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

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.

Keywords: PII-like protein SbtB; cAMP; cyanobacteria; inorganic carbon signaling; signal transduction.

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.

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.

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.

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.