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Intracellular cyanophycin (A), arginine (B), phycocyanin (C), glycogen (D) contents, gene expression levels (E), and enzyme activities (F) of Synechocystis sp. PCC 6803 wild-type and nrrA mutant strains during the nitrogen deprivation and replenishment experiment. 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 S.D. of three independent cultures.
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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 c...
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... 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 Synechocys- tis sp. PCC 6803 uses both cyanophycin and phycobilisome as nitrogen-storage reservoirs (5). As shown in Fig. 7A, cyanophy- cin content in the wild-type was decreased 18-fold after nitro- gen deprivation, whereas upon nitrogen replenishment the cya- nophycin 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 ...
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... exponentially grown under photoautotrophic con- ditions (time 0). Although the nrrA mutant also accumulated cyanophycin following nitrate upshift, the formation rate of cyanophycin was decreased by 50% compared with the wild- type. The intracellular arginine concentration was also signifi- cantly 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 ...
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... rate of cyanophycin was decreased by 50% compared with the wild- type. The intracellular arginine concentration was also signifi- cantly 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 rela- tively stable phycocyanin to chlorophyll ratio throughout the experiment. In addition, determination of glycogen content revealed that ...
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... after nitrogen deprivation and then increased to 7.3 upon nitrogen upshift, whereas the wild-type had a rela- tively 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 compari- son of transcript levels of the genes and activities of the enzymes involved in glycogen ...
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... biosyn- thesis 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 ...
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... 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 replen- ishment experiment (Fig. ...
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... of argG and argD genes during nitrogen starvation could con- tribute 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 nitro- gen 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 ...
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... as a dynamic nitro- gen 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 ...
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... 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 S.D. of three independent cultures. depletion and a significantly decreased rate of glycogen degra- dation 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 Syn- echocystis (33). It is noteworthy that SigE probably induces expression of the pentose phosphate pathway genes and other copies of glgP and glgX ...
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Citations
... In Synechocystis 6803, the gene encoding argininosuccinate synthetase has been annotated as argG (Kaneko et al. 1996). A previous report has suggested that NrrA, a nitrogen-modulated response regulator, regulates ArgG levels for arginine synthesis under nitrogen excess in Synechocystis 6803 (Liu and Yang 2014). Argininosuccinate synthetase of Synechocystis 6803 from crude cell extracts shows specific activities approximately 3-4-fold lower in the NrrA deletion strain than in the wild-type strain. ...
... Argininosuccinate synthetase of Synechocystis 6803 from crude cell extracts shows specific activities approximately 3-4-fold lower in the NrrA deletion strain than in the wild-type strain. (Liu and Yang 2014). However, the biochemical properties of Synechocystis 6803 ArgG (SyArgG), such as the regulation by metabolites and catalytic efficiency, have not been reported in detail. ...
... In addition, the activity of the arginine biosynthesis key enzyme of Synechocystis 6803, N-acetylornithine aminotransferase, for N-acetylornithine (k cat /K m : 19.3 s − 1 mM − 1 ) was approximately 10-fold higher than that of SyArgG (k cat /K m : 1.97 s − 1 mM − 1 ) (Li et al. 2023; Table 1). Synechocystis 6803 cell crude extract experiments also show that N-acetylornithine aminotransferase activity is 2-fold higher than argininosuccinate synthetase activity (Liu and Yang 2014). The k cat /K m of SyArgG for the substrate (citrulline: 1.97 s − 1 mM − 1 , aspartate: 2.16 s − 1 mM − 1 ) was lower than that of the last-step enzyme in arginine biosynthesis (SyArgH: 37.3 s − 1 mM − 1 ) (Katayama and Osanai 2022). ...
Cyanobacteria are oxygen-evolving photosynthetic prokaryotes that affect the global carbon and nitrogen turnover. Synechocystis sp. PCC 6803 (Synechocystis 6803) is a model cyanobacterium that has been widely studied and can utilize and uptake various nitrogen sources and amino acids from the outer environment and media. l-arginine is a nitrogen-rich amino acid used as a nitrogen reservoir in Synechocystis 6803, and its biosynthesis is strictly regulated by feedback inhibition. Argininosuccinate synthetase (ArgG; EC 6.3.4.5) is the rate-limiting enzyme in arginine biosynthesis and catalyzes the condensation of citrulline and aspartate using ATP to produce argininosuccinate, which is converted to l-arginine and fumarate through argininosuccinate lyase (ArgH). We performed a biochemical analysis of Synechocystis 6803 ArgG (SyArgG) and obtained a Synechocystis 6803 mutant overexpressing SyArgG and ArgH of Synechocystis 6803 (SyArgH). The specific activity of SyArgG was lower than that of other arginine biosynthesis enzymes and SyArgG was inhibited by arginine, especially among amino acids and organic acids. Both arginine biosynthesis enzyme-overexpressing strains grew faster than the wild-type Synechocystis 6803. Based on previous reports and our results, we suggest that SyArgG is the rate-limiting enzyme in the arginine biosynthesis pathway in cyanobacteria and that arginine biosynthesis enzymes are similarly regulated by arginine in this cyanobacterium. Our results contribute to elucidating the regulation of arginine biosynthesis during nitrogen metabolism.
... The resulting plasmids for production of the C-terminal hexahistidine-tagged proteins were transformed into Escherichia coli BL21 (DE3) (Novagen). Protein overexpression and purification were performed as described previously (66). ...
... Various combinations of CAPADH, CAPADC, and APAUH were tested to confirm that the presence of all three enzymes is required. Polyamines produced by the enzymatic reactions were identified by HPLC following derivatization with phenylisothiocyanate (66) and were further validated by LC-MS/MS as described above. ...
Spermidine, a ubiquitous polyamine, is known to be required for critical physiological functions in bacteria. Two principal pathways are known for spermidine biosynthesis, both of which involve aminopropylation of putrescine. Here, we identified a spermidine biosynthetic pathway via a previously unknown metabolite, carboxyaminopropylagmatine (CAPA), in a model cyanobacterium Synechocystis sp. PCC 6803 through an approach combining ¹³ C and ¹⁵ N tracers, metabolomics, and genetic and biochemical characterization. The CAPA pathway starts with reductive condensation of agmatine and l -aspartate-β-semialdehyde into CAPA by a previously unknown CAPA dehydrogenase, followed by decarboxylation of CAPA to form aminopropylagmatine, and ends with conversion of aminopropylagmatine to spermidine by an aminopropylagmatine ureohydrolase. Thus, the pathway does not involve putrescine and depends on l -aspartate-β-semialdehyde as the aminopropyl group donor. Genomic, biochemical, and metagenomic analyses showed that the CAPA-pathway genes are widespread in 15 different phyla of bacteria distributed in marine, freshwater, and other ecosystems.
... For PCC 7002, glycogen production is induced by different forms of nutrient starvation including, most commonly, nitrogen starvation . Theglobal nitrogen control factor, ntcA, controls this response and can be triggered by other forms of nutrient or light limitation (Liu and Yang, 2014;Forchhammer and Selim, 2019). Historically, the glgC node has seen the most manipulation in PCC 7002. ...
Current sources of fermentation feedstocks, i.e., corn, sugar cane, or plant biomass, fall short of demand for liquid transportation fuels and commodity chemicals in the United States. Aquatic phototrophs including cyanobacteria have the potential to supplement the supply of current fermentable feedstocks. In this strategy, cells are engineered to accumulate storage molecules including glycogen, cellulose, and/or lipid oils that can be extracted from harvested biomass and fed to heterotrophic organisms engineered to produce desired chemical products. In this manuscript, we examine the production of glycogen in the model cyanobacteria, Synechococcus sp. strain PCC 7002, and subsequent conversion of cyanobacterial biomass by an engineered Escherichia coli to octanoic acid as a model product. In effort to maximize glycogen production, we explored the deletion of catabolic enzymes and overexpression of GlgC, an enzyme that catalyzes the first committed step toward glycogen synthesis. We found that deletion of glgP increased final glycogen titers when cells were grown in diurnal light. Overexpression of GlgC led to a temporal increase in glycogen content but not in an overall increase in final titer or content. The best strains were grown, harvested, and used to formulate media for growth of E. coli. The cyanobacterial media was able to support the growth of an engineered E. coli and produce octanoic acid at the same titer as common laboratory media.
... While on the other, the elucidation of the cyanobacterial regulatory network under various environmental stimuli would be beneficial for the identification and development of additional native inducible promoters. Currently, various TFs and RRs are involved deciphering the mechanism of stresses such as ion, solvent, temperature, nutrition, and pH (López-Redondo et al., 2010;Ehira and Ohmori, 2012;Liu and Yang, 2014), and a few inducible promoters are listed in Table 9.2. Besides, this potential inducible promoters for cyanobacteria can be obtained from the cTFbase database and also other microalgal prediction tools possess putative TFs belonging to 26 cyanobacterial genomes (Wu et al., 2007;Thiriet-Rupert et al., 2016;). ...
... Another layer of regulation of arginine biosynthesis with an impact on the production of cyanophycin is exerted by NrrA (All4312, Sll1330), a response regulator that mediates the induction of some genes under nitrogen deprivation [58,59]. In Synechocystis, inactivation of nrrA impairs induction of arginine biosynthesis genes argD and argG with a negative impact on the production of arginine and cyanophycin [60], and overexpression of NrrA increases the expression of argD, argJ and the carbamoyl phosphate synthase genes carA and carB [61]. It has been suggested that NrrA-mediated induction of arginine biosynthesis genes during nitrogen deprivation could contribute to the immediate production of arginine and cyanophycin once nitrogen becomes available again [60]. ...
... In Synechocystis, inactivation of nrrA impairs induction of arginine biosynthesis genes argD and argG with a negative impact on the production of arginine and cyanophycin [60], and overexpression of NrrA increases the expression of argD, argJ and the carbamoyl phosphate synthase genes carA and carB [61]. It has been suggested that NrrA-mediated induction of arginine biosynthesis genes during nitrogen deprivation could contribute to the immediate production of arginine and cyanophycin once nitrogen becomes available again [60]. ...
Cyanobacteria are oxygenic phoautotrophs that can utilize inorganic nitrogen salts, atmospheric nitrogen and some amino acids such as arginine as nitrogen source. Under unbalanced growth in the presence of sufficient nitrogen, many cyanobacteria accumulate cyanophycin, a co-polymer of aspartate and arginine that serves as a nitrogen reservoir. Cyanophycin metabolism enzymes include cyanophycin synthetases, cyanophycinase and isoaspartyl dipeptidase. The latter splits β‑aspartyl arginine released from cyanophycin by cyanophycinase into aspartate and arginine. The arginine catabolic pathway of cyanobacteria has been recently elucidated and consists of two bifunctional enzymes, arginine-guanidine removing enzyme (AgrE) and proline oxidase (PutA). This pathway makes available to metabolism the four nitrogen atoms of arginine, three as ammonia and one as glutamate. A variant of the pathway cycles ornithine (an intermediate in the AgrE-catalyzed reactions) back to arginine incorporating aspartate and, hence, recovering its nitrogen atom for metabolism. Many cyanobacteria also make use of this pathway to utilize arginine taken up from the outer medium through a high-affinity ABC transporter. An analysis of co-occurrence in cyanobacteria of genes encoding cyanophycin metabolism and arginine catabolism enzymes and arginine and aspartate transporters indicates a strong correlation between the presence of cyanophycin and the AgrE/PutA pathway.
... An NrrA-deicient mutant in Synechocystis sp. PCC 6803 shows reduced intracellular arginine levels and, consequently, reduced CGP amount [70]. ...
... The nitrogen response regulator NrrA has been shown to regulate expression of genes involved in arginine synthesis, glycogen degradation, and glycolysis [158]. The expression of nrrA is activated by the global regulator NtcA during nitrogen starvation [158], and P nrrA was one of the promoters activated by nitrogen depriva- tion [72]. ...
... The nitrogen response regulator NrrA has been shown to regulate expression of genes involved in arginine synthesis, glycogen degradation, and glycolysis [158]. The expression of nrrA is activated by the global regulator NtcA during nitrogen starvation [158], and P nrrA was one of the promoters activated by nitrogen depriva- tion [72]. Promoters that are activated by NrrA were tested for their response to nitrate in Synechocystis sp. ...
... ATCC 51142's NrrA was chosen for the system since its DNA-binding domain is identical to that of Synechocystis' nitrogen response regu- lator. When the NrrA-activated promoter for the icfG operon (P slr1852 ) [158] expressed nrrA, and the FbFP reporter was expressed from P gapI , the fluorescent response to nitrogen deprivation became stronger but more gradual [72]. When P gapI regulated transcription of both the reporter (fbfp) and nitrogen response regulator (nrrA), the sensor maintained digital nitrogen responsiveness with a weaker maximum output than the sensor without positive autoregulation of nrrA. ...
Cyanobacteria are appealing hosts for green chemical synthesis due to their use of light and carbon dioxide. To optimize product yields and titers, specific and tunable regulation of the metabolic pathways is needed. Synthetic biology has increased and diversified the genetic tools available for biological process control. While early tool development focused on commonly used heterotrophs, there has been a recent expansion of tools for cyanobacteria. CRISPR-Cas9 has been used to edit the genome of cyanobacterial strains, while transcriptional regulation has been accomplished with CRISPR interference and RNA riboswitches. Promoter development has produced a significant number of transcriptional regulators, including those that respond to chemicals, environmental signals, and metabolic states. Trans-acting RNAs have been utilized for posttranscriptional and translational control. The regulation of translation initiation is beginning to be explored with ribosome binding sites and riboswitches, while protein degradation tags have been used to control expression levels. Devices built from multiple parts have also been developed to create more complex behaviors. These advances in development of synthetic cyanobacterial regulatory parts provide the groundwork for creation of new, even more sophisticated bioprocess control devices, bolstering the viability of cyanobacteria as sustainable biotechnology platforms.
... To identify the arginine-degrading activity of ArgZ, in vitro assays were performed using Synechocystis ArgZ after histidine-tag purification. After incubation of the purified enzyme with 5 mM l-arginine, the reaction products were identified by HPLC as described previously 47 . Ornithine produced by the enzymatic reaction was further confirmed with LC-MS as described above. ...
... Arginine effluxes to biomass, cyanophycin, and polyamines. The flux of arginine to cyanophycin was quantified from the experimentally measured accumulation rate of cyanophycin 47 . The arginine efflux to biomass was calculated on the basis of previously reported Synechocystis biomass composition 49 and experimentally determined cell growth rate. ...
Living organisms have evolved mechanisms for adjusting their metabolism to adapt to environmental nutrient availability. Terrestrial animals utilize the ornithine–urea cycle to dispose of excess nitrogen derived from dietary protein. Here, we identified an active ornithine–ammonia cycle (OAC) in cyanobacteria through an approach combining dynamic 15N and 13C tracers, metabolomics, and mathematical modeling. The pathway starts with carbamoyl phosphate synthesis by the bacterial- and plant-type glutamine-dependent enzyme and ends with conversion of arginine to ornithine and ammonia by a novel arginine dihydrolase. An arginine dihydrolase–deficient mutant showed disruption of OAC and severely impaired cell growth when nitrogen availability oscillated. We demonstrated that the OAC allows for rapid remobilization of nitrogen reserves under starvation and a high rate of nitrogen assimilation and storage after the nutrient becomes available. Thus, the OAC serves as a conduit in the nitrogen storage-and-
... On the other hand, elucidation of regulatory network of cyanobacteria under various environmental conditions will benefit the efforts of identifying and developing more native inducible promoters. Currently, various RRs and TFs involved in solvent, ion, nutrition, temperature and pH stress responses have been identified and elucidated in cyanobacteria (Ehira and Ohmori, 2012;López-Redondo et al., 2010;Liu and Yang, 2014), and some with potential application in promoter development were summarized in Table 2. Moreover, the cTFbase database and other prediction tools developed for microalgae also contain the putative TFs in up to 26 cyanobacterial genomes, thus could also be useful for defining potential inducible promoters for cyanobacteria (Thiriet-Rupert et al., 2016;Wu et al., 2007). ...
Photosynthetic cyanobacteria are important primary producers and model organisms for studying photosynthesis and elements cycling on earth. Due to the ability to absorb sunlight and utilize carbon dioxide, cyanobacteria have also been proposed as renewable chassis for carbon-neutral "microbial cell factories". Recent progresses on cyanobacterial synthetic biology have led to the successful production of more than two dozen of fuels and fine chemicals directly from CO2, demonstrating their potential for scale-up application in the future. However, compared with popular heterotrophic chassis like Escherichia coli and Saccharomyces cerevisiae, where abundant genetic tools are available for manipulations at levels from single gene, pathway to whole genome, limited genetic tools are accessible to cyanobacteria. Consequently, this significant technical hurdle restricts both the basic biological researches and further development and application of these renewable systems. Though still lagging the heterotrophic chassis, the vital roles of genetic tools in tuning of gene expression, carbon flux re-direction as well as genome-wide manipulations have been increasingly recognized in cyanobacteria. In recent years, significant progresses on developing and introducing new and efficient genetic tools have been made for cyanobacteria, including promoters, riboswitches, ribosome binding site engineering, clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease (CRISPR/Cas) systems, small RNA regulatory tools and genome-scale modeling strategies. In this review, we critically summarize recent advances on development and applications as well as technical limitations and future directions of the genetic tools in cyanobacteria. In addition, toolboxes feasible for using in large-scale cultivation are also briefly discussed.
... In cyanobacteria, the glgP gene, encoding glycogen phosphorylase (GP) that is involved in glycogen breakdown, is upregulated by nitrogen deprivation, and activation of glycogen catabolism is suggested to be a primitive response to nitrogen deprivation (Ehira et al. 2017). Expression of glgP is regulated by the nitrogen-regulated response regulator A (NrrA) Ohmori 2011, Liu andYang 2014). NrrA is a highly conserved transcriptional regulator among b-cyanobacteria and its expression is induced by nitrogen deprivation Ohmori 2006a, Muro-Pastor et al. 2006). ...
... It is noteworthy that glycogen contents of the nrrA mutant after nitrogen deprivation were not higher than those of the WT in Synechococcus PCC 7002 (Fig. 2C). In Synechocystis PCC 6803 and Anabaena PCC 7120, disruption of nrrA increases glycogen contents under nitrogen-deprived conditions Ohmori 2011, Liu andYang 2014). This discrepancy could reflect differences in the methods for determination of glycogen contents, but the possibility that glycogen degradation activity in Synechococcus PCC 7002 might be relatively low compared with Synechocystis PCC 6803 and Anabaena PCC 7120 could not be ruled out. ...
... Substrate-dependent changes in NADPH concentration were monitored by measuring absorbance at 340 nm with a EnSpire 2300 Multimode Plate Reader (PerkinElmer) to determine enzyme activities. GP activities were measured according to Liu and Yang (2014). G6PD and 6PGD activities were measured according to the protocol provided by Oriental Yeast Co. ...
Cyanobacteria respond to nitrogen deprivation by changing cellular metabolism. Glycogen is accumulated within cells to assimilate excess carbon and energy during nitrogen starvation, and inhibition of glycogen synthesis results in impaired nitrogen response and decreased survivability. In spite of glycogen accumulation, genes related to glycogen catabolism are upregulated by nitrogen deprivation. In this study, we found that glycogen catabolism was also involved in acclimation to nitrogen deprivation in the cyanobacterium Synechococcus sp. PCC 7002. The glgP2 gene, encoding glycogen phosphorylase, was induced by nitrogen deprivation, and its expression was regulated by the nitrogen-regulated response regulator A (NrrA), which is a highly conserved transcriptional regulator in cyanobacteria. Activation of glycogen phosphorylase under nitrogen-deprived conditions was abolished by disruption of the nrrA gene, and survivability of the nrrA mutant declined. In addition, a glgP2 mutant was highly susceptible to nitrogen starvation. NrrA also regulated expression of tal-zwf-opcA operon, encoding enzymes of the oxidative pentose phosphate (OPP) pathway, and inactivation of glucose-6-phosphate dehydrogenase, the first enzyme of the OPP pathway, decreased survivability under nitrogen starvation. It was concluded that NrrA facilitates cell survival by activating glycogen degradation and the OPP pathway under nitrogen-deprived conditions.
