.     

Extracellular degradation of phenol by the cyanobacterium Synechococcus PCC 7002

Investigations of the unicellular marine cyanobacterium Synechococcus PCC 7002 revealed its ability to metabolize phenol under non-photosynthetic conditions up to 100 mg L^–1. Under continuous light, photoautotrophic growth was reduced only slightly in the presence of this phenol concentration, but no transformation was observed. However neither under photoautotrophic nor heterotrophic conditions were the cells able to use phenol for growth. During the degradation of phenol in the dark cis,cis-muconic acid was produced as the major product, which was identified by gas chromatography/mass spectrometry. This result was confirmed by an identical absorption spectrum and an identical retention time in high performance liquid chromatographic analysis with authentic muconic acid as standard. This provides the first record for an ortho-fission of a phenolic substance by cyanobacteria. Further investigations of the breakdown mechanism of phenol have shown that the transformation is an extracellular process inhibited by heat, proteases and metal ions and is probably catalyzed by a protein.     

Glutamine synthetase from the green alga Monoraphidium braunii is regulated by oxidative modification

Monoraphidium braunii glutamine synthetase is inactivated by several mixed-function oxidation systems. Inactivation requires oxygen and a metal cation as it does not take place under anaerobic conditions or in the presence of EDTA. Glutamine synthetase can be protected against that inactivation by peroxidase and catalase but not by superoxide dismutase indicating that hydrogen peroxide is involved in the process, although hydrogen peroxide is not itself effective. The oxidative modification of glutamine synthetase renders the protein more sensitive to temperature and susceptible to proteolytic attack. This has been demonstrated by measuring by quantitative immunoelectrophoresis the levels of glutamine synthetase antigen, in enzymatic preparations treated with different oxidation systems. Besides, immunoblotting of crude extracts in the presence of mixed-function oxidation systems shows the disappearance of material cross-reacting with anti-glutamine synthetase antibodies. Other results show that glutamine synthetase from Chlamydomonas reinhardtii could be subjected to the same kind of oxidative inactivation. The possible regulatory role of oxidative modification of glutamine synthetase in green algae is discussed.     

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.