PII-like signaling protein SbtB links cAMP sensing with cyanobacterial inorganic carbon response

Khaled A. Selim; Florian Haase; Marcus D. Hartmann; Martin Hagemann; Karl Forchhammer

doi:10.1073/pnas.1803790115

Edited by Susan S. Golden, University of California, San Diego, La Jolla, CA, and approved April 9, 2018 (received for review March 7, 2018)

Significance

Life on Earth depends on photosynthetic CO₂ fixation to form organic carbon. This process evolved in cyanobacteria and was later conveyed to eukaryotes, giving rise to plastids in algae and plants. To cope with low atmospheric CO₂ concentrations that developed over the course of evolution, cyanobacteria evolved a CO₂-concentrating mechanism (CCM), which elevates CO₂ levels in the vicinity of RubisCO, the key enzyme of CO₂ fixation. Here we describe a conserved cyclic AMP receptor protein, SbtB, which participates in the sensing of fluctuating C_i levels to regulate the cyanobacterial CCM system. SbtB represents a new principle of C_i sensing, which is important for acclimation to varying C_i regimes in the ecological niches of cyanobacteria.

Abstract

Cyanobacteria are phototrophic prokaryotes that evolved oxygenic photosynthesis ∼2.7 billion y ago and are presently responsible for ∼10% of total global photosynthetic production. To cope with the evolutionary pressure of dropping ambient CO₂ concentrations, they evolved a CO₂-concentrating mechanism (CCM) to augment intracellular inorganic carbon (C_i) levels for efficient CO₂ fixation. However, how cyanobacteria sense the fluctuation in C_i is poorly understood. Here we present biochemical, structural, and physiological insights into SbtB, a unique P_II-like signaling protein, which provides new insights into C_i sensing. SbtB is highly conserved in cyanobacteria and is coexpressed with CCM genes. The SbtB protein from the cyanobacterium Synechocystis sp. PCC 6803 bound a variety of adenosine nucleotides, including the second messenger cAMP. Cocrystal structures unraveled the individual binding modes of trimeric SbtB with AMP and cAMP. The nucleotide-binding pocket is located between the subunit clefts of SbtB, perfectly matching the structure of canonical P_II proteins. This clearly indicates that proteins of the P_II superfamily arose from a common ancestor, whose structurally conserved nucleotide-binding pocket has evolved to sense different adenyl nucleotides for various signaling functions. Moreover, we provide physiological and biochemical evidence for the involvement of SbtB in C_i acclimation. Collectively, our results suggest that SbtB acts as a C_i sensor protein via cAMP binding, highlighting an evolutionarily conserved role for cAMP in signaling the cellular carbon status.

Cyanobacteria evolved oxygenic photosynthesis about 2.7 billion y ago. This ability was later transmitted to eukaryotes via endosymbiosis, giving rise to plastids in algae and plants (1). The global proliferation of oxygenic photosynthetic conversion of C_i (CO₂ and HCO₃⁻) into organic matter using light energy and electrons from water splitting led over time to a strong decline in available C_i on Earth. As a consequence, cyanobacteria evolved a CO₂-concentrating mechanism (CCM) that allows them to grow in the present-day atmosphere of the Earth, which contains only 0.04% CO₂ (2, 3).

The cyanobacterial CCM consists of several systems for C_i uptake as well as of the carboxysome, a proteinaceous bacterial cellular compartment in which the CO₂-fixing enzyme RubisCO and carbonic anhydrase (CA) are localized. The C_i-uptake systems concentrate massive amounts of bicarbonate inside the cyanobacterial cells, which then diffuses into the carboxysome, where CA converts it to CO₂. This strategy increases the CO₂ concentration in the vicinity of RubisCO, thereby saturating the carboxylation activity and minimizing the competing oxygenation reaction of this enzyme (2, 3). Therefore, the ability to respond to fluctuating C_i supply is of key importance, but the underlying mechanisms are still poorly understood.

The cyanobacterial bicarbonate transporter SbtA (4), as part of the CCM, is encoded in a bicistronic operon, together with the downstream gene sbtB; this operon is highly expressed with other CCM genes under C_i-limiting conditions (5, 6). In Escherichia coli cells expressing cyanobacterial sbtA and sbtB, SbtB inhibits SbtA-mediated HCO₃⁻ uptake (7). SbtB has been identified as a noncanonical member of the P_II signal transduction superfamily (8). P_II proteins are among the most widespread signaling proteins in nature. Canonical P_II proteins regulate nitrogen assimilation reactions by sensing the metabolic state of the cells through binding of small effector molecules [2-oxoglutarate (2-OG) and ATP/ADP]. This induces conformational changes in the large T loop that changes P_II interactions with target proteins, thereby modulating their activities. However, although members of the P_II superfamily are highly conserved in structure, they are very divergent in sequence, such that P_II-like proteins usually have low sequence identity and lack multiple consensus sequence motifs of canonical P_II proteins (8). The cognate effector molecules and biological functions of most P_II-like proteins, such as SbtB, are unknown. Here we analyzed the sensing properties and regulatory features of SbtB from Synechocystis sp. PCC 6803 (ScSbtB).

Results

Biochemical Properties of ScSbtB.

Analysis of the ScSbtB amino acid sequence (SI Appendix, Bioinformatics Analysis and Fig. S1 A and B) revealed the absence of virtually all residues known to interact with 2-OG or the β- and γ-phosphates of the effector nucleotides (ATP/ADP) of canonical P_II proteins, which suggests that SbtB evolved sensory properties distinct from those of canonical P_II proteins. To identify potential sensing properties and regulatory features of SbtB, we prepared a recombinant ScSbtB protein in its native trimeric state (SI Appendix, Fig. S1 C–E) and set out to address the question of potential metabolite binding using isothermal titration calorimetry (ITC) and microscale thermophoresis (MST).

We first tested the binding of the typical P_II effectors ATP, ADP, and 2-OG. While 2-OG binding was not detected (SI Appendix, Fig. S2), both ATP and ADP bound with comparably high affinities in the micromolar range (Table 1), even though SbtB lacks the canonical core residues for β- and γ-phosphate coordination. This led us to test for the binding of adenosine and AMP, which represent the core moiety for which large parts of the binding site appeared to be conserved. Binding of adenosine was weak (SI Appendix, Fig. S2), and AMP bound with an affinity only slightly lower than that of ATP and ADP, which suggested that AMP is the minimal binding moiety that is specifically recognized (SI Appendix, Fig. S2 A–C). We then checked for the binding of cAMP, which surprisingly bound with the highest affinity of all compounds tested (SI Appendix, Fig. S2D). Moreover, the binding enthalpy for cAMP was higher than that of the other nucleotides at the same concentration (compare SI Appendix, Fig. S2E with SI Appendix, Fig. S2 A–C), which indicated that binding cAMP is preferred over the other nucleotides. The guanosine nucleotides GTP and cGMP did not bind to ScSbtB (SI Appendix, Fig. S2). For all nucleotides, a comparison of the calculated affinities using a model of three binding sites showed that the first two sites have similar affinities, whereas the third site is occupied with an approximately 10-fold lower affinity, which is indicative of anticooperativity (Table 1), like the canonical P_II protein (9, 10). The K_d values obtained by the MST measurements were in good qualitative agreement with the ITC data (Fig. 1A and Table 1).

View this table:

Table 1.

Dissociation constants calculated for binding of the adenyl nucleotides ATP, ADP, AMP, cAMP, and adenosine to the SbtB protein measured by ITC and MST

Fig. 1.

Structural and binding properties of the ScSbtB protein. (A) Binding of nucleotides monitored by MST (n = 3). (A, Upper) Titration series of ATP, ADP, AMP, and adenosine. (A, Lower) Titration series of cAMP. (B) Overall architecture of the SbtB protein. The structure of the trimeric SbtB–cAMP complex is shown as an electrostatic potential surface (Upper) and as a cartoon (Lower). The red–white–blue color gradient corresponds to surface potential values ranging from −10 to +10 k_BT/e. The structure of SbtB reveals the typical ferredoxin-like fold of the P_II superfamily, with nucleotide-binding pockets situated between the clefts of the subunits. (C) Superposition of monomeric subunits of SbtB (violet) with SeP_II (brown; PDB ID code 2XUL), yielding a 0.93-Å rmsd, with secondary structure elements and characteristic structural motifs indicated. (C, Inset) Highlighting of the hairpin-loop (CGPEGC) motif of SbtB with the disulfide bond between C105 and C110 and the hydrogen bond to the N terminus. (D–G) Close-up of the nucleotide binding site of the apo-SbtB, SbtB–AMP, SbtB–cAMP, and SeP_II–ATP structures, respectively. Relevant residues for nucleotide binding are shown as sticks, and H bonds are indicated by thin lines. (H) Superposition of the SbtB–AMP (yellow–violet) and SeP_II–ATP (brown) nucleotide binding sites, with relevant residues labeled in orange and dark brown, respectively.

The recent characterization of the P_II-like protein associated with carboxysomes from Thiomonas intermedia (TiCP_II), whose gene is located near the sbtAB operon in many chemoautotrophic bacteria, provided clues that bicarbonate, AMP, and ADP are ligands (11). In addition, the binding of the adenosine nucleotides to TiCP_II is stimulated 15-fold by 50 mM bicarbonate (11). Therefore, we also tested the effect of bicarbonate on ScSbtB. Bicarbonate (50 mM) did not bind to ScSbtB (SI Appendix, Fig. S2) or significantly influence the binding properties of the verified ligands (Table 1). To ensure that binding of cAMP is unique to ScSbtB and not an overlooked property of canonical P_II proteins, we titrated cAMP and AMP against the Synechococcus elongatus P_II protein (SeP_II) without detecting any binding (SI Appendix, Fig. S2 L and M).

Structural Characterization of ScSbtB.

To determine the structural basis for ligand binding, ScSbtB in apo form and in complex with AMP, ADP, or cAMP was crystallized, all rewarding well-diffracting crystals. The apo protein yielded a trigonal crystal form of space group P3₂. We solved its structure by molecular replacement using the Anabaena variabilis SbtB structure [AvSbtB; Protein Data Bank (PDB) ID code 3DFE] as a search model (SI Appendix, Structural Characterization of ScSbtB). As expected, the refined apo-ScSbtb structure shows a canonical P_II ferredoxin-like fold (Fig. 1 B and C). All three subunits of the trimer are essentially in the same conformation, and the entire ScSbtB trimer superimposes with Cα rmsd values of 0.53 Å on the AvSbtB trimer and 0.93 Å on the canonical SeP_II trimer (PDB ID code 2XUL) (SI Appendix, Figs. S3 and S4A). As in essentially all other crystal structures of isolated trimers of the P_II superfamily, the flexible T loop was mostly disordered (Fig. 1C).

Notable overall differences from the structures of canonical P_II proteins are found at the N and C termini. At the N terminus, the first β-strand runs antiparallel along the full length of the β4-strand to face the C terminus (Fig. 1C). The C terminus features a unique extension, which was unresolved in the AvSbtB structure but is fully ordered here. It forms a single α-helical turn flanking the canonical nucleotide-binding cleft, followed by a short hairpin loop formed by a CGPxGC motif, which is conserved in SbtB proteins (SI Appendix, Fig. S1 A and B), in which the two cysteines form a disulfide bridge (Fig. 1C). The carboxyl terminus of the last cysteine forms a hydrogen bond with the amino terminus. This mutually stabilizing interaction between the ScSbtB N and C termini is shown in detail in Fig. 1C. The whole structural motif is invariant in all three chains of the trimer, which underpins its relevance.

Structural Characterization of Ligand Binding.

Cocrystals of ScSbtB with AMP, ADP, or cAMP yielded the same trigonal crystal form as for apo-ScSbtB, and the same crystals were obtained in the presence and absence of bicarbonate. We solved the structures on the basis of the apo-ScSbtB coordinates; in all cocrystal structures, the electron density of the respective nucleotide was clear (SI Appendix, Fig. S4B). Structures of crystals grown in the presence or absence of bicarbonate did not differ, in agreement with the negligible influence of bicarbonate on the ITC measurements, which further confirms that the sensory properties of ScSbtB differ from those of TiCP_II (11).

To our surprise, however, not only in the ScSbtB–AMP structure but also in the putative ScSbtB–ADP structure, only AMP was complexed, with clearly no electron density for a β-phosphate. Consequently, these two structures are virtually identical. The AMP moiety is bound as in most other known nucleotide-bound structures of the P_II superfamily (Fig. 1 D–H). Specifically, the adenosine moiety forms hydrogen bonds to the backbone of L65 and the T33 side chain, and the phosphate forms a hydrogen bond to G89. Additional ScSbtB-specific hydrogen bonds are formed between the ribose hydroxyl groups and the side chain of N59, which is only conserved in ScSbtB, in the β-strand (β3) following the T loop. In two chains of the trimer, another bond is observed between the phosphate and a histidine (H102) in the C extension (Fig. 1E).

Due to the cyclic nature of cAMP, its phosphate binding mode differs from that of AMP. While the interactions of the adenine and ribose moieties of cAMP are the same as in the ScSbtB–AMP structure, no bonds are formed between the phosphate and G89 or H102. Instead, at least in one binding site, the phosphate forms an additional hydrogen bond with N59 (Fig. 1F). All residues involved in nucleotide binding are shown in detail in SI Appendix, Fig. S4B.

ScSbtB Reversibly Associates with Membranes in an SbtA-Dependent Manner.

ScSbtB-specific antibodies (SI Appendix, Fig. S5A) were raised to reveal the subcellular localization and expression pattern of ScSbtB in response to different treatments, showing that ScSbtB accumulates in stationary-phase and carbon-limited conditions (SI Appendix, Conditions Triggering SbtB Accumulation and Fig. S5). It has been reported that when sbtB and sbtA are coexpressed in E. coli, SbtB tunes down SbtA-dependent HCO₃⁻ uptake (7). If the two proteins interact in wild-type Synechocystis sp. PCC 6803 cells (hereinafter WT), then SbtB should colocalize with the integral membrane protein SbtA. In WT cultures grown with low carbon (ambient air; hereinafter LC), SbtB was equally distributed between the membrane and soluble (cytosolic) fractions (Fig. 2A, Left). When cells were exposed to harsh C_i starvation by omitting any gas exchange for 24 h (stationary culture flasks without aeration), SbtB was mainly in the membrane fraction (Fig. 2A, Right). By contrast, in an SbtA-deficient mutant, SbtB was exclusively soluble and never membrane-associated (Fig. 2B), which indicates that SbtA is required for SbtB membrane localization. To determine the dynamics of SbtB association/dissociation to/from the membrane, a WT culture grown with high carbon (5% CO₂; hereinafter HC) was shifted to LC conditions. At the start of the experiment (T_zero), SbtB was detected exclusively in the soluble fraction. One hour after the shift, the first traces of membrane-localized SbtB were observed, and the membrane-bound SbtB fraction gradually increased over 6 h (Fig. 2C).

Fig. 2.

Immunoblot analysis of SbtA-dependent ScSbtB membrane localization. (A) Localization of SbtB in soluble (S) and membrane (M) fractions of cells grown with ambient air, that is, LC (0.04% CO₂; Left), or in carbon-starved cells (stationary culture flasks without aeration for 24 h; Right). (B) Distribution of SbtB between soluble and membrane fractions of WT and ΔsbtA mutant cells grown with LC (0.04% CO₂). SbtA antibodies were used to determine the quality of membrane isolation and the presence of SbtA. (C) Dynamics of SbtB membrane localization in WT cultures shifted from HC (5% CO₂) to LC (0.04% CO₂) for 1 to 6 h; T_zero represents HC conditions before the shift. (D) Influence of added nucleotides on SbtB localization. Crude cell extracts of cells grown under LC were prepared, the indicated nucleotide (2 mM) was then added separately to the extracts, and membrane and soluble fractions were separated. (E) SbtB localization in WT and Δcya1 mutant cells grown under LC (air-grown) and HC (2% CO₂) conditions as indicated. See SI Appendix, Materials and Methods for a detailed description of membrane fractionation.

To determine whether the ligands cAMP, AMP, and ADP affect the membrane localization of SbtB, each ligand (2 mM) was separately added to extracts of WT cells grown with LC and the extracts were then fractionated into soluble and membrane fractions. In WT extracts without added ligand, SbtB was partially localized in the membrane. Addition of ADP or AMP clearly and markedly triggered membrane localization of SbtB, whereas cAMP did not cause membrane localization of SbtB (Fig. 2D). Hence, binding of ADP or AMP, in contrast to binding of cAMP, would be advantageous for SbtA–SbtB complex formation in the membrane.

We then created a cAMP-free mutant by knocking out slr1991 (designated cya1), which encodes the soluble adenylyl cyclase (12) (SI Appendix, Fig. S6 A and B), and tested the effect on SbtB localization upon shifting from LC to HC conditions. Under LC conditions, the amount of SbtB associated with the membrane was similar in the Δcya1 mutant and the WT. However, after shifting to HC conditions, SbtB dissociated from the membrane in the WT but not in the Δcya1 mutant (Fig. 2E). This result indicates that SbtB in the Δcya1 mutant does not sense the HC signal, which suggests that cAMP represents the HC signal that leads to membrane dissociation of SbtB.

ScSbtB Is Required for Efficient C_i Acclimation.

To characterize the role of SbtB in the CCM, we generated a completely segregated sbtB knockout mutant (ΔsbtB) (SI Appendix, Figs. S5A and S6 A and C). On solid BG₁₁ agar medium at pH 8 and 9, the ΔsbtB mutant grew almost like the WT under HC/LC conditions, but growth was negatively affected only at pH 7 under LC conditions, where more C_i is present in the form of CO₂ (Fig. 3A, Left). Similarly, the ΔsbtB mutant grew significantly slower than the WT in liquid cultures at pH 8 under LC conditions, but such a difference was not observed under HC conditions (Fig. 3A, Right). Importantly, ΔsbtB cells were highly sensitive to sudden changes in carbon supply; when slowly growing ΔsbtB mutant cells (LC and low light of 30 µE) were suddenly exposed to 2% CO₂ at the same light intensity, the culture temporarily ceased growth and only slowly recovered. This effect became even more pronounced at higher light intensity and with further CO₂ supplementation (5% CO₂, 120 µE). By contrast, WT cultures immediately increased growth at HC levels under low and high light intensity (Fig. 3B and SI Appendix, Physiological Function of ScSbtB and Fig. S7A).

Fig. 3.

Phenotypic characterization of the ΔsbtB knockout mutant. (A) Growth of WT or ΔsbtB mutant cells on solid BG₁₁ medium at different pH values under LC conditions (Left) or in liquid BG₁₁ medium at pH 8.0 under LC or HC conditions (Right, error bars represent standard deviations from three independent cultivation experiments). (B) Effect of sudden change in carbon supply in the form of growth yield. WT and the ΔsbtB mutant were shifted from slow-growing cultures (LC/low light, 30 µE) either to HC (2% CO₂) or LC (air) under the initial light intensity (30 µE), or to HC (5% CO₂) and LC (air) with increased light intensity (120 µE). (C) Levels of fluorescent SbtB-sfGFP protein in WT and the ΔsbtB complemented mutant in response to HC and LC growth (standard deviation; n = 3). (D) Bicarbonate-dependent photosynthetic rates per chlorophyll a (Chla) of WT and the ΔsbtB mutant as a function of increasing HCO₃⁻ concentrations. Cells were acclimated to either HC or LC conditions (n = 3). (E) Gene expression of selected CCM genes in WT and the ΔsbtB mutant analyzed using semiquantitative RT-PCR. Cells were cultivated under LC conditions (0 h) and then shifted to HC conditions (5% CO₂) for 24 h; the constitutively expressed rnpB gene served as loading control.

To complement the growth defect of ∆sbtB, a plasmid expressing the gene encoding an ScSbtB-sfGFP (superfolded GFP) fusion protein under the control of the native promoter of the sbt operon was transformed into WT and ∆sbtB cells to generate the strains WT+SbtB-sfGFP and ∆sbtB+SbtB-sfGFP (SI Appendix, Fig. S6 A and D). To check the expression of the SbtB-sfGFP construct, we quantified SbtB-sfGFP fluorescence under HC and LC conditions (Fig. 3C). The GFP signal was much lower under HC conditions than under LC conditions, in agreement with the expression pattern of native SbtB in response to C_i (5, 6). Under LC, the SbtB-sfGFP signal was lower in the WT+SbtB-sfGFP strain than in the ∆sbtB+SbtB-sfGFP strain, which indicated that the genomic copies of sbtB (slr1513) in WT cells quench the expression of SbtB-sfGFP. Next, we tested whether the ScSbtB-sfGFP construct complements the phenotype of the ΔsbtB mutant by analyzing acclimation to a shift in CO₂ levels as described above. Indeed, the SbtB-sfGFP construct complemented the growth defect of the ∆sbtB mutant (SI Appendix, Fig. S7 A–D), which demonstrated that SbtB-sfGFP was functionally active and that the growth defect of ∆sbtB was not caused by polar effects. Last, we determined the cellular localization of SbtB under HC and LC conditions by monitoring the sfGFP signals by fluorescence microscopy. Under HC conditions, the SbtB-sfGFP signal was mainly cytoplasmic. However, 1 h after the shift to LC conditions, SbtB-sfGFP partially relocalized to the cytoplasmic membrane (SI Appendix, Fig. S8). Together, these data suggest that SbtB is required to acclimate to sudden changes in CO₂ levels, which highlights its sensory/regulatory role in C_i acclimation.

To obtain further evidence for the proposed functional link between SbtB and control of C_i metabolism, we determined C_i-uptake affinity from HC- and LC-acclimated cells by measuring the amount of oxygen evolved from HCO₃⁻-dependent photosynthesis. Intriguingly, the maximal photosynthetic rates of LC-acclimated ΔsbtB mutant cells were lower than those of the WT (Fig. 3D). The lower V_max indicates an impaired function of CO₂-fixing Calvin–Benson cycle activity, which limits the photosynthetic activity at saturating C_i amounts; this result is consistent with the slower growth of ΔsbtB under LC conditions. In contrast, the affinity toward bicarbonate was almost similar in WT and mutant ΔsbtB cells under LC conditions, since the initial rise in the photosynthetic activity was indistinguishable between these two strains. Strikingly, only WT cells showed the expected acclimation to HC conditions by lowering bicarbonate affinity (HCO₃⁻ K_m: LC, 48 µM; HC, 95 µM), whereas ΔsbtB cells retained the high C_i affinity under HC conditions (HCO₃⁻ K_m: LC, 52 µM; HC, 68 µM) (Fig. 3D). This behavior implies that ΔsbtB resides in the LC-adapted state even under HC conditions, and provides a rationale for the inability of the ΔsbtB mutant to rapidly acclimate to a CO₂ upshift (see above).

The inability of the ΔsbtB mutant to adjust the C_i affinity in response to the ambient CO₂ supply suggests that the SbtB protein plays a role in the regulation of the entire CCM, similar to the role of the canonical P_II protein in N assimilation (8, 13). To test whether the ΔsbtB mutation affects the expression of other C_i-controlled genes, we analyzed the transcript levels of CCM-related genes upon shifting cells from LC to HC. The genes coding for various C_i-uptake systems were highly expressed in WT and ΔsbtB mutant cells grown under LC conditions (Fig. 3E, time 0), and expression of sbtA and ndhF3 markedly decreased upon shifting to HC conditions; ndhF3 encodes a subunit of the inducible CO₂-uptake system NDH-I₃ (3, 14) (Fig. 3E, time points 6 and 24 h after shift). Since the abundance of sbtA mRNA was similar in the WT and ΔsbtB mutant, we can safely assume that the downstream sbtB gene mutation does not affect the expression of the upstream sbtA gene; this result agrees with a previous report that the inactivation of sbtB in Synechococcus sp. PCC 7942 does not affect the bicarbonate-uptake activity of SbtA (15). Surprisingly, the expression of another HCO₃⁻ transporter, bicA (16), was deregulated completely in ΔsbtB, whereas in the WT the expression was like that of sbtA (Fig. 3E), which indicates that the sbtB mutation might have a broader impact on C_i regulation than only the direct regulation of SbtA. The expression of the LC-inducible gene bicA under HC conditions agrees with the preacclimatization of the ΔsbtB mutant to LC conditions, and might be the cause of the inability of the ΔsbtB mutant to adapt C_i affinity to HC conditions.

cAMP/AMP as a C_i Signal Compete for ScSbtB.

To address whether the stably bound ScSbtB effector molecules cAMP and AMP are related to fluctuations in C_i supply, we measured the cellular levels of cAMP and AMP in WT cells as a function of C_i. An HC-adapted culture was harvested, resuspended in fresh medium, and divided into two subcultures. One subculture was reincubated again under HC conditions, and the other subculture was incubated under LC conditions. After 30 min and 4 h, samples were taken, and the levels of cAMP and AMP were quantified. The AMP levels were 18- to 20-fold higher under LC conditions than under HC conditions. By contrast, cAMP showed the opposite trend, namely cAMP levels were 3- to 4-fold higher under HC conditions than under LC conditions (Fig. 4A).

Fig. 4.

Response of cAMP and AMP to cellular C_i levels, and competitive binding assay of cAMP and AMP to ScSbtB. (A) Mean cellular levels and standard deviations (n = 3) of cAMP and AMP were determined under LC or HC conditions after 30 min and 4 h. (B and C) ITC titration of 33.3 µM SbtB (trimeric concentration) in the presence of (B) 300 µM cAMP against 2 mM AMP and (C) 300 µM AMP against 2 mM cAMP. (B and C, Upper) Raw ITC data in the form of the heat produced during the titration. (B and C, Lower) Binding isotherms and the best-fit curves according to the one–binding-site model for trimeric SbtB.

To reveal whether these physiological changes in cAMP relative to AMP are functionally relevant for ScSbtB signaling, we determined the competitive binding of cAMP and AMP to ScSbtB using ITC. Under saturating concentrations of cAMP (300 µM), binding of AMP (2 mM) to ScSbtB was completely inhibited (Fig. 4B). In the presence of 100 µM cAMP, AMP was able to bind only to the available unoccupied sites of ScSbtB with low affinity (SI Appendix, Fig. S2N). The determined K_d value of 240.9 µM is similar to the third (low-affinity) binding site of AMP in trimeric ScSbtB (compare with Table 1). However, when ScSbtB was preincubated with 300 µM AMP, cAMP was still able to bind to ScSbtB, with each injection (corresponding to ∼14 µM cAMP) producing a strong exothermic signal. The resulting isotherm could be fitted to an apparent K_d of 42.2 µM (Fig. 4C). Therefore, under HC conditions, with elevated levels of cAMP and highly diminished levels of AMP, we expect that ScSbtB will accommodate cAMP. By contrast, under LC conditions, the increasing levels of AMP and concomitantly decreasing levels of cAMP will give rise to the AMP-bound state of ScSbtB.

Discussion

Common properties of canonical P_II proteins are the competitive binding of the adenyl nucleotides ATP or ADP to sense the energy status of cells (8, 17) and the ATP-dependent binding of 2-OG as a status indicator of the cellular carbon–nitrogen balance. By contrast, the larger family of P_II-like proteins has different adenyl-nucleotide binding modes and does not generally bind 2-OG (8, 18). SbtB extends the range of sensing in the P_II superfamily. ScSbtB is a unique member of the P_II superfamily for which specific binding of ATP, ADP, AMP, and cAMP has been described. In contrast to canonical PII proteins, the β- and γ-phosphates of the adenyl nucleotides seem of minor importance for SbtB binding and, accordingly, the interacting residues, such as the RxR motif in the C loop, are not conserved (SI Appendix, Fig. S1). However, the location of the nucleotide-binding pocket in the intersubunit clefts and the recognition mode of the nucleoside moiety are perfectly conserved with other members of the P_II superfamily (8) (Fig. 1H). This agrees with the assumption that proteins of the P_II superfamily arose from a common nucleotide-binding ancestor, whose binding pocket has been structurally conserved but whose binding mode has evolved to sense different adenyl nucleotides to fulfill various signaling functions (8).

Interestingly, when we solved the structure of the putative ScSbtB–ADP complex, AMP was bound and there was clearly no electron density for a β-phosphate. A similar phenomenon has been reported for other members of the P_II superfamily (11, 19), but whether ADP is enzymatically hydrolyzed by ScSbtB to AMP remains unclear.

An intriguing property of ScSbtB is the presence of a conserved CGPxGC motif at the C terminus. This extension forms a small helix–hairpin loop structure, which forms a disulfide bond between cysteines C105 and C110. An identical C-terminal segment, CGPxGC, is also present in a subgroup of thiol disulfide oxidoreductases (DsbA). The members of this subgroup form dimers in which the CGPxGC terminus of one monomer is in close proximity to the catalytic CPxC motif. The structure of such a C terminus of a DsbA family protein (PDB ID code 3GL5) is almost identical to the hairpin-loop extension of ScSbtB. Furthermore, many of the plant Calvin–Benson–cycle enzymes have evolved similar redox-regulated C-terminal extensions, C(V/I)VxVC, to modulate their activity through oxidation/reduction of intramolecular disulfide bridges under the different light conditions of day/night cycles (20, 21). Therefore, the hairpin-loop extension of SbtB might be involved in a redox-sensory function.

One of the most striking features of ScSbtB is the binding of cAMP. Increasing evidence suggests that cAMP signaling plays an important role in C_i perception. The increased levels of cAMP under HC conditions agree with the finding that cyanobacterial soluble adenylyl cyclase activity is stimulated by elevated levels of C_i, which causes increased cAMP production (22 ⇓⇓–25). In diatoms, which possess CCMs, increasing CO₂ concentrations cause elevated cAMP levels, which induce down-regulation of the CCMs (26). Therefore, cAMP can be considered an evolutionarily conserved second messenger molecule that responds to C_i, thereby regulating CCMs in deeply branched phyla of cyanobacteria and algae. Another well-known regulatory nucleotide, the stringent control nucleotide ppGpp, was recently identified in cyanobacteria to control global gene expression during day/night switches (27, 28), emphasizing a fundamental role of secondary messenger nucleotides in the cyanobacteria lifestyle.

Although it has been recognized that cAMP is involved in carbon sensing, our results imply that the level of AMP might also play a role in C_i sensing. The levels of cAMP and AMP were similar in cells grown under HC conditions, whereas the cAMP-to-AMP ratio decreased ∼60- to 80-fold when cultures were shifted to LC conditions (Fig. 4). From this, we expect that under HC conditions, SbtB will preferentially bind cAMP over AMP, whereas under LC conditions, AMP will outcompete cAMP. In vitro, AMP favored the membrane-associated state of SbtB, whereas cAMP favored the soluble state. This perfectly agrees with the prevalence of cytoplasmic SbtB under HC conditions (high cAMP-to-AMP ratio) as opposed to membrane-associated SbtB in the LC state (low cAMP-to-AMP ratio). Structurally, there is no considerable difference between the AMP- and cAMP-bound SbtB conformations. However, it is conceivable that the bound nucleotides directly contribute to the interface between SbtA and SbtB, such that the open phosphate group of AMP stabilizes the complex, while cAMP disrupts the interface with its differently oriented phosphate.

ScSbtB and the recently described TiCP_II protein (PDB ID code 5DS7) both sense ADP and AMP (11), and superimpose closely with an rmsd of 0.9 Å (SI Appendix, Figs. S3C and S4A). However, TiCP_II directly responds to HCO₃⁻ and thereby increases the affinity to the adenyl nucleotides (11), whereas ScSbtB does not respond to HCO₃⁻, its affinity to adenyl nucleotides is not influenced by high amounts of external bicarbonate levels (Table 1), and it senses cAMP as a second messenger of the cellular carbon supply. Since both P_II-like proteins respond to ADP/AMP, it appears that a central, yet not understood, task of these proteins is to link C_i assimilation to the energy and carbon states of the cells. However, the sensing of the C_i status occurs via distinct mechanisms. In the case of TiCP_II, HCO₃⁻ is directly bound in the effector molecule binding site, thereby increasing the affinity by two orders of magnitude. By contrast, ScSbtB perceives the carbon status indirectly, via the binding of the second messenger cAMP.

The phenotypic changes of the SbtB-deficient mutant suggest that SbtB is involved in the regulation of the cyanobacterial CCM, similar to the role of the canonical P_II protein in N assimilation (8, 13). Cells of the SbtB-deficient mutant seem to be locked in the LC-acclimated state even under HC conditions. Gene expression analysis revealed that the sbtB deletion had no impact on HC-induced repression of the upstream sbtA gene, whereas expression of the bicA gene encoding another bicarbonate transporter differed in WT and mutant cells. Currently, we have no mechanistic explanation for this finding, but it indicates that different factors are involved in the regulation of bicA and the sbt operon, with bicA expression being dependent on SbtB. Therefore, in addition to a direct regulation of SbtA transport activity (7), SbtB might be involved in a more general regulation of the CCM, in analogy to the multiple functions of canonical cyanobacterial P_II proteins in metabolic regulation (29).

Under conditions of C_i starvation, SbtA and SbtB were produced at the highest levels, and SbtB was bound to the membrane via SbtA. Under these conditions, maximal activity of SbtA is required to keep the intracellular bicarbonate level high. Consequently, this SbtA–SbtB complex must be active in bicarbonate transport and, therefore, binding of SbtB does not necessarily inhibit SbtA activity. This clearly distinguishes SbtB from the P_II protein GlnK, which associates with the ammonium channel protein Amt to close the pores and shut down the uptake of ammonia (30, 31). It is, however, possible that SbtB switches off SbtA activity during the night, possibly involving the C terminus of ScSbtB, which is likely redox-regulated. Another purpose of SbtB binding to SbtA could be to stabilize SbtA or to sequester SbtB to the membrane and thereby exert a regulatory function, similar to that shown for the regulation of nitrogenase-associated DraG/DraT by P_II proteins in Azospirillum brasilense (32). If soluble SbtB interacts with a regulator that mediates acclimation to high CO₂ levels, then deletion of SbtB could lock the cells in the LC-adapted state.

Collectively, our findings extend the knowledge on cAMP sensing. While the sensing of organic carbon (glucose) by cAMP is well-understood, a mechanistic link for the thus far unclear relationship between cAMP and C_i sensing has been enigmatic. The P_II-like signaling protein SbtB represents a novel type of cAMP receptor protein and perceives C_i status via binding of the second messenger cAMP. In eukaryotes, cAMP transmits signals that originate at G protein-coupled receptors and modulate cellular activities mainly through cAMP-dependent protein kinases (33), thereby affecting gene expression or central metabolism, in particular glycogen, sugar, and lipid metabolism. In bacteria, cAMP mediates manifold cellular responses, mainly via cAMP-responsive transcription factors, such as CRP (34, 35), but also via cAMP-controlled protein modification enzymes, such as protein kinases and GNAT-related protein lysine acetylases (36). With SbtB, a novel mechanism of cAMP-mediated signaling in cyanobacterial carbon regulation has been unraveled, and a functional link between CO₂ acquisition and cAMP sensing has been revealed. However, further studies are required to mechanistically understand the interaction with the target SbtA as well as the interplay between the various adenyl-nucleotide effector molecules for energy and carbon sensing.

Materials and Methods

Full protocols are available in SI Appendix, Materials and Methods.

Purification of Recombinant Proteins.

Recombinant C-terminal StrepII-tagged proteins (ScSbtB and canonical SeP_II) were purified as previously described (9, 10).

Determination of ScSbtB Binding Constants.

ScSbtB binding constants were determined using ITC (VP-ITC microcalorimeter; MicroCal) and MST (Monolith NT.LabelFree; NanoTemper Technologies). The ITC binding isotherms were calculated from the recorded data and fitted to one-site and three-site binding models using MicroCal Origin software (10). MST single-site fitting was done using NanoTemper data analysis software.

Crystallization, Data Collection, and Structure Elucidation.

Proteins were crystallized via vapor diffusion at 20 °C in 96-well sitting-drop plates. The structure of apo-ScSbtB was solved by molecular replacement using MOLREP (37) and the AvSbtB structure (PDB ID code 3DFE) as a search model. Other structures were solved subsequently on the basis of the apo-ScSbtB coordinates (PDB ID code 5O3P). Structural representations were prepared using UCSF Chimera (https://www.cgl.ucsf.edu/chimera/).

Generation of Mutants.

Knockout deletion mutants were constructed by cloning the upstream and downstream regions of the ORF slr1513 (designated sbtB) (4, 5) and the ORF slr1991 (designated cya1) (12) with the erythromycin resistance cassette (primers are listed in SI Appendix, Table S1). For complementation, SbtB-sfGFP strains were generated by introducing the sbtB gene (slr1513) fused to the gene encoding sfGFP into ΔsbtB and WT backgrounds using the self-replicating plasmid pVZ322.

CO₂-Shift Experiments.

CO₂-shift experiments were performed according to ref. 38. Briefly, cells were precultured in standard BG₁₁ medium (pH 8) with bubbling of air-enriched CO₂ [2 to 5% (vol/vol); defined as high-carbon conditions] at a light intensity of about 100 µE. Cells were shifted to low CO₂ by bubbling with ambient air [0.04% (vol/vol) CO₂; defined as low-carbon conditions]. For a harsh carbon shift, cells were transferred to BG₁₁ medium free of HCO₃⁻ and CO₃⁻² and kept stationary. For a shift to HC conditions, the gas stream of LC-grown cells was switched from ambient air to CO₂-supplemented air.

Phenotypic Characterization of the ∆sbtB Mutant.

Wild-type Synechocystis sp. PCC 6803 and ΔsbtB cells were grown under various CO₂ conditions as mentioned above. The survival of cells was checked in standard BG₁₁ medium in a drop-dilution assay on agar plates of various pH and by measuring growth rates in liquid medium. The rate of C_i-dependent oxygen evolution as a function of increasing HCO₃⁻ supply was determined using a Clark-type oxygen electrode (Hansatech).

Transcriptional Analysis.

Total RNA was isolated using the PGTX method (39). Reverse transcription was done using the RevertAid RT Kit (Thermo Fisher). Aliquots of the generated cDNA were used for the RT-PCR assays of selected C_i-regulated genes using gene-specific primers (SI Appendix, Table S1).

Quantification of cAMP and AMP.

cAMP and AMP were extracted according to ref. 40. The intracellular levels of cAMP and AMP were analyzed and determined using the LCMS-8050 System and LC/MS/MS Method Package for Primary Metabolites version 2 (Shimadzu). For quantification of cAMP and AMP, authentic substances in appropriate concentrations were used for calibration.

Immunoblot Analysis of the Cellular Localization of ScSbtB.

For immunoblot analysis, the crude cell extract from WT cells grown in or shifted to different CO₂ conditions as indicated was prepared using a ribolyser for four cycles at 4 °C. For localization experiments, total cell extracts were fractionated into membranes and soluble fractions according to ref. 30.

Acknowledgments

The authors are grateful to Reinhard Albrecht for crystallographic sample preparation and assistance with diffraction data collection, the staff of beamline X10SA/SLS, and Karen Brune for critical linguistic editing of the manuscript. M.D.H. gratefully acknowledges Andrei Lupas for continued support. K.A.S. and K.F. thank all members of the Organismic Interactions Department, especially Jan Lüddecke and Carolina Nishi, for technical assistance. This work was supported by Deutsche Forschungsgemeinschaft Grant Fo195/9-2, Graduiertenkolleg Grant 1708-2, and Deutsch Akademischer Austauschdienst (DAAD); the Hochschulbauförderungsgesetz (HBFG) program financed the LC/MS equipment (GZ: INST 264/125-1 FUGG).

Footnotes

↵¹To whom correspondence may be addressed. Email: khaled.selim{at}uni-tuebingen.de or karl.forchhammer{at}uni-tuebingen.de.

Author contributions: K.A.S., M.D.H., M.H., and K.F. designed research; K.A.S. performed biochemical, structural, and biophysical experiments; K.A.S. and F.H. performed physiological experiments; K.A.S., M.D.H., M.H., and K.F. analyzed data; and K.A.S., M.D.H., M.H., and K.F. wrote the manuscript.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: Crystallography, atomic coordinates, and structure factors have been deposited in the Protein Data Bank, www.wwpdb.org (PDB ID codes 5O3P, 5O3Q, 5O3R, and 5O3S).
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1803790115/-/DCSupplemental.

Published under the PNAS license.

References

↵
1. Hohmann-Marriott MF,
2. Blankenship RE
(2011) Evolution of photosynthesis. Annu Rev Plant Biol 62:515–548.
OpenUrl CrossRef PubMed
↵
1. Rae BD,
2. Long BM,
3. Badger MR,
4. Price GD
(2013) Functions, compositions, and evolution of the two types of carboxysomes: Polyhedral microcompartments that facilitate CO₂ fixation in cyanobacteria and some proteobacteria. Microbiol Mol Biol Rev 77:357–379.
OpenUrl Abstract/FREE Full Text
↵
1. Burnap RL,
2. Hagemann M,
3. Kaplan A
(2015) Regulation of CO₂ concentrating mechanism in cyanobacteria. Life (Basel) 5:348–371.
OpenUrl CrossRef PubMed
↵
1. Shibata M, et al.
(2002) Genes essential to sodium-dependent bicarbonate transport in cyanobacteria: Function and phylogenetic analysis. J Biol Chem 277:18658–18664.
OpenUrl Abstract/FREE Full Text
↵
1. Wang HL,
2. Postier BL,
3. 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
↵
1. Schwarz D,
2. Schubert H,
3. Georg J,
4. Hess WR,
5. Hagemann M
(2013) The gene sml0013 of Synechocystis species strain PCC 6803 encodes for a novel subunit of the NAD(P)H oxidoreductase or complex I that is ubiquitously distributed among cyanobacteria. Plant Physiol 163:1191–1202.
OpenUrl Abstract/FREE Full Text
↵
1. Du J,
2. Förster B,
3. Rourke L,
4. Howitt SM,
5. Price GD
(2014) Characterisation of cyanobacterial bicarbonate transporters in E. coli shows that SbtA homologs are functional in this heterologous expression system. PLoS One 9:e115905.
OpenUrl CrossRef PubMed
↵
1. Forchhammer K,
2. Lüddecke J
(2016) Sensory properties of the P_II signalling protein family. FEBS J 283:425–437.
OpenUrl CrossRef PubMed
↵
1. Fokina O,
2. Chellamuthu VR,
3. Zeth K,
4. Forchhammer K
(2010) A novel signal transduction protein P(II) variant from Synechococcus elongatus PCC 7942 indicates a two-step process for NAGK-P(II) complex formation. J Mol Biol 399:410–421.
OpenUrl CrossRef PubMed
↵
1. Lapina T,
2. Selim KA,
3. Forchhammer K,
4. Ermilova E
(2018) The P_II signaling protein from red algae represents an evolutionary link between cyanobacterial and Chloroplastida P_II proteins. Sci Rep 8:790.
OpenUrl CrossRef
↵
1. Wheatley NM, et al.
(2016) A PII-like protein regulated by bicarbonate: Structural and biochemical studies of the carboxysome-associated CP_II protein. J Mol Biol 428:4013–4030.
OpenUrl
↵
1. Terauchi K,
2. Ohmori M
(1999) An adenylate cyclase, Cya1, regulates cell motility in the cyanobacterium Synechocystis sp. PCC 6803. Plant Cell Physiol 40:248–251.
OpenUrl CrossRef PubMed
↵
1. Huergo LF,
2. Chandra G,
3. Merrick M
(2013) P(II) signal transduction proteins: Nitrogen regulation and beyond. FEMS Microbiol Rev 37:251–283.
OpenUrl CrossRef PubMed
↵
1. Shibata M, et al.
(2001) Distinct constitutive and low-CO₂-induced CO₂ uptake systems in cyanobacteria: Genes involved and their phylogenetic relationship with homologous genes in other organisms. Proc Natl Acad Sci USA 98:11789–11794.
OpenUrl Abstract/FREE Full Text
↵
1. Price GD
(2011) Inorganic carbon transporters of the cyanobacterial CO₂ concentrating mechanism. Photosynth Res 109:47–57.
OpenUrl CrossRef PubMed
↵
1. Price GD,
2. Woodger FJ,
3. Badger MR,
4. Howitt SM,
5. Tucker L
(2004) Identification of a SulP-type bicarbonate transporter in marine cyanobacteria. Proc Natl Acad Sci USA 101:18228–18233.
OpenUrl Abstract/FREE Full Text
↵
1. Lüddecke J,
2. Forchhammer K
(2015) Energy sensing versus 2-oxoglutarate dependent ATPase switch in the control of Synechococcus P_II interaction with its targets NAGK and PipX. PLoS One 10:e0137114.
OpenUrl
↵
1. Fokina O,
2. Chellamuthu VR,
3. Forchhammer K,
4. Zeth K
(2010) Mechanism of 2-oxoglutarate signaling by the Synechococcus elongatus P_II signal transduction protein. Proc Natl Acad Sci USA 107:19760–19765.
OpenUrl Abstract/FREE Full Text
↵
1. Radchenko MV,
2. Thornton J,
3. Merrick M
(2013) P(II) signal transduction proteins are ATPases whose activity is regulated by 2-oxoglutarate. Proc Natl Acad Sci USA 110:12948–12953.
OpenUrl Abstract/FREE Full Text
↵
1. Michelet L, et al.
(2013) Redox regulation of the Calvin-Benson cycle: Something old, something new. Front Plant Sci 4:470.
OpenUrl CrossRef PubMed
↵
1. Tamoi M,
2. Shigeoka S
(2015) Diversity of regulatory mechanisms of photosynthetic carbon metabolism in plants and algae. Biosci Biotechnol Biochem 79:870–876.
OpenUrl
↵
1. Chen Y, et al.
(2000) Soluble adenylyl cyclase as an evolutionarily conserved bicarbonate sensor. Science 289:625–628.
OpenUrl Abstract/FREE Full Text
↵
1. Cann MJ,
2. Hammer A,
3. Zhou J,
4. Kanacher T
(2003) A defined subset of adenylyl cyclases is regulated by bicarbonate ion. J Biol Chem 278:35033–35038.
OpenUrl Abstract/FREE Full Text
↵
1. Steegborn C,
2. Litvin TN,
3. Levin LR,
4. Buck J,
5. Wu H
(2005) Bicarbonate activation of adenylyl cyclase via promotion of catalytic active site closure and metal recruitment. Nat Struct Mol Biol 12:32–37.
OpenUrl CrossRef PubMed
↵
1. Hammer A,
2. Hodgson DR,
3. Cann MJ
(2006) Regulation of prokaryotic adenylyl cyclases by CO₂. Biochem J 396:215–218.
OpenUrl Abstract/FREE Full Text
↵
1. Hennon GMM, et al.
(2015) Diatom acclimation to elevated CO₂ via cAMP signalling and coordinated gene expression. Nat Clim Chang 5:761–765.
OpenUrl
↵
1. Hood RD,
2. Higgins SA,
3. Flamholz A,
4. Nichols RJ,
5. Savage DF
(2016) The stringent response regulates adaptation to darkness in the cyanobacterium Synechococcus elongatus. Proc Natl Acad Sci USA 113:E4867–E4876.
OpenUrl Abstract/FREE Full Text
↵
1. Puszynska AM,
2. O’Shea EK
(2017) ppGpp controls global gene expression in light and in darkness in S. elongatus. Cell Rep 21:3155–3165.
OpenUrl CrossRef
↵
1. Schwarz D, et al.
(2011) Metabolic and transcriptomic phenotyping of inorganic carbon acclimation in the cyanobacterium Synechococcus elongatus PCC 7942. Plant Physiol 155:1640–1655.
OpenUrl Abstract/FREE Full Text
↵
1. Heinrich A, et al.
(2006) Interaction of the membrane-bound GlnK-AmtB complex with the master regulator of nitrogen metabolism TnrA in Bacillus subtilis. J Biol Chem 281:34909–34917.
OpenUrl Abstract/FREE Full Text
↵
1. Radchenko MV,
2. Thornton J,
3. Merrick M
(2010) Control of AmtB-GlnK complex formation by intracellular levels of ATP, ADP, and 2-oxoglutarate. J Biol Chem 285:31037–31045.
OpenUrl Abstract/FREE Full Text
↵
1. Huergo LF, et al.
(2006) Interactions between P_II proteins and the nitrogenase regulatory enzymes DraT and DraG in Azospirillum brasilense. FEBS Lett 580:5232–5236.
OpenUrl CrossRef PubMed
↵
1. Sassone-Corsi P
(2012) The cyclic AMP pathway. Cold Spring Harb Perspect Biol 4:a011148.
OpenUrl FREE Full Text
↵
1. Ohmori M,
2. Okamoto S
(2004) Photoresponsive cAMP signal transduction in cyanobacteria. Photochem Photobiol Sci 3:503–511.
OpenUrl CrossRef PubMed
↵
1. Forcada-Nadal A,
2. Forchhammer K,
3. Rubio V
(2014) SPR analysis of promoter binding of Synechocystis PCC6803 transcription factors NtcA and CRP suggests cross-talk and sheds light on regulation by effector molecules. FEBS Lett 588:2270–2276.
OpenUrl CrossRef PubMed
↵
1. Nambi S,
2. Basu N,
3. Visweswariah SS
(2010) cAMP-regulated protein lysine acetylases in mycobacteria. J Biol Chem 285:24313–24323.
OpenUrl Abstract/FREE Full Text
↵
1. Vagin A,
2. Teplyakov A
(2010) Molecular replacement with MOLREP. Acta Crystallogr D Biol Crystallogr 66:22–25.
OpenUrl CrossRef PubMed
↵
1. Orf I, et al.
(2015) Integrated analysis of engineered carbon limitation in a quadruple CO₂/HCO₃⁻ uptake mutant of Synechocystis sp. PCC 6803. Plant Physiol 169:1787–1806.
OpenUrl Abstract/FREE Full Text
↵
1. Pinto FL,
2. Thapper A,
3. Sontheim W,
4. Lindblad P
(2009) Analysis of current and alternative phenol based RNA extraction methodologies for cyanobacteria. BMC Mol Biol 10:79.
OpenUrl CrossRef PubMed
↵
1. Imashimizu M,
2. Yoshimura H,
3. Katoh H,
4. Ehira S,
5. Ohmori M
(2005) NaCl enhances cellular cAMP and upregulates genes related to heterocyst development in the cyanobacterium, Anabaena sp. strain PCC 7120. FEMS Microbiol Lett 252:97–103.
OpenUrl CrossRef PubMed

Article Alerts

Email Article

Citation Tools

Request Permissions

Article Classifications

Biological Sciences
Microbiology

Proceedings of the National Academy of Sciences: 115 (21)

Table of Contents

Submit

[1] ↵
Hohmann-Marriott MF,
Blankenship RE
(2011) Evolution of photosynthesis. Annu Rev Plant Biol 62:515–548.
OpenUrl CrossRef PubMed

[2] Hohmann-Marriott MF,

[3] Blankenship RE

[4] ↵
Rae BD,
Long BM,
Badger MR,
Price GD
(2013) Functions, compositions, and evolution of the two types of carboxysomes: Polyhedral microcompartments that facilitate CO₂ fixation in cyanobacteria and some proteobacteria. Microbiol Mol Biol Rev 77:357–379.
OpenUrl Abstract/FREE Full Text

[5] Rae BD,

[6] Long BM,

[7] Badger MR,

[8] Price GD

[9] ↵
Burnap RL,
Hagemann M,
Kaplan A
(2015) Regulation of CO₂ concentrating mechanism in cyanobacteria. Life (Basel) 5:348–371.
OpenUrl CrossRef PubMed

[10] Burnap RL,

[11] Hagemann M,

[12] Kaplan A

[13] ↵
Shibata M, et al.
(2002) Genes essential to sodium-dependent bicarbonate transport in cyanobacteria: Function and phylogenetic analysis. J Biol Chem 277:18658–18664.
OpenUrl Abstract/FREE Full Text

[14] Shibata M, et al.

[15] ↵
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

[16] Wang HL,

[17] Postier BL,

[18] Burnap RL

[19] ↵
Schwarz D,
Schubert H,
Georg J,
Hess WR,
Hagemann M
(2013) The gene sml0013 of Synechocystis species strain PCC 6803 encodes for a novel subunit of the NAD(P)H oxidoreductase or complex I that is ubiquitously distributed among cyanobacteria. Plant Physiol 163:1191–1202.
OpenUrl Abstract/FREE Full Text

[20] Schwarz D,

[21] Schubert H,

[22] Georg J,

[23] Hess WR,

[24] Hagemann M

[25] ↵
Du J,
Förster B,
Rourke L,
Howitt SM,
Price GD
(2014) Characterisation of cyanobacterial bicarbonate transporters in E. coli shows that SbtA homologs are functional in this heterologous expression system. PLoS One 9:e115905.
OpenUrl CrossRef PubMed

[26] Du J,

[27] Förster B,

[28] Rourke L,

[29] Howitt SM,

[30] Price GD

[31] ↵
Forchhammer K,
Lüddecke J
(2016) Sensory properties of the P_II signalling protein family. FEBS J 283:425–437.
OpenUrl CrossRef PubMed

[32] Forchhammer K,

[33] Lüddecke J

[34] ↵
Fokina O,
Chellamuthu VR,
Zeth K,
Forchhammer K
(2010) A novel signal transduction protein P(II) variant from Synechococcus elongatus PCC 7942 indicates a two-step process for NAGK-P(II) complex formation. J Mol Biol 399:410–421.
OpenUrl CrossRef PubMed

[35] Fokina O,

[36] Chellamuthu VR,

[37] Zeth K,

[38] Forchhammer K

[39] ↵
Lapina T,
Selim KA,
Forchhammer K,
Ermilova E
(2018) The P_II signaling protein from red algae represents an evolutionary link between cyanobacterial and Chloroplastida P_II proteins. Sci Rep 8:790.
OpenUrl CrossRef

[40] Lapina T,

[41] Selim KA,

[42] Forchhammer K,

[43] Ermilova E

[44] ↵
Wheatley NM, et al.
(2016) A PII-like protein regulated by bicarbonate: Structural and biochemical studies of the carboxysome-associated CP_II protein. J Mol Biol 428:4013–4030.
OpenUrl

[45] Wheatley NM, et al.

[46] ↵
Terauchi K,
Ohmori M
(1999) An adenylate cyclase, Cya1, regulates cell motility in the cyanobacterium Synechocystis sp. PCC 6803. Plant Cell Physiol 40:248–251.
OpenUrl CrossRef PubMed

[47] Terauchi K,

[48] Ohmori M

[49] ↵
Huergo LF,
Chandra G,
Merrick M
(2013) P(II) signal transduction proteins: Nitrogen regulation and beyond. FEMS Microbiol Rev 37:251–283.
OpenUrl CrossRef PubMed

[50] Huergo LF,

[51] Chandra G,

[52] Merrick M

[53] ↵
Shibata M, et al.
(2001) Distinct constitutive and low-CO₂-induced CO₂ uptake systems in cyanobacteria: Genes involved and their phylogenetic relationship with homologous genes in other organisms. Proc Natl Acad Sci USA 98:11789–11794.
OpenUrl Abstract/FREE Full Text

[54] Shibata M, et al.

[55] ↵
Price GD
(2011) Inorganic carbon transporters of the cyanobacterial CO₂ concentrating mechanism. Photosynth Res 109:47–57.
OpenUrl CrossRef PubMed

[56] Price GD

[57] ↵
Price GD,
Woodger FJ,
Badger MR,
Howitt SM,
Tucker L
(2004) Identification of a SulP-type bicarbonate transporter in marine cyanobacteria. Proc Natl Acad Sci USA 101:18228–18233.
OpenUrl Abstract/FREE Full Text

[58] Price GD,

[59] Woodger FJ,

[60] Badger MR,

[61] Howitt SM,

[62] Tucker L

[63] ↵
Lüddecke J,
Forchhammer K
(2015) Energy sensing versus 2-oxoglutarate dependent ATPase switch in the control of Synechococcus P_II interaction with its targets NAGK and PipX. PLoS One 10:e0137114.
OpenUrl

[64] Lüddecke J,

[65] Forchhammer K

[66] ↵
Fokina O,
Chellamuthu VR,
Forchhammer K,
Zeth K
(2010) Mechanism of 2-oxoglutarate signaling by the Synechococcus elongatus P_II signal transduction protein. Proc Natl Acad Sci USA 107:19760–19765.
OpenUrl Abstract/FREE Full Text

[67] Fokina O,

[68] Chellamuthu VR,

[69] Forchhammer K,

[70] Zeth K

[71] ↵
Radchenko MV,
Thornton J,
Merrick M
(2013) P(II) signal transduction proteins are ATPases whose activity is regulated by 2-oxoglutarate. Proc Natl Acad Sci USA 110:12948–12953.
OpenUrl Abstract/FREE Full Text

[72] Radchenko MV,

[73] Thornton J,

[74] Merrick M

[75] ↵
Michelet L, et al.
(2013) Redox regulation of the Calvin-Benson cycle: Something old, something new. Front Plant Sci 4:470.
OpenUrl CrossRef PubMed

[76] Michelet L, et al.

[77] ↵
Tamoi M,
Shigeoka S
(2015) Diversity of regulatory mechanisms of photosynthetic carbon metabolism in plants and algae. Biosci Biotechnol Biochem 79:870–876.
OpenUrl

[78] Tamoi M,

[79] Shigeoka S

[80] ↵
Chen Y, et al.
(2000) Soluble adenylyl cyclase as an evolutionarily conserved bicarbonate sensor. Science 289:625–628.
OpenUrl Abstract/FREE Full Text

[81] Chen Y, et al.

[82] ↵
Cann MJ,
Hammer A,
Zhou J,
Kanacher T
(2003) A defined subset of adenylyl cyclases is regulated by bicarbonate ion. J Biol Chem 278:35033–35038.
OpenUrl Abstract/FREE Full Text

[83] Cann MJ,

[84] Hammer A,

[85] Zhou J,

[86] Kanacher T

[87] ↵
Steegborn C,
Litvin TN,
Levin LR,
Buck J,
Wu H
(2005) Bicarbonate activation of adenylyl cyclase via promotion of catalytic active site closure and metal recruitment. Nat Struct Mol Biol 12:32–37.
OpenUrl CrossRef PubMed

[88] Steegborn C,

[89] Litvin TN,

[90] Levin LR,

[91] Buck J,

[92] Wu H

[93] ↵
Hammer A,
Hodgson DR,
Cann MJ
(2006) Regulation of prokaryotic adenylyl cyclases by CO₂. Biochem J 396:215–218.
OpenUrl Abstract/FREE Full Text

[94] Hammer A,

[95] Hodgson DR,

[96] Cann MJ

[97] ↵
Hennon GMM, et al.
(2015) Diatom acclimation to elevated CO₂ via cAMP signalling and coordinated gene expression. Nat Clim Chang 5:761–765.
OpenUrl

[98] Hennon GMM, et al.

[99] ↵
Hood RD,
Higgins SA,
Flamholz A,
Nichols RJ,
Savage DF
(2016) The stringent response regulates adaptation to darkness in the cyanobacterium Synechococcus elongatus. Proc Natl Acad Sci USA 113:E4867–E4876.
OpenUrl Abstract/FREE Full Text

[100] Hood RD,

[101] Higgins SA,

[102] Flamholz A,

[103] Nichols RJ,

[104] Savage DF

[105] ↵
Puszynska AM,
O’Shea EK
(2017) ppGpp controls global gene expression in light and in darkness in S. elongatus. Cell Rep 21:3155–3165.
OpenUrl CrossRef

[106] Puszynska AM,

[107] O’Shea EK

[108] ↵
Schwarz D, et al.
(2011) Metabolic and transcriptomic phenotyping of inorganic carbon acclimation in the cyanobacterium Synechococcus elongatus PCC 7942. Plant Physiol 155:1640–1655.
OpenUrl Abstract/FREE Full Text

[109] Schwarz D, et al.

[110] ↵
Heinrich A, et al.
(2006) Interaction of the membrane-bound GlnK-AmtB complex with the master regulator of nitrogen metabolism TnrA in Bacillus subtilis. J Biol Chem 281:34909–34917.
OpenUrl Abstract/FREE Full Text

[111] Heinrich A, et al.

[112] ↵
Radchenko MV,
Thornton J,
Merrick M
(2010) Control of AmtB-GlnK complex formation by intracellular levels of ATP, ADP, and 2-oxoglutarate. J Biol Chem 285:31037–31045.
OpenUrl Abstract/FREE Full Text

[113] Radchenko MV,

[114] Thornton J,

[115] Merrick M

[116] ↵
Huergo LF, et al.
(2006) Interactions between P_II proteins and the nitrogenase regulatory enzymes DraT and DraG in Azospirillum brasilense. FEBS Lett 580:5232–5236.
OpenUrl CrossRef PubMed

[117] Huergo LF, et al.

[118] ↵
Sassone-Corsi P
(2012) The cyclic AMP pathway. Cold Spring Harb Perspect Biol 4:a011148.
OpenUrl FREE Full Text

[119] Sassone-Corsi P

[120] ↵
Ohmori M,
Okamoto S
(2004) Photoresponsive cAMP signal transduction in cyanobacteria. Photochem Photobiol Sci 3:503–511.
OpenUrl CrossRef PubMed

[121] Ohmori M,

[122] Okamoto S

[123] ↵
Forcada-Nadal A,
Forchhammer K,
Rubio V
(2014) SPR analysis of promoter binding of Synechocystis PCC6803 transcription factors NtcA and CRP suggests cross-talk and sheds light on regulation by effector molecules. FEBS Lett 588:2270–2276.
OpenUrl CrossRef PubMed

[124] Forcada-Nadal A,

[125] Forchhammer K,

[126] Rubio V

[127] ↵
Nambi S,
Basu N,
Visweswariah SS
(2010) cAMP-regulated protein lysine acetylases in mycobacteria. J Biol Chem 285:24313–24323.
OpenUrl Abstract/FREE Full Text

[128] Nambi S,

[129] Basu N,

[130] Visweswariah SS

[131] ↵
Vagin A,
Teplyakov A
(2010) Molecular replacement with MOLREP. Acta Crystallogr D Biol Crystallogr 66:22–25.
OpenUrl CrossRef PubMed

[132] Vagin A,

[133] Teplyakov A

[134] ↵
Orf I, et al.
(2015) Integrated analysis of engineered carbon limitation in a quadruple CO₂/HCO₃⁻ uptake mutant of Synechocystis sp. PCC 6803. Plant Physiol 169:1787–1806.
OpenUrl Abstract/FREE Full Text

[135] Orf I, et al.

[136] ↵
Pinto FL,
Thapper A,
Sontheim W,
Lindblad P
(2009) Analysis of current and alternative phenol based RNA extraction methodologies for cyanobacteria. BMC Mol Biol 10:79.
OpenUrl CrossRef PubMed

[137] Pinto FL,

[138] Thapper A,

[139] Sontheim W,

[140] Lindblad P

[141] ↵
Imashimizu M,
Yoshimura H,
Katoh H,
Ehira S,
Ohmori M
(2005) NaCl enhances cellular cAMP and upregulates genes related to heterocyst development in the cyanobacterium, Anabaena sp. strain PCC 7120. FEMS Microbiol Lett 252:97–103.
OpenUrl CrossRef PubMed

[142] Imashimizu M,

[143] Yoshimura H,

[144] Katoh H,

[145] Ehira S,

[146] Ohmori M

Main menu

User menu

Search

P_II-like signaling protein SbtB links cAMP sensing with cyanobacterial inorganic carbon response

Significance

Abstract

Results

Biochemical Properties of ScSbtB.

Structural Characterization of ScSbtB.

Structural Characterization of Ligand Binding.

ScSbtB Reversibly Associates with Membranes in an SbtA-Dependent Manner.

ScSbtB Is Required for Efficient C_i Acclimation.

cAMP/AMP as a C_i Signal Compete for ScSbtB.

Discussion

Materials and Methods

Purification of Recombinant Proteins.

Determination of ScSbtB Binding Constants.

Crystallization, Data Collection, and Structure Elucidation.

Generation of Mutants.

CO₂-Shift Experiments.

Phenotypic Characterization of the ∆sbtB Mutant.

Transcriptional Analysis.

Quantification of cAMP and AMP.

Immunoblot Analysis of the Cellular Localization of ScSbtB.

Acknowledgments

Footnotes

References

Citation Manager Formats

Article Classifications

You May Also be Interested in

Similar Articles

Articles

PNAS Portals

Information

Main menu

User menu

Search

Significance

Abstract

Results

Biochemical Properties of ScSbtB.

Structural Characterization of ScSbtB.

Structural Characterization of Ligand Binding.

ScSbtB Reversibly Associates with Membranes in an SbtA-Dependent Manner.

ScSbtB Is Required for Efficient Ci Acclimation.

cAMP/AMP as a Ci Signal Compete for ScSbtB.

Discussion

Materials and Methods

Purification of Recombinant Proteins.

Determination of ScSbtB Binding Constants.

Crystallization, Data Collection, and Structure Elucidation.

Generation of Mutants.

CO2-Shift Experiments.

Phenotypic Characterization of the ∆sbtB Mutant.

Transcriptional Analysis.

Quantification of cAMP and AMP.

Immunoblot Analysis of the Cellular Localization of ScSbtB.

Acknowledgments

Footnotes

References

Citation Manager Formats

Article Classifications

Sign up for Article Alerts

Jump to section

You May Also be Interested in

Similar Articles

Articles

PNAS Portals

Information

ScSbtB Is Required for Efficient C_i Acclimation.

cAMP/AMP as a C_i Signal Compete for ScSbtB.

CO₂-Shift Experiments.