# Biosensors-Based In Vivo Quantification of 2-Oxoglutate in Cyanobacteria and Proteobacteria
## Results
### The 2-OG Biosensors Are Responsive to 2-OG Changes In Vivo in Anabaena and E. coli  
We finally tested whether the biosensors are sensitive enough to detect 2-OG variations in vivo. Based on the in vitro experiments described above, the F-mT_PII, that is more sensitive to low 2-OG concentrations, was used for studies in _Anabaena_. To avoid heterogeneous expression form replicative plasmids [47], the F-mT_PII and or F-mT_PIIK58M constructs were inserted into the chromosome of _Anabaena_. FRET was then measured over time after addition of dimethyl-2-OG, a membrane-permeable 2-OG analogue that is processed into 2-OG by cytoplasmic esterases [22]. The addition of 10 mM of dimethyl-2-OG induced a significant increase of the reference FRET ratio in cells bearing the F-mT_PII by contrast to cells bearing the F-mT_PIIK58M construct, demonstrating that the biosensor responds to 2-OG changes in vivo (Figure 3C). We observed a decrease of the FRET ratio in the control (0 mM dimethyl-2-OG, gold line), likely due to photobleaching induced by the large number of excitation of the fluorophores during the experiment. Next, we followed 2-OG changes in _Anabaena_ after the deprivation of combined nitrogen. It is well described that in these conditions, the internal 2-OG concentration will increase to a peak level 1.5 to 2 h after depletion, and then decreases gradually to the basal level [3]. Figure 3D shows that the FRET ratio followed a similar pattern, with an increase at 2 h after nitrogen deprivation, followed by a decrease measured at time 6 h after nitrogen step down. In contrast, we did not detect any variation for cells producing the K58M biosensor variant that indicates that the FRET ratio measured actually reflects 2-OG binding. For _E. coli_, we used the F-mT-NtcA biosensor. Recombinant _E. coli_ strains producing F-mT_NtcA or its R88E mutant were grown in M9 medium supplemented with glucose to the exponential phase. Cells were then transferred into carbon-free M9 medium for 4 h to reduce the intracellular 2-OG level. FRET was then measured over time after addition of 10 mM of glucose or of dimethyl-2-OG. While we did not detect any significant FRET variation in cells depleted of carbon sources, the addition of dimethyl-2-OG or of glucose (which increases 2-OG levels through the TCA cycle) induced a significant increase in the FRET ratio (Figure 3E). This response was due to 2-OG-induced NtcA conformational change, but not to changes in plasmid copy number or biosensor production, as no FRET variation was observed with the mutant version of the biosensor (Figure 3F). The relationship between ammonium abundance in the medium and intracellular 2-OG levels was then examined. It is well described that the presence of nitrogen decreases 2-OG levels. Figure 3G shows that a decrease in ammonium concentration in the medium from 10 mM to 2 mM (Figure 3G, time T1) induces a rapid increase in the FRET ratio, whereas the FRET ratio diminished when ammonium was added at the concentration of 10 mM (Figure 3G, time T2). This opposite correlation between the intracellular concentration of 2-OG and the ammonium level in the medium measured in vivo is consistent with the previous results of 2-OG measurement on cell extracts using HPLC-MS [27] or the PROBS biosensor [35], even if an absolute comparison of the levels obtained by these different methodologies is not accurate. Taken together, we conclude that the two mono-protein biosensors adequately report 2-OG levels variation in vivo. Figure 3: _In vitro_ and in vivo features of the 2-OG FRET biosensors. (**A**,**B**) Time-lapse recordings of F-mT_PII (**A**) and F-mT_NtCa (**B**) responses upon addition of different concentrations of 2-OG. The relative FRET ratio was plotted against time (in seconds). The emission data were recorded every 30 s for 11 min with 0.5 uM of protein at 30 \({}^{\circ}\)C, pH7.4.