Roles of second messengers in the regulation of cyanobacterial physiology: the carbon-concentrating mechanism and beyond

Abstract

Second messengers are a fundamental category of small molecules and ions that are involved in the regulation of many processes in all domains of life. Here we focus on cyanobacteria, prokaryotes playing important roles as primary producers in the geochemical cycles due to their capability of oxygenic photosynthesis and carbon and nitrogen fixation. Of particular interest is the inorganic carbon-concentrating mechanism (CCM), which allows cyanobacteria to concentrate CO₂ near RubisCO. This mechanism needs to acclimate toward fluctuating conditions, such as inorganic carbon availability, intracellular energy levels, diurnal light cycle, light intensity, nitrogen availability, and redox state of the cell. During acclimation to such changing conditions, second messengers play a crucial role, particularly important is their interaction with the carbon control protein SbtB, a member of the PII regulator protein superfamily. SbtB is capable of binding several second messengers, uniquely adenyl nucleotides, to interact with different partners in a variety of responses. The main identified interaction partner is the bicarbonate transporter SbtA, which is regulated via SbtB depending on the energy state of the cell, the light conditions, and different CO₂ availability, including cAMP signaling. The interaction with the glycogen branching enzyme, GlgB, showed a role for SbtB in the c-di-AMP-dependent regulation of glycogen synthesis during the diurnal life cycle of cyanobacteria. SbtB has also been shown to impact gene expression and metabolism during acclimation to changing CO₂ conditions. This review summarizes the current knowledge about the complex second messenger regulatory network in cyanobacteria, with emphasis on carbon metabolism.

Keywords: CCM; SbtB; Synechocystis; c-di-AMP; cAMP; glycogen.

Figure 1.

Schematic representation of reported environmental stimuli on the concentrations of the most important second messengers in cyanobacteria (as shown in text). Above and below the cyanobacterial cell, the specific stimuli, first messengers are represented. The effect of each is shown inside the cell, with green and red highlights indicating an increase and decrease in the concentration of the specific second messengers, respectively. In the top, from the left, the effect of blue light, UV light, day/night cycle, and temperature stress are shown. In the bottom, from the left, the signaling of high/low inorganic carbon, nitrogen starvation, low/high pH, and osmotic stress are displayed. In the center of the cell, DNA and carboxysomes are depicted as targets of second messenger signaling.

Figure 2.

Schematic representation of the cyanobacterial inorganic carbon-concentrating mechanism (CCM). Three bicarbonate transporters (SbtA, BicA, and BCT1) and the Na⁺-gradient restoring different Na⁺/H⁺ antiporters (NhaS1-6) are located at the plasma membrane. Together with two CO₂-hydrating systems on thylakoid membranes they accumulate high internal bicarbonate levels. Bicarbonate is diffusing into the carboxysome, in which CO₂ is released by carbonic anhydrase (CA) thereby promoting the carboxylation reaction of RuBisCO.

Figure 3.

Structural insights and sensing properties of SbtB protein. (A) Top and side view on the trimeric architecture of the SbtB-c-di-AMP complex (PDB:7OBJ; in ribbon representation with different color for each monomer). The SbtB structure reveals the typical ferredoxin-like fold of the PII superfamily, with nucleotide-binding pockets located between the clefts of the subunits. The characteristic structural motifs (T- and R-loops) are indicated. The large flexible T-loop is partially ordered and therefore not fully resolved. The oxidized R-loop forms a hairpin via disulfide bond between Cys₁₀₅ and Cys₁₁₀ residues. (B) Superposition of monomeric subunits of SbtB complexed with either AMP (violet; PDB:5O3R), c-di-AMP (brown; PDB:7OBJ), ATP (green; PDB:7R31), or SbtA (yellow; PDB:7EGK), showing the high flexibility of the T-loop with different conformations. The T-loop is the major target-binding element of PII superfamily proteins. The secondary structure elements and characteristic structural motifs are indicated. (Inset) The oxidized SbtB R-loop (C₁₀₅GPEGC₁₁₀) motif with the disulfide bond is highlighted. The reduced R-loop in the SbtB-ATP complex (green; PDB:7R31) is disordered, while the T-loop is fully structured. (C–F) Close-up of the nucleotide binding site of SbtB-ATP (C), SbtB-AMP (D), SbtB-cAMP (E), and SbtB-c-di-AMP (F). The relevant residues for nucleotide binding and the nucleotides are shown in stick representation, with O, N, and P atoms colored in red, blue, and orange, respectively. H-bonds are indicated by black lines. The R₄₃xxR₄₆ motif coordinates the ATP phosphate groups. For more details, see Selim et al. (2018; 2021b; 2023a).

Figure 4.

Schematic representation of the regulatory mechanism of SbtB and second messengers in Synechocystis sp. PCC 6803. On the left, the mechanism during the night is shown. The absence of photosynthesis causes the cell to be in an oxidized state, leading the R-loop of SbtB (shown in red) to be oxidized (note the closed drawing of the SbtB tail), activating its apyrase (ATPase/ADPase) activity to reach to the AMP-state, which stabilizes SbtA-SbtB complex formation. SbtB’s role is probably to prevent wasteful bicarbonate uptake in the night by interacting with SbtA (shown in green). The night-active ATPase/ADPase activity keeps SbtB constantly in the AMP state, maintaining its inhibitory function. On the right, the mechanism during the day is shown in cells exposed either to low or high Ci conditions. Under photosynthetic conditions, the R-loop becomes reduced, inhibiting SbtB apyrase activity. Now, SbtB responds to steady state level changes of the various adenyl-nucleotides. Low CO₂ conditions correlate with increased AMP levels and low cAMP levels, conditions that favor SbtA/SbtB complex formation. Despite being complexed by SbtB, the bicarbonate transporter SbtA is active and highly expressed and thus, the intracellular concentrations of inorganic carbon (Ci) increase. When exposed to high CO₂ conditions, the bicarbonate transporter SbtA is inactivated probably involving changes in the effector binding state of SbtB mainly via cAMP. Apart from the regulation of SbtA during the day, SbtB performs other regulatory functions. Via the interaction with c-di-AMP, it modulates the activity of the glycogen branching enzyme GlgB, to regulate the synthesis of glycogen. The changing c-di-AMP concentrations also perform a regulatory role on the gene regulation of the CCM, through interaction with SbtB and other unidentified transcription factors. cAMP concentrations increase upon entering high CO₂ conditions, and causes the inhibition of the CCM through the action of SbtB and other regulators, likely including the transcription factor SyCRP.