The Nitrogen-regulated Response Regulator NrrA Controls Cyanophycin Synthesis and Glycogen Catabolism in the Cyanobacterium Synechocystis sp. PCC 6803*

Background: Cyanobacterial metabolism is extensively regulated in response to nitrogen limitation. Results: The regulon of transcriptional factor NrrA was reconstructed in the genomes of diverse cyanobacteria and experimentally characterized in Synechocystis. Conclusion: NrrA controls cyanophycin accumulation and glycogen catabolism in Synechocystis. Significance: A molecular mechanism coordinately regulating synthesis and degradation of nitrogen and carbon reserves in cyanobacteria is identified. The cellular metabolism in cyanobacteria is extensively regulated in response to changes of environmental nitrogen availability. Multiple regulators are involved in this process, including a nitrogen-regulated response regulator NrrA. However, the regulatory role of NrrA in most cyanobacteria remains to be elucidated. In this study, we combined a comparative genomic reconstruction of NrrA regulons in 15 diverse cyanobacterial species with detailed experimental characterization of NrrA-mediated regulation in Synechocystis sp. PCC 6803. The reconstructed NrrA regulons in most species included the genes involved in glycogen catabolism, central carbon metabolism, amino acid biosynthesis, and protein degradation. A predicted NrrA-binding motif consisting of two direct repeats of TG(T/A)CA separated by an 8-bp A/T-rich spacer was verified by in vitro binding assays with purified NrrA protein. The predicted target genes of NrrA in Synechocystis sp. PCC 6803 were experimentally validated by comparing the transcript levels and enzyme activities between the wild-type and nrrA-inactivated mutant strains. The effect of NrrA deficiency on intracellular contents of arginine, cyanophycin, and glycogen was studied. Severe impairments in arginine synthesis and cyanophycin accumulation were observed in the nrrA-inactivated mutant. The nrrA inactivation also resulted in a significantly decreased rate of glycogen degradation. Our results indicate that by directly up-regulating expression of the genes involved in arginine synthesis, glycogen degradation, and glycolysis, NrrA controls cyanophycin accumulation and glycogen catabolism in Synechocystis sp. PCC 6803. It is suggested that NrrA plays a role in coordinating the synthesis and degradation of nitrogen and carbon reserves in cyanobacteria.

Cyanobacteria are a large group of oxygenic photosynthetic prokaryotes that are found in diverse ecological habitats. In many of these habitats, nitrogen is limiting and cyanobacteria are exposed to periods of severe nitrogen starvation (1). To survive under such conditions, they have evolved sophisticated mechanisms to sense and respond to nitrogen limitation, including induction of the systems for high-affinity uptake of nitrogen-containing compounds (2). During nitrogen starvation, non-diazotrophic cyanobacteria may consume internal stores of nitrogen to prolong their growth. For example, the unicellular Synechocystis sp. PCC 6803 uses cyanophycin (multi-L-arginyl-poly-[L-aspartic acid]), a non-ribosomally synthesized peptide consisting of equimolar quantities of arginine and aspartic acid, as a nitrogen source upon nitrogen starvation (3). After cyanophycin is exhausted, cells degrade the phycobilisomes that are light-harvesting antennae composed of rod and core proteins to provide nitrogen, which leads to a color change of cells from blue-green to yellow-green, known as bleaching (4). Upon reintroduction of nitrogen, cyanophycin is synthesized immediately, thus cyanophycin is considered as a dynamic nitrogen reservoir in Synechocystis sp. PCC 6803 and many other cyanobacteria (5). Some cyanobacteria are able to fix dinitrogen in the absence of combined nitrogen such as nitrate or ammonium. The filamentous Anabaena sp. PCC 7120 produces heterocysts that are specialized cells for nitrogen fixation (6), whereas the unicellular Cyanothece sp. ATCC 51142 fixes nitrogen and accumulates cyanophycin granules under dark conditions (7). Nitrogen depletion also impacts glycogen accumulation in cyanobacteria (8). Previous studies have shown that during nitrogen starvation, glycogen is accumulated in Synechocystis sp. PCC 6803, whereas the expression of sugar catabolic genes is widely up-regulated (9).
The NtcA protein is the global nitrogen regulator in cyanobacteria (10). It senses intracellular 2-oxoglutarate levels and regulates many genes including those involved in nitrogen assimilation. In non-diazotrophic Synechocystis sp. PCC 6803, NtcA directly regulates transcription of the nrrA gene (referred to as rre37 in Ref. 11), encoding a nitrogen-regulated response regulator of the OmpR family, which has a response regulator domain at the N terminus and a DNA-binding domain at the C terminus. NrrA is involved in induction of sugar catabolic genes in Synechocystis sp. PCC 6803 during nitrogen starvation (11), however, it remains unclear whether NrrA directly regulates transcription of these genes and other genes up-regulated by nitrogen deprivation. In diazotrophic Anabaena sp. PCC 7120, expression of nrrA is up-regulated by nitrogen deprivation under the control of NtcA (12), and NrrA is required for full induction of the hetR gene (13), encoding a master regulator of heterocyst differentiation (14). The NrrA-binding site of the hetR promoter has been determined by a DNase footprinting assay (13). NrrA also controls glycogen catabolism in Anabaena sp. PCC 7120 by directly regulating expression of the glgP gene encoding a glycogen phosphorylase and a sigE gene encoding a group 2 factor of RNA polymerase (15). Although NrrA seems to be widely distributed in cyanobacteria (12), nothing was known about its function in the species other than Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120.
The main goal of this study was to investigate the regulatory role of NrrA in diverse cyanobacteria. We used a comparative genomic approach (16) to identify NrrA-binding DNA motifs and reconstruct NrrA regulons in 15 diverse cyanobacterial species. The predicted members of NrrA regulons in most species are involved in glycogen catabolism, central carbon metabolism, amino acid biosynthesis, and protein degradation. A combination of in vivo and in vitro experimental techniques was used to validate the predicted direct target genes of NrrA in Synechocystis sp. PCC 6803. Furthermore, the effect of NrrA deficiency on intracellular levels of arginine, cyanophycin, and glycogen was studied. Our results indicate that NrrA controls cyanophycin accumulation and glycogen catabolism in Synechocystis sp. PCC 6803.

EXPERIMENTAL PROCEDURES
Bioinformatics Approaches and Tools-Genome sequences of cyanobacteria analyzed in this study were obtained from GenBank TM . Identification of orthologs was performed using the BLASTP tool provided by NCBI (17). Orthologs of the NrrA proteins from Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120 were identified with a 50% protein sequence identity threshold. The ClustalX (version 2.1) program (18) was used for protein sequence alignments, and the PhyML 3.0 program (19) for phylogenetic analysis. A phylogenetic tree of NrrA proteins was built using the maximum likelihood method, with calculation of bootstraps from 1000 replications.
Regulon reconstruction was performed using an established comparative genomics method based on identification of candidate regulator-binding sites in closely related prokaryotic genomes (16). For identification of the conserved DNA-binding motif for NrrA, we started from training sets of known NrrAregulated genes in Synechocystis sp. PCC 6803 (11) and Anabaena sp. PCC 7120 (15) and their orthologs in multiple cyanobacterial genomes. An iterative motif detection algorithm implemented in the RegPredict web-server (20) was used to identify common regulatory DNA motifs in upstream regions of these genes. For each clade of NrrA proteins on the phylogenetic tree, a separate training gene set was used. A positional weight matrix was constructed for each identified motif and used to scan the genomes in this clade. Candidate NrrA-binding sites were identified using the RegPredict (20) and Genome-Explorer tools (21). Scores of candidate sites were calculated as the sum of positional nucleotide weights. The score threshold was defined as the lowest score observed in the training set. Genes with candidate upstream binding sites that are high scored and/or conserved in two or more genomes were included in the NrrA regulon. Candidate sites associated with new regulon members were added to the training set, and the respective positional weight matrix describing a group-specific NrrA motif was rebuilt to improve search accuracy. The NrrAbinding DNA motifs were visualized as sequence logos using WebLogo (22). Functional annotations of the predicted regulon members were based on CyanoBase (23).
Strains and Growth Conditions-Synechocystis sp. PCC 6803 and its derivative with the nrrA gene inactivation were used in this study. Synechocystis strains were routinely grown in the BG-11 medium (24) containing 18 mM NaNO 3 under continuous white light (ϳ70 mol of photons m Ϫ2 s Ϫ1 ). Kanamycin (30 g/ml) was added when needed. The photo-mixotrophic cultures were started with an optical density at 730 nm (A 730 ) of about 0.05, and grown at 30°C under continuous illumination in triplicates in 300-ml glass flasks with 100 ml of BG-11 liquid medium containing 5 mM NaNO 3 and supplemented with 10 mM glucose (BG-11 G ). Cell growth was monitored spectrophotometrically at 730 nm. For nitrogen deprivation and replenishment experiments, cells were grown in 100 ml of BG-11 medium to an A 730 of about 1.0, washed with the nitrogen-free minimal medium (BG-11 N0 ), and resuspended in 100 ml of BG-11 N0 medium. The cultures were grown in triplicates in 300-ml glass flasks with shaking at 30°C under light condition. After 12 h of nitrogen deprivation, 5 mM NaNO 3 with or without 5 mM arginine was added to the culture, and cells were grown for another 12 h. Aliquots of the culture were harvested in the course of time for metabolite analyses.
Mutant Construction-To construct the nrrA gene-inactivated mutant of Synechocystis sp. PCC 6803, DNA fragments immediately upstream and downstream of the nrrA gene (sll1330) were amplified by PCR using the primers shown in supplemental Table S1. The upstream fragment was cloned between the SacI and BamHI sites of pBluescript KS ϩ (Agilent Technologies), and the downstream fragment was cloned between the EcoRI and SalI sites. A kanamycin resistance cassette from the plasmid pUC4K (25) was inserted between the upstream and downstream fragments to form plasmid pKSnrrA. This plasmid was introduced into Synechocystis sp. PCC 6803 according to Ref. 26. The mutant was selected on BG-11 plates supplemented with kanamycin, and segregation was confirmed by PCR using the primers shown in supplemental Table S1. In the mutant, the region from ϩ95 to ϩ450 with respect to the translation start site of the nrrA gene (753 bp long) was replaced with the kanamycin resistance cassette.
RNA Isolation and Real-time PCR Analysis-Synechocystis sp. PCC 6803 cells were harvested by centrifugation, frozen immediately in liquid nitrogen, and ground into powder. RNA was isolated using TRIzol reagent (Invitrogen). Contaminant DNA was removed by DNase I (Takara) digestion. RNA (1 g) was transcribed into cDNA with random primers using the ReverTra-Plus kit from TOYOBO. The product was quantified via real-time PCR using the Applied Biosystems 7300 PCR system. The reaction mixture (20 l) contained Power SYBR Green PCR master mix (Takara) and 0.3 M gene-specific primers (as shown in supplemental Table S1). The PCR parameters were 1 cycle of 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 31 s. The accuracy of the PCR product was checked by melting curve analysis. The expression level of each gene was normalized with the value for the rnpB gene encoding RNase P subunit B, which was used as a reference gene with constitutive expression (27). Data were presented as the average of six measurements from two biological replicates, with the corresponding standard deviation.
Protein Overexpression and Purification-The nrrA genes (sll1330 and all4312) were PCR-amplified from the genomic DNA of Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120, respectively, using the primers shown in supplemental  Table S1. The PCR fragment was ligated into the expression vector pET28a cleaved by NdeI and BamHI. The resulting plasmid pET28a-nrrA was sequenced to exclude unwanted mutations in the nrrA gene and used to produce NrrA protein with an N-terminal His 6 tag. For overproduction of NrrA proteins, Escherichia coli BL21(DE3) was transformed with plasmid pET28a-nrrA and cultivated in LB medium at 37°C to an optical density at 600 nm (A 600 ) of 0.8. Protein expression was induced by the addition of 0.2 mM isopropyl ␤-D-thiogalactopyranoside, and the culture was incubated for another 18 h at 16°C. After the cells were harvested, purification of NrrA by nickel-nitrilotriacetic acid affinity chromatography was performed as described previously (28). The purified protein was run on a 12% SDS-PAGE to monitor its size and purity.
Electrophoretic Mobility Shift Assay (EMSA)-The 200-bp DNA fragments in the promoter regions of individual genes of Synechocystis sp. PCC 6803 and the sigE gene (alr4249) of Anabaena sp. PCC 7120 were PCR-amplified using the primers shown in supplemental Table S1. Both forward and reverse primers were Cy5 fluorescence labeled at the 5Ј-end (Sangong Corp., Shanghai, China), and the PCR products were purified with a PCR purification kit (AXYGEN). Purified NrrA protein was incubated with the fluorescence-labeled DNA fragment (1 nM) in 20 l of binding buffer containing 20 mM Tris (pH 7.5), 0.25 mM DTT, 10 mM MgCl 2 , 5% glycerol, 0.8 g of bovine serum albumin (BSA), and 1 g of salmon sperm DNA (nonspecific random-sequence competitor). After incubation at room temperature for 20 min, the reaction mixture was electrophoresed at 4°C on a 6% native polyacrylamide gel in 0.5ϫ Tris borate-EDTA for 1.5 h at 100 V. Fluorescence-labeled DNA on the gel was then detected by the Starion FLA-9000 (FujiFilm, Japan). Specificity of the NrrA-DNA interactions was tested by including a 200-fold excess of unlabeled target DNA (specific competitor) in binding reaction mixtures.
Analysis of Enzyme Activities-Enzyme activities were measured in crude cell extracts from 25-ml culture aliquots. The cell pellets were washed and resuspended in 100 mM Tris-HCl buffer (pH 7.5). After sonication, cell debris was removed by centrifugation, and the supernatant was used for determination of enzyme activities and protein concentration.
Glycogen phosphorylase activity was measured by monitoring the increase in NADPH concentration using phosphoglucomutase and glucose-6-P dehydrogenase as coupling enzymes (29). Briefly, 10 l of the cell extract was added to 200 l of 100 mM potassium phosphate buffer (pH 7.5) containing 2.5 mM EDTA, 2.5 mM MgCl 2 , 2 mM NADP ϩ , 1 unit of phosphoglucomutase, 6 units of glucose-6-P dehydrogenase, and 1 g/liter of glycogen. The change in NADPH concentration was monitored at 340 nm using a Beckman DU-800 spectrophotometer.
Glyceraldehyde-3-P dehydrogenase activity was measured by adding 10 l of the cell extract to 200 l of 100 mM potassium phosphate buffer (pH 7.5) containing 4 mM glyceraldehyde-3-P, 10 mM EDTA, and 2 mM NAD ϩ . The formation of NADH was monitored spectrophotometrically at 340 nm.
Argininosuccinate synthetase activity was assayed by coupling the formation of AMP to the oxidation of NADH to NAD ϩ through adenylate kinase, pyruvate kinase, and lactate dehydrogenase (31). Briefly, 10 l of the cell extract was added to 200 l of 100 mM Tris-HCl buffer (pH 7.5) containing 1 mM ATP, 5 mM MgCl 2 , 2 mM KCl, 16 mM phosphoenolpyruvate, 0.2 mM NADH, 7.5 mM citrulline, 7.5 mM aspartate, 10 units of inorganic pyrophosphatase, 10 units of adenylate kinase, 4 units of pyruvate kinase, and 4 units of lactate dehydrogenase. The change in NADH absorbance was monitored at 340 nm.
Metabolite Measurements-For analysis of extracellular metabolites, culture samples were harvested by centrifugation at 15,000 ϫ g for 10 min at 4°C. The glucose concentration was determined with an enzymatic test kit (r-Biopharm, Darmstadt, Germany). The nitrate concentration was measured with a colorimetric assay kit (Roche Applied Science).
For determination of the intracellular arginine concentration, cells were harvested by centrifuging 20 ml of culture broth at 9,000 ϫ g and 4°C for 10 min and resuspended in 10 ml of 80% (v/v) ethanol. Norleucine was added as an internal standard. After heating at 65°C for 3 h, cell debris was removed from extracts by centrifugation at 18,000 ϫ g for 15 min. The extracts were dried in a vacuum centrifuge. Arginine in the extracts was derivatized with phenylisothiocyanate by incubating with 200 l of the derivative reagent (Sigma) at room temperature for 45 min (32). The resulting phenylisothiocyanate-arginine was quantitated by high pressure liquid chromatography (HPLC) using an Agilent model 1260 instrument equipped with an Ultimate Amino Acid Column (4.6 ϫ 250 mm; Welch, Shanghai, China) and a UV detector (Agilent) operated at 254 nm. The mobile phase solutions were pumped at a flow rate of 1.0 ml/min, and the temperature of the column was kept at 40°C.
Determination of Glycogen, Cyanophycin, and Phycocyanin Levels-For determination of intracellular glycogen levels, cell pellets were harvested by centrifuging 1 ml of culture aliquots, resuspended in 100 l of 3.5% (v/v) sulfuric acid and boiled for 40 min. The amount of glucose in the hydrolysate was deter-mined using o-toluidine reagent and reading the absorbance at 635 nm (33).
Cyanophycin was isolated and purified using a previously published method with minor modifications (5). Cells were harvested by centrifuging 25 ml of culture aliquots and resuspending in 2 ml of Tris-HCl buffer (pH 7.0). After sonication, the suspension was centrifuged and the supernatant was discarded. The pellet was washed twice with distilled water and extracted by two successive treatments with 0.5 ml of 0.1 M HCl for 30 min at room temperature. The suspension was centrifuged and the supernatant was neutralized with 0.1 M NaOH. Cyanophycin that is insoluble at neutral pH was collected by centrifugation, washed with distilled water, and solubilized in 0.1 ml of 0.1 M HCl. The cyanophycin content was measured with the Bradford reagent using bovine serum albumin as the standard.
The ratio of phycocyanin to chlorophyll levels was used as a measure of the phycocyanin content. Phycocyanin and chlorophyll levels were obtained spectrophotometry as described previously (34).

Genomic Reconstruction of NrrA Regulons in Cyanobacteria
Phylogenetic Distribution of NrrA Proteins-Orthologs of NrrA proteins from Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120 were identified by BLASTP searches in the reference protein database (refseq-protein). NrrA orthologs were detected in 6 cyanobacterial orders including Chroococcales, Oscillatoriales, Nostocales, Gloeobacterales, Pleurocapsales, and Stigonematales but not in the Prochlorales order (supplemental Table S2). A single copy of nrrA is present in the genomes of 39 unicellular, 4 baeocytous, 20 filamentous, 15 heterocystous, and 1 ramified species. Thus NrrA proteins are widely distributed in cyanobacteria, independent of morphology and taxonomy of species. A maximum likelihood phylogenetic tree was constructed for NrrA proteins identified in cyanobacteria (supplemental Fig. S1 and Fig. 1), which largely coincides with the phylogeny of cyanobacterial species (35). The NrrA proteins from Chroococcales and Pleurocapsales are similar, whereas they are distantly related to NrrA from Nostocales. The major subclade of NrrA proteins from Oscillatoriales is split on the tree into two separated groups (supplemental Fig.  S1), which may reflect the functional divergence, e.g. by the set of target genes or by the DNA recognition motifs (see next section).
Identification of NrrA-binding Motifs and Regulons-To reconstruct the NrrA regulons in cyanobacteria, we applied the integrative comparative genomics approach that combines identification of candidate transcription factor-binding sites with cross-genomic comparison of regulons and with the functional context analysis of candidate target genes (16 and metabolisms (e.g. diazotroph and non-diazotroph). For identification of the conserved DNA-binding motif for NrrA, we started from training sets of known NrrA-regulated genes in Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120 and their orthologs in other genomes. The upstream regions of these genes were analyzed using a motif recognition program to identify conserved NrrA-binding motifs. After construction of a positional weight matrix for each identified motif, we searched for additional NrrA-binding sites in the analyzed genomes and finally performed a cross-species comparison of the predicted sets of potentially co-regulated genes to define the NrrA regulon for each species.
Based on the identified NrrA-binding motifs and regulons, the analyzed cyanobacterial species can be divided into two groups. As shown in Fig. 1A, a highly conserved signal consisting of two direct repeats of TG(T/A)CA separated by an 8-bp A/T-rich spacer was identified as a candidate NrrA-binding motif in the first group including 8 species of Chroococcales, Pleurocapsa sp. PCC 7327 (Pleurocapsales), and Leptolyngbya sp. PCC 7376 (Oscillatoriales). This motif is similar to the pho box, the specific DNA target sequence of E. coli PhoB regulator that also belongs to the OmpR family (36). A slightly different binding motif with consensus TGTCATCNNAANTTNACA was detected for NrrA from the second group including 4 species of Nostocales and T. erythraeum IMS101 (Oscillatoriales). This result is in accordance with the experimentally determined NrrA-binding sequence of the hetR promoter in Anabaena sp. PCC 7120 (13). The obtained NrrA-binding motifs were used to detect candidate members of the NrrA regulons in the 15 cyanobacterial genomes (Table 1). Detailed information about the predicted DNA-binding sites and downstream regulated genes is provided in supplemental Table S3.
Predicted Members of NrrA Regulons-The reconstructed NrrA regulons control the central carbon metabolism in most of the analyzed cyanobacteria (Fig. 1B). However, the specific content of NrrA regulons is highly variable between different species (Table 1). Most of the predicted members of NrrA regulons in the 10 species of the first group are involved in glycogen catabolism, central carbon metabolism, amino acid biosynthesis, and protein degradation. For example, candidate NrrAbinding sites were identified in the promoter regions of the genes coding for glycogen phosphorylase (glgP) and two glycolytic enzymes (pfkA and gap1) in the Synechocystis sp. PCC 6803 genome, suggesting that these genes are direct targets of NrrA in Synechocystis. A putative NrrA-binding site was also identified to be located upstream of the icfG gene cluster, which encodes a glycogen isoamylase (glgX) and IcfG protein phosphatase participating in the regulation of glucose metabolism in Synechocystis (37). Moreover, the predicted NrrA targets in Synechocystis include genes encoding proteases (prp1, prp2, and pfpI) and the enzymes of arginine biosynthesis (argG and argD) ( Table 1). Arginine can be polymerized with aspartic acid to form cyanophycin, a nitrogen reserve present in most cyanobacteria (38).
Similar to that in Synechocystis, the reconstructed NrrA regulon in Cyanothece sp. ATCC 51142 contains genes from glycogen catabolism (glgP), central carbon metabolism (pfkA), arginine biosynthesis (argG), and protein degradation (prp1-prp2, clpS-cce_2239) ( Table 1). We found that the nrrA gene is preceded by a putative NrrA-binding site in all the four Cyanothece species analyzed (ATCC 51142, PCC 8801, PCC 7822, and PCC 7424), indicating that NrrA could regulate expression of its own gene in these Cyanothece species. Additionally, a candidate NrrA-binding site was identified upstream of the pipX gene in the genomes of Cyanothece sp. PCC 8801 and M.
A decrease in the size of the reconstructed NrrA regulons was observed for the 5 species of the second group (Fig. 1B). For Carbon metabolism regulator, glycogen debranching enzyme Hypothetic  Table 1). The identified binding sites were found within the experimentally determined NrrA-binding regions of glgP and sigE promoters (15). We found that the hetR gene encoding a master regulator of heterocyst differentiation (14) is preceded by a candidate NrrA-binding site not only in Anabaena sp. PCC 7120 but also in Nostoc sp. PCC 7107, N. punctiforme PCC 73102, and T. erythraeum IMS101 (Table 1). This suggests that NrrA may also directly regulate expression of the hetR gene in the latter three diazotrophic species. The presence of a putative NrrA-binding site upstream of peptidoglycan synthetic genes murD and murE was detected for multiple genomes including Anabaena sp. PCC 7120, N. spumigena CCY 9414, and T. erythraeum IMS101 (Table 1).
In summary, the comparative genomics analysis allowed us to identify the conserved NrrA-binding motifs and reconstruct the NrrA regulons in 15 diverse cyanobacterial species. Among these species, Synechocystis sp. PCC 6803 has one of the largest set of predicted NrrA targets, including 19 genes organized in 10 putative operons that are involved in glycogen catabolism, glycolysis, arginine biosynthesis, and protein degradation. We then performed experimental validation of the predicted NrrAbinding motif and characterization of the NrrA regulon in Synechocystis sp. PCC 6803 as described below.

Experimental Characterization of NrrA Regulon in Synechocystis sp. PCC 6803
NrrA Binds Its Cognate DNA Sites in Vitro-To validate the predicted NrrA regulon, EMSAs were performed using the recombinant NrrA protein from Synechocystis sp. PCC 6803, which was overexpressed in E. coli with the N-terminal His 6 tag and purified with a nickel-chelating affinity column. For all predicted NrrA target genes or operons in Synechocystis sp. PCC 6803, DNA fragments (200 bp) in the promoter regions containing candidate NrrA-binding sites were tested in EMSAs (Fig. 2). A shifted band was observed upon incubation of the NrrA protein with each promoter fragment, and its intensity was enhanced in the presence of increasing amounts of NrrA protein. The promoter fragments of glgP, icfG operon (slr1852-slr1861), argG, prp1, flv3, pilL, and sll0185 genes were completely shifted with 500 nM NrrA protein ( Fig. 2A). For the upstream fragments of gap1, pfkA, and argD, only incomplete shifts were achieved using the same concentration of NrrA, suggesting that NrrA exhibited a lower affinity for these binding sites (Fig. 2B). No specific shift was observed with the promoter regions of Synechocystis sigE (sll1689) and cysA (slr1455) genes (Fig. 2C). The sigE gene of Synechocystis lacks a predicted NrrA-binding site in the upstream region, whereas the cysA gene is preceded by a sequence bearing some resemblance to a NrrA-binding site but with one mismatch in the direct repeat.
Both sigE and cysA genes show unaltered mRNA levels in the nrrA-inactivated mutant (11). The formation of the NrrA-DNA complex was suppressed in the presence of 200-fold excess unlabeled DNA fragments containing a NrrA-binding site but not in the presence of nonspecific competitors (Fig. 2D). These observations confirm that NrrA binds specifically to the pro-moter regions of predicted targets in Synechocystis sp. PCC 6803.
To characterize the NrrA-binding DNA motif in Synechocystis sp. PCC 6803 and other species of the first group, site-directed mutagenesis was performed on the promoter regions of three Synechocystis genes, including glgP, argG, and pilL genes, encoding a sensory transduction histidine kinase. These three genes are involved in glycogen catabolism, arginine biosynthe-  fifth lanes). C, the promoter regions of Synechocystis sigE (sll1689) and cysA (slr1455) genes, which lack a putative NrrA-binding site, were used as negative controls. A sequence upstream of the cysA gene, which bears some resemblance to the NrrA-binding motif but with one mismatch in the direct repeat (underlined), is shown. D, specificity of the NrrA-DNA interactions was tested by competition with 10-, 100-, or 200-fold of non-labeled target DNA (specific comp). The DNA fragment from the upstream region of the rplW gene (sll1801), which lacks a predicted NrrA-binding site, was added at the same concentrations as a nonspecific competitor (nonspecific comp).
sis, and other functions, respectively (Table 1), and their promoter fragments showed a substantial shift in the presence of 200 nM Synechocystis NrrA protein (Fig. 2). Nineteen mutated pilL promoter fragments, each with one or two nucleotide substitutions upstream, downstream, or within the two direct repeats, were tested in EMSAs (Fig. 3A). In the case of fragments M3-M7 and M13-M17, the mutations prevented the binding of NrrA, indicating that the nucleotides within the direct repeat region were required for binding (Fig. 3A). Exchange of the nucleotides between the two repeats showed that an optimal spacer has to be A/T-rich (Fig. 3A, M8 -M12). The mutations outside of the predicted NrrA-binding sequence showed no significant effect (Fig. 3A, M1, M2, M18, and M19). For mutation analysis of the predicted NrrA-binding sites upstream of glgP and argG genes, eight DNA fragments with a single base substitution in the direct repeat region were amplified by PCR. As shown in Fig. 3B, no binding of NrrA was observed for the mutated fragments. These results confirm the predicted NrrA-binding sites and DNA motif in Synechocystis sp. PCC 6803.
In addition, to provide support for the identified NrrA-binding motif in species of the second group, we performed sitedirected mutagenesis on the upstream region of the Anabaena sp. PCC 7120 sigE gene (alr4249). NrrA directly regulates sigE expression in Anabaena sp. PCC 7120 (15), however, its NrrAbinding site has not been identified. The binding affinity of the Anabaena NrrA protein was tested by EMSAs for DNA fragments with a single base substitution on the predicted NrrAbinding sequence. As shown in Fig. 4, nucleotide substitutions within the direct repeat region remarkably reduced binding activity of NrrA to the fragments (M30 -M35, M39, M40, and M42-M44) except for fragment M41. The binding of NrrA remained largely unaffected when nucleotides in the spacer region and outside of the predicted site were exchanged (M28, M29, M36 -M38, M45, and M46). This result is consistent with the previously reported NrrA-binding sequence of the hetR promoter in Anabaena sp. PCC 7120 (13).
NrrA Positively Regulate Expression of Its Target Genes in Vivo-To validate the predicted regulation of NrrA on gene expression in vivo, the coding region of the nrrA gene (sll1330) was partly deleted from the chromosome of Synechocystis sp. PCC 6803, resulting in the nrrA-inactivated mutant (⌬nrrA) (Fig. 5A). Complete segregation of the mutant was confirmed by PCR (Fig. 5A). The transcript levels of the predicted NrrA target genes in the ⌬nrrA mutant were compared with those in the wild-type by using quantitative RT-PCR. The two strains were cultivated photo-mixotrophically under continuous illumination in BG-11 liquid medium supplemented with 10 mM glucose. Despite a longer lag phase for the ⌬nrrA mutant, both strains grew at a similar growth rate during the exponential growth phase (Fig. 5B). For comparison of transcript levels, cells were harvested in the exponential growth phase at an A 730 of 1.2 and a growth rate of 0.13 h Ϫ1 for both strains, and total RNA was isolated. Six quantitative RT-PCR measurements from two independent cultures were performed. As shown in Table 2, the relative mRNA levels of 17 genes were decreased more than 1.8-fold in the ⌬nrrA mutant compared with the wild-type strain. The most prominent effect of nrrA mutation was observed for the two glycolytic genes pfkA and gap1, which showed a Ն19-fold reduced mRNA level in the ⌬nrrA mutant. The genes with a strongly decreased expression also include the icfG gene cluster, which contains glgX, icfG, and pfpI genes. The prp1-prp2 operon, glgP, argD, argG, and pilL genes showed a 1.8 -6-fold decreased transcript level in the ⌬nrrA mutant. Expression of the sll0185 and flv3 genes was not significantly affected by nrrA mutation, which may be explained by possible involvement in their regulation of other still unknown regulatory mechanisms. Hence, the quantitative RT-PCR results confirm that NrrA is a positive regulator of glgP, glgX, pfkA, gap1, icfG, argG, argD, prp1, prp2, pfpI, and pilL genes involved in glycogen catabolism, glycolysis, arginine biosynthesis, and protein degradation in Synechocystis sp. PCC 6803.
To investigate the effect of NrrA deficiency on the protein level, enzyme activities were determined in crude cell extracts of Synechocystis sp. PCC 6803 wild-type and ⌬nrrA mutant strains. As shown in Table 3, glycogen phosphorylase and glyceraldehyde-3-P dehydrogenase (GAPDH) exhibited ϳ4 -6fold decreased activities in crude extract of the ⌬nrrA mutant as compared with the wild-type. Moreover, the activities of two enzymes of arginine biosynthesis, N-acetylornithine aminotransferase and argininosuccinate synthetase, were about 3-4fold lower in the ⌬nrrA mutant than in the wild-type strain.
NrrA Controls Cyanophycin Accumulation and Glycogen Catabolism in Synechocystis-Argininosuccinate synthetase catalyzes the rate-limiting step in the arginine synthesis pathway, and N-acetylornithine aminotransferase (AcOAT) 2 is also a key enzyme in this pathway (30,41). Arginine can serve as a nitrogen buffer in cyanobacteria, storing excess nitrogen in the form of cyanophycin. A previous study has suggested that cyanophycin accumulation in Synechocystis sp. PCC 6803 is controlled by arginine synthesis under conditions of nitrogen excess (42). Based on our observations on the NrrA regulation of argininosuccinate synthetase and AcOAT levels and the key functions of these two enzymes in arginine synthesis, we hypothesized that NrrA might activate arginine synthesis and play a role in the accumulation of cyanophycin under nitrogenexcess conditions. To test this hypothesis, we investigated the effect of NrrA deficiency on intracellular arginine concentration and cyanophycin accumulation as well as on glycogen catabolism.
Synechocystis sp. PCC 6803 wild-type and ⌬nrrA mutant strains were cultivated photo-mixotrophically, and the culture samples were harvested in the course of time for analysis of glucose and nitrate consumption (Fig. 6A). Based on the residual nitrate concentration in the medium, five time points during the change from nitrogen-excess to nitrogen-depleted conditions were selected for determination of intracellular arginine and cyanophycin levels. Similarly, seven time points were selected for quantification of glycogen content based on the residual glucose concentration in the medium. As shown in Fig.  6, B and C, the intracellular arginine concentration and cyanophycin content in the wild-type strain were increased about 5and 3-fold, respectively, as the extracellular nitrate was consumed and reached the maximum levels when nitrate was depleted in the medium. Then the arginine concentration and cyanophycin content were decreased to low levels within 15 h after nitrate depletion. By contrast, the intracellular arginine concentration in the ⌬nrrA mutant was kept at a very low level and the cyanophycin synthesis before nitrate depletion was almost absent. These results suggest that NrrA activates arginine synthesis and cyanophycin accumulation through up-regulation of argininosuccinate synthetase and AcOAT levels. On the other hand, quantification of glycogen amounts revealed that the glycogen level in the ⌬nrrA mutant was increased almost 2-fold compared with that in the wild-type when glucose was present in the medium (Fig. 6D). After glucose was depleted, the glycogen content was decreased more slowly in the ⌬nrrA mutant than in the wild-type. 2 The abbreviation used is: AcOAT, N-acetylornithine aminotransferase. To provide further evidence to NrrA activation of cyanophycin accumulation and glycogen degradation, we performed nitrogen deprivation and replenishment experiments. Synechocystis sp. PCC 6803 wild-type and ⌬nrrA mutant strains exponentially grown in BG-11 medium were transferred to the nitrogen-deficient medium. After 12 h nitrate was added to a final concentration of 5 mM and cells were grown for another 12 h. The intracellular arginine concentration and cyanophycin and glycogen contents were measured throughout the nitrogen deprivation and replenishment experiments. The change in phycocyanin content was also monitored, because Synechocystis sp. PCC 6803 uses both cyanophycin and phycobilisome as nitrogen-storage reservoirs (5). As shown in Fig. 7A, cyanophycin content in the wild-type was decreased 18-fold after nitrogen deprivation, whereas upon nitrogen replenishment the cyanophycin content was rapidly increased from 0.05 to 2.3% of the total protein. Compared with the wild-type, the cyanophycin amount in the ⌬nrrA mutant was reduced by 93% when both strains were exponentially grown under photoautotrophic conditions (time 0). Although the ⌬nrrA mutant also accumulated cyanophycin following nitrate upshift, the formation rate of cyanophycin was decreased by ϳ50% compared with the wildtype. The intracellular arginine concentration was also significantly lower in the ⌬nrrA mutant than in the wild-type (Fig.  7B). Quantification of phycocyanin content revealed that the ratio of phycocyanin to chlorophyll levels was higher in the ⌬nrrA mutant than in the wild-type (Fig. 7C). Moreover, the phycocyanin to chlorophyll ratio in the ⌬nrrA mutant declined from 7.1 to 6.5 after nitrogen deprivation and then increased to 7.3 upon nitrogen upshift, whereas the wild-type had a relatively stable phycocyanin to chlorophyll ratio throughout the experiment. In addition, determination of glycogen content revealed that the ⌬nrrA mutant accumulated higher amounts of glycogen than the wild-type during nitrogen starvation (Fig.  7D). Following nitrate replenishment, glycogen content in the wild-type was rapidly reduced by 70% with 12 h and a notable decrease in the rate of glycogen degradation was observed for the ⌬nrrA mutant compared with the wild-type. For comparison of transcript levels of the genes and activities of the enzymes involved in glycogen catabolism and arginine biosynthesis between the wild-type and ⌬nrrA mutant, samples were prepared after 4 h of nitrogen starvation and after 4 h following nitrogen replenishment. The quantitative RT-PCR analyses showed that transcript levels of glgP, glgX, gap1, pfkA, argD, and argG genes were decreased drastically in the ⌬nrrA mutant compared with the wild-type (Fig. 7E). Particularly, these genes showed a 4 -51-fold reduced mRNA level in the ⌬nrrA mutant under nitrogen starvation conditions. Determination of enzyme activities revealed that the ⌬nrrA mutant exhibited 3-5-fold decreased activities of glycogen phosphorylase, GAPDH, AcOAT, and argininosuccinate synthetase compared with the wild-type during the nitrogen deprivation and replenishment experiment (Fig. 7F).
The above results strongly suggest that NrrA activates synthesis of arginine, which then leads to cyanophycin accumulation. To verify that impaired cyanophycin synthesis in the ⌬nrrA mutant is indeed due to limiting arginine concentrations and not caused by reduced cyanophycin synthetase levels, nitrate replenishment experiments in the presence of 5 mM arginine were carried out with wild-type and ⌬nrrA mutant. As shown in Fig. 8, after arginine and nitrate were added to the medium, the intracellular arginine concentration in the wildtype and ⌬nrrA mutant was increased to similar levels, and a

DISCUSSION
In this work, we performed comparative genomic reconstruction of NrrA regulons in 15 diverse cyanobacterial species by combining the identification of candidate NrrA-binding sites with cross-genomic comparison of regulons. A conserved NrrA-binding motif consisting of two direct repeats of TG(T/ A)CA separated by an 8-bp A/T-rich spacer was identified for the 10 species of the first group. The combined bioinformatics, in vitro and in vivo characterization of the NrrA regulon in Synechocystis sp. PCC 6803 revealed that NrrA directly regulates the expression of glgP, glgX, pfkA, gap1, argG, argD, prp1, prp2, and pfpI genes involved in glycogen catabolism, glycolysis, arginine biosynthesis, and protein degradation (Fig. 9). These NrrA target genes have been shown to be up-regulated under nitrogen depletion (9). Moreover, we demonstrated that NrrA-regulated arginine synthesis controls cyanophycin accu-mulation, and NrrA also plays a pivotal role in the regulation of glycogen catabolism in Synechocystis sp. PCC 6803.
During nitrogen starvation, expression of the nrrA gene in Synechocystis sp. PCC 6803 is induced (11). The NrrA regulator binds to its operator sites, leading to activation of expression of argG and argD genes coding for two key enzymes (argininosuccinate synthetase and AcOAT) of arginine synthesis. The availability of arginine appears to limit cyanophycin synthesis in Synechocystis (Fig. 8) (43). Thus, NrrA-mediated up-regulation of argG and argD genes during nitrogen starvation could contribute to the immediate synthesis of arginine and cyanophycin in Synechocystis once nitrogen is replenished. In fact, we found that arginine synthesis and cyanophycin accumulation upon nitrogen upshift were significantly impaired in the ⌬nrrA mutant when compared with the wild-type (Fig. 7). According to previous reports (5), cyanophycin serves as a dynamic nitrogen reservoir, whereas phycobilisomes appear to be the main nitrogen reserve in non-diazotrophic unicellular strains such as Synechocystis sp. PCC 6803. Here we noticed that the ⌬nrrA mutant exhibited a more variable phycocyanin to chlorophyll ratio than the wild-type during the nitrogen deprivation and replenishment experiment (Fig. 7), suggesting that the mutant has to degrade and resynthesize phycobilisomes to respond to transient changes in environmental nitrogen availability.
On the other hand, NrrA directly up-regulates expression of glgP (slr1367), glgX (slr1857), pfkA, and gap1 genes involved in glycogen degradation and glycolysis in Synechocystis sp. PCC 6803. Glycogen, the carbon sink of most cyanobacteria, is utilized as carbon and energy reserves to cope with transient starvation and stress conditions (44). We found that the ⌬nrrA mutant exhibited a high abundance of glycogen during nitrogen Both strains were cultured photo-autotrophically in BG-11 medium to exponential growth phase and then transferred to nitrogen-deficient medium (time 0). After 12 h nitrate was added to a final concentration of 5 mM and cells were grown for another 12 h. The intracellular cyanophycin, arginine, phycocyanin, and glycogen contents were measured at different time points as indicated throughout the experiment. The transcript levels of the genes and the activities of the enzymes involved in glycogen catabolism and arginine biosynthesis were determined after 4 h of nitrogen starvation (4 h) and after 4 h following nitrogen replenishment (16 h). The data points and error bars represent mean Ϯ S.D. of three independent cultures. depletion and a significantly decreased rate of glycogen degradation after nitrogen replenishment (Fig. 7), indicating that NrrA controls glycogen catabolism in Synechocystis. Earlier studies have shown that the group 2 factor SigE is also involved in the regulation of sugar catabolic genes in Synechocystis (33). It is noteworthy that SigE probably induces expression of the pentose phosphate pathway genes and other copies of glgP and glgX genes (sll1356 and slr0237, respectively) (33). Based on our results (Fig. 2) and previous reports (11), regulation of sigE gene expression is probably independent of NrrA in Synechocystis. Thus, it appears that NrrA and SigE may independently regulate different genes of sugar catabolism in Synechocystis during nitrogen starvation. This is in contrast to the situation in Anabaena sp. PCC 7120, where NrrA directly activates sigE expression and SigE up-regulates the genes of glycolysis and the pentose phosphate pathway. Further studies are required to elucidate the contribution of NrrA and SigE to regulation of the sugar catabolic genes and metabolic flux responses in Synechocystis. Therefore, our results revealed that by directly regulating expression of the genes involved in glyco-gen catabolism, glycolysis, and arginine biosynthesis, NrrA may have an important role in coordinating the synthesis and degradation of nitrogen and carbon reserves in Synechocystis.
In addition to nitrogen regulation by NtcA, expression of the nrrA gene in Synechocystis is enhanced by glucose and high salt (45,46). It is tempting to speculate that NrrA may also control cyanophycin accumulation and sugar catabolism under glucose and high salt conditions. In fact, we observed that NrrA deficiency resulted in remarkable changes in intracellular levels of arginine, cyanophycin, and glycogen under the photo-mixotrophic, nitrogen-excess condition (Fig. 6). Further work is needed to clarify the potential role of NrrA in Synechocystis grown under glucose, high salt, and various unbalanced nutrient conditions. Other than transcriptional control, post-translational regulation of NrrA may also occur. NrrA is a response regulator belonging to the OmpR family. The activity of NrrA is probably regulated by phosphorylation, however, the sensory histidine kinase that phosphorylates NrrA has not yet been identified.
Similar to that in Synechocystis, the predicted NrrA regulon in Cyanothece sp. ATCC 51142 contains genes from glycogen catabolism (glgP), glycolysis (pfkA), arginine biosynthesis (argG), and protein degradation (prp1-prp2, clpS-cce_2239) as well as its own gene nrrA. The unicellular diazotrophic Cyanothece strains perform photosynthesis during the day and nitrogen fixation during the night (7). Accumulation and degradation of glycogen and cyanophycin granules, which occur concomitantly with photosynthesis and nitrogen fixation, are a very important feature of their metabolism, and a strong coordination of correlated processes at the transcriptional level has been proposed (47). Based on the published transcriptome data of Cyanothece sp. ATCC 51142 during dark-light cycles (47), we  found that expression profiles of the nrrA gene and NrrA candidate target genes glgP, pfkA, and argG are very similar and their Pearson correlation coefficients are larger than 0.9. Therefore, the nrrA, glgP, pfkA, and argG genes are coregulated, which is consistent with our prediction. These genes are upregulated at the beginning of the dark period (47), suggesting that NrrA may be involved in glycogen degradation and cyanophycin accumulation in Cyanothece sp. ATCC 51142 during the night.
Based on identification of the NrrA-binding motif, we predicted the NrrA regulons in the 5 species of the second group. Previous reports have shown that in the filamentous diazotrophic Anabaena sp. PCC 7120, NrrA controls heterocyst differentiation by directly regulating expression of the hetR gene (13). Here we predicted a candidate NrrA-binding site located upstream of the hetR gene in the genomes of Nostoc sp. PCC 7107, N. punctiforme PCC 73102, and T. erythraeum IMS101, suggesting that regulation of hetR expression by NrrA may also occur in these three diazotrophic species. HetR is a master regulator of heterocyst development and the basic mechanism of heterocyst development seems to be conserved among various heterocystous strains (6). It is worth noting that unlike Anabaena sp. PCC 7120, Nostoc sp. PCC 7107, and N. punctiforme PCC 73102, T. erythraeum IMS101 differentiates diazocytes instead of heterocysts for nitrogen fixation, and a possible involvement of HetR in diazocyte differentiation has been implicated (48). Therefore, it remains an interesting question whether NrrA may have a general role in the regulation of development of specialized cells for nitrogen fixation in filamentous diazotrophic cyanobacteria.

CONCLUDING REMARKS
This study provided a comprehensive bioinformatic analysis of the NrrA regulons in 15 diverse cyanobacterial species. By integrating experimental characterization of the predicted NrrA-binding motif and regulon, the regulatory function of NrrA in Synechocystis sp. PCC 6803 was elucidated. Moreover, this study gains an insight into the potential regulatory role of NrrA in other species by predicting its candidate targets and provides a framework for further studies of the NrrA-dependent regulation in diverse cyanobacteria.