# Biosensors-Based In Vivo Quantification of 2-Oxoglutate in Cyanobacteria and Proteobacteria
## 1 Introduction  
Within a cell, the coordination of distinct metabolic pathways is mediated by fluctuations in the concentrations of specific metabolites, allowing the allosteric transfer of this information to sensor proteins, thereby generating the appropriated metabolic response. Metabolites that act as key players in microbial signal transduction include glutamine, 2-oxoglutarate (\(\alpha\)-ketoglutarate, 2-OG), cAMP, and ppGpp [1]. Each of these metabolites is dedicated to the transfer of information upon the availability of a specific class of nutrient. 2-OG is an intermediate metabolite of the tricarboxylic acid (TCA or Krebs) cycle and is used as a carbon skeleton for nitrogen assimilation. It therefore coordinates carbon and nitrogen metabolisms. The TCA cycle is an essential metabolic pathway conserved in all living organisms and provides not only precursors for biosynthesis of a large number of biomolecules, but also reducing power for the production of the biological form of energy, ATP, through respiration. The 2-OG level reflects also the carbon and nitrogen balance, a signaling characteristic that is conserved throughout organisms. An inverse correlation between ammonium availability and 2-OG accumulation has been demonstrated in various microorganisms such as _Escherichia coli_, archaea, cyanobacteria, and budding yeast [2; 3; 4]. The signaling role of 2-OG is well described in cyanobacteria and especially in the filamentous heterocystous cyanobacterium _Anabaena_ sp. strain PCC 7120 (hereafter _Anabaena_). In _Anabaena_, combined-nitrogen depletion leads to the accumulation of 2-OG, which acts as a trigger to elicit heterocyst differentiation [3; 5; 6]. Heterocysts are cells specialized in nitrogen fixation, which differentiate from vegetative cells. Heterocysts and vegetative cells are interdependent: heterocysts provide fixed nitrogen from atmospheric N\({}_{2}\) in the formof amino acids to vegetative cells, while the latter use photosynthesis to fix carbon from atmospheric CO\({}_{2}\) and provide sucrose to heterocysts [5; 7; 8]. For its function as signaling molecule, 2-OG binds to the regulatory proteins, PII and NtAcA. The crystal structures of these two regulators, alone or complexed to 2-OG, showed that they undergo significant conformational changes when binding to their ligands [9; 10; 11; 12; 13]. PII, encoded by the \(glnB\) gene, is conserved in bacteria and plants. It assembles as homotrimer and performs its regulatory function through protein-protein interactions [14]. In cyanobacteria, the activity of PII is regulated by phosphorylation, and by the binding of effectors such as ATP and 2-OG, enabling PII to adopt different conformations, and to interact with different protein partners under different conditions [15]. In _Anabaena_, the PII trimer binds to 3 molecules of PipX in conditions of combined nitrogen deprivation. Interestingly, PipX and 2-OG share the same binding site on PII, and are therefore mutually exclusive. When the ammonium cell concentration decreases, 2-OG level increases and chases PipX from PII. In addition, PipX is a co-activator of the cyanobacterial-specific transcriptional factor NtAcA which belongs to the CRP/CAP family of homodimeric transcriptional regulators, and uses 2-OG as the inducer molecule [16; 17]. One molecule of 2-OG binds to each monomer of the NtAcA dimer in a pocket located in the N-terminal region. 2-OG binding on NtAcA reduces the distance between the two DNA-binding F-helixes of the NtAcA dimer from 37 A in the apo form to 34 A in the 2-OG-NtAcA complex, an optimal distance for DNA binding [3; 9]. The activity of NtAcA is also controlled by its interaction with PipX. Each NtAcA subunit binds one subunit of PipX in the complex and this interaction is required for NtAcA-mediated gene expression. It has been proposed that PipX stabilizes the active form of NtAcA and helps NtAcA to recruit the RNA polymerase [4; 10; 16; 17; 18]. In _Anabaena_, an _ntcA_ mutant is unable to initiate heterocyst differentiation [5; 19].