Surviving antibiotic stress: The essential GTPase ObgE controls persistence in Escherichia coli through toxin-mediated membrane depolarization

Upon encountering the sudden, lethal activity of antibiotics, survival of a bacterial population largely depends on the presence of a small fraction of transiently stressresistant cells that protect the population from eradication. These so-called persisters are genetically identical to other cells in the population and are therefore phenotypic variants. As a bet-hedging strategy, persistence levels are determined both by stochastic induction and by environmental stimuli called responsive diversification. For unknown reasons, persister cells can switch back to the non-persistent state, implying that it is a reversible phenotype. Notably, persistence has been held responsible for recurrent outbreaks of bacterial infections once the antibiotic pressure decreases. Research by our group identified ObgE as a major regulator of persistence in Escherichia coli. Additionally, its function is conserved in the nosocomial pathogen Pseudomonas aeruginosa. ObgE is an essential GTPase involved in numerous major cellular processes like DNA replication, ribosome biogenesis and the stringent response. Furthermore, as a sensor of cellular GTP concentrations, ObgE controls morphological differentiation of Streptomyces as well as the formation of stress-resistant spores in Bacillus subtilis. Our results support a model in which naturally occurring cell-to-cell variations in obgE expression contribute to the formation of persisters in E. coli. In this work we show that ObgE-mediated persistence requires the stringent response. Triggered upon amino acid starvation and deprivation of carbon sources, phosphate and fatty acids, the stringent response governs the formation of (p)ppGpp resulting in the inhibition of protein synthesis and growth by stimulating or inhibiting expression of approximately 500 genes. This alarmone has been implicated in persistence by numerous studies and opens perspectives for ObgE-controlled persister cell formation through stochastically generated subpopulations of cells adapting to growth under new conditions following environmental cues. A previously performed transcriptome analysis on cells overexpressing or downregulating obgE implicated HokB in ObgE-mediated persistence. hokB encodes a small poreforming peptide, part of a chromosomally encoded toxin/antitoxin (TA) module. To corroborate the transcriptome data, we performed flow cytometric analyses of cells overexpressing ObgE translationally fused to Venus. In these experiments, hokB was controlle