Biology subjects

Levin, P. A.

Publications and source records attributed to Levin, P. A..

17 recordsLinked to original sources

bioRxiv2026

Multiple non-redundant functions underlie the essentiality of lipopolysaccharide in Gram-negative bacteria

Lipopolysaccharide (LPS) is well known for its immunostimulatory activity and its contribution to the permeability barrier of the Gram-negative outer membrane. Yet LPS is essential for growth in nearly all Gram-negative bacteria, even under laboratory conditions where its barrier function should be less critical. LPS contributes to envelope mechanics, membrane biogenesis, and cell division, but whether any of these functions accounts for its essentiality remains unclear. Here, we systematically tested these possibilities by depleting LPS in Escherichia coli and Klebsiella pneumoniae. Osmoprotection largely preserved cell-envelope integrity but not viability, demonstrating that the mechanical contribution of LPS alone cannot explain its essentiality. Genetic perturbations that increase peptidoglycan rigidity or alter phospholipid homeostasis partially improved growth, supporting independent roles for LPS in envelope mechanics and membrane biogenesis. An unbiased suppressor screen further identified activation of the MarA regulon as a potent mechanism for surviving LPS depletion. Protection required transport by the AcrAB-TolC multidrug efflux pump, revealing an unexpected connection between efflux and cell-envelope homeostasis. Thus, LPS essentiality emerges from multiple non-redundant contributions to Gram-negative cell-envelope physiology.

microbiology↗

bioRxiv2026

Diverse stresses converge on bacterial periplasmic expansion through distinct physiological mechanisms

Diverse environmental and antibiotic stresses induce localized periplasmic expansion, the separation of the inner membrane from the cell wall and outer membrane, in Gram-negative bacteria, yet whether these morphologically similar responses arise through a common mechanism remains unclear. Here, we compare periplasmic expansion induced by sudden starvation, ciprofloxacin, mitomycin C, and globomycin in Escherichia coli. We find that these perturbations produce similar morphologies through distinct physiological and mechanical routes. Ciprofloxacin- and mitomycin C-induced periplasmic expansion coincides with loss of turgor, whereas starvation requires water efflux, and globomycin leads to unequal expansion of the inner and outer membrane as a result of aberrant inner membrane-cell wall crosslinking. Outer membrane stiffness modulates expansion during ciprofloxacin treatment. Despite these distinct mechanisms, robust periplasmic expansion during starvation and treatment with ciprofloxacin or globomycin requires UDP-glucose synthesis. Together, these findings establish periplasmic expansion as a convergent physical outcome of diverse stresses and reveal the remarkable plasticity of the Gram-negative cell envelope.

microbiology↗

bioRxiv2025

Enzymatic Activity of PBP1B Compensates for β-lactam Mediated Inhibition of PBP2 in Cells Overproducing ppGpp

The alarmone (p)ppGpp (ppGpp) accumulates in response to starvation and other stress, leading to inhibition of multiple biosynthetic pathways and, at high concentrations, suppression of bacterial growth. Growth suppression by ppGpp is implicated in the formation of persister cells, which survive antibiotic challenge only to regrow once the drug is removed. However, there is also evidence that low levels of ppGpp contribute to resistance to certain cell wall-active antibiotics in actively growing cells. To characterize ppGpps contribution to antibiotic resistance, we measured MICs of a panel of {beta}-lactams in actively growing Escherichia coli cells overexpressing a ppGpp synthase (relA*). Cells engineered to modestly overproduce ppGpp exhibited up to 64-fold increases in resistance to PBP2-targeting {beta}-lactams only, with mecillinam the most dramatically affected. Resistance required the transcription factor DksA and the class A penicillin binding protein (PBP) PBP1B. PBP1B variants defective for transpeptidase activity, glycosyltransferase activity, or both were incapable of mediating resistance, suggesting the full enzymatic activity of PBP1B is required for resistance. Transcriptomics revealed that ppGpp overproduction leads to increased expression of lpoB, which encodes an activator of PBP1B. LpoB was required for mecillinam resistance, with an lpoB deletion mutant exhibiting a loss of ppGpp-dependent resistance. Resistance was partially lost in an lpoB deletion strain expressing an LpoB-bypass variant of PBP1B (mrcB*). Together these results support a model wherein overproduction of ppGpp favors cell wall synthesis by PBP1B, in part via increased expression of lpoB, allowing PBP1B to substitute for PBP2 when it is inhibited by {beta}-lactams. ImportanceAntimicrobial resistance is an increasing global health threat, but the molecular mechanisms underlying resistance remain incompletely understood. This work clarifies the role of the alarmone ppGpp in mediating antibiotic resistance in the model bacterium Escherichia coli. Elevated levels of ppGpp were found to cause resistance to {beta}-lactam antibiotics targeting the cell wall synthesis enzyme PBP2. Resistance required transcriptional regulation by ppGpp and enzymatic activity of the cell wall enzyme PBP1B. This work suggests that ppGpp causes resistance in part by increasing expression of lpoB, which encodes an activator of PBP1B. Because ppGpp levels are controlled by nutritional conditions, this work suggests that nutritional availability could impact the efficacy of antibiotics. Furthermore, this study adds to a number of studies demonstrating that cell wall synthesizing PBP enzymes can compensate for one another under certain conditions, leading to {beta}-lactam resistance.

microbiology↗

bioRxiv2025

pH-dependent beta-lactam resistance in Klebsiella pneumoniae is mediated by paralogous class B PBPs and the class A PBP, PBP1b.

Klebsiella pneumoniae is a leading cause of global deaths due to antibiotic resistance. Of particular concern is the rapid expansion of resistance to beta-lactam antibiotics within K. pneumoniae lineages. The environmental factors that influence pathogen physiology and, subsequently, antibiotic resistance remain poorly understood. Here we demonstrate that physiologically-relevant reductions in pH increased K. pneumoniae beta-lactam resistance as much as 64-fold, with the most dramatic increase observed for beta-lactams that specifically inhibit cell division. We identified two genes that contribute to acid-dependent beta-lactam resistance, the class A PBP, PBP1b, and the paralogous class B PBP, PBP3PARA. Loss of either PBP1b or PBP3PARA increases K. pneumoniae susceptibility to beta-lactams at low pH. Altogether these data emphasize the importance of functional redundancy among cell wall synthesis enzymes which allows for specialization and ensures robust cell wall synthesis across a range of environmental conditions. ImportanceBeta-lactams are the most prescribed class of antibiotics, but their effectiveness is threatened by a global rise in antimicrobial resistance. How the environment within a host or infection site shapes pathogen response to antibiotics is frequently overlooked in assessments of antibiotic effectiveness. We demonstrate that growth at physiologically-relevant low pH substantially increases Klebsiella pneumoniae resistance to clinically important beta-lactams. An important finding of this study is that during growth in acidic pH K. pneumoniae has a different repertoire of cell wall synthesis genes available than during growth at neutral pH due to the presence of acid-inducible paralogous copies of essential cell wall synthesis enzymes, PBP2 and PBP3. An additional functionally-redundant enzyme, PBP1b, also contributes to acid-dependent beta-lactam resistance. Together, these findings expand our understanding of how bacteria maintain cell wall synthesis across diverse physiochemical environments and highlight potential new therapeutic targets.

microbiology↗

bioRxiv2023

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 (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. Surprisingly, 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. ImportanceCell 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.

microbiology↗

bioRxiv2022

Connecting sequence features within the disordered C-terminal linker of B. subtilis FtsZ to function and bacterial cell division

Intrinsically disordered regions (IDRs) can function as autoregulators of folded enzymes to which they are tethered. One example is the bacterial cell division protein, FtsZ. This includes a folded core and a C-terminal tail (CTT) that encompasses a poorly conserved, disordered C-terminal linker (CTL) and a well-conserved 17-residue C-terminal peptide (CT17). Sites for GTPase activity of FtsZs are formed at the interface between GTP binding sites and T7 loops on cores of adjacent subunits within dimers. Here, we explore the basis of autoregulatory functions of the CTT in Bacillus subtilis FtsZ (Bs-FtsZ). Molecular simulations show that the CT17 of Bs- FtsZ makes statistically significant CTL-mediated contacts with the T7 loop. Statistical Coupling Analysis of more than 103 sequences from FtsZ orthologs reveals clear covariation of the T7 loop and the CT17 with most of the core domain whereas the CTL is under independent selection. Despite this, we discover the conservation of non-random sequence patterns within CTLs across orthologs. To test how the non-random patterns of CTLs mediate CTT-core interactions and modulate FtsZ functionalities, we designed Bs-FtsZ variants by altering the patterning of oppositely charged residues within the CTL. Such alterations disrupt the core-CTT interactions, lead to anomalous assembly and inefficient GTP hydrolysis in vitro and protein degradation, aberrant assembly, and disruption of cell division in vivo. Our findings suggest that viable CTLs in FtsZs are likely to be IDRs that encompass non-random, functionally relevant sequence patterns that also preserve three-way covariation of the CT17, the T7 loop, and core domain. Significance StatementZ-ring formation by the protein FtsZ controls cell division in rod-shaped bacteria. The C-terminus of FtsZ encompasses a disordered C-terminal linker (CTL) and a conserved CT17 motif. Both modules are essential for Z-ring formation and proper localization of FtsZ in cells. Previous studies suggested that generic intrinsically disordered regions (IDRs) might be suitable functional replacements for naturally occurring CTLs. Contrary to this suggestion, we find that the sequence-encoded conformational properties of CTLs help mediate autoregulatory interactions between covarying regions within FtsZ. Functional properties of the CTL are encoded via evolutionarily conserved, non-random sequence patterns. Disruption of these patterns impair molecular functions and cellular phenotypes. Our findings have broad implications for discovering functionally consequential sequence features within IDRs of other proteins.

biophysics↗

bioRxiv2020

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. colis inner membrane from the cell wall. Shrinkage was accompanied by a [~]17% reduction in cytoplasmic volume and a concurrent increase in periplasmic volume. Inner membrane retraction 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. Robust recovery from starvation required the Tol-Pal system, highlighting the importance of envelope coupling during 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. Significance statementBacterial cells constantly face nutrient fluctuations in their natural environments. While previous studies have identified gene expression changes upon nutrient depletion, it is much less well known how cellular morphology and cytoplasmic properties respond to shifts in nutrient availability. Here, we discovered that switching fast-growing Escherichia coli cells to nutrient-free conditions results in substantial shrinkage of the inner membrane away from the cell wall, especially at the new pole. Shrinkage was primarily driven by loss of cytoplasmic water contents. Shrinkage was also exhibited by cells naturally entering stationary phase, highlighting its biological relevance across starvation conditions. The membrane-spanning Tol-Pal system was critical for robust entry into and recovery from shrinkage, indicating the importance of cell-envelope homeostasis in surviving nutrient starvation.

microbiology↗

bioRxiv2020

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

Nearly all bacteria are encased in a peptidoglycan cell wall, an essential crosslinked matrix of polysaccharide strands and short peptide stems. In the Gram-negative model organism Escherichia coli, more than forty cell wall synthases and autolysins coordinate the growth and division of the peptidoglycan sacculus in the periplasm. The precise contribution of many of these enzymes to cell wall metabolism remains unclear due to significant apparent redundancy, particularly among the cell wall 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 exhibit growth medium-dependent changes in activity. Specifically, we report acidic growth conditions stimulate AmiB--and to a lesser extent, AmiC--activity. Combining computational and genetic analysis, we demonstrate that low pH-dependent stimulation of AmiB requires three periplasmic amidase activators: EnvC, NlpD, and YgeR. Altogether, our findings support overlapping, but not redundant, roles for the E. coli amidases in cell separation and illuminate the physiochemical environment as an important mediator of cell wall enzyme activity. IMPORTANCEPenicillin and related {beta}-lactam antibiotics targeting the bacterial cell wall synthesis are among the most commonly prescribed antimicrobials worldwide. However, rising rates of antibiotic resistance and tolerance jeopardize their continued clinical use. Development of new cell wall active therapeutics, including those targeting cell wall autolysins, has been stymied in part due to high levels of apparent enzymatic redundancy. In this study, we report a subset of E. coli amidases involved in cell separation during cell division are not redundant and instead are preferentially active during growth in distinct pH environments. Specifically, we discover E. coli amidases AmiB and AmiC are activated by acidic pH. Three semi-redundant periplasmic regulators--NlpD, EnvC, and YgeR--collectively mediate low pH-dependent stimulation of amidase activity. This discovery contributes to our understanding of how the cell wall remains robust across diverse environmental conditions and reveals opportunities for the development of condition-specific antimicrobial agents.

microbiology↗

bioRxiv2020

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 the host immune response, antibiotic treatment and 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 non-canonical 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. We propose that PG editing has evolved as an effective strategy for bacteria to overcome T6SS attacks. 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 possessed 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. One sentence summaryAcinetobacter baumannii attains immunity against nonkin competitors during T6SS warfare by incorporating D-Lysine into its peptidoglycan.

microbiology↗

bioRxiv2020

Bacteriophage SP01 Gene Product 56 (gp56) Inhibits Bacillus subtilis Cell Division by Interacting with DivIC/FtsL to Prevent Pbp2B/FtsW Recruitment

Previous work identified gp56, encoded by the lytic bacteriophage SP01, as 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 gene product 56 (gp56) of SP01 inhibits latter stages of B. subtilis cell division without altering FtsZ ring assembly. GFP-tagged gp56 localizes to the membrane at the site of division. While its localization permits 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 analysis suggest that gp56 localization and activity depends on its interaction with mid-recruited proteins DivIC and/or FtsL. Together these data support a model where gp56 interacts with a central part of the division machinery to disrupt late recruitment of the division proteins involved in septal cell wall synthesis. IMPORTANCEResearch over the past decades has uncovered bacteriophage-encoded factors that interfere with host cell shape or cytokinesis during viral infection. Phage factors that cause 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 mechanism of several identified phage factors that inhibit cytokinesis remain unexplored, including gp56 of bacteriophage SP01 of Bacillus subtilis. Here, we show that unlike related published examples of phage inhibition of cyotkinesis, gp56 blocks B. subtilis cell division without targeting FtsZ. Rather, it utilizes the assembled FtsZ cytokinetic ring to localize to the division machinery and block recruitment of proteins needed for the septal cell wall synthesis.

microbiology↗

bioRxiv2019

Environmental pH impacts division assembly and cell size in Escherichia coli

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 key 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 machinery and activate division. Acidic environments lead to enrichment of FtsN at the septum and activation of division at a reduced cell length, while alkaline pH inhibits FtsN localization and suppress division activation. Altogether, our work reveals a previously unappreciated role for pH in bacterial cell size control.

microbiology↗

bioRxiv2018

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 division and replication are independently controlled, and that division processes exclusively drive cell-size homeostasis in bacteria.

systems biology↗

bioRxiv2018

Plasticity in Escherichia coli cell wall metabolism promotes fitness and mediates intrinsic 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 PBP1 a and PBP1 b; 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 differential activity across pH environments significantly alters intrinsic resistance to cell wall active antibiotics. Together, our findings reveal previously thought to be redundant enzymes are instead specialized for distinct environmental niches, thereby ensuring robust growth and cell wall integrity in a wide range of conditions.

microbiology↗

bioRxiv2018

Evolved sequence features within the intrinsically disordered tail influence FtsZ assembly and bacterial cell division

Intrinsically disordered regions (IDRs) challenge the well-established sequence-structure-function paradigm for describing protein function and evolution. Here, we direct a combination of biophysical and cellular studies to further our understanding of how the intrinsically disordered C-terminal tail of FtsZ contributes to cell division in rod-shaped bacteria. FtsZ is a modular protein that encompasses a conserved GTPase domain and a highly variable intrinsically disordered C-terminal tail (CTT). The CTT is essential for forming the cytokinetic Z-ring. Despite poor sequence conservation of the CTT, the patterning of oppositely charged residues, which refers to the extent of linear mixing / segregation of oppositely charged residues within CTT sequences is bounded within a narrow range. To assess the impact of evolutionary bounds on charge patterning within CTT sequences we performed experiments, aided by sequence design, to quantify the impact of changing the patterning of oppositely charged residues within the CTT on the functions of FtsZ from B. subtilis. Z-ring formation is robust if and only if the extent of linear mixing / segregation of oppositely charged residues within the CTT sequences is within evolutionarily observed bounds. Otherwise, aberrant, CTT-mediated, FtsZ assemblies impair Z-ring formation. The complexities of CTT sequences also have to be above a threshold value because FtsZ variants with low complexity CTTs are not tolerated in cells. Taken together, our results suggest that CTT sequences have evolved to be \"just right\" and that this is achieved through an optimal extent of charge patterning while maintaining the sequence complexity above a threshold value.

biophysics↗

bioRxiv2017

A nutrient-dependent division antagonist is regulated post-translationally by the Clp proteases in Bacillus subtilis

Changes in nutrient availability have dramatic and well-defined impacts on both transcription and translation in bacterial cells. At the same time, the role of post-translational control in adaptation to nutrient-poor environments is poorly understood. Here we report a role for the bacterial Clp proteases in degradation of the division inhibitor UgtP during growth in nutrient-poor medium. Under nutrient-rich conditions, interactions with its substrate UDP-glucose promote interactions between UgtP and the tubulin-like cell division protein FtsZ in Bacillus subtilis, inhibiting maturation of the cytokinetic ring and increasing cell size. In nutrient-poor medium, reductions in UDP-glucose availability favor UgtP oligomerization, sequestering it from FtsZ and allowing division to occur at a smaller cell mass. Intriguingly, in nutrient-poor conditions UgtP levels are reduced [~] 3-fold independent of UDP-glucose, suggesting an additional layer of regulation. B. subtilis cells cultured under different nutrient conditions indicate that UgtP accumulation is controlled through a nutrient-dependent post-translational mechanism dependent on the Clp proteases. Notably, all three B. subtilis Clp chaperones appeared able to target UgtP for degradation during growth in nutrient-poor conditions. Together these findings highlight conditional proteolysis as a mechanism for bacterial adaptation to a rapidly changing nutritional landscape.

microbiology↗

bioRxiv2016

Flux through lipid synthesis dictates bacterial cell size.

Nutrients--and by extension biosynthetic capacity--positively impact cell size in organisms throughout the tree of life. In bacteria, cell size is reduced three-fold in response to nutrient starvation or accumulation of the alarmone ppGpp, a global inhibitor of biosynthesis. However, whether biosynthetic capacity as a whole determines cell size or if particular anabolic pathways are more important than others remains an open question. Utilizing a top-down approach, here we identify flux through lipid synthesis as the primary biosynthetic determinant of Escherichia coli cell size. Altering flux through lipid synthesis recapitulated the impact of altering nutrients on cell size and morphology, while defects in other biosynthetic pathways either did not impact size or altered size in a lipid-dependent manner. Together our findings support a model in which lipid availability dictates cell envelope capacity and ppGpp functions as a linchpin linking surface area expansion with cytoplasmic volume to maintain cellular integrity.

microbiology↗

bioRxiv2016

High levels of antibiotic tolerance and persistence are induced by the commercial anti-microbial triclosan

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 US 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.\n\nImportanceAdded as a prophylactic to a wide range of consumer products, the fatty acid synthesis inhibitor triclosan accumulates to high levels in humans and the environment. Based on links between defects in fatty acid synthesis and accumulation of the alarmone ppGpp, we hypothesized that triclosan would render cells tolerant to bactericidal compounds due to ppGpp-mediated inhibition of biosynthetic capacity. Our data indicate that clinically relevant concentrations of triclosan induces higher tolerance of E. coli and methicillin resistant S. aureus (MRSA) to a panel of bactericidal antibiotics up to 10,000-fold. In a urinary tract infection model, mice exposed to triclosan exhibited bacterial loads ~100-fold higher in the bladder than control animals following ciprofloxacin challenge. These findings highlight an unexpected consequence of antimicrobials in consumer products and support an urgent need to reevaluate the costs and benefits of the prophylactic use of triclosan and other bacteriostatic compounds.

microbiology↗