UNRAVELING CRP/cAMP-MEDIATED METABOLIC REGULATION IN ESCHERICHIA COLI PERSISTER CELLS.

A substantial gap persists in our comprehension of how bacterial metabolism undergoes rewiring during the transition to a persistent state. Also, it remains unclear which metabolic mechanisms become indispensable for persister cell survival. To address these questions, we directed our efforts towards persister cells in Escherichia coli that emerge during the late stationary phase. These cells have been recognized for their exceptional resilience and are commonly believed to be in a dormant state. Our results demonstrate that the global metabolic regulator Crp/cAMP redirects the metabolism of these antibiotic-tolerant cells from anabolism to oxidative phosphorylation. Although our data indicates that persisters exhibit a reduced metabolic rate compared to rapidly growing exponential-phase cells, their survival still relies on energy metabolism. Extensive genomic-level analyses of metabolomics, proteomics, and single-gene deletions consistently emphasize the critical role of energy metabolism, specifically the tricarboxylic acid (TCA) cycle, electron transport chain (ETC), and ATP synthase, in sustaining the viability of persisters. Altogether, this study provides much-needed clarification regarding the role of energy metabolism in antibiotic tolerance and highlights the importance of using a multipronged approach at the genomic level to obtain a broader picture of the metabolic state of persister cells.

Exploring the links between SOS response, mutagenesis, and resistance during the recovery period.

Although the mechanistic connections between SOS-induced mutagenesis and antibiotic resistance are well established, our current understanding of the impact of SOS response levels, recovery durations, and transcription/translation activities on mutagenesis remains relatively limited. In this study, when bacterial cells were exposed to mutagens like ultraviolet light for defined time intervals, a compelling connection between the rate of mutagenesis and the RecA-mediated SOS response levels became evident. Our observations also indicate that mutagenesis primarily occurs during the subsequent recovery phase following the removal of the mutagenic agent. When transcription/translation was inhibited or energy molecules were depleted at the onset of treatment or during the early recovery phase, there was a noticeable decrease in SOS response activation and mutagenesis. However, targeting these processes later in the recovery phase does not have the same effect in reducing mutagenesis, suggesting that the timing of inhibiting transcription/translation or depleting energy molecules is crucial for their efficacy in reducing mutagenesis. Active transcription, translation, and energy availability within the framework of SOS response and DNA repair mechanisms appear to be conserved attributes, supported by their consistent manifestation across diverse conditions, including the use of distinct mutagens such as fluoroquinolones and various bacterial strains.

METABOLIC DISRUPTION IMPAIRS RIBOSOMAL PROTEIN LEVELS, RESULTING IN ENHANCED AMINOGLYCOSIDE TOLERANCE.

Aminoglycoside antibiotics display broad-spectrum activity against Gram-negative and Grampositive bacteria by targeting their ribosomes. Herein, we have demonstrated that energy metabolism plays a crucial role in aminoglycoside tolerance, as knockout strains associated with the tricarboxylic acid cycle (TCA) and the electron transport chain (ETC) exhibited increased tolerance to aminoglycosides in the mid-exponential growth phase of Escherichia coli cells. Given that aminoglycoside uptake relies on the energy-driven electrochemical potential across the cytoplasmic membrane, our initial expectation was that these genetic perturbations would decrease the proton motive force (PMF), subsequently affecting the uptake of aminoglycosides. However, our results did not corroborate this assumption. We found no consistent metabolic changes, ATP levels, cytoplasmic pH variations, or membrane potential differences in the mutant strains compared to the wild type. Additionally, intracellular concentrations of fluorophore-labeled gentamicin remained similar across all strains. To uncover the mechanism responsible for the observed tolerance in mutant strains, we employed untargeted mass spectrometry to quantify the proteins within these mutants and subsequently compared them to their wild-type counterparts. Our comprehensive analysis, which encompassed protein-protein association networks and functional enrichment, unveiled a noteworthy upregulation of proteins linked to the TCA cycle in the mutant strains during the mid-exponential growth phase, suggesting that these strains compensate for the perturbation in their energy metabolism by increasing TCA cycle activity to maintain their membrane potential and ATP levels. Furthermore, our pathway enrichment analysis shed light on local network clusters displaying downregulation across all mutant strains, which were associated with both large and small ribosomal binding proteins, ribosome biogenesis, translation factor activity, and the biosynthesis of ribonucleoside monophosphates. These findings offer a plausible explanation for the observed tolerance of aminoglycosides in the mutant strains. Altogether, this research has the potential to uncover mechanisms behind aminoglycoside tolerance, paving the way for novel strategies to combat such cells.

Systematic design of pulse dosing to eradicate persister bacteria.

A small fraction of infectious bacteria use persistence as a strategy to survive exposure to antibiotics. Periodic pulse dosing of antibiotics has long been considered a potentially effective strategy towards eradication of persisters. Recent studies have demonstrated through in vitro experiments that it is indeed feasible to achieve such effectiveness. However, systematic design of periodic pulse dosing regimens to treat persisters is currently lacking. Here we rigorously develop a methodology for the systematic design of optimal periodic pulse dosing strategies for rapid eradication of persisters. A key outcome of the theoretical analysis, on which the proposed methodology is based, is that bactericidal effectiveness of periodic pulse dosing depends mainly on the ratio of durations of the corresponding on and off parts of the pulse. Simple formulas for critical and optimal values of this ratio are derived. The proposed methodology is supported by computer simulations and in vitro experiments.

Suppression of amyloid-ß fibril growth by drug-engineered polymorph transformation.

Fibrillization of the protein amyloid ß is assumed to trigger Alzheimer's pathology. Approaches that target amyloid plaques, however, have garnered limited clinical success, and their failures may relate to the scarce understanding of the impact of potential drugs on the intertwined stages of fibrillization. Here, we demonstrate that bexarotene, a T-cell lymphoma medication with known antiamyloid activity both in vitro and in vivo, suppresses amyloid fibrillization by promoting an alternative fibril structure. We employ time-resolved in situ atomic force microscopy to quantify the kinetics of growth of individual fibrils and supplement it with structure characterization by cryo-EM. We show that fibrils with structure engineered by the drug nucleate and grow substantially slower than "normal" fibrils; remarkably, growth remains stunted even in drug-free solutions. We find that the suppression of fibril growth by bexarotene is not because of the drug binding to the fibril tips or to the peptides in the solution. Kinetic analyses attribute the slow growth of drug-enforced fibril polymorph to the distinctive dynamics of peptide chain association to their tips. As an additional benefit, the bexarotene fibrils kill primary rat hippocampal neurons less efficiently than normal fibrils. In conclusion, the suggested drug-driven polymorph transformation presents a mode of action to irreversibly suppress toxic aggregates not only in Alzheimer's but also potentially in myriad diverse pathologies that originate with protein condensation.

Proton Motive Force Inhibitors Are Detrimental to Methicillin-Resistant Staphylococcus aureus Strains.

Methicillin-resistant Staphylococcus aureus (MRSA) strains are tolerant of conventional antibiotics, making them extremely dangerous. Previous studies have shown the effectiveness of proton motive force (PMF) inhibitors at killing bacterial cells; however, whether these agents can launch a new treatment strategy to eliminate antibiotic-tolerant cells mandates further investigation. Here, using known PMF inhibitors and two different MRSA isolates, we showed that the bactericidal potency of PMF inhibitors seemed to correlate with their ability to disrupt PMF and permeabilize cell membranes. By screening a small chemical library to verify this correlation, we identified a subset of chemicals (including nordihydroguaiaretic acid, gossypol, trifluoperazine, and amitriptyline) that strongly disrupted PMF in MRSA cells by dissipating either the transmembrane electric potential (ΔΨ) or the proton gradient (ΔpH). These drugs robustly permeabilized cell membranes and reduced MRSA cell levels below the limit of detection. Overall, our study further highlights the importance of cellular PMF as a target for designing new bactericidal therapeutics for pathogens. IMPORTANCE Methicillin-resistant Staphylococcus aureus (MRSA) emerged as a major hypervirulent pathogen that causes severe health care-acquired infections. These pathogens can be multidrug-tolerant cells, which can facilitate the recurrence of chronic infections and the emergence of diverse antibiotic-resistant mutants. In this study, we aimed to investigate whether proton motive force (PMF) inhibitors can launch a new treatment strategy to eliminate MRSA cells. Our in-depth analysis showed that PMF inhibitors that strongly dissipate either the transmembrane electric potential or the proton gradient can robustly permeabilize cell membranes and reduce MRSA cell levels below the limit of detection.

Pleiotropic actions of phenothiazine drugs are detrimental to Gram-negative bacterial persister cells.

Bacterial persister cells are temporarily tolerant to bactericidal antibiotics but are not necessarily dormant and may exhibit physiological activities leading to cell damage. Based on the link between fluoroquinolone-mediated SOS responses and persister cell recovery, we screened chemicals that target fluoroquinolone persisters. Metabolic inhibitors (e.g., phenothiazines) combined with ofloxacin (OFX) perturbed persister levels in metabolically active cell populations. When metabolically stimulated, intrinsically tolerant stationary phase cells also became OFX-sensitive in the presence of phenothiazines. The effects of phenothiazines on cell metabolism and physiology are highly pleiotropic: at sublethal concentrations, phenothiazines reduce cellular metabolic, transcriptional, and translational activities; impair cell repair and recovery mechanisms; transiently perturb membrane integrity; and disrupt proton motive force by dissipating the proton concentration gradient across the cell membrane. Screening a subset of mutant strains lacking membrane-bound proteins revealed the pleiotropic effects of phenothiazines potentially rely on their ability to inhibit a wide range of critical metabolic proteins. Altogether, our study further highlights the complex roles of metabolism in persister cell formation, survival and recovery, and suggests metabolic inhibitors such as phenothiazines can be selectively detrimental to persister cells.

High-Throughput Screening of a Promoter Library Reveals New Persister Mechanisms in Escherichia Coli.

Persister cells are a small subpopulation of phenotypic variants that survive high concentrations of bactericidal antibiotics. Their survival mechanisms are not heritable and can be formed stochastically or triggered by environmental stresses such as antibiotic treatment. In this study, high-throughput screening of an Escherichia coli promoter library and subsequent validation experiments identified several genes whose expression was upregulated by antibiotic treatment. Among the identified genes, waaG, guaA, and guaB were found to be important in persister cell formation in E. coli as their deletion significantly enhanced the sensitivity of cells to various antibiotics. The GuaA and GuaB enzymes form the upstream reactions of ppGpp (a global persister molecule) biosynthesis, and the deletion of guaA and guaB drastically perturbs the ppGpp regulon in E. coli. WaaG, a lipopolysaccharide glucosyltransferase, plays an important role in shaping the outer membrane structure, and the deletion of waaG dissipates the proton gradient (ΔpH) component of cellular proton motive force (PMF), perturbs cellular ATP production, and reduces type I persister formation in stationary phase. Active respiration in the stationary phase, which drives the PMF, was previously shown to play a critical role in type I persister formation, and our results associated with the waaG deficient strain further corroborate these findings. IMPORTANCE Persistence is a nonheritable trait by which normal growing cells switch phenotypically to antibiotic tolerant persister cells. This transient state enables persister cells to recover and grow into an antibiotic-sensitive population. Persister cells have been observed in many pathogenic and nonpathogenic bacteria. Previous studies highlight the complexity and diversity of bacterial persister-cell mechanisms, many of which still remain to be elucidated. Here, using promoter and knockout cell libraries in Escherichia coli, we have identified genes that reveal novel persister mechanisms. As persistence is a critical survival strategy that evolved in many bacteria, our study will enhance the current molecular-level understanding of this conserved mechanism.

lon Deletion Impairs Persister Cell Resuscitation in Escherichia coli.

Bacterial persisters are nongrowing cells highly tolerant to bactericidal antibiotics. However, this tolerance is reversible and not mediated by heritable genetic changes. Lon, an ATP-dependent protease, has repeatedly been shown to play a critical role in fluoroquinolone persistence in Escherichia coli. Although lon deletion (Δlon) is thought to eliminate persister cells via accumulation of the cell division inhibitor protein SulA, the exact mechanism underlying this phenomenon is not yet elucidated. Here, we show that Lon is an important regulatory protein for the resuscitation of the fluoroquinolone persisters in E. coli, and lon deletion impairs the ability of persister cells to form colonies during recovery through a sulA- and ftsZ-dependent mechanism. Notably, this observed "viable but nonculturable" state of antibiotic-tolerant Δlon cells is transient, as environmental conditions, such as starvation, can restore their culturability. Our data further indicate that starvation-induced SulA degradation or expression of Lon during recovery facilitates Z-ring formation in Δlon persisters, and Z-ring architecture is important for persister resuscitation in both wild-type and Δlon strains. Our in-depth image analysis clearly shows that the ratio of cell length to number of FtsZ rings for each intact ofloxacin-treated cell predicts the probability of resuscitation and, hence, can be used as a potential biomarker for persisters. IMPORTANCE The ATP-dependent Lon protease is one of the most studied bacterial proteases. Although deletion of lon has been frequently shown to reduce fluoroquinolone persistence, the proposed mechanisms underlying this phenomenon are highly controversial. Here, we have shown that lon deletion in Escherichia coli impairs the ability of persister cells to form colonies during recovery and that this reduction of persister levels in lon-deficient cells can be transient. We also found that altered Z-ring architecture is a key biomarker in both wild-type and lon-deficient persister cells transitioning to a normal cell state. Collectively, our findings highlight the importance of differentiating persister formation mechanisms from resuscitation mechanisms and underscore the critical role of the nonculturable cell state in antibiotic tolerance.

Cellular Self-Digestion and Persistence in Bacteria.

Cellular self-digestion is an evolutionarily conserved process occurring in prokaryotic cells that enables survival under stressful conditions by recycling essential energy molecules. Self-digestion, which is triggered by extracellular stress conditions, such as nutrient depletion and overpopulation, induces degradation of intracellular components. This self-inflicted damage renders the bacterium less fit to produce building blocks and resume growth upon exposure to fresh nutrients. However, self-digestion may also provide temporary protection from antibiotics until the self-digestion-mediated damage is repaired. In fact, many persistence mechanisms identified to date may be directly or indirectly related to self-digestion, as these processes are also mediated by many degradative enzymes, including proteases and ribonucleases (RNases). In this review article, we will discuss the potential roles of self-digestion in bacterial persistence.

BRAF-Inhibitor-Induced Metabolic Alterations in A375 Melanoma Cells.

Acquired drug tolerance has been a major challenge in cancer therapy. Recent evidence has revealed the existence of slow-cycling persister cells that survive drug treatments and give rise to multi-drug-tolerant mutants in cancer. Cells in this dynamic persister state can escape drug treatment by undergoing various epigenetic changes, which may result in a transient metabolic rewiring. In this study, with the use of untargeted metabolomics and phenotype microarrays, we characterize the metabolic profiles of melanoma persister cells mediated by treatment with vemurafenib, a BRAF inhibitor. Our findings demonstrate that metabolites associated with phospholipid synthesis, pyrimidine, and one-carbon metabolism and branched-chain amino acid metabolism are significantly altered in vemurafenib persister cells when compared to the bulk cancer population. Our data also show that vemurafenib persisters have higher lactic acid consumption rates than control cells, further validating the existence of a unique metabolic reprogramming in these drug-tolerant cells. Determining the metabolic mechanisms underlying persister cell survival and maintenance will facilitate the development of novel treatment strategies that target persisters and enhance cancer therapy.

Monitoring Persister Resuscitation with Flow Cytometry.

Persister cells are defined as a small fraction of phenotypic variants in a cell population that are temporarily tolerant to bactericidal antibiotics. Persisters are not mutant cells; they generally survive lethal concentrations of antibiotics due to their transient nongrowing state. Persister cells have the ability to resuscitate after the end of antibiotic treatment. Despite significant advancements in the understanding of the molecular mechanisms underlying persister formation, we still have little information about their resuscitation mechanisms. In this chapter, we describe a method to detect and monitor persister resuscitation at the single-cell level using flow cytometry analysis. This method enables us to not only assess the resuscitation characteristics of persisters but also determine and quantify various subpopulations in antibiotic-treated cultures, including viable but nonculturable (VBNC) and dead cells.

High-throughput Screening of Chemical Compounds to Elucidate Their Effects on Bacterial Persistence.

Bacterial persisters are defined as a small subpopulation of phenotypic variants with the capability of tolerating high concentrations of antibiotics. They are an important health concern as they have been associated with recurrent chronic infections. Although stochastic and deterministic dynamics of stress-related mechanisms are known to play a significant role in persistence, mechanisms underlying the phenotypic switch to/from the persistence state are not completely understood. While persistence factors triggered by environmental signals (e.g., depletion of carbon, nitrogen and oxygen sources) have been extensively studied, the impacts of osmolytes on persistence are yet to be determined. Using microarrays (i.e., 96 well plates containing various chemicals), we have designed an approach to elucidate the effects of various osmolytes on Escherichia coli persistence in a high throughput manner. This approach is transformative as it can be readily adapted for other screening arrays, such as drug panels and gene knockout libraries.

A Transient Metabolic State in Melanoma Persister Cells Mediated by Chemotherapeutic Treatments.

Persistence is a transient state that poses an important health concern in cancer therapy. The mechanisms associated with persister phenotypes are highly diverse and complex, and many aspects of persister cell physiology remain to be explored. We applied a melanoma cell line and panel of chemotherapeutic agents to show that melanoma persister cells are not necessarily preexisting dormant cells; in fact, they may be induced by cancer chemotherapeutics. Our metabolomics analysis and phenotype microarray assays further demonstrated a transient upregulation in Krebs cycle metabolism in persister cells. We also verified that targeting electron transport chain activity can significantly reduce melanoma persister levels. The reported metabolic remodeling feature seems to be a conserved characteristic of melanoma persistence, as it has been observed in various melanoma persister subpopulations derived from a diverse range of chemotherapeutics. Elucidating a global metabolic mechanism that contributes to persister survival and reversible switching will ultimately foster the development of novel cancer therapeutic strategies.

Flow-cytometry analysis reveals persister resuscitation characteristics.

BACKGROUND: Persisters and viable but non-culturable (VBNC) cells are two phenotypic variants known to be highly tolerant to antibiotics. Although both cell types are stained as live and often appear as nongrowing during antibiotic treatment, the only distinguishing feature is the ability of persisters to recolonize in standard culture media in the absence of antibiotics. Despite considerable progress in the characterization of persister formation mechanisms, their resuscitation mechanisms remain unclear due to technical limitations in detecting and isolating these cell types in culture environments that are highly heterogeneous. RESULTS: In this study, we used a methodology integrating flow cytometry, fluorescent protein expression systems and ampicillin-mediated cell lysing technique to monitor persister resuscitation at the single-cell level. With this method, we were able to investigate the effects of various culture conditions (e.g., antibiotic treatment time, the length of the stationary phase in overnight pre-cultures, or pretreatment of cells with a metabolic inhibitor) on persister resuscitation. Although we observed long-term pre-cultures have many more VBNC cells compared to short-term pre-cultures, only a small fraction of non-lysed cells was able to resuscitate in all conditions tested. Regardless of pre-culturing and ampicillin treatment times, these persister cells started to resuscitate within 1 hour, after they were transferred to fresh liquid media, with the same doubling time that normal cells have. Our analysis further showed that ampicillin was not able to lyse the cells in the presence of arsenate, a metabolic inhibitor commonly used to increase bacterial persistence. However, the removal of arsenate during antibiotic treatment resulted in cell lysis and a reduction in persister levels despite the significant decrease in ATP levels in the cells. CONCLUSIONS: The strategy presented in this study helps us monitor persister resuscitation at the single-cell level, and simultaneously quantify persister, VBNC and dead cell subpopulations in ampicillin-treated cultures. Our results indicate that the characterization of persister resuscitation with flow cytometry will enhance the current molecular-level understanding of persistence and its evolution.

Identifying Metabolic Inhibitors to Reduce Bacterial Persistence.

Bacterial persisters are rare phenotypic variants that are temporarily tolerant to high concentrations of antibiotics. We have previously discovered that stationary-phase-cell subpopulations exhibiting high redox activities were less capable of producing proteins and resuming growth upon their dilution into fresh media. The redox activities of these cells were maintained by endogenous protein and RNA degradation, resulting in self-inflicted damage that transiently repressed the cellular functions targeted by antibiotics. Here, we showed that pretreatment of stationary-phase cells with an ATP synthase inhibitor, chlorpromazine hydrochloride (CPZ), significantly reduced stationary-phase-redox activities and protein degradation, and yielded cells that were more susceptible to cell death when exposed to antibiotics in fresh media. Leveraging this knowledge, we developed an assay integrating a degradable fluorescent protein system and a small library, containing FDA-approved drugs and antibiotics, to detect medically relevant drugs that potentially target persister metabolism. We identified a subset of chemical inhibitors, including polymyxin B, poly-L-lysine and phenothiazine anti-psychotic drugs, that were able to reduce the persistence phenotype in Escherichia coli. These chemical inhibitors also reduced Pseudomonas aeruginosa persistence, potentially verifying the existence of similar mechanisms in a medically relevant organism.

Investigating the Effects of Osmolytes and Environmental pH on Bacterial Persisters.

Bacterial persisters are phenotypic variants that temporarily demonstrate an extraordinary tolerance toward antibiotics. Persisters have been linked to the recalcitrance of biofilm-related infections; hence, a complete understanding of their physiology can lead to improvement of therapeutic strategies for such infections. Mechanisms pertaining to persister formation are thought to be associated with stress response pathways triggered by intra- or extracellular stress factors. Unfortunately, studies demonstrating the effects of osmolyte- and/or pH-induced stresses on bacterial persistence are largely missing. To fill this knowledge gap within the field, we studied the effects of various osmolytes and pH conditions on Escherichia coli persistence with the use of phenotype microarrays and antibiotic tolerance assays. Although we found that a number of chemicals and pH environments, including urea, sodium nitrite, and acidic pH, significantly reduced persister formation in E. coli compared to no-osmolyte/no-buffer controls, this reduction in persister levels was less pronounced in late-stationary-phase cultures. Our results further demonstrated a positive correlation between cell growth and persister formation, which challenges the general notion in the field that slow-growing cultures have more persister cells than fast-growing cultures.

Stationary phase persister formation in Escherichia coli can be suppressed by piperacillin and PBP3 inhibition.

BACKGROUND: Persisters are rare phenotypic variants within a bacterial population that are capable of tolerating lethal antibiotic concentrations. Passage through stationary phase is associated with the formation of persisters (type I), and a major physiological response of Escherichia coli during stationary phase is cell wall restructuring. Given the concurrence of these processes, we sought to assess whether perturbation to cell wall synthesis during stationary phase impacts type I persister formation. RESULTS: We tested a panel of cell wall inhibitors and found that piperacillin, which primarily targets penicillin binding protein 3 (PBP3 encoded by ftsI), resulted in a significant reduction in both ß-lactam (ampicillin, carbenicillin) and fluoroquinolone (ofloxacin, ciprofloxacin) persister levels. Further analyses showed that piperacillin exposure through stationary phase resulted in cells with more ATP, DNA, RNA, and protein (including PBPs) than untreated controls; and that their physiology led to more rapid resumption of DNA gyrase supercoiling activity, translation, and cell division upon introduction into fresh media. Previously, PBP3 inhibition had been linked to antibiotic efficacy through the DpiBA two component system; however, piperacillin suppressed persister formation in ΔdpiA to the same extent as it did in wild-type, suggesting that DpiBA is not required for the phenomenon reported here. To test the generality of PBP3 inhibition on persister formation, we expressed FtsI Ser307Ala to genetically inhibit PBP3, and suppression of persister formation was also observed, although not to the same magnitude as that seen for piperacillin treatment. CONCLUSIONS: From these data we conclude that stationary phase PBP3 activity is important to type I persister formation in E. coli.

Burn trauma disrupts circadian rhythms in rat liver.

Circadian rhythms play an important role in maintaining homeostasis and solid organ function. The purpose of this study is to assess the implications of burn injury in rats on the underlying circadian patterns of gene expression in liver. Circadian-regulated genes and burn-induced genes were identified by applying consensus clustering methodology to temporally differentially expressed probe sets obtained from burn and sham-burn data sets. Of the liver specific genes which we hypothesize that exhibit circadian rhythmicity, 88% are not differentially expressed following the burn injury. Specifically, the vast majority of the circadian regulated-genes representing central carbon and nitrogen metabolism are "up-regulated" after the burn injury, indicating the onset of hypermetabolism. In addition, cell-cell junction and membrane structure related genes showing rhythmic behavior in the control group were not differentially expressed across time in the burn group, which could be an indication of hepatic damage due to the burn. Finally, the suppression of the immune function related genes is observed in the postburn phase, implying the severe "immunosuppression". Our results demonstrated that the short term response (24-h post injury) manifests a loss of circadian variability possibly compromising the host in terms of subsequent challenges.

Persister formation in Escherichia coli can be inhibited by treatment with nitric oxide.

Bacterial persisters are phenotypic variants that survive extraordinary concentrations of antibiotics, and are thought to underlie the propensity of biofilm infections to relapse. Unfortunately many aspects of persister physiology remain ill-defined, which prevents progress toward eradicating the phenotype. Recently, we identified respiration within non-growing Escherichia coli populations as a potential target for the elimination type I persisters, which are those that arise from passage through stationary phase. Here we discovered that nitric oxide (NO) treatment at the onset of stationary phase significantly reduced type I persister formation through its ability to inhibit respiration. NO decreased protein and RNA degradation in stationary phase cells, and produced populations that were more fit for protein synthesis and growth resumption upon introduction into fresh media than untreated controls. Overall, this data shows that NO, which is a therapeutically-relevant compound, has the potential to decrease the incidence of recurrent infections from persisters.