ИСТИНА

ATeam is a fluorescent ATP indicator based on FRET and composed of the ε subunit of bacterial FOF1-ATP-synthase sandwiched by the cyan and yellow fluorescent proteins [1]. It turned to be convenient for visualizing of ATP levels inside living eukaryotic cells, but to our knowledge it was never used in experiments on bacteria. The intracellular ATP level is a key parameter in bacterial bioenergetics, so we consider ATeam as an efficient tool in this field. Using ATeam probe, we developed a new method for ATP level measurement in bacterial living cells. We monitored the probe response in wild type Escherichia coli cells expressing ATeam, and tested its adequacy with standard chemiluminescence method. In this system, as expected, the signal rate increased after glucose addition and was reduced after addition of ATP synthesis inhibitor potassium arsenate. The addition of respiratory substrate succinate also led to signal rate increase, and the following addition of protonophore resulted in decrease of signal rate. When uncoupling preceded the succinate addition, the signal rate increase was not observed. With new approach, we also studied dynamics of ATP levels in Escherichia coli Δunc mutant with deleted ATP synthase operon. Glucose and arsenate additions influenced the signal rate the same way as in wild type. Addition of succinate to mutant cells unexpectedly led to increase of signal rate, which, however, was half less than that after glucose addition. In general, our data correspond well to general knowledge of bacterial energetic metabolism, so our method seems suitable for bacterial bioenergetic studies.