CBASS is an immune pathway that recognizes phage infection and generates cyclic nucleotide signals, which directly activate effectors that stop phage replication. Membrane-acting effectors are proposed to induce cell death to prevent phage replication; however, this mechanism has not been assessed with endogenous expression levels of the effector. We therefore sought to determine the cell viability outcomes of the CBASS phospholipase effector (CapV) upon activation with 3',3'-cGAMP signals in Pseudomonas aeruginosa. Here, we surprisingly observe that constitutive 3',3'-cGAMP signaling from the synthase (CdnA) enables robust cell growth and viability while effectively abolishing phage production in a CapV-dependent manner. Exogenous 3',3'-cGAMP also enhances CBASS anti-phage activity and cell growth. Moreover, constitutive activation of the CapV effector induces no cell fitness cost, and blocks replication of many, but not all, phages. This demonstrates that a cyclic nucleotide-activated CBASS effector possess a degree of phage specificity that has been previously overlooked. When CBASS is active, phage transcription and initiation of DNA replication proceed normally, but phages do not reach maximum DNA levels and fewer mature virions are produced. Based on these findings, we propose that CapV interferes with the early stages of phage capsid assembly at the cell membrane and resultantly disrupts DNA packaging. Collectively, we demonstrate that a successful CBASS response antagonizes a late-stage of the phage replication cycle while maintaining cell viability.

The swimming motility of is a virulence factor that aids in breaching the mucus layer of the small intestine. cells have a curved cell shape and previous work demonstrated that loss of curvature decreases infectivity. Here we investigate the mechanism by which 's curvature affects single-cell motility within mucus-mimicking environments. Using a multiscale chemotaxis assay, we compared the chemotactic performance of wild-type curved cells (O1 El Tor C6706) and straight mutants under linear chemical gradients in liquid solutions, viscous solutions, and soft agar hydrogels. Our findings reveal that curved and straight exhibit similar swimming properties in liquid and purely viscous solutions but significantly differ in hydrogels, with curved cells demonstrating an 86% increase in average chemotactic drift compared to straight mutants in the same chemical gradients. Trajectory analysis indicates that swimming speeds are comparable, but straight mutants experience more frequent stalls, reducing the total time spent swimming. We also found that stalls further reduce chemotactic performances by imposing an average reorientation of bacteria down the chemical gradient, regardless of cell shape. In-silico coarse-grained molecular dynamics simulations corroborate these results and extend them over the wide range of intestinal mucus hydrogel stiffnesses. This model also identifies an optimal curvature for enhanced movement through hydrogel-like meshes that is close to the real median curvature of the pathogen. Our findings thus highlight the mechanisms underpinning cell shape's role in 's pathogenicity and underscore the necessity of studying bacterial behaviors under conditions that simulate host environments.

Genetic screens are powerful approaches to unveiling new biological insights and ordered redundant transposon libraries have emerged as a primary tool for performing screens with known genetic saturation. Newer sequencing approaches based on combinatorial pooling have lowered the cost and time required to generate these libraries. is a gram-negative bacterium that has served as a model for understanding bacterial physiology with a myriad of genetic tools. To add to this collection of tools, we created CauloKO-the first ordered, transposon library in . CauloKO includes insertion mutants in 86% of all non-essential genes and 77% of all open reading frames of strain CB15. CauloKO insertion mutants were validated using Sanger sequencing. We also present phenotypic analysis of the CauloKO library using a crystal violet screen for biofilm mutants, which both confirmed previous results and identified new mutants for future studies. This combined approach revealed that the CauloKO library shows promise for screening applications, particularly for phenotypes that require monoclonal populations of cells.IMPORTANCEOrdered transposon mutant libraries have dramatically advanced the ability to study other systems, and the potential for combining such a library with the other genome-wide tools available in makes such a library particularly important for this system. Our work also establishes a novel method for analyzing sequencing data for ordered library construction purposes, and a first-of-its-kind dynamic, online platform for cataloging mutants within the library. Furthermore, our findings further confirmed results of previously published studies, but also identified potential novel regulators of biofilm formation in that will be important starting points for future investigations.

Fluid flows are dominant features of many bacterial environments, and flow can often impact bacterial behaviors in unexpected ways. For example, the most common type of cardiovascular infection is heart valve colonization by gram-positive bacteria like and (endocarditis). This behavior is counterintuitive because heart valves experience high shear rates that would naively be expected to reduce colonization. To determine whether these bacteria preferentially colonize higher shear rate environments, we developed a microfluidic system to quantify the effect of flow conditions on the colonization of and . We find that the preferential colonization in high flow of both species is not specific to heart valves and can be found in simple configurations lacking any host factors. This behavior enables bacteria that are outcompeted in low flow to dominate in high flow. Surprisingly, experimental and computational studies reveal that the two species achieve this behavior via distinct mechanisms. grows in cell clusters and produces a dispersal signal whose transport is affected by shear rate. Meanwhile, grows in linear chains whose mechanical properties result in less dispersal in the presence of higher shear force. In addition to establishing two divergent mechanisms by which these bacteria each preferentially colonize high-flow environments, our findings highlight the importance of understanding bacterial behaviors at the level of collective interactions among cells. These results suggest that distinct multicellular nanocolony morphologies have previously unappreciated costs and benefits in different environments, like those introduced by fluid flow.

In its natural habitat, must distinguish friend from foe. are abundant in the worm's environment and can be nutritious or pathogenic. Previously, we found that worms learn to avoid and through a small RNA (sRNA)-mediated pathway targeting the gene , and this behavior is inherited for four generations. Here, we show that learns to transgenerationally avoid another pathogenic bacteria 15 (PF15). The PF15 sRNA, Pfs1, targets the ephrin receptor through 16 nt of perfect match, suggesting the evolution of a distinct bacterial sRNA/ gene target pair. Knockdown of both induce PF15 avoidance, and loss reduces expression, placing both genes in the sRNA-targeted pathogenic avoidance pathway. Thus, multiple genes in this avoidance pathway can act as targets for bacterial sRNAs, expanding the possibilities for evolution of trans-kingdom regulation of behavior.

Understanding the mechanisms that dictate the localization of cytoskeletal filaments is crucial for elucidating cell shape regulation in prokaryotes. The actin homolog MreB plays a pivotal role in maintaining the shape of many rod-shaped bacteria such as by directing cell-wall synthesis according to local curvature cues. However, the basis of MreB's curvature-dependent localization has remained elusive. Here, we develop a biophysical model for the energetics of a filament binding to a surface that integrates the complex interplay between filament twist and bending and the two-dimensional surface geometry. Our model predicts that the spatial localization of a filament like MreB with substantial intrinsic twist is governed by both the mean and Gaussian curvatures of the cell envelope, which strongly covary in rod-shaped cells. Using molecular dynamics simulations to estimate the mechanical properties of MreB filaments, we show that their thermodynamic preference for regions with lower mean and Gaussian curvatures matches experimental observations for physiologically relevant filament lengths of ~50 nm. We find that the experimentally measured statistical curvature preference is maintained in the absence of filament motion and after a cycle of depolymerization, repolymerization, and membrane rebinding, indicating that equilibrium energetics can explain MreB localization. These findings provide critical insights into the physical principles underlying cytoskeletal filament localization and suggest design principles for synthetic shape-sensing nanomaterials.

In microbial communities, viruses compete for host cells and have evolved diverse mechanisms to inhibit competitors. One strategy is superinfection exclusion, whereby an established viral infection prevents a secondary infection of the same cell. This phenomenon has been shown to have an important role in the spread of eukaryotic viruses. Here we determine that superinfection exclusion proteins in bacterial viruses (bacteriophages, hereafter phages) perform a similar role, promoting viral spread through the bacterial community. We characterize a phage protein that alters the dynamics of a common phage receptor, the type IV pilus. This protein, known as Zip, does not abrogate pilus activity, but fine-tunes it, providing a strong phage defence without a fitness cost. Notably, Zip also prevents internalization and destruction of newly released phage progeny, a phenomenon that we call the anti-Kronos effect after the Greek god who consumed his offspring. Zip activity promotes the accumulation of free phages in bacterial lysogen communities, thereby enhancing viral spread. We further demonstrate that the anti-Kronos effect is conserved across diverse prophage-encoded superinfection exclusion systems. Our results identify the mechanistic basis of a superinfection exclusion system that safeguards phage progeny and provide insights into the conservation of viral defence mechanisms among bacterial and eukaryotic systems.

The crowded bacterial cytoplasm is composed of biomolecules that span several orders of magnitude in size and electrical charge. This complexity has been proposed as the source of the rich spatial organization and apparent anomalous diffusion of intracellular components, although this has not been tested directly. Here, we use biplane microscopy to track the 3D motion of self-assembled bacterial genetically encoded multimeric nanoparticles (bGEMs) with tunable size (20 to 50 nm) and charge (-3,240 to +2,700 e) in live cells. To probe intermolecular details at spatial and temporal resolutions beyond experimental limits, we also developed a colloidal whole-cell model that explicitly represents the size and charge of cytoplasmic macromolecules and the porous structure of the bacterial nucleoid. Combining these techniques, we show that bGEMs spatially segregate by size, with small 20-nm particles enriched inside the nucleoid, and larger and/or positively charged particles excluded from this region. Localization is driven by entropic and electrostatic forces arising from cytoplasmic polydispersity, nucleoid structure, geometrical confinement, and interactions with other biomolecules including ribosomes and DNA. We observe that at the timescales of traditional single molecule tracking experiments, motion appears subdiffusive for all particle sizes and charges. However, using computer simulations with higher temporal resolution, we find that the apparent anomalous exponents are governed by the region of the cell in which bGEMs are located. Molecular motion does not display anomalous diffusion on short time scales and the apparent subdiffusion arises from geometrical confinement within the nucleoid and by the cell boundary.

Pseudomonas aeruginosa is a leading cause of hospital-acquired infections for which the development of antibiotics is urgently needed. Unlike most enteric bacteria, P. aeruginosa lacks enzymes required to scavenge exogenous thymine. An appealing strategy to selectively target P. aeruginosa is to disrupt thymidine synthesis while providing exogenous thymine. However, known antibiotics that perturb thymidine synthesis are largely inactive against P. aeruginosa.Here we characterize fluorofolin, a dihydrofolate reductase (DHFR) inhibitor derived from Irresistin-16, that exhibits significant activity against P. aeruginosa in culture and in a mouse thigh infection model. Fluorofolin is active against a wide range of clinical P. aeruginosa isolates resistant to known antibiotics. Metabolomics and in vitro assays using purified folA confirm that fluorofolin inhibits P. aeruginosa DHFR. Importantly, in the presence of thymine supplementation, fluorofolin activity is selective for P. aeruginosa. Resistance to fluorofolin can emerge through overexpression of the efflux pumps MexCD-OprJ and MexEF-OprN, but these mutants also decrease pathogenesis. Our findings demonstrate how understanding species-specific genetic differences can enable selective targeting of important pathogens while revealing trade-offs between resistance and pathogenesis.

Protein turnover is critical for proteostasis, but turnover quantification is challenging, and even in well-studied E. coli, proteome-wide measurements remain scarce. Here, we quantify the turnover rates of ~3200 E. coli proteins under 13 conditions by combining heavy isotope labeling with complement reporter ion quantification and find that cytoplasmic proteins are recycled when nitrogen is limited. We use knockout experiments to assign substrates to the known cytoplasmic ATP-dependent proteases. Surprisingly, none of these proteases are responsible for the observed cytoplasmic protein degradation in nitrogen limitation, suggesting that a major proteolysis pathway in E. coli remains to be discovered. Lastly, we show that protein degradation rates are generally independent of cell division rates. Thus, we present broadly applicable technology for protein turnover measurements and provide a rich resource for protein half-lives and protease substrates in E. coli, complementary to genomics data, that will allow researchers to study the control of proteostasis.

Detecting chemical signals is important for identifying food sources and avoiding harmful agents. Like many animals, C. elegans use olfaction to chemotax towards their main food source, bacteria. However, little is known about the bacterial compounds governing C. elegans attraction to bacteria and the physiological importance of these compounds to bacteria. Here, we address these questions by investigating the function of a small RNA, P11, in the pathogen, Pseudomonas aeruginosa, that was previously shown to mediate learned pathogen avoidance. We discovered that this RNA also affects the attraction of untrained C. elegans to P. aeruginosa and does so by controlling production of ammonia, a volatile odorant produced during nitrogen assimilation. We describe the complex regulation of P. aeruginosa nitrogen assimilation, which is mediated by a partner-switching mechanism involving environmental nitrates, sensor proteins, and P11. In addition to mediating C. elegans attraction, we demonstrate that nitrogen assimilation mutants perturb bacterial fitness and pathogenesis during C. elegans infection by P. aeruginosa. These studies define ammonia as a major mediator of trans-kingdom signaling, implicate nitrogen assimilation as important for both bacteria and host organisms, and highlight how a bacterial metabolic pathway can either benefit or harm a host in different contexts.

Detecting chemical signals is important for identifying food sources and avoiding harmful agents. Like many animals, C. elegans use olfaction to chemotax towards their main food source, bacteria. However, little is known about the bacterial compounds governing C. elegans attraction to bacteria and the physiological importance of these compounds to bacteria. Here, we address these questions by investigating the function of a small RNA, P11, in the pathogen, Pseudomonas aeruginosa, that was previously shown to mediate learned pathogen avoidance. We discovered that this RNA also affects the attraction of untrained C. elegans to P. aeruginosa and does so by controlling production of ammonia, a volatile odorant produced during nitrogen assimilation. We describe the complex regulation of P. aeruginosa nitrogen assimilation, which is mediated by a partner-switching mechanism involving environmental nitrates, sensor proteins, and P11. In addition to mediating C. elegans attraction, we demonstrate that nitrogen assimilation mutants perturb bacterial fitness and pathogenesis during C. elegans infection by P. aeruginosa. These studies define ammonia as a major mediator of trans-kingdom signaling, implicate nitrogen assimilation as important for both bacteria and host organisms, and highlight how a bacterial metabolic pathway can either benefit or harm a host in different contexts.

Surface-attached cells can sense and respond to shear flow, but planktonic (free-swimming) cells are typically assumed to be oblivious to any flow that carries them. Here, we find that planktonic bacteria can transcriptionally respond to flow, inducing expression changes that are beneficial in flow. Specifically, we use microfluidic experiments and quantitative modeling to show that in the presence of flow, planktonic induce shear rate-dependent genes that promote growth in low-oxygen environments. Untangling this mechanism revealed that in flow, motile spatially redistribute, leading to cell density changes that activate quorum sensing, which in turn enhances the oxygen uptake rate. In diffusion-limited environments, including those commonly encountered by bacteria, flow-induced cell density gradients also independently generate oxygen gradients that alter gene expression. Mutants deficient in this flow-responsive mechanism exhibit decreased fitness in flow, suggesting that this dynamic coupling of biological and mechanical processes can be physiologically significant.

C. elegans can learn to avoid pathogenic bacteria through several mechanisms, including bacterial small RNA-induced learned avoidance behavior, which can be inherited transgenerationally. Previously, we discovered that a small RNA from a clinical isolate of Pseudomonas aeruginosa, PA14, induces learned avoidance and transgenerational inheritance of that avoidance in C. elegans. Pseudomonas aeruginosa is an important human pathogen, and there are other Pseudomonads in C. elegans' natural habitat, but it is unclear whether C. elegans ever encounters PA14-like bacteria in the wild. Thus, it is not known if small RNAs from bacteria found in C. elegans' natural habitat can also regulate host behavior and produce heritable behavioral effects. Here we screened a set of wild habitat bacteria, and found that a pathogenic Pseudomonas vranovensis strain isolated from the C. elegans microbiota, GRb0427, regulates worm behavior: worms learn to avoid this pathogenic bacterium following exposure, and this learned avoidance is inherited for four generations. The learned response is entirely mediated by bacterially-produced small RNAs, which induce avoidance and transgenerational inheritance, providing further support that such mechanisms of learning and inheritance exist in the wild. We identified Pv1, a small RNA expressed in P. vranovensis, that has a 16-nucleotide match to an exon of the C. elegans gene maco-1. Pv1 is both necessary and sufficient to induce learned avoidance of Grb0427. However, Pv1 also results in avoidance of a beneficial microbiome strain, P. mendocina. Our findings suggest that bacterial small RNA-mediated regulation of host behavior and its transgenerational inheritance may be functional in C. elegans' natural environment, and that this potentially maladaptive response may favor reversal of the transgenerational memory after a few generations. Our data also suggest that different bacterial small RNA-mediated regulation systems evolved independently, but define shared molecular features of bacterial small RNAs that produce transgenerationally-inherited effects.

C. elegans can learn to avoid pathogenic bacteria through several mechanisms, including bacterial small RNA-induced learned avoidance behavior, which can be inherited transgenerationally. Previously, we discovered that a small RNA from a clinical isolate of Pseudomonas aeruginosa, PA14, induces learned avoidance and transgenerational inheritance of that avoidance in C. elegans. Pseudomonas aeruginosa is an important human pathogen, and there are other Pseudomonads in C. elegans' natural habitat, but it is unclear whether C. elegans ever encounters PA14-like bacteria in the wild. Thus, it is not known if small RNAs from bacteria found in C. elegans' natural habitat can also regulate host behavior and produce heritable behavioral effects. Here we screened a set of wild habitat bacteria, and found that a pathogenic Pseudomonas vranovensis strain isolated from the C. elegans microbiota, GRb0427, regulates worm behavior: worms learn to avoid this pathogenic bacterium following exposure, and this learned avoidance is inherited for four generations. The learned response is entirely mediated by bacterially-produced small RNAs, which induce avoidance and transgenerational inheritance, providing further support that such mechanisms of learning and inheritance exist in the wild. We identified Pv1, a small RNA expressed in P. vranovensis, that has a 16-nucleotide match to an exon of the C. elegans gene maco-1. Pv1 is both necessary and sufficient to induce learned avoidance of Grb0427. However, Pv1 also results in avoidance of a beneficial microbiome strain, P. mendocina. Our findings suggest that bacterial small RNA-mediated regulation of host behavior and its transgenerational inheritance may be functional in C. elegans' natural environment, and that this potentially maladaptive response may favor reversal of the transgenerational memory after a few generations. Our data also suggest that different bacterial small RNA-mediated regulation systems evolved independently, but define shared molecular features of bacterial small RNAs that produce transgenerationally-inherited effects.

are exposed to a variety of pathogenic and non-pathogenic bacteria species in their natural environment. Correspondingly, has evolved an ability to discern between nutritive and infectious bacterial food sources. Here we show that can learn to avoid the pathogenic bacteria (PF15), and that this learned avoidance behavior is passed on to progeny for four generations, as we previously demonstrated for (PA14) and , using similar mechanisms, including the involvement of both the TGF-β ligand DAF-7 and retrotransposon-encoded virus-like particles. PF15 small RNAs are both necessary and sufficient to induce this transgenerational avoidance behavior. Unlike PA14 or , PF15 does not use P11, Pv1, or a small RNA with homology for this avoidance; instead, an unrelated PF15 small RNA, Pfs1, that targets the Ephrin receptor gene is necessary and sufficient for learned avoidance, suggesting the evolution of yet another bacterial sRNA/ gene target pair involved in transgenerational inheritance of pathogen avoidance. As VAB-2 Ephrin receptor ligand and MACO-1 knockdown also induce PF15 avoidance, we have begun to understand the genetic pathway involved in small RNA targeted pathogenic avoidance. Moreover, these data show that axon guidance pathway genes (VAB-1 and VAB-2) have previously unknown adult roles in regulating neuronal function. may have evolved multiple bacterial specificity-encoded small RNA-dependent mechanisms to avoid different pathogenic bacteria species, thereby providing progeny with a survival advantage in a dynamic environment.

are exposed to a variety of pathogenic and non-pathogenic bacteria species in their natural environment. Correspondingly, has evolved an ability to discern between nutritive and infectious bacterial food sources. Here we show that can learn to avoid the pathogenic bacteria (PF15), and that this learned avoidance behavior is passed on to progeny for four generations, as we previously demonstrated for (PA14) and , using similar mechanisms, including the involvement of both the TGF-β ligand DAF-7 and retrotransposon-encoded virus-like particles. PF15 small RNAs are both necessary and sufficient to induce this transgenerational avoidance behavior. Unlike PA14 or , PF15 does not use P11, Pv1, or a small RNA with homology for this avoidance; instead, an unrelated PF15 small RNA, Pfs1, that targets the Ephrin receptor gene is necessary and sufficient for learned avoidance, suggesting the evolution of yet another bacterial sRNA/ gene target pair involved in transgenerational inheritance of pathogen avoidance. As VAB-2 Ephrin receptor ligand and MACO-1 knockdown also induce PF15 avoidance, we have begun to understand the genetic pathway involved in small RNA targeted pathogenic avoidance. Moreover, these data show that axon guidance pathway genes (VAB-1 and VAB-2) have previously unknown adult roles in regulating neuronal function. may have evolved multiple bacterial specificity-encoded small RNA-dependent mechanisms to avoid different pathogenic bacteria species, thereby providing progeny with a survival advantage in a dynamic environment.

UNLABELLED: Understanding the mechanisms that dictate the localization of cytoskeletal filaments is crucial for elucidating cell shape regulation in prokaryotes. The actin homolog MreB plays a pivotal role in maintaining the shape of many rod-shaped bacteria such as by directing cell-wall synthesis according to local curvature cues. However, the basis of MreB's curvature-dependent localization has remained elusive. Here, we develop a biophysical model for the energetics of filament binding to a surface that integrates the complex interplay between filament twist and bending and the two-dimensional surface geometry. Our model predicts that the spatial localization of a filament like MreB with substantial intrinsic twist is governed by both the mean and Gaussian curvatures of the cell envelope, which strongly covary in rod-shaped cells. Using molecular dynamics simulations to estimate the mechanical properties of MreB filaments, we show that their thermodynamic preference for regions with lower mean and Gaussian curvatures matches experimental observations for physiologically relevant filament lengths of ∼50 nm. We find that the experimentally measured statistical curvature preference is maintained in the absence of filament motion and after a cycle of depolymerization, repolymerization, and membrane rebinding, indicating that equilibrium energetics can explain MreB localization. These findings provide critical insights into the physical principles underlying cytoskeletal filament localization, and suggest new design principles for synthetic shape sensing nanomaterials.

SIGNIFICANCE STATEMENT: The protein MreB, a homolog of eukaryotic actin, regulates the shape of bacteria like by guiding new cell-wall insertion based on local curvature cues. However, the mechanism by which a nanometer-scale MreB filament "senses" the micron-scale curvature of the cell wall has remained a mystery. We introduce a biophysical model of the energetics of twisted and bent filaments bound to curved surfaces, which predicts that localization of filaments like MreB is sensitive to both mean and Gaussian curvature. The model captures experimentally measured curvature enrichment patterns and explains how MreB naturally localizes to saddle-shaped regions without energy-consuming processes. Beyond cell shape regulation, our work suggests design principles for synthetic systems that can sense and respond to surface shape.

UNLABELLED: Understanding the mechanisms that dictate the localization of cytoskeletal filaments is crucial for elucidating cell shape regulation in prokaryotes. The actin homolog MreB plays a pivotal role in maintaining the shape of many rod-shaped bacteria such as by directing cell-wall synthesis according to local curvature cues. However, the basis of MreB's curvature-dependent localization has remained elusive. Here, we develop a biophysical model for the energetics of filament binding to a surface that integrates the complex interplay between filament twist and bending and the two-dimensional surface geometry. Our model predicts that the spatial localization of a filament like MreB with substantial intrinsic twist is governed by both the mean and Gaussian curvatures of the cell envelope, which strongly covary in rod-shaped cells. Using molecular dynamics simulations to estimate the mechanical properties of MreB filaments, we show that their thermodynamic preference for regions with lower mean and Gaussian curvatures matches experimental observations for physiologically relevant filament lengths of ∼50 nm. We find that the experimentally measured statistical curvature preference is maintained in the absence of filament motion and after a cycle of depolymerization, repolymerization, and membrane rebinding, indicating that equilibrium energetics can explain MreB localization. These findings provide critical insights into the physical principles underlying cytoskeletal filament localization, and suggest new design principles for synthetic shape sensing nanomaterials.

SIGNIFICANCE STATEMENT: The protein MreB, a homolog of eukaryotic actin, regulates the shape of bacteria like by guiding new cell-wall insertion based on local curvature cues. However, the mechanism by which a nanometer-scale MreB filament "senses" the micron-scale curvature of the cell wall has remained a mystery. We introduce a biophysical model of the energetics of twisted and bent filaments bound to curved surfaces, which predicts that localization of filaments like MreB is sensitive to both mean and Gaussian curvature. The model captures experimentally measured curvature enrichment patterns and explains how MreB naturally localizes to saddle-shaped regions without energy-consuming processes. Beyond cell shape regulation, our work suggests design principles for synthetic systems that can sense and respond to surface shape.

The retractile type IV pilus (T4P) is important for virulence of the opportunistic human pathogen . The single-stranded RNA (ssRNA) phage PP7 binds to T4P and is brought to the cell surface through pilus retraction. Using fluorescence microscopy, we discovered that PP7 detaches T4P, which impairs cell motility and restricts the pathogen's virulence. Using cryo-electron microscopy, mutagenesis, optical trapping, and Langevin dynamics simulation, we resolved the structure of PP7, T4P, and the PP7/T4P complex and showed that T4P detachment is driven by the affinity between the phage maturation protein and its bound pilin, plus the pilus retraction force and speed, and pilus bending. Pilus detachment may be widespread among other ssRNA phages and their retractile pilus systems and offers new prospects for antibacterial prophylaxis and therapeutics.

In vertebrate spindles, most microtubules are formed via branching microtubule nucleation, whereby microtubules nucleate along the side of pre-existing microtubules. Hepatoma up-regulated protein (HURP) is a microtubule-associated protein that has been implicated in spindle assembly, but its mode of action is yet to be defined. In this study, we show that HURP is necessary for RanGTP-induced branching microtubule nucleation in Xenopus egg extract. Specifically, HURP stabilizes the microtubule lattice to promote microtubule formation from γ-TuRC. This function is shifted to promote branching microtubule nucleation through enhanced localization to TPX2 condensates, which form the core of the branch site on microtubules. Lastly, we provide a high-resolution cryo-EM structure of HURP on the microtubule, revealing how HURP binding stabilizes the microtubule lattice. We propose a model in which HURP stabilizes microtubules during their formation, and TPX2 preferentially enriches HURP to microtubules to promote branching microtubule nucleation and thus spindle assembly.

The crowded bacterial cytoplasm is comprised of biomolecules that span several orders of magnitude in size and electrical charge. This complexity has been proposed as the source of the rich spatial organization and apparent anomalous diffusion of intracellular components, although this has not been tested directly. Here, we use biplane microscopy to track the 3D motion of self-assembled bacterial Genetically Encoded Multimeric nanoparticles (bGEMs) with tunable size (20 to 50 nm) and charge (-2160 to +1800 e) in live cells. To probe intermolecular details at spatial and temporal resolutions beyond experimental limits, we also developed a colloidal whole-cell model that explicitly represents the size and charge of cytoplasmic macromolecules and the porous structure of the bacterial nucleoid. Combining these techniques, we show that bGEMs spatially segregate by size, with small 20-nm particles enriched inside the nucleoid, and larger and/or positively charged particles excluded from this region. Localization is driven by entropic and electrostatic forces arising from cytoplasmic polydispersity, nucleoid structure, geometrical confinement, and interactions with other biomolecules including ribosomes and DNA. We observe that at the timescales of traditional single molecule tracking experiments, motion appears sub-diffusive for all particle sizes and charges. However, using computer simulations with higher temporal resolution, we find that the apparent anomalous exponents are governed by the region of the cell in which bGEMs are located. Molecular motion does not display anomalous diffusion on short time scales and the apparent sub-diffusion arises from geometrical confinement within the nucleoid and by the cell boundary.

The skin microbiome provides vital contributions to human health. However, the spatial organization and viability of its bacterial components remain unclear. Here, we apply culturing, imaging, and molecular approaches to human and mouse skin samples, and find that the skin surface is colonized by fewer viable bacteria than predicted by bacterial DNA levels. Instead, viable skin-associated bacteria are predominantly located in hair follicles and other cutaneous invaginations. Furthermore, we show that the skin microbiome has a uniquely low fraction of viable bacteria compared to other human microbiome sites, indicating that most bacterial DNA on the skin surface is not associated with viable cells Additionally, a small number of bacterial families dominate each skin site and traditional sequencing methods overestimate both the richness and diversity of the skin microbiome. Finally, we performed an in vivo skin microbiome perturbation-recovery study using human volunteers. Bacterial 16S rRNA gene sequencing revealed that, while the skin microbiome is remarkably stable even in the wake of aggressive perturbation, repopulation of the skin surface is driven by the underlying viable population. Our findings help explain the dynamics of skin microbiome perturbation as bacterial DNA on the skin surface can be transiently perturbed but is replenished by a stable underlying viable population. These results address multiple outstanding questions in skin microbiome biology with significant implications for future efforts to study and manipulate it.

Microtubules are generated at centrosomes, chromosomes, and within spindles during cell division. Whereas microtubule nucleation at the centrosome is well characterized, much remains unknown about where, when, and how microtubules are nucleated at chromosomes. To address these questions, we reconstitute microtubule nucleation from purified chromosomes in meiotic Xenopus egg extract and find that chromosomes alone can form spindles. We visualize microtubule nucleation near chromosomes using total internal reflection fluorescence microscopy to find that this occurs through branching microtubule nucleation. By inhibiting molecular motors, we find that the organization of the resultant polar branched networks is consistent with a theoretical model where the effectors for branching nucleation are released by chromosomes, forming a concentration gradient that spatially biases branching microtbule nucleation. In the presence of motors, these branched networks are ultimately organized into functional spindles, where the number of emergent spindle poles scales with the number of chromosomes and total chromatin area.

Detecting chemical signals is important for identifying food sources and avoiding harmful agents. Like most animals, use olfaction to chemotax towards their main food source, bacteria. However, little is known about the bacterial compounds governing attraction to bacteria and the physiological importance of these compounds to bacteria. Here, we address these questions by investigating the function of a small RNA, P11, in the pathogen, that was previously shown to mediate learned pathogen avoidance. We discovered that this RNA also affects the attraction of untrained to and does so by controlling production of ammonia, a volatile odorant produced during nitrogen assimilation. We untangle the complex regulation of nitrogen assimilation, which is mediated by a partner-switching mechanism involving environmental nitrates, sensor proteins, and P11. In addition to mediating attraction, nitrogen assimilation is important for bacterial fitness and pathogenesis during infection by . These studies define ammonia as a major mediator of trans-kingdom signaling, reveal the physiological importance of nitrogen assimilation for both bacteria and host organisms, and highlight how a bacterial metabolic pathway can either benefit or harm a host in different contexts.

Previously, we discovered that a small RNA from a clinical isolate of PA14, induces learned avoidance and its transgenerational inheritance in . is an important human pathogen, and there are other in natural habitat, but it is unclear whether ever encounters PA14-like bacteria in the wild. Thus, it is not known if small RNAs from bacteria found in natural habitat can also regulate host behavior and produce heritable behavioral effects. Here we found that a pathogenic strain isolated from the microbiota, GRb0427, like PA14, regulates worm behavior: worms learn to avoid this pathogenic bacterium following exposure to GRb0427, and this learned avoidance is inherited for four generations. The learned response is entirely mediated by bacterially-produced small RNAs, which induce avoidance and transgenerational inheritance, providing further support that such mechanisms of learning and inheritance exist in the wild. Using bacterial small RNA sequencing, we identified Pv1, a small RNA from GRb0427, that matches the sequence of . We find that Pv1 is both necessary and sufficient to induce learned avoidance of Grb0427. However, Pv1 also results in avoidance of a beneficial microbiome strain, ; this potentially maladaptive response may favor reversal of the transgenerational memory after a few generations. Our findings suggest that bacterial small RNA-mediated regulation of host behavior and its transgenerational inheritance are functional in natural environment, and that different bacterial small RNA-mediated regulation systems evolved independently but define shared molecular features of bacterial small RNAs that produce transgenerationally-inherited effects.

Bacterial pathogenicity relies on both firm surface adhesion and cell dissemination. How twitching bacteria resolve the fundamental contradiction between adhesion and migration is unknown. To address this question, we employ live-cell imaging of type-IV pili (T4P) and therewith construct a comprehensive mathematical model of migration. The data show that only 10% to 50% of T4P bind to substrates and contribute to migration through random extension and retraction. Individual T4P do not display a measurable sensory response to surfaces, but their number increases on cellular surface contact. Attachment to surfaces is mediated, besides T4P, by passive adhesive forces acting on the cell body. Passive adhesions slow down cell migration and result in local random motion on short time scales, which is followed by directionally persistent, superdiffusive motion on longer time scales. Moreover, passive adhesions strongly enhance surface attachment under shear flow. Δ mutants, which produce no T4P, robustly stick to surfaces under shear flow. In contrast, rapidly migrating Δ cells, which produce an excessive number of T4P, are easily detached by shear. Wild-type cells sacrifice migration speed for robust surface attachment by maintaining a low number of active pili. The different cell strains pertain to disjunct regimes in a generic adhesion-migration trait space. Depending on the nature of the adhesion structures, adhesion and migration are either compatible or a trade-off is required for efficient bacterial surface colonization under different conditions.

Bacterial pathogenicity relies on both firm surface adhesion and cell dissemination. How twitching bacteria resolve the fundamental contradiction between adhesion and migration is unknown. To address this question, we employ live-cell imaging of type-IV pili (T4P) and therewith construct a comprehensive mathematical model of migration. The data show that only 10% to 50% of T4P bind to substrates and contribute to migration through random extension and retraction. Individual T4P do not display a measurable sensory response to surfaces, but their number increases on cellular surface contact. Attachment to surfaces is mediated, besides T4P, by passive adhesive forces acting on the cell body. Passive adhesions slow down cell migration and result in local random motion on short time scales, which is followed by directionally persistent, superdiffusive motion on longer time scales. Moreover, passive adhesions strongly enhance surface attachment under shear flow. Δ mutants, which produce no T4P, robustly stick to surfaces under shear flow. In contrast, rapidly migrating Δ cells, which produce an excessive number of T4P, are easily detached by shear. Wild-type cells sacrifice migration speed for robust surface attachment by maintaining a low number of active pili. The different cell strains pertain to disjunct regimes in a generic adhesion-migration trait space. Depending on the nature of the adhesion structures, adhesion and migration are either compatible or a trade-off is required for efficient bacterial surface colonization under different conditions.

Bacterial populations are highly adaptive. They can respond to stress and survive in shifting environments. How the behaviours of individual bacteria vary during stress, however, is poorly understood. To identify and characterize rare bacterial subpopulations, technologies for single-cell transcriptional profiling have been developed. Existing approaches show some degree of limitation, for example, in terms of number of cells or transcripts that can be profiled. Due in part to these limitations, few conditions have been studied with these tools. Here we develop massively-parallel, multiplexed, microbial sequencing (M3-seq)-a single-cell RNA-sequencing platform for bacteria that pairs combinatorial cell indexing with post hoc rRNA depletion. We show that M3-seq can profile bacterial cells from different species under a range of conditions in single experiments. We then apply M3-seq to hundreds of thousands of cells, revealing rare populations and insights into bet-hedging associated with stress responses and characterizing phage infection.

In mammals, subcellular protein localization of factors like planar cell polarity proteins is a key driver of the multicellular organization of tissues. Bacteria also form organized multicellular communities, but these patterns are largely thought to emerge from regulation of whole-cell processes like growth, motility, cell shape, and differentiation. Here we show that a unique intracellular patterning of appendages known as type IV pili (T4P) can drive multicellular development of complex bacterial communities. Specifically, dynamic T4P appendages localize in a line along the long axis of the cell in the bacterium Acinetobacter baylyi. This long-axis localization is regulated by a functionally divergent chemosensory Pil-Chp system, and an atypical T4P protein homologue (FimV) bridges Pil-Chp signaling and T4P positioning. We further demonstrate through modeling and empirical approaches that subcellular T4P localization controls how individual cells interact with one another, independently of T4P dynamics, with different patterns of localization giving rise to distinct multicellular architectures. Our results reveal how subcellular patterning of single cells regulates the development of multicellular bacterial communities.

Fibronectin (FN), an essential component of the extracellular matrix (ECM), is assembled via a cell-mediated process in which integrin receptors bind secreted FN and mediate its polymerization into fibrils that extend between cells, ultimately forming an insoluble matrix. Our previous work using mutant Chinese hamster ovary (CHO) cells identified the glycosaminoglycan heparan sulfate (HS) and its binding to FN as essential for the formation of insoluble FN fibrils. In this study, we investigated the contributions of HS at an early stage of the assembly process using knockdown of exostosin-1 (EXT1), one of the glycosyltransferases required for HS chain synthesis. NIH 3T3 fibroblasts with decreased EXT1 expression exhibited a significant reduction in both FN and type I collagen in the insoluble matrix. We show that FN fibril formation is initiated at matrix assembly sites, and while these sites were formed by cells with EXT1 knockdown, their growth was stunted compared with wild-type cells. The most severe defect observed was in the polymerization of nascent FN fibrils, which was reduced 2.5-fold upon EXT1 knockdown. This defect was rescued by the addition of exogenous soluble heparin chains long enough to simultaneously bind multiple FN molecules. The activity of soluble heparin in this process indicates that nascent fibril formation depends on HS more so than on the protein component of a specific HS proteoglycan. Together, our results suggest that heparin or HS is necessary for concentrating and localizing FN molecules at sites of early fibril assembly.

The ability of eukaryotic cells to differentiate surface stiffness is fundamental for many processes like stem cell development. Bacteria were previously known to sense the presence of surfaces, but the extent to which they could differentiate stiffnesses remained unclear. Here we establish that the human pathogen Pseudomonas aeruginosa actively measures surface stiffness using type IV pili (TFP). Stiffness sensing is nonlinear, as induction of the virulence factor regulator is peaked with stiffness in a physiologically important range between 0.1 kPa (similar to mucus) and 1,000 kPa (similar to cartilage). Experiments on surfaces with distinct material properties establish that stiffness is the specific biophysical parameter important for this sensing. Traction force measurements reveal that the retraction of TFP is capable of deforming even stiff substrates. We show how slow diffusion of the pilin PilA in the inner membrane yields local concentration changes at the base of TFP during extension and retraction that change with substrate stiffness. We develop a quantitative biomechanical model that explains the transcriptional response to stiffness. A competition between PilA diffusion in the inner membrane and a loss/gain of monomers during TFP extension/retraction produces substrate stiffness-dependent dynamics of the local PilA concentration. We validated this model by manipulating the ATPase activity of the TFP motors to change TFP extension and retraction velocities and PilA concentration dynamics, altering the stiffness response in a predictable manner. Our results highlight stiffness sensing as a shared behavior across biological kingdoms, revealing generalizable principles of environmental sensing across small and large cells.

Pancreatic cancer cells with limited access to free amino acids can grow by scavenging extracellular protein. In a murine model of pancreatic cancer, we performed a genome-wide CRISPR screen for genes required for scavenging-dependent growth. The screen identified key mediators of macropinocytosis, peripheral lysosome positioning, endosome-lysosome fusion, lysosomal protein catabolism, and translational control. The top hit was GCN2, a kinase that suppresses translation initiation upon amino acid depletion. Using isotope tracers, we show that GCN2 is not required for protein scavenging. Instead, GCN2 prevents ribosome stalling but without slowing protein synthesis; cells still use all of the limiting amino acids as they emerge from lysosomes. GCN2 also adapts gene expression to the nutrient-poor environment, reorienting protein synthesis away from ribosomes and toward lysosomal hydrolases, such as cathepsin L. GCN2, cathepsin L, and the other genes identified in the screen are potential therapeutic targets in pancreatic cancer.

Pseudomonas aeruginosa is a significant threat in healthcare settings where it deploys a wide host of virulence factors to cause disease. Many virulence-related phenotypes such as pyocyanin production, biofilm formation, and twitching motility have been implicated in causing disease in a number of hosts. In this study, we investigate these three virulence factors in a collection of 22 clinical strains isolated from blood stream infections. Despite the fact that all 22 strains caused disease and came from the same body site of different patients, they show significant variability in assays for each of the three specific phenotypes examined. There was no significant correlation between the strength of the three phenotypes across our collection, suggesting that they can be independently modulated. Furthermore, strains deficient in each of the virulence-associated phenotypes examined could be identified. To understand the genetic basis of this variability we sequenced the genomes of the 22 strains. We found that the majority of genes responsible for pyocyanin production, biofilm formation, and twitching motility were highly conserved among the strains despite their phenotypic variability, suggesting that the phenotypic variability is likely due to regulatory changes. Our findings thus demonstrate that no one lab-assayed phenotype of pyocyanin production, biofilm production, and twitching motility is necessary for a P. aeruginosa strain to cause blood stream infection and that additional factors may be needed to fully predict what strains will lead to specific human diseases.

The marine alpha-proteobacterium Phaeobacter inhibens engages in intermittent symbioses with microalgae. The symbiosis is biphasic and concludes in a parasitic phase, during which the bacteria release algaecidal metabolites in response to algal p-coumaric acid (pCA). The cell-wide effects of pCA on P. inhibens remain unknown. Herein, we report a microarray-based transcriptomic study and find that genes related to the oxidative stress response and secondary metabolism are upregulated most, while those associated with energy production and motility are downregulated in the presence of pCA. Among genes upregulated is a previously unannotated biosynthetic gene cluster and, using a combination of gene deletions and metabolic profiling, we show that it gives rise to an unreported siderophore, roseobactin. The simultaneous production of algaecides and roseobactin in the parasitic phase allows the bacteria to take up any iron that is released from dying algal cells, thereby securing a limited micronutrient.

How well mRNA transcript levels represent protein abundances has been a controversial issue. Particularly across different environments, correlations between mRNA and protein exhibit remarkable variability from gene to gene. Translational regulation is likely to be one of the key factors contributing to mismatches between mRNA level and protein abundance in bacteria. Here, we quantified genome-wide transcriptome and relative translation efficiency (RTE) under 12 different conditions in Escherichia coli. By quantifying the mRNA-RTE correlation both across genes and across conditions, we uncovered a diversity of gene-specific translational regulations, cooperating with transcriptional regulations, in response to carbon (C), nitrogen (N), and phosphate (P) limitations. Intriguingly, we found that many genes regulating translation are themselves subject to translational regulation, suggesting possible feedbacks. Furthermore, a random forest model suggests that codon usage partially predicts a gene's cross-condition variability in translation efficiency; such cross-condition variability tends to be an inherent quality of a gene, independent of the specific nutrient limitations. These findings broaden the understanding of translational regulation under different environments and provide novel strategies for the control of translation in synthetic biology. In addition, our data offers a resource for future multi-omics studies.

Double-stranded RNA (dsRNA), a hallmark viral material that activates antiviral interferon (IFN) responses, can appear in human cells also in the absence of viruses. We identify phosphorothioate DNAs (PS DNAs) as triggers of such endogenous dsRNA (endo-dsRNA). PS DNAs inhibit decay of nuclear RNAs and induce endo-dsRNA via accumulation of high levels of intronic and intergenic inverted retroelements (IIIR). IIIRs activate endo-dsRNA responses distinct from antiviral defense programs. IIIRs do not turn on transcriptional RIG-I/MDA5/IFN signaling, but they trigger the dsRNA-sensing pathways of OAS3/RNase L and PKR. Thus, nuclear RNA decay and nuclear-cytosolic RNA sorting actively protect from these innate immune responses to self. Our data suggest that the OAS3/RNase L and PKR arms of innate immunity diverge from antiviral IFN responses and monitor nuclear RNA decay by sensing cytosolic escape of IIIRs. OAS3 provides a receptor for IIIRs, whereas RNase L cleaves IIIR-carrying introns and intergenic RNAs.

Bacteria use extracellular appendages called type IV pili (T4P) for diverse behaviors including DNA uptake, surface sensing, virulence, protein secretion, and twitching motility. Dynamic extension and retraction of T4P is essential for their function, and T4P extension is thought to occur through the action of a single, highly conserved motor, PilB. Here, we develop Acinetobacter baylyi as a model to study T4P by employing a recently developed pilus labeling method. By contrast to previous studies of other bacterial species, we find that T4P synthesis in A. baylyi is dependent not only on PilB but also on an additional, phylogenetically distinct motor, TfpB. Furthermore, we identify a protein (CpiA) that inhibits T4P extension by specifically binding and inhibiting PilB but not TfpB. These results expand our understanding of T4P regulation and highlight how inhibitors might be exploited to disrupt T4P synthesis.

Type IV pili (TFP) function through cycles of extension and retraction. The coordination of these cycles remains mysterious due to a lack of quantitative measurements of multiple features of TFP dynamics. Here, we fluorescently label TFP in the pathogen and track full extension and retraction cycles of individual filaments. Polymerization and depolymerization dynamics are stochastic; TFP are made at random times and extend, pause, and retract for random lengths of time. TFP can also pause for extended periods between two extension or two retraction events in both wild-type cells and a slowly retracting PilT mutant. We developed a biophysical model based on the stochastic binding of two dedicated extension and retraction motors to the same pilus machine that predicts the observed features of the data with no free parameters. We show that only a model in which both motors stochastically bind and unbind to the pilus machine independent of the piliation state of the machine quantitatively explains the experimentally observed pilus production rate. In experimental support of this model, we show that the abundance of the retraction motor dictates the pilus production rate and that PilT is bound to pilus machines even in their unpiliated state. Together, the strong quantitative agreement of our model with a variety of experiments suggests that the entire repetitive cycle of pilus extension and retraction is coordinated by the competition of stochastic motor binding to the pilus machine, and that the retraction motor is the major throttle for pilus production.

Several essential components of the electron transport chain, the major producer of ATP in mammalian cells, are encoded in the mitochondrial genome. These 13 proteins are translated within mitochondria by 'mitoribosomes'. Defective mitochondrial translation underlies multiple inborn errors of metabolism and has been implicated in pathologies such as aging, metabolic syndrome and cancer. Here, we provide a detailed ribosome profiling protocol optimized to interrogate mitochondrial translation in mammalian cells (MitoRiboSeq), wherein mitoribosome footprints are generated with micrococcal nuclease and mitoribosomes are separated from cytosolic ribosomes and other RNAs by ultracentrifugation in a single straightforward step. We highlight critical steps during library preparation and provide a step-by-step guide to data analysis accompanied by open-source bioinformatic code. Our method outputs mitoribosome footprints at single-codon resolution. Codons with high footprint densities are sites of mitoribosome stalling. We recently applied this approach to demonstrate that defects in mitochondrial serine catabolism or in mitochondrial tRNA methylation cause stalling of mitoribosomes at specific codons. Our method can be applied to study basic mitochondrial biology or to characterize abnormalities in mitochondrial translation in patients with mitochondrial disorders.

Bacterial species have diverse cell shapes that enable motility, colonization and virulence. The cell wall defines bacterial shape and is primarily built by two cytoskeleton-guided synthesis machines, the elongasome and the divisome. However, the mechanisms producing complex shapes, like the curved-rod shape of Vibrio cholerae, are incompletely defined. Previous studies have reported that species-specific regulation of cytoskeleton-guided machines enables formation of complex bacterial shapes such as cell curvature and cellular appendages. In contrast, we report that CrvA and CrvB are sufficient to induce complex cell shape autonomously of the cytoskeleton in V. cholerae. The autonomy of the CrvAB module also enables it to induce curvature in the Gram-negative species Escherichia coli, Pseudomonas aeruginosa, Caulobacter crescentus and Agrobacterium tumefaciens. Using inducible gene expression, quantitative microscopy and biochemistry, we show that CrvA and CrvB circumvent the need for patterning via cytoskeletal elements by regulating each other to form an asymmetrically localized, periplasmic structure that binds directly to the cell wall. The assembly and disassembly of this periplasmic structure enables dynamic changes in cell shape. Bioinformatics indicate that CrvA and CrvB may have diverged from a single ancestral hybrid protein. Using fusion experiments in V. cholerae, we find that a synthetic CrvA/B hybrid protein is sufficient to induce curvature on its own, but that expression of two distinct proteins, CrvA and CrvB, promotes more rapid curvature induction. We conclude that morphological complexity can arise independently of cell-shape specification by the core cytoskeleton-guided synthesis machines.

Animals face both external and internal dangers: pathogens threaten from the environment, and unstable genomic elements threaten from within. C. elegans protects itself from pathogens by "reading" bacterial small RNAs, using this information to both induce avoidance and transmit memories for four generations. Here, we found that memories can be transferred from either lysed animals or from conditioned media to naive animals via Cer1 retrotransposon-encoded virus-like particles. Moreover, Cer1 functions internally at the step of transmission of information from the germline to neurons and is required for learned avoidance. The presence of the Cer1 retrotransposon in wild C. elegans strains correlates with the ability to learn and inherit small-RNA-induced pathogen avoidance. Together, these results suggest that C. elegans has co-opted a potentially dangerous retrotransposon to instead protect itself and its progeny from a common pathogen through its inter-tissue signaling ability, hijacking this genomic element for its own adaptive immunity benefit.

Hepatitis E virus (HEV) causes 14 million infections and 60,000 deaths per year globally, with immunocompromised persons and pregnant women experiencing severe symptoms. Although ribavirin can be used to treat chronic hepatitis E, toxicity in pregnant patients and the emergence of resistant strains are major concerns. Therefore there is an imminent need for effective HEV antiviral agents. The aims of this study were to develop a drug screening platform and to discover novel approaches to targeting steps within the viral life cycle. We developed a screening platform for molecules inhibiting HEV replication and selected a candidate, isocotoin. Isocotoin inhibits HEV replication through interference with heat shock protein 90 (HSP90), a host factor not previously known to be involved in HEV replication. Additional work is required to understand the compound's translational potential, however this suggests that HSP90-modulating molecules, which are in clinical development as anti-cancer agents, may be promising therapies against HEV.

The microtubule-based mitotic spindle is responsible for equally partitioning the genome during each cell division, and its assembly is executed via several microtubule nucleation pathways. Targeting Protein for XKlp2 (TPX2) stimulates the branching microtubule nucleation pathway, where new microtubules are nucleated from preexisting ones within mitotic or meiotic spindles. TPX2, like other spindle assembly factors, is sequestered by binding to nuclear importins-α/β until the onset of mitosis, yet the molecular nature of this regulation remains unclear. Here we demonstrate that TPX2 interacts with importins-α/β with nanomolar affinity in a 1:1:1 monodispersed trimer. We also identify a new nuclear localization sequence in TPX2 that contributes to its high-affinity interaction with importin-α. In addition, we establish that TPX2 interacts with importin-β via dispersed, weak interactions. We show that interactions of both importin-α and -β with TPX2 inhibit its ability to undergo phase separation, which was recently shown to enhance the kinetics of branching microtubule nucleation. In summary, our study informs how importins regulate TPX2 to facilitate spindle assembly, and provides novel insight into the functional regulation of protein phase separation.

Pseudomonas aeruginosa is a significant threat in both healthcare and industrial biofouling. Surface attachment of P. aeruginosa is particularly problematic as surface association induces virulence and is necessary for the ensuing process of biofilm formation, which hampers antibiotic treatments. Previous efforts have searched for dispersal agents of mature biofilm collectives, but there are no known factors that specifically disperse individual surface-attached P. aeruginosa. In this study, we develop a quantitative single-cell surface-dispersal assay and use it to show that P. aeruginosa itself produces factors that can stimulate its dispersal. Through bioactivity-guided fractionation, mass spectrometry, and nuclear magnetic resonance, we elucidated the structure of one such factor, 2-methyl-4-hydroxyquinoline (MHQ). MHQ is an alkyl quinolone with a previously unknown activity and is synthesized by the PqsABC enzymes. Pure MHQ is sufficient to disperse P. aeruginosa, but the dispersal activity of natural P. aeruginosa conditioned media requires additional factors. Whereas other alkyl quinolones have been shown to act as antibiotics or membrane depolarizers, MHQ lacks these activities and known antibiotics do not induce dispersal. In contrast, we show that MHQ inhibits the activity of Type IV Pili (TFP) and that TFP targeting can explain its dispersal activity. Our work thus identifies single-cell surface dispersal as a new activity of P. aeruginosa-produced small molecules, characterizes MHQ as a promising dispersal agent, and establishes TFP inhibition as a viable mechanism for P. aeruginosa dispersal.

The spread of pathogenic bacteria in unsaturated porous media, where air and liquid coexist in pore spaces, is the major cause of soil contamination by pathogens, soft rot in plants, food spoilage, and many pulmonary diseases. However, visualization and fundamental understanding of bacterial transport in unsaturated porous media are currently lacking, limiting the ability to address the above contamination- and disease-related issues. Here, we demonstrate a previously unreported mechanism by which bacterial cells are transported in unsaturated porous media. We discover that surfactant-producing bacteria can generate flows along corners through surfactant production that changes the wettability of the solid surface. The corner flow velocity is on the order of several millimeters per hour, which is the same order of magnitude as bacterial swarming, one of the fastest known modes of bacterial surface translocation. We successfully predict the critical corner angle for bacterial corner flow to occur based on the biosurfactant-induced change in the contact angle of the bacterial solution on the solid surface. Furthermore, we demonstrate that bacteria can indeed spread by producing biosurfactants in a model soil, which consists of packed angular grains. In addition, we demonstrate that bacterial corner flow is controlled by quorum sensing, the cell-cell communication process that regulates biosurfactant production. Understanding this previously unappreciated bacterial transport mechanism will enable more accurate predictions of bacterial spreading in soil and other unsaturated porous media.

Caenorhabditis elegans must distinguish pathogens from nutritious food sources among the many bacteria to which it is exposed in its environment. Here we show that a single exposure to purified small RNAs isolated from pathogenic Pseudomonas aeruginosa (PA14) is sufficient to induce pathogen avoidance in the treated worms and in four subsequent generations of progeny. The RNA interference (RNAi) and PIWI-interacting RNA (piRNA) pathways, the germline and the ASI neuron are all required for avoidance behaviour induced by bacterial small RNAs, and for the transgenerational inheritance of this behaviour. A single P. aeruginosa non-coding RNA, P11, is both necessary and sufficient to convey learned avoidance of PA14, and its C. elegans target, maco-1, is required for avoidance. Our results suggest that this non-coding-RNA-dependent mechanism evolved to survey the microbial environment of the worm, use this information to make appropriate behavioural decisions and pass this information on to its progeny.

Microbes face intense competition in the natural world, and so need to wisely allocate their resources to multiple functions, in particular to metabolism. Understanding competition among metabolic strategies that are subject to trade-offs is therefore crucial for deeper insight into the competition, cooperation, and community assembly of microorganisms. In this work, we evaluate competing metabolic strategies within an ecological context by considering not only how the environment influences cell growth, but also how microbes shape their chemical environment. Utilizing chemostat-based resource-competition models, we exhibit a set of intuitive and general procedures for assessing metabolic strategies. Using this framework, we are able to relate and unify multiple metabolic models, and to demonstrate how the fitness landscape of strategies becomes intrinsically dynamic due to species-environment feedback. Such dynamic fitness landscapes produce rich behaviors, and prove to be crucial for ecological and evolutionarily stable coexistence in all the models we examined.

The rise of antibiotic resistance and declining discovery of new antibiotics has created a global health crisis. Of particular concern, no new antibiotic classes have been approved for treating Gram-negative pathogens in decades. Here, we characterize a compound, SCH-79797, that kills both Gram-negative and Gram-positive bacteria through a unique dual-targeting mechanism of action (MoA) with undetectably low resistance frequencies. To characterize its MoA, we combined quantitative imaging, proteomic, genetic, metabolomic, and cell-based assays. This pipeline demonstrates that SCH-79797 has two independent cellular targets, folate metabolism and bacterial membrane integrity, and outperforms combination treatments in killing methicillin-resistant Staphylococcus aureus (MRSA) persisters. Building on the molecular core of SCH-79797, we developed a derivative, Irresistin-16, with increased potency and showed its efficacy against Neisseria gonorrhoeae in a mouse vaginal infection model. This promising antibiotic lead suggests that combining multiple MoAs onto a single chemical scaffold may be an underappreciated approach to targeting challenging bacterial pathogens.

The rise of antibiotic resistance and declining discovery of new antibiotics has created a global health crisis. Of particular concern, no new antibiotic classes have been approved for treating Gram-negative pathogens in decades. Here, we characterize a compound, SCH-79797, that kills both Gram-negative and Gram-positive bacteria through a unique dual-targeting mechanism of action (MoA) with undetectably low resistance frequencies. To characterize its MoA, we combined quantitative imaging, proteomic, genetic, metabolomic, and cell-based assays. This pipeline demonstrates that SCH-79797 has two independent cellular targets, folate metabolism and bacterial membrane integrity, and outperforms combination treatments in killing methicillin-resistant Staphylococcus aureus (MRSA) persisters. Building on the molecular core of SCH-79797, we developed a derivative, Irresistin-16, with increased potency and showed its efficacy against Neisseria gonorrhoeae in a mouse vaginal infection model. This promising antibiotic lead suggests that combining multiple MoAs onto a single chemical scaffold may be an underappreciated approach to targeting challenging bacterial pathogens.