Rapid Transcriptional Reprogramming Triggered by Alteration of the Carbon/Nitrogen Balance Has an Impact on Energy Metabolism in Nostoc sp. PCC 7120

Nostoc (Anabaena) sp. PCC 7120 is a filamentous cyanobacterial species that fixes N2 to nitrogenous compounds using specialised heterocyst cells. Changes in the intracellular ratio of carbon to nitrogen (C/N balance) is known to trigger major transcriptional reprogramming of the cell, including initiating the differentiation of vegetative cells to heterocysts. Substantial transcriptional analysis has been performed on Nostoc sp. PCC 7120 during N stepdown (low to high C/N), but not during C stepdown (high to low C/N). In the current study, we shifted the metabolic balance of Nostoc sp. PCC 7120 cultures grown at 3% CO2 by introducing them to atmospheric conditions containing 0.04% CO2 for 1 h, after which the changes in gene expression were measured using RNAseq transcriptomics. This analysis revealed strong upregulation of carbon uptake, while nitrogen uptake and metabolism and early stages of heterocyst development were downregulated in response to the shift to low CO2. Furthermore, gene expression changes revealed a decrease in photosynthetic electron transport and increased photoprotection and reactive oxygen metabolism, as well a decrease in iron uptake and metabolism. Differential gene expression was largely attributed to change in the abundances of the metabolites 2-phosphoglycolate and 2-oxoglutarate, which signal a rapid shift from fluent photoassimilation to glycolytic metabolism of carbon after transition to low CO2. This work shows that the C/N balance in Nostoc sp. PCC 7120 rapidly adjusts the metabolic strategy through transcriptional reprogramming, enabling survival in the fluctuating environment.


Introduction
Cyanobacteria use light energy to fix inorganic carbon (C i ) and nitrogen (N), harvested from their aquatic environment, into the metabolic components required for growth and propagation. Environmental sources of C i include dissolved CO 2 and bicarbonate (HCO 3 − ), while N can be supplied by nitrate (NO 3 − ), nitrite (NO 2 − ), ammonium (NH 4 + ), urea or N 2 (in diazotrophic cyanobacteria; reviewed in [1]). The tight coupling of the concentrations of C i and N taken up from the environment prevents metabolic imbalance within the cell, which allows cyanobacteria to thrive amidst varying nutritional conditions. This is achieved in large part by transcriptional modifications that are triggered by fluctuations in the cellular homeostasis of organic carbon (C) and N, which are represented by changes in the relative abundances of key metabolite signals (reviewed in [2][3][4]). One such metabolite is 2-oxoglutarate (2OG), also known as α-ketoglutarate (αKG), which is a product of the tricarboxylic acid (TCA) cycle. The metabolite 2OG provides the inorganic carbohydrate skeleton for glutamate synthesis that occurs by the incorporation of NH 4 + in the glutamine synthetase/glutamine-oxoglutarate aminotransferase (GS/GOGAT) cycle. Cellular 2OG levels therefore represent the abundances of both C and N (reviewed in [5,6]), making 2OG a central signalling metabolite that triggers transcriptional adjustments to restore C/N balance [7][8][9]. 2-phosphoglycolate (2PG) is another metabolite that controls transcriptional reprogramming in response to C/N balance [10], and 2PG is formed when Rubisco catalyses the oxygenation of RuBP (photorespiration), instead of the favoured carboxylation reaction between RuBP and CO 2 (reviewed in [4,11]). An increase in 2PG concentration therefore represents CO 2 deficiency, triggering transcriptional reprogramming designed to upregulate C i import into the cell [12][13][14][15]. Nostoc (Anabaena) sp. PCC 7120 is a filamentous, diazotrophic cyanobacterium wherein C/N balance controls the formation of heterocyst cells specialised for fixing N 2 into NH 4 + , while photosynthetic CO 2 fixation is restricted to vegetative cells (reviewed in [16,17]). In Nostoc sp. PCC 7120 and other heterocystous cyanobacteria, differentiation in cell structure and function is triggered by changes in C/N homeostasis and enacted by massive transcriptional reprogramming [1]. Emphasis on the impact of N concentration on heterocyst differentiation has revealed the central roles of 2OG and several regulatory proteins in instigating transcriptional and physiological responses to N deficit [6]. However, the transcriptional response of heterocystous cyanobacteria to C i availability has drawn only little attention [18], in sharp contrast to that in non-diazotrophic species [12,13,15,[19][20][21][22]. In the current study of the global transcriptome of Nostoc sp. PCC 7120, we found that a shift from 3% CO 2 to 0.04% CO 2 for 1 h can be largely attributed to changes in the levels of the metabolites 2PG and 2OG, which has a strong effect on genes involved in the import and metabolism of C i and N. This study also identified that genes encoding factors involved in photosynthetic electron transport, glycolysis and iron homeostasis are regulated by C/N homeostasis, which is suggested to trigger a transition from efficient photoautotrophic growth and energy storage to photoinhibition and glycolysis.

Growth and CO 2 Stepdown
Nostoc sp. PCC 7120 cultures were grown in BG11 medium [23] buffered with 10 mM TES-KOH (pH 8.0) at 30 • C under constant illumination of 50 µmol photons m −2 s −1 with 120 rpm agitation, in air enriched with 3% (v/v) CO 2 . During the exponential growth phase (OD 750 = 1.0), 2 mL samples were taken from three individual replicate cultures and frozen for RNA isolation. For CO 2 stepdown, the cultures were pelleted and the pellets washed once with fresh BG11, before resuspension in fresh BG11 and growth in air containing 0.04% (v/v) CO 2 . After 1 h, 2 mL samples were collected from three replicates and frozen for RNA isolation.

RNA Isolation and Transcriptomics
Total RNA was isolated as described in [24]. Total RNA samples were submitted to the Beijing Genomics Institute (China) for library construction and RNA sequencing using Illumina HiSeq2500. RNA reads were aligned using Strand NGS 2.7 software (Avadis) using the Nostoc sp. PCC 7120 reference genome and annotations downloaded from Ensembl (EBI). Aligned reads were normalised and quantified using the DESeq package (R). Significantly differentially expressed genes were identified using a 2-way ANOVA. p-Values were adjusted for false discovery rate (FDR) using the Benjamini-Hochberg procedure.

Results
The transcriptome of Nostoc sp. PCC 7120 grown in BG11 under 3% CO 2 -enriched air was compared with that of the same strain shifted from 3% CO 2 to 0.04% CO 2 for 1 h, revealing 230 genes to be upregulated >2-fold and 211 genes to be downregulated >2-fold, in the low CO 2 condition. The RNAseq data are available at the NCBI Sequence Read Archive (submission SUB8244772). As expected, given the short period under a new metabolic condition, no differences in the growth rates, lengths of filaments or frequencies of heterocysts (approximately 4% of all cells under 3% CO 2 ) were observed between the cultures exposed to 0.04% CO 2 conditions, compared to those grown at 3% CO 2 . Therefore, statistical tests of these parameters in the different cultures were not performed.

Uptake and Metabolism of Carbon and Nitrogen Are Inversely Responsive to Low CO 2 Conditions
The operons encoding three plasma membrane-localised HCO 3 − uptake systems were among the most strongly upregulated entities in Nostoc sp. PCC 7120 following CO 2 stepdown (Table 1). In cyanobacteria, the Cmp (BCT1) system is powered by ATP hydrolysis [25], while the SbtA and BicA systems depend on Na + ions for HCO 3 − symport [21,26]. The upregulation of the operon encoding the Mrp Na + :H + antiporter upon CO 2 stepdown may also be linked to HCO 3 − uptake through the extrusion of Na + to support SbtA and BicA activity [27,28]. HCO 3 − uptake in cyanobacteria forms a major part of the carbon concentration mechanism (CCM), which also involves the concentration of cellular CO 2 into HCO 3 − by a customised NAD(P)H dehydrogenase (NDH-1) complex [29]. Genes ndhF3, ndhD3 and cupA, which encode subunits that specialise the NDH1-MS complex for inducible CO 2 uptake, were upregulated after CO 2 stepdown (Table 1), as were several other genes encoding the core NDH-1M complex (see below). Notably, a putative cupS orthologue (alr1320), which is encoded separately from the ndhF3/ndhD3/cupA cluster in the Nostoc sp. PCC 7120 genome [30], was not differentially expressed (DE) in the current work. Genes encoding Rubisco and some carboxysome subunits were mildly upregulated in low CO 2 (1.3 to 1.7 fold change (FC); not shown), while other CCM components were not DE, suggesting that these components were already in sufficient abundance before CO 2 stepdown, whereas C i uptake from the environment, especially HCO 3 − uptake, was apparently a primary concern for survival after 1 h under low CO 2 . The decrease in CO 2 was found to cause downregulation of the nir operon, which encodes subunits of an ATP-dependent nitrate (NO 3 − ) transporter, as well as NO 3 − and nitrite (NO 2 − ) reductases [31,32].
The nir operon is responsible for NO 3 − uptake and reduction to ammonia (NH 3 ), and is broadly conserved across cyanobacteria (reviewed in [5]). Unlike the nir operon, the majority of nif genes that encode subunits for the assembly and function of nitrogenase, which reduces atmospheric N 2 to NH 3 , were not DE in the current work. Exceptions were nifB and nifH2, which were downregulated (Table 1). A gene that encodes a protein similar to the C-terminus of Mo-like nitrogenase (alr1713) was strongly downregulated, along with its neighbour (asr1714); however, the function of the encoded proteins is not known. Since heterocyst development is upregulated by a high C/N ratio (reviewed in [33]), it was not surprising to see many genes involved in the structural development of heterocysts repressed by the shift to low CO 2 . In particular, the hpd, hgl and dev gene clusters that encode many components for the synthesis and export of glycolipids, which form the oxygen-impermeable heterocyst envelope (reviewed in [34,35]), and were downregulated in the current data. Notably, the hep genes encoding heterocyst outer layer polysaccharides were only moderately downregulated by the shift to low CO 2 (average FC −1.5, data not shown).

Expression of Genes Encoding Photosynthetic and Respiratory Components Responds to Low CO 2 Conditions
Expression of genes encoding photosystem II (PSII) core proteins D1 and D2 was upregulated after the shift to low CO 2 ( Table 2), indicating an increase in the damage and turnover of PSII reaction centres [36,37]. In keeping with this, the genes encoding the two FtsH proteases involved in the degradation and turnover of damaged D1 protein were also upregulated [38,39]. We also found strongly induced expression of the flv2-flv4 operon, as well as several genes encoding orange carotenoid proteins (OCPs) and early light-inducible proteins (ELIPs), all of which are associated with PSII photoprotection [40][41][42][43][44]. These expression data suggest that the shift to low CO 2 led to the over-reduction, damage and repair of PSII. Given this evidence for PSII over-reduction, it was surprising to find the gene encoding the "plastid" terminal oxidase (PTOX) as one of the most strongly downregulated in the current study (Table 2), being highly expressed under 3% CO 2 and strongly repressed in 0.04% CO 2 . PTOX is part of a water-water cycle that moves electrons from reduced plastoquinone (PQ) to O 2 , and is thought to be an electron valve for balancing the photosynthetic redox state [45], which would presumably be important under low CO 2 (discussed below).
Virtually all genes encoding subunits of photosystem I (PSI) were substantially downregulated in the current study, which is in contrast to their increased expression in Synechocystis sp. PCC 6803 and unchanged expression Synechococcus elongatus PCC 7942 in low CO 2 [13,20]. PSI downregulation in Nostoc sp. PCC 7120 points towards a decrease in PSI electron transport that may be related to the diminution of the terminal electron acceptor CO 2 . These conditions would also be expected to upregulate O 2 reduction and the subsequent formation of toxic superoxide radicals (O 2 •− ); indeed, genes encoding a superoxide dismutase (SodB; alr2938) and peroxiredoxin (all2375), involved in reactive oxygen species (ROS) scavenging, were upregulated 3.2-fold and 2.3-fold, respectively (not shown). The expression of isiB (alr2405), which encodes a flavodoxin (Fld) that accepts electrons from PSI via a flavin mononucleotide cofactor [46], was also upregulated after low CO 2 treatment ( Table 2), suggesting a shortage of oxidised ferredoxin (Fd) acceptors [47]. The upregulated expression of genes encoding F 0 -F 1 ATP synthase points to an increased demand for energy in low CO 2 , required for HCO 3 − import and CO 2 hydration (reviewed in [2]). Notably, the gene alr1004 encoding an enzyme that converts glyoxylate to glycine for the detoxification of 2PG [18] was found to be downregulated after CO 2 stepdown, while other enzymes in the glycolate metabolism pathway were not DE. Uncharacterised protein −5.8 <0.001 1 Shaded cells represent operons or clusters of neighbouring genes; 2 Fold changes of genes upregulated or downregulated in low CO 2 , compared to high CO 2 , are coloured orange or green, respectively. In cases of multiple genes, average fold changes are shown; 3 p-values determined by moderated t-test. In cases of multiple genes, largest p-value is shown. The downregulation of PSI abundance in response to low CO 2 can partially clarify the apparent PSII over-reduction discussed above. Lower PSI levels may also be linked to a decrease in the number of heterocysts, which contain a higher PSI:PSII ratio than in vegetative cells [16]. Although such a decrease in heterocysts was not observed after 1 h in low CO 2 , suppressed heterocyst development was evident in the downregulation of hgl and dev clusters (Table 1), and this can also explain the suppression of genes encoding cytochrome c6, Flv1B and Flv3B ( Table 2) that operate predominately [48] or exclusively [49] in heterocysts. The gene encoding the small subunit of the heterocyst-specific uptake hydrogenase (HupS) was also downregulated here, reflecting the downregulation of the heterocystous nitrogenase activity under low CO 2 .
We observed downregulation of the pec cluster that encodes the phycoerythrocyanin (PEC) parts of the light-harvesting phycobilisome (PBS) complex [50,51], while genes encoding the other components of the PBS were not DE, indicating that the light-harvesting cross-section of PBS in Nostoc sp. PCC 7120 is altered in response to low CO 2 . An operon encoding a subunit of the light-independent protochlorophyllide reductase (DPOR) was also downregulated after 1 h in low CO 2 , while the expression of chlG and hemH genes, involved in later stages of chlorophyll and haem synthesis, respectively, were upregulated ( Table 2).
Most genes encoding subunits of the NDH-1 complex were upregulated under low CO 2 ( Table 2), probably to fulfil their role in CO 2 uptake as part of the NDH-1MS complex described above. In contrast, ndbA encoding NDH-2 was downregulated in the current study, suggesting a decrease in NDH-2-mediated respiration after the shift to low CO 2 . In addition, the gene encoding phosphoenolpyruvate (PEP) synthase, which converts pyruvate to PEP that is consumed in the TCA cycle, was strongly downregulated ( Table 2). Many genes encoding subunits of the bidirectional hydrogenase (Hox) were among the most strongly downregulated in response to low CO 2 conditions (Table 2), being both highly expressed in 3% CO 2 and strongly repressed in low CO 2 . Hox reversibly catalyses the reduction of H + to form H 2 , powered by reduced Fd/Fld with electrons derived from either PSI or pyruvate, the latter route by way of pyruvate ferredoxin/flavodoxin oxidoreductase (PFOR), which converts pyruvate to acetyl-CoA (reviewed in [52]). The nifJ gene encoding PFOR followed a similar expression profile to Hox subunits, being another of the most strongly downregulated genes in the current study. The physiological role of the Hox enzyme is not known, but has been described as an electron valve that can maintain redox balance and store reducing power as H 2 during excess photosynthesis or fermentation [52][53][54][55]. The expression profile of Hox and PFOR genes suggests that pyruvate-powered hydrogen production is active under 3% CO 2 and inactivated by the shift to low CO 2 .

Expression of Transcription Regulators Responds to Changes in CO 2 Conditions
The current study revealed substantial changes in the expression of several genes encoding transcription regulators, providing evidence of an ongoing cascade of transcriptional reprogramming after 1 h under low CO 2 (Table 3). Upregulated expression of the LysR-type regulator (LTTR) cmpR corresponds to the strong upregulation of its target, the cmp cluster encoding the BCT1 HCO 3 − transporter (Table 1), as previously shown in Synechocystis sp. PCC 6803, Synechococcus sp. PCC 7942 [56] and Nostoc sp. PCC 7120 [57]. Two sigB-type group 2 sigma factors, which have roles in the transcriptional response to low CO 2 and C/N balance [58][59][60], were upregulated after the shift to low CO 2 (Table 3). SigB has also been implicated in response to environmental stress and resistance to photoinhibition in Synechocystis sp. PCC 6803 [61,62], which is in line with the upregulation in this study of groES and groEL genes (4.3 to 5.5 FC; not shown) and photoprotective factors such as OCPs and the flv2-flv4 operon ( Table 2). Two homologous two-component response regulator clusters, which each comprise a histidine kinase and a DNA-binding regulator, were found to be upregulated by low CO 2 . Of the two, the chromosomal hik31 (C-hik31) operon was more highly upregulated, and has been found to be involved in the responses to oxygen concentration, light and metabolism [63,64]. A TetR-family transcription regulator with unknown function was also upregulated by low CO 2 (Table 3).
Another LTTR gene that was highly expressed under high CO 2 and was strongly downregulated after low CO 2 transition (Table 3) shared substantial sequence homology with the ndhR transcription repressor (also called ccmR) of Synechocystis sp. PCC 6803 [12,27]. In other cyanobacteria, NdhR represses the expression of CCM genes, including the sbt and bicA HCO 3 − importers, the mrp cluster and the ndhF3/ndhD3/cupA cluster [12,15,20,27,65]. The rapid downregulation of a putative ndhR orthologue in Nostoc sp. PCC 7120 may reveal the mechanism behind the strong upregulation of CCM genes after CO 2 stepdown in the current study ( Table 1). Downregulation of the transcription enhancer ntcB, which increases N metabolism through upregulation of the nir operon [66], also correlates with downregulation of other N-related genes in the current work ( Table 1). Expression of ntcB is controlled by NtcA [1]. As ntcA was not DE in the current study, downregulation of ntcB and other NtcA regulons may be due to the low CO 2 -induced inactivation of NtcA (discussed below). Similarly, downregulated transcriptional regulator genes patB (also called cnfR), devH and nrrA can also be attributed to inhibited NtcA activity [67][68][69][70][71]. These genes are expressed in heterocysts, where PatB upregulates the expression of nifB [72], DevH regulates heterocyst glycolipids [48,68,73] and NrrA induces expression of both the heterocyst regulator hetR and fraF encoding a filament integrity protein [70,74]. Both nifB and the hgl cluster were downregulated in this study (Table 1), while hetR and fraF were not DE (not shown).

Low CO 2 Conditions Influence the Expression of Metal Homeostasis Genes
A number of genes and gene clusters related to cellular homeostasis of iron (Fe) and other metals were found to be DE after CO 2 stepdown (Table 4). A gene cluster encoding subunits of a periplasmic ferrous Fe (Fe(II)) transporter [75,76] was strongly downregulated in the current study (Table 4), indicating a lower uptake of Fe from the environment under low CO 2 . The suf cluster, encoding proteins involved in Fe mobilization and Fe-S cluster assembly, was also downregulated. The expression of both the Fe(II) transporter and the suf cluster is upregulated by Fe deprivation [77][78][79], suggesting a surplus of cellular Fe after low CO 2 treatment. Several neighbouring clusters of genes encoding subunits of metal cation efflux systems such as copper, nickel, zinc, cadmium and cobalt, were upregulated after low CO 2 treatment (Table 4). These genes have been implicated in heavy metal resistance [80,81], although the link to the current conditions is not clear. Taken together, low CO 2 appears to induce an active decrease in cellular metal content, which may be a strategy to avoid oxidative stress during the reducing conditions induced by an insufficient availability of photosynthetic electron acceptors.

Transcriptional Regulation in Response to CO 2 Stepdown is Triggered by Metabolites
Induction of the most strongly upregulated genes, the HCO 3 − transporters (Table 1), after CO 2 stepdown, suggests a rapid transcriptional response that is highly sensitive to cellular C i levels.
In some unicellular cyanobacteria, repression of the CCM genes by NdhR (also called CcmR) can be modulated by both 2OG and 2PG [12,15,65,82]. Increased cellular 2PG concentration caused by increased photorespiration in low CO 2 leads to increased abundance of the NdhR-2PG complex that is unable to bind DNA to repress expression [4]. Additionally, declining 2OG levels due to lower CO 2 fixation and potentially lower TCA cycle activity decrease the abundance of the NdhR-2OG repressor complex, although it is unclear whether 2OG levels would actually decrease after only 1 h in low CO 2 due to the mobilisation of stored glycogen into the TCA cycle [2,15,82,83]. NADP + , another co-repressor of NdhR [82], is also theoretically far less abundant after CO 2 stepdown, due to decreased CO 2 fixation and lower NADPH consumption despite constant light conditions. Although a putative NdhR in Nostoc sp. PCC 7120 has not been studied, it appears that the LTTR encoded by all4986 represents such an orthologue, and that the strong downregulation of all4986 after CO 2 stepdown led to de-repression of the NdhR regulon, which includes the ndhR gene itself [84]. Previous transcriptomics studies have shown ndhR expression to be upregulated in Synechocystis sp. PCC 6803 after >3 h in low C i conditions [12,13], which is in contrast to the strong downregulation of all4986 seen here after 1 h (Table 3). This suggests that NdhR de-repression in response to CO 2 stepdown may be transient, and/or that expression of the NdhR regulon is also controlled by other transcription factors [18]. In the current study, de-repression by the putative NdhR is proposed to have caused a rapid and strong increase in HCO 3 − and CO 2 uptake under C limitation, with the upregulated cmp operon (Table 1) inducing further increase in HCO 3 − uptake. The cmp inducer CmpR is activated by 2PG or RuBP [82,85], both of which are in higher concentrations after CO 2 stepdown due to decreased CO 2 fixation. Furthermore, cmpR expression is also auto-upregulated (Table 3) [57]. In Synechococcus sp. PCC 7942 CmpR additionally upregulates the expression of PSII core subunits [86,87], found upregulated also in the current study alongside factors for PSII photoprotection and turnover, and downregulation of most PSI genes ( Table 2). The overlap between cellular responses to either low CO 2 or high light stress is well documented [12,20,86,88], highlighting insufficient electron sinks similarly created by both high light and low CO 2 , and resulting in the over-reduction of photosynthetic electron carriers [2]. Notably, several PSII photoprotection factors encoded by genes that were DE in the current study, including psbAIII, flv4 and sodB, were shown to be regulated together with Rubisco and other CCM genes by another LTTR in Nostoc sp. PCC 7120 called PacR [18], suggesting the likely involvement of PacR in the transcriptional reprogramming seen here. In addition to the LTTR transcription factors, the transcriptional response to low CO 2 is also regulated by LexA and the cyAbrB paralogues [89][90][91][92]. The vast change in expression of the hox operon after CO 2 stepdown (Table 2) may be related to the activity of LexA [93] and/or cyAbrB [90,[94][95][96]. In Synechocystis sp. PCC 6803, cyAbrB2 controls the expression of many CCM components that were likewise found to be upregulated in this study (Table 1) [90], while the cyAbrB2 orthologue in Nostoc sp. PCC 7120 regulates the expression of FeSOD [96], also upregulated here. Nostoc sp. PCC 7120 cyAbrB1 has been recently implicated in transcriptional regulation of heterocyst differentiation [97], demonstrating a role close to the interface of C i and N availability that suggests cyAbrB1/2 were likely active in the transcription regulation observed in the current study.
Given the transcriptional activation of NtcA, the master regulator of N metabolism, by high levels of 2OG upon a shift to low N (high C/N ratio) [7,98,99], it is widely assumed that a decrease in CO 2 leads to a decline in NtcA activity by decreasing the abundance of the 2OG-NtcA-PipX activator complex [100]. In the current study, lower NtcA activity was indeed evident in the downregulation of nir genes encoding NO 3 − uptake and metabolism (Table 1), as well as downregulation of the nir co-activator ntcB (Table 3; reviewed in [101]). This transcriptional regulation would effectively bring N metabolism into alignment with decreased C metabolism after CO 2 stepdown, despite the presence of N sources in the BG11 media. Overall, nearly 50% of genes downregulated in the current study have NtcA-binding promoters [71], although many NtcA-regulated genes, such as those involved in the uptake of NH 3 and urea, regulation of GS-GOGAT enzymes, as well as NtcA itself [71,[102][103][104], were not DE after 1 h in low CO 2 . The current work may therefore include only the early NtcA regulon. The NtcA-activated differentiation of vegetative cells to heterocysts occurs over approximately 24 h, via a cascade of transcriptional regulation that includes early upregulation of the co-activator nrrA [33]. The rapid downregulation in the current work of some heterocyst regulators, including nrrA, may block the commencement of heterocyst differentiation in response to relative N excess over C after CO 2 stepdown, and may signal an eventual decrease in the small number of heterocysts that are known to occur under high C/N [105]. Downregulation of NtcA activity in the current study appears to indicate a decline in 2OG levels after only 1 h in low CO 2 , although an increased concentration of NH 3 derived from 2PG metabolism can also explain suppression the NtcA regulon under low CO 2 (low C/N ratio) [83]. N excess under low CO 2 is also evident in the −2.2 FC downregulated expression of cyanophycinase chb2 (all0571; not shown), which is regulated by NrrA, suggesting a decrease in the mobilisation of stored N under low CO 2 (reviewed in [106]). It can also be argued that an increase in the ADP/ATP ratio under CO 2 stepdown, caused by decreased photosynthetic electron transport and rapid changes in metabolism, increases the abundance of both the ADP-PII-PipX complex and the inactive form of NtcA [6]. The strong downregulation of operons involved in ferrous Fe import and Fe-S cluster assembly in the current work (Table 4) suggests a connection between cellular Fe homeostasis and C/N balance in Nostoc sp. PCC 7120, which has been explored [107]. The current results indicate a CO 2 stepdown-induced cellular excess of Fe and other transition metals, which may be due to downregulation of Fe-rich PSI complexes (Table 2) [108] and/or an excess of reductant caused by insufficient electron sinks. Both conditions pose the danger of ROS formation, evidenced by upregulated expression of SOD, peroxiredoxin and protein chaperones.

Altered C/N Balance Modulates the Energetic Strategy of Nostoc sp. PCC 7120
The current study shows that a stepdown from 3% CO 2 in enriched air to 0.04% CO 2 (atmospheric) for just 1 h led to substantial reprogramming of global gene expression in Nostoc sp. PCC 7120 cultures. As discussed above, most of the transcriptional changes observed here can be directly attributed to metabolite signalling instigated by the alteration of the cellular C/N balance (Figure 1), initiated by the decrease in CO 2 concentration. Furthermore, these results highlight rapid transcriptional reprogramming of photosynthesis and energy metabolism in Nostoc sp. PCC 7120 in response to CO 2 levels (Figure 2). High CO 2 in light promotes a high rate of photosynthesis and the storage of photosynthate in the form of glycogen [13,[109][110][111]. Strong expression of PEP synthase and PFOR under these conditions indicate glycolysis/gluconeogenesis through the metabolism of pyruvate, which is consumed in the TCA cycle to facilitate respiratory electron transport and to provide 2OG for amino acid synthesis (reviewed in [112,113]). Therefore, growth under 3% CO 2 somewhat resembles photomixotrophy, with photosynthetic and glycolytic metabolisms occurring concomitantly, even though glucose was not externally provided to cultures. Under these conditions, the cells experience a high C/N, which is evident in the relatively high expression of N metabolic genes, reflecting a cellular excess of 2OG [7,98,99]. In high CO 2 , strong expression of Hox, which is important under mixotrophy and N deprivation [55], and PTOX, may provide a system to maintain redox poise [45], and in the case of Hox, also store surplus energy as H 2 [53]. The transfer of Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942 cultures from high to low CO 2 showed that the toxic effects of 2PG transiently block Calvin-Benson-Bassham (CBB) activity [15,20,83] and this is also evident here in the upregulated expression of PSII repair, photoprotection and ROS scavenging enzymes in Nostoc sp. PCC 7120, which indicate over-reduction of the photosynthetic electron transport chain. Interestingly, detoxification of 2PG appeared to be downregulated through repression of alr1004 during CO 2 stepdown (Table 2), perhaps highlighting the importance of the metabolite for signalling during the early stages of C i deprivation. We also observed downregulation of the terminal proteins in the phycobilisomes, suggesting modified harvesting of light energy to alleviate excitation pressure on the photosynthetic system. Under these conditions, inhibition of photoassimilation is compensated by glycolysis of stored glucose and CBB intermediates, providing an important supply of substrates for anaplerosis of the CBB and TCA cycles during acclimation to the transition [83,114,115]. In the current work, enhanced glycolytic activity is indicated by the upregulation of NDH-1, suggesting an increased reliance on respiratory electron transport for ATP generation, while strong downregulation of PEP synthase and PFOR after CO 2 stepdown may prevent diversion of pyruvate away from the TCA cycle. Notably, PEP abundance increased substantially in Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942 after a shift from high to low CO 2 [20,83], while genes encoding both pyruvate kinase and PFOR were highly expressed in Synechococcus elongatus PCC 7942 after long-term acclimation to low CO 2 , but not directly after the transition [20]. These findings support the results of this study and suggest that the metabolism of PEP and pyruvate are tightly regulated after the transition to low CO 2 . This may be linked to the role of PEP and pyruvate as substrates to anaplerotic carbon fixation to produce TCA cycle intermediates oxaloacetate and malate (reviewed in [116]). During the adjustment to a low C/N ratio, TCA cycle activity generates 2OG for amino acid synthesis, utilising excess NH 4 + generated through 2PG detoxification [11,83].
This work has revealed rapid transcriptional reprogramming in Nostoc sp. PCC 7120 in response to a decrease in C i availability, namely strong upregulation of CCM components and photoprotection, and downregulation of N uptake and early stages of heterocyst differentiation. Despite the vast increase in HCO 3 − uptake, glycolysis of stored C apparently plays an important role in energy metabolism at low CO 2 , likely due to 2PG-induced inhibition of the CBB cycle. A majority of the transcriptional effects induced by low CO 2 in Nostoc sp. PCC 7120 can be attributed to 2PG modulation of CmpR and a putative NdhR homologue; however, the effects of changing abundance of 2OG and NtcA activity after 1 h in 0.04% CO 2 , as well as the roles of other transcriptional regulators cannot be discounted. Finally, this work highlights the sensitivity of Nostoc sp. PCC 7120 to factors that influence the cellular C/N balance and demonstrates the speed at which genetic and metabolic reprogramming can take place, allowing rapid acclimation for surviving and thriving in the fluctuating environment.
Life 2020, 10, x FOR PEER REVIEW 11 of 20 may be linked to the role of PEP and pyruvate as substrates to anaplerotic carbon fixation to produce TCA cycle intermediates oxaloacetate and malate (reviewed in [116]). During the adjustment to a low C/N ratio, TCA cycle activity generates 2OG for amino acid synthesis, utilising excess NH4 + generated through 2PG detoxification [11,83].   cycle, which produces reductant, ATP and succinate to drive respiratory electron transport through NAD(P)H-dehydrogenase (NDH) and succinate dehydrogenase (SDH), as well as other cellular processes. The TCA cycle also generates 2-oxoglutarate (2OG), which accumulates under high CO 2 due to a relative shortage of NH 4 + and glutamine. 2OG operates as a signal for upregulating genes involved in N uptake and metabolism. Strong expression of phosphoenolpyruvate (PEP) synthase converts pyruvate to PEP. Pyruvate:ferredoxin/flavodoxin oxidoreductase (PFOR) converts pyruvate to acetyl-CoA, reducing oxidised ferredoxin/flavodoxin (Fd/Flv ox ) that is consumed by bidirectional hydrogenase (Hox) for storage of excess energy as H 2 . Strong expression of plastoquinone terminal oxidase (PTOX) maintains redox homeostasis of the plastoquinone (PQ) pool during high photosynthetic electron transport, while cytochrome c6 oxidase (COX) maintains the redox poise of lumenal electron carriers cytochrome c 6 (c 6 ) and plastocyanin (PC). Genes encoding factors coloured orange are highly expressed under 3% CO 2 and strongly downregulated by the shift to 0.04% CO 2 . (B) After 1 h in 0.04% CO 2 , a deficiency of CO 2 electron acceptors leads to oxygenation of Rubisco, causing photorespiration that produces 2-phosphoglycolate (2PG). The CBB cycle and other metabolic pathways are inhibited by 2PG, which also signals upregulation of the transcriptomic response to low CO 2 . Low CBB activity causes over-reduction of the photosynthetic electron transport chain, leading to the production of reactive oxygen species (ROS) at photosystem I (PSI) and downregulation of PSI subunits. Increased reducing pressure on photosystem II (PSII) also causes upregulation of the PSII repair cycle and upregulation of PSII photoprotection by flavoproteins (Flv2/4) and orange carotenoid proteins (OCPs), as well as downregulation of phycoerythrocyanin in the phycobilisome (PBS). Glycolysis triggered by decreased CO 2 assimilation provides substrates for anaplerotic supplementation of the CBB and TCA cycles, including the production of oxaloacetate from PEP and bicarbonate (HCO 3 − ). TCA cycle activity also produces 2OG for glutamate production from excess ammonium (NH 4 + ) resulting from the shift from 3% to 0.04% CO 2 . Genes encoding factors coloured orange or green are upregulated or downregulated, respectively, 1 h after the shift from 3% to 0.04% CO 2 . Black arrows indicate the movement of electrons or ATP, dashed arrows summarise multiple enzymatic reactions in carbohydrate metabolism, blue arrows indicate the movement of protons. The red arrow in (A) shows the production of 2OG by TCA cycle activity, the red arrow in (B) shows the production of 2PG by Rubisco oxygenation in the CBB cycle and the black bar in (B) indicates CBB inhibition by 2PG.