Fluctuations in intracellular molecule abundance can lead to distinct, coexisting phenotypes in isogenic populations. Although metabolism continuously adapts to unpredictable environmental changes, and although bistability was found in certain substrate-uptake pathways, central carbon metabolism is thought to operate deterministically. Here, we combine experiment and theory to demonstrate that a clonal Escherichia coli population splits into two stochastically generated phenotypic subpopulations after glucose-gluconeogenic substrate shifts. Most cells refrain from growth, entering a dormant persister state that manifests as a lag phase in the population growth curve. The subpopulation-generating mechanism resides at the metabolic core, overarches the metabolic and transcriptional networks, and only allows the growth of cells initially achieving sufficiently high gluconeogenic flux. Thus, central metabolism does not ensure the gluconeogenic growth of individual cells, but uses a population-level adaptation resulting in responsive diversification upon nutrient changes.

While persisters are a health threat due to their transient antibiotic tolerance, little is known about their phenotype and what actually causes persistence. Using a new method for persister generation and high‐throughput methods, we comprehensively mapped the molecular phenotype of Escherichia coli during the entry and in the state of persistence in nutrient‐rich conditions. The persister proteome is characterized by σS‐mediated stress response and a shift to catabolism, a proteome that starved cells tried to but could not reach due to absence of a carbon and energy source. Metabolism of persisters is geared toward energy production, with depleted metabolite pools. We developed and experimentally verified a model, in which persistence is established through a system‐level feedback: Strong perturbations of metabolic homeostasis cause metabolic fluxes to collapse, prohibiting adjustments toward restoring homeostasis. This vicious cycle is stabilized and modulated by high ppGpp levels, toxin/anti‐toxin systems, and the σS‐mediated stress response. Our system‐level model consistently integrates past findings with our new data, thereby providing an important basis for future research on persisters.

In recent years, our understanding about bacterial persistence has significantly advanced: we comprehend the persister phenotype better, more triggers for persistence entry have been found, and more insights in the involvement and role of toxin-antitoxin systems and other molecular mechanisms have been unravelled. In this review, we attempt to put these findings into an integrated, system-level perspective. From this point of view, persistence can be seen as a response to a strong perturbation of metabolic homeostasis, either triggered environmentally, or by means of intracellular stochasticity. Metabolic-flux-regulated resource allocation ensures stress protection, and several feedback mechanisms stabilize the cells in this protected state. We hope that this novel view can advance our understanding about persistence.

Antibiotic persistence describes the presence of phenotypic variants within an isogenic bacterial population that are transiently tolerant to antibiotic treatment. Perturbations of metabolic homeostasis can promote antibiotic persistence, but the precise mechanisms are not well understood. Here, we use laboratory evolution, population-wide sequencing and biochemical characterizations to identify mutations in respiratory complex I and discover how they promote persistence in Escherichia coli. We show that persistence-inducing perturbations of metabolic homeostasis are associated with cytoplasmic acidification. Such cytoplasmic acidification is further strengthened by compromised proton pumping in the complex I mutants. While RpoS regulon activation induces persistence in the wild type, the aggravated cytoplasmic acidification in the complex I mutants leads to increased persistence via global shutdown of protein synthesis. Thus, we propose that cytoplasmic acidification, amplified by a compromised complex I, can act as a signaling hub for perturbed metabolic homeostasis in antibiotic persisters.

This commentary responds to the Covid-19 pandemic which poses a serious challenge for the delivery of “wet laboratory” practical education. We discuss the “Chemical Kitchen” project, which teaches laboratory skills and the scientific method through the parallel discipline of gastronomy and its transformation into a home-based program: how it came to be, its successes to date, and our plans to deploy it for remote delivery in the uncertain future.