Coordinating carbon and nitrogen metabolic signaling through the cyanobacterial global repressor NdhR

Yong-Liang Jiang; Xue-Ping Wang; Hui Sun; Shu-Jing Han; Wei-Fang Li; Ning Cui; Gui-Ming Lin; Ju-Yuan Zhang; Wang Cheng; Dong-Dong Cao; Zhi-Yong Zhang; Cheng-Cai Zhang; Yuxing Chen; Cong-Zhao Zhou

doi:10.1073/pnas.1716062115

Edited by Susan S. Golden, University of California, San Diego, La Jolla, CA, and approved December 8, 2017 (received for review September 14, 2017)

Significance

The homeostasis of carbon and nitrogen metabolism needs to be tightly regulated for cell acclimation to fluctuating environments. The related metabolic pathways are ultimately coordinated by the master transcription factors that sense the intracellular metabolites. We report here biochemical, structural, and functional studies of the fine regulation of the transcriptional repressor NAD(P)H dehydrogenase regulator (NdhR). Two key metabolites, 2-OG from the Krebs cycle and 2-PG from photorespiration, have opposite effects on the regulatory activity of NdhR. We propose that 2-OG and 2-PG function together as indicators of intracellular carbon/nitrogen status and that NdhR senses these two effectors in a mutually exclusive manner. Our findings together with previous studies provide a model for the fine coordination of carbon and nitrogen metabolic signaling.

Abstract

The coordination of carbon and nitrogen metabolism is essential for bacteria to adapt to nutritional variations in the environment, but the underlying mechanism remains poorly understood. In autotrophic cyanobacteria, high CO₂ levels favor the carboxylase activity of ribulose 1,5 bisphosphate carboxylase/oxygenase (RuBisCO) to produce 3-phosphoglycerate, whereas low CO₂ levels promote the oxygenase activity of RuBisCO, leading to 2-phosphoglycolate (2-PG) production. Thus, the 2-PG level is reversely correlated with that of 2-oxoglutarate (2-OG), which accumulates under a high carbon/nitrogen ratio and acts as a nitrogen-starvation signal. The LysR-type transcriptional repressor NAD(P)H dehydrogenase regulator (NdhR) controls the expression of genes related to carbon metabolism. Based on genetic and biochemical studies, we report here that 2-PG is an inducer of NdhR, while 2-OG is a corepressor, as found previously. Furthermore, structural analyses indicate that binding of 2-OG at the interface between the two regulatory domains (RD) allows the NdhR tetramer to adopt a repressor conformation, whereas 2-PG binding to an intradomain cleft of each RD triggers drastic conformational changes leading to the dissociation of NdhR from its target DNA. We further confirmed the effect of 2-PG or 2-OG levels on the transcription of the NdhR regulon. Together with previous findings, we propose that NdhR can sense 2-OG from the Krebs cycle and 2-PG from photorespiration, two key metabolites that function together as indicators of intracellular carbon/nitrogen status, thus representing a fine sensor for the coordination of carbon and nitrogen metabolism in cyanobacteria.

Carbon and nitrogen are the two most abundant nutrient elements for living organisms. Therefore, the homeostasis of carbon and nitrogen metabolisms, which are fundamental to all forms of life to allow acclimation to fluctuating conditions, needs to be tightly controlled via diverse mechanisms. In autotrophic microorganisms such as cyanobacteria, the control of carbon metabolism is particularly important because of their limited choice of carbon sources to keep pace with nitrogen assimilation. Therefore, cyanobacteria constitute an ideal model for investigating the coordination of carbon and nitrogen metabolisms. Cyanobacteria are able to perform oxygen-evolving photosynthesis and use different forms of nitrogen sources such as nitrate, ammonium, and/or N₂ for diazotrophic strains (1). These nitrogen sources are converted into ammonium, which is then incorporated into the carbon skeleton 2-oxoglutarate (2-OG) for biosynthesis of various biomolecules (1). Thus, the accumulation of 2-OG in vivo constitutes the nitrogen-starvation signal and elicits a series of cellular responses, including the formation of nitrogen-fixing heterocysts in some filamentous cyanobacterial strains (2). The trimeric protein PII and the transcription factor NtcA constitute two well-established receptors of 2-OG in the nitrogen-starvation–signaling pathways in cyanobacteria (3). Each monomer of PII possesses a flexible T-loop protruding outwards to interact with the partner proteins (4). The binding of 2-OG and/or other effectors enables the T-loop to adopt various conformations to interact with diverse partners (5). NtcA is a transcription factor belonging to the cAMP receptor protein and fumarate/nitrate reductase regulator (Crp-Fnr) family, and the binding of 2-OG leads to a conformational change that enhances the DNA-binding ability of NtcA (6, 7).

As autotrophic organisms, cyanobacteria use inorganic carbon (Ci), including CO₂ and bicarbonate, as the major carbon source for growth. They are capable of acclimating and growing under a wide range of ambient CO₂ concentrations, thanks to the CO₂-concentrating mechanism (CCM). The CCM is the primary carbon-acquisition pathway for actively accumulating Ci into CO₂ fixation by ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) within microcompartments termed “carboxysomes” (8). To guarantee the high efficiency of the Calvin–Benson cycle, the cyanobacterial CCM could functionally compensate for the poor affinity and selectivity of RuBisCO toward CO₂ (9, 10). The CCM requires a number of genes involved in bicarbonate transport, CO₂ uptake, and other related energetic or regulatory processes and thus its activities are highly coordinated (8, 11). In Synechocystis sp. PCC 6803 (hereafter “Synechocystis”), the CCM is mainly under the control of two LysR-type transcriptional regulators (LTTRs), namely NAD(P)H dehydrogenase regulator (NdhR)/Sll1594 (also known as “CcmR”) (12 ⇓⇓–15) and CmpR/Sll0030 (16, 17). CmpR functions as a transcriptional activator to up-regulate the expression of the Ci-responsive cmp operon that encodes the HCO₃⁻ transporter CmpABCD under Ci-limiting conditions (16). In contrast, despite sharing a sequence identity of 54% with CmpR, NdhR acts as a repressor to globally control the expression of several CCM-related genes, including the gene clusters containing ndhF3/ndhD3/cupA/sll1735 (the ndh-I3 operon), which encode the high-affinity CO₂-uptake system proteins, the sbtA/B genes encoding the Na⁺/HCO₃⁻ symporter, the mnh operon, and bicA (13). Moreover, similar to most LTTR proteins, NdhR also negatively autoregulates the expression of its own gene ndhR (12). The promoter region recognized by NdhR usually consists of two tandem repetitive motifs with a consensus sequence of ATAG-N₈-CTAT that overlap the transcription start site (18). A previous report found that in Synechocystis, NADP⁺ and the metabolic intermediate 2-OG enhance the binding of NdhR to its own promoter as well as to the promoter region of the ndh-I3 operon and thereby appear to act as corepressors (19). In addition, binding of CmpR to the cmp operon could be significantly elevated in the presence of ribulose-1,5-bisphosphate (RuBP) or 2-phosphoglycolate (2-PG) in Synechococcus (17). However, no direct evidence is available to support the effector function of these metabolites, and the binding pattern and regulatory mechanism remain unknown.

RuBisCO is a bifunctional enzyme that can use either CO₂ (carboxylase) or O₂ (oxygenase) as the substrate (20). Under high CO₂ (HC) levels, it acts as a carboxylase to convert CO₂ into 3-phosphoglycerate to feed the Calvin–Benson cycle, thus providing carbon sources for cell growth. When the CO₂ levels become limiting, the oxygenase activity is favored, which leads to the synthesis of both 3-phosphoglycerate and 2-PG, which is then metabolized in the photorespiratory cycle (21). During the primary response, a carbon limitation in the cells can also be considered as a nitrogen oversupply which will drain the carbon skeleton 2-OG for nitrogen assimilation through the glutamine synthetase–glutamate synthase pathway (3), thus most likely leading to a lower level of 2-OG. Therefore, the level of 2-PG is somewhat inversely related to that of 2-OG, and vice versa. These findings raise a number of questions. How do cyanobacteria sense the intracellular carbon status? Does 2-OG alone suffice to reflect the nitrogen metabolic status as well as the N/C balance? Are other receptors of the 2-OG signal, in addition to NtcA and PII, involved in the control of nitrogen or carbon metabolism? To answer these questions, we characterized NdhR, a transcriptional repressor involved in the control of carbon metabolism, including that of the CCM in cyanobacteria. We solved the full-length structure of NdhR from Synechocystis in complex with 2-OG as well as the complex structure of the NdhR regulatory domain with 2-PG. Further biochemical and genetic experiments suggested that the metabolites 2-PG and 2-OG act as an inducer and a corepressor, respectively, to cooperatively regulate CCM via NdhR. Our findings provide structural insights into the regulatory mechanism of LTTR repressors and reveal a connection between the carbon and nitrogen metabolisms.

Results

2-OG and 2-PG Are the Effectors of NdhR.

The transcriptional activity of an LTTR is usually modulated upon binding of small molecule(s) (22). NdhR is a repressor that regulates genes or gene clusters involved in the CCM (13), in which several metabolites, such as 2-PG, 2-OG, RuBP, and NADP⁺, have been suggested to act as internal signals for the deprivation of inorganic carbon (8, 17, 23). Of note, RuBP and 2-PG were found to enhance the binding of CmpR to the promoter region of the cmp operon (17) but did not affect the DNA-binding activity of NdhR (19). By contrast, 2-OG and NADP⁺ were reported to enhance the binding of NdhR to its own gene promoter as well as to that of ndh-I3 and thereby seemed to be corepressors (19). To identify the effectors of NdhR, we applied surface plasmon resonance (SPR) assays by immobilizing the NdhR proteins on the chip to detect the binding affinity of NdhR toward the potential metabolites 2-PG, 2-OG, RuBP, and NADP⁺. The results showed that 2-OG and 2-PG, but not RuBP and NADP⁺, could bind to NdhR (Fig. 1A). Further experiments enabled us to calculate the dissociation constants (K_ds) at 0.55 mM for 2-OG (Fig. 1B) and at 0.43 mM for 2-PG (Fig. 1C), which are comparable to the intracellular concentrations of these metabolites under physiological conditions (24). Notably, the binding affinity of NdhR toward 2-OG is comparable to that of previously reported interactions between repressors and corepressors, such as Escherichia coli MetJ and Trp (25, 26). These results strongly indicated that 2-OG and 2-PG are effectors of NdhR.

Fig. 1.

Binding affinity assays of NdhR toward small molecules or target DNA. (A) The SPR difference curves of NdhR in the presence of different molecules at a final concentration of 1 mM. The NdhR proteins were immobilized on the surface of the CM5 chip to test the binding of different ligands. RU, response units. One RU represents the binding of 1 pg of protein per square mm. (B and C) The binding of NdhR with 2-OG (B) and 2-PG (C) ranging from 0.125–2 mM. K_d values are shown. (D–F)The DNA-binding affinity of NdhR alone (D) and NdhR in the presence of 2-OG (E) or 2-PG (F). The 140-bp DNA was derived from the promoter region of the ndhR gene and then was immobilized on the chip. NdhR was diluted at a series of concentrations from 6–1,405 nM. 2-OG or 2-PG was added at a final concentration of 5 mM.

To further investigate if 2-OG and 2-PG indeed alter the DNA-binding affinity of NdhR, we applied the SPR assays with DNA immobilized on the chip to determine the binding affinity of NdhR toward the 140-bp ndhR promoter sequence (18). NdhR displays a strong binding affinity toward the target DNA, at a K_d of 45 nM (Fig. 1D). The DNA-binding affinity of NdhR is increased to a K_d of 27 nM upon the addition of 2-OG (Fig. 1E), which is in accordance with the previous results (19). By contrast, the addition of 2-PG sharply reduced the DNA-binding affinity of NdhR, resulting in a K_d of 5,210 nM (Fig. 1F). These results suggested that 2-PG significantly alleviates the interaction of NdhR with the ndhR promoter region, whereas 2-OG has the opposite effect. Thus, 2-PG acts as an inducer of NdhR, and the effect of 2-OG is consistent with its corepressor function, as previous suggested (19).

Crystal Structure of NdhR.

To provide evidence of the effector function of 2-OG and 2-PG and to gain insight into the regulatory mechanism of NdhR, we solved the crystal structure of the full-length NdhR at 2.60 Å. Each asymmetric unit contains four subunits assembling into a tetramer (Fig. 2A). Similar to typical LTTRs, the NdhR tetramer consists of two compact subunits and two extended subunits (Fig. 2B). Each subunit is composed of a DNA-binding domain (DBD) and a regulatory domain (RD) connected by a short hinge region (Fig. 2C). The DBD consists of four α-helices (α1–α4) flanked by two β-strands (β1–β2) (Fig. 2B), with a winged helix-turn-helix (wHTH) DNA-binding region (27, 28). The RD could be further divided into two subdomains, RD1 and RD2, both of which adopt an α/β Rossman-like fold and are connected to each other with two crossover β-strands, β6 and β11 (Fig. 2B). RD1 and RD2 pack against each other to form a cleft 10 Å in depth and 20 Å in width.

Fig. 2.

The tetrameric structure of NdhR. (A) The tetrameric structure of NdhR, shown in two different views. Each tetramer is composed of two compact subunits (in cyan) and two extended subunits (in orange). (B) Cartoon presentation of the compact and extended subunits of NdhR, with the secondary structural elements labeled sequentially. (C and D) DBD-mediated (C) and RD-mediated (D) interfaces of NdhR. (Insets) The detailed interaction networks. The interacting residues are shown as sticks, and the polar interactions are indicated by dashed lines.

The two compact subunits are quite similar to each other, with an rmsd of 0.44 Å over 266 Cα atoms, whereas the two similar extended subunits share an rmsd of 0.31 Å over 289 Cα atoms. In the compact subunit, the DBD and RD are close to each other, with an interdomain angle of ∼37° (Fig. 2B). Of note, Asp86 from the DBD forms two salt bridges with Arg94 from RD1. However, in the extended subunit, the DBD and RD stay away from each other, with an interdomain angle of 134° (Fig. 2B). Nevertheless, either the individual DBDs or RDs are almost the same in the compact and extended subunits, which differ from each other due to rigid-body rotation.

In the tetrameric assembly, four DBDs of NdhR project outward and form a V-shaped surface along the DNA-binding helices of the tetramer, with an angle of ∼142° (Fig. 2A). This arrangement of DBDs indicated that the NdhR tetramer most likely binds to and bends the target DNA. The tetrameric assembly of NdhR can be regarded as a dimer of dimers in which each dimer comprises one compact and one extended subunit. The helices α4 and α4′ from two DBDs form an antiparallel coiled-coil that contributes to the majority of dimeric interface (Fig. 2C). In detail, the ∼1,700-Å² dimeric interface is stabilized mainly by hydrophobic interactions in addition to several hydrogen bonds (Fig. 2C). This type of DBD-mediated dimer is highly conserved among all LTTRs of known structure (28 ⇓⇓⇓⇓⇓⇓–35). The dimer could be further assembled into a tetramer via the interactions between two RDs (Fig. 2D). The two RDs contact each other in an antiparallel, side-by-side alignment, forming an interface of 1,900 Å². α8 and β8 from RD2 of one subunit interact with β4′ and α5′ from RD1 of the symmetric subunit, and vice versa (Fig. 2D).

Typical LTTRs usually have a conserved structure with an N-terminal DBD and a C-terminal RD (36). To date, the crystal structures of LTTRs revealed a tetramer composed of two compact and two extended subunits (28 ⇓⇓⇓⇓⇓⇓–35), except for CrgA, which forms an octamer comprising only compact subunits (30). Despite sharing a tetramer structure, NdhR differs from other LTTRs in the relative positions and rotations of the DBDs against the RDs, in addition to the lengths of the linker helix. Another difference can be found at the variations of the RD domains in NdhR, especially the effector-binding cleft, suggesting that NdhR senses molecules that are different from those sensed by other LTTRs of known structure.

2-OG Binds at the Interface Between Two RDs and Acts as a Corepressor of NdhR.

In the full-length structure of NdhR, two pieces of electron density were found at the interfaces between the RD domains. However, none of the molecules from the purification and crystallization buffers could fit the density. Instead, the density fits well with a molecule of 2-OG (Fig. 2A), an effector of NdhR according to our biochemical data and a previous report (Fig. 1B) (19). 2-OG is most likely incorporated during the heterogeneous expression of NdhR in E. coli, which possess an intracellular 2-OG concentration in the millimolar range (37). 2-OG binds to the residues Ser224, Asn225, Tyr105, and Lys104 from one RD and to the counterpart residues from the symmetric RD (Fig. 3A). Under conditions of carbon oversupply or nitrogen starvation, the intracellular concentration of 2-OG increases in cyanobacteria (38, 39); such conditions should favor the binding of 2-OG to the NdhR tetramer. The 2-OG–complexed NdhR adopts a conformation complementary to the DNA major groove, in agreement with the distance of 29 Å between the two recognition helices binding to the DNA palindrome (Fig. 3A) and therefore leading to repression of the NdhR regulon.

Fig. 3.

2-OG is a corepressor of NdhR. (A) The binding site of 2-OG. One 2-OG molecule binds at the interface between two RD domains. (Inset) The 2-OG molecule and the interacting residues are shown as sticks, and the polar interactions are marked by dashed lines. The 2Fo- Fc electron density map of 2-OG is shown as a blue mesh, countered at 1.5 σ. (B) The transcript abundance of NdhR-regulated genes in the wild-type strain (white bars) or ndhR-deletion strain (∆ndhR, black bars). The cells were grown at ambient air conditions. (C) The transcript abundance of the NdhR-regulated genes without (control, white bars) or with the addition of dimethyl-ketoglutarate (dmKG, black bars). Data are presented as the means ± SD from three independent assays. A two-tailed Student’s t test was used for the comparison of statistical significance; ***P < 0.001.

To confirm the repressor function of NdhR and the effect of 2-OG, we first constructed an ndhR-deletion strain. We found that the expression levels of NdhR-regulated genes, such as ndhF3, sbtA, and bicA, are significantly up-regulated (Fig. 3B), in agreement with a previous report (13). Afterwards, to check the in vivo effect of 2-OG on the activity of NdhR, we increased the intracellular 2-OG level by adding to the medium 20 mM dimethyl-ketoglutarate (dmKG), a membrane-permeable ester that is cleaved by intracellular esterase to produce 2-OG (40). Upon the increase of the intracellular 2-OG concentration, the expression levels of ndhR, ndhF3, sbtA, and bicA were significantly down-regulated compared with the control (Fig. 3C). Together, these findings demonstrate that 2-OG is an intrinsic corepressor that keeps NdhR bound to the target DNA to repress gene expression.

2-PG Binds at the Cleft of RDs of NdhR and Induces Gene Expression.

The in vitro binding experiments indicated that 2-PG dramatically weakens the DNA-binding capability of NdhR toward its DNA targets (Fig. 1F). To elucidate the mechanism of 2-PG interaction with NdhR, we initially attempted to solve the full-length structure of NdhR in complex with 2-PG, but we failed to do so after extensive trials. Alternatively, we solved the structure of the RD domain of NdhR in complex with 2-PG at 2.20 Å. In each RD subunit, 2-PG binds to the cleft between RD1 and RD2 (Fig. 4A). The oxygen atoms of the phosphate group of 2-PG form hydrogen bonds with the side chains of Arg195, His130, Thr201, and Thr101. The carboxyl group of 2-PG is hydrogen-bonded with Thr102, Asn164, and Arg267 (Fig. 4A). Sequence alignment revealed that all these 2-PG–binding residues are highly conserved among NdhR and homologs. Compared with the structure of NdhR complexed with 2-OG, binding of 2-PG triggers a significant induced fit of the intradomain cleft between RD1 and RD2. In detail, upon 2-PG binding, the residues Asn164 from β6 and Arg267 from β11 at the bottom of the binding cleft form hydrogen bonds with 2-PG, thus disrupting the previous β6 and β11 that cross over RD1 and RD2 (Fig. 4B). In consequence, RD1 and RD2 undergo a rigid-body rotation toward 2-PG, resulting in a rather compact conformation of the RD. With the two RD1 subdomains superimposed, RD2 rotates about ∼25° toward the cleft upon 2-PG binding (Fig. 4B). The conformational changes at the RD domains upon 2-PG binding further trigger the conformational changes in the overall structure of the NdhR tetramer, resulting in the altered DNA-binding capability of NdhR, as confirmed by the SPR assays. Notably, binding of 2-OG will stabilize the interface between the two RDs, thus excluding the binding of 2-PG to the intradomain cleft between RD1 and RD2, whereas the induced fit upon binding to 2-PG will disrupt the 2-OG–binding site (Fig. 4B). Thus, the binding of 2-PG or 2-OG to NdhR should be mutually exclusive.

Fig. 4.

2-PG is an inducer of NdhR. (A) The binding site of 2-PG. The 2-PG molecule and the interacting residues are shown as sticks, and the polar interactions are marked by dashed lines. The 2Fo-Fc electron density map of 2-PG is shown as a blue mesh, countered at 1.5 σ. (B) Comparison of the RD domains in the presence of 2-PG (yellow) or 2-OG (cyan) with RD1 superimposed. (C) Comparison of the transcript abundance of slr0458 in the wild-type (white bar) and POE (black bar) strains. For all in vivo experiments, total mRNA was extracted 30 min after the shift from the HC to the LC condition, and qPCR assays were used to assess the relative level of relevant genes. The cells were cultured in ambient air for the LC condition and in ambient air supplemented with 5% (vol/vol) CO₂ for the HC condition. (D) Transcript abundance of NdhR-regulated genes in the wild-type (white bars) and POE (black bars) strains. (E) Comparison of the transcript abundance of slr0458 in the ndhR-deletion strain without Slr0458 (∆ndhR, white bar) or with overexpressed Slr0458 (∆ndhR-POE, black bar). (F) Transcript abundance of NdhR-regulated genes in the ndhR-deletion strain without (∆ndhR, white bars) or with (∆ndhR-POE, black bars) overexpressed Slr0458. Mean values represent at least three independent experiments. A two-tailed Student’s t test was used for the comparison of statistical significance; ***P < 0.001.

To further examine the possible roles of 2-PG in NdhR’s transcriptional regulation of its target genes, we constructed a recombinant strain overexpressing slr0458, which encodes a phosphoglycolate phosphatase (PGPase) [hereafter, the “PGPase-overexpression” (POE) strain]. This PGPase was reported to be responsible for the catabolism of intracellular 2-PG, which usually accumulates in the cell at a high level after 30 min after the shift from an HC to a limiting CO₂ level (LC) (23). qPCR analyses showed that the transcription level of the slr0458 gene increased by ∼25-fold in the POE strain compared with the wild type (Fig. 4C). In the POE strain, due to the high level of PGPase that degrades the intracellular 2-PG, the transcription of NdhR target genes ndhR, ndhF3, sbtA, and bicA is significantly down-regulated, to about 20–60% of that in the wild-type strain (Fig. 4D), in agreement with the previous report on ndhF3 and sbtA genes (23). To validate that the effect of 2-PG is mediated by NdhR in vivo, we also overexpressed slr0458 in the ndhR-deletion strain. Although the transcription of slr0458 is also up-regulated by ∼35-fold (Fig. 4E), the expression levels of NdhR target genes are comparable to those without the overexpression of slr0458 (Fig. 4F). Taken together, these results further confirmed that 2-PG is an inducer that directly interacts with NdhR in vivo.

Discussion

LTTRs are the most ubiquitous transcriptional factors in prokaryotes, regulating various biological processes such as the fixation of CO₂ and N₂, cell division, oxidative response, and the synthesis or degradation of amino acids (41). They can act as either activators or repressors in transcriptional regulation. So far, the structures of only a few activator-type LTTRs have been reported, and a sliding dimer model was proposed to explain how effectors activate LTTRs (28 ⇓⇓⇓⇓⇓⇓–35). The recent structures of the ligand-binding domain of CysB from Salmonella typhimurium reveal two distinct binding sites that are allosterically coupled by two inducers (42). The overall structures of the RDs of NdhR and CysB are similar, with an rmsd of 2.90 Å over 167 Cα atoms, although CysB has two additional C-terminal helices and an unstructured tail. Moreover, the two proteins display distinct features in their ligand-binding mode. To the best of our knowledge, NdhR has only one inducer, namely 2-PG, binding to the intradomain cleft of RD, which corresponds to the primary binding site of CysB. In addition, the corepressor 2-OG of NdhR binds to the interface between the two RDs, which is different from the 2-PG–binding site. The present structural and biochemical analyses enabled us to propose a model for the regulation mechanism of the LTTR repressor NdhR, which is distinct from the allosteric coupling mechanism of CysB activation or that of other LTTR activators.

In the present study, we found that 2-OG and 2-PG, not RuBP or NADP⁺, are effector molecules of NdhR. These two effectors display similar affinity toward NdhR but bind to different sites of the protein in a mutually exclusive manner. Structural analyses and biochemical characterizations combined with genetic studies indicate that the interactions between the two effectors and NdhR provoke different conformational changes, hence leading to opposite effects on the transcriptional activity of NdhR. As the levels of 2-PG and 2-OG are reversely correlated, their relative changes synergistically coordinate the NdhR-regulated transcription (Fig. 5A). Our present data together with previous findings (3, 8, 12, 13, 23) enable us to propose a detailed model of the molecular mechanism allowing cyanobacteria to adapt to relative changes in the status of carbon and nitrogen metabolism mediated by NdhR (Fig. 5A). Noticeably, the coordination of carbon and nitrogen balance is achieved through a network of regulators, such as PII, NtcA (7, 43), and the phosphotransferase system (44). Moreover, the cyanobacterial CCM is regulated by a transcriptional network of NdhR, CmpR, and an AbrB family member, Sll0822 (45) for acclimation to fluctuating environments (8). The model presented here is simplified for clarity and emphasizes only the role of NdhR in coordinating carbon and nitrogen balance. Nevertheless, our model provides structural insights into the regulation of NdhR allowing the coordinated response of part of the high-affinity Ci-uptake system and also expands previous findings on the regulation of CCM by NdhR and CmpR (19).

Fig. 5.

NdhR-coordinated balance of carbon and nitrogen metabolisms. (A) A putative transcriptional regulation mechanism of NdhR. Upon the binding of 2-OG (red rectangle) at the RD interface, the NdhR tetramer (cyan for the compact subunits and orange for the extended subunits) binds to the flanking region of the −10 site (red box) through DBDs, leading to repressed transcription. Upon binding of 2-PG (green triangle) at the intradomain cleft of the RD, the NdhR tetramer undergoes drastic conformational changes and dissociates from the target DNA, enabling the recruitment of RNA polymerase (RNAP) to the −10 site and induction of transcription. (B) Coordinated carbon and nitrogen metabolisms via repression and induction of NdhR/CmpR. At the high-Ci condition, the newly fixed organic triose produced in the Calvin–Benson cycle feeds the Krebs cycle to create the pentose 2-OG, which links the glutamate/glutamine metabolism shunt. At the low-Ci condition, the predominant reaction of RuBisCO shifts from carboxylation to oxygenation of RuBP, leading to the production of 2-PG. 2-OG is an indicator of high C/N and keeps NdhR in the repressive state, whereas 2-PG indicates a high N/C level and triggers the induction/activation of NdhR/CmpR.

The cyanobacterial CCM elevates the CO₂ concentration near RuBisCO to enhance CO₂ fixation in the carboxysome, which is the primary carbon source for the Calvin–Benson cycle in photosynthesis (8). When environmental Ci is sufficient, the higher CO₂/O₂ ratio around the catalytic site of RuBisCO activates its carboxylation activity, which assimilates CO₂ to produce 3-phophoglycerate (46); meanwhile, the oxygenase function of Rubisco is inactive, leading to a low concentration of 2-PG in the cell (Fig. 5B). The newly fixed organic triose can feed the Krebs cycle (Fig. 5B), in which the 2-OG dehydrogenase is replaced by two alternative enzymes, 2-OG decarboxylase and succinic semialdehyde dehydrogenase, to convert the intermediate 2-OG to succinate (39). In addition, 2-OG is also a primary carbon skeleton for the assimilation of ammonia that bridges the Krebs cycle and the glutamate/glutamine metabolism shunt (Fig. 5B). The central metabolite 2-OG has been shown to be a signal of nitrogen starvation, and its binding to PII and NtcA enables the cells to respond to nitrogen deficiency (3). Thus, when the 2-PG level is low due to high CO₂ input in the cells, the relative level of 2-OG increases to augment nitrogen assimilation through NtcA and PII (7, 43); at the same time, the NdhR tetramer binds to two molecules of the corepressor 2-OG at the interface between two adjacent RDs (Fig. 5A). Due to its high affinity for two tandem consensus motifs, ATAG-N₈-CTAT, at the core promoter region around −10 sites (18), the 2-OG–complexed NdhR is a strong repressor that blocks the binding site of RNA polymerase, causing a decrease in the transcription of CCM-related genes. Thus, increased nitrogen assimilation and decreased carbon input enable the cells to rebalance the carbon and nitrogen metabolism. On the contrary, when CO₂ input is low, the 2-PG concentration in the cells increases because of the oxygenase activity of RuBisCO; this will also lead to a lower level of 2-OG because of reduced carbon input into the Krebs cycle through the Calvin–Benson cycle. In such situations, four molecules of 2-PG bind to a NdhR tetramer at the intradomain cleft of the RD, which triggers a significant induced fit that alters the interface between the two adjacent RDs, thus excluding the binding of 2-OG. Moreover, molecular dynamics simulations indicated that, upon 2-PG binding, the RD domains undergo drastic conformational changes and trigger the rearrangements of the DBDs (Fig. S1). In the 2-OG–bound state, the distance between the DNA-recognition helices of adjacent DBDs is 29 Å, which matches a continuous DNA major groove, thus providing a stronger DNA binding and repression effect. By contrast, upon binding to 2-PG, the corresponding distance between DBDs is shifted to 25 Å, which is hard to fit into the DNA major groove. Accordingly, the DNA-binding affinity is sharply decreased, as shown by the SPR assays (Fig. 1F). In consequence, dissociation of NdhR enables the binding of RNA polymerase to the promoter region of NdhR-suppressed target genes (Fig. 5A). Thus, a high 2-PG level relieves the repressor effect of NdhR, allowing the transcription of genes related to CCM to increase carbon input, while the native forms of NtcA and PII lower nitrogen assimilation, so that carbon and nitrogen metabolisms are kept in balance. Additionally, the accumulation of 2-PG is also sensed by the activator CmpR (17), which in turn initiates the transcription of the cmp operon and thus further accelerates the carbon uptake rate. Considering the 54% sequence identity with NdhR, we speculate that CmpR binds to 2-PG in a pattern similar to 2-PG binding by NdhR, although CmpR and NdhR regulate gene expression in an opposite manner. Furthermore, our result strengthens the previous finding of the role of 2-PG as an internal signal of Ci deprivation (23). Sensing of the intracellular 2-PG level by both NdhR and CmpR suggests a positive feedback circuit for a rapid response to Ci deprivation. Overall, our findings together with the previous result regarding the role of 2-OG (19) further provide a structural basis explaining the coordination of global carbon and nitrogen assimilation during changes in Ci availability (13, 47, 48).

In summary, a balanced supply of carbon and nitrogen is necessary for the optimal growth of cells under varying environmental conditions. The accumulation of intracellular 2-OG is an indicator of a high C/N ratio (3), whereas the accumulation of the photorespiratory metabolite 2-PG is an indicator of a high N/C ratio (Fig. 5B). Imbalanced C/N metabolism will result in the intracellular accumulation of either 2-OG or 2-PG, and their interaction with global sensors, such as NtcA, CmpR, and NdhR, switches on or off the downstream genes involved in carbon and nitrogen metabolism and eventually leads to the restoration of balance. The opposite effects of 2-OG and 2-PG on the activity of NdhR provide an example of the integration of the metabolic signaling necessary for cyanobacterial adaptation in response to the cellular status of carbon or nitrogen metabolism.

Materials and Methods

The ndhR gene was cloned into the pET28a-derived vector and then was expressed in the E. coli BL21 strain. The proteins were purified by nickel-affinity purification followed by size-exclusion chromatography. The structure of NdhR was solved by the single-wavelength anomalous dispersion phasing method. The ndhR-deletion strain was constructed by replacing the ndhR gene with the spectinomycin resistance gene via homologous recombination. The real-time qPCR experiments were carried out according to the protocols provided (Roche). For full details of all these processes, see Supporting Information.

Acknowledgments

We thank the staff at the Shanghai Synchrotron Radiation Facility for technical assistance. This work is supported by National Natural Science Foundation of China (www.nsfc.gov.cn) Grants 31630001, 31500598, and 21573205, Strategic Priority Research Program of the Chinese Academy of Sciences Grant XDB08020300, Key Research Program of Frontier Sciences of the Chinese Academy of Sciences Grant QYZDJ-SSW-SMC016, and the Innovation Center for Cell Signaling Network.

Footnotes

↵¹Y.-L.J., X.-P.W., and H.S. contributed equally to this work.
↵²To whom correspondence may be addressed. Email: zcz{at}ustc.edu.cn, cczhang{at}ihb.ac.cn, or cyxing{at}ustc.edu.cn.

Author contributions: Y.-L.J., C.-C.Z., Y.C., and C.-Z.Z. designed research; Y.-L.J., X.-P.W., H.S., S.-J.H., N.C., G.-M.L., J.-Y.Z., D.-D.C., and Z.-Y.Z. performed research; Y.-L.J., X.-P.W., H.S., W.-F.L., W.C., C.-C.Z., Y.C., and C.-Z.Z. analyzed data; and Y.-L.J., C.-C.Z., and C.-Z.Z. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.wwpdb.org (PDB ID codes 5Y2V for the full-length NdhR complexed with 2-OG and 5Y2W for the NdhR RD domain complexed with 2-PG).
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1716062115/-/DCSupplemental.

Published under the PNAS license.

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Proceedings of the National Academy of Sciences: 115 (2)

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Submit

[1] ↵
Flores E,
Herrero A
(2005) Nitrogen assimilation and nitrogen control in cyanobacteria. Biochem Soc Trans 33:164–167.
OpenUrl Abstract/FREE Full Text

[2] Flores E,

[3] Herrero A

[4] ↵
Laurent S, et al.
(2005) Nonmetabolizable analogue of 2-oxoglutarate elicits heterocyst differentiation under repressive conditions in Anabaena sp. PCC 7120. Proc Natl Acad Sci USA 102:9907–9912.
OpenUrl Abstract/FREE Full Text

[5] Laurent S, et al.

[6] ↵
Huergo LF,
Dixon R
(2015) The emergence of 2-oxoglutarate as a master regulator metabolite. Microbiol Mol Biol Rev 79:419–435.
OpenUrl Abstract/FREE Full Text

[7] Huergo LF,

[8] Dixon R

[9] ↵
Xu Y, et al.
(2003) The structures of the PII proteins from the cyanobacteria Synechococcus sp. PCC 7942 and Synechocystis sp. PCC 6803. Acta Crystallogr D Biol Crystallogr 59:2183–2190.
OpenUrl CrossRef PubMed

[10] Xu Y, et al.

[11] ↵
Forchhammer K
(2008) P(II) signal transducers: Novel functional and structural insights. Trends Microbiol 16:65–72.
OpenUrl CrossRef PubMed

[12] Forchhammer K

[13] ↵
Zhao MX, et al.
(2010) 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.
OpenUrl Abstract/FREE Full Text

[14] Zhao MX, et al.

[15] ↵
Llácer JL, et al.
(2010) Structural basis for the regulation of NtcA-dependent transcription by proteins PipX and PII. Proc Natl Acad Sci USA 107:15397–15402.
OpenUrl Abstract/FREE Full Text

[16] Llácer JL, et al.

[17] ↵
Burnap RL,
Hagemann M,
Kaplan A
(2015) Regulation of CO₂ concentrating mechanism in cyanobacteria. Life (Basel) 5:348–371.
OpenUrl CrossRef PubMed

[18] Burnap RL,

[19] Hagemann M,

[20] Kaplan A

[21] ↵
Price GD,
Sultemeyer D,
Klughammer B,
Ludwig M,
Badger MR
(1998) The functioning of the CO₂ concentrating mechanism in several cyanobacterial strains: A review of general physiological characteristics, genes, proteins, and recent advances. Can J Bot 76:973–1002.
OpenUrl CrossRef

[22] Price GD,

[23] Sultemeyer D,

[24] Klughammer B,

[25] Ludwig M,

[26] Badger MR

[27] ↵
Badger MR,
Price GD
(2003) CO₂ concentrating mechanisms in cyanobacteria: Molecular components, their diversity and evolution. J Exp Bot 54:609–622.
OpenUrl CrossRef PubMed

[28] Badger MR,

[29] Price GD

[30] ↵
Kaplan A
(2017) On the cradle of CCM research: Discovery, development, and challenges ahead. J Exp Bot 68:3785–3796.
OpenUrl

[31] Kaplan A

[32] ↵
Figge RM,
Cassier-Chauvat C,
Chauvat F,
Cerff R
(2001) Characterization and analysis of an NAD(P)H dehydrogenase transcriptional regulator critical for the survival of cyanobacteria facing inorganic carbon starvation and osmotic stress. Mol Microbiol 39:455–468.
OpenUrl CrossRef PubMed

[33] Figge RM,

[34] Cassier-Chauvat C,

[35] Chauvat F,

[36] Cerff R

[37] ↵
Wang HL,
Postier BL,
Burnap RL
(2004) Alterations in global patterns of gene expression in Synechocystis sp. PCC 6803 in response to inorganic carbon limitation and the inactivation of ndhR, a LysR family regulator. J Biol Chem 279:5739–5751.
OpenUrl Abstract/FREE Full Text

[38] Wang HL,

[39] Postier BL,

[40] Burnap RL

[41] ↵
Woodger FJ,
Bryant DA,
Price GD
(2007) Transcriptional regulation of the CO₂-concentrating mechanism in a euryhaline, coastal marine cyanobacterium, Synechococcus sp. strain PCC 7002: Role of NdhR/CcmR. J Bacteriol 189:3335–3347.
OpenUrl Abstract/FREE Full Text

[42] Woodger FJ,

[43] Bryant DA,

[44] Price GD

[45] ↵
Burnap RL,
Nambudiri R,
Holland S
(2013) Regulation of the carbon-concentrating mechanism in the cyanobacterium Synechocystis sp. PCC6803 in response to changing light intensity and inorganic carbon availability. Photosynth Res 118:115–124.
OpenUrl CrossRef PubMed

[46] Burnap RL,

[47] Nambudiri R,

[48] Holland S

[49] ↵
Omata T,
Gohta S,
Takahashi Y,
Harano Y,
Maeda S
(2001) Involvement of a CbbR homolog in low CO₂-induced activation of the bicarbonate transporter operon in cyanobacteria. J Bacteriol 183:1891–1898.
OpenUrl Abstract/FREE Full Text

[50] Omata T,

[51] Gohta S,

[52] Takahashi Y,

[53] Harano Y,

[54] Maeda S

[55] ↵
Nishimura T, et al.
(2008) Mechanism of low CO₂-induced activation of the cmp bicarbonate transporter operon by a LysR family protein in the cyanobacterium Synechococcus elongatus strain PCC 7942. Mol Microbiol 68:98–109.
OpenUrl CrossRef PubMed

[56] Nishimura T, et al.

[57] ↵
Klähn S, et al.
(2015) Integrated transcriptomic and metabolomic characterization of the low-carbon response using an ndhR mutant of Synechocystis sp. PCC 6803. Plant Physiol 169:1540–1556.
OpenUrl Abstract/FREE Full Text

[58] Klähn S, et al.

[59] ↵
Daley SM,
Kappell AD,
Carrick MJ,
Burnap RL
(2012) Regulation of the cyanobacterial CO2-concentrating mechanism involves internal sensing of NADP+ and α-ketogutarate levels by transcription factor CcmR. PLoS One 7:e41286.
OpenUrl CrossRef PubMed

[60] Daley SM,

[61] Kappell AD,

[62] Carrick MJ,

[63] Burnap RL

[64] ↵
Kent SS,
Young JD
(1980) Simultaneous kinetic analysis of ribulose 1,5-bisphosphate carboxylase/oxygenase activities. Plant Physiol 65:465–468.
OpenUrl Abstract/FREE Full Text

[65] Kent SS,

[66] Young JD

[67] ↵
Hagemann M, et al.
(2013) Evolution of the biochemistry of the photorespiratory C2 cycle. Plant Biol (Stuttg) 15:639–647.
OpenUrl CrossRef PubMed

[68] Hagemann M, et al.

[69] ↵
Schell MA
(1993) Molecular biology of the LysR family of transcriptional regulators. Annu Rev Microbiol 47:597–626.
OpenUrl CrossRef PubMed

[70] Schell MA

[71] ↵
Haimovich-Dayan M,
Lieman-Hurwitz J,
Orf I,
Hagemann M,
Kaplan A
(2015) Does 2-phosphoglycolate serve as an internal signal molecule of inorganic carbon deprivation in the cyanobacterium Synechocystis sp. PCC 6803? Environ Microbiol 17:1794–1804.
OpenUrl CrossRef

[72] Haimovich-Dayan M,

[73] Lieman-Hurwitz J,

[74] Orf I,

[75] Hagemann M,

[76] Kaplan A

[77] ↵
Schwarz D,
Orf I,
Kopka J,
Hagemann M
(2014) Effects of inorganic carbon limitation on the metabolome of the Synechocystis sp. PCC 6803 mutant defective in glnB encoding the central regulator PII of cyanobacterial C/N acclimation. Metabolites 4:232–247.
OpenUrl CrossRef PubMed

[78] Schwarz D,

[79] Orf I,

[80] Kopka J,

[81] Hagemann M

[82] ↵
Saint-Girons I, et al.
(1986) Interactions of the Escherichia coli methionine repressor with the metF operator and with its corepressor, S-adenosylmethionine. J Biol Chem 261:10936–10940.
OpenUrl Abstract/FREE Full Text

[83] Saint-Girons I, et al.

[84] ↵
Arvidson DN,
Bruce C,
Gunsalus RP
(1986) Interaction of the Escherichia coli trp aporepressor with its ligand, L-tryptophan. J Biol Chem 261:238–243.
OpenUrl Abstract/FREE Full Text

[85] Arvidson DN,

[86] Bruce C,

[87] Gunsalus RP

[88] ↵
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Coordinating carbon and nitrogen metabolic signaling through the cyanobacterial global repressor NdhR

Significance

Abstract

Results

2-OG and 2-PG Are the Effectors of NdhR.

Crystal Structure of NdhR.

2-OG Binds at the Interface Between Two RDs and Acts as a Corepressor of NdhR.

2-PG Binds at the Cleft of RDs of NdhR and Induces Gene Expression.

Discussion

Materials and Methods

Acknowledgments

Footnotes

References

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Search

Significance

Abstract

Results

2-OG and 2-PG Are the Effectors of NdhR.

Crystal Structure of NdhR.

2-OG Binds at the Interface Between Two RDs and Acts as a Corepressor of NdhR.

2-PG Binds at the Cleft of RDs of NdhR and Induces Gene Expression.

Discussion

Materials and Methods

Acknowledgments

Footnotes

References

Citation Manager Formats

Article Classifications

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