# Biosensors-Based In Vivo Quantification of 2-Oxoglutate in Cyanobacteria and Proteobacteria
## 1 Introduction
Based on the observation that 2-OG binds to NtAcA and that an increase in the levels of NtAcA and 2-OG triggers heterocyst differentiation, it has been proposed that the interaction between 2-OG and NtAcA constitutes the very first step in heterocyst development [3; 5]. However, the role of NtAcA is not limited to the initiation of heterocyst development, as it has been shown to have a broad regulatory function. The NtAcA regulon comprises several hundreds of genes, including targets involved in both carbon and nitrogen metabolism, stress response, and in different steps of heterocyst differentiation and functioning [5; 20]. In addition to PII and NtAcA, 2-OG levels also modulate the activity of many regulatory proteins and pathways, predominantly related to nitrogen or carbon metabolism in bacteria, archaea, algae, plants and animals [1; 11; 15; 21; 22; 23]. Despite the central role of 2-OG and its extensive regulatory repertoire, we still lack tools to appropriately measure its concentrations and dynamics in vivo. Enzymatic assays, mass spectrometry or high-pressure liquid chromatography (HPLC) approaches have been developed to estimate 2-OG levels in cell extracts [4; 24; 25; 26; 27; 28; 29; 30]. However, 2-OG such as many metabolites, turns over very quickly and may be lost or degraded during extraction. As a consequence, 2-OG concentration measurements are non-reproducible and underestimated [30; 31; 32]. In addition, none of these approaches allow to follow 2-OG dynamics. To circumvent these disadvantages, 2-OG biosensors based on fluorescence resonance energy transfer (FRET) have been recently developed. FRET relies on the distance-dependent interaction between two fluorescent molecules, and on the transfer of the excited state of the donor molecule to the acceptor when these two molecules are in close proximity [33; 34]. We have constructed a 2-OG biosensor based on the interaction between the _Anabaena_ PII and PipX proteins, whose interaction is disrupted by 2-OG binding to PII. The FRET ratio (fluorescence of the acceptor YFP/fluorescence of the donor CFP) has been shown to be inversely proportional to 2-OG levels in vitro in a 0.01-10 mM range [35]. A second biosensor, based on the 2-OG-dependent interaction between the _Synechococcus elongatus_ PII and \(N\)-acetyl-glutamate kinase (NAGK) proteins, has been shown to measure 2-OG levels in the 0.01-1 mM range [36]. Although such biosensors have been proved to be highly efficient in vitro or ex vivo, they failed to measure 2-OG variations within living cells [35; 36; 37]. One of the limitations is that it was difficult to achieve similar levels of production for the two fusion proteins in _E. coli_ and _Anabaena_. For this reason, 2-OG biosensors based on single proteins need to be developed to measure 2-OG levels in vitro and in vivo. In this study, we report the engineering of two new biosensors, based on 2-OG-dependentconformational changes of PII or NtcA. We show that they could both report 2-OG changes, with a different concentration range in vitro and in vivo in two bacteria belonging to separate phyla.