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J. Biol. Chem., Vol. 276, Issue 41, 38320-38328, October 12, 2001

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Cyanobacteria Perceive Nitrogen Status by Sensing Intracellular 2-Oxoglutarate Levels^*

M. Isabel Muro-Pastor, José C. Reyes, and Francisco J. Florencio

From the Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla-Consejo Superior de Investigaciones Científicas, Centro de Investigaciones Científicas Isla de la Cartuja, Avenida Américo Vespucio s/n, Isla de la Cartuja, E-41092 Sevilla, Spain

Received for publication, June 8, 2001, and in revised form, July 27, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The regulatory circuits that control nitrogen metabolism are relatively well known in several bacterial model groups. However, much less is understood about how the nitrogen status of the cell is perceived in vivo. In cyanobacteria, the transcription factor NtcA is required for regulation (activation or repression) of an extensive number of genes involved in nitrogen metabolism. In contrast, how NtcA activity is regulated is largely unknown. Assimilation of ammonium by most microorganisms occurs through the sequential action of two enzymes: glutamine synthetase (GS) and glutamate synthase. Interestingly, regulation of the expression of NtcA-dependent genes in the cyanobacterium Synechocystis sp. PCC 6803 is altered in mutants with modified levels of GS activity. Two types of mutants were analyzed: glnA null mutants that lack GS type I and gif mutants unable to inactivate GS in the presence of ammonium. Changes in the intracellular pools of 19 different amino acids and the keto acid 2-oxoglutarate were recorded in wild-type and mutant strains under different nitrogen conditions. Our data strongly indicate that the nitrogen status in cyanobacteria is perceived as changes in the intracellular 2-oxoglutarate pool.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

To maintain their metabolic homeostasis, bacteria must coordinate the flow rates of carbon and nitrogen assimilation. This coordination is achieved by signal transduction systems that integrate signals from the status of the carbon and nitrogen metabolism and regulate the level of glutamine synthetase (GS)¹ activity and the transcription of genes involved in the utilization of different nitrogen sources (reviewed in Refs. 1 and 2). GS catalyzes the ATP-dependent amidation of glutamate to yield glutamine. GS operates sequentially with the enzyme glutamate synthase (GOGAT), which catalyzes the transfer of the amide group from glutamine to 2-oxoglutarate to yield two molecules of glutamate. This pathway (commonly known as the GS-GOGAT cycle) represents the connecting step between carbon and nitrogen metabolism. Ammonium is the preferred nitrogen source for most of the microorganisms. In the presence of abundant carbon sources, ammonium deficiency results in a high level of GS activity, presumably to warrant the assimilation of the small amount of free ammonium available. At the same time, genes that encode enzymes or transport proteins involved in the utilization of alternative nitrogen sources are activated. On the contrary, in the presence of ammonium, GS activity is down-regulated, and genes involved in utilization of alternative sources of nitrogen are repressed (1, 2). Although the general rules outlined above are shared by most of the prokaryotes, the regulatory networks controlling nitrogen metabolism differ between the different bacterial groups.

In enteric bacteria, the global Ntr system is responsible for the control of GS activity at the transcriptional and post-transcriptional levels (reviewed in Refs. 1-5). The two-component regulatory system composed of the histidine kinase NtrB and the response regulator NtrC controls the transcription of the glnA gene. Phosphorylation/dephosphorylation of NtrC by NtrB is controlled by the signal-transducing protein PII and a uridylyltransferase (Utase/UR) that modifies the PII protein, by uridylylation, in response to the nitrogen status of the cell. Uridylyltransferase and PII proteins also control the activity of the adenylyltransferase. This enzyme carries out the covalent modification of GS by adenylylation. The adenylylated GS form is highly sensitive to feedback inhibition by a number of metabolites that contain a nitrogen atom derived from glutamine and that accumulate under nitrogen-rich conditions. Therefore, adenylylation determines that GS activity decreases dramatically. In addition to GS levels, expression of genes involved in transport and metabolism of alternative nitrogen sources is also dependent on the Ntr system.

Gram-positive bacteria lack a typical Ntr system, and there is no evidence for a post-transcriptional control of GS activity (6, 7). In Bacillus subtilis, transcription of the bicistronic operon glnRA is repressed by the GlnR transcription factor under conditions of nitrogen excess (reviewed in Ref. 7). Among other genes, GlnR also represses the expression of the transcription factor TnrA. Under conditions of nitrogen limitation, repression of glnRA and tnrA expression by GlnR is released; GS activity increases; and TnrA induces the expression of a number of genes involved in utilization of alternative nitrogen sources. Both GlnR and TnrA belong to the MerR family of DNA-binding regulatory proteins. None of them has been shown to be modified in response to nitrogen limitation or excess.

In cyanobacteria, transcription of nitrogen-regulated genes is under the control of the DNA-binding protein NtcA (recently reviewed in Ref. 8). NtcA belongs to the CRP (catabolic repressor protein) family of bacterial transcription factors. Transcription of the glnA gene is activated by NtcA in the presence of poor nitrogen sources (nitrate or dinitrogen) and under conditions of nitrogen starvation. In addition to control glnA expression, NtcA also activates transcription at a set of promoters induced in the absence of ammonium and involved in the utilization of alternative nitrogen sources. Like GlnR or TnrA, NtcA modification in response to the nitrogen status has not been shown; and therefore, how NtcA activity is controlled is unknown. In the cyanobacterium Synechocystis sp. PCC 6803, GS is inactivated upon ammonium upshift. In this case, GS activity is modulated by protein-protein interaction with two polypeptides (IF7 and IF17). Direct binding of IF7 or IF17 to GS results in the formation of an inactive GS·IF complex (9). IF7 and IF17 are homologous proteins encoded by two unlinked genes, gifA and gifB. Synechocystis gifA and gifB mutant strains are severely impaired in GS inactivation, and the double mutant gifAgifB is completely deficient in GS inactivation. Formation of the GS·IF complex seems to be determined only by the intracellular concentration of IFs. Expression of IF7 and IF17 is repressed by the transcription factor NtcA under conditions of nitrogen deficiency (10). Therefore, derepression of the gifA and gifB genes in the presence of ammonium determines the synthesis of IF7 and IF17 and the inactivation of GS.

Although the molecular mechanisms that control GS activity and synthesis are relatively well known in several bacterial model systems, such as those that have been discussed above, much less is understood about how the nitrogen status of the cell is sensed and the molecules that are involved in signaling. Many different studies with purified signal transduction components of the adenylylation cascade suggest that 2-oxoglutarate and glutamine are the signaling molecules in Escherichia coli. However, in vivo data are scarce. Early studies by Senior (11) in E. coli also indicated that intracellular levels of 2-oxoglutarate and glutamine correlate with the degree of adenylylation of GS. An exhaustive study by Kustu and co-workers (12) suggests that nitrogen limitation is perceived by Salmonella typhimurium as a decrease in the intracellular concentration of the glutamine pool. This was deduced from the direct correlation between nitrogen-limited growth rate and the intracellular concentration of glutamine. This correlation is far less obvious in B. subtilis, suggesting that other metabolites are probably involved in nitrogen sensing (13). Unfortunately, the two previously cited works did not report data on the intracellular 2-oxoglutarate pool. In cyanobacteria, it was demonstrated long ago that the ammonium-promoted repression of nitrate or dinitrogen utilization does not occur in the presence of inhibitors of the GS-GOGAT pathway (14, 15), also suggesting that metabolites related to the GS-GOGAT cycle are involved in signaling. In this work, we used two different Synechocystis mutants with altered levels of GS to study the metabolic signals involved in controlling NtcA-dependent genes. Our data strongly indicate that Synechocystis senses nitrogen status by detecting changes in the intracellular pool of 2-oxoglutarate irrespective of the intracellular glutamine and glutamate concentrations.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Strains and Growth Conditions-- Synechocystis sp. strain PCC 6803 was grown photoautotrophically at 30 °C in BG11 medium (16) supplemented with 1 g/liter NaHCO₃ (BG11C) and bubbled with a continuous stream of 1% (v/v) CO₂ in air under continuous fluorescent illumination (50 µmol of photons·m² s¹, white light). To achieve nitrogen starvation, cells were harvested at room temperature by centrifugation at 5000 × g for 10 min, washed, and resuspended in fresh medium lacking the nitrogen source (BG11₀C). For plate cultures, BG11C liquid medium was solidified by 1% (w/v) agar. When ammonium was used as nitrogen source, nitrate was replaced by 10 mM NH₄Cl, and the medium was buffered with 20 mM TES (pH 7.0).

RNA Isolation and Northern Blot Hybridization-- Total RNA was isolated from 25-ml samples of Synechocystis cultures at the mid-exponential phase (3-5 µg/ml chlorophyll). Extractions were performed by vortexing cells in the presence of phenol/chloroform and acid-washed baked glass beads (0.25-0.3-mm diameter; Braun, Melsungen, Germany) as previously described (17).

For Northern blotting, 15 µg of total RNA was loaded per lane and electrophoresed on denaturing formaldehyde-containing 1.2% agarose gels. Transfer to nylon membranes (Hybond N-plus, Amersham Pharmacia Biotech), prehybridization, hybridization, and washes were performed as recommended by the manufacturer. Probes for the genes gifA, gifB, icd, glnA, glnN, and glnB were obtained as previously described (9, 17-19). A nirA gene probe was generated by polymerase chain reaction using oligonucleotides nit1 (5'-CGTATTCCCCACGGCTTGCTCACC-3') and nit2 (5'-GACACCACTGACCGTTGCAGATTG-3'). An nblA gene probe was generated by polymerase chain reaction using oligonucleotides nbl1 (5'-GAAAGGTAGTCGCCTTGGAGGGC-3') and nbl2 (5'-CCTGTTGCAAACACTGCAGTTG-3'). As a control, in all cases, the filters were reprobed with a 580-base pair HindIII-BamHI probe from plasmid pAV1100 containing the constitutively expressed RNase P RNA gene from Synechocystis (20). To determine the cpm of radioactive areas in Northern blot hybridizations, an InstantImager electronic autoradiography apparatus (Packard Instrument Co.) was used.

GS Assay-- GS activity was determined in situ using the Mn²⁺-dependent -glutamyltransferase assay in cells permeabilized with mixed alkyltrimethylammonium bromide (21). One unit of GS activity corresponds to the amount of enzyme that catalyzes the synthesis of 1 µmol of -glutamyl hydroxamate/min.

Amino Acid Determination-- Cells from 2 ml of culture were recovered by centrifugation, and cell lysates were obtained by adding 0.45 ml of 0.2 N HCl, followed by vigorous shaking and incubation for 15 min on ice. After centrifugation, supernatant was filtered through an Ultrafree-MC 10,000 NMWL filter unit (Millipore Corp.) for deproteinization. The amino acid concentration of the deproteinized lysate was determined by HPLC using a 3.9 × 150-mm Nova-Pak C₁₈ column. Amino acids were separated using a linear gradient from 50 mM phosphate-acetate buffer (pH 7.5)/tetrahydrofuran/methanol (96:2:2) to methanol/water (65:35). The following 19 amino acids were determined (retention times given in parentheses): Asp (2.23 min), Glu (3.69 min), Asn (7.91 min), Ser (9.48 min), Gln (10.93 min), Gly (13.18 min), Arg (14.24 min), Thr (14.86 min), Ala (18.66 min), GABA (20.07 min), Tyr (22.00 min), Met (30.75 min), Val (31.91 min), Trp (33.00 min), Phe (36.48 min), Ile (39.75 min), Leu (41.28 min), Orn (46.83 min), and Lys (48.18 min).

2-Oxoglutarate Determination-- For the determination of the intracellular 2-oxoglutarate concentration, cells from 150-200-ml cultures (~1 mg of chlorophyll) were harvested by centrifugation at 10,000 × g for 8 min at 4 °C. The pellet was resuspended in 1.5 ml (final volume) of cold 0.3 M HClO₄ and incubated for 15 min on ice. The cell lysates were centrifuged at 12,000 × g for 15 min at 4 °C, and the 2-oxoglutarate in the supernatant was analyzed spectrophotometrically using glutamate dehydrogenase as described previously (11).

Western Blotting-- Glass beads crude extracts from Synechocystis grown under different conditions were made as previously described (22). Samples of cell-free extracts were subjected to SDS-polyacrylamide gel electrophoresis according to the method of Laemmli (23). Proteins were electrotransferred to a nitrocellulose sheet, and Western blotting was carried out as described (24). Purified polyclonal antibodies obtained against Synechococcus sp. PCC 6301 glutamine synthetase were used (25).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

GS Type I Is Required for Ammonium Signaling-- Two different GS-encoding genes have been found in a number of unicellular non-nitrogen-fixing cyanobacteria: glnA and glnN (26, 27). The glnA gene encodes the typical prokaryotic dodecameric GS (known as GS type I (GSI)). In the presence of a nitrogen source, Synechocystis GSI accounts for >95% of the total GS activity. The glnN gene encodes a hexameric GS (known as GS type III). GS type III is synthesized preferentially under nitrogen deprivation, and its activity is fully inhibited in the presence of ammonium (27, 28). Therefore, in ammonium-containing medium, Synechocystis glnA cells completely lack GS activity. As expected, glnA cells are unable to grow in ammonium-containing medium. As previously discussed, the transcription factor NtcA is required for regulation (activation or repression) of an extensive number of genes involved in nitrogen metabolism. To investigate the role of GSI in ammonium sensing, we analyzed the expression of three NtcA-dependent genes upon ammonium addition to nitrate-grown WT and glnA mutant cells. We selected genes that strongly change its level of expression upon ammonium upshift, such as the gifA, gifB, and nirA genes. The gifA and gifB genes encode IF7 and IF17, respectively, the inactivating factors of Synechocystis GSI. Both genes are subject to repression by NtcA in the absence of ammonium (10). Therefore, ammonium upshift provoked strong derepression of both promoters in WT cells (Fig. 1). Interestingly, ammonium-promoted derepression of both genes was severely impaired in the glnA cells. We also analyzed the expression of the nirA gene (structural gene for nitrite reductase). Transcription of the nirA gene is activated by NtcA in the absence of ammonium.² Ammonium upshift provoked a decrease in the level of nirA transcript in the case of the WT cells, but not in the case of glnA mutant cells. These results suggest that GSI activity is required for the transduction of the ammonium-promoted signal to the NtcA protein.

View larger version (42K): [in this window] [in a new window]   Fig. 1.   Absence of GSI alters NtcA-dependent gene expression. A, ammonium chloride (10 mM) was added to nitrate-grown Synechocystis WT and glnA cultures at t = 0, and samples were taken at the indicated times (minutes) for total RNA isolation. 15 µg of total RNA was denatured; separated by electrophoresis on 1.2% agarose gel; blotted; and hybridized with gifA, gifB, or nirA probes (see "Experimental Procedures"). B, the filters were stripped and rehybridized with an rnpB gene probe. The levels of gifA, gifB, and nirA mRNAs in the WT () and in mutant () cells were quantified and normalized with the rnpB signal, and plots of relative mRNA levels versus time were drawn. Each experiment was repeated at least twice. One representative experiment is shown.

Ammonium-dependent Hyperrepression of NtcA-dependent Genes in gifAgifB Mutants-- Thereafter, we examined the behavior of NtcA-dependent genes in a strain with constitutive levels of GSI activity. We have previously shown that gifAgifB cells are unable to inactivate GSI in response to ammonium upshift (9). However, gifA, gifB, and gifAgifB mutant cells showed little growth defect using either nitrate (a poor nitrogen source for Synechocystis) or ammonium as nitrogen source (data not shown). We have previously shown that the level of GSI activity in steady-state ammonium-grown WT cells is ~5-10% of the level in nitrate-grown cells. In contrast, glnA mRNA and GSI protein levels found in ammonium-grown cells were ~50% of the level found in nitrate-grown cells (Fig. 2, B and C) (19, 21, 29). These data indicate that most of the GSI is inactive in WT cells grown in ammonium-containing medium. Since gifAgifB cells lack the GSI inactivation system, a high GSI activity level was expected under these conditions. However, gifAgifB cells displayed only ~2-fold more GSI activity than WT cells (Fig. 2A). To investigate how gifAgifB cells are able to down-regulate GSI activity levels in the presence of ammonium, Northern and Western blot experiments were carried out to determine the amount of the glnA transcript and GSI protein, respectively. As shown in Fig. 2 (B and C), the levels of both the glnA transcript and GSI protein were strongly down-regulated in gifAgifB cells. A clear but less severe reduction in the level of the glnA transcript was also observed in gifA and gifB cells.

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Altered levels of glnA expression in gifAgifB mutants. GS activity and glnA transcript and GS protein levels were determined in nitrate- or ammonium-grown Synechocystis WT, gifA, gifB, and gifAgifB cells. Samples for the three determinations were taken from the same cultures. A, GS transferase activity from the different cultures was determined in situ as described under "Experimental Procedures." The average of three independent determinations is shown. 100% corresponds to 1.3 units/mg of total protein. Error bars correspond to S.D. B, the level of glnA mRNA was determined by Northern blotting. For this, total RNA from the different cultures was isolated, processed as described for Fig. 1, and hybridized with a glnA probe. The filter was stripped and rehybridized with an rnpB gene probe. The levels of glnA mRNA were quantified, normalized with the rnpB signal, and plotted. C, the level of GS protein was determined by Western blotting using anti-GSI antibodies.

The addition of ammonium to nitrate-grown cultures provoked a significant decrease in the growth rate of gifAgifB cells, but not in WT cells (Fig. 3A). Fig. 3B shows the kinetics of the glnA transcript level upon ammonium upshift. In WT cells, the glnA transcript dropped dramatically to almost undetectable levels and then quickly recovered to ~40% of the original level. However, in gifAgifB cells, the level of the glnA transcript remained undetectable 24 h after the ammonium shift. To verify whether the ammonium-promoted hyperrepression found for the glnA gene in gifAgifB cells occurred also for other NtcA-dependent genes, we analyzed the ammonium-promoted repression of the nirA and icd (encoding isocitrate dehydrogenase) genes. Interestingly, expression of the nirA gene was also hyperrepressed by ammonium in the gifAgifB strain. In contrast, the levels of the icd transcript did not change significantly upon ammonium upshift in either WT or gifAgifB cells (Fig. 3, B and C). In fact, it has previously been shown that NtcA induces transcription of the icd gene only under conditions of nitrogen deprivation (18) (see below).

View larger version (31K): [in this window] [in a new window]   Fig. 3.   Ammonium-promoted hyperrepression of NtcA-dependent genes in gifAgifB cells. A, shown is the effect on growth of the addition of 10 mM ammonium chloride to nitrate-grown Synechocystis WT and gifAgifB cells. Samples for chlorophyll determination were taken at the indicated times. B, the transcript levels of ammonium-repressed genes upon ammonium upshift were determined. Samples for total RNA isolation were taken at the indicated times upon ammonium addition to nitrate (NO)-grown WT and gifAgifB mutant cells. Total RNA was processed as described for Fig. 1 and hybridized with DNA probes for the glnA, nirA, and icd genes (see "Experimental Procedures"). C, the filters were stripped and rehybridized with an rnpB gene probe. The levels of gifA, gifB, and nirA mRNAs in the WT () and mutant () cells were quantified and normalized with the rnpB signal, and plots of relative mRNA levels versus time were drawn. The levels of the rnpB transcript are also plotted as a control. Each experiment was repeated at least twice. One representative experiment is shown.

Alteration of Metabolite Pools in gifAgifB and glnA Mutants-- To investigate whether alterations in the expression patterns of NtcA-dependent genes can be correlated with changes in the intracellular concentration of metabolites, we determined the intracellular pools of several amino acids and of 2-oxoglutarate. We have previously shown that the addition of ammonium to nitrate-grown Synechocystis cells provokes a quick and dramatic change in the intracellular pools of glutamate and glutamine (21). A few seconds after the addition of ammonium, the glutamine concentration increases ~30-40-fold with a concomitant decrease in the glutamate pool. About 5-10 min later, the glutamate and glutamine pools start to revert to the steady-state level, and normal levels are completely restored ~30 min after ammonium shift. It is worth noting that changes in the glutamate and glutamine pools are dramatically attenuated in glnA mutant cells, indicating that GSI is responsible for the changes in concentration observed in the WT strain (28). We have previously proposed that restoration of the glutamate and glutamine concentrations to the steady-state levels is the consequence of the GSI inactivation mechanism (21). If this is true, the amount of glutamine in gifAgifB cells should increase endlessly upon the ammonium upshift. We have monitored the intracellular concentration of 19 amino acids (see "Experimental Procedures") and the keto acid 2-oxoglutarate after ammonium addition to nitrate-grown cells. 1 min after ammonium shift, the level of glutamine increased ~30-fold in both WT and gifAgifB cells. At the same time, the glutamate and GABA pool sizes decreased ~3- and 6-fold, respectively (Fig. 4A). In the WT cells, the pools of glutamate, glutamine, and GABA were restored gradually during the following hour. However, in gifAgifB cells, the pools of these three amino acids remained altered at least during the following 24 h. The glutamine pool decreased partially over a few minutes after reaching the top level and then increased continuously during the 4-h post-ammonium shift. The intracellular glutamine pool reached concentrations as high as 197.9 nmol/mg of protein (~40 mM), but glutamine was not significantly excreted into the medium. 16 h post-ammonium shift, the glutamine pool initiated a very slow decrease, but 24 h later, the concentration was still ~50 nmol/mg of protein (~17-fold higher concentration than in WT cells under the same conditions) (data not shown). The glutamate and GABA pools in gifAgifB cells remained at low levels (12.3 ± 2.2 and 13.0 ± 1.6 nmol/mg of protein, respectively) at least during the first 24 h post-ammonium shift (Fig. 4 and data not shown). These data demonstrate that at least one of the physiological functions of the GSI inactivation system in cyanobacteria is to restore amino acid homeostasis upon strong changes in the availability of nitrogen.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Change in intracellular glutamine, glutamate, GABA, and 2-oxoglutarate pools upon ammonium upshift. Intracellular amino acid (aa) pools were determined from the same cultures that were used for RNA isolation in Fig. 3. For this, 2-ml samples were taken before ammonium addition (t = 0) and at the indicated times after ammonium addition. Amino acid pools were determined as described under "Experimental Procedures." Intracellular concentrations of glutamine (GLN) (), glutamate (GLU) (), and GABA () relative to total protein in WT (A) and gifAgifB (B) cells are shown. For 2-oxoglutarate determination, 200-ml culture samples were taken before ammonium addition (NO) and 15 min and 1 h after ammonium addition (C). The 2-oxoglutarate concentration was determined as described under "Experimental Procedures." The intracellular concentration of 2-oxoglutarate relative to total protein is shown. Values plotted are the average of at least two independent experiments. S.D. values were never higher than 10% of the value.

Thereafter, we determined the change in the intracellular pool of 2-oxoglutarate in the WT, gifAgifB, and glnA strains upon the addition of ammonium to nitrate-grown cells. In WT cells, the intracellular concentration of 2-oxoglutarate decreased dramatically 15 min upon ammonium upshift. However, 1 h after the shift, the 2-oxoglutarate concentration recovered to ~40% of the original level. In the gifAgifB cells, the level of 2-oxoglutarate was low (0.22 ± 0.05 nmol/mg of protein) even in nitrate-grown cells. As in the WT strain, the addition of ammonium provoked a decrease in the intracellular pool of 2-oxoglutarate. However and in contrast to the WT strain, the steady-state levels of the keto acid were not recovered 1 h later in this strain. Finally, in the glnA strain, the 2-oxoglutarate pool remained high at least 1 h after ammonium upshift.

Induction of NtcA-dependent Genes during Nitrogen Deprivation in WT and gifAgifB Mutant Cells-- To investigate the role of the glutamine pool in nitrogen signaling, we analyzed the induction of NtcA-dependent genes in the presence of a high intracellular pool of glutamine. For this, we took advantage of the elevated glutamine concentration of the gifAgifB cells upon ammonium upshift. Nitrate-grown WT and gifAgifB cells were subjected to ammonium upshift over 4 h and subsequently transferred to nitrogen-free medium. The kinetics of induction of five NtcA-activated genes were analyzed by Northern blotting. In this case, we selected genes that are strongly induced under nitrogen deficiency, such as glnA, glnN (encoding GS type III) (19), glnB (encoding the signaling protein PII) (17), icd (18), and nblA (encoding a small polypeptide required for phycobiliprotein degradation during nitrogen starvation) (30, 31).² For all the tested genes, accumulation of mRNA was significantly or severely delayed in the gifAgifB cells compared with the WT cells (Fig. 5). Furthermore, none of the analyzed transcripts reached the WT levels even 4 h after induction. We also studied the intracellular pools of metabolites under these conditions (Fig. 6). After the first 15 min of nitrogen deprivation, only the glutamine pool changed significantly in the WT strain, decreasing from 3.17 to 0.76 nmol/mg of protein. In the gifAgifB cells, nitrogen starvation provoked a fast mobilization of the large glutamine pool. Thus, the glutamine concentration decreased from 92.85 ± 7.01 to 2.76 ± 0.78 nmol/mg of protein in only 15 min. The concomitant increase in the glutamate, GABA, and arginine pools suggests that glutamine was quickly transformed into glutamate by the GOGAT enzyme and that the glutamate was then metabolized to GABA and arginine. Interestingly, 2 h after nitrogen deprivation, the level of induction of NtcA-dependent genes was lower in gifAgifB cells than in WT cells; however, the glutamine pool was similar in both strains at that time (see Fig. 5). This argues against glutamine as the signaling metabolite. We then analyzed the level of 2-oxoglutarate under these conditions. As shown in Fig. 6C, the 2-oxoglutarate pool increased significantly after transferring the WT cells to nitrogen-free medium. Importantly, the level of 2-oxoglutarate remained almost unchanged in the gifAgifB cells, at least during the first 2 h of nitrogen deprivation. This result suggests that an increase in the 2-oxoglutarate pool is involved in signaling the nitrogen deficiency status.

View larger version (38K): [in this window] [in a new window]   Fig. 5.   Induction of NtcA-dependent genes in WT and gifAgifB mutant cells under conditions of nitrogen starvation. A, 10 mM ammonium chloride was added to exponentially growing WT and gifAgifB cells using nitrate as the nitrogen source. After 4 h, cells were recovered by centrifugation and washed with and resuspended in nitrogen-free BG110C medium. Samples for total RNA isolation were taken before transferring the cells to nitrogen-free medium (NH) and at the indicated times (minutes) of nitrogen starvation (N). Total RNA was processed as described for Fig. 1 and hybridized with probes of the genes glnA, glnB, glnN, icd, and nblA. B, the filters were stripped and rehybridized with an rnpB gene probe. The levels of the different transcripts in the WT () and mutant () cells were quantified and normalized with the rnpB signal, and plots of relative mRNA levels versus time were drawn. Each experiment was repeated at least twice. One representative experiment is shown.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Change in the intracellular pools of glutamine, glutamate, GABA, arginine, and 2-oxoglutarate under conditions of nitrogen deficiency. Intracellular amino acid (aa) pools were determined from the same cultures that were used for RNA isolation in Fig. 5. For this, 2-ml samples were taken before transferring the cells to nitrogen-free medium (t = 0) and at the indicated times of nitrogen starvation. Amino acid pools were determined as described under "Experimental Procedures." Intracellular concentrations of glutamine (GLN) (), glutamate (GLU) (), arginine (ARG) (), and GABA () relative to total protein in the WT (A) and gifAgifB (B) cells are shown. For 2-oxoglutarate determination, 200-ml culture samples were taken before transferring the cells to nitrogen-free medium (NH) and 15 min and 2 h after nitrogen deprivation (N) (C). The 2-oxoglutarate concentration was determined as described under "Experimental Procedures." The intracellular concentration of 2-oxoglutarate relative to total protein is shown. Values plotted are the average of at least two independent experiments. S.D. values were never higher than 10% of the value.

Although the previous experiment excluded glutamine as the sole nitrogen-signaling molecule in cyanobacteria, we could not rule out the possibility that the 2-oxoglutarate/glutamine ratio is involved in sensing. We also analyzed the induction of NtcA-dependent genes in WT and gifAgifB cells under conditions in which the 2-oxoglutarate pool was similar in both strains, but the 2-oxoglutarate/glutamine ratio was very different. For this, glutamine mobilization was abolished by inhibition of the GOGAT enzyme with the glutamine analog DON. Thus, nitrate-grown WT and gifAgifB cells were subjected to ammonium upshift over 4 h and subsequently transferred to nitrogen-free medium in the presence of DON. As expected, 2-oxoglutarate accumulated quickly and similarly in both WT and gifAgifB cells (Fig. 7C). However, the glutamine pool was 4-fold higher in the gifAgifB cells than in the WT cells; and therefore, the 2-oxoglutarate/glutamine ratio was 4-fold lower in the gifAgifB cells than in the WT cells (Fig. 7, A and C). Remarkably, induction of the NtcA-dependent genes was very similar in both WT and gifAgifB cells under these conditions (Fig. 8, A and B). Thus, the NtcA-dependent genes were strongly and quickly induced despite the high concentration of glutamine found in the gifAgifB cells. These results suggest that cyanobacteria sense nitrogen deprivation as an increase in the intracellular pool of 2-oxoglutarate irrespective of the intracellular glutamine concentration.

View larger version (17K): [in this window] [in a new window]   Fig. 7.   Change in the intracellular pools of glutamine, glutamate, GABA, arginine, and 2-oxoglutarate under conditions of nitrogen deficiency in the presence of DON. Intracellular amino acid (aa) pools were determined from the same cultures that were used for RNA isolation in Fig. 8. For this, 2-ml samples were taken before transferring the cells to nitrogen-free medium (t = 0) and at the indicated times after transferring the cells to BG110C medium containing 100 µM DON. Amino acid pools were determined as described under "Experimental Procedures." Intracellular concentrations of (GLN) (), glutamate (GLU) (), arginine (ARG) (), and GABA () relative to total protein in the WT (A) and gifAgifB (B) cells are shown. For 2-oxoglutarate determination, 200-ml culture samples were taken before transferring the cells to nitrogen-free medium (NH) and after 15 min and 2 h of DON treatment in BG110C medium (C). The 2-oxoglutarate concentration was determined as described under "Experimental Procedures." The intracellular concentration of 2-oxoglutarate relative to total protein is shown. N, nitrogen starvation.

View larger version (36K): [in this window] [in a new window]   Fig. 8.   Effect of DON on the induction of NtcA-dependent genes under conditions of nitrogen deficiency. A, 10 mM ammonium chloride was added to exponentially growing WT and gifAgifB cells using nitrate as the nitrogen source. 4 h later, cells were recovered by centrifugation, washed with nitrogen-free medium (BG110C), and resuspended in BG110C medium supplemented with 100 µM DON. Samples for total RNA isolation were taken before DON treatment (NH) and at the indicated times (minutes and hours) after transferring the cells to DON-containing medium. Total RNA was processed as described for Fig. 1 and hybridized with probes of the genes glnA, glnB, glnN, and nblA. B, the filters were stripped and rehybridized with an rnpB gene probe. The levels of the different transcripts in the WT () and mutant () cells were quantified and normalized with the rnpB signal, and plots of relative mRNA levels versus time were drawn. Each experiment was repeated at least twice. One representative experiment is shown. N, nitrogen starvation.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

To quickly perceive and adapt to changes in the environment, microorganisms have efficient systems for sensing and signal transduction. Many of these sensing elements are placed in the cytoplasmic membrane, which seems a logical location to monitor changes in the external environment (32). Interestingly, what we know at the moment about the nitrogen-sensing systems in bacteria indicates that perception of the nitrogen availability is carried out intracellularly. This is probably due to the fact that nitrogen control systems integrate signals from carbon and nitrogen metabolism. Therefore, in the process of nitrogen sensing, changes in the extracellular nitrogen concentration (or in the nitrogen form) provoke changes in the concentration of intracellular metabolites that are perceived by the sensing systems. Determination of the metabolite(s) involved in nitrogen sensing has been a focus of intense research efforts for >3 decades (2, 12, 13). In enteric bacteria, an extensive number of studies carried out with purified signal transduction components and a small number of in vivo studies indicate that both 2-oxoglutarate and glutamine are the most important signaling molecules.

We have previously shown that in a Synechocystis glnA mutant, nitrate and nitrite reductase activities are not repressed by ammonium, and the short-term ammonium-promoted inhibition of nitrate uptake is impaired (28). Here we show that ammonium-dependent derepression of genes repressed by NtcA such as gifA and gifB and ammonium-dependent repression of a gene activated by NtcA such as nirA are severely impaired in the same glnA mutant strain. Previous studies have reported that expression of the nirAnrtABCDnarB operon from Synechococcus sp. PCC 7942, which is activated by NtcA, is also deregulated in cells treated with the GS inhibitor L-methionine, D-L-sulfoximine (33). Taken together, these data suggest that transduction of the ammonium-promoted signal to NtcA requires ammonium incorporation into carbon skeletons through the GS reaction. These data point to glutamine or some metabolite enzymatically linked to glutamine as the nitrogen-signaling molecule in cyanobacteria.

We have reported that in the absence of GSI, ammonium upshift causes a minor change in the glutamine and glutamate pools (28). This is in agreement with the minor change that we observe in the 2-oxoglutarate pool for the glnA mutant under the same conditions (Fig. 4b). Thus, in the glnA strain, the shift from a poor nitrogen source to ammonium, a rich nitrogen source, provokes little alteration of the metabolite pools and, as a consequence, deficient nitrogen signaling. Similar signaling defects have been previously reported for glnA mutants in enteric bacteria (see, for example, Ref. 12) and in B. subtilis (reviewed in Ref. 7).

In contrast to the ammonium insensitivity of the glnA strain, the gifAgifB cells displayed a strong ammonium-promoted hyperrepression of NtcA-dependent genes. In the WT strain, the levels of the glnA and nirA transcripts decreased 10- and 5-fold, respectively, 15 min after ammonium shift. However, the mRNA levels of both genes recovered to almost 40% of the original level 45 min later, and this level was maintained at least during the following 24 h. Interestingly, in the gifAgifB cells, the levels of both transcripts remained very low not only during the first 15 min, but also during the subsequent 24 h. Therefore, it seems that the metabolic state found in the WT cells during the first 15 min is frozen in the gifAgifB cells for at least 24 h.

Hence, we analyzed the concentration of metabolites in the first 15 min upon the ammonium shift. 2 min after the shift, glutamine had reached the maximum concentration that coincided with the minimal glutamate and GABA concentrations in the WT cells. Afterward, pools of these three amino acids evolved in the sense of recovering the original levels. Therefore, glutamate was synthesized from glutamine and 2-oxoglutarate by the GOGAT reaction. This also caused a strong decrease in the 2-oxoglutarate pool (Fig. 4). After 1 h, when the amino acid pools were restored, the 2-oxoglutarate pool started to increase. This situation is the consequence of the inactivation of GSI by IF7 and IF17. This was proven by the fact that in the gifAgifB strain, amino acid pools were not recovered, and glutamine accumulated to high levels. In these cells, the GOGAT reaction also worked efficiently, but the glutamate concentration did not increase because GSI could not be inactivated. In fact, gifAgifB cells consumed ammonium at higher rate than WT cells (data not shown). Since ammonium was not limiting and GSI was not down-regulated, the limiting substrate for the GS-GOGAT cycle under these conditions was the 2-oxoglutarate. As a consequence, the 2-oxoglutarate concentration remained low several hours after the shift (Fig. 4). Therefore, the metabolic situation characterized by a high concentration of glutamine and a low concentration of glutamate, GABA, and 2-oxoglutarate correlates with a low level of expression of NtcA-induced genes.

NtcA-activated genes were induced within the first 15 min of nitrogen starvation (Fig. 5). In fact, metabolites involved in nitrogen signaling should change within this short period. Most of the monitored amino acids, including glutamate and GABA, did not change significantly during this interval of time. However, the glutamine pool decreased, and the 2-oxoglutarate pool increased dramatically under these conditions, suggesting that they may be involved in signaling. The use of the gifAgifB strain allowed us to study nitrogen signaling in cells with a pool of glutamine 30-40-fold higher than that in WT cells. When these cells were subjected to ammonium upshift to increase the concentration of glutamine and then to nitrogen starvation, the glutamine concentration dropped quickly to levels similar to those found in the WT strain; however, accumulation of NtcA-induced transcripts was clearly retarded. Glutamine was immediately mobilized to glutamate, indicating a large demand of 2-oxoglutarate for the GOGAT enzyme. As a consequence, 2-oxoglutarate did not accumulate in the gifAgifB cells, in contrast to what happened in the WT cells (see Fig. 6). Furthermore, when mobilization of glutamine to glutamate was prevented by the use of a GOGAT inhibitor and, hence, 2-oxoglutarate equally accumulated in WT and gifAgifB cells, the kinetics of accumulation of NtcA-induced transcripts were very similar in both strains, despite the high level of glutamine in the gifAgifB cells. We reached two conclusions from these data. First, 2-oxoglutarate is the metabolite related to the GS-GOGAT pathway involved in signaling. Thus, nitrogen deficiency is perceived as an increase in the intracellular 2-oxoglutarate concentration; and inversely, nitrogen excess is perceived as a decrease in the intracellular 2-oxoglutarate pool. Second, the intracellular glutamine pool is not involved in nitrogen signaling.

These results contrast with the enterobacterial and Bacillus systems, where both 2-oxoglutarate and glutamine pools seem to be involved in nitrogen sensing (12, 13). The cyanobacterial signaling system seems to differ also from the fungi and plants systems, where the glutamine pool has also been implicated in nitrogen signaling (34, 35). In which way do cyanobacteria differ concerning 2-oxoglutarate metabolism? 2-Oxoglutarate is the product of the reaction catalyzed by isocitrate dehydrogenase, an enzyme of the tricarboxylic acid cycle. Cyanobacteria have an incomplete tricarboxylic acid cycle, lacking the 2-oxoglutarate dehydrogenase enzyme complex (36). Therefore, the 2-oxoglutarate produced in the isocitrate dehydrogenase reaction cannot be further oxidized, and it directly enters the GS-GOGAT cycle as the carbon skeleton for nitrogen incorporation. Interestingly, expression of the icd gene is induced by NtcA only under conditions of nitrogen starvation (Ref. 18; see also Fig. 5), raising the question as to why the synthesis of carbon skeletons is increased when nitrogen is scarce. One possibility, supported by the results presented in this work, is that an increase in the isocitrate dehydrogenase activity under nitrogen deprivation is a positive feedback mechanism intended for generating higher and higher levels of the nitrogen starvation-signaling molecule, 2-oxoglutarate.

How changes in the 2-oxoglutarate pool affect the activity of NtcA is presently unknown. Importantly, Herrero et al. (8) have reported, in a recent review, preliminary data suggesting that the affinity of NtcA for the Synechococcus sp. PCC 7942 glnA promoter increases in the presence of 2-oxoglutarate in vitro. However, a role of 2-oxoglutarate as an allosteric effector of another signal transduction protein that modifies NtcA cannot be ruled out. An obvious candidate is the PII protein, which is one of the key components of the enterobacterial Ntr system and which is known to bind 2-oxoglutarate (37). In cyanobacteria, the PII protein has been shown to be involved in controlling the ammonium-mediated inhibition of nitrate uptake in the cyanobacterium Synechococcus sp. PCC 7942 (38, 39). However, the phenotype of a glnB mutant of Synechococcus sp. PCC 7942 suggests that the PII protein does not modulate the activity of NtcA. Further experiments are required to determine how changes in the 2-oxoglutarate pool modulate the transcriptional activity of NtcA.

	ACKNOWLEDGEMENTS

We thank Rocío Rodríguez for amino acid determination by HPLC. We are grateful to Marika Lindahl and Mario García-Domínguez for critical reading of the manuscript.

	FOOTNOTES

^* This work was supported by Grant PB97-0732 from Direccion General de Enseñanza Superior e Investigacion Científica and by Junta de Andalucía (Group CV1-0112).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 34-5-4489518; Fax: 34-5-4620154; E-mail: floren@cica.es.

Published, JBC Papers in Press, July 30, 2001, DOI 10.1074/jbc.M105297200

² M. I. Muro-Pastor, unpublished data.

	ABBREVIATIONS

The abbreviations used are: GS, glutamine synthetase; GSI, glutamine synthetase type I; GOGAT, glutamate synthase; IF, inactivating factor; TES, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid; HPLC, high pressure liquid chromatography; GABA, -aminobutyric acid; WT, wild-type; DON, 6-diazo-5-oxo-L-norleucine; Ntr, nitrogen regulatory.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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