Biology subjects

Gitai, Z.

Publications and source records attributed to Gitai, Z..

At least 19 recordsLinked to original sources

bioRxiv2026

Release of the Staphylococcus aureus autoinducing peptide is enabled by active export of the biosynthetic intermediate

Staphylococcus aureus is a species of Gram-positive bacteria that uses the accessory gene regulator (agr) quorum sensing system to regulate many aspects of cell behavior such as virulence and toxin production. Quorum sensing is activated in dense S. aureus populations by the extracellular accumulation of a signaling molecule called the autoinducing peptide (AIP). While the biosynthetic enzymes that produce AIP have been elucidated in certain subgroups of S. aureus, the mechanism of AIP secretion is unknown. Here, we show that the biosynthetic intermediate known as AgrD (1-32) thiolactone is actively exported into the surrounding environment in certain S. aureus subgroups. Using a high-throughput transposon mutagenesis screen, we identify the integral membrane transporters, AbcA and PmtCD, as being involved in the biosynthesis of certain AIPs. Subsequent genetic complementation and biochemical experiments reveal that these proteins actively transport AgrD (1-32) thiolactone out of the cell. This enables the production of AIP directly on the cell surface following the action of MroQ, an integral membrane protease demonstrated here to possess an active site facing the extracellular milieu. Furthermore, we show that media supplementation with catalytically-active MroQ nanodiscs rescues AIP production in an mroQ knockout strain. This observation suggests the possibility that some proportion of AgrD (1-32) thiolactone exported by one cell may travel to neighboring cell surfaces for further processing into the diffusive AIP molecule. Collectively, our studies shed light on a poorly understood step in the agr quorum sensing pathway with clear implications for the design of novel therapeutic strategies.

biochemistry↗

bioRxiv2026

Counting to two: how phages decide between lysis and lysogeny

Upon infecting a bacterium, temperate phages must decide between killing the cell to reproduce (lysis) and entering a symbiotic lifestyle (lysogeny). This choice is often informed by the cells state and by the number of infecting phage particles, or multiplicity of infection (MOI). Because the copy numbers of all phage genes scale identically with MOI, an MOI-dependent decision requires a rapidly established asymmetry between the lytic and lysogenic pathways. We introduce a minimal model showing how a simple coupling to host machinery (e.g. a protease) can generate such an asymmetry. Our analytical model yields scaling laws that predict how the lysislysogeny decision responds to perturbations of the host. It also quantitatively predicts stochastic features of infection outcomes, such as lysogeny probabilities, consistent with experimental data. By reducing complex regulatory networks to their essential components, our framework reveals the organizing principles of lysislysogeny decisions across phage species.

biophysics↗

bioRxiv2026

Rational Design of Resistance-Suppressing Phage-Antibiotic Cocktails via Receptor Tradeoffs

Bacteriophages (phages) are promising antibiotic alternatives, yet their utility is limited by rapid resistance evolution. Current phage-antibiotic combination strategies rely on empirical screening for efficacy in killing, which often fails to prevent resistance. Here, we establish a mechanistic framework to rationally design phage-antibiotic cotreatments that specifically suppress resistance by leveraging evolutionary tradeoffs. We show that traditional killing efficacy metrics do not predict resistance suppression. Instead, we identify predictable evolutionary trade-offs, including species-specific targets like TolC (Escherichia coli) and Type IV pili (Pseudomonas aeruginosa). We also identify a universal vulnerability for Gram-negative bacteria, lipopolysaccharide (LPS). We find that LPS-targeting phages are ubiquitous and that phage-steered LPS perturbation creates a predictable hypersensitivity to lipophilic antibiotics. This mechanistic trade-off, governed by physicochemical rules rather than drug-specific targets, directly correlates with resistance suppression. This framework shifts combination therapy from empirical screening to the a priori design of evolutionarily stable cocktails.

microbiology↗

bioRxiv2026

Pseudomonas aeruginosa deploys competitor-specific antagonistic strategies

1Microbial competition shapes polymicrobial communities, yet it remains unclear whether bacteria deploy fixed or specific antagonistic strategies against different rivals. Here we show that Pseudomonas aeruginosa deploys distinct strategies to outcompete two clinically relevant species, Burkholderia cenocepacia and Staphylococcus aureus. Under matched conditions, competition with B. cenocepacia is mediated by contact-dependent Type VI secretion, whereas competition with S. aureus follows a staged diffusible program of alkyl quinolone-mediated growth inhibition followed by LasA-dependent lysis. Transcriptomic analysis supports a "Swiss Army knife" model of antagonism in which different competitive context triggers distinct subsets of P. aeruginosas arsenal. Minimal dynamical models verify that contact-dependent killing is effective against slower-growing competitors, whereas a staged strategy of growth inhibition followed by killing via diffusible factors is preferable against faster-growing rivals. Together, these results show that the competitive success of P. aeruginosa depends on competitor-specific antagonistic strategies.

microbiology↗

bioRxiv2026

CBASS limits bacteriophage production while maintaining cell viability in Pseudomonas aeruginosa

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.

microbiology↗

bioRxiv2026

Cell-body curvature modulates stall frequency to enhance Vibrio cholerae swimming and chemotaxis through hydrogels

The swimming motility of Vibrio cholerae is a virulence factor that aids in breaching the mucus layer of the small intestine. V. cholerae cells have a curved cell shape and previous work demonstrated that loss of curvature decreases infectivity. Here we investigate the mechanism by which V. choleraes 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 V. cholerae 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 shapes role in V. choleraes pathogenicity and underscore the necessity of studying bacterial behaviors under conditions that simulate host environments.

microbiology↗

bioRxiv2024

Sensing the shape of a surface by intracellular filaments

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 Escherichia coli by directing cell-wall synthesis according to local curvature cues. However, the basis of MreBs 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 statementThe protein MreB, a homolog of eukaryotic actin, regulates the shape of bacteria like Escherichia coli 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.

biophysics↗

bioRxiv2024

Macromolecular interactions and geometrical confinement determine the 3D diffusion of ribosome-sized particles in live Escherichia coli cells

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 Escherichia coli 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.

biophysics↗

bioRxiv2024

Prophages block cell surface receptors to ensure survival of their viral progeny

In microbial communities, viruses compete for host cells to infect, and thus evolved diverse ways to inhibit their competitors. One mechanism is Superinfection exclusion (Sie), whereby a virus that has established an infection prevents a secondary infection. We identified a Pseudomonas prophage Sie protein that alters pilus dynamics through the pilus assembly chaperone, PilZ. This protein, known as Zip for PilZ interacting protein, does not abrogate pilus activity, but fine tunes it, providing strong phage resistance without a fitness cost. This tuning is modulated through quorum sensing, which coordinates Zip production in concert with bacterial cell density to ensure maximal protection when bacterial populations are at the highest risk of phage infection. Most notably, Zip activity prevents internalization and destruction of phage progeny. We refer to this as the "anti-Kronos effect" after the Greek god who devoured his own children and show that it is a conserved feature of diverse prophage-encoded Sie systems.

microbiology↗

bioRxiv2023

Free-floating Bacteria Transcriptionally Respond to Shear Flow

Planktonic (free-floating) cells are typically assumed to be oblivious to any flow that carries them. Here we discover that planktonic bacteria can sense flow to induce gene expression changes that are beneficial in flow. Specifically, planktonic P. aeruginosa induce shear-rate-dependent genes that promote growth in low oxygen environments. Untangling this mechanism revealed that in flow, motile P. aeruginosa spatially redistribute, leading to cell density changes that activate quorum sensing, which in turn enhances 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 newly-discovered flow sensing mechanism exhibit decreased fitness in flow, suggesting that this dynamic coupling of biological and mechanical processes can be physiologically significant. One-Sentence SummaryFree-floating bacteria integrate biological and mechanical responses to adaptively alter gene expression in flow environments

microbiology↗

bioRxiv2023

P. aeruginosa controls both C. elegans attraction and pathogenesis by regulating nitrogen assimilation

Detecting chemical signals is important for identifying food sources and avoiding harmful agents. Like most 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 untangle 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, nitrogen assimilation is important for bacterial fitness and pathogenesis during C. elegans infection by P. aeruginosa. 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.

microbiology↗

bioRxiv2023

Community behaviors of bacteria in flow: Divergent roles for transport and collective morphology

Fluid flows can impact bacterial behaviors in unexpected ways (1-3). The high shear rate in heart valves should reduce colonization, but in endocarditis, valves are often counter-intuitively colonized by Staphylococcus aureus and Enterococcus faecalis (4, 5). Here we discover bacteria-specific mechanisms for preferential surface colonization in higher shear rate environments. This behavior enables bacteria that are outcompeted in low flow to dominate in high flow. Flow-dependent colonization by S. aureus and E. faecalis are mediated by distinct mechanisms that depend on each species microcolony morphologies: transport of a dispersal signaling molecule for clustered S. aureus and mechanical forces for linear chains of E. faecalis. These results suggest that microcolony morphologies have previously unappreciated costs and benefits in different environments, like those introduced by flow. One-Sentence SummaryBacterial surface colonization in high fluid flow depends upon the species clustered or chained microcolony morphologies.

microbiology↗

bioRxiv2023

A small RNA from a natural bacterial pathogen of C. elegans induces transgenerational inheritance of learned avoidance

Previously, we discovered that a small RNA from a clinical isolate of Pseudomonas aeruginosa, PA14, induces learned avoidance and its transgenerational inheritance 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 found that a pathogenic Pseudomonas vranovensis strain isolated from the C. elegans 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 C. elegans maco-1. 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, P. mendocina; 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 C. elegans 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.

genetics↗

bioRxiv2023

Type-IV pili tune an adhesion-migration trade-off during surface colonization of Pseudomonas aeruginosa

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 Pseudomonas aeruginosa 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. {Delta}pilA mutants, which produce no T4P, robustly stick to surfaces under shear flow. In contrast, rapidly migrating {Delta}pilH 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.

biophysics↗

bioRxiv2022

CauloKO: an ordered transposon mutant library in Caulobacter crescentus

Genetic screens are powerful approaches to unveiling new biological insight and ordered redundant transposon libraries have emerged as a primary tool for performing screens of known genetic saturation. Newer sequencing methods based on combinatorial pooling have lowered the cost and time required to generate these libraries. Caulobacter crescentus is a gramnegative 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 C. crescentus. 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.

microbiology↗

bioRxiv2022

Massively-parallel Microbial mRNA Sequencing (M3-Seq) reveals heterogenous behaviors in bacteria at single-cell resolution

Bacterial populations are highly adaptive. They can respond to stress and survive in shifting environments. How the behaviors 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, though, are all limited in some technical capacity (e.g., number of cells or transcripts that can be profiled). Due in part to these limitations, few conditions have yet been studied with these tools. Here, we develop Massively-parallel Microbial mRNA 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 hundreds of thousands of bacterial cells from different species under a range of conditions in single experiments. We then apply M3-Seq to reveal rare populations, insights into bet hedging strategies during stress responses, and host responses to phage infection.

microbiology↗

bioRxiv2022

Global Protein-Turnover Quantification in Escherichia coli Reveals Cytoplasmic Recycling under Nitrogen-Limitation

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 degradation rates of [~]3.2k E. coli proteins under 12 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 introduce 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 decipher the control of proteostasis.

systems biology↗

bioRxiv2022

Global and gene-specific translational regulation in Escherichia coli across different conditions

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 genes 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.

genomics↗