Intracellular ATP concentration is a key regulator of bacterial cell fate.

ATP, most widely known as the primary energy source for numerous cellular processes, also exhibits the characteristics of a biological hydrotrope. The viable but nonculturable (VBNC) and persister states are two prevalent dormant phenotypes employed by bacteria to survive challenging environments, both of which are associated with low metabolic activity. Here, we investigate the intracellular ATP concentration of individual VBNC and persister cells using a sensitive ATP biosensor QUEEN-7μ and reveal that both types of cells possess a lower intracellular ATP concentration than culturable and sensitive cells, although there is a certain overlap in the intracellular ATP concentrations between antibiotic-sensitive cells and persisters. Moreover, we successfully separated VBNC cells from culturable cells using fluorescence-activated cell sorting based on the intracellular ATP concentration threshold of 12.5 µM. Using an enriched VBNC cell population, we confirm that the precipitation of proteins involved in key biological processes promotes VBNC cell formation. Notably, using green light-illuminated proteorhodopsin (PR), we demonstrate that VBNC cells can be effectively resuscitated by elevating their intracellular ATP concentration. These findings highlight the crucial role of intracellular ATP concentration in the regulation of bacterial cell fate and provide new insights into the formation of VBNC and persister cells.IMPORTANCEThe viable but nonculturable (VBNC) and persister states are two dormant phenotypes employed by bacteria to counter stressful conditions and play a crucial role in chronic and recurrent bacterial infections. However, the lack of precise detection methods poses significant threats to public health. Our study reveals lower intracellular ATP concentrations in these states and establishes an ATP threshold for distinguishing VBNC from culturable cells. Remarkably, we revive VBNC cells by elevating their intracellular ATP levels. This echoes recent eukaryotic studies where modulating metabolism impacts outcomes like osteoarthritis treatment and lifespan extension in