Bacterial persisters are phenotypic variants that survive antibiotic treatment in a dormant state and can be formed by multiple pathways. We recently proposed that the second messenger (p)ppGpp drives Escherichia coli persister formation through protease Lon and activation of toxin-antitoxin (TA) modules. This model found considerable support among researchers studying persisters but also generated controversy as part of recent debates in the field. In this study, we therefore used our previous work as a model to critically examine common experimental procedures to understand and overcome the inconsistencies often observed between results of different laboratories. Our results show that seemingly simple antibiotic killing assays are very sensitive to variations in culture conditions and bacterial growth phase. Additionally, we found that some assay conditions cause the killing of antibiotic-tolerant persisters via induction of cryptic prophages. Similarly, the inadvertent infection of mutant strains with bacteriophage ϕ80, a notorious laboratory contaminant, apparently caused several of the phenotypes that we reported in our previous studies. We therefore reconstructed all infected mutants and probed the validity of our model of persister formation in a refined assay setup that uses robust culture conditions and unravels the dynamics of persister cells through all bacterial growth stages. Our results confirm the importance of (p)ppGpp and Lon but no longer support a role of TA modules in E. coli persister formation under unstressed conditions. We anticipate that the results and approaches reported in our study will lay the ground for future work in the field.

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Gene arrays and operons that encode functionally linked proteins form the most basic unit of transcriptional regulation in bacteria. Rules that govern the order and orientation of genes in these systems have been defined; however, these were based on a small set of genomes that may not be representative. The growing availability of large genomic datasets presents an opportunity to test these rules, to define the full range and diversity of these systems, and to understand their evolution. Here we present SLING, a tool to Search for LINked Genes by searching for a single functionally essential gene, along with its neighbours in a rule-defined proximity (https://github.com/ghoresh11/sling/wiki). Examining this subset of genes enables us to understand the basic diversity of these genetic systems in large datasets. We demonstrate the utility of SLING on a clinical collection of enteropathogenic Escherichia coli for two relevant operons: toxin antitoxin (TA) systems and RND efflux pumps. By examining the diversity of these systems, we gain insight on distinct classes of operons which present variable levels of prevalence and ability to be lost or gained. The importance of this analysis is not limited to TA systems and RND pumps, and can be expanded to understand the diversity of many other relevant gene arrays.

Antibiotic‐tolerant persisters are often implicated in treatment failure of chronic and relapsing bacterial infections, but the underlying molecular mechanisms have remained elusive. Controversies revolve around the relative contribution of specific genetic switches called toxin–antitoxin (TA) modules and global modulation of cellular core functions such as slow growth. Previous studies on uropathogenic Escherichia coli observed impaired persister formation for mutants lacking the pasTI locus that had been proposed to encode a TA module. Here, we show that pasTI is not a TA module and that the supposed toxin PasT is instead the bacterial homolog of mitochondrial protein Coq10 that enables the functionality of the respiratory electron carrier ubiquinone as a “lipid chaperone.” Consistently, pasTI mutants show pleiotropic phenotypes linked to defective electron transport such as decreased membrane potential and increased sensitivity to oxidative stress. We link impaired persister formation of pasTI mutants to a global distortion of cellular stress responses due to defective respiration. Remarkably, the ectopic expression of human coq10 largely complements the respiratory defects and decreased persister levels of pasTI mutants. Our work suggests that PasT/Coq10 has a central role in respiratory electron transport that is conserved from bacteria to humans and sustains bacterial tolerance to antibiotics.