Abstract
BackgroundAdenosine triphosphate (ATP) is the main energy carrier in living organisms, critical for metabolism and essential physiological processes. In humans, abnormal regulation of energy levels (ATP concentration) and power consumption (ATP consumption flux) in cells is associated with numerous diseases from cancer, to viral infection and immune dysfunction, while in microbes it influences their responses to drugs and other stresses. The measurement and modeling of ATP dynamics in cells is therefore a critical component in understanding fundamental physiology and its role in pathology. Despite the importance of ATP, our current understanding of energy dynamics and homeostasis in living cells has been limited by the lack of easy-to-use ATP sensors and the lack of models that enable accurate estimates of energy and power consumption related to these ATP dynamics. Here we describe a dynamic model and an ATP reporter that tracks ATP in E. coli over different growth phases.ResultsThe reporter is made by fusing an ATP-sensing rrnB P1 promoter with a fast-folding and fast-degrading GFP. Good correlations between reporter GFP and cellular ATP were obtained in E. coli growing in both minimal and rich media and in various strains. The ATP reporter can reliably monitor bacterial ATP dynamics in response to nutrient availability. Fitting the dynamics of experimental data corresponding to cell growth, glucose, acetate, dissolved oxygen, and ATP yielded a mathematical and circuit model. This model can accurately predict cellular energy and power consumption under various conditions. We found that cellular power consumption varies significantly from approximately 0.8 and 0.2 million ATP/s for a tested strain during lag and stationary phases to 6.4 million ATP/s during exponential phase, indicating ~ 8–30-fold changes of metabolic rates among different growth phases. Bacteria turn over their cellular ATP pool a few times per second during the exponential phase and slow this rate by ~ 2–5-fold in lag and stationary phases.ConclusionOur rrnB P1-GFP reporter and kinetic circuit model provide a fast and simple way to monitor and predict energy and power consumption dynamics in bacterial cells, which can impact fundamental scientific studies and applied medical treatments in the future.
Highlights
Adenosine triphosphate (ATP) is the main energy carrier in living organisms, critical for metabolism and essential physiological processes
Regardless of many factors that might affect fluorescence-reporter dynamics that are common to almost all biosensors, good correlations were found between GFP and ATP under many experimentally tested conditions
We found that our ATP reporter can reliably monitor cellular ATP dynamics in response to nutrient availability
Summary
Adenosine triphosphate (ATP) is the main energy carrier in living organisms, critical for metabolism and essential physiological processes. Conventional methods, such as luciferase assays, require efficient lysis of cells and preclude real-time and continuous intracellular ATP measurements [13] To this end, several genetically encoded ATP biosensors have been developed, such as the fluorescence resonance energy transfer FRET-based ATeam biosensor [14], the bioluminescence resonance energy transfer BRET-based BTeam biosensor [13], and the new ATeam3.10 biosensor [15]. To monitor cellular ATP in fast-growing bacteria, Yaginuma et al developed a QUEEN ATP sensor but wider applications of this sensor in bacteria have not been reported, possibly due to its relatively dim signal and sensitivity to temperature [16] These biosensors require expensive fluorescence microscopes and time-consuming procedures for sample preparation and image analysis. Monitoring such ATP dynamics can predict nutrients, cellular stresses, disease states, or efficacy of drug treatments [2,3,4,5,6,7, 9, 11] and might be used to modulate or actuate therapeutic molecular release in response to cellular energetics
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