Transcriptional Activation of Glycogen Catabolism and the Oxidative Pentose Phosphate Pathway by NrrA Facilitates Cell Survival Under Nitrogen Starvation in the Cyanobacterium Synechococcus sp. Strain PCC 7002

Transcriptional Activation of Glycogen Catabolism and the Oxidative Pentose Phosphate Pathway by... Abstract 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 ability to survive. In spite of glycogen accumulation, genes related to glycogen catabolism are up-regulated 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 survival of the nrrA mutant declined. In addition, a glgP2 mutant was highly susceptible to nitrogen starvation. NrrA also regulated expression of the 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 the ability to survive under nitrogen starvation. It was concluded that NrrA facilitates cell survival by activating glycogen degradation and the OPP pathway under nitrogen-deprived conditions. Introduction Cyanobacteria are a diverse group of bacteria that perform oxygenic photosynthesis. Cyanobacteria inhabit almost all of the biosphere where light is available and play an important role in ecosystems as primary producers. Their growth affects the entire ecosystem and is often limited by the deprivation of nutrients, such as nitrogen and phosphorus (Vitousek and Howarth 1991). In the absence of combined nitrogen sources, many cyanobacteria carry out nitrogen fixation to utilize atmospheric nitrogen as a nitrogen source. Meanwhile, responses to nitrogen deprivation of non-nitrogen-fixing cyanobacteria include cessation of cell proliferation and changes of cellular metabolism and morphology (Schwarz and Forchhammer 2005). The color of cyanobacterial cultures changes from blue-green to yellow, and this phenomenon is known as bleaching or chlorosis (Collier and Grossman 1992). During chlorosis, phycobilisomes, light-harvesting antennae protein complexes, were degraded, and then Chl was lost, with a decline of photosynthetic activity (Görl et al. 1998). Degradation of phycobilisomes balances production of ATP and NADPH by photosynthesis with utilization of energy for cell maintenance during nitrogen starvation (Schwarz and Grossman 1998). Imbalances between energy acquisition and utilization could result in production of reactive oxygen species (Latifi et al. 2009). Thus, the pigment degradation is crucial for cell survival under conditions of nitrogen deprivation. Glycogen accumulation is another response to nitrogen deprivation in cyanobacteria (Gründel et al. 2012, Jackson et al. 2015). Glycogen amounts to 40–60% of the dry cell weight under nitrogen-deprived conditions (Hasunuma et al. 2013). Glycogen is synthesized from glucose-1-phosphate by the series of reactions catalyzed by ADP-glucose pyrophosphorylase and glycogen synthase (Suzuki et al. 2010). Under nitrogen-deprived conditions, cell growth ceases, and demands for building blocks of cellular components and energy for cellular activity are decreased. Hence, excess carbon and energy are stored as glycogen. Inhibition of glycogen synthesis perturbs carbon and energy homeostasis and reduces cell viability during nitrogen starvation (Gründel et al. 2012). Moreover, mutants in glycogen synthesis are unable to degrade phycobilisomes, indicating that glycogen synthesis is involved in regulation of the nitrogen response (Gründel et al. 2012, Hickman et al. 2013, Jackson et al. 2015). In cyanobacteria, the glgP gene, encoding glycogen phosphorylase (GP) that is involved in glycogen breakdown, is up-regulated 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) (Ehira and Ohmori 2011, Liu and Yang 2014). NrrA is a highly conserved transcriptional regulator among β-cyanobacteria and its expression is induced by nitrogen deprivation (Ehira and Ohmori 2006a, Muro-Pastor et al. 2006). In the unicellular cyanobacterium Synechocystis sp. PCC 6803, NrrA facilitates heterotrophic growth with glucose in the dark by up-regulating expression of genes involved in glycogen catabolism (Azuma et al. 2011). In the nitrogen-fixing, heterocystous cyanobacterium Anabaena sp. PCC 7120, NrrA regulates nitrogen fixation activity by activating glycogen catabolism (Ehira and Ohmori 2011). NrrA is shown to be a regulator of glycogen catabolism, but it still remains to be unraveled whether NrrA and glycogen catabolism are involved in acclimation to nitrogen starvation. In this study, we characterized an nrrA mutant of the non-nitrogen-fixing cyanobacterium Synechococcus sp. PCC 7002 and indicated that the ability of the nrrA mutant to survive during nitrogen starvation was lower than that of the wild-type (WT) strain. In addition, disruption of the glgP2 gene, which was regulated by NrrA, resulted in a drastic decline of survival. Thus, glycogen catabolism is crucial for survival under nitrogen starvation. In contrast to the mutant of glycogen synthesis, the nrrA and glgP2 mutants responded normally to nitrogen deprivation, i.e. photosynthetic pigments were degraded and glycogen was accumulated within cells, indicating that glycogen degradation plays different roles from glycogen synthesis in acclimation to nitrogen deprivation. Results NrrA plays an important role in cell survival under nitrogen-deprived conditions Expression of nrrA is induced by nitrogen deprivation in β-cyanobacteria including Synechococcus PCC 7002 (Ehira et al. 2017). To investigate a role for nrrA in acclimation to nitrogen starvation, an insertion mutant of the nrrA gene in Synechococcus PCC 7002 was created, and cellular viability after incubation under nitrogen-deprived conditions was determined by calculating the colony-forming units (CFU) (Fig. 1). In the WT, the CFU were reduced to 15% after 1 d of nitrogen starvation and to 4% after 2 d. The CFU of the nrrA mutant immediately after shift to nitrogen-free medium were comparable with those of the WT; however they were reduced to 6% after 1 d and, after 2 d, only 0.6% of cells were able to regrow. Thus, the nrrA mutant was susceptible to nitrogen starvation, indicating that the nrrA gene facilitates cell survival under nitrogen starvation. Fig. 1 View largeDownload slide Viability under nitrogen-deprived conditions. (A) Cells of the WT and the nrrA mutant (ΔnrrA) grown with nitrate were transferred to nitrogen-free medium at an OD750 of 0.5. After 0, 1 or 2 d, 10 µl aliquots of cultures were plated on medium A+ agar at a 10-fold serial dilution and the plates were incubated under continuous illumination of 100 µmol photons m–2 s–1 for 3 d. (B) A viability test was conducted with the WT (filled circles), the nrrA mutant (open circles), the glgP2 mutant (filled triangles) and the opcA mutant (open triangles). Colonies that appeared on the plates after 2 or 3 d incubation were counted. The means ± SD (error bar) of three independent experiments are shown. Fig. 1 View largeDownload slide Viability under nitrogen-deprived conditions. (A) Cells of the WT and the nrrA mutant (ΔnrrA) grown with nitrate were transferred to nitrogen-free medium at an OD750 of 0.5. After 0, 1 or 2 d, 10 µl aliquots of cultures were plated on medium A+ agar at a 10-fold serial dilution and the plates were incubated under continuous illumination of 100 µmol photons m–2 s–1 for 3 d. (B) A viability test was conducted with the WT (filled circles), the nrrA mutant (open circles), the glgP2 mutant (filled triangles) and the opcA mutant (open triangles). Colonies that appeared on the plates after 2 or 3 d incubation were counted. The means ± SD (error bar) of three independent experiments are shown. Under nitrogen-deprived conditions, photosynthetic pigments are degraded, photosynthetic activity declines and glycogen is accumulated within cells, which are important responses to acclimate to nitrogen starvation (Gründel et al. 2012, Jackson et al. 2015). We investigated whether the nrrA mutant was able to respond adequately to nitrogen deprivation (Fig. 2). Photosynthetic activity was assayed by measuring oxygen generation by PSII with bicarbonate as an electron acceptor (Fig. 2A), and glycogen levels within cells were determined by measuring glucose that was liberated from glycogen by glucoamylase (Fig. 2C). Changes in photosynthetic activity and contents of Chl and glycogen in the nrrA mutant were comparable with those in the WT, indicating that the nrrA mutant was capable of responding normally to nitrogen deprivation at least with respect to suppression of photosynthesis and accumulation of glycogen. Thus, NrrA is likely to support cell survival by hitherto unidentified mechanisms. Fig. 2 View largeDownload slide Cellular responses to nitrogen deprivation. Cells of the WT (filled circles), the nrrA mutant (open circles) and the glgP2 mutant (filled triangles) that were subjected to nitrogen deficiency for the indicated time were collected, and then photosynthetic activities (A), Chl (B) and glycogen contents (C) were determined. The means ± SD (error bar) of at least three independent experiments are shown. Fig. 2 View largeDownload slide Cellular responses to nitrogen deprivation. Cells of the WT (filled circles), the nrrA mutant (open circles) and the glgP2 mutant (filled triangles) that were subjected to nitrogen deficiency for the indicated time were collected, and then photosynthetic activities (A), Chl (B) and glycogen contents (C) were determined. The means ± SD (error bar) of at least three independent experiments are shown. NrrA activates glycogen catabolism and respiration under nitrogen-deprived conditions To identify genes regulated by NrrA, RNA-sequencing analysis was conducted in the WT and the nrrA mutant. In the WT, the transcript levels of 404 genes were increased 3 h after nitrogen deprivation (Supplementary Table S1). The up-regulated genes included glnA, glnN, nblA, the ctaI operon, the ctaII operon, amt and ndhD2, which was consistent with the previous report by Ludwig and Bryant (2012). NrrA-regulated genes were identified by comparing the gene expression profile 3 h after nitrogen deprivation between the WT and the nrrA mutant. The transcript levels of 25 genes that were induced by nitrogen deprivation were decreased by disruption of nrrA (Table 1). As is the case in other β-cyanobacteria (Ehira et al. 2017), glgP2 that encodes GP was down-regulated by nrrA disruption. In addition, the tal-zwf-opcA operon that encodes enzymes of the oxidative pentose phosphate (OPP) pathway and the ctaI (ctaCI-DI-EI) operon that encodes Cyt oxidase were down-regulated. Because the gene expression profile of the nrrA mutant was analyzed by RNA-sequencing only once without making biological replicates, we confirmed the differences in gene expression by quantitative reverse transcription–PCR (qRT–PCR). It was shown that the transcript levels of glgP2, tal, zwf, opcA and ctaCI after 3 h of nitrogen deprivation were decreased in the nrrA mutant (Fig. 3; Supplementary Fig. S1). The gnd gene that encodes 6-phosphogluconate dehydrogenase (6PGD), another enzyme of the OPP pathway, was also up-regulated by nitrogen deprivation (Supplementary Table S1). The difference between the WT and the nrrA mutant was not significant in the RNA-sequencing analysis, but qRT–PCR showed that the transcript level of gnd was decreased by disruption of nrrA (Fig. 3). Table 1 Nitrogen-induced genes that are down-regulated by nrrA disruption       WT3/WT0a  ΔnrrA3/WT3b  Gene-ID  Gene  Product  Ratio  qc  Ratio  qc  A0330  malQ  4-α-Glucanotransferase  1.5  4.8E-06  –1.7  5.9E-03  A0331    Conserved hypothetical protein  1.8  1.2E-08  –1.9  3.8E-03  A0378    Conserved hypothetical protein  1.8  1.1E-05  –1.9  4.7E-02  A0479    Conserved hypothetical protein  1.9  1.1E-11  –1.6  2.8E-03  A0481  glgP2  Glycogen phosphorylase  1.5  7.1E-08  –1.7  3.4E-03  A0496  nrrA  Two-component response regulator  3.8  3.0E-31  –9.9  5.7E-49  A0497    ATP-binding protein of ABC transporter  3.1  6.1E-28  –4.6  3.0E-21  A0832    Acetyltransferase, GNAT family  2.0  4.6E-14  –1.5  1.4E-03  A0922    Conserved hypothetical protein  2.1  7.7E-15  –2.4  1.0E-06  A1071  putA  Proline oxidase  1.5  9.5E-07  –1.7  2.9E-04  A1162  ctaCI  Cytochrome oxidase subunit II  2.0  7.5E-12  –1.6  2.0E-03  A1163  ctaDI  Cytochrome oxidase subunit I  1.8  4.7E-09  –1.7  7.6E-04  A1164  ctaEI  Cytochrome oxidase subunit III  1.4  9.7E-07  –1.3  1.1E-02  A1458  opcA  Putative OxPP cycle protein opcA  2.2  7.5E-13  –1.3  2.5E-02  A1459  zwf  Glucose-6-phosphate dehydrogenase  2.4  3.2E-16  –1.5  3.3E-03  A1460  tal  Transaldolase  2.6  3.7E-16  –1.6  2.8E-03  A1933    NblA-related protein  6.4  6.9E-67  –1.9  5.8E-04  A1934    Universal stress protein-like protein  2.6  2.3E-20  –1.4  2.1E-02  A2278    Hypothetical protein  2.6  1.9E-23  –1.2  2.2E-02  A2357  rsuA  RNA pseudouridylate synthase  2.3  1.4E-20  –1.2  2.8E-02  A2405  cphA  Cyanophycin synthetase  1.2  1.6E-05  –1.8  2.6E-04  A2490    Transglutaminase domain protein  1.7  4.7E-13  –1.6  2.9E-04  A2771    Succinate-semialdehyde dehydrogenase  2.4  9.7E-17  –1.4  1.0E-02  A2772    Conserved hypothetical protein  5.9  6.2E-65  –2.2  1.7E-05  B0001    Hypothetical protein  1.1  3.0E-03  –1.6  1.0E-02        WT3/WT0a  ΔnrrA3/WT3b  Gene-ID  Gene  Product  Ratio  qc  Ratio  qc  A0330  malQ  4-α-Glucanotransferase  1.5  4.8E-06  –1.7  5.9E-03  A0331    Conserved hypothetical protein  1.8  1.2E-08  –1.9  3.8E-03  A0378    Conserved hypothetical protein  1.8  1.1E-05  –1.9  4.7E-02  A0479    Conserved hypothetical protein  1.9  1.1E-11  –1.6  2.8E-03  A0481  glgP2  Glycogen phosphorylase  1.5  7.1E-08  –1.7  3.4E-03  A0496  nrrA  Two-component response regulator  3.8  3.0E-31  –9.9  5.7E-49  A0497    ATP-binding protein of ABC transporter  3.1  6.1E-28  –4.6  3.0E-21  A0832    Acetyltransferase, GNAT family  2.0  4.6E-14  –1.5  1.4E-03  A0922    Conserved hypothetical protein  2.1  7.7E-15  –2.4  1.0E-06  A1071  putA  Proline oxidase  1.5  9.5E-07  –1.7  2.9E-04  A1162  ctaCI  Cytochrome oxidase subunit II  2.0  7.5E-12  –1.6  2.0E-03  A1163  ctaDI  Cytochrome oxidase subunit I  1.8  4.7E-09  –1.7  7.6E-04  A1164  ctaEI  Cytochrome oxidase subunit III  1.4  9.7E-07  –1.3  1.1E-02  A1458  opcA  Putative OxPP cycle protein opcA  2.2  7.5E-13  –1.3  2.5E-02  A1459  zwf  Glucose-6-phosphate dehydrogenase  2.4  3.2E-16  –1.5  3.3E-03  A1460  tal  Transaldolase  2.6  3.7E-16  –1.6  2.8E-03  A1933    NblA-related protein  6.4  6.9E-67  –1.9  5.8E-04  A1934    Universal stress protein-like protein  2.6  2.3E-20  –1.4  2.1E-02  A2278    Hypothetical protein  2.6  1.9E-23  –1.2  2.2E-02  A2357  rsuA  RNA pseudouridylate synthase  2.3  1.4E-20  –1.2  2.8E-02  A2405  cphA  Cyanophycin synthetase  1.2  1.6E-05  –1.8  2.6E-04  A2490    Transglutaminase domain protein  1.7  4.7E-13  –1.6  2.9E-04  A2771    Succinate-semialdehyde dehydrogenase  2.4  9.7E-17  –1.4  1.0E-02  A2772    Conserved hypothetical protein  5.9  6.2E-65  –2.2  1.7E-05  B0001    Hypothetical protein  1.1  3.0E-03  –1.6  1.0E-02  aRatios of transcript levels 3 h after relative to before nitrogen deprivation in the WT are shown in the base 2 logarithm. bRatios of transcript levels in the nrrA mutant relative to the WT 3 h after nitrogen deprivation are shown in the base 2 logarithm. cFalse discovery rate or the rate of type I errors with consideration of multiple testing. Table 1 Nitrogen-induced genes that are down-regulated by nrrA disruption       WT3/WT0a  ΔnrrA3/WT3b  Gene-ID  Gene  Product  Ratio  qc  Ratio  qc  A0330  malQ  4-α-Glucanotransferase  1.5  4.8E-06  –1.7  5.9E-03  A0331    Conserved hypothetical protein  1.8  1.2E-08  –1.9  3.8E-03  A0378    Conserved hypothetical protein  1.8  1.1E-05  –1.9  4.7E-02  A0479    Conserved hypothetical protein  1.9  1.1E-11  –1.6  2.8E-03  A0481  glgP2  Glycogen phosphorylase  1.5  7.1E-08  –1.7  3.4E-03  A0496  nrrA  Two-component response regulator  3.8  3.0E-31  –9.9  5.7E-49  A0497    ATP-binding protein of ABC transporter  3.1  6.1E-28  –4.6  3.0E-21  A0832    Acetyltransferase, GNAT family  2.0  4.6E-14  –1.5  1.4E-03  A0922    Conserved hypothetical protein  2.1  7.7E-15  –2.4  1.0E-06  A1071  putA  Proline oxidase  1.5  9.5E-07  –1.7  2.9E-04  A1162  ctaCI  Cytochrome oxidase subunit II  2.0  7.5E-12  –1.6  2.0E-03  A1163  ctaDI  Cytochrome oxidase subunit I  1.8  4.7E-09  –1.7  7.6E-04  A1164  ctaEI  Cytochrome oxidase subunit III  1.4  9.7E-07  –1.3  1.1E-02  A1458  opcA  Putative OxPP cycle protein opcA  2.2  7.5E-13  –1.3  2.5E-02  A1459  zwf  Glucose-6-phosphate dehydrogenase  2.4  3.2E-16  –1.5  3.3E-03  A1460  tal  Transaldolase  2.6  3.7E-16  –1.6  2.8E-03  A1933    NblA-related protein  6.4  6.9E-67  –1.9  5.8E-04  A1934    Universal stress protein-like protein  2.6  2.3E-20  –1.4  2.1E-02  A2278    Hypothetical protein  2.6  1.9E-23  –1.2  2.2E-02  A2357  rsuA  RNA pseudouridylate synthase  2.3  1.4E-20  –1.2  2.8E-02  A2405  cphA  Cyanophycin synthetase  1.2  1.6E-05  –1.8  2.6E-04  A2490    Transglutaminase domain protein  1.7  4.7E-13  –1.6  2.9E-04  A2771    Succinate-semialdehyde dehydrogenase  2.4  9.7E-17  –1.4  1.0E-02  A2772    Conserved hypothetical protein  5.9  6.2E-65  –2.2  1.7E-05  B0001    Hypothetical protein  1.1  3.0E-03  –1.6  1.0E-02        WT3/WT0a  ΔnrrA3/WT3b  Gene-ID  Gene  Product  Ratio  qc  Ratio  qc  A0330  malQ  4-α-Glucanotransferase  1.5  4.8E-06  –1.7  5.9E-03  A0331    Conserved hypothetical protein  1.8  1.2E-08  –1.9  3.8E-03  A0378    Conserved hypothetical protein  1.8  1.1E-05  –1.9  4.7E-02  A0479    Conserved hypothetical protein  1.9  1.1E-11  –1.6  2.8E-03  A0481  glgP2  Glycogen phosphorylase  1.5  7.1E-08  –1.7  3.4E-03  A0496  nrrA  Two-component response regulator  3.8  3.0E-31  –9.9  5.7E-49  A0497    ATP-binding protein of ABC transporter  3.1  6.1E-28  –4.6  3.0E-21  A0832    Acetyltransferase, GNAT family  2.0  4.6E-14  –1.5  1.4E-03  A0922    Conserved hypothetical protein  2.1  7.7E-15  –2.4  1.0E-06  A1071  putA  Proline oxidase  1.5  9.5E-07  –1.7  2.9E-04  A1162  ctaCI  Cytochrome oxidase subunit II  2.0  7.5E-12  –1.6  2.0E-03  A1163  ctaDI  Cytochrome oxidase subunit I  1.8  4.7E-09  –1.7  7.6E-04  A1164  ctaEI  Cytochrome oxidase subunit III  1.4  9.7E-07  –1.3  1.1E-02  A1458  opcA  Putative OxPP cycle protein opcA  2.2  7.5E-13  –1.3  2.5E-02  A1459  zwf  Glucose-6-phosphate dehydrogenase  2.4  3.2E-16  –1.5  3.3E-03  A1460  tal  Transaldolase  2.6  3.7E-16  –1.6  2.8E-03  A1933    NblA-related protein  6.4  6.9E-67  –1.9  5.8E-04  A1934    Universal stress protein-like protein  2.6  2.3E-20  –1.4  2.1E-02  A2278    Hypothetical protein  2.6  1.9E-23  –1.2  2.2E-02  A2357  rsuA  RNA pseudouridylate synthase  2.3  1.4E-20  –1.2  2.8E-02  A2405  cphA  Cyanophycin synthetase  1.2  1.6E-05  –1.8  2.6E-04  A2490    Transglutaminase domain protein  1.7  4.7E-13  –1.6  2.9E-04  A2771    Succinate-semialdehyde dehydrogenase  2.4  9.7E-17  –1.4  1.0E-02  A2772    Conserved hypothetical protein  5.9  6.2E-65  –2.2  1.7E-05  B0001    Hypothetical protein  1.1  3.0E-03  –1.6  1.0E-02  aRatios of transcript levels 3 h after relative to before nitrogen deprivation in the WT are shown in the base 2 logarithm. bRatios of transcript levels in the nrrA mutant relative to the WT 3 h after nitrogen deprivation are shown in the base 2 logarithm. cFalse discovery rate or the rate of type I errors with consideration of multiple testing. Fig. 3 View largeDownload slide Changes in the transcript levels of the NrrA regulon after nitrogen deprivation. The relative transcript levels of glgP2, tal, catCI and gnd were determined by qRT–PCR in the WT (filled circles) and the nrrA mutant (open circles). RNA samples were prepared from three independently grown cultures. The transcript level at 0 h of the WT was taken as 1. Data that represent a significant difference (P < 0.01) by applying the t-test between the WT and the nrrA mutant are marked with asterisks. Fig. 3 View largeDownload slide Changes in the transcript levels of the NrrA regulon after nitrogen deprivation. The relative transcript levels of glgP2, tal, catCI and gnd were determined by qRT–PCR in the WT (filled circles) and the nrrA mutant (open circles). RNA samples were prepared from three independently grown cultures. The transcript level at 0 h of the WT was taken as 1. Data that represent a significant difference (P < 0.01) by applying the t-test between the WT and the nrrA mutant are marked with asterisks. Although the transcript levels of glgP2, tal, ctaCI and gnd were significantly decreased 3 h after nitrogen deprivation in the nrrA mutant, expression of these genes was increased after 6 h (Fig. 3). To analyze the consequence of the delayed induction in the nrrA mutant, enzyme activities of GP and two enzymes of the OPP pathway, glucose-6-phosphate dehydrogenase (G6PD) encoded by zwf and 6PGD at 9 h after nitrogen deprivation were determined. Consistent with changes in the transcript levels, activities of these enzymes were increased by nitrogen deprivation in the WT (Fig. 4A). In the nrrA mutant, activities of these enzymes before nitrogen deprivation were the same as those in the WT, but an increase after nitrogen deprivation was not observed or was less than that in the WT (Fig. 4A). These results indicate that the delayed induction of gene expression in the nrrA mutant significantly reduces the enzyme activities after nitrogen deprivation. Next, oxygen consumption rates were determined. Oxygen consumption rates of the WT were increased about 3-fold 3 h after nitrogen deprivation, and then gradually decreased to the level before nitrogen deprivation (Fig. 4B). In the nrrA mutant, oxygen consumption rates were also increased by 85% after 3 h, but the activity was 40% lower than that of the WT (Fig. 4B). Thus, NrrA activates glycogen degradation, the OPP pathway and respiration in response to nitrogen deprivation. Fig. 4 View largeDownload slide Changes in enzyme activities after nitrogen deprivation. (A) Activities of glycogen phosphorylase (GP), glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) were determined before (open bars) and 9 h after nitrogen deprivation (filled bars) in the WT and the nrrA mutant (ΔnrrA). P-values in the t-test are indicated. (B) Respiration activities of cells subjected to nitrogen deficiency for the indicated time were determined in the WT (filled circles), the nrrA mutant (open circles) and the ctaCI mutant (black triangles). Data that represent a significant difference (P < 0.05) by applying the t-test between the WT and the mutants are marked with asterisks. Measurement was repeated at least three times with cells from independently grown cultures. Fig. 4 View largeDownload slide Changes in enzyme activities after nitrogen deprivation. (A) Activities of glycogen phosphorylase (GP), glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) were determined before (open bars) and 9 h after nitrogen deprivation (filled bars) in the WT and the nrrA mutant (ΔnrrA). P-values in the t-test are indicated. (B) Respiration activities of cells subjected to nitrogen deficiency for the indicated time were determined in the WT (filled circles), the nrrA mutant (open circles) and the ctaCI mutant (black triangles). Data that represent a significant difference (P < 0.05) by applying the t-test between the WT and the mutants are marked with asterisks. Measurement was repeated at least three times with cells from independently grown cultures. Glycogen degradation and the OPP pathway facilitate cell survival under nitrogen-deprived conditions The nrrA disruptant showed reduced ability to survive under nitrogen-deprived conditions (Fig. 1). To reveal which genes regulated by NrrA were related to survival under nitrogen starvation, the glgP2, opcA and ctaCI genes were inactivated and cell viability of the mutants was determined. The viability of the glgP2 and opcA mutants was lower than that of the WT (Fig. 1B). The decrease of viability in the opcA mutant was comparable with that in the nrrA mutant, while the glgP2 mutant was much more susceptible to nitrogen starvation than the nrrA mutant. The CFU of the glgP2 mutant after 24 h of nitrogen deprivation were <1% of those of the nrrA mutant. Because disruption of glgP2 might affect glycogen synthesis, which is important for cell survival under nitrogen starvation (Gründel et al. 2012), glycogen contents of the glgP2 disruptant were determined. The glycogen level was increased 10-fold within 6 h after nitrogen deprivation as well as in the WT, though the levels after 24 and 48 h were 25% lower than those of the WT (Fig. 2C). Thus, glycogen synthesis was not inhibited in the glgP2 mutant, suggesting that the decrease of viability in the glgP2 mutant was not caused by impairment of glycogen synthesis. Glycogen degradation catalyzed by GP could allow cells to survive under nitrogen starvation. OpcA is an allosteric activator of G6PD and its disruption results in the loss of G6PD activity (Summers et al. 1995, Hagen and Meeks 2001). G6PD, which is the first enzyme of the OPP pathway, controls the entire activity of the pathway. Thus, it was shown that the OPP pathway also supports survival under nitrogen starvation. Meanwhile, disruption of ctaCI did not affect cell viability (data not shown). In Synechococcus PCC 7002, there are two operons (ctaI and ctaII) that encode Cyt oxidases (Nomura et al. 2006a). Although both operons were up-regulated by nitrogen deprivation, only ctaI was regulated by NrrA (Table 1; Supplementary Table S1). Oxygen consumption rates of the ctaCI mutant were lower than those of the WT even before nitrogen deprivation, but were increased about 3-fold after nitrogen deprivation as well as in the WT (Fig. 4B). Thus, the ctaI operon was not required for activation of respiration and survival under nitrogen starvation. These results indicate that up-regulation of the glgP2 gene and the tal-zwf-opcA operon by NrrA plays an important role in acclimation to nitrogen deprivation. NrrA directly regulates expression of the glgP2 and gnd genes, and the tal-zwf-opcA and ctaI operons Interaction between NrrA and upstream regions of glgP2, tal, ctaCI and gnd was analyzed by electrophoretic mobility shift assay. His-tagged NrrA protein was mixed with Cy3-labeled DNA probes containing the upstream region of glgP2, tal, ctaCI or gnd, and then the mixtures were subjected to electrophoresis. NrrA decreased the electrophoretic mobility of DNA probes in a concentration-dependent manner (Fig. 5A). Addition of non-labeled probe diminished the interaction of NrrA and DNA probes, but the DNA fragment PhetR2, which does not bind to NrrA of Anabaena PCC 7120 (Ehira and Ohmori 2014), did not affect the interaction. NrrA recognizes the inverted repeat sequence GTCAN8TGAC (Ehira et al. 2017). The NrrA recognition sequences were found within the upstream regions of glgP2, tal, ctaCI and gnd (Fig. 5B). These results strongly support the idea that NrrA directly regulates expression of these genes. Fig. 5 View largeDownload slide DNA binding assays of NrrA protein. (A) Cy3-labeled probes (3 nM) including the promoter regions of glgP2, tal, ctaCI and gnd were mixed with NrrA proteins in the amount indicated above each lane, and the mixtures were subjected to electrophoresis. Non-labeled fragments (PglgP2, Ptal, PctaCI or Pgnd, and PhetR2) were added at a final concentration of 30 nM (lanes 5 and 7) or 90 nM (lanes 6 and 8). Open arrows, probe alone; filled arrows, complexes with NrrA and the Cy3-labeled probes. (B) Alignment of the promoter regions of glgP2, tal, ctaCI and gnd. The consensus sequence recognized by NrrA is shown at the bottom, and the putative NrrA-binding sites that have been found within the promoter regions are shown byin bold. Fig. 5 View largeDownload slide DNA binding assays of NrrA protein. (A) Cy3-labeled probes (3 nM) including the promoter regions of glgP2, tal, ctaCI and gnd were mixed with NrrA proteins in the amount indicated above each lane, and the mixtures were subjected to electrophoresis. Non-labeled fragments (PglgP2, Ptal, PctaCI or Pgnd, and PhetR2) were added at a final concentration of 30 nM (lanes 5 and 7) or 90 nM (lanes 6 and 8). Open arrows, probe alone; filled arrows, complexes with NrrA and the Cy3-labeled probes. (B) Alignment of the promoter regions of glgP2, tal, ctaCI and gnd. The consensus sequence recognized by NrrA is shown at the bottom, and the putative NrrA-binding sites that have been found within the promoter regions are shown byin bold. Discussion In this study, we demonstrated that the nitrogen-regulated response regulator NrrA played an important role in survival under nitrogen starvation in Synechococcus PCC 7002. NrrA directly regulated expression of glgP2, gnd, tal-zwf-opcA and the ctaI operon in response to nitrogen deprivation. Activation of GP, enzymes of the OPP pathway and respiration under nitrogen-deprived conditions was diminished by nrrA disruption, which led to a decline in the ability to survive under nitrogen starvation. The glgP2 and opcA mutants were also susceptible to nitrogen starvation, indicating that NrrA-dependent activation of glycogen degradation and the OPP pathway would be crucial for acclimation to nitrogen starvation. NrrA is a highly conserved transcriptional regulator among β-cyanobacteria (Ehira et al. 2017). It has been shown that NrrA regulates genes for glycogen catabolism and respiration in Synechococcus elongatus PCC 7942 and Synechocystis PCC 6803, as well as in Synechococcus PCC 7002 (Azuma et al. 2011, Ehira et al. 2017). Activation of glycogen catabolism and respiration by NrrA would be a universal response to nitrogen starvation in cyanobacteria. 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 (Ehira and Ohmori 2011, Liu and Yang 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. In cyanobacteria, glucose derived from glycogen degradation is mainly catabolized though the OPP pathway (Jansén et al. 2010, Nakajima et al. 2014). The OPP pathway produces NADPH, and NADPH is used as the electron donor for respiration in cyanobacteria (Ogawa and Mi 2007). The activity of the OPP pathway is regulated by the cellular redox state and is usually suppressed under photoautotrophic conditions to prevent futile cycling, which occurs if the Calvin–Benson and OPP pathways operate simultaneously (Udvardy et al. 1984). However, the OPP pathway enhanced survival under nitrogen starvation (Fig. 1), indicating that the OPP pathway was operative in those conditions. Photosynthetic activity was decreased to one-third within 6 h after nitrogen deprivation, with a further decline to one-tenth after 24 h, when the increase in glycogen level ceased (Fig. 2). ATP and NADPH supply from photosynthesis would be insufficient to sustain cell viability when nitrogen starvation was prolonged, and glycogen degradation and the subsequent catabolism through the OPP pathway could compensate for the shortage of ATP and NADPH to support long-term survival. The glgP2 mutant was much more sensitive to nitrogen starvation than the nrrA and opcA mutants (Fig. 1). The difference in tha ability to survive between the glgP2 and opcA mutants implies that the OPP pathway is not the sole route of glucose catabolism. The gap and pyk genes, encoding key enzymes of the Embden–Meyerhof–Parnass (EMP) pathway, are up-regulated by nitrogen deprivation in Synechococcus elongatus PCC 7942, Synechocystis PCC 6803 and Anabaena PCC 7120 (Osanai et al. 2006, Ehira and Ohmori 2011, Ehira et al. 2017). Glucose could also be catabolized via the EMP pathway. In addition, the Entner–Doudoroff (ED) pathway has been shown to operate in cyanobacteria (Chen et al. 2016). Compared with the EMP pathway, the ED pathway has lower ATP yield, but requires less enzymatic protein to achieve the same glycolytic flux (Flamholz et al. 2013). Thus, the ED pathway would be preferable to the EMP pathway under nitrogen-deprived conditions, when protein synthesis is limited. Further research is needed to clarify the contribution of each glucose degradation pathway to acclimation to nitrogen starvation. In the nrrA disruptant, expression of glgP2 was not increased until 3 h after nitrogen deprivation, but it was induced at 6 h (Fig. 3). The nitrogen-dependent induction of glgP2 would be controlled by multiple transcriptional regulators including NrrA. In Anabaena PCC 7120 and Synechocystis PCC 6803, a group 2 sigma factor, SigE, is involved in regulation of glycogen catabolism (Azuma et al. 2011, Ehira and Ohmori 2011). The sigC gene, encoding a SigE ortholog in Synechococcus PCC 7002, was up-regulated by nitrogen deprivation (Supplementary Table S1). The collaborative regulation of glycogen catabolism by NrrA and SigC would also function in Synechococcus PCC 7002. Although the glgP2 transcript level was increased with a delay of 3 h in the nrrA mutant, the activity of GP was not increased (Fig. 4A). This was the same for G6PD and 6PGD. It is likely that a delay in the transcriptional induction results in disconnection of transcription and translation. Intracellular levels of some amino acids, such as alanine, aspartate, asparagine, glutamate and valine, are decreased with time during nitrogen starvation (Hasunuma et al. 2013), and the shortage of amino acids would hinder de novo protein synthesis. Thus, NrrA enables cells to respond rapidly to nitrogen deprivation, and a rapid response is crucial for adjusting the cellular proteome to survival under nitrogen starvation. Materials and Methods Bacterial strains and culture conditions Synechococcus sp. PCC 7002 and its derivatives were grown at 38°C with continuous illumination at 200 µmol photons m–2 s–2 in A+ medium as described previously (Ehira et al. 2017). Liquid cultures were bubbled with air containing 1% (v/v) CO2. To shift cells into conditions of nitrogen deprivation, cells were washed with sterilized water three times and then resuspended in A medium without combined nitrogen sources at an OD750 of 0.5, followed by cultivation under the same conditions as mentioned above. Kanamycin (50 μg ml–1) was added to cultures of the nrrA and ctaCI mutants, and spectinomycin (50 μg ml–1) was added to cultures of the glgP2 and opcA mutants when required. Mutant construction To inactivate the glgP2 gene, a DNA fragment containing the glgP2 gene was amplified by PCR using the primer pair sypglgP2-F and sypglgP2-R (Supplementary Table S2). The fragment was cloned at the EcoRV site of pPCR-Script Amp SK+ (Agilent Technologies). A spectinomycin resistance cassette from the plasmid pDW9 (Golden and Wiest 1988) was inserted into the XbaI site within the glgP2 coding region. To inactivate nrrA, opcA and ctaCI, upstream and downstream regions of each gene were amplified by PCR using primer pairs 5F and 5R, and 3F and 3R for each gene, respectively (Supplementary Table S2), and a spectinomycin or kanamycin cassette was inserted between the upstream and downstream fragments. These plasmids were used for transformation of Synechococcus PCC 7002. Glycogen determination The cellular glycogen content was determined according to Jackson et al. (2015). Briefly, glycogen was extracted from cells by boiling in 30% (w/v) KOH for 1 h, and then precipitated by mixing with ethanol. Glycogen content was determined by measuring glucose liberated by treatment of α-amyloglucosidase using a glycogen colorimetric/fluorometric assay kit (BioVision). Oxygen evolution assay Oxygen evolution and consumption activities were determined using a Clark-type electrode (Hansatech Instruments) according to Nomura et al. (2006b). To determine photosynthetic activities, 1 ml of culture (OD750 of 0.5–1.0) was placed into an electrode chamber and 5 mM NaHCO3 was added. The samples were kept in the dark for 5 min without a lid to saturate the samples with air levels of oxygen. Oxygen evolution was recorded for 5 min at 38°C with a saturating amount of light (4,000 µmol photons m–2 s–1). Photosynthetic activities were determined by adding values of oxygen consumption in the dark to those of oxygen evolution. To determine respiration activities, 1 ml of culture concentrated to OD750 of about 4.0 was used. Oxygen consumption was measured in the dark for 5 min and then 1 mM KCN was added. Respiration activities were determined by subtracting values of oxygen consumption with KCN from those in the dark. The Chl content of cultures was determined according to Mackinney (1941). RNA extraction and qRT–PCR analysis RNA extraction and qRT–PCR analysis were conducted according to Ehira and Ohmori (2011) using GoTaq qPCR Master Mix (Promega). Primers used for qRT–PCR are listed in Supplementary Table S2. RNA-sequencing analysis Libraries for RNA-sequencing were constructed using 1 µg of total RNA as follows. rRNA was depleted from total RNA by the Ribo-Zero rRNA Removal kit (Bacteria) (Illumina) according to the manufacturer’s protocol. Sequencing libraries were prepared by an NEBNext mRNA library prep kit from Illumina (NEB) with the following modifications. The random hexamer primer was used for reverse transcription. After second-strand synthesis, double-stranded cDNA was fragmented to an average length of 300 bp using a Covaris S2 sonication system (Covaris). Three biological replicates were made for each condition (before nitrogen deprivation and 3 h after nitrogen deprivation) of the WT, whereas no biological replicate was made for each condition of the nrrA mutant. One hundred base pairs from both ends of each fragment were then sequenced on the HiSeq2500 system platform (Illumina). After the sequencing reactions were complete, the Illumina analysis pipeline (CASAVA 1.8.0) was used to process the raw sequencing data. Original read data are available under the accession numbers DRX114943–DRX114950 in the DDBJ/ENA/GenBank. All reads obtained were first processed with Cutadapt version 1.14 (Martin 2011) to remove adaptor sequences. The processed reads were further treated with SolexaQA++ version 3.1.7.1 (Cox et al. 2010) to select pairs of sequences with a quality score ≥25 for ≥50 consecutive nucleotides for both forward and reverse reads. The processed high quality read pairs were mapped onto the reference genome (GCA_000019485.1_ASM1948v1_genomic.fna) using Bowtie 2 version 2.3.3 (Langmead and Salzberg 2012). Using HTSeq (Anders et al. 2015), the number of reads mapped onto each gene was then counted, providing a gff file (GCA_000019485.1_ASM1948v1_genomic.gff) as the reference gene annotation. Based on the information of the raw read counts, differentially expressed genes between conditions were detected by using TCC (Sun et al. 2013). Gel mobility shift assay His-tagged NrrA proteins were prepared as described previously (Ehira et al. 2017). DNA fragments of the promoter regions for each gene were generated by PCR using the primer pairs listed in Supplementary Table S2. The fragments were cloned in the HinCII site of pHSG396 (TAKARA BIO INC.) and the resultant plasmids were used as templates of PCR with a Cy3-labeled primer Cy3-M13-F. A gel mobility shift assay was conducted using 3 nM Cy3-labeled probes as described previously (Ehira and Ohmori 2006b), and the probes were detected with the FLA9000image scanner (Fuji Film). Analysis of enzyme activities Cells were harvested by centrifugation and then the cell pellets were washed and resuspended in phosphate-buffered saline (PBS). After cell disruption by vigorously mixing with zirconia/silica beads (0.1 mm in diameter; Bio Spec Products), cell debris was removed by centrifugation. The supernatants were used for analysis of enzyme activities and determination of protein concentration with a Protein Assay (Bio-Rad). 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. Supplementary Data Supplementary data are available at PCP online. Funding This work was supported by the Japan Science and Technology Agency [Core Research for Evolutional Science and Technology]; the Japan Society for the Promotion of Science [Grant-in-aid for Young Scientists (B) 26870472 to S.E.]; and NODAI Genome Research Center, Tokyo University of Agriculture [Cooperative Research Grant of the Genome Research for BioResource]. Disclosures The authors have no conflicts of interest to declare. References Anders S., Pyl P.T., Huber W. ( 2015) HTSeq—a Python framework to work with high-throughput sequencing data. Bioinformatics  31: 166– 169. Google Scholar CrossRef Search ADS PubMed  Azuma M., Osanai T., Hirai M.Y., Tanaka K. ( 2011) A response regulator Rre37 and an RNA polymerase sigma factor SigE represent two parallel pathways to activate sugar catabolism in a cyanobacterium Synechocystis sp. PCC 6803. Plant Cell Physiol . 52: 404– 412. 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Google Scholar CrossRef Search ADS   Abbreviations Abbreviations CFU colony-forming units ED Entner–Doudoroff EMP Embden–Meyerhof–Parnass GP glycogen phosphorylase G6PD glucose-6-phosphate dehydrogenase NrrA nitrogen-regulated response regulator A OPP oxidative pentose phosphate 6PGD 6-phosphogluconate dehydrogenase qRT–PCR quantitative reverse transcription–PCR WT wild type © The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Plant and Cell Physiology Oxford University Press

Transcriptional Activation of Glycogen Catabolism and the Oxidative Pentose Phosphate Pathway by NrrA Facilitates Cell Survival Under Nitrogen Starvation in the Cyanobacterium Synechococcus sp. Strain PCC 7002

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Abstract

Abstract 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 ability to survive. In spite of glycogen accumulation, genes related to glycogen catabolism are up-regulated 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 survival of the nrrA mutant declined. In addition, a glgP2 mutant was highly susceptible to nitrogen starvation. NrrA also regulated expression of the 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 the ability to survive under nitrogen starvation. It was concluded that NrrA facilitates cell survival by activating glycogen degradation and the OPP pathway under nitrogen-deprived conditions. Introduction Cyanobacteria are a diverse group of bacteria that perform oxygenic photosynthesis. Cyanobacteria inhabit almost all of the biosphere where light is available and play an important role in ecosystems as primary producers. Their growth affects the entire ecosystem and is often limited by the deprivation of nutrients, such as nitrogen and phosphorus (Vitousek and Howarth 1991). In the absence of combined nitrogen sources, many cyanobacteria carry out nitrogen fixation to utilize atmospheric nitrogen as a nitrogen source. Meanwhile, responses to nitrogen deprivation of non-nitrogen-fixing cyanobacteria include cessation of cell proliferation and changes of cellular metabolism and morphology (Schwarz and Forchhammer 2005). The color of cyanobacterial cultures changes from blue-green to yellow, and this phenomenon is known as bleaching or chlorosis (Collier and Grossman 1992). During chlorosis, phycobilisomes, light-harvesting antennae protein complexes, were degraded, and then Chl was lost, with a decline of photosynthetic activity (Görl et al. 1998). Degradation of phycobilisomes balances production of ATP and NADPH by photosynthesis with utilization of energy for cell maintenance during nitrogen starvation (Schwarz and Grossman 1998). Imbalances between energy acquisition and utilization could result in production of reactive oxygen species (Latifi et al. 2009). Thus, the pigment degradation is crucial for cell survival under conditions of nitrogen deprivation. Glycogen accumulation is another response to nitrogen deprivation in cyanobacteria (Gründel et al. 2012, Jackson et al. 2015). Glycogen amounts to 40–60% of the dry cell weight under nitrogen-deprived conditions (Hasunuma et al. 2013). Glycogen is synthesized from glucose-1-phosphate by the series of reactions catalyzed by ADP-glucose pyrophosphorylase and glycogen synthase (Suzuki et al. 2010). Under nitrogen-deprived conditions, cell growth ceases, and demands for building blocks of cellular components and energy for cellular activity are decreased. Hence, excess carbon and energy are stored as glycogen. Inhibition of glycogen synthesis perturbs carbon and energy homeostasis and reduces cell viability during nitrogen starvation (Gründel et al. 2012). Moreover, mutants in glycogen synthesis are unable to degrade phycobilisomes, indicating that glycogen synthesis is involved in regulation of the nitrogen response (Gründel et al. 2012, Hickman et al. 2013, Jackson et al. 2015). In cyanobacteria, the glgP gene, encoding glycogen phosphorylase (GP) that is involved in glycogen breakdown, is up-regulated 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) (Ehira and Ohmori 2011, Liu and Yang 2014). NrrA is a highly conserved transcriptional regulator among β-cyanobacteria and its expression is induced by nitrogen deprivation (Ehira and Ohmori 2006a, Muro-Pastor et al. 2006). In the unicellular cyanobacterium Synechocystis sp. PCC 6803, NrrA facilitates heterotrophic growth with glucose in the dark by up-regulating expression of genes involved in glycogen catabolism (Azuma et al. 2011). In the nitrogen-fixing, heterocystous cyanobacterium Anabaena sp. PCC 7120, NrrA regulates nitrogen fixation activity by activating glycogen catabolism (Ehira and Ohmori 2011). NrrA is shown to be a regulator of glycogen catabolism, but it still remains to be unraveled whether NrrA and glycogen catabolism are involved in acclimation to nitrogen starvation. In this study, we characterized an nrrA mutant of the non-nitrogen-fixing cyanobacterium Synechococcus sp. PCC 7002 and indicated that the ability of the nrrA mutant to survive during nitrogen starvation was lower than that of the wild-type (WT) strain. In addition, disruption of the glgP2 gene, which was regulated by NrrA, resulted in a drastic decline of survival. Thus, glycogen catabolism is crucial for survival under nitrogen starvation. In contrast to the mutant of glycogen synthesis, the nrrA and glgP2 mutants responded normally to nitrogen deprivation, i.e. photosynthetic pigments were degraded and glycogen was accumulated within cells, indicating that glycogen degradation plays different roles from glycogen synthesis in acclimation to nitrogen deprivation. Results NrrA plays an important role in cell survival under nitrogen-deprived conditions Expression of nrrA is induced by nitrogen deprivation in β-cyanobacteria including Synechococcus PCC 7002 (Ehira et al. 2017). To investigate a role for nrrA in acclimation to nitrogen starvation, an insertion mutant of the nrrA gene in Synechococcus PCC 7002 was created, and cellular viability after incubation under nitrogen-deprived conditions was determined by calculating the colony-forming units (CFU) (Fig. 1). In the WT, the CFU were reduced to 15% after 1 d of nitrogen starvation and to 4% after 2 d. The CFU of the nrrA mutant immediately after shift to nitrogen-free medium were comparable with those of the WT; however they were reduced to 6% after 1 d and, after 2 d, only 0.6% of cells were able to regrow. Thus, the nrrA mutant was susceptible to nitrogen starvation, indicating that the nrrA gene facilitates cell survival under nitrogen starvation. Fig. 1 View largeDownload slide Viability under nitrogen-deprived conditions. (A) Cells of the WT and the nrrA mutant (ΔnrrA) grown with nitrate were transferred to nitrogen-free medium at an OD750 of 0.5. After 0, 1 or 2 d, 10 µl aliquots of cultures were plated on medium A+ agar at a 10-fold serial dilution and the plates were incubated under continuous illumination of 100 µmol photons m–2 s–1 for 3 d. (B) A viability test was conducted with the WT (filled circles), the nrrA mutant (open circles), the glgP2 mutant (filled triangles) and the opcA mutant (open triangles). Colonies that appeared on the plates after 2 or 3 d incubation were counted. The means ± SD (error bar) of three independent experiments are shown. Fig. 1 View largeDownload slide Viability under nitrogen-deprived conditions. (A) Cells of the WT and the nrrA mutant (ΔnrrA) grown with nitrate were transferred to nitrogen-free medium at an OD750 of 0.5. After 0, 1 or 2 d, 10 µl aliquots of cultures were plated on medium A+ agar at a 10-fold serial dilution and the plates were incubated under continuous illumination of 100 µmol photons m–2 s–1 for 3 d. (B) A viability test was conducted with the WT (filled circles), the nrrA mutant (open circles), the glgP2 mutant (filled triangles) and the opcA mutant (open triangles). Colonies that appeared on the plates after 2 or 3 d incubation were counted. The means ± SD (error bar) of three independent experiments are shown. Under nitrogen-deprived conditions, photosynthetic pigments are degraded, photosynthetic activity declines and glycogen is accumulated within cells, which are important responses to acclimate to nitrogen starvation (Gründel et al. 2012, Jackson et al. 2015). We investigated whether the nrrA mutant was able to respond adequately to nitrogen deprivation (Fig. 2). Photosynthetic activity was assayed by measuring oxygen generation by PSII with bicarbonate as an electron acceptor (Fig. 2A), and glycogen levels within cells were determined by measuring glucose that was liberated from glycogen by glucoamylase (Fig. 2C). Changes in photosynthetic activity and contents of Chl and glycogen in the nrrA mutant were comparable with those in the WT, indicating that the nrrA mutant was capable of responding normally to nitrogen deprivation at least with respect to suppression of photosynthesis and accumulation of glycogen. Thus, NrrA is likely to support cell survival by hitherto unidentified mechanisms. Fig. 2 View largeDownload slide Cellular responses to nitrogen deprivation. Cells of the WT (filled circles), the nrrA mutant (open circles) and the glgP2 mutant (filled triangles) that were subjected to nitrogen deficiency for the indicated time were collected, and then photosynthetic activities (A), Chl (B) and glycogen contents (C) were determined. The means ± SD (error bar) of at least three independent experiments are shown. Fig. 2 View largeDownload slide Cellular responses to nitrogen deprivation. Cells of the WT (filled circles), the nrrA mutant (open circles) and the glgP2 mutant (filled triangles) that were subjected to nitrogen deficiency for the indicated time were collected, and then photosynthetic activities (A), Chl (B) and glycogen contents (C) were determined. The means ± SD (error bar) of at least three independent experiments are shown. NrrA activates glycogen catabolism and respiration under nitrogen-deprived conditions To identify genes regulated by NrrA, RNA-sequencing analysis was conducted in the WT and the nrrA mutant. In the WT, the transcript levels of 404 genes were increased 3 h after nitrogen deprivation (Supplementary Table S1). The up-regulated genes included glnA, glnN, nblA, the ctaI operon, the ctaII operon, amt and ndhD2, which was consistent with the previous report by Ludwig and Bryant (2012). NrrA-regulated genes were identified by comparing the gene expression profile 3 h after nitrogen deprivation between the WT and the nrrA mutant. The transcript levels of 25 genes that were induced by nitrogen deprivation were decreased by disruption of nrrA (Table 1). As is the case in other β-cyanobacteria (Ehira et al. 2017), glgP2 that encodes GP was down-regulated by nrrA disruption. In addition, the tal-zwf-opcA operon that encodes enzymes of the oxidative pentose phosphate (OPP) pathway and the ctaI (ctaCI-DI-EI) operon that encodes Cyt oxidase were down-regulated. Because the gene expression profile of the nrrA mutant was analyzed by RNA-sequencing only once without making biological replicates, we confirmed the differences in gene expression by quantitative reverse transcription–PCR (qRT–PCR). It was shown that the transcript levels of glgP2, tal, zwf, opcA and ctaCI after 3 h of nitrogen deprivation were decreased in the nrrA mutant (Fig. 3; Supplementary Fig. S1). The gnd gene that encodes 6-phosphogluconate dehydrogenase (6PGD), another enzyme of the OPP pathway, was also up-regulated by nitrogen deprivation (Supplementary Table S1). The difference between the WT and the nrrA mutant was not significant in the RNA-sequencing analysis, but qRT–PCR showed that the transcript level of gnd was decreased by disruption of nrrA (Fig. 3). Table 1 Nitrogen-induced genes that are down-regulated by nrrA disruption       WT3/WT0a  ΔnrrA3/WT3b  Gene-ID  Gene  Product  Ratio  qc  Ratio  qc  A0330  malQ  4-α-Glucanotransferase  1.5  4.8E-06  –1.7  5.9E-03  A0331    Conserved hypothetical protein  1.8  1.2E-08  –1.9  3.8E-03  A0378    Conserved hypothetical protein  1.8  1.1E-05  –1.9  4.7E-02  A0479    Conserved hypothetical protein  1.9  1.1E-11  –1.6  2.8E-03  A0481  glgP2  Glycogen phosphorylase  1.5  7.1E-08  –1.7  3.4E-03  A0496  nrrA  Two-component response regulator  3.8  3.0E-31  –9.9  5.7E-49  A0497    ATP-binding protein of ABC transporter  3.1  6.1E-28  –4.6  3.0E-21  A0832    Acetyltransferase, GNAT family  2.0  4.6E-14  –1.5  1.4E-03  A0922    Conserved hypothetical protein  2.1  7.7E-15  –2.4  1.0E-06  A1071  putA  Proline oxidase  1.5  9.5E-07  –1.7  2.9E-04  A1162  ctaCI  Cytochrome oxidase subunit II  2.0  7.5E-12  –1.6  2.0E-03  A1163  ctaDI  Cytochrome oxidase subunit I  1.8  4.7E-09  –1.7  7.6E-04  A1164  ctaEI  Cytochrome oxidase subunit III  1.4  9.7E-07  –1.3  1.1E-02  A1458  opcA  Putative OxPP cycle protein opcA  2.2  7.5E-13  –1.3  2.5E-02  A1459  zwf  Glucose-6-phosphate dehydrogenase  2.4  3.2E-16  –1.5  3.3E-03  A1460  tal  Transaldolase  2.6  3.7E-16  –1.6  2.8E-03  A1933    NblA-related protein  6.4  6.9E-67  –1.9  5.8E-04  A1934    Universal stress protein-like protein  2.6  2.3E-20  –1.4  2.1E-02  A2278    Hypothetical protein  2.6  1.9E-23  –1.2  2.2E-02  A2357  rsuA  RNA pseudouridylate synthase  2.3  1.4E-20  –1.2  2.8E-02  A2405  cphA  Cyanophycin synthetase  1.2  1.6E-05  –1.8  2.6E-04  A2490    Transglutaminase domain protein  1.7  4.7E-13  –1.6  2.9E-04  A2771    Succinate-semialdehyde dehydrogenase  2.4  9.7E-17  –1.4  1.0E-02  A2772    Conserved hypothetical protein  5.9  6.2E-65  –2.2  1.7E-05  B0001    Hypothetical protein  1.1  3.0E-03  –1.6  1.0E-02        WT3/WT0a  ΔnrrA3/WT3b  Gene-ID  Gene  Product  Ratio  qc  Ratio  qc  A0330  malQ  4-α-Glucanotransferase  1.5  4.8E-06  –1.7  5.9E-03  A0331    Conserved hypothetical protein  1.8  1.2E-08  –1.9  3.8E-03  A0378    Conserved hypothetical protein  1.8  1.1E-05  –1.9  4.7E-02  A0479    Conserved hypothetical protein  1.9  1.1E-11  –1.6  2.8E-03  A0481  glgP2  Glycogen phosphorylase  1.5  7.1E-08  –1.7  3.4E-03  A0496  nrrA  Two-component response regulator  3.8  3.0E-31  –9.9  5.7E-49  A0497    ATP-binding protein of ABC transporter  3.1  6.1E-28  –4.6  3.0E-21  A0832    Acetyltransferase, GNAT family  2.0  4.6E-14  –1.5  1.4E-03  A0922    Conserved hypothetical protein  2.1  7.7E-15  –2.4  1.0E-06  A1071  putA  Proline oxidase  1.5  9.5E-07  –1.7  2.9E-04  A1162  ctaCI  Cytochrome oxidase subunit II  2.0  7.5E-12  –1.6  2.0E-03  A1163  ctaDI  Cytochrome oxidase subunit I  1.8  4.7E-09  –1.7  7.6E-04  A1164  ctaEI  Cytochrome oxidase subunit III  1.4  9.7E-07  –1.3  1.1E-02  A1458  opcA  Putative OxPP cycle protein opcA  2.2  7.5E-13  –1.3  2.5E-02  A1459  zwf  Glucose-6-phosphate dehydrogenase  2.4  3.2E-16  –1.5  3.3E-03  A1460  tal  Transaldolase  2.6  3.7E-16  –1.6  2.8E-03  A1933    NblA-related protein  6.4  6.9E-67  –1.9  5.8E-04  A1934    Universal stress protein-like protein  2.6  2.3E-20  –1.4  2.1E-02  A2278    Hypothetical protein  2.6  1.9E-23  –1.2  2.2E-02  A2357  rsuA  RNA pseudouridylate synthase  2.3  1.4E-20  –1.2  2.8E-02  A2405  cphA  Cyanophycin synthetase  1.2  1.6E-05  –1.8  2.6E-04  A2490    Transglutaminase domain protein  1.7  4.7E-13  –1.6  2.9E-04  A2771    Succinate-semialdehyde dehydrogenase  2.4  9.7E-17  –1.4  1.0E-02  A2772    Conserved hypothetical protein  5.9  6.2E-65  –2.2  1.7E-05  B0001    Hypothetical protein  1.1  3.0E-03  –1.6  1.0E-02  aRatios of transcript levels 3 h after relative to before nitrogen deprivation in the WT are shown in the base 2 logarithm. bRatios of transcript levels in the nrrA mutant relative to the WT 3 h after nitrogen deprivation are shown in the base 2 logarithm. cFalse discovery rate or the rate of type I errors with consideration of multiple testing. Table 1 Nitrogen-induced genes that are down-regulated by nrrA disruption       WT3/WT0a  ΔnrrA3/WT3b  Gene-ID  Gene  Product  Ratio  qc  Ratio  qc  A0330  malQ  4-α-Glucanotransferase  1.5  4.8E-06  –1.7  5.9E-03  A0331    Conserved hypothetical protein  1.8  1.2E-08  –1.9  3.8E-03  A0378    Conserved hypothetical protein  1.8  1.1E-05  –1.9  4.7E-02  A0479    Conserved hypothetical protein  1.9  1.1E-11  –1.6  2.8E-03  A0481  glgP2  Glycogen phosphorylase  1.5  7.1E-08  –1.7  3.4E-03  A0496  nrrA  Two-component response regulator  3.8  3.0E-31  –9.9  5.7E-49  A0497    ATP-binding protein of ABC transporter  3.1  6.1E-28  –4.6  3.0E-21  A0832    Acetyltransferase, GNAT family  2.0  4.6E-14  –1.5  1.4E-03  A0922    Conserved hypothetical protein  2.1  7.7E-15  –2.4  1.0E-06  A1071  putA  Proline oxidase  1.5  9.5E-07  –1.7  2.9E-04  A1162  ctaCI  Cytochrome oxidase subunit II  2.0  7.5E-12  –1.6  2.0E-03  A1163  ctaDI  Cytochrome oxidase subunit I  1.8  4.7E-09  –1.7  7.6E-04  A1164  ctaEI  Cytochrome oxidase subunit III  1.4  9.7E-07  –1.3  1.1E-02  A1458  opcA  Putative OxPP cycle protein opcA  2.2  7.5E-13  –1.3  2.5E-02  A1459  zwf  Glucose-6-phosphate dehydrogenase  2.4  3.2E-16  –1.5  3.3E-03  A1460  tal  Transaldolase  2.6  3.7E-16  –1.6  2.8E-03  A1933    NblA-related protein  6.4  6.9E-67  –1.9  5.8E-04  A1934    Universal stress protein-like protein  2.6  2.3E-20  –1.4  2.1E-02  A2278    Hypothetical protein  2.6  1.9E-23  –1.2  2.2E-02  A2357  rsuA  RNA pseudouridylate synthase  2.3  1.4E-20  –1.2  2.8E-02  A2405  cphA  Cyanophycin synthetase  1.2  1.6E-05  –1.8  2.6E-04  A2490    Transglutaminase domain protein  1.7  4.7E-13  –1.6  2.9E-04  A2771    Succinate-semialdehyde dehydrogenase  2.4  9.7E-17  –1.4  1.0E-02  A2772    Conserved hypothetical protein  5.9  6.2E-65  –2.2  1.7E-05  B0001    Hypothetical protein  1.1  3.0E-03  –1.6  1.0E-02        WT3/WT0a  ΔnrrA3/WT3b  Gene-ID  Gene  Product  Ratio  qc  Ratio  qc  A0330  malQ  4-α-Glucanotransferase  1.5  4.8E-06  –1.7  5.9E-03  A0331    Conserved hypothetical protein  1.8  1.2E-08  –1.9  3.8E-03  A0378    Conserved hypothetical protein  1.8  1.1E-05  –1.9  4.7E-02  A0479    Conserved hypothetical protein  1.9  1.1E-11  –1.6  2.8E-03  A0481  glgP2  Glycogen phosphorylase  1.5  7.1E-08  –1.7  3.4E-03  A0496  nrrA  Two-component response regulator  3.8  3.0E-31  –9.9  5.7E-49  A0497    ATP-binding protein of ABC transporter  3.1  6.1E-28  –4.6  3.0E-21  A0832    Acetyltransferase, GNAT family  2.0  4.6E-14  –1.5  1.4E-03  A0922    Conserved hypothetical protein  2.1  7.7E-15  –2.4  1.0E-06  A1071  putA  Proline oxidase  1.5  9.5E-07  –1.7  2.9E-04  A1162  ctaCI  Cytochrome oxidase subunit II  2.0  7.5E-12  –1.6  2.0E-03  A1163  ctaDI  Cytochrome oxidase subunit I  1.8  4.7E-09  –1.7  7.6E-04  A1164  ctaEI  Cytochrome oxidase subunit III  1.4  9.7E-07  –1.3  1.1E-02  A1458  opcA  Putative OxPP cycle protein opcA  2.2  7.5E-13  –1.3  2.5E-02  A1459  zwf  Glucose-6-phosphate dehydrogenase  2.4  3.2E-16  –1.5  3.3E-03  A1460  tal  Transaldolase  2.6  3.7E-16  –1.6  2.8E-03  A1933    NblA-related protein  6.4  6.9E-67  –1.9  5.8E-04  A1934    Universal stress protein-like protein  2.6  2.3E-20  –1.4  2.1E-02  A2278    Hypothetical protein  2.6  1.9E-23  –1.2  2.2E-02  A2357  rsuA  RNA pseudouridylate synthase  2.3  1.4E-20  –1.2  2.8E-02  A2405  cphA  Cyanophycin synthetase  1.2  1.6E-05  –1.8  2.6E-04  A2490    Transglutaminase domain protein  1.7  4.7E-13  –1.6  2.9E-04  A2771    Succinate-semialdehyde dehydrogenase  2.4  9.7E-17  –1.4  1.0E-02  A2772    Conserved hypothetical protein  5.9  6.2E-65  –2.2  1.7E-05  B0001    Hypothetical protein  1.1  3.0E-03  –1.6  1.0E-02  aRatios of transcript levels 3 h after relative to before nitrogen deprivation in the WT are shown in the base 2 logarithm. bRatios of transcript levels in the nrrA mutant relative to the WT 3 h after nitrogen deprivation are shown in the base 2 logarithm. cFalse discovery rate or the rate of type I errors with consideration of multiple testing. Fig. 3 View largeDownload slide Changes in the transcript levels of the NrrA regulon after nitrogen deprivation. The relative transcript levels of glgP2, tal, catCI and gnd were determined by qRT–PCR in the WT (filled circles) and the nrrA mutant (open circles). RNA samples were prepared from three independently grown cultures. The transcript level at 0 h of the WT was taken as 1. Data that represent a significant difference (P < 0.01) by applying the t-test between the WT and the nrrA mutant are marked with asterisks. Fig. 3 View largeDownload slide Changes in the transcript levels of the NrrA regulon after nitrogen deprivation. The relative transcript levels of glgP2, tal, catCI and gnd were determined by qRT–PCR in the WT (filled circles) and the nrrA mutant (open circles). RNA samples were prepared from three independently grown cultures. The transcript level at 0 h of the WT was taken as 1. Data that represent a significant difference (P < 0.01) by applying the t-test between the WT and the nrrA mutant are marked with asterisks. Although the transcript levels of glgP2, tal, ctaCI and gnd were significantly decreased 3 h after nitrogen deprivation in the nrrA mutant, expression of these genes was increased after 6 h (Fig. 3). To analyze the consequence of the delayed induction in the nrrA mutant, enzyme activities of GP and two enzymes of the OPP pathway, glucose-6-phosphate dehydrogenase (G6PD) encoded by zwf and 6PGD at 9 h after nitrogen deprivation were determined. Consistent with changes in the transcript levels, activities of these enzymes were increased by nitrogen deprivation in the WT (Fig. 4A). In the nrrA mutant, activities of these enzymes before nitrogen deprivation were the same as those in the WT, but an increase after nitrogen deprivation was not observed or was less than that in the WT (Fig. 4A). These results indicate that the delayed induction of gene expression in the nrrA mutant significantly reduces the enzyme activities after nitrogen deprivation. Next, oxygen consumption rates were determined. Oxygen consumption rates of the WT were increased about 3-fold 3 h after nitrogen deprivation, and then gradually decreased to the level before nitrogen deprivation (Fig. 4B). In the nrrA mutant, oxygen consumption rates were also increased by 85% after 3 h, but the activity was 40% lower than that of the WT (Fig. 4B). Thus, NrrA activates glycogen degradation, the OPP pathway and respiration in response to nitrogen deprivation. Fig. 4 View largeDownload slide Changes in enzyme activities after nitrogen deprivation. (A) Activities of glycogen phosphorylase (GP), glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) were determined before (open bars) and 9 h after nitrogen deprivation (filled bars) in the WT and the nrrA mutant (ΔnrrA). P-values in the t-test are indicated. (B) Respiration activities of cells subjected to nitrogen deficiency for the indicated time were determined in the WT (filled circles), the nrrA mutant (open circles) and the ctaCI mutant (black triangles). Data that represent a significant difference (P < 0.05) by applying the t-test between the WT and the mutants are marked with asterisks. Measurement was repeated at least three times with cells from independently grown cultures. Fig. 4 View largeDownload slide Changes in enzyme activities after nitrogen deprivation. (A) Activities of glycogen phosphorylase (GP), glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) were determined before (open bars) and 9 h after nitrogen deprivation (filled bars) in the WT and the nrrA mutant (ΔnrrA). P-values in the t-test are indicated. (B) Respiration activities of cells subjected to nitrogen deficiency for the indicated time were determined in the WT (filled circles), the nrrA mutant (open circles) and the ctaCI mutant (black triangles). Data that represent a significant difference (P < 0.05) by applying the t-test between the WT and the mutants are marked with asterisks. Measurement was repeated at least three times with cells from independently grown cultures. Glycogen degradation and the OPP pathway facilitate cell survival under nitrogen-deprived conditions The nrrA disruptant showed reduced ability to survive under nitrogen-deprived conditions (Fig. 1). To reveal which genes regulated by NrrA were related to survival under nitrogen starvation, the glgP2, opcA and ctaCI genes were inactivated and cell viability of the mutants was determined. The viability of the glgP2 and opcA mutants was lower than that of the WT (Fig. 1B). The decrease of viability in the opcA mutant was comparable with that in the nrrA mutant, while the glgP2 mutant was much more susceptible to nitrogen starvation than the nrrA mutant. The CFU of the glgP2 mutant after 24 h of nitrogen deprivation were <1% of those of the nrrA mutant. Because disruption of glgP2 might affect glycogen synthesis, which is important for cell survival under nitrogen starvation (Gründel et al. 2012), glycogen contents of the glgP2 disruptant were determined. The glycogen level was increased 10-fold within 6 h after nitrogen deprivation as well as in the WT, though the levels after 24 and 48 h were 25% lower than those of the WT (Fig. 2C). Thus, glycogen synthesis was not inhibited in the glgP2 mutant, suggesting that the decrease of viability in the glgP2 mutant was not caused by impairment of glycogen synthesis. Glycogen degradation catalyzed by GP could allow cells to survive under nitrogen starvation. OpcA is an allosteric activator of G6PD and its disruption results in the loss of G6PD activity (Summers et al. 1995, Hagen and Meeks 2001). G6PD, which is the first enzyme of the OPP pathway, controls the entire activity of the pathway. Thus, it was shown that the OPP pathway also supports survival under nitrogen starvation. Meanwhile, disruption of ctaCI did not affect cell viability (data not shown). In Synechococcus PCC 7002, there are two operons (ctaI and ctaII) that encode Cyt oxidases (Nomura et al. 2006a). Although both operons were up-regulated by nitrogen deprivation, only ctaI was regulated by NrrA (Table 1; Supplementary Table S1). Oxygen consumption rates of the ctaCI mutant were lower than those of the WT even before nitrogen deprivation, but were increased about 3-fold after nitrogen deprivation as well as in the WT (Fig. 4B). Thus, the ctaI operon was not required for activation of respiration and survival under nitrogen starvation. These results indicate that up-regulation of the glgP2 gene and the tal-zwf-opcA operon by NrrA plays an important role in acclimation to nitrogen deprivation. NrrA directly regulates expression of the glgP2 and gnd genes, and the tal-zwf-opcA and ctaI operons Interaction between NrrA and upstream regions of glgP2, tal, ctaCI and gnd was analyzed by electrophoretic mobility shift assay. His-tagged NrrA protein was mixed with Cy3-labeled DNA probes containing the upstream region of glgP2, tal, ctaCI or gnd, and then the mixtures were subjected to electrophoresis. NrrA decreased the electrophoretic mobility of DNA probes in a concentration-dependent manner (Fig. 5A). Addition of non-labeled probe diminished the interaction of NrrA and DNA probes, but the DNA fragment PhetR2, which does not bind to NrrA of Anabaena PCC 7120 (Ehira and Ohmori 2014), did not affect the interaction. NrrA recognizes the inverted repeat sequence GTCAN8TGAC (Ehira et al. 2017). The NrrA recognition sequences were found within the upstream regions of glgP2, tal, ctaCI and gnd (Fig. 5B). These results strongly support the idea that NrrA directly regulates expression of these genes. Fig. 5 View largeDownload slide DNA binding assays of NrrA protein. (A) Cy3-labeled probes (3 nM) including the promoter regions of glgP2, tal, ctaCI and gnd were mixed with NrrA proteins in the amount indicated above each lane, and the mixtures were subjected to electrophoresis. Non-labeled fragments (PglgP2, Ptal, PctaCI or Pgnd, and PhetR2) were added at a final concentration of 30 nM (lanes 5 and 7) or 90 nM (lanes 6 and 8). Open arrows, probe alone; filled arrows, complexes with NrrA and the Cy3-labeled probes. (B) Alignment of the promoter regions of glgP2, tal, ctaCI and gnd. The consensus sequence recognized by NrrA is shown at the bottom, and the putative NrrA-binding sites that have been found within the promoter regions are shown byin bold. Fig. 5 View largeDownload slide DNA binding assays of NrrA protein. (A) Cy3-labeled probes (3 nM) including the promoter regions of glgP2, tal, ctaCI and gnd were mixed with NrrA proteins in the amount indicated above each lane, and the mixtures were subjected to electrophoresis. Non-labeled fragments (PglgP2, Ptal, PctaCI or Pgnd, and PhetR2) were added at a final concentration of 30 nM (lanes 5 and 7) or 90 nM (lanes 6 and 8). Open arrows, probe alone; filled arrows, complexes with NrrA and the Cy3-labeled probes. (B) Alignment of the promoter regions of glgP2, tal, ctaCI and gnd. The consensus sequence recognized by NrrA is shown at the bottom, and the putative NrrA-binding sites that have been found within the promoter regions are shown byin bold. Discussion In this study, we demonstrated that the nitrogen-regulated response regulator NrrA played an important role in survival under nitrogen starvation in Synechococcus PCC 7002. NrrA directly regulated expression of glgP2, gnd, tal-zwf-opcA and the ctaI operon in response to nitrogen deprivation. Activation of GP, enzymes of the OPP pathway and respiration under nitrogen-deprived conditions was diminished by nrrA disruption, which led to a decline in the ability to survive under nitrogen starvation. The glgP2 and opcA mutants were also susceptible to nitrogen starvation, indicating that NrrA-dependent activation of glycogen degradation and the OPP pathway would be crucial for acclimation to nitrogen starvation. NrrA is a highly conserved transcriptional regulator among β-cyanobacteria (Ehira et al. 2017). It has been shown that NrrA regulates genes for glycogen catabolism and respiration in Synechococcus elongatus PCC 7942 and Synechocystis PCC 6803, as well as in Synechococcus PCC 7002 (Azuma et al. 2011, Ehira et al. 2017). Activation of glycogen catabolism and respiration by NrrA would be a universal response to nitrogen starvation in cyanobacteria. 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 (Ehira and Ohmori 2011, Liu and Yang 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. In cyanobacteria, glucose derived from glycogen degradation is mainly catabolized though the OPP pathway (Jansén et al. 2010, Nakajima et al. 2014). The OPP pathway produces NADPH, and NADPH is used as the electron donor for respiration in cyanobacteria (Ogawa and Mi 2007). The activity of the OPP pathway is regulated by the cellular redox state and is usually suppressed under photoautotrophic conditions to prevent futile cycling, which occurs if the Calvin–Benson and OPP pathways operate simultaneously (Udvardy et al. 1984). However, the OPP pathway enhanced survival under nitrogen starvation (Fig. 1), indicating that the OPP pathway was operative in those conditions. Photosynthetic activity was decreased to one-third within 6 h after nitrogen deprivation, with a further decline to one-tenth after 24 h, when the increase in glycogen level ceased (Fig. 2). ATP and NADPH supply from photosynthesis would be insufficient to sustain cell viability when nitrogen starvation was prolonged, and glycogen degradation and the subsequent catabolism through the OPP pathway could compensate for the shortage of ATP and NADPH to support long-term survival. The glgP2 mutant was much more sensitive to nitrogen starvation than the nrrA and opcA mutants (Fig. 1). The difference in tha ability to survive between the glgP2 and opcA mutants implies that the OPP pathway is not the sole route of glucose catabolism. The gap and pyk genes, encoding key enzymes of the Embden–Meyerhof–Parnass (EMP) pathway, are up-regulated by nitrogen deprivation in Synechococcus elongatus PCC 7942, Synechocystis PCC 6803 and Anabaena PCC 7120 (Osanai et al. 2006, Ehira and Ohmori 2011, Ehira et al. 2017). Glucose could also be catabolized via the EMP pathway. In addition, the Entner–Doudoroff (ED) pathway has been shown to operate in cyanobacteria (Chen et al. 2016). Compared with the EMP pathway, the ED pathway has lower ATP yield, but requires less enzymatic protein to achieve the same glycolytic flux (Flamholz et al. 2013). Thus, the ED pathway would be preferable to the EMP pathway under nitrogen-deprived conditions, when protein synthesis is limited. Further research is needed to clarify the contribution of each glucose degradation pathway to acclimation to nitrogen starvation. In the nrrA disruptant, expression of glgP2 was not increased until 3 h after nitrogen deprivation, but it was induced at 6 h (Fig. 3). The nitrogen-dependent induction of glgP2 would be controlled by multiple transcriptional regulators including NrrA. In Anabaena PCC 7120 and Synechocystis PCC 6803, a group 2 sigma factor, SigE, is involved in regulation of glycogen catabolism (Azuma et al. 2011, Ehira and Ohmori 2011). The sigC gene, encoding a SigE ortholog in Synechococcus PCC 7002, was up-regulated by nitrogen deprivation (Supplementary Table S1). The collaborative regulation of glycogen catabolism by NrrA and SigC would also function in Synechococcus PCC 7002. Although the glgP2 transcript level was increased with a delay of 3 h in the nrrA mutant, the activity of GP was not increased (Fig. 4A). This was the same for G6PD and 6PGD. It is likely that a delay in the transcriptional induction results in disconnection of transcription and translation. Intracellular levels of some amino acids, such as alanine, aspartate, asparagine, glutamate and valine, are decreased with time during nitrogen starvation (Hasunuma et al. 2013), and the shortage of amino acids would hinder de novo protein synthesis. Thus, NrrA enables cells to respond rapidly to nitrogen deprivation, and a rapid response is crucial for adjusting the cellular proteome to survival under nitrogen starvation. Materials and Methods Bacterial strains and culture conditions Synechococcus sp. PCC 7002 and its derivatives were grown at 38°C with continuous illumination at 200 µmol photons m–2 s–2 in A+ medium as described previously (Ehira et al. 2017). Liquid cultures were bubbled with air containing 1% (v/v) CO2. To shift cells into conditions of nitrogen deprivation, cells were washed with sterilized water three times and then resuspended in A medium without combined nitrogen sources at an OD750 of 0.5, followed by cultivation under the same conditions as mentioned above. Kanamycin (50 μg ml–1) was added to cultures of the nrrA and ctaCI mutants, and spectinomycin (50 μg ml–1) was added to cultures of the glgP2 and opcA mutants when required. Mutant construction To inactivate the glgP2 gene, a DNA fragment containing the glgP2 gene was amplified by PCR using the primer pair sypglgP2-F and sypglgP2-R (Supplementary Table S2). The fragment was cloned at the EcoRV site of pPCR-Script Amp SK+ (Agilent Technologies). A spectinomycin resistance cassette from the plasmid pDW9 (Golden and Wiest 1988) was inserted into the XbaI site within the glgP2 coding region. To inactivate nrrA, opcA and ctaCI, upstream and downstream regions of each gene were amplified by PCR using primer pairs 5F and 5R, and 3F and 3R for each gene, respectively (Supplementary Table S2), and a spectinomycin or kanamycin cassette was inserted between the upstream and downstream fragments. These plasmids were used for transformation of Synechococcus PCC 7002. Glycogen determination The cellular glycogen content was determined according to Jackson et al. (2015). Briefly, glycogen was extracted from cells by boiling in 30% (w/v) KOH for 1 h, and then precipitated by mixing with ethanol. Glycogen content was determined by measuring glucose liberated by treatment of α-amyloglucosidase using a glycogen colorimetric/fluorometric assay kit (BioVision). Oxygen evolution assay Oxygen evolution and consumption activities were determined using a Clark-type electrode (Hansatech Instruments) according to Nomura et al. (2006b). To determine photosynthetic activities, 1 ml of culture (OD750 of 0.5–1.0) was placed into an electrode chamber and 5 mM NaHCO3 was added. The samples were kept in the dark for 5 min without a lid to saturate the samples with air levels of oxygen. Oxygen evolution was recorded for 5 min at 38°C with a saturating amount of light (4,000 µmol photons m–2 s–1). Photosynthetic activities were determined by adding values of oxygen consumption in the dark to those of oxygen evolution. To determine respiration activities, 1 ml of culture concentrated to OD750 of about 4.0 was used. Oxygen consumption was measured in the dark for 5 min and then 1 mM KCN was added. Respiration activities were determined by subtracting values of oxygen consumption with KCN from those in the dark. The Chl content of cultures was determined according to Mackinney (1941). RNA extraction and qRT–PCR analysis RNA extraction and qRT–PCR analysis were conducted according to Ehira and Ohmori (2011) using GoTaq qPCR Master Mix (Promega). Primers used for qRT–PCR are listed in Supplementary Table S2. RNA-sequencing analysis Libraries for RNA-sequencing were constructed using 1 µg of total RNA as follows. rRNA was depleted from total RNA by the Ribo-Zero rRNA Removal kit (Bacteria) (Illumina) according to the manufacturer’s protocol. Sequencing libraries were prepared by an NEBNext mRNA library prep kit from Illumina (NEB) with the following modifications. The random hexamer primer was used for reverse transcription. After second-strand synthesis, double-stranded cDNA was fragmented to an average length of 300 bp using a Covaris S2 sonication system (Covaris). Three biological replicates were made for each condition (before nitrogen deprivation and 3 h after nitrogen deprivation) of the WT, whereas no biological replicate was made for each condition of the nrrA mutant. One hundred base pairs from both ends of each fragment were then sequenced on the HiSeq2500 system platform (Illumina). After the sequencing reactions were complete, the Illumina analysis pipeline (CASAVA 1.8.0) was used to process the raw sequencing data. Original read data are available under the accession numbers DRX114943–DRX114950 in the DDBJ/ENA/GenBank. All reads obtained were first processed with Cutadapt version 1.14 (Martin 2011) to remove adaptor sequences. The processed reads were further treated with SolexaQA++ version 3.1.7.1 (Cox et al. 2010) to select pairs of sequences with a quality score ≥25 for ≥50 consecutive nucleotides for both forward and reverse reads. The processed high quality read pairs were mapped onto the reference genome (GCA_000019485.1_ASM1948v1_genomic.fna) using Bowtie 2 version 2.3.3 (Langmead and Salzberg 2012). Using HTSeq (Anders et al. 2015), the number of reads mapped onto each gene was then counted, providing a gff file (GCA_000019485.1_ASM1948v1_genomic.gff) as the reference gene annotation. Based on the information of the raw read counts, differentially expressed genes between conditions were detected by using TCC (Sun et al. 2013). Gel mobility shift assay His-tagged NrrA proteins were prepared as described previously (Ehira et al. 2017). DNA fragments of the promoter regions for each gene were generated by PCR using the primer pairs listed in Supplementary Table S2. The fragments were cloned in the HinCII site of pHSG396 (TAKARA BIO INC.) and the resultant plasmids were used as templates of PCR with a Cy3-labeled primer Cy3-M13-F. A gel mobility shift assay was conducted using 3 nM Cy3-labeled probes as described previously (Ehira and Ohmori 2006b), and the probes were detected with the FLA9000image scanner (Fuji Film). Analysis of enzyme activities Cells were harvested by centrifugation and then the cell pellets were washed and resuspended in phosphate-buffered saline (PBS). After cell disruption by vigorously mixing with zirconia/silica beads (0.1 mm in diameter; Bio Spec Products), cell debris was removed by centrifugation. The supernatants were used for analysis of enzyme activities and determination of protein concentration with a Protein Assay (Bio-Rad). 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. 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Google Scholar CrossRef Search ADS   Abbreviations Abbreviations CFU colony-forming units ED Entner–Doudoroff EMP Embden–Meyerhof–Parnass GP glycogen phosphorylase G6PD glucose-6-phosphate dehydrogenase NrrA nitrogen-regulated response regulator A OPP oxidative pentose phosphate 6PGD 6-phosphogluconate dehydrogenase qRT–PCR quantitative reverse transcription–PCR WT wild type © The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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Plant and Cell PhysiologyOxford University Press

Published: Mar 15, 2018

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