Nitrogen Availability Affects the Metabolic Profile in Cyanobacteria

Nitrogen is essential for the biosynthesis of various molecules in cells, such as amino acids and nucleotides, as well as several types of lipids and sugars. Cyanobacteria can assimilate several forms of nitrogen, including nitrate, ammonium, and urea, and the physiological and genetic responses to these nitrogen sources have been studied previously. However, the metabolic changes in cyanobacteria caused by different nitrogen sources have not yet been characterized. This study aimed to elucidate the influence of nitrate and ammonium on the metabolic profiles of the cyanobacterium Synechocystis sp. strain PCC 6803. When supplemented with NaNO3 or NH4Cl as the nitrogen source, Synechocystis sp. PCC 6803 grew faster in NH4Cl medium than in NaNO3 medium. Metabolome analysis indicated that some metabolites in the CBB cycle, glycolysis, and TCA cycle, and amino acids were more abundant when grown in NH4Cl medium than NaNO3 medium. 15N turnover rate analysis revealed that the nitrogen assimilation rate in NH4Cl medium was higher than in NaNO3 medium. These results indicate that the mechanism of nitrogen assimilation in the GS-GOGAT cycle differs between NaNO3 and NH4Cl. We conclude that the amounts and biosynthetic rate of cyanobacterial metabolites varies depending on the type of nitrogen.


Introduction
Nitrogen is an important element for living things, being used in amino acids, nucleotides, lipids, and sugars, which are building blocks of all forms of life. Cyanobacteria are Gram-negative bacteria and are known to be oxygenic photosynthetic microorganisms that utilize solar energy to generate chemical energy (ATP and NADPH). Such chemical energy is used in the Calvin-Benson-Bassham cycle (CBB cycle), glycolysis, and tricarboxylic acid (TCA cycle). Similar to other organisms, non-diazotrophic cyanobacteria, such as Synechocystis sp. PCC 6803 can take up nitrogen as NO 3 − , NO 2 − , NH 4 + , CO(NH 2 ) 2 (urea), and arginine [1,2]. Diazotrophic cyanobacteria (for example, Anabaena sp. PCC 7120) can fix N 2 in heterocyst cells using nitrogenase [3]. Cyanobacteria require reducing power when using the former nitrogen sources (NO 3 − and NO 2 − ), but not when using the latter (NH 4 + , urea, and arginine). NO 3 − and NO 2 − transporters are already identified as NrtA-D. NH 4 + is transported by ammonium transporters Amt1-3 and urea by urea transporters UrtA-E [4][5][6]. NO 3 − in the cell is reduced to NO 2 − by ferredoxin-nitrate reductase, NarB, and finally reduced to NH 4 + by NirA [7,8]. Urea in the cell is converted by UreA-B to NH 3 [6,9]. NH 4 + is produced from arginine by arginine dihydrolase [2]. The GS (glutamine synthetase)-GOGAT (glutamate synthase or glutamine-oxoglutarate cycle amido transferase) cycle synthesizes glutamate and glutamine from NH 4 + and 2OG [10]. The resulting Glu is mainly consumed as a nitrogen source.
Although cyanobacteria can assimilate nitrogen compounds as mentioned above, the choice of nitrogen sources is important for cultivation because photosynthesis and growth are affected by the type of nitrogen source [11][12][13]. In addition, there are differences in gene expression associated with utilizing the nitrogen sources and changes in activity of enzymes involved in nitrogen assimilation when cyanobacteria are exposed to each of the nitrogen sources, NO 3 − , NH 4 + , and urea [14][15][16]. The physiological response of cyanobacteria to nitrogen sources also varies according to the species [13,17]. We previously reported that when Synechocystis sp. PCC 6803 was phototrophically cultivated in the presence of NH 4 Cl, it produced a higher content of intracellular organic acids including malate, fumarate, and succinate under dark anoxic fermentation than cells grown in NaNO 3 , resulting in increased succinate secretion [18]. In this way, the influences of different types of nitrogen sources have been investigated to reveal the physiological responses of cyanobacteria and their application. However, metabolites produced with different types of nitrogen sources have not been fully investigated, in contrast to the effects of nitrogen starvation [19,20]. Moreover, in our previous report, we revealed that the different metabolic profiles produced during dark anoxic cultivation after a transfer from phototrophic cultivation with different nitrogen sources enhanced the production of succinate. These findings prompted us to investigate the metabolic profiles under phototrophic conditions.
In this study, we aimed to clarify the metabolic responses to different nitrogen sources. For this purpose, we performed a combination of in vivo 15 N-labeling of metabolites and metabolome analysis. The 15 N-labeling technique is applied to detect metabolites of interest in cyanobacteria or to examine the metabolic behavior of a few metabolites [2,17,21,22]. This technique enables us to compare the metabolic turnover under different nitrogen sources by calculating the 15 N labeling rate at each time point. Using this technique and metabolome analysis, we compared the metabolic profiles and synthesis rates of amino acids in Synechocystis sp. PCC 6803 when grown in NaNO 3 or NH 4 Cl, revealing distinct metabolic profiles between the different nitrogen sources.

Growth in Different Types of Nitrogen Source
Synechocystis sp. PCC 6803 (hereafter Synechocystis) was cultivated in BG11 medium with 5 mM NaNO 3 or NH 4 Cl (hereafter NaNO 3 medium or NH 4 Cl medium) under phototrophic growth conditions (Figures 1 and S1). The growth rate of Synechocystis was also calculated based on the growth data by 48 h when there are the residual nitrogen sources. The growth rate of Synechocystis grown in NaNO 3 medium or NH 4 Cl medium was 0.028 ± 0.002 h −1 and 0.036 ± 0.002 h −1 . The growth rate of Synechocystis in NH 4 Cl medium was significantly faster than that in NaNO 3 medium throughout the cultivation. In contrast, there was no significant difference in the residual amounts of NaNO 3 and NH 4 Cl in either medium.

Metabolome Analysis with Different Types of Nitrogen Source
As nitrogen is thought to be assimilated mainly through the GS-GOGAT cycle, which synthesizes glutamate (Glu) and glutamine (Gln) from NH 4 − and 2OG, we first compared the amino acid levels with different nitrogen sources (Figures 2 and S2). The pool sizes of serine (Ser), glycine (Gly), threonine (Thr), alanine (Ala), aspartate (Asp), asparagine (Asn), lysine (Lys), valine (Val), and isoleucine (Ile) when grown in NH 4 Cl medium were higher than those in NaNO 3 medium 24 h after the start of cultivation. The pool sizes of methionine (Met) were still high 48 h after the start of cultivation. The pool sizes of Gln and Glu were the same between the NaNO 3 and NH 4 Cl media. On the other hand, the pool size of tryptophan (Trp) when grown in NH 4 Cl medium was lower than that in NaNO 3 .
Since Thr and Lys are synthesized from Asp, the pool sizes of Thr and Lys increased with an increase in Asp.
etabolites 2021, 11, x FOR PEER REVIEW Figure 1. Cell growth of Synechocystis and the residual nitrogen concentration unde of nitrogen. The cell growth of Synechocystis cultivated in NaNO3 medium or NH4C phototrophic conditions was compared. Blue lines, NaNO3 medium; red lines, NH4C lines, the cellular biomass (dried cell weight); dotted lines, the residual nitrogen c the medium. Error bars indicate the standard deviation of three replicate experim significance was determined using Welch's t-test (* <0.05, ** <0.01).

Metabolome Analysis with Different Types of Nitrogen Source
As nitrogen is thought to be assimilated mainly through the GS-GOGA synthesizes glutamate (Glu) and glutamine (Gln) from NH4 − and 2OG, we the amino acid levels with different nitrogen sources (Figures 2 and S2). Th serine (Ser), glycine (Gly), threonine (Thr), alanine (Ala), aspartate (As (Asn), lysine (Lys), valine (Val), and isoleucine (Ile) when grown in NH4Cl higher than those in NaNO3 medium 24 h after the start of cultivation. Th methionine (Met) were still high 48 h after the start of cultivation. The po and Glu were the same between the NaNO3 and NH4Cl media. On the o pool size of tryptophan (Trp) when grown in NH4Cl medium was lowe NaNO3. Since Thr and Lys are synthesized from Asp, the pool sizes of T creased with an increase in Asp.

15 N-Turnover Analysis with Different Types of Nitrogen
The growth rates and pool sizes of the metabolites were different in NaNO3 and NH4Cl. However, the nitrogen flow in the cell when grown in NaNO3 or NH4Cl media remains unclear. To reveal nitrogen flow with different nitrogen sources, we measured the time-resolved labeling rate of amino acids by 15

15 N-Turnover Analysis with Different Types of Nitrogen
The growth rates and pool sizes of the metabolites were different in NaNO 3 and NH 4 Cl. However, the nitrogen flow in the cell when grown in NaNO 3 or NH 4 Cl media remains unclear. To reveal nitrogen flow with different nitrogen sources, we measured the time-resolved labeling rate of amino acids by 15 N stable isotope labeling (Figure 4 and Figure S3). For this purpose, Synechocystis cells were taken after 24 h of cultivation and transferred to fresh BG11 medium containing 15 NH 4 Cl or Na 15 NO 3 (labeling time = 0 h). The labeling rates of Ala, Ser, and Gly, which are synthesized from 3PGA, were significantly higher in NH 4 Cl medium than in NaNO 3 medium. The larger pool sizes of Ala, Ser, and Gly when Synechocystis are grown in NH 4 Cl medium might result from the higher 15 N labeling rate. The labeling rate of Glu and Gln were significantly higher in NH 4 Cl medium than in NaNO 3 medium, which is consistent with a previous report [17]. However, the pool sizes of Glu and Gln did not change, as shown in Figure 2. This indicates that when Synechocystis is grown in NaNO 3 medium, one of the two nitrogen atoms in Gln is 14 NH 4 + , which can result from cellular nitrogen sources such as amino acids.
Metabolites 2021, 11, x FOR PEER REVIEW 6 of 14 higher in NH4Cl medium than in NaNO3 medium. The larger pool sizes of Ala, Ser, and Gly when Synechocystis are grown in NH4Cl medium might result from the higher 15 N labeling rate. The labeling rate of Glu and Gln were significantly higher in NH4Cl medium than in NaNO3 medium, which is consistent with a previous report [17]. However, the pool sizes of Glu and Gln did not change, as shown in Figure 2. This indicates that when Synechocystis is grown in NaNO3 medium, one of the two nitrogen atoms in Gln is 14 NH4 + , which can result from cellular nitrogen sources such as amino acids.

15 N Labelling Rate and Order in Glutamine Synthesis
To elucidate the mechanism underlying the lower labeling rate of glutamine in the NH 4 Cl medium, the position of the 15 N-labeled nitrogen atom in the Gln molecule was examined using liquid chromatography-tandem mass spectrometry with the multiple reaction monitoring method (LC-MS/MS MRM) ( Figure 5). 15 N labeling conditions were the same as those for the experiments performed in Figure 4 (see Materials and Methods). The 15 N labeling rate of Gln when cultured in Na 15 NO 3 was almost constant for 24 h after Synechocystis inoculation ( Figure 5A,B). On the other hand, when cultured in 15 NH 4 Cl, the rate of 15 N labeling of one of the two nitrogen atoms in Gln gradually decreased, and the unlabeled rate of Gln was 0.23% (2.5% in the presence of Na 15 NO 3 ). As shown in Figure 4, the total 15 N labeling rate of the two nitrogen atoms in Gln was 71% in Na 15

15 N Labelling Rate and Order in Glutamine Synthesis
To elucidate the mechanism underlying the lower labeling rate of glutamine in the NH4Cl medium, the position of the 15 N-labeled nitrogen atom in the Gln molecule was examined using liquid chromatography-tandem mass spectrometry with the multiple reaction monitoring method (LC-MS/MS MRM) ( Figure 5). 15 N labeling conditions were the same as those for the experiments performed in Figure 4 (see Materials and Methods). The 15 N labeling rate of Gln when cultured in Na 15 NO3 was almost constant for 24 h after Synechocystis inoculation ( Figure 5A,B). On the other hand, when cultured in 15 NH4Cl, the rate of 15 N labeling of one of the two nitrogen atoms in Gln gradually decreased, and the unlabeled rate of Gln was 0.23% (2.5% in the presence of Na 15 NO3). As shown in Figure 4, the total 15 N labeling rate of the two nitrogen atoms in Gln was 71% in Na 15 NO3 and 89% in 15 NH4Cl. Next, the position of the 15

Nitrogen Assimilation Rate by Glutamine Synthase and Glutamate Dehydrogenase
From these results, the enzymatic activity for Gln synthesis was assumed to be affected by the type of nitrogen source. To test this hypothesis, we measured the catalytic activities of glutamine synthetase (GS) and glutamate dehydrogenase (GDH) from wholecell lysates ( Figure 6). The catalytic activity of GS was 1.1 U/mg-protein when Synechocystis was grown in NaNO3 medium and 0.73 U/mg-protein when grown in NH4Cl medium. In a previous report, the expression level of GS was higher in NaNO3 medium than in NH4Cl medium [14]. When considered with the results from this previous report, the difference in the catalytic activity of GS in Figure 6B reflects

Nitrogen Assimilation Rate by Glutamine Synthase and Glutamate Dehydrogenase
From these results, the enzymatic activity for Gln synthesis was assumed to be affected by the type of nitrogen source. To test this hypothesis, we measured the catalytic activities of glutamine synthetase (GS) and glutamate dehydrogenase (GDH) from whole-cell lysates ( Figure 6). The catalytic activity of GS was 1.1 U/mg-protein when Synechocystis was grown in NaNO 3 medium and 0.73 U/mg-protein when grown in NH 4 Cl medium. In a previous report, the expression level of GS was higher in NaNO 3 medium than in NH 4 Cl medium [14]. When considered with the results from this previous report, the difference in the catalytic activity of GS in Figure 6B reflects the difference in the expression level of GS itself. On the other hand, the activity of GDH was 3.5 mU/mg-protein when Synechocystis was grown in NH 4 Cl medium and 2.3 mU/mg-protein in NaNO 3 medium; the catalytic activity of GDH in NH 4 Cl was higher than that in NaNO 3 . GS itself. On the other hand, the activity of GDH was 3.5 mU/mg-protein when Synechocystis was grown in NH4Cl medium and 2.3 mU/mg-protein in NaNO3 medium; the catalytic activity of GDH in NH4Cl was higher than that in NaNO3.

Different Assimilation Mechanisms for Nitrogen Depending on the Nitrogen Source
In this study, we wished to elucidate the mechanisms by which Synechocystis grown under different nitrogen sources assimilates nitrogen, and more generally, to clarify how it responds to different nitrogen sources. Different types of nitrogen altered the growth rate, pool sizes of metabolites, and nitrogen assimilation rate of Synechocystis (Figures 1-5). In addition, the catalytic activities of GS and GDH were different for different types of nitrogen sources ( Figure 6).
Based on the findings of this study, we propose a model for nitrogen assimilation by Synechocystis grown in different nitrogen sources (Figure 7). The model was prompted by 15 N labeling experiments. In contrast to the assimilation of NH4 + , Synechocystis was unable to assimilate NO3 − by itself and required an additional reducing power to convert it to NH4 + ( Figure 7A). This means that the reduction of NO3 − appears to be the rate-limiting step for nitrogen assimilation. Therefore, at the beginning of the log phase, when there is sufficient photosynthetic reducing power, GS synthesizes Gln with 15 NH4 + , resulting in the rapid labeling of the side chain of Gln with 15 N (Reaction 1 in Figure 7A) [23]. This hypothesis is consistent with the finding that the preferentially 15 N-labeled nitrogen was in position 5 until 1 h later ( Figure 5C). Subsequently, the 15 N labelled NH2 group is rapidly transferred to 2OG by GOGAT, resulting in two Glu molecules (Reaction 2 in Figure  7A) [24]. GS can also synthesize doubly 15 N-labeled Gln from newly reduced 15 NH4 + and 15 N-labeled Glu (Reaction 1 in Figure 7A). Furthermore, 15 N-labeled Glu can be used as a nitrogen source in another pathway to generate another amino acid (Reaction 4 in Figure  7A). However, the reducing power gradually decreased as the light transmittance of the culture medium decreased during the transition from the early log phase ( Figure 7A) to the late log phase ( Figure 7B). It has been previously shown that light is necessary to reduce NO3 − , and Fd, which accepts the reducing power from the photosystem, reduces NO3 − and NO2 − to NH4 + [7,8,23,24]. Thus, GS cannot use the newly reduced 15 NH4 + but reuses 14 NH4 + , which derived from amino acids due to the decrease in light ( Figure 7B). As a result, Reaction 1 with 15 NH4 + in Figure 7 would not occur. In fact, the 15 N labeling rate of position 2 in Figure 5C gradually increased and became equal to that of position 5 after

Different Assimilation Mechanisms for Nitrogen Depending on the Nitrogen Source
In this study, we wished to elucidate the mechanisms by which Synechocystis grown under different nitrogen sources assimilates nitrogen, and more generally, to clarify how it responds to different nitrogen sources. Different types of nitrogen altered the growth rate, pool sizes of metabolites, and nitrogen assimilation rate of Synechocystis (Figures 1-5). In addition, the catalytic activities of GS and GDH were different for different types of nitrogen sources ( Figure 6).
Based on the findings of this study, we propose a model for nitrogen assimilation by Synechocystis grown in different nitrogen sources (Figure 7). The model was prompted by 15 N labeling experiments. In contrast to the assimilation of NH 4 + , Synechocystis was unable to assimilate NO 3 − by itself and required an additional reducing power to convert it to NH 4 + ( Figure 7A). This means that the reduction of NO 3 − appears to be the rate-limiting step for nitrogen assimilation. Therefore, at the beginning of the log phase, when there is sufficient photosynthetic reducing power, GS synthesizes Gln with 15 NH 4 + , resulting in the rapid labeling of the side chain of Gln with 15 N (Reaction 1 in Figure 7A) [23]. This hypothesis is consistent with the finding that the preferentially 15 N-labeled nitrogen was in position 5 until 1 h later ( Figure 5C). Subsequently, the 15 N labelled NH 2 group is rapidly transferred to 2OG by GOGAT, resulting in two Glu molecules (Reaction 2 in Figure 7A) [24]. GS can also synthesize doubly 15 N-labeled Gln from newly reduced 15 NH 4 + and 15 N-labeled Glu (Reaction 1 in Figure 7A). Furthermore, 15 N-labeled Glu can be used as a nitrogen source in another pathway to generate another amino acid (Reaction 4 in Figure 7A). However, the reducing power gradually decreased as the light transmittance of the culture medium decreased during the transition from the early log phase ( Figure 7A) to the late log phase ( Figure 7B). It has been previously shown that light is necessary to reduce NO 3 − , and Fd, which accepts the reducing power from the photosystem, reduces NO 3 − and NO 2 − to NH 4 + [7,8,23,24]. Thus, GS cannot use the newly reduced 15 NH 4 + but reuses 14 NH 4 + , which derived from amino acids due to the decrease in light ( Figure 7B). As a result, Reaction 1 with 15 NH 4 + in Figure 7 would not occur. In fact, the 15 N labeling rate of position 2 in Figure 5C gradually increased and became equal to that of position 5 after 24 h. This can also explain why the 15 N labeling rate of Gln was 50% ( Figure 4) and 71% ( Figure 5A).
is not labeled [25] and GS activity was much higher than the activity of GDH ( Figure 6).
The catalytic activity of GS was about 1000-fold higher than that of GDH in Figure  6B,C. For Synechocystis, GDH is not essential for growth under the high CO2 conditions [26]. In Escherichia coli, GDH supplies glutamine in the absence of a carbon source because GDH can produce it without ATP originating from carbon utilization [27]. This means that the high CO2 conditions which were adopted in our experiments might have caused the 1000-fold change differences between the catalytic activity of GS and GDH.  In contrast to NO 3 − , reducing power is not required to assimilate NH 4 + . Figure 5D illustrates that position 5 in Gln is always dominant throughout the 24 h period, and Figure 5B indicates that 90% of nitrogen in all Gln molecules was labeled. The light conditions cannot be the rate-determining step to assimilate NH 4 + , and Reaction 1 in Figure 7C continues unless NH 4 + is lacking. Therefore, we assumed that the 15 N labeling rate quickly approached 90%, as shown in Figures 4 and 5B.
We also considered the possibility that the higher labeling rate with 15 NH 4 Cl in Figures 4 and 5 resulted from the higher activity of the GDH pathway, as shown in Figure 6C. However, such possibilities can be excluded because, in Figure 5D,E, position 2 should be preferentially labeled if the GDH pathway is activated when Synechocystis is cultivated in NH 4 Cl medium. GDH synthesizes Glu from 2OG and NH 4 + , suggesting that position 5 is not labeled [25] and GS activity was much higher than the activity of GDH ( Figure 6). The catalytic activity of GS was about 1000-fold higher than that of GDH in Figure 6B,C. For Synechocystis, GDH is not essential for growth under the high CO 2 conditions [26]. In Escherichia coli, GDH supplies glutamine in the absence of a carbon source because GDH can produce it without ATP originating from carbon utilization [27]. This means that the high CO 2 conditions which were adopted in our experiments might have caused the 1000-fold change differences between the catalytic activity of GS and GDH.
In this study, we found that the type of nitrogen sources can affect the metabolic profile of Synechocystis. The altered metabolic profile and labeling rate of Synechocytstis in Figures 2-5 when Synechocystis was grown in NaNO 3 or NH 4 Cl media came from the availability of nitrogen. When Synechocystis was grown in NH 4 Cl medium, NH 4 + could be used without limitation unless NH 4 Cl was absent in the medium. On the other hand, additional reducing power is required to use NaNO 3 because Synechocystis cannot use NO 3 − directly. This means that the amount of light transmitted, which is the source of reducing power, can be the rate-determining step. Thus, Synechocystis switch the main nitrogen sources from the external to internal nitrogen sources which arise from internal amino acids or proteins to produce glutamine during the change of growth stage when grown in NaNO 3 medium. This switch between external and internal nitrogen sources might enable Synechocystis to grow at the constant rate. When grown in NH 4 Cl medium, the residual additional reducing power for Synechocystis to assimilate nitrogen enables it to supply reducing power to other pathways. The pool size of 3PGA increased and that of S7P and R5P decreased, as shown in Figure 3. The altered pool sizes of these metabolites might be caused by the enhanced CBB cycle, consuming the residual additional reducing power that was not used in the reduction of NO 3 − . Accordingly, the abundance of some metabolites in glycolysis and TCA cycle, and amino acids including 3PGA itself increased in Figures 2 and 3. We suspect the high cell growth rate was accomplished by higher abundance of those metabolites.

The Choice of Nitrogen Source: NO 3
− or NH 4 + ?
So far, we revealed that Synechocystis switches the main nitrogen sources from the external to internal nitrogen sources during the change of growth stage when grown in NaNO 3 medium and does not need to switch the nitrogen sources in NH 4 Cl medium. This speculation suggests that NH 4 + is ideal nitrogen sources for Synechocystis. However, there is a side effect of using NH 4 + as a nitrogen source. The electron transport rate in Synechocystis decreased in the presence of over 15 mM NH 4 Cl according to a previous report [11]. This means that excess NH 4 + can potentially inhibit photosynthesis in Synechocystis. In addition, GS-GOGAT cycle also requires the reducing power to assimilate nitrogen sources [1]. If the amount of NH 4 + supplied exceeds the reducing power from photosynthesis, the accumulation of NH 4 + would inhibit photosynthesis. On the other hand, it is confirmed that there was no influence on photosynthesis over 15 mM NaNO 3 in the same previous report. We guess the reason the excess amount of NH 4 + cannot accumulate in the cell because the production of NH 4 + is dependent on the availability of reducing power arisen from photosynthesis. This means that all the nitrogen assimilation pathway is dependent on the photosynthesis when grown in NaNO 3 , and it is beneficial for Synechocystis to perform the nitrogen assimilation in concert with photosynthesis and cell growth, preventing the excess accumulation of NH 4 + in the cell. The proper type of nitrogen depends on the species of cyanobacteria, given that they can assimilate various types of nitrogen sources, such as NO 3 − and NH 4 + , as shown in this paper. NH 4 + stimulated the growth of Synechocystis, as shown in Figure 1. In contrast, it attenuated the growth of Arthrospira (Spirulina) sp. [12,16] and did not affect the growth of Microcystis aeruginosa NIES-843 [17]. The reasons for the differing responses of cyanobacteria species to different types of nitrogen sources remain unclear. However, we revealed that the enhancement of metabolite pool size and nitrogen turnover by NH 4 + stimulated the growth of Synechocystis in this study. A comparison of the comprehensive metabolic profiles of these cyanobacteria species under different types of nitrogen might reveal the reasons for the different responses of cyanobacteria species.

Measurement of the Intracellular Metabolite Concentration
Extraction and analysis of the intracellular metabolites was performed as previously reported [18]. The procedure is described briefly. For the analysis of the intracellular metabolite concentration, the culture medium corresponding to 5 mg of dry cell weight was recovered at 0, 24, 48, and 72 h after inoculation with Synechocystis. After filtration, the collected cells were washed with 20 mM (NH 4 ) 2 CO 3 . The intracellular metabolite was extracted using pre-cooled methanol containing the internal standard, and the watersoluble phase was collected by mixing chloroform. The soluble protein was removed by filtration, and the resultant water phase containing the metabolite was evaporated under vacuum. The dried metabolites were dissolved in pure water and subjected to CE-MS analysis.

15 N-Metabolic Turnover Analysis
The assimilation ratio of newly added nitrogen sources at each time point was determined using stable isotope 15 N-labelled Na 15 NO 3 or 15 NH 4 Cl. Synechocystis was transferred to the modified BG11 medium containing 5 mM Na 15 NO 3 or 15 NH 4 Cl 24 h after inoculation and cultivated under 1% (v/v) CO 2 and 100 µ mol photons m −2 s −1 at 30 • C. The culture medium corresponding to 5 mg of dry cell weight was recovered at 0, 4, and 24 h, and the intracellular metabolites were analyzed by CE-MS as described above or by LC-MS/MS MRM. The procedure of sample preparation was the same as described above Section 4.2 (measurement of the intracellular metabolite concentration) and LC-MS/MS MRM analysis was performed by employing Nexera X2 high-performance liquid chromatography system and a LCMS-8060 triple quadrupole mass spectrometer (Shimadzu Corporation, Kyoto, Japan), as described previously [28]. The 15 N labeling rate was calculated as performed in 13 C labeling experiments in previous reports [18]. The relative isotopomer abundance (m i ) for each metabolite in which the i 15 N atoms were incorporated is calculated as follows: where M i represents the isotopomer abundance of metabolite incorporating i 15 N atoms, and n is the number of nitrogen atoms in the metabolite. Statistical analysis was conducted using Welch's t-test (* <0.05, ** <0.01).

Enzymatic Assay of Whole Cell Lysate
Synechocystis cells that were cultured in the presence of NaNO 3 or NH 4 Cl were collected by centrifugation (3000× g, 4 • C, 10 min) and washed with nitrogen-free BG11