Carbon metabolism shapes FtsZ levels and cell division in the cyanobacterium Anabaena PCC 7120

Ran, Wen-Shuo; Zeng, Xiaoli; Zhang, Cheng-Cai

doi:10.1038/s42003-025-08849-4

Download PDF

Article
Open access
Published: 08 October 2025

Carbon metabolism shapes FtsZ levels and cell division in the cyanobacterium Anabaena PCC 7120

Communications Biology volume 8, Article number: 1440 (2025) Cite this article

Subjects

Abstract

Cyanobacteria, as photoautotrophs, play key ecological roles and are widely used in synthetic biology research. While metabolism profoundly influences cellular processes like cell division, the regulatory mechanisms linking these activities remain poorly understood in cyanobacteria. Here, we perturb the tricarboxylic acid (TCA) pathway by introducing an extra CO2 fixation module in the model cyanobacterium Anabaena sp. PCC 7120. This modification alters the levels of the cell division protein FtsZ, impairing cell division under varying light conditions. We found that 2-oxoglutarate, an intermediate of the TCA pathway known as a metabolic signal, regulates ftsZ expression via the transcription factor NtcA. Furthermore, metabolic status modulates cell division in a NtcA-dependent manner, directly integrating metabolic control and cell division. Our findings uncover a coordination mechanism between metabolism and cell division in cyanobacteria, providing mechanistic insights for synthetic biology engineering and understanding of metabolic regulation.

Introduction

How different cell activities are orderly and effectively coordinated is a central question in biology. For a bacterial cell facing constantly changing environments, coordinating various metabolic and physiological processes is essential for survival and adaptation. Each bacterial species can maintain its distinct cell size and morphology by integrating the external environmental input and internal metabolic state^1,2,3,4,5. By integrating informational cues, bacterial cell division and the other cell cycle events, such as cell growth, DNA replication, segregation, etc., are thus coordinated⁶. However, because of the complexity of such regulation, the mechanisms required are mostly poorly understood.

Bacterial cell division is a highly controlled, dynamic process involving numerous proteins. The prokaryotic tubulin homolog FtsZ is the central component for cell division in most bacteria, as well as plastids in algae and plants, and in some mitochondria^6,7. FtsZ can assemble into a ring-like structure called the Z-ring at the division site (mid-cell). The Z-ring then recruits the downstream proteins to form a multiprotein complex termed the divisome that drives cell constriction^6,7,8. Thus, the amount and the localization of the FtsZ protein in the cell must be tightly regulated. Changes in ftsZ expression level or FtsZ localization can strongly impact cytokinesis, which in turn can alter cell morphology, cell size, or even affect cell viability^9,10,11. Several factors regulating the activity or polymerization state of FtsZ have been reported¹²; in contrast, much less is known about the mechanism that controls FtsZ levels in response to internal or external cues. Multiple promoters are found at the ftsZ genes in various bacteria¹³, but few transcriptional regulators have been identified, and the underlying mechanism of the transcription regulation of ftsZ expression remains unclear. Recently, an increasing number of sRNAs, including cis- and trans-acting sRNAs, such as DinR, StfZ¹⁴, DicF¹⁵, and OxyS¹⁶, have been found to regulate FtsZ levels post-transcriptionally. In addition, the amount of FtsZ levels was shown to be regulated by the ClpXP protease in different bacteria, such as E. coli¹⁷, Bacillus subtilis¹⁸, Caulobacter crescentus¹⁹, and Synechocystis sp. PCC6803²⁰.

As one of the most fundamental biological processes, cell division is strongly linked to central carbon metabolism in different heterotrophic bacteria^21,22. In E. coli and B. subtilis, the accumulation of uridine diphosphate (UDP)-glucose stimulates the interaction between the glucosyltransferase OpgH/ UgtP and FtsZ, delaying the division machinery assembly, and hence, cell division^23,24. It was also reported that pyruvate mediates Z-ring localization and assembly in B. subtilis by regulating the localization of the pyruvate dehydrogenase²⁵. In E. coli, perturbations of acetyl-CoA metabolism impact cell size and division through changes in fatty acid synthesis²⁶. Moreover, other metabolic enzymes, such as glutamate dehydrogenase in C. crescentus and Brucella abortus²⁷, KidO (oxidoreductase) in C. crescentus²⁸, and aceE, ackA, ccr, pta, tktA in E. coli²⁹, have also been identified for their function in cell division. The absence of these metabolic enzymes causes cell division abnormalities to varying degrees, but their underlying mechanisms remain unknown.

Cyanobacteria are the only prokaryotes able to perform oxygenic photosynthesis, using light energy to convert water and CO₂ into biomass³⁰. Its carbon metabolism is characterized by the Calvin–Benson cycle (carbon fixation) and photorespiration³¹. Furthermore, cyanobacteria have an incomplete TCA cycle due to the lack of 2-oxoglutarate dehydrogenase (2-OGDH), which catalyzes succinyl-CoA production³². Although some different bypass pathways were found to close the TCA cycle in different cyanobacteria species^33,34, biochemical analyses revealed that the contribution of these bypass pathways in terms of metabolic flux and metabolite pools to the TCA pathway is limited³⁵. A few studies suggest that the inorganic carbon and nitrogen regime³ or nutrient metabolism can affect cell division in cyanobacteria³⁶, but the underlying regulation mechanism is unknown.

Since the relationship between metabolism and cell division remains unknown in cyanobacteria, we sought to disturb the TCA metabolic pathway by engineering a strain harboring an additional carboxylation module, using the model cyanobacterium Anabaena sp. PCC 7120 (hereafter Anabaena). This module contains the crotonyl-CoA carboxylase/reductase (Ccr), the most efficient carboxylase known in nature, to redirect metabolic flux into the TCA pathway. Anabaena is a filamentous cyanobacterium capable of diazotrophic growth following deprivation of combined nitrogen. Nitrogen fixation occurs exclusively in heterocysts, cells differentiated from vegetative cells within 20–24 h after nitrogen starvation³⁷. The process of heterocyst development is triggered by the accumulation of 2-oxoglutarate (2-OG), a signal of carbon-nitrogen metabolic balance^37,38. 2-OG then binds to NtcA to initiate the process of heterocyst differentiation^38,39,40,41. Our data reported here found that the addition of the extra module of carboxylation in Anabaena affects the pool of the TCA metabolites, 2-OG, which controls FtsZ levels through NtcA. This study highlights a distinct coordination mechanism between carbon metabolism and cell division in cyanobacteria, offering critical insights into the metabolic engineering of these photoautotrophic organisms.

Results

Introduction of an additional carboxylation module into Anabaena

One way to investigate the relationship between carbon metabolism and cell division is to disturb metabolic pools and examine their consequence on cell division. To do so, we integrated an extra carboxylation module (named PCEM), containing the genes pco (propionyl-CoA oxidase, Pco), ccr (crotonyl-CoA carboxylase, Ccr), epi (emC/mmC epimerase, Epi), and mcm (methylmalonyl-CoA isomerase, Mcm) into the Anabaena genome with the substitution of the alr2634-all2640 gene cluster (encoding peptide synthetases and polyketide synthases) by the Cpf1-based gene editing technique⁴². The four genes of the module were in one artificial operon under the control of a previously characterized CT promoter (P_CT, inducible by Cu²⁺ and theophylline) (Fig. 1A, B)⁵. Pco is the acyl-CoA oxidase ACX4 from Arabidopsis thaliana with a T134L mutation and a strong activity for oxidizing propionyl-CoA into acrylyl-CoA⁴³. Ccr is from Methylobacterium extorques and can produce methylmalonyl-CoA via reductive carboxylation of acrylyl-CoA with high efficiency⁴³. Mcm and Epi are from Rhodobacter sphaeroides, which together catalyze the methylmalonyl-CoA to succinyl-CoA⁴³. The sequential reactions catalyzed by these enzymes result in succinyl-CoA formation from propionyl-CoA, fixing one extra carbon (CO₂) at the expense of one NADPH. This CO₂ fixation pathway provides a direct metabolic link to the incomplete TCA cycle in Anabaena (Fig. 1A and Supplementary Fig. 1). The resulting cyanobacterial strain (gPCEM) was verified by PCR and sequencing (Fig. 1C). Further western blotting experiments showed that each of the 4 genes was expressed in the gPCEM strain in BG11 48 h after the addition of 1 mM theophylline and 0.3 µM Cu²⁺ (determined as the optimal inducer concentrations, which will be used in the following studies unless otherwise indicated) with continuous standard light (SL) illumination (Fig. 1D). These results showed that the PCEM module could be well expressed in Anabaena.

Fig. 1: Engineering the PCEM carboxylation module in *Anabaena.*

Metabolic pool analysis of the gPCEM strain under different light conditions

How does this extra carboxylation module affect the TCA metabolite pools in Anabaena? To answer this question, the products of the PCEM module, and the TCA pathway, including propionyl-CoA, pyruvate, succinate, fumarate, malate, citrate, 2-OG, and acetyl- CoA of the gPCEM strain at different light conditions (HL, high light; SL, standard light; LL, low light, see details in “Methods” section) were analyzed by Liquid Chromatography-Mass Spectrometry (LC-MS)⁴⁴. As shown in Fig. 2, propionyl-CoA, the first substrate of the PCEM module, exhibited a dramatic reduction in the gPCEM strain under different light conditions. These results indicated that the heterologous Pco has strong catalytic activity in Anabaena (Fig. 2). On the contrary, metabolites of the TCA pathway in the gPCEM strain varied significantly under different light conditions (Fig. 2). Under LL, the intracellular levels of fumarate, malate, and 2-OG in the gPCEM strain exhibited significant reduction, whereas the amounts of succinate and citrate remained relatively stable when compared to the WT. However, under SL and HL, the levels of most of the TCA cycle metabolites in the gPCEM strain increased significantly, as compared to the WT strain, such as succinate, fumarate, and malate under SL, and fumarate, malate, citrate, and 2-OG under HL. These results suggested that introducing the PCEM module into Anabaena could cause different disturbances to the metabolic pools of the TCA pathway under different light conditions. Notably, pyruvate, the end product of the glycolysis pathway, and acetyl-CoA, a central metabolic intermediate, remained in the gPCEM strain similar to those in the WT strain across all light conditions, except for pyruvate, which showed a reduction in the gPCEM strain under low light (LL).

**Fig. 2: Changes of the selected metabolite pools in the gPCEM under different light conditions.**

The presence of the PCEM module affects cell differentiation in Anabaena

One intermediate from the TCA pathway, 2-OG, acts as a nitrogen-carbon balancing signal in cyanobacteria^37,38. In Anabaena, the transient accumulation of 2-OG signals nitrogen starvation and triggers heterocyst differentiation^37,38,39,40. 2-OG levels in the gPCEM strain under different light conditions were changed significantly, except under SL. However, even under SL conditions, a reproducible increase in 2-OG level was found (mean ± SEM: WT 1.11 ± 0.18 vs gPCEM 1.47 ± 0.09 normalized units), with a significant score of 0.09. We expected that heterocyst development would be impacted. Therefore, we examined their effect on heterocyst development in BG11₀, a culture medium free of combined nitrogen. The WT strain was analyzed in parallel as a control. When observed under a microscope, the gPCEM strain under different light conditions was affected in heterocyst frequency (Supplementary Fig. 2A). After 48 h of growth in BG11₀ medium, the gPCEM strain could form mature heterocysts under all three different light conditions; however, the percentage of heterocysts showed a decrease under LL (6% ± 0.31%), an increase under SL (11.7% ± 0.62%) and under HL (15.6% ± 0.55%), as compared to the WT at each of the corresponding conditions (7.2% ± 0.32%; 9.4% ± 0.32%, 9.4% ± 0.56%) (Supplementary Fig. 2A). Moreover, an Mch (multiple-contiguous-heterocysts) phenotype was occasionally observed under SL and HL (Supplementary Fig. 2A). To confirm this phenotype, we further quantified the pattern of heterocysts along the filaments. The heterocyst intervals of the WT strain showed regularity under different light conditions, with 18–20 vegetative cells, 12–13 vegetative cells, and 9–11 vegetative cells as the most representative intervals under LL, SL, and HL, respectively. In the gPCEM strain, heterocyst distributions kept a regularity under LL, but the intervals increased to 24–25 vegetative cells, consistent with a lower heterocyst frequency. Under SL and HL, heterocyst patterns of the gPCEM strain were changed, since a high frequency of Mch was observed and the intervals were shortened to 9–11 vegetative cells and 5–7 vegetative cells as the most representative intervals under SL and HL, respectively (Supplementary Fig. 2B). These results are consistent with the changes in the 2-OG levels observed in the gPCEM strain under different light conditions, further confirming the metabolite disturbances caused by the PCEM module as measured by the LC-MS/MS.

The presence of the PCEM module affects the cell morphology of Anabaena

To evaluate the effects following the metabolic disturbances of the TCA pathway caused by the PCEM module in Anabaena, we first tested cell growth under different light conditions in BG11 medium in the presence of inducers as described above. As shown in Fig. 3A, the growth rates of the gPCEM strain were slightly slower than that of the wild type under different light conditions, and the difference increased with increasing light intensities (Fig. 3A). When observed under a microscope, the gPCEM strain exhibited significant cell morphology defects under LL and HL, and the phenotype became more pronounced with the extension of culture time, while under SL conditions, its cell morphology was comparable to that of the WT (Fig. 3B, C). Under LL, over time, the average cell length of the gPCEM strain gradually increased while the cell width remained constant, suggesting a delay in cell division (Fig. 3C). On the 6th day, the average cell length of the gPCEM strain was 8.73 ± 2.57 µm as compared to 4.28 ± 0.72 µm found for cells of the WT strain (Fig. 3C). Under HL, the cell morphology of the gPCEM strain becomes more and more irregular over time, as both cell length and width gradually increase with a large variation (Fig. 3B, C). As a control, we confirmed that the markless deletion of the all2634-all2640 gene cluster did not cause any cell morphology defects in Anabaena under similar conditions (Supplementary Fig. 3A), consistent with previous reports⁴². These results all suggested that the expression of the extra carboxylation module may affect cell growth and cell division in Anabaena.

**Fig. 3: Phenotype of the gPCEM strain.**

To rule out potential side effects from the introduction of multiple genes as the cause of the observed cell division and morphological changes, we constructed a series of strains related to the PCEM module in the WT background, including four strains expressing each of the four single genes (strain WT::OE_CT-pco, WT::OE_CT-ccr, WT::OE_CT-epi, WT::OE_CT-mcm), and three strains expressing two, three, or all fours genes of the whole module (strain WT::OE_CT-PC with the pco-ccr genes, strain WT::OE_CT-PCE with the pco-ccr-epi triple genes, and strain WT::OE_CT-PCEM with all four genes of the PCEM module). All these constructs were carried on a replicative plasmid PCT, and gene expression was under the control of the CT promoter, inducible by copper and theophylline^4,5,45. After conjugation, we successfully obtained colonies for all strains, except WT::OE_CT-PCEM. Finally, a few colonies of WT::OE_CT-PCEM, validated by PCR, could be obtained only when a trace amount of copper was removed from glassware with acid treatment. These colonies grew poorly on plates without inducers. However, when inoculated in liquid BG11 medium, the strain WT::OE_CT-PCEM was unable to grow, in contrast to other strains and the WT. We further observed the filament of the strain WT::OE_CT-PCEM from the colonies under a microscope and found that its cells displayed significant cell morphology defects, much stronger than those of the gPCEM strain under HL (Supplementary Fig. 3B). These observations suggest that even without inducers, basal-level expression of the whole module derived from a high-copy plasmid leads to cell growth defects. The other strains were further inoculated in BG11 medium with 1 mM theophylline and 0.3 µM Cu²⁺ under SL. Western blotting results confirmed that Pco, Ccr, Mcm, and Epi proteins were present in the strains containing the corresponding genes (Supplementary Fig. 3C). When observed under a microscope, the cell morphology of all these strains was comparable to that of the WT (Supplementary Fig. 3D). Similar results were obtained when these strains were grown under LL and HL. Together, these results indicate that the metabolic disturbances and phenotypic changes in the gPCEM strain arise from the integrated activity of the entire PCEM module, rather than from the individual expression or function of its constituent enzymes.

ΔsucCD suppresses cell morphology defects in the gPCEM strain

The final product of the carboxylation PCEM module is presumed to be succinyl-CoA (Fig. 1B), which could potentially enter and disrupt the TCA pathway (Fig. 1B). While the 2-OGDH (2-oxoglutarate dehydrogenase) is absent in cyanobacteria³², previous studies in Synechocystis sp. PCC 6803³³ and Synechococcus sp. PCC 7002³⁵ has shown that succinyl-CoA is still converted to succinate via succinyl-CoA synthetase (encoded by sucC and sucD). Our sequence alignment analysis confirmed the presence of SucC and SucD homologs in Anabaena (Supplementary Fig. 4), supporting the possibility that PCEM-generated succinyl-CoA may enter the TCA pathway via succinate, potentially causing metabolite disturbances and, hence, cell morphology defects in the gPCEM strain. To test this hypothesis, we deleted the sucC and sucD genes together (Δsuc) in the gPCEM strain backgrounds, leading to strain Δsuc-gPCEM. We first checked the cell morphology of this strain under different light conditions (Fig. 4). While the gPCEM strain exhibited significant cell morphology changes under LL and HL, these phenotypes were largely suppressed in the Δsuc-gPCEM strain (Fig. 4A). The average cell length and width of the Δsuc-gPCEM strain are similar to those of the WT, independent of light conditions (Fig. 4B). Furthermore, heterocysts in Δsuc-gPCEM also exhibit similar frequencies and distribution patterns as the WT strain (Supplementary Fig. 2). LC-MS metabolite profiling showed that while the propionyl-CoA was reduced similarly to the gPCEM strain, most of the TCA pathway metabolites in the Δsuc-gPCEM strain showed similar variation patterns as the WT strain under comparable light conditions (Fig. 2). We did observe changes in the levels of a few metabolites under certain conditions, including acetyl-CoA (slight reduction under HL), citrate (a decrease under LL and SL), succinate (a slight reduction under SL), malate (a significant increase under SL), and 2-OG (although the change was considered as being nonsignificant (p = 0.32 by t-test, n = 3 biological replicates) under SL, the reduction was visible (mean ± SEM: WT 1.11 ± 0.18 vs 0.87 ± 0.27 normalized units)) (Fig. 2). Importantly, these changes did not cause a particular phenotype in cell division and morphology in this strain. These observations suggest that SucC/D-mediated metabolism plays a central role in the morphological abnormalities of the gPCEM strain.

**Fig. 4: *ΔsucCD* suppresses the cell morphology defects of the gPCEM strain.**

The gPCEM strain has different FtsZ levels in vivo under different light conditions

Given that in bacteria, FtsZ dosage critically regulates cell division, with reduced levels causing filamentation and excess levels triggering minicell formation^9,11, we hypothesized therefore the expression of the PCEM module under different light conditions may affect the FtsZ levels in the gPCEM strain, and cause the corresponding cell morphology defects. To validate this hypothesis, the intracellular FtsZ levels of the gPCEM strain under different light conditions were tested in comparison to the WT under similar conditions. As shown in Fig. 5A, under SL, the FtsZ levels of the gPCEM and the WT strains remained the same, independent of the incubation time (Fig. 5A). However, the FtsZ levels of the gPCEM strain decreased significantly under LL over time but increased significantly under HL over time (Fig. 5A). These results indicated that the cell morphology defects of the gPCEM strain under LL and HL are correlated with the changes in the FtsZ levels. We further analyzed the FtsZ levels of the Δsuc-gPCEM strain at different light conditions (Fig. 5B). As expected, they all showed FtsZ levels similar to those of the WT strain, independent of the light conditions. These results suggested that the changes in FtsZ levels in the gPCEM strain under LL and HL require the connection between the PCEM module and the TCA pathway.

**Fig. 5: The FtsZ levels in the gPCEM strain vary under different light conditions.**

To evaluate whether the transcriptional levels of ftsZ also change in the gPCEM strain under LL and HL, quantitative Real-Time PCR (qRT-PCR) was performed using the gPCEM, the Δsuc-gPCEM, and the WT strains. Under different tested conditions, the transcript levels of ftsZ remained relatively stable in all strains, except for the gPCEM strain cultured under HL, which showed a significant increase, consistent with the increase in FtsZ protein levels under similar conditions (Fig. 5C). These results indicate that transcriptional regulation of ftsZ expression contributes to the increase of FtsZ protein levels in the presence of the PCEM module under HL. However, under LL, the decrease in the FtsZ levels observed in the gPCEM strain was likely caused by posttranscriptional regulations that remain to be determined.

NtcA regulates the transcription of the ftsZ gene in Anabaena

How do the TCA metabolite pools affect the transcriptional level of ftsZ? Under HL conditions, the gPCEM strain exhibits elevated levels of fumarate, malate, citrate, and 2-oxoglutarate (2-OG) (Fig. 2). Since fumarate and malate levels are also significantly elevated in the gPCEM strain under SL conditions (Fig. 2), while FtsZ levels remain unchanged (Fig. 5), the elevated levels of 2-OG and citrate are likely the main factors for the enhanced transcriptional activity of the ftsZ gene under HL conditions. Previous studies indicated that the 2-OG signal in Anabaena could be perceived by the global transcription factor NtcA, which regulates the transcription of genes involved in different processes^{37,38,39,40,41}, and could regulate the expression of the mre genes (encoding MreB, MreC, and MreD, cell elongation) related to cell division³. The promoters of the NtcA regulon usually contain the consensus palindromic sequence GTAN8TAC at the upstream of the transcription start site (TSS)⁴¹. We wondered whether the ftsZ gene could also be regulated by NtcA in Anabaena. We first checked the promoter region of ftsZ for the presence of potential NtcA-binding motifs by FIMO. Interestingly, two putative NtcA-binding sites (GTAN8TAT and GTN10AC, −142 bp and −470 bp to the start codon) at the ftsZ promoter region could be identified (p-value < 0.001, Fig. 6A). According to the published transcriptomic data⁴⁶, six TSS sites could be found at the ftsZ promoter region, and the two putative NtcA-binding sites were located in front of these two TSSs (Fig. 6A). To confirm that NtcA could recognize the two putative NtcA-binding sites, electrophoretic mobility shift assays (EMSA) were performed. Two PCR-amplified DNA fragments of 200 bp from the ftsZ promoter region, each containing one of the two putative binding sites, respectively, were obtained. As shown in Fig. 6B, C, NtcA alone could not bind to the two tested DNA fragments, even with increasing amounts of NtcA (Fig. 6B, C). However, with the presence of 2-OG, both DNA fragments formed a DNA-protein complex with NtcA even at a low protein concentration (Fig. 6B, C). The addition of increasing concentrations of 2-OG significantly promoted the formation of the NtcA-DNA complex (Fig. 6B, C). These results indicated that NtcA could bind to the promoter region of ftsZ in response to the 2-OG signal in vitro.

**Fig. 6: NtcA binds to the promoter region of the *ftsZ* gene in the presence of 2-OG (2-oxoglutarate).**

To investigate whether the two newly identified NtcA motifs are responsible for the transcriptional regulation of ftsZ in vivo, the promoter regions, each containing one NtcA motif, were fused to gfp. The fusions carried on a replicative plasmid were introduced into the WT and the gPCEM strains, respectively. The GFP fluorescence from these fusions in different strains under different light conditions was determined. Under LL, no significant GFP fluorescence could be detected for all tested strains (Fig. 6D, Supplementary Figs. 5 and 6). Under SL, the GFP fluorescence from both fusions in the gPCEM strain was a little bit brighter than that of the WT strains, suggesting a transcriptional activation of ftsZ under such conditions in the WT is similar to the gPCEM strain (Fig. 6, Supplementary Figs. 5 and 6). Under HL, the GFP fluorescence from both fusions in the gPCEM strain was much brighter than that of the WT strain (Fig. 6D, Supplementary Figs. 5 and 6). These results are consistent with the changes in the transcriptional levels of ftsZ analyzed by qRT-PCR above, and the increase of the 2-OG levels under similar conditions in gPCEM (Fig. 5C). They also indicated that the two promoter regions were involved in the ftsZ transcriptional regulation in vivo.

The NtcA mutant displays morphological defects

Following the finding that elevated 2-OG levels in gPCEM affect cell morphology through NtcA, we wondered whether ntcA controls ftsZ expression under physiological conditions in the wild type. In our analysis of cell length, cell width, FtsZ levels, and 2-OG levels in the gPCEM strain under varying light conditions, we used the WT as a control for a similar analysis. We observed that the average cell length of WT Anabaena gradually decreased as light intensity increased, while cell width remained largely unchanged (Supplementary Fig. 7A). This phenotype correlated with elevated levels of 2-OG and increased expression of FtsZ (Supplementary Fig. 7B–D). Although the 2-OG levels in WT Anabaena showed no significant change from SL to HL, we noted that the increased NtcA protein levels under HL led to further enhancement of FtsZ expression (Supplementary Fig. 7D). These findings provide additional evidence for NtcA-mediated FtsZ regulation in Anabaena.

If NtcA regulates ftsZ expression, we expect that an ntcA mutant would display a cell-division-related phenotype. A previous study already reported that the ntcA mutant (CSE2) in ammonium-supplemented BG11₀ medium exhibits a higher cell aspect ratio than the wild-type strain³. NtcA regulates the expression of the mre operon, and the Mre proteins control peptidoglycan synthesis along the bacterial lateral cell wall, hence cell elongation; therefore, the phenotype of aspect ratio was proposed to be caused by elevated levels of the Mre proteins in the ntcA mutant⁴⁷. Our findings corroborate this observation, as we also detected a higher cell aspect ratio in the ntcA mutant under the same conditions (Fig. 6E), with a significant increase in cell length and a reduction in cell width (Fig. 6E). Further analysis revealed that both the level of ftsZ transcripts and that of the FtsZ protein were significantly elevated in the ntcA mutant (Fig. 6F, G). These results indicate that NtcA regulates the expression of FtsZ in Anabaena. Taking all results together, we conclude that NtcA regulates the transcription of the ftsZ gene in Anabaena in a 2-OG-dependent manner. The observed morphological abnormalities in the ntcA mutant can be attributed to a synthetic effect caused by decreased FtsZ levels and increased Mre levels, which together account for changes in the aspect ratio.

Discussion

In heterotrophic bacteria, central carbon metabolism—including the glycolytic pathway, the pentose phosphate pathway, and the TCA cycle—is closely linked to cell division^{22,24,25,28,29}. The molecular mechanisms by which different carbon metabolites coordinate the regulation of cell division vary significantly, highlighting the complexity of the network between carbon metabolism and cell division. Despite the significant differences in metabolic modes as compared to heterotrophic bacteria, cyanobacteria also modulate cell size and morphology according to nutrient regimes (such as carbon supply) and light intensity^2,3,47; however, the mechanism remains largely unexplored. In this study, we disturbed the functioning of the TCA pathway in the model cyanobacterium Anabaena by introducing a highly efficient, artificial CO₂ fixation module (PCEM module) (Figs. 1, 2, and 4). We observed changes in the levels of FtsZ (Fig. 5) as well as cell morphology (Figs. 3 and 4). Combining genetics and biochemistry techniques, we confirmed that the TCA cycle intermediate 2-oxoglutarate (2-OG) and the transcription factor NtcA play a crucial role in coordinating cell division with growth and carbon metabolism in cyanobacteria (Fig. 6), which provides a distinct mechanism in contrast to model heterotrophic bacteria.

The PCEM module could convert propionyl-CoA to succinyl-CoA, fixing one extra carbon (CO₂) and consuming one additional NADPH in the process (Fig. 1)⁴³. Since the succinyl-CoA can integrate into the TCA pathway via succinyl-CoA synthetase (SucC and SucD) in cyanobacteria (Figs. 2 and 4)³⁵, the gPCEM strain may redirect propionyl-CoA flux via the engineered pathway. While the observed cell division defects in the gPCEM strain could theoretically result from depletion of propionyl-CoA precursors - which remain uncharacterized in cyanobacteria - three lines of experimental evidence argue against this possibility: Firstly, control strains expressing individual PCEM components (the WT::OE_CT-pco strain, the WT::OE_CT-PC strain, as well as the WT::OE_CT-PCE strain), all exhibited normal cell division (Supplementary Fig. 3), excluding nonspecific effects of protein expression or potential propionyl-CoA consumption. Secondly, genetic disruption of the metabolic steps connecting the PCEM-TCA cycle in the Δsuc-gPCEM completely suppressed the cell division phenotype (Figs. 4 and 5), demonstrating the involvement of a functional metabolic coupling in the alteration of cell division. Finally, we measured the acetyl-CoA levels under different conditions in the gPCEM strain and found no evidence of significant alteration of this metabolite (Fig. 2). The only condition where a drop in acetyl-CoA levels was observed was under high light (HL) in the Δsuc-gPCEM strain, where decreased acetyl-CoA showed no phenotypic consequence.

The PCEM module displays varying efficiency in carbon source input in Anabaena under various light conditions, affecting the TCA cycle differently (Fig. 2). This suggests that the PCEM-TCA coupled metabolic pathway in the gPCEM strain is light-dependent. This effect may be explained by the effect of light intensity on the production of NADPH and CO₂ limitation in cyanobacteria^48,49. NADPH is a reducing power required for CO₂ fixation by PCEM. High light intensity may enhance the production of NADPH through photosynthesis in Anabaena, conditions in favor of the PCEM-TCA coupled metabolism. In contrast, low light intensity limits the energy supply and substrates available for the PCEM-TCA coupled pathway, ultimately affecting its overall activity. Furthermore, HL-induced faster growth may lead to relative CO₂ limitation of cyanobacterial cells, which could be alleviated by the carbon input through the activity of the PCEM module - a function not required under low light (LL) or standard light (SL) conditions when CO₂ is sufficient. In addition, the TCA pathway may be directly or indirectly regulated by light so that carbon metabolism operates in response to photosynthetic activities. However, the artificial and synthetic PCEM pathway itself is unlikely to be regulated by light, consistent with the observation that consumption of propinyl-CoA (Fig. 2), the first substrate of this pathway, is similar under different light conditions.

Based on the impact of the PCEM module on the TCA pathway (Fig. 2), we found that FtsZ levels are controlled through distinct mechanisms under different light conditions in Anabaena (Figs. 5, 6, and 7). Under LL conditions, the gPCEM strain exhibits low levels of several metabolites and the FtsZ protein compared to the WT (Fig. 2). However, the transcriptional levels of the ftsZ gene remain comparable to those in the WT (Fig. 5C). This observation suggests that these TCA metabolites may directly or indirectly influence the FtsZ amount at posttranscriptional levels and Z-ring assembly through an unknown mechanism. Consistent with these results, posttranscriptional control of ftsZ expression^14,15,16, or FtsZ degradation^17,18,19, has been suggested previously, although the underlying mechanism remains unknown.

Fig. 7: A model for NtcA-regulated FtsZ levels and cell division in *Anabaena.*

Our studies also confirm a mechanism that accounts for the relationship between the TCA metabolite 2-OG levels and the transcriptional control of ftsZ (Fig. 7C). Combining the results of the EMSA assay, the P_ftsz-gfp reporter expression analyses, and the ntcA mutant analyses, we proposed that the global transcription factor NtcA regulates the transcription of the ftsZ gene in a 2-OG-dependent manner in Anabaena (Figs. 6 and 7C). The accumulation of 2-OG and the NtcA protein level in the gPCEM strain under highlight led to an increased transcript level of ftsZ, resulting in irregular cell division (Figs. 2, 7C, and S7). High levels of FtsZ will initiate FtsZ ring formation in an uncontrolled manner, causing ectopic cell division sites, and thus the formation of cells with varying sizes and shapes (Fig. 3). Such a phenotype is similar to that observed in E. coli when FtsZ levels increase with minicell formation⁹. In the WT Anabaena, we observed a significant reduction in cell length (indicating higher cell division rates) with increasing light intensity. This correlated with a rise in the intracellular 2-OG levels from low light (LL) to standard light (SL) or high light (HL), as well as a consistent increase in NtcA protein levels in cells shifted from LL to HL (Fig. S7). Based on these findings, we propose that varying light intensities lead to differential intracellular 2-OG levels, which in turn modulate NtcA-dependent cell division (Fig. 7A, B). However, the physiological advantages of this light-dependent morphological regulation remain unclear and warrant further investigation. Furthermore, the presence of multiple TSS, including two NtcA-recognized promoters (Fig. 6), suggests that the expression of ftsZ is under complex control with the possibility of integrating various environmental inputs. Notably, citrate- a key metabolite in the TCA pathway that accumulates alongside 2-OG in the gPCEM strain under HL- may also contribute to the regulation of ftsZ expression, though this possibility requires further validation.

Previous studies have shown that NtcA negatively regulates the expression of the mre genes (encoding MreB, MreC, and MreD)³. FtsZ controls cell width by regulating peptidoglycan synthesis at the division site, whereas MreB controls peptidoglycan synthesis at the lateral cell wall^8,10. Since both mre and ftsZ are under the regulation of NtcA, the observed cell morphological defects in the gPCEM strain could be the consequence of the misregulation of both systems. These findings suggest that NtcA, as a global transcription factor, coordinates cell division and elongation in response to C-to-N balance in cyanobacteria. 2-OG accumulates transiently and serves as a signal at the early phase of heterocyst differentiation. Whether 2-OG/NtcA coordinately regulates cell division during the developmental process remains an open question.

Methods

Strains and cultural conditions

The strains used in this study are listed in Supplementary Table 1. Anabaena WT and its derivatives were cultivated at 30 °C in BG11 medium with continuous illumination (standard light, 30 μmol/m² s), and no additional CO₂ was supplemented. When needed, 1 mM of theophylline (TP) and 0.3 μM Cu²⁺ were added to induce gene expression under indicated light conditions (LL, low light, 7 μmol/m² s; SL, standard light, 30 μmol/m² s; HL, high light, 70 μmol/m² s). For heterocyst induction, cells grown to the logarithmic phase in BG11 were transferred to BG11₀ (BG11 without combined nitrogen) medium, and heterocysts were observed under a light microscope. To make the data comparable, TP and Cu²⁺ were added to both the WT and the gPCEM strains before and during heterocyst induction. To compare the growth of the WT and the gPCEM strains, the absorbance at 750 nm was measured at the indicated time points after inoculation in BG11 liquid medium with TP and Cu²⁺ under the indicated light conditions. The ntcA mutant, as described in ref. ⁴², was first cultured at 30 °C in BG11 medium with 5 mM NH4⁺ under SL to log phase and then transferred to BG11₀ medium with 5 mM NH4⁺ under SL for further analysis.

Construction of plasmids and a cyanobacterial recombinant strain

All plasmids and primers used in this study are listed in Supplementary Tables 2 and 3, respectively. All Anabaena variants, including the Δalr2634-all2640 strain, the gPCEM strain, and the Δsuc-gPCEM strain, were generated by the genome editing technique based on CRISPR-Cpf1, and the plasmids were constructed as previously described⁴². The plasmids for gene overexpression in Anabaena were constructed based on the PCT vector, which was derived from the well-characterized high-copy-number plasmid pDU1, allowing control of gene expression by the artificial CT promoter^4,5,45. The plasmids for P_ftsZ1/2-gfp transcription fusion in Anabaena WT or in the gPCEM strain were constructed with a modified PCT vector in which the CT promoter elements were deleted so that the fusion is only under the control of the native ftsZ promoter^4,5. To generate recombinant strains or mutants, the corresponding plasmid was transferred into Anabaena by conjugation as described⁴. All mutants and overexpression strains were confirmed by PCR, and the corresponding PCR products were verified by Sanger sequencing.

Microscopy

Bright-field images were taken by an SDPTOP EX30 microscope, and fluorescence images were taken by an SDPTOP EX40 epifluorescence microscope. A filter (EX379-401, DM420LP, EM435-485) was used to image fluorescence (exposure time of 500 ms) GFP. Fluorescence images were taken with an oil immersion lens objective (100/1.28). All images were processed and analyzed using ImageJ. Statistical tests and plotting of data were performed with the GraphPad Prism software.

Western blots

Cells were collected by filtration and then broken with FastPrep-24 (6.0 m/s, 60 s) in a protein-loading buffer as described⁵. Cell extracts were heated at 100 °C for 5–10 min, followed by centrifugation at 12,000 rpm for 5 min. The supernatant was collected and separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to the nitrocellulose membranes. Blots were probed with the corresponding protein rabbit antiserum (1:1000) and HRP-conjugated secondary antibody (1:5000, goat anti-rabbit, Thermo Fisher, CAT#: A16172).

Quantitative real-time PCR

The WT, gPCEM, and Δsuc-gPCEM strains were cultivated in BG11 with 1 mM of theophylline (TP) and 0.3 μM Cu²⁺ under the indicated light conditions. Cultures after 4 days under LL, and 2 days under SL and HL were harvested by rapid filtration. The total RNA was extracted using a Plant Total RNA Isolation Kit (#RE-05011, Foregene, China), and the genomic DNA was removed by using the RNase-free DNase I (Promega, USA). The obtained total RNA was reverse transcribed using the HiScript®II Q RT SuperMix (#R223-0, Vazyme, China). Quantitative Real-Time PCR was performed by using ChamQ SYBR qPCR Master Mix (Vazyme). The housekeeping gene rnpB was used as an internal control. The data were analyzed by ABI 7500 SDS software. The transcriptional levels of the ftsZ gene were quantified with the comparative CT method (2-ΔΔCT method). Primers used for qPCR are listed in Supplementary Table 3.

Electrophoretic mobility shift assays (EMSA)

The His-tagged NtcA protein (NtcA-His6) for EMSA was prepared as previously described⁴⁰. The DNA fragments were amplified from the genome of Anabaena using the primers Palr3858F260m / Palr3858R1 m and Palr3858F526m / Palr3858R242m, respectively (Supplementary Table 3). The two DNA fragments were further labeled with fluorescent 6-carboxyfluorescein (FAM) tag at 5′ end. 2 pmol of the labeled probes were incubated with varying amounts of NtcA-His6 and 2-OG in a binding buffer (50 mM Tris-HCl pH7.5, 60 mM KCl, 5 mM MgCl₂, 0.25 M NaCl, 0.5 mM EDTA, 5 mM Beta-mercaptoethanol, 12% glycerol,40 μg/mL BSA) for 30 min at 25 °C. The samples were subjected to a 5% polyacrylamide gel electrophoresis and run in 1× PAGE buffer (50 mM Tris-HCl, pH 8.0, 2 mM EDTA, 380 mM glycine) at constant 20 mA for 3 h in an ice bath. Imaging and data analysis were performed using Amersham Typhon.

Extraction and quantification of cellular metabolites by LC-MS/MS

The WT Anabaena, the gPCEM stain, and the Δsuc-gPCEM strain, cultured as indicated above, were harvested by rapid filtration and resuspended in 500 μL of ice-cold extraction buffer (80% methanol). After incubating at −20 °C for 20 min, the cells were broken by ultrasonication at 90 W amplitude for 1 min. The samples were further incubated at −20 °C for 15 min and then centrifuged at 13,000 rpm for 10 min at 4 °C. The supernatant was transferred to a new 1.5 mL tube, evaporated, and dried by freeze-vacuum drying (Eppendorf Concentrator Plus).

To quantify the organic acids in the samples, the pellets were resuspended in 200 μL of the initial mobile phase (1:9 = solvent A: B). All samples were filtered with a 0.22 µm Ultra free-MC membrane before being subjected to the column. 1 μL of each sample was analyzed by LC-MS/MS on an Agilent 6550 iFunnel Q-TOF system, equipped with an Atlantis Premier BEH Z-HILIC column⁴⁴. The samples were separated at 40 °C with a flow rate of 0.3 mL/min. The chromatography process was used as the following: gradient of solvent A (20 mM ammonium formate pH = 9.0) to solvent B (methanol): t = 1.0 min, A-10%: B-90%; t = 5.0 min, A-40%: B-60%; t = 6.0 min, A-70%: B-30%, t = 8.0 min, A-70%: B-30%; t = 8.5 min, A-10%: B-90%; t = 13 min (end of gradient). MS data were scanned over a range of 50–500 m/z at a scan rate of 1 spectra/s. The parameters for mass spectrum were as follows: capillary voltage, 3.5 kV; nitrogen atomization (35 psig), drying (8 L/min, 200 °C), and sheath gas (12 L/min, 400 °C).

To quantify the CoA esters in the samples, the pellets were resuspended in 80 μL of 50%. All samples were filtered with a 0.22 µm Ultra free-MC membrane before being subjected to the column. 20 μL of each sample was analyzed by LC-MS/MS on an Agilent 6550 iFunnel Q-TOF system, equipped with a 50 × 2.1 mm C18 column (Kinetex 1.7 µm EVO C18 100 Å). The samples were separated at 25 °C with a flow rate of 0.2 mL/min. The chromatography process was used as follows: gradient of solvent A (50 mM ammonium formate pH = 8.0) to solvent B (methanol): t = 2.0 min, A-95%: B-5%; t = 10.0 min, A-80%: B-20%; t = 12.0 min, A-80%: B-20%; t = 13.0 min, A-20%: B-80%; t = 15.0 min, A-20%: B-80%; t = 15.1 min, A-95%: B-5%; t = 19.0 min (end of gradient). MS data were scanned over a range of 80-1000 m/z at a scan rate of 1 spectra/s. The parameters for mass spectrum were as follows: capillary voltage, 3.5 kV; nitrogen atomization (35 psig), drying (8 L/min, 320 °C), and sheath gas (12 L/min, 400 °C).

The corresponding standard solutions, prepared at 50 μg/mL concentration in the initial mobile phase, were analyzed in parallel. All standard stock solutions were prepared daily just before running samples in the LC-MS/MS system.

All LC/MS/MS peak data were collected on an Agilent Mass Hunter Data Acquisition software, and analyzed using Qualitative Analysis Navigator B.08.00 and Quantitative Analysis B.09.00. The signal intensity of each metabolite was integrated by the corresponding peak area and normalized to the WT strain under the same conditions. The differences (P < 0.05, indicating statistical significance) between the WT and the other strains were evaluated using the Student’s t-test.

Statistics and reproducibility

All statistical analyses and data visualization were performed using GraphPad Prism 8 (www.graphpad.com). Each data point in the plots represents an independent measurement derived from at least three biologically independent experiments. For comparisons between two independent groups, unpaired two-tailed Student′s t-tests were applied. Multiple comparisons were done using One-Way ANOVA followed by Sidak’s multiple comparisons test in Figs. 3C, 4B, 6E, and 7A; Two-Way ANOVA followed by Sidak’s multiple comparisons test in Fig. 6D; Two-Way ANOVA followed by Dunnett’s Multiple Comparison test in Figs. 2 and 5C; Two-Way ANOVA followed by Tukey’s multiple comparisons test in Fig. 7B. Significance thresholds (p-values) are provided in the figure legends. Complete statistical details—including means, standard deviations, exact p-values, and t-statistics—can be found in Supplementary Data 1.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

Data are available in the published article and its online supplemental material. All source data underlying the graphics and charts in the main and supplemental figures have been included as Supplementary Data 1. Unedited gel, blot, and micrograph images for the main and supplemental figures are available as Supplementary Fig. 8–16 in the Supplementary Information file. Plasmid pCpf1b-sp was deposited at Addgene with ID number #122188. Plasmid pCT was submitted to GenBank with ID number MK948095. For any further inquiries about the work, please contact the corresponding author.

References

Young, K. D. The selective value of bacterial shape. Microbiol. Mol. Biol. Rev. 70, 660–703 (2006).
Article PubMed PubMed Central Google Scholar
Martins, B. M. C., Tooke, A. K., Thomas, P. & Locke, J. C. W. Cell size control driven by the circadian clock and environment in cyanobacteria. Proc. Natl. Acad. Sci. USA 115, E11415–E11424 (2018).
Article CAS PubMed PubMed Central Google Scholar
Velazquez-Suarez, C., Luque, I. & Herrero, A. The inorganic nutrient regime and the mre genes regulate cell and filament size and morphology in the phototrophic multicellular bacterium Anabaena. mSphere https://doi.org/10.1128/mSphere.00747-20 (2020).
Sun, Q. X., Huang, M., Zhang, J. Y., Zeng, X. & Zhang, C. C. Control of cell size by c-di-GMP requires a two-component signaling system in the cyanobacterium anabaena sp. strain PCC 7120. Microbiol. Spectr. 11, e0422822 (2023).
Article PubMed Google Scholar
Zeng, X. et al. A c-di-GMP binding effector controls cell size in a cyanobacterium. Proc. Natl. Acad. Sci. USA 120, e2221874120 (2023).
Article CAS PubMed PubMed Central Google Scholar
Egan, A. J. & Vollmer, W. The physiology of bacterial cell division. Ann. N. Y Acad. Sci. 1277, 8–28 (2013).
Article CAS PubMed Google Scholar
Barrows, J. M. & Goley, E. D. FtsZ dynamics in bacterial division: what, how, and why?. Curr. Opin. Cell Biol. 68, 163–172 (2021).
Article CAS PubMed Google Scholar
Cameron, T. A. & Margolin, W. Insights into the assembly and regulation of the bacterial divisome. Nat. Rev. Microbiol 22, 33–45 (2024).
Article CAS PubMed Google Scholar
Ward, J. E. Jr & Lutkenhaus, J. Overproduction of FtsZ induces minicell formation in E. coli. Cell 42, 941–949 (1985).
Article CAS PubMed Google Scholar
den Blaauwen, T., Hamoen, L. W. & Levin, P. A. The divisome at 25: the road ahead. Curr. Opin. Microbiol 36, 85–94 (2017).
Article Google Scholar
Dai, K. & Lutkenhaus, J. The proper ratio of Ftsz to Ftsa is required for cell-division to occur in Escherichia coli. J. Bacteriol. 174, 6145–6151 (1992).
Article CAS PubMed PubMed Central Google Scholar
Ortiz, C., Natale, P., Cueto, L. & Vicente, M. The keepers of the ring: regulators of FtsZ assembly. FEMS Microbiol. Rev. 40, 57–67 (2016).
Article CAS PubMed Google Scholar
Aldea, M., Garrido, T., Pla, J. & Vicente, M. Division genes in Escherichia-Coli are expressed coordinately to cell septum requirements by gearbox promoters. EMBO J. 9, 3787–3794 (1990).
Article CAS PubMed PubMed Central Google Scholar
Anand, D. et al. Complete identity and expression of StfZ, the cis-antisense RNA to the mRNA of the cell division gene ftsZ, in Escherichia coli. Front. Microbiol. 13, 920117 (2022).
Article PubMed PubMed Central Google Scholar
Tetart, F. & Bouche, J. P. Regulation of the expression of the cell-cycle gene ftsZ by DicF antisense RNA - division does not require a fixed number of Ftsz molecules. Mol. Microbiol. 6, 615–620 (1992).
Article CAS PubMed Google Scholar
Barshishat, S. et al. OxyS small RNA induces cell cycle arrest to allow DNA damage repair. EMBO J. 37, 413–426 (2018).
Article CAS PubMed Google Scholar
Camberg, J. L., Hoskins, J. R. & Wickner, S. ClpXP protease degrades the cytoskeletal protein, FtsZ, and modulates FtsZ polymer dynamics. Proc. Natl. Acad. Sci. USA 106, 10614–10619 (2009).
Article CAS PubMed PubMed Central Google Scholar
Weart, R. B., Nakano, S., Lane, B. E., Zuber, P. & Levin, P. A. The ClpX chaperone modulates assembly of the tubulin-like protein FtsZ. Mol. Microbiol. 57, 238–249 (2005).
Article CAS PubMed PubMed Central Google Scholar
Williams, B., Bhat, N., Chien, P. & Shapiro, L. ClpXP and ClpAP proteolytic activity on divisome substrates is differentially regulated following the Caulobacter asymmetric cell division. Mol. Microbiol. 93, 853–866 (2014).
Article CAS PubMed PubMed Central Google Scholar
Kohga, H., Saito, Y., Kanamaru, M., Uchiyama, J. & Ohta, H. The lack of the cell division protein FtsZ induced generation of giant cells under acidic stress in cyanobacterium sp. PCC6803. Photosynth. Res. 150, 343–356 (2021).
Article CAS PubMed Google Scholar
Galinier, A., Foulquier, E. & Pompeo, F. Metabolic control of cell elongation and cell division in Bacillus subtilis. Front. Microbiol. 12 (2021).
Sperber, A. M. & Herman, J. K. Metabolism shapes the cell. J. Bacteriol. 199, e00039 (2017).
Weart, R. B. et al. A metabolic sensor governing cell size in bacteria. Cell 130, 335–347 (2007).
Article CAS PubMed PubMed Central Google Scholar
Hill, N. S., Buske, P. J., Shi, Y. & Levin, P. A. A moonlighting enzyme links Escherichia coli cell size with central metabolism. PLoS Genet. 9, e1003663 (2013).
Article CAS PubMed PubMed Central Google Scholar
Monahan, L. G., Hajduk, I. V., Blaber, S. P., Charles, I. G. & Harry, E. J. Coordinating bacterial cell division with nutrient availability: a role for glycolysis. mBio 5, e00935–00914 (2014).
Article PubMed PubMed Central Google Scholar
Yao, Z. Z., Davis, R. M., Kishony, R., Kahne, D. & Ruiz, N. Regulation of cell size in response to nutrient availability by fatty acid biosynthesis in. Proc. Natl. Acad. Sci. USA 109, E2561–E2568 (2012).
Article CAS PubMed PubMed Central Google Scholar
Beaufay, F., De Bolle, X. & Hallez, R. Metabolic control of cell division in alpha-proteobacteria by a NAD-dependent glutamate dehydrogenase. Commun. Integr. Biol. 9, e1125052 (2016).
Article PubMed PubMed Central Google Scholar
Radhakrishnan, S. K., Pritchard, S. & Viollier, P. H. Coupling prokaryotic cell fate and division control with a bifunctional and oscillating oxidoreductase homolog. Dev. Cell 18, 90–101 (2010).
Article CAS PubMed Google Scholar
Westfall, C. S. & Levin, P. A. Comprehensive analysis of central carbon metabolism illuminates connections between nutrient availability, growth rate, and cell morphology in Escherichia coli. PLoS Genet. 14, e1007205 (2018).
Article PubMed PubMed Central Google Scholar
Stanier, R. Y. & Cohen-Bazire, G. Phototrophic prokaryotes: the cyanobacteria. Annu. Rev. Microbiol. 31, 225–274 (1977).
Article CAS PubMed Google Scholar
Lucius, S. & Hagemann, M. The primary carbon metabolism in cyanobacteria and its regulation. Front. Plant Sci. 15, 1417680 (2024).
Article PubMed PubMed Central Google Scholar
Zhang, S. Y. & Bryant, D. A. The tricarboxylic acid cycle in cyanobacteria. Science 334, 1551–1553 (2011).
Article CAS PubMed Google Scholar
Xiong, W., Brune, D. & Vermaas, W. F. J. The γ-aminobutyric acid shunt contributes to closing the tricarboxylic acid cycle in sp PCC 6803. Mol. Microbiol. 93, 786–796 (2014).
Article CAS PubMed Google Scholar
Zhang, S. Y., Qian, X., Chang, S. N., Dismukes, G. C. & Bryant, D. A. Natural and synthetic variants of the tricarboxylic acid cycle in cyanobacteria: introduction of the GABA shunt into Synechococcus sp. PCC 7002. Front. Microbiol. 7, 1972 (2016).
Article PubMed PubMed Central Google Scholar
Hendry, J. I., Prasannan, C. B., Joshi, A., Dasgupta, S. & Wangikar, P. P. Metabolic model of Synechococcus sp. PCC 7002: prediction of flux distribution and network modification for enhanced biofuel production. Bioresour. Technol. 213, 190–197 (2016).
Article CAS PubMed Google Scholar
Goclaw-Binder, H. et al. Nutrient-associated elongation and asymmetric division of the cyanobacterium PCC 7942. Environ. Microbiol. 14, 680–690 (2012).
Article CAS PubMed Google Scholar
Xing, W. Y., Liu, J., Zhang, J. Y., Zeng, X. L. & Zhang, C. C. A proteolytic pathway coordinates cell division and heterocyst differentiation in the cyanobacterium sp. PCC 7120. Proc. Natl. Acad. Sci. USA 119, e2207963119 (2022).
Article CAS PubMed PubMed Central Google Scholar
Zhang, C. C., Zhou, C. Z., Burnap, R. L. & Peng, L. Carbon/nitrogen metabolic balance: lessons from cyanobacteria. Trends Plant Sci. 23, 1116–1130 (2018).
Article CAS PubMed Google Scholar
Laurent, S. et al. Nonmetabolizable analogue of 2-oxoglutarate elicits heterocyst differentiation under repressive conditions in Anabaena sp. PCC 7120. Proc. Natl. Acad. Sci. USA 102, 9907–9912 (2005).
Article CAS PubMed PubMed Central Google Scholar
Zhao, M. X. et al. Structural basis for the allosteric control of the global transcription factor NtcA by the nitrogen starvation signal 2-oxoglutarate. Proc. Natl. Acad. Sci. USA 107, 12487–12492 (2010).
Article CAS PubMed PubMed Central Google Scholar
Picossi, S., Flores, E. & Herrero, A. ChIP analysis unravels an exceptionally wide distribution of DNA binding sites for the NtcA transcription factor in a heterocyst-forming cyanobacterium. BMC Genom. 15, 22 (2014).
Article Google Scholar
Niu, T. C. et al. Expanding the potential of CRISPR-Cpf1-based genome editing technology in the cyanobacterium anabaena PCC 7120. ACS Synth. Biol. 8, 170–180 (2019).
Article CAS PubMed Google Scholar
Schwander, T., von Borzyskowski, L. S., Burgener, S., Cortina, N. S. & Erb, T. J. A synthetic pathway for the fixation of carbon dioxide in vitro. Science 354, 900–904 (2016).
Article CAS PubMed PubMed Central Google Scholar
Wang, Z.-Q. et al. Investigation on cyanobacterial production of the proposed neurotoxin β-N-methylamino-L-alanine (BMAA). Water Biol. Secur. 2, 100208 (2023).
Article Google Scholar
Yang, Y. et al. Phenotypic variation caused by variation in the relative copy number of pDU1-based plasmids expressing the GAF domain of Pkn41 or Pkn42 in sp PCC 7120. Res. Microbiol. 164, 127–135 (2013).
Article CAS PubMed Google Scholar
Mitschke, J., Vioque, A., Haas, F., Hess, W. R. & Muro-Pastor, A. M. Dynamics of transcriptional start site selection during nitrogen stress-induced cell differentiation in sp PCC7120. Proc. Natl. Acad. Sci. USA 108, 20130–20135 (2011).
Article CAS PubMed PubMed Central Google Scholar
Xing, W. Y. et al. HetF protein is a new divisome component in a filamentous and developmental cyanobacterium. Mbio 12, e01382–21 (2021).
Article CAS PubMed PubMed Central Google Scholar
Mi, H. L., Klughammer, C. & Schreiber, U. Light-induced dynamic changes of NADPH fluorescence in PCC 6803 and its-defective mutant M55. Plant Cell Physiol. 41, 1129–1135 (2000).
Article CAS PubMed Google Scholar
Kauny, J. & Sétif, P. NADPH fluorescence in the cyanobacterium sp PCC 6803: A versatile probe for measurements of rates, yields and pools. Biochim. Biophys. Acta 1837, 792–801 (2014).
Article CAS PubMed Google Scholar

Download references

Acknowledgements

This research was supported by the CAS Project for Young Scientists in Basic Research (Grant No: #YSBR-015), the Strategic Priority Research Program of the Chinese Academy of Sciences (Grant No. XDB0480000), the Key Program of the National Natural Science Foundation of China (Grant No. 32330003) and the Youth Innovation Promotion Association CAS (Grant No. 2022342). We thank Amel Latifi (Aix-Marseille University) for providing the ntcA mutant strain.

Author information

Authors and Affiliations

Key Laboratory of Algal Biology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei, People’s Republic of China
Wen-Shuo Ran, Xiaoli Zeng & Cheng-Cai Zhang
University of Chinese Academy of Sciences, Beijing, People’s Republic of China
Wen-Shuo Ran
State Key Laboratory of Lake and Watershed Science for Water Security, Chinese Academy of Sciences, Nanjing, People’s Republic of China
Xiaoli Zeng & Cheng-Cai Zhang
Hubei Hongshan Laboratory, Wuhan, People’s Republic of China
Cheng-Cai Zhang

Authors

Wen-Shuo Ran
View author publications
Search author on:PubMed Google Scholar
Xiaoli Zeng
View author publications
Search author on:PubMed Google Scholar
Cheng-Cai Zhang
View author publications
Search author on:PubMed Google Scholar

Contributions

Conceptualization: CZ.Z. and X.Z.; Methodology: WS.R.; Investigation: WS.R.; Formal Analysis: WS.R. and X.Z.; Visualization: WS.R. and X.Z.; Writing – Original Draft: X.Z., Writing – Review & Editing: X.Z. and CC.Z.; Funding Acquisition: CC.Z. and X.Z.

Corresponding authors

Correspondence to Xiaoli Zeng or Cheng-Cai Zhang.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Communications Biology thanks Conrad Mullineaux and the other anonymous reviewers for their contribution to the peer review of this work. Primary Handling Editors: Xiaoling Xu and Johannes Stortz. A peer review file is available.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Description of Additional Supplementary Files

Supplementary Data 1

Reporting Summary

Transparent Peer Review file

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

Reprints and permissions

About this article

Cite this article

Ran, WS., Zeng, X. & Zhang, CC. Carbon metabolism shapes FtsZ levels and cell division in the cyanobacterium Anabaena PCC 7120. Commun Biol 8, 1440 (2025). https://doi.org/10.1038/s42003-025-08849-4

Download citation

Received: 15 June 2025
Accepted: 04 September 2025
Published: 08 October 2025
DOI: https://doi.org/10.1038/s42003-025-08849-4