Multisite phosphorylation drives phenotypic variation in (p)ppGpp synthetase-dependent antibiotic tolerance.

Isogenic populations of cells exhibit phenotypic variability that has specific physiological consequences. Individual bacteria within a population can differ in antibiotic tolerance, but whether this variability can be regulated or is generally an unavoidable consequence of stochastic fluctuations is unclear. Here we report that a gene encoding a bacterial (p)ppGpp synthetase in Bacillus subtilis, sasA, exhibits high levels of extrinsic noise in expression. We find that sasA is regulated by multisite phosphorylation of the transcription factor WalR, mediated by a Ser/Thr kinase-phosphatase pair PrkC/PrpC, and a Histidine kinase WalK of a two-component system. This regulatory intersection is crucial for controlling the appearance of outliers; rare cells with unusually high levels of sasA expression, having increased antibiotic tolerance. We create a predictive model demonstrating that the probability of a given cell surviving antibiotic treatment increases with sasA expression. Therefore, multisite phosphorylation can be used to strongly regulate variability in antibiotic tolerance.