Regulation of the cyanobacterial CO2-concentrating mechanism involves internal sensing of NADP+ and α-ketogutarate levels by transcription factor CcmR.

Inorganic carbon is the major macronutrient required by organisms utilizing oxygenic photosynthesis for autotrophic growth. Aquatic photoautotrophic organisms are dependent upon a CO(2) concentrating mechanism (CCM) to overcome the poor CO(2)-affinity of the major carbon-fixing enzyme, ribulose-bisphosphate carboxylase/oxygenase (Rubisco). The CCM involves the active transport of inorganic forms of carbon (C(i)) into the cell to increase the CO(2) concentration around the active site of Rubisco. It employs both bicarbonate transporters and redox-powered CO(2)-hydration enzymes coupled to membranous NDH-type electron transport complexes that collectively produce C(i) concentrations up to a 1000-fold greater in the cytoplasm compared to the external environment. The CCM is regulated: a high affinity CCM comprised of multiple components is induced under limiting external Ci concentrations. The LysR-type transcriptional regulator CcmR has been shown to repress its own expression along with structural genes encoding high affinity C(i) transporters distributed throughout the genome of Synechocystis sp. PCC 6803. While much has been learned about the structural genes of the CCM and the identity of the transcriptional regulators controlling their expression, little is known about the physiological signals that elicit the induction of the high affinity CCM. Here CcmR is studied to identify metabolites that modulate its transcriptional repressor activity. Using surface plasmon resonance (SPR) α-ketoglutarate (α-KG) and the oxidized form of nicotinamide adenine dinucleotide phosphate (NADP(+)) have been identified as the co-repressors of CcmR. Additionally, ribulose-1,5-bisphosphate (RuBP) and 2-phosphoglycolate (2-PG) have been confirmed as co-activators of CmpR which controls the expression of the ABC-type bicarbonate transporter

Diagram of the proposed regulatory network within Synechocystis sp. PCC6803 showing both CcmR and CmpR, along with their ligand molecules and the relevant metabolic pathways.

<p>Enzyme/complex or metabolic pathways involved in the given step are indicated in solid lines, regulatory interactions are indicated in dotted lines. [HCO₃⁻]_cyt,, cytosolic bicarbonate; CCA, Carboxysome Carbonic Anhydrase; PDC, Pyruvate Dehydrogenase Complex; TCA, Tricarboxylic Acid Cycle; PSII, Photosystem II; B₆f, Cytochrome B₆f complex; PSI, Photosystem I. Ligand molecules for transcriptional repressor CcmR (NADP⁺ and α-KG) are indicated in red boxes, while those of transcriptional activator CmpR (RuBP, 2-PG) are indicated in white boxes. For C_i uptake genes repressed by CcmR, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041286#pone-0041286-g001" target="_blank">Figure 1</a> and reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041286#pone.0041286-Wang1" target="_blank">[11]</a>. For more information on the CmpR effectors, see reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041286#pone.0041286-Nishimura1" target="_blank">[20]</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041286#pone.0041286.s004" target="_blank">Data S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041286#pone.0041286.s002" target="_blank">Figure S2</a>.</p

SPR difference curves showing destabilization of CcmR binding to non-specific DNA (<i>rimM</i>) due to interaction with effector molecule, NADP⁺.

<p>DNA immobilization as described in Fig. 2, except that a biotinylated 150 bp DNA fragment the <i>rimM</i> gene (non-specific DNA) was immobilized upon the SPR surface. All curves are double referenced as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041286#pone-0041286-g003" target="_blank">Figure 3 </a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041286#pone.0041286-Myszka1" target="_blank">[33]</a>. The CcmR protein was injected at a concentration of 1.5 µM following incubation with ligand molecule on ice for at least 5 minutes before injection.</p

Surface Plasmon Resonance curves illustrating binding of CcmR to promoter regions of the <i>ccmR</i> and <i>ndhF3</i>.

<p>Biotinylated upstream duplex DNA ∼150 bp of the pccmR (A) and (B) pndhF3. Each DNA fragment was immobilized to a Neutravidin-coated SPR chip (Nomadics). The upstream sequences for <i>ccmR</i> and <i>ndhF3</i> that bind CcmR has been previously determined <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041286#pone.0041286-Figge1" target="_blank">[17]</a>, (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041286#pone.0041286.s004" target="_blank">Data S1</a> for primers used to generate DNA fragments). Heterologously expressed CcmR concentration for each target is as follows, markers are for visualization only; 0 nM (Closed Square), 250 nM (Open Square), 500 nM (Open Diamond), 750 nM (X), 1000 nM (+), 2000 nM (Open Triangle), 3000 nM (Closed Circle).</p

Effects of metabolic inhibitors on the redox state of the plastoquinone and pyridine nucleotide pools.

<p>Simultaneous measurements of chlorophyll <i>a</i> fluorescence (left panel) and NAD(P)H fluorescence (right panel) were made during exposure to light with an intensity approximating growth illumination (∼80 µmoles photons m⁻² s⁻¹) with <i>Synechocystis</i> cells treated C_i-uptake inhibitor EZ (ethoxyzolamide), red traces or no addition, black traces. Note that the data are presented using a log scale for the time axis. Measurements were made using a pulse amplitude modulated (PAM) fluorometer (DUAL-PAM-100, Walz) and an emitter-detection-cuvette assembly (ED-101US) with a DUAL-ENADPH emitter (Walz) detection of chlorophyll and NADPH fluorescence (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041286#s4" target="_blank">Methods</a> for details).</p

Surface Plasmon Resonance difference curves (double reference) illustrating protein binding to immobilized biotinylated DNA fragments with increasing concentrations of ligand molecule.

<p>DNA immobilization as described in Fig. 2. CcmR binding to pndhF3 fragments as the binding target. For assay method see Fig. 2. All curves are double referenced such that the curve corresponding to CcmR without added effector is subtracted from the curves corresponding to CcmR with the tested effector <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041286#pone.0041286-Myszka1" target="_blank">[33]</a>. Proteins where incubated with the indicated ligand molecule on ice for at least 5 minutes before injection. Injections testing contain 1.5 µM of CcmR and 10 µM (Black), 100 µM (Red), or 500 µM (Blue) of the indicated ligand molecule in the case of NADP⁺ and α-KG, or 500 µM for the non-effector molecules tested. These results have allowed for the identification of NADP⁺ and α-KG as the ligand molecules for CcmR. Based on their affect on CcmR (increasing signal) these molecules function as co-repressors within the regulation system.</p