# Biosensors-Based In Vivo Quantification of 2-Oxoglutate in Cyanobacteria and Proteobacteria
## 4 Discussion  
In this work, we show that PII protein or a domain of NtcA containing the 2-OG binding region could be used as 2-OG FRET mono-protein biosensors when fused to both YFP and CFP/mTurquoise. The two constructs, named F-mT_PII and F-mT_NtcA (with mTurquoise at the C-terminus and YFP midway through the T-loop of PII or close to the N-terminus in NtcA) were based on 2-OG binding dependent conformational changes of the T-loop and of the C-helices, respectively. Variant biosensors, bearing mutations that abolish binding of 2-OG on PII and NtcA, have also been engineered for specificity controls. We show that these mono-protein biosensors are as efficient as the two-partner based biosensors previously developed for in vitro studies [35; 36]. However, these biosensors present additional advantages: by being single polypeptide, only a single purification is required for in vitro studies. They also increase the easy in vivo use as mono-protein biosensors prevent issues regarding differential regulation and/or plasmid copy number when using two constructs. Our results demonstrate that 2-OG binding specifically modifies the FRET ratio of both biosensors, which is not observed with the two mutant versions. One of the most significant properties of the two biosensors tested in our study is their different sensitivity to 2-OG concentrations and therefore their responsiveness to different 2-OG level ranges (Figure 2A,B). This variation of affinity can be inherent to the large difference between the 2-OG binding pockets and the residues involved in 2-OG binding in PII and NtcA [10; 12]. 2-OG binding to PII occurs at the micromolar range to fine tune the cellular nitrogen fixation ability as a function of the Carbon/Nitrogen balance [48], whereas 2-OG binds NtcA at high levels during cellular combined nitrogen deprivation [3]. This difference of affinity and binding pocket could explain why the 2-methylenepentanedioic acid (2-MPA) a non-metabolizable analogue of 2-OG, is able to modify the FRET ratio of F-mT_PII but not that of F-mT_NtcA (Figure 2G) [46]. We also noted that the biosensor concentrations did not have any impact on 2-OG level measurements (Figure 2A,B). This observation, as well as the fact that the two biosensors respond to different 2-OG level ranges, represent an advantage to measuring in vivo 2-OG fluctuations in a wide range of organisms. The tools developed here can be therefore applied to many biological systems such as cyanobacteria, bacteria, fungi, plants, and animals. Importantly, we showed that the two biosensors are able to detect variations of 2-OG levels within minutes (Figure 3A,B), which is particularly crucial when probing 2-OG fluctuations during time-lapse experiments is needed or after a change in the experimental conditions. By modulating glucose, 2-OG and ammonium concentrations in the medium, we observed that the NtcA-based biosensor F-mT_NtcA can detect 2-OG fluctuations in _E. coli_ populations as a function of Carbon or Nitrogen availability. Even if our aim is to determine 2-OG variations in vivo and not to quantify the absolute 2-OG concentrations, the data obtained could be considered as consistent with previous results available in the literature. HPLC measurements of the intracellular concentration of 2-OG showed that it increases from 0.35 mM to 2.6 mM after the addition of 10 mM glucose to a carbon-free M9 medium [37]. 2-OG levels have also been shown to significantly decrease after the addition of 10-200 mM NH\({}_{4}\)Cl to an _E. coli_ cell culture cultivated under nitrogen starvation conditions [27]. Conversely, _E. coli_ cultivated in presence of high ammonium level (10 mM NH\({}_{4}\)Cl) exhibited an increase in intracellular 2-OG from 0.5 mM to 2.5 mM under nitrogen starvation [30]. Regarding the PII-based biosensor, it was found to detect 2-OG levels within the physiological range (10\({}^{-2}\)-10\({}^{-5}\) M) in vitro (Figure 2A). We further showed that it is efficient to measure 2-OG levels in vivo in _Anabaena_ cells (Figure 3C,D).