The transcription factor DksA exerts opposing effects on cell division depending on the presence of ppGpp.

Bacterial cell size is a multifactorial trait that is influenced by variables including nutritional availability and the timing of cell division. Prior work revealed a negative correlation between concentration of the alarmone (p)ppGpp (ppGpp) and cell length in Escherichia coli, suggesting that ppGpp may promote assembly of the division machinery (divisome) and cytokinesis in this organism. To clarify this counterintuitive connection between a starvation-induced stress response effector and cell proliferation, we undertook a systematic analysis of growth and division in E. coli cells defective in ppGpp synthesis and/or engineered to overproduce the alarmone. Our data indicate that ppGpp acts indirectly on divisome assembly through its role as a global mediator of transcription. Loss of either ppGpp (ppGpp0) or the ppGpp-associated transcription factor DksA led to increased average length, with ppGpp0 mutants also exhibiting a high frequency of extremely long filamentous cells. Using heat-sensitive division mutants and fluorescently labeled division proteins, we confirmed that ppGpp and DksA are cell division activators. We found that ppGpp and DksA regulate division through their effects on transcription, although the lack of known division genes or regulators in available transcriptomics data strongly suggests that this regulation is indirect. We also found that DksA inhibits division in ppGpp0 cells, contrary to its role in a wild-type background. We propose that the ability of ppGpp to switch DksA from a division inhibitor to a division activator helps tune cell length across different concentrations of ppGpp. IMPORTANCE Cell division is a key step in the bacterial lifecycle that must be appropriately regulated to ensure survival. This work identifies the alarmone (p)ppGpp (ppGpp) as a general regulator of cell division, extending our understanding of the role of ppGpp beyond a signal for starvation and other stress. Even in nutrient-replete conditions, basal levels of ppGpp are essential for division to occur appropriately and for cell size to be maintained. This study establishes ppGpp as a "switch" that controls whether the transcription factor DksA behaves as a division activator or inhibitor. This unexpected finding enhances our understanding of the complex regulatory mechanisms employed by bacteria to coordinate division with diverse aspects of cell growth and stress response. Because division is an essential process, a better understanding of the mechanisms governing the assembly and activation of the division machinery could contribute to the development of novel therapeutics to treat bacterial infections.

The transcription factor DksA exerts opposing effects on cell division depending on the presence of ppGpp.

Bacterial cell size is a multifactorial trait that is influenced by variables including nutritional availability and the timing of cell division. Prior work revealed a negative correlation between the alarmone (p)ppGpp (ppGpp) and cell length in Escherichia coli , suggesting that ppGpp may promote assembly of the division machinery (divisome) and cytokinesis in this organism. To clarify this counterintuitive connection between a starvation induced stress response effector and cell proliferation, we undertook a systematic analysis of growth and division in E. coli cells defective in ppGpp synthesis and/or engineered to overproduce the alarmone. Our data indicate that ppGpp acts indirectly on divisome assembly through its role as a global mediator of transcription. Loss of either ppGpp (ppGpp 0 ) or the ppGpp-associated transcription factor DksA led to increased average length, with ppGpp 0 mutants also exhibiting a high frequency of extremely long filamentous cells. Using heat-sensitive division mutants and fluorescently labeled division proteins, we confirmed that ppGpp and DksA are cell division activators. We found that ppGpp and DksA regulate division through their effects on transcription, although the lack of known division genes or regulators in available transcriptomics data strongly suggests that this regulation is indirect. Surprisingly, we also found that DksA inhibits division in ppGpp 0 cells, contrary to its role in a wild-type background. We propose that the ability of ppGpp to switch DksA from a division inhibitor to a division activator helps tune cell length across different concentrations of ppGpp. Importance: Cell division is a key step in the bacterial lifecycle that must be appropriately regulated to ensure survival. This work identifies the alarmone ppGpp as a general regulator of cell division, extending our understanding of the role of ppGpp beyond a signal for starvation and other stress. Even in nutrient replete conditions, basal levels of ppGpp are essential for division to occur appropriately and for cell size to be maintained. This study establishes ppGpp as a "switch" that controls whether the transcription factor DksA behaves as a division activator or inhibitor. This unexpected finding enhances our understanding of the complex regulatory mechanisms employed by bacteria to coordinate division with diverse aspects of cell growth and stress response. Because division is an essential process, a better understanding the mechanisms governing assembly and activation of the division machinery could contribute to the development of novel therapeutics to treat bacterial infections.

Physiological and regulatory convergence between osmotic and nutrient stress responses in microbes.

Bacterial cells are regularly confronted with simultaneous changes in environmental nutrient supply and osmolarity. Despite the importance of osmolarity and osmoregulation in bacterial physiology, the relationship between the cellular response to osmotic perturbations and other stresses has remained largely unexplored. Bacteria cultured in hyperosmotic conditions and bacteria experiencing nutrient stress exhibit similar physiological changes, including metabolic shutdown, increased protein instability, dehydration, and condensation of chromosomal DNA. In this review, we highlight overlapping molecular players between osmotic and nutrient stresses. These connections between two seemingly disparate stress response pathways reinforce the importance of central carbon metabolism as a control point for diverse aspects of homeostatic regulation. We identify important open questions for future research, emphasizing the pressing need to develop and exploit new methods for probing how osmolarity affects phylogenetically diverse species.

Beyond the average: An updated framework for understanding the relationship between cell growth, DNA replication, and division in a bacterial system.

Our understanding of the bacterial cell cycle is framed largely by population-based experiments that focus on the behavior of idealized average cells. Most famously, the contributions of Cooper and Helmstetter help to contextualize the phenomenon of overlapping replication cycles observed in rapidly growing bacteria. Despite the undeniable value of these approaches, their necessary reliance on the behavior of idealized average cells masks the stochasticity inherent in single-cell growth and physiology and limits their mechanistic value. To bridge this gap, we propose an updated and agnostic framework, informed by extant single-cell data, that quantitatively accounts for stochastic variations in single-cell dynamics and the impact of medium composition on cell growth and cell cycle progression. In this framework, stochastic timers sensitive to medium composition impact the relationship between cell cycle events, accounting for observed differences in the relationship between cell cycle events in slow- and fast-growing cells. We conclude with a roadmap for potential application of this framework to longstanding open questions in the bacterial cell cycle field.

Nutrient availability as an arbiter of cell size.

Pioneering work carried out over 60 years ago discovered that bacterial cell size is proportional to the growth rate set by nutrient availability. This relationship is traditionally referred to as the 'growth law'. Subsequent studies revealed the growth law to hold across all orders of life, a remarkable degree of conservation. However, recent work suggests the relationship between growth rate, nutrients, and cell size is far more complicated and less deterministic than originally thought. Focusing on bacteria and yeast, here we review efforts to understand the molecular mechanisms underlying the relationship between growth rate and cell size.

A bacterium that is not a microbe.

A new discovery challenges the prevailing view of the boundaries of bacterial cell size.

Inactivation of multidrug-resistant bacteria and bacterial spores and generation of high-potency bacterial vaccines using ultrashort pulsed lasers.

Multidrug-resistant organisms (MDROs) represent a continuing healthcare crisis with no definitive solution to date. An alternative to antibiotics is the development of therapies and vaccines using biocompatible physical methods such as ultrashort pulsed (USP) lasers, which have previously been shown to inactivate pathogens while minimizing collateral damage to human cells, blood proteins, and vaccine antigens. Here we demonstrate that visible USP laser treatment results in bactericidal effect (≥3-log load reduction) against clinically significant MDROs, including methicillin-resistant Staphylococcus aureus and extended spectrum beta-lactamase-producing Escherichia coli. Bacillus cereus endospores, which are highly resistant to conventional chemical and physical treatments, were also shown to be effectively inactivated by USP laser treatment, resulting in sporicidal (≥3-log load reduction) activity. Furthermore, we demonstrate that administration of USP laser-inactivated E. coli whole-cell vaccines at dosages as low as 105 cfu equivalents without adjuvant was able to protect 100% of mice against subsequent lethal challenge. Our findings open the possibility for application of USP lasers in disinfection of hospital environments, therapy of drug-resistant bacterial infections in skin or bloodstream via pheresis modalities, and in the production of potent bacterial vaccines.

Localization, Assembly, and Activation of the Escherichia coli Cell Division Machinery.

Decades of research, much of it in Escherichia coli, have yielded a wealth of insight into bacterial cell division. Here, we provide an overview of the E. coli division machinery with an emphasis on recent findings. We begin with a short historical perspective into the discovery of FtsZ, the tubulin homolog that is essential for division in bacteria and archaea. We then discuss assembly of the divisome, an FtsZ-dependent multiprotein platform, at the midcell septal site. Not simply a scaffold, the dynamic properties of polymeric FtsZ ensure the efficient and uniform synthesis of septal peptidoglycan. Next, we describe the remodeling of the cell wall, invagination of the cell envelope, and disassembly of the division apparatus culminating in scission of the mother cell into two daughter cells. We conclude this review by highlighting some of the open questions in the cell division field, emphasizing that much remains to be discovered, even in an organism as extensively studied as E. coli.

Starvation induces shrinkage of the bacterial cytoplasm.

Environmental fluctuations are a common challenge for single-celled organisms; enteric bacteria such as Escherichia coli experience dramatic changes in nutrient availability, pH, and temperature during their journey into and out of the host. While the effects of altered nutrient availability on gene expression and protein synthesis are well known, their impacts on cytoplasmic dynamics and cell morphology have been largely overlooked. Here, we discover that depletion of utilizable nutrients results in shrinkage of E. coli's inner membrane from the cell wall. Shrinkage was accompanied by an ∼17% reduction in cytoplasmic volume and a concurrent increase in periplasmic volume. Inner membrane retraction after sudden starvation occurred almost exclusively at the new cell pole. This phenomenon was distinct from turgor-mediated plasmolysis and independent of new transcription, translation, or canonical starvation-sensing pathways. Cytoplasmic dry-mass density increased during shrinkage, suggesting that it is driven primarily by loss of water. Shrinkage was reversible: upon a shift to nutrient-rich medium, expansion started almost immediately at a rate dependent on carbon source quality. A robust entry into and recovery from shrinkage required the Tol-Pal system, highlighting the importance of envelope coupling during shrinkage and recovery. Klebsiella pneumoniae also exhibited shrinkage when shifted to carbon-free conditions, suggesting a conserved phenomenon. These findings demonstrate that even when Gram-negative bacterial growth is arrested, cell morphology and physiology are still dynamic.

The active repertoire of Escherichia coli peptidoglycan amidases varies with physiochemical environment.

Nearly all bacteria are encased in peptidoglycan, an extracytoplasmic matrix of polysaccharide strands crosslinked through short peptide stems. In the Gram-negative model organism Escherichia coli, more than 40 synthases and autolysins coordinate the growth and division of the peptidoglycan sacculus in the periplasm. The precise contribution of many of these enzymes to peptidoglycan metabolism remains unclear due to significant apparent redundancy, particularly among the autolysins. E. coli produces three major LytC-type-N-acetylmuramoyl-L-alanine amidases, which share a role in separating the newly formed daughter cells during cytokinesis. Here, we reveal two of the three amidases that exhibit growth medium-dependent changes in activity. Specifically, we report acidic growth conditions stimulate AmiB-and to a lesser extent, AmiC-amidase activity. Combining genetic, biochemical, and computational analyses, we demonstrate that low pH-dependent stimulation of AmiB is mediated through the periplasmic amidase activators NlpD, EnvC, and ActS (formerly known as YgeR). Although NlpD and EnvC promote amidase activity across pH environments, ActS preferentially stimulates AmiB activity in acidic conditions. Altogether, our findings support partially overlapping roles for E. coli amidases and their regulators in cell separation and illuminate the physiochemical environment as an important mediator of cell wall enzyme activity.

Bacteriophage SP01 Gene Product 56 Inhibits Bacillus subtilis Cell Division by Interacting with FtsL and Disrupting Pbp2B and FtsW Recruitment.

Previous work identified gene product 56 (gp56), encoded by the lytic bacteriophage SP01, as being responsible for inhibition of Bacillus subtilis cell division during its infection. Assembly of the essential tubulin-like protein FtsZ into a ring-shaped structure at the nascent site of cytokinesis determines the timing and position of division in most bacteria. This FtsZ ring serves as a scaffold for recruitment of other proteins into a mature division-competent structure permitting membrane constriction and septal cell wall synthesis. Here, we show that expression of the predicted 9.3-kDa gp56 of SP01 inhibits later stages of B. subtilis cell division without altering FtsZ ring assembly. Green fluorescent protein-tagged gp56 localizes to the membrane at the site of division. While its localization does not interfere with recruitment of early division proteins, gp56 interferes with the recruitment of late division proteins, including Pbp2b and FtsW. Imaging of cells with specific division components deleted or depleted and two-hybrid analyses suggest that gp56 localization and activity depend on its interaction with FtsL. Together, these data support a model in which gp56 interacts with a central part of the division machinery to disrupt late recruitment of the division proteins involved in septal cell wall synthesis.IMPORTANCE Studies over the past decades have identified bacteriophage-encoded factors that interfere with host cell shape or cytokinesis during viral infection. The phage factors causing cell filamentation that have been investigated to date all act by targeting FtsZ, the conserved prokaryotic tubulin homolog that composes the cytokinetic ring in most bacteria and some groups of archaea. However, the mechanisms of several phage factors that inhibit cytokinesis, including gp56 of bacteriophage SP01 of Bacillus subtilis, remain unexplored. Here, we show that, unlike other published examples of phage inhibition of cytokinesis, gp56 blocks B. subtilis cell division without targeting FtsZ. Rather, it utilizes the assembled FtsZ cytokinetic ring to localize to the division machinery and to block recruitment of proteins needed for septal cell wall synthesis.

Bacterial Cell Wall Quality Control during Environmental Stress.

Single-celled organisms must adapt their physiology to persist and propagate across a wide range of environmental conditions. The growth and division of bacterial cells depend on continuous synthesis of an essential extracellular barrier: the peptidoglycan cell wall, a polysaccharide matrix that counteracts turgor pressure and confers cell shape. Unlike many other essential processes and structures within the bacterial cell, the peptidoglycan cell wall and its synthesis machinery reside at the cell surface and are thus uniquely vulnerable to the physicochemical environment and exogenous threats. In addition to the diversity of stressors endangering cell wall integrity, defects in peptidoglycan metabolism require rapid repair in order to prevent osmotic lysis, which can occur within minutes. Here, we review recent work that illuminates mechanisms that ensure robust peptidoglycan metabolism in response to persistent and acute environmental stress. Advances in our understanding of bacterial cell wall quality control promise to inform the development and use of antimicrobial agents that target the synthesis and remodeling of this essential macromolecule.IMPORTANCE Nearly all bacteria are encased in a peptidoglycan cell wall, an essential polysaccharide structure that protects the cell from osmotic rupture and reinforces cell shape. The integrity of this protective barrier must be maintained across the diversity of environmental conditions wherein bacteria replicate. However, at the cell surface, the cell wall and its synthesis machinery face unique challenges that threaten their integrity. Directly exposed to the extracellular environment, the peptidoglycan synthesis machinery encounters dynamic and extreme physicochemical conditions, which may impair enzymatic activity and critical protein-protein interactions. Biotic and abiotic stressors-including host defenses, cell wall active antibiotics, and predatory bacteria and phage-also jeopardize peptidoglycan integrity by introducing lesions, which must be rapidly repaired to prevent cell lysis. Here, we review recently discovered mechanisms that promote robust peptidoglycan synthesis during environmental and acute stress and highlight the opportunities and challenges for the development of cell wall active therapeutics.

Peptidoglycan editing provides immunity to Acinetobacter baumannii during bacterial warfare.

Peptidoglycan (PG) is essential in most bacteria. Thus, it is often targeted by various assaults, including interbacterial attacks via the type VI secretion system (T6SS). Here, we report that the Gram-negative bacterium Acinetobacter baumannii strain ATCC 17978 produces, secretes, and incorporates the noncanonical d-amino acid d-lysine into its PG during stationary phase. We show that PG editing increases the competitiveness of A. baumannii during bacterial warfare by providing immunity against peptidoglycan-targeting T6SS effectors from various bacterial competitors. In contrast, we found that d-Lys production is detrimental to pathogenesis due, at least in part, to the activity of the human enzyme d-amino acid oxidase (DAO), which degrades d-Lys producing H2O2 toxic to bacteria. Phylogenetic analyses indicate that the last common ancestor of A. baumannii had the ability to produce d-Lys. However, this trait was independently lost multiple times, likely reflecting the evolution of A. baumannii as a human pathogen.

The Absence of (p)ppGpp Renders Initiation of Escherichia coli Chromosomal DNA Synthesis Independent of Growth Rates.

The initiation of Escherichia coli chromosomal DNA replication starts with the oligomerization of the DnaA protein at repeat sequences within the origin (ori) region. The amount of ori DNA per cell directly correlates with the growth rate. During fast growth, the cell generation time is shorter than the time required for complete DNA replication; therefore, overlapping rounds of chromosome replication are required. Under these circumstances, the ori region DNA abundance exceeds the DNA abundance in the termination (ter) region. Here, high ori/ter ratios are found to persist in (p)ppGpp-deficient [(p)ppGpp0] cells over a wide range of balanced exponential growth rates determined by medium composition. Evidently, (p)ppGpp is necessary to maintain the usual correlation of slow DNA replication initiation with a low growth rate. Conversely, ori/ter ratios are lowered when cell growth is slowed by incrementally increasing even low constitutive basal levels of (p)ppGpp without stress, as if (p)ppGpp alone is sufficient for this response. There are several previous reports of (p)ppGpp inhibition of chromosomal DNA synthesis initiation that occurs with very high levels of (p)ppGpp that stop growth, as during the stringent starvation response or during serine hydroxamate treatment. This work suggests that low physiological levels of (p)ppGpp have significant functions in growing cells without stress through a mechanism involving negative supercoiling, which is likely mediated by (p)ppGpp regulation of DNA gyrase.IMPORTANCE Bacterial cells regulate their own chromosomal DNA synthesis and cell division depending on the growth conditions, producing more DNA when growing in nutritionally rich media than in poor media (i.e., human gut versus water reservoir). The accumulation of the nucleotide analog (p)ppGpp is usually viewed as serving to warn cells of impending peril due to otherwise lethal sources of stress, which stops growth and inhibits DNA, RNA, and protein synthesis. This work importantly finds that small physiological changes in (p)ppGpp basal levels associated with slow balanced exponential growth incrementally inhibit the intricate process of initiation of chromosomal DNA synthesis. Without (p)ppGpp, initiations mimic the high rates present during fast growth. Here, we report that the effect of (p)ppGpp may be due to the regulation of the expression of gyrase, an important enzyme for the replication of DNA that is a current target of several antibiotics.

pH-dependent activation of cytokinesis modulates Escherichia coli cell size.

Cell size is a complex trait, derived from both genetic and environmental factors. Environmental determinants of bacterial cell size identified to date primarily target assembly of cytosolic components of the cell division machinery. Whether certain environmental cues also impact cell size through changes in the assembly or activity of extracytoplasmic division proteins remains an open question. Here, we identify extracellular pH as a modulator of cell division and a significant determinant of cell size across evolutionarily distant bacterial species. In the Gram-negative model organism Escherichia coli, our data indicate environmental pH impacts the length at which cells divide by altering the ability of the terminal cell division protein FtsN to localize to the cytokinetic ring where it activates division. Acidic environments lead to enrichment of FtsN at the septum and activation of division at a reduced cell length. Alkaline pH inhibits FtsN localization and suppresses division activation. Altogether, our work reveals a previously unappreciated role for pH in bacterial cell size control.

Mechanistic Origin of Cell-Size Control and Homeostasis in Bacteria.

Evolutionarily divergent bacteria share a common phenomenological strategy for cell-size homeostasis under steady-state conditions. In the presence of inherent physiological stochasticity, cells following this "adder" principle gradually return to their steady-state size by adding a constant volume between birth and division, regardless of their size at birth. However, the mechanism of the adder has been unknown despite intense efforts. In this work, we show that the adder is a direct consequence of two general processes in biology: (1) threshold-accumulation of initiators and precursors required for cell division to a respective fixed number-and (2) balanced biosynthesis-maintenance of their production proportional to volume growth. This mechanism is naturally robust to static growth inhibition but also allows us to "reprogram" cell-size homeostasis in a quantitatively predictive manner in both Gram-negative Escherichia coli and Gram-positive Bacillus subtilis. By generating dynamic oscillations in the concentration of the division protein FtsZ, we were able to oscillate cell size at division and systematically break the adder. In contrast, periodic induction of replication initiator protein DnaA caused oscillations in cell size at initiation but did not alter division size or the adder. Finally, we were able to restore the adder phenotype in slow-growing E. coli, the only known steady-state growth condition wherein E. coli significantly deviates from the adder, by repressing active degradation of division proteins. Together, these results show that cell division and replication initiation are independently controlled at the gene-expression level and that division processes exclusively drive cell-size homeostasis in bacteria. VIDEO ABSTRACT.

One is Nothing without the Other: Theoretical and Empirical Analysis of Cell Growth and Cell Cycle Progression.

Small, fast-growing bacteria make ideal subjects for genetic and quantitative analysis alike. Long the darling of theoreticians, efforts to understand the relationship between cell growth and cell cycle progression in bacterial systems have been propelled by modelers and empiricist in equal measure. Taking a historical approach, here we break down early work in this area, the impact it had on how the bacterial cell cycle is understood and interrogated, and changes brought by the advent of high-throughput techniques for the analysis of individual bacterial cells in culture.

Plasticity of Escherichia coli cell wall metabolism promotes fitness and antibiotic resistance across environmental conditions.

Although the peptidoglycan cell wall is an essential structural and morphological feature of most bacterial cells, the extracytoplasmic enzymes involved in its synthesis are frequently dispensable under standard culture conditions. By modulating a single growth parameter-extracellular pH-we discovered a subset of these so-called 'redundant' enzymes in Escherichia coli are required for maximal fitness across pH environments. Among these pH specialists are the class A penicillin binding proteins PBP1a and PBP1b; defects in these enzymes attenuate growth in alkaline and acidic conditions, respectively. Genetic, biochemical, and cytological studies demonstrate that synthase activity is required for cell wall integrity across a wide pH range and influences pH-dependent changes in resistance to cell wall active antibiotics. Altogether, our findings reveal previously thought to be redundant enzymes are instead specialized for distinct environmental niches. This specialization may ensure robust growth and cell wall integrity in a wide range of conditions. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

A Chemical Inhibitor of Cell Growth Reduces Cell Size in Bacillus subtilis.

Bacteria exhibit complex responses to biologically active small molecules. These responses include reductions in transcriptional and translational efficiency, alterations in metabolic flux, and in some cases, dramatic changes in growth and morphology. Here, we describe Min-1, a novel small molecule that inhibits growth of Gram-positive bacteria by targeting the cell envelope. Subinhibitory levels of Min-1 inhibits sporulation in Streptomyces venezuelae and reduces growth rate and cell length in Bacillus subtilis. The effect of Min-1 on B. subtilis cell length is significant at high growth rates sustained by nutrient-rich media but drops off when growth rate is reduced during growth on less energy-rich carbon sources. In each medium, Min-1 has no impact on the proportion of cells containing FtsZ-rings, suggesting that Min-1 reduces the mass at which FtsZ assembly is initiated. The effect of Min-1 on size is independent of UDP-glucose, which couples cell division to carbon availability, and the alarmone ppGpp, which reduces cell size via its impact on fatty acid synthesis. Min-1 activates the LiaRS stress response, which is sensitive to disruptions in the lipid II cycle and the cell membrane, and also compromises cell membrane integrity. Therefore, this novel synthetic molecule inhibits growth at high concentrations and induces a short-cell phenotype at subinhibitory concentrations that is independent of known systems that influence cell length, highlighting the complex interactions between small molecules and cell morphology.

The Widely Used Antimicrobial Triclosan Induces High Levels of Antibiotic Tolerance In Vitro and Reduces Antibiotic Efficacy up to 100-Fold In Vivo.

The antimicrobial triclosan is used in a wide range of consumer products ranging from toothpaste, cleansers, socks, and baby toys. A bacteriostatic inhibitor of fatty acid synthesis, triclosan is extremely stable and accumulates in the environment. Approximately 75% of adults in the United States have detectable levels of the compound in their urine, with a sizeable fraction of individuals (>10%) having urine concentrations equal to or greater than the minimal inhibitory concentration for Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA). Previous work has identified connections between defects in fatty acid synthesis and accumulation of the alarmone guanosine tetraphosphate (ppGpp), which has been repeatedly associated with antibiotic tolerance and persistence. Based on these data, we hypothesized that triclosan exposure may inadvertently drive bacteria into a state in which they are able to tolerate normally lethal concentrations of antibiotics. Here we report that clinically relevant concentrations of triclosan increased E. coli and MRSA tolerance to bactericidal antibiotics as much as 10,000-fold in vitro and reduced antibiotic efficacy up to 100-fold in a mouse urinary tract infection model. Genetic analysis indicated that triclosan-mediated antibiotic tolerance requires ppGpp synthesis but is independent of growth. These data highlight an unexpected and certainly unintended consequence of adding high concentrations of antimicrobials in consumer products, supporting an urgent need to reevaluate the costs and benefits of the prophylactic use of triclosan and other bacteriostatic compounds.