A shared alarmone-GTP switch underlies triggered and spontaneous persistence

Persisters describe phenotypically switched cells refractory to antibiotic killing in a genetically susceptible population, while preserving the ability to resume growth when antibiotics are discontinued. Since its proposal 70 years ago, great strides were made to build the framework regarding persistence, including defining triggered, spontaneous and antibiotic-induced persisters. However, challenges remain in characterizing the molecular determinants underlying the phenotypic switch into persistence. Here we document triggered, spontaneous and antibioticinduced persistence in a Gram-positive bacterium, all through a common switch involving the alarmone (p)ppGpp and each stemming from a different alarmone synthesis pathway. Starvation triggered persistence is mediated by Rel synthetase, and spontaneous persistence is through selfamplification via allosteric enzyme activation of alarmone synthetases Rel and SasB, whereas lethal and sublethal concentrations of cell wall antibiotics induce alarmones through an antibioticinduced alarmone synthetase SasA, consequently enabling adaptive persistence that promotes survival. (p)ppGpp accumulation promotes persistence by depleting intracellular GTP and antagonizing its action. 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 modest drop of GTP in bulk population slows down growth and promotes antibiotic tolerance, (p)ppGpp drives persistence by allowing the switch-like dynamics to drop GTP beneath the persister threshold in single cells. Persistence through alarmone-GTP antagonism is likely a widespread mechanism to survive antibiotics in Gram positive bacteria and possibly beyond.