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

Abstract

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.

1. 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 CO₂ and bicarbonate (HCO₃⁻), while N can be supplied by nitrate (NO₃⁻), nitrite (NO₂⁻), ammonium (NH₄⁺), urea or N₂ (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 NH₄⁺ 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 CO₂ (reviewed in [4,11]). An increase in 2PG concentration therefore represents CO₂ 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 N₂ into NH₄⁺, while photosynthetic CO₂ 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% CO₂ to 0.04% CO₂ 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.

2. Materials and Methods

2.1. Growth and CO₂ 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) CO₂. During the exponential growth phase (OD₇₅₀ = 1.0), 2 mL samples were taken from three individual replicate cultures and frozen for RNA isolation. For CO₂ 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) CO₂. After 1 h, 2 mL samples were collected from three replicates and frozen for RNA isolation.

2.2. 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.

3. Results

The transcriptome of Nostoc sp. PCC 7120 grown in BG11 under 3% CO₂-enriched air was compared with that of the same strain shifted from 3% CO₂ to 0.04% CO₂ for 1 h, revealing 230 genes to be upregulated >2-fold and 211 genes to be downregulated >2-fold, in the low CO₂ 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% CO₂) were observed between the cultures exposed to 0.04% CO₂ conditions, compared to those grown at 3% CO₂. Therefore, statistical tests of these parameters in the different cultures were not performed.

3.1. Uptake and Metabolism of Carbon and Nitrogen Are Inversely Responsive to Low CO₂ Conditions

The operons encoding three plasma membrane-localised HCO₃⁻ uptake systems were among the most strongly upregulated entities in Nostoc sp. PCC 7120 following CO₂ stepdown (

Table 1. Differentially Expressed (DE) Genes Involved in Carbon and Nitrogen Metabolism.

Name 1	Gene ID 1	Description	Process	Fold Change 2	p-Value 3
sbt operon	all2133–all2134	Na+-dependent bicarbonate permease, PII-like regulatory protein	Bicarbonate import	78.8	<0.001
cmp operon	alr2877–alr2880	ATP-dependent bicarbonate uptake subunits		36.4	<0.001
bicA operon	all1303–all1304	Na+-dependent bicarbonate permease, Na+:H+ antiporter		8.7	<0.001
mrp operon	all1837–all1843	Na+:H+ antiporter subunits	Na+ extrusion, pH regulation	5.4	<0.001
ndhF3	alr4156	NDH–1MS subunit 5	CO2 uptake	2.5	0.002
ndhD3	alr4157	NDH–1MS subunit 4		2.0	<0.001
cupA	alr4158	NDH–1MS CO2 uptake subunit		5.3	<0.001
nir operon	alr0607–alr0612	Nitrate/nitrite reductase, ATP-dependent nitrate permease	Nitrate/nitrite import and metabolism	−3.5	0.017
nirB	all0605	Nitrate-dependent expression of nir cluster		−2.9	<0.001
nifB	all1517	Fe–Mo cofactor biosynthesis subunit	N2 fixation, heterocyst development and function	−2.0	0.002
nifH2	alr0874	Fe–S cluster-binding nitrogenase reductase		−3.2	0.024
hgd, hgl clusters	all5341–alr5359	Heterocyst glycolipid layer biosynthesis		−2.6	0.004
dev operon	alr3710–alr3712	ATP-binding subunit, membrane transport subunits		−2.1	0.004
alr1713	alr1713	Similar to Mo-dependent nitrogenase, C-terminus	Unknown	−5.6	0.002
asr1714	asr1714	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 CO₂, compared to high CO₂, 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.

). In cyanobacteria, the Cmp (BCT1) system is powered by ATP hydrolysis [25], while the SbtA and BicA systems depend on Na⁺ ions for HCO₃⁻ symport [21,26]. The upregulation of the operon encoding the Mrp Na⁺:H⁺ antiporter upon CO₂ stepdown may also be linked to HCO₃⁻ uptake through the extrusion of Na⁺ to support SbtA and BicA activity [27,28]. HCO₃⁻ uptake in cyanobacteria forms a major part of the carbon concentration mechanism (CCM), which also involves the concentration of cellular CO₂ into HCO₃⁻ 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 CO₂ uptake, were upregulated after CO₂ 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 CO₂ (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 CO₂ stepdown, whereas C_i uptake from the environment, especially HCO₃⁻ uptake, was apparently a primary concern for survival after 1 h under low CO₂.

The decrease in CO₂ was found to cause downregulation of the nir operon, which encodes subunits of an ATP-dependent nitrate (NO₃⁻) transporter, as well as NO₃⁻ and nitrite (NO₂⁻) reductases [31,32]. The nir operon is responsible for NO₃⁻ uptake and reduction to ammonia (NH₃), 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 N₂ to NH₃, 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 CO₂. 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 CO₂ (average FC −1.5, data not shown).

3.2. Expression of Genes Encoding Photosynthetic and Respiratory Components Responds to Low CO₂ Conditions

Expression of genes encoding photosystem II (PSII) core proteins D1 and D2 was upregulated after the shift to low CO₂ (

Table 2. Differentially Expressed (DE) Genes Involved in Photosynthesis and Respiration.

Name 1	Gene ID 1	Description	Process	Fold Change 2	p Value 3
psbAII	alr3727	Photosystem II D1 protein	PSII electron transport	6.9	<0.001
psbAIII	alr4592	Photosystem II D1 protein		1.9	0.003
psbAIV	all3572	Photosystem II D1 protein		3.7	<0.001
psbD	alr4548	Photosystem II D2 protein		3.4	<0.001
psaA	alr5154	Photosystem I core protein A1	PSI electron transport	−1.9	0.035
psaB1	alr5155	Photosystem I core protein A2		−1.9	0.038
psaB2	alr5314	Photosystem I core protein A2		−2.2	0.031
psaC	asr3463	Photosystem I Fe-S subunit		−2.2	<0.001
psaD	all0329	Photosystem I reaction centre subunit 2		−2.7	<0.001
psaE	asr4319	Photosystem I subunit E		−2.2	0.001
psaI	asl3849	Photosystem I subunit I		−2.5	0.012
pasK	asr4775	Photosystem I subunit K		−3.1	0.004
psaM	asr4657	Photosystem I subunit M		−2.3	0.005
flv2	all4444	Flavodiiron protein	Other photosynthetic/respiratory electron transport	23.7	<0.001
all4445	all4445	Unknown protein		31.8	<0.001
flv4	all4446	Flavodiiron protein		16.7	<0.001
isiB	alr2405	Flavodoxin		2.9	0.003
cytA	alr4251	Cytochrome c6		−2.2	0.011
flv1B-flv3B	all0177–all0178	Flavodiiron protein (heterocyst-specific)		−2.0	0.004
ptox	all2096	Alternative plastoquinone oxidase		−65.0	<0.001
ftsH	alr1261	FtsH protease	PSII turnover	2.3	<0.001
ftsH2	all3642	FtsH protease		2.4	<0.001
pec operon	alr0523–alr0527	Phycoerythrocyanin synthesis	Metabolism/binding of light-harvesting pigments	−3.1	0.015
chlL, chlN operon	all5076–all5078	Protochlorophyllide reductase, ATP-binding protein		−3.3	<0.001
chlG	all4480	Chlorophyll synthase 33 kDa subunit		2.2	0.003
hemH	alr4616	Ferrochelatase		8.6	<0.001
ocp	all3149	Orange carotenoid-binding protein		23.9	<0.001
ocp-like	all4941	Orange carotenoid protein-like		3.1	0.010
asl3726	asl3726	CAB/ELIP/HLIP superfamily		9.8	<0.001
asr5262	asr5262	CAB/ELIP/HLIP superfamily		8.6	0.005
atpase cluster	all0004–all0010	ATP synthase subunits	ATP synthesis	2.7	<0.001
ndh-1 operon	alr0223–alr0227	NDH-1 complex subunits	Electron and proton transport	2.3	0.005
ndh-1 operon	all3840–all3842			2.1	0.001
ndhB	all4883			2.5	0.003
ndhN	alr4216			1.9	0.001
alr1004	alr1004	Alanine-glyoxylate transaminase	Glycolate metabolim	−2.7	<0.001
ndbA	all1553	NDH-2 NAD(P)H:PQ reductase	Respiration	−2.1	<0.001
hupS	all0688	Uptake hydrogenase, small subunit	H2 uptake/evolution	−2.5	0.002
nifJ/PFOR	alr1911	Pyruvate-ferredoxin/flavodoxin oxidoreductase		−42.4	<0.001
hox clusters	alr0750–all0752alr0760–alr0766	Bidirectional hydrogenase subunits, assembly and regulation		−73.9	0.002
ppsA	all0635	Phophoenolpyruvate synthase	Glycolysis	−107.4	<0.001

¹ Shaded cells represent operons or clusters of neighbouring genes; ² Fold changes of genes upregulated or downregulated in low CO₂, compared to high CO₂, 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.

), 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 CO₂ 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% CO₂ and strongly repressed in 0.04% CO₂. PTOX is part of a water–water cycle that moves electrons from reduced plastoquinone (PQ) to O₂, and is thought to be an electron valve for balancing the photosynthetic redox state [45], which would presumably be important under low CO₂ (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 CO₂ [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 CO₂. These conditions would also be expected to upregulate O₂ reduction and the subsequent formation of toxic superoxide radicals (O₂^•−); 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 CO₂ treatment (Table 2), suggesting a shortage of oxidised ferredoxin (Fd) acceptors [47]. The upregulated expression of genes encoding F₀-F₁ ATP synthase points to an increased demand for energy in low CO₂, required for HCO₃⁻ import and CO₂ 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 CO₂ stepdown, while other enzymes in the glycolate metabolism pathway were not DE.

The downregulation of PSI abundance in response to low CO₂ 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 CO₂, 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 CO₂.

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 CO₂. An operon encoding a subunit of the light-independent protochlorophyllide reductase (DPOR) was also downregulated after 1 h in low CO₂, 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 CO₂ (Table 2), probably to fulfil their role in CO₂ 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 CO₂. 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 CO₂ conditions (Table 2), being both highly expressed in 3% CO₂ and strongly repressed in low CO₂. Hox reversibly catalyses the reduction of H⁺ to form H₂, 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 H₂ 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% CO₂ and inactivated by the shift to low CO₂.

3.3. Expression of Transcription Regulators Responds to Changes in CO₂ 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 CO₂ (

Table 3. Differentially Expressed (DE) Genes Encoding Transcription Regulators.

Name	Gene ID	Description	Process	Fold Change 1	p Value 2
cmpR	all0862	LysR-type transcriptional regulator	Regulates cmp cluster	3.1	<0.001
sigB	all7615	Group 2 sigma factor	Response to stress	4.6	<0.001
sigB3	all7608	Group 2 sigma factor		3.8	<0.001
C-hik31 operon	all7583–all7584	Two-component sensor His kinase, response regulator	Regulation of central metabolism in response to glucose, low O2	2.8	<0.001
P-hik31 operon	alr1170–alr1171			1.6	0.001
all7523	all7523	TetR family regulator	Unknown	2.4	0.046
putative ndhR orthologue	all4986	LysR-type transcriptional regulator	Repression of CCM expression	−146.2	<0.001
ntcB	all0602	LysR-type transcriptional regulator	Co-activation of nir operon	−2.4	0.002
devH	alr3952	CRP family transcriptional regulator	Het glycolipid biosynthesis	−2.2	0.006
patB/cnfR	all2512	Heterocyst patterning	Heterocyst development	−2.4	0.001
nrrA	all4312	OmpR family regulator		−2.1	<0.001

¹ Fold changes of genes upregulated or downregulated in low CO₂, compared to high CO₂, are coloured orange or green, respectively. In cases of multiple genes, average fold changes are shown; ² p-values determined by moderated t-test. In cases of multiple genes, largest p-value is shown.

). Upregulated expression of the LysR-type regulator (LTTR) cmpR corresponds to the strong upregulation of its target, the cmp cluster encoding the BCT1 HCO₃⁻ 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 CO₂ and C/N balance [58,59,60], were upregulated after the shift to low CO₂ (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 CO₂. 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 CO₂ (Table 3).

Another LTTR gene that was highly expressed under high CO₂ and was strongly downregulated after low CO₂ 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 HCO₃⁻ 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 CO₂ 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 CO₂-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).

3.4. Low CO₂ 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 CO₂ stepdown (

Table 4. Differentially Expressed (DE) Genes Involved in the Transport and Metabolism of Metals.

Name	Gene ID	Description	Process	Fold Change 1	p Value 2
Fe(II) transport operon	alr2118–asr2120	Ferrous iron transporter subunits	Periplasmic iron import	−42.6	<0.001
suf operon	alr2492–alr2496	ATPase, iron and sulphur transfer	Fe–S cluster assembly, transfer	−3.8	<0.001
Metal efflux cluster	all7606–all7611	Proton extrusion, cation efflux	Metal cation efflux, cellular metal homeostasis	3.1	<0.001
Metal efflux cluster	all7616–all7619	Cadmium/nickel/zinc/cobalt efflux system		3.9	<0.001
Metal efflux cluster	all7629–all7633	Cadmium/nickel/zinc/cobalt efflux system		5.0	<0.001
Cu2+ efflux cluster	alr7634–alr7636	Putative copper efflux		5.1	0.002

¹ Fold changes of genes upregulated or downregulated in low CO₂, compared to high CO₂, are coloured orange or green, respectively. In cases of multiple genes, average fold changes are shown; ² p-values determined by moderated t-test. In cases of multiple genes, largest p-value is shown.

). 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 CO₂. 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 CO₂ 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 CO₂ 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 CO₂ 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.

4. Discussion

4.1. Transcriptional Regulation in Response to CO₂ Stepdown is Triggered by Metabolites

Induction of the most strongly upregulated genes, the HCO₃⁻ transporters (Table 1), after CO₂ 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 CO₂ 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 CO₂ 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 CO₂ 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 CO₂ stepdown, due to decreased CO₂ 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 CO₂ 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 CO₂ 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 HCO₃⁻ and CO₂ uptake under C limitation, with the upregulated cmp operon (Table 1) inducing further increase in HCO₃⁻ uptake. The cmp inducer CmpR is activated by 2PG or RuBP [82,85], both of which are in higher concentrations after CO₂ stepdown due to decreased CO₂ 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 CO₂ or high light stress is well documented [12,20,86,88], highlighting insufficient electron sinks similarly created by both high light and low CO₂, 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 CO₂ is also regulated by LexA and the cyAbrB paralogues [89,90,91,92]. The vast change in expression of the hox operon after CO₂ 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 CO₂ 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 NO₃⁻ 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 CO₂ 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 NH₃ and urea, regulation of GS-GOGAT enzymes, as well as NtcA itself [71,102,103,104], were not DE after 1 h in low CO₂. 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 CO₂ 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 CO₂, although an increased concentration of NH₃ derived from 2PG metabolism can also explain suppression the NtcA regulon under low CO₂ (low C/N ratio) [83]. N excess under low CO₂ 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 CO₂ (reviewed in [106]). It can also be argued that an increase in the ADP/ATP ratio under CO₂ 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 CO₂ 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.

4.2. Altered C/N Balance Modulates the Energetic Strategy of Nostoc sp. PCC 7120

The current study shows that a stepdown from 3% CO₂ in enriched air to 0.04% CO₂ (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 CO₂ concentration. Furthermore, these results highlight rapid transcriptional reprogramming of photosynthesis and energy metabolism in Nostoc sp. PCC 7120 in response to CO₂ levels (Figure 2). High CO₂ 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% CO₂ 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 CO₂, 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 H₂ [53]. The transfer of Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942 cultures from high to low CO₂ 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 CO₂ 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 CO₂ 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 CO₂ [20,83], while genes encoding both pyruvate kinase and PFOR were highly expressed in Synechococcus elongatus PCC 7942 after long-term acclimation to low CO₂, 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 CO₂. 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 NH₄⁺ 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 HCO₃⁻ uptake, glycolysis of stored C apparently plays an important role in energy metabolism at low CO₂, likely due to 2PG-induced inhibition of the CBB cycle. A majority of the transcriptional effects induced by low CO₂ 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% CO₂, 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.