Escherichia coli self-organizes developmental rosettes.

Rosettes are self-organizing, circular multicellular communities that initiate developmental processes, like organogenesis and embryogenesis, in complex organisms. Their formation results from the active repositioning of adhered sister cells and is thought to distinguish multicellular organisms from unicellular ones. Though common in eukaryotes, this multicellular behavior has not been reported in bacteria. In this study, we found that Escherichia coli forms rosettes by active sister-cell repositioning. After division, sister cells "fold" to actively align at the 2- and 4-cell stages of clonal division, thereby producing rosettes with characteristic quatrefoil configuration. Analysis revealed that folding follows an angular random walk, composed of ~1 µm strokes and directional randomization. We further showed that this motion was produced by the flagellum, the extracellular tail whose rotation generates swimming motility. Rosette formation was found to require de novo flagella synthesis suggesting it must balance the opposing forces of Ag43 adhesion and flagellar propulsion. We went on to show that proper rosette formation was required for subsequent morphogenesis of multicellular chains, rpoS gene expression, and formation of hydrostatic clonal-chain biofilms. Moreover, we found self-folding rosette-like communities in the standard motility assay, indicating that this behavior may be a general response to hydrostatic environments in E. coli. These findings establish self-organization of clonal rosettes by a prokaryote and have implications for evolutionary biology, synthetic biology, and medical microbiology.

Resuscitation dynamics reveal persister partitioning after antibiotic treatment.

Bacteria can survive antibiotics by forming dormant, drug-tolerant persisters. Persisters can resuscitate from dormancy after treatment and prolong infections. Resuscitation is thought to occur stochastically, but its transient, single-cell nature makes it difficult to investigate. We tracked the resuscitation of individual persisters by microscopy after ampicillin treatment and, by characterizing their dynamics, discovered that Escherichia coli and Salmonella enterica persisters resuscitate exponentially rather than stochastically. We demonstrated that the key parameters controlling resuscitation map to the ampicillin concentration during treatment and efflux during resuscitation. Consistently, we observed many persister progeny have structural defects and transcriptional responses indicative of cellular damage, for both ß-lactam and quinolone antibiotics. During resuscitation, damaged persisters partition unevenly, generating both healthy daughter cells and defective ones. This persister partitioning phenomenon was observed in S. enterica, Klebsiella pneumoniae, Pseudomonas aeruginosa, and an E. coli urinary tract infection (UTI) isolate. It was also observed in the standard persister assay and after in situ treatment of a clinical UTI sample. This study reveals novel properties of resuscitation and indicates that persister partitioning may be a survival strategy in bacteria that lack genetic resistance.

Bacteria as control engineers.

Bacteria encounter fluctuations in both their external and internal environments, and to manage these conditions, they employ various control mechanisms. In this issue of Molecular Cell, Hart et al. (2011) investigate how E. coli robustly controls nitrogen assimilation.

Metabolite-enabled eradication of bacterial persisters by aminoglycosides.

Bacterial persistence is a state in which a sub-population of dormant cells, or 'persisters', tolerates antibiotic treatment. Bacterial persisters have been implicated in biofilms and in chronic and recurrent infections. Despite this clinical relevance, there are currently no viable means for eradicating persisters. Here we show that specific metabolic stimuli enable the killing of both Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) persisters with aminoglycosides. This potentiation is aminoglycoside-specific, it does not rely on growth resumption and it is effective in both aerobic and anaerobic conditions. It proceeds by the generation of a proton-motive force which facilitates aminoglycoside uptake. Our results demonstrate that persisters, although dormant, are primed for metabolite uptake, central metabolism and respiration. We show that aminoglycosides can be used in combination with specific metabolites to treat E. coli and S. aureus biofilms. Furthermore, we demonstrate that this approach can improve the treatment of chronic infections in a mouse urinary tract infection model. This work establishes a strategy for eradicating bacterial persisters that is based on metabolism, and highlights the importance of the metabolic environment to antibiotic treatment.

Antibiotics induce redox-related physiological alterations as part of their lethality.

Deeper understanding of antibiotic-induced physiological responses is critical to identifying means for enhancing our current antibiotic arsenal. Bactericidal antibiotics with diverse targets have been hypothesized to kill bacteria, in part by inducing production of damaging reactive species. This notion has been supported by many groups but has been challenged recently. Here we robustly test the hypothesis using biochemical, enzymatic, and biophysical assays along with genetic and phenotypic experiments. We first used a novel intracellular H2O2 sensor, together with a chemically diverse panel of fluorescent dyes sensitive to an array of reactive species to demonstrate that antibiotics broadly induce redox stress. Subsequent gene-expression analyses reveal that complex antibiotic-induced oxidative stress responses are distinct from canonical responses generated by supraphysiological levels of H2O2. We next developed a method to quantify cellular respiration dynamically and found that bactericidal antibiotics elevate oxygen consumption, indicating significant alterations to bacterial redox physiology. We further show that overexpression of catalase or DNA mismatch repair enzyme, MutS, and antioxidant pretreatment limit antibiotic lethality, indicating that reactive oxygen species causatively contribute to antibiotic killing. Critically, the killing efficacy of antibiotics was diminished under strict anaerobic conditions but could be enhanced by exposure to molecular oxygen or by the addition of alternative electron acceptors, indicating that environmental factors play a role in killing cells physiologically primed for death. This work provides direct evidence that, downstream of their target-specific interactions, bactericidal antibiotics induce complex redox alterations that contribute to cellular damage and death, thus supporting an evolving, expanded model of antibiotic lethality.

Salmonella typhimurium intercepts Escherichia coli signaling to enhance antibiotic tolerance.

Bacterial communication plays an important role in many population-based phenotypes and interspecies interactions, including those in host environments. These interspecies interactions may prove critical to some infectious diseases, and it follows that communication between pathogenic bacteria and commensal bacteria is a subject of growing interest. Recent studies have shown that Escherichia coli uses the signaling molecule indole to increase antibiotic tolerance throughout its population. Here, we show that the intestinal pathogen Salmonella typhimurium increases its antibiotic tolerance in response to indole, even though S. typhimurium does not natively produce indole. Increased antibiotic tolerance can be induced in S. typhimurium by both exogenous indole added to clonal S. typhimurium populations and indole produced by E. coli in mixed-microbial communities. Our data show that indole-induced tolerance in S. typhimurium is mediated primarily by the oxidative stress response and, to a lesser extent, by the phage shock response, which were previously shown to mediate indole-induced tolerance in E. coli. Further, we find that indole signaling by E. coli induces S. typhimurium antibiotic tolerance in a Caenorhabditis elegans model for gastrointestinal infection. These results suggest that the intestinal pathogen S. typhimurium can intercept indole signaling from the commensal bacterium E. coli to enhance its antibiotic tolerance in the host intestine.

Wisdom of crowds for robust gene network inference.

Reconstructing gene regulatory networks from high-throughput data is a long-standing challenge. Through the Dialogue on Reverse Engineering Assessment and Methods (DREAM) project, we performed a comprehensive blind assessment of over 30 network inference methods on Escherichia coli, Staphylococcus aureus, Saccharomyces cerevisiae and in silico microarray data. We characterize the performance, data requirements and inherent biases of different inference approaches, and we provide guidelines for algorithm application and development. We observed that no single inference method performs optimally across all data sets. In contrast, integration of predictions from multiple inference methods shows robust and high performance across diverse data sets. We thereby constructed high-confidence networks for E. coli and S. aureus, each comprising ~1,700 transcriptional interactions at a precision of ~50%. We experimentally tested 53 previously unobserved regulatory interactions in E. coli, of which 23 (43%) were supported. Our results establish community-based methods as a powerful and robust tool for the inference of transcriptional gene regulatory networks.

Signaling-mediated bacterial persister formation.

Here we show that bacterial communication through indole signaling induces persistence, a phenomenon in which a subset of an isogenic bacterial population tolerates antibiotic treatment. We monitor indole-induced persister formation using microfluidics and identify the role of oxidative-stress and phage-shock pathways in this phenomenon. We propose a model in which indole signaling 'inoculates' a bacterial subpopulation against antibiotics by activating stress responses, leading to persister formation.

Evidence of a possible multicellular life cycle in Escherichia coli.

Biofilms are surface-attached multicellular microbial communities. Their genetics have been extensively studied, but the cell-scale morphogenetic events of their formation are largely unknown. Here, we recorded the entirety of morphogenesis in Escherichia coli, and discovered a previously unknown multicellular self-assembly process. Unattached, single-cells formed 4-cell rosettes which grew into constant-width chains. After ∼10 cell generations, these multicellular chains attached to surfaces and stopped growing. Chains remained clonal throughout morphogenesis. We showed that this process generates biofilms, which we found are composed of attached clonal chains, aligned in parallel. We investigated genetics of chain morphogenesis: Ag43 facilitates rosette formation and clonality; type-1 fimbriae and curli promote stability and configuration; and extracellular polysaccharide production facilitates attachment. Our study establishes that E. coli, a unicellular organism, can follow a multistage, clonal, genetically-regulated, rosette-initiated multicellular life cycle. These findings have implications for synthetic biology, multicellular development, and the treatment and prevention of bacterial diseases.

Fluorescence-based protocol for revealing cellular arrangement in biofilms.

Standardized assays have greatly advanced the understanding of multicellular bacterial biofilms, but they lack cell-scale detail. Here, we present a fluorescence-based protocol that builds on past assays to reveal the cellular-scale arrangement within biofilms. We describe steps for growing biofilms on cover glass, followed by imaging and visualization of cellular arrangements in biofilms. We have applied this protocol to study Escherichia coli biofilms, though it could also be adapted to study biofilm formation in other species. For complete details on the use and execution of this protocol, please refer to Puri et al. (2023).1.

Potentiating aminoglycoside antibiotics to reduce their toxic side effects.

The lack of new antibiotics necessitates the improvement of existing ones, many of which are limited by toxic side effects. Aminoglycosides, antibiotics with excellent activity and low bacterial resistance, are hampered by dose-dependent toxic effects in patients (nephrotoxicity, ototoxicity). High antibiotic concentrations are often required to treat dormant, non-dividing bacteria, though previous studies show that aminoglycosides can be activated against such bacteria by specific metabolites. Here, we employed this mechanism to greatly boost the activity of low concentrations of aminoglycosides against prevalent Gram-negative pathogens (Escherichia coli, Salmonella enterica, and Klebsiella pneumoniae), suggesting that less toxic drug concentrations might be used effectively in patients. We go on to show that this effect improved treatment of biofilms, did not increase aminoglycoside resistance, and was due to the generation of proton-motive force (PMF). By single-cell microscopy, we demonstrate that stationary-phase cells, while non-dividing, actively maintain a growth-arrested state that is not reversed by metabolite addition. Surprisingly, within starved populations, we observed rare cells (3%) that divided without added nutrients. Additionally, we discovered that mannitol could directly protect human kidney cells from aminoglycoside cytotoxicity, independent of the metabolite's effect on bacteria. This work forwards a mechanism-based strategy to improve existing antibiotics by mitigating their toxic side effects.

Heterogeneous bacterial persisters and engineering approaches to eliminate them.

Bacterial persistence is a state in which a subpopulation of cells (persisters) survives antibiotic treatment, and has been implicated in the tolerance of clinical infections and the recalcitrance of biofilms. There has been a renewed interest in the role of bacterial persisters in treatment failure in light of a wealth of recent findings. Here we review recent laboratory studies of bacterial persistence. Further, we pose the hypothesis that each bacterial population may contain a diverse collection of persisters and discuss engineering strategies for persister eradication.