Abstract
Transient antibiotic treatment typically eradicates most sensitive bacteria except a few survivors called persisters. The second messenger (p)ppGpp plays a key role in persister formation in Escherichia coli populations but the underlying mechanisms have remained elusive. In this study we induced (p)ppGpp synthesis by modulating tRNA charging and then directly observed the stochastic appearance, antibiotic tolerance, and resuscitation of persister cells using live microscopy. Different physiological parameters of persister cells as well as their regularly growing ancestors and sisters were continuously monitored using fluorescent reporters. Our results confirmed previous findings that high (p)ppGpp levels are critical for persister formation, but the phenomenon remained strikingly stochastic without any correlation between (p)ppGpp levels and antibiotic tolerance on the single-cell level. We could not confirm previous notions that persisters exhibit markedly low concentrations of intracellular ATP or were linked to post-transcriptional effects of (p)ppGpp through the activation of small genetic elements known as toxin-antitoxin (TA) modules. Instead, we suggest that persister cell formation under regular conditions is driven by the transcriptional response to increased (p)ppGpp levels.
Highlights
Persister formation of Escherichia coli largely depends on the alarmone (p)ppGpp, which controls the stringent response and is connected to toxin-antitoxin (TA) modules through different pathways[12,13,14]
We explored the formation of E. coli persisters upon induction of (p)ppGpp signaling in response to amino acid starvation, a physiological setup that is known to ramp up persister formation and has been well-studied with regard to other aspects of bacterial cell biology[8,25]
Many bacteria including the model organism Escherichia coli K-12 MG1655 respond to amino acid starvation with the production of (p)ppGpp to induce a well-studied physiological program known as the stringent response[25]
Summary
Persister formation of Escherichia coli largely depends on the alarmone (p)ppGpp, which controls the stringent response and is connected to toxin-antitoxin (TA) modules through different pathways[12,13,14]. The stochasticity of persister cell formation can be caused (i) by the heterogeneity of cellular (p)ppGpp levels among different cells in the population or (ii) by the molecular noise in the regulatory circuit that connects the (p)ppGpp level and the gene expression program controlling the phenotypic transition into the persister state. To distinguish these possibilities, we induced (p)ppGpp signaling in E. coli by limiting tRNA charging and followed the sequence of birth, antibiotic survival, and resuscitation of persister cells directly by live microscopy. Persister formation was often preceded by TA module activation, we did not observe a critical role of previously implicated TA modules in persister formation
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