A shared alarmone–GTP switch controls persister formation in bacteria

Persisters are phenotypically switched bacteria that survive antibiotic exposure despite being genetically susceptible. Three pathways to persistence-triggered, spontaneous and antibiotic-induced-have been described, but the underlying molecular mechanisms are poorly understood. Here, we used antibiotic time-kill assays as well as single-cell approaches to show that all of the pathways depend on a common switch involving the alarmone guanosine tetra/penta-phosphate ((p)ppGpp) in Bacillus subtilis, each stemming from different alarmone synthetase(s). The accumulation of (p)ppGpp promotes persistence through depletion of intracellular GTP. We developed a fluorescent GTP reporter to visualize rare events of persister formation in wild-type bacteria, revealing a rapid switch from growth to dormancy in single cells as their GTP levels drop beneath a threshold. While a decrease in GTP in the bulk population slows growth and promotes antibiotic tolerance, (p)ppGpp drives persistence by driving rapid, switch-like decreases in GTP levels beneath the persister threshold in single cells. Persistence through alarmone-GTP antagonism is probably a widespread mechanism to survive antibiotics in B. subtilis and potentially beyond.