Biology subjects

Bassler, B. L.

Publications and source records attributed to Bassler, B. L..

At least 19 recordsLinked to original sources

bioRxiv2026

Clonal microcolonies recruit individual planktonic colonizers using extracellular matrix factors to assemble Vibrio cholerae pellicles

Pellicles, expansive bacterial communities that form on liquid surfaces, contain residents that incorporate asynchronously and exist in distinct physiological states. How interactions between cell populations convert segregated founder pellicle microcolonies into a contiguous, macroscale structure is unknown. Using high resolution timelapse microscopy of Vibrio cholerae pellicle formation, we show that the confluent community forms through the recruitment of individual planktonic cells to regions between founder microcolonies. In addition to Type IV MSHA pili, individual planktonic V. cholerae cells attach to the air-liquid interface using their cell-surface-bound vibrio polysaccharide (VPS), which binds to the Bap1 and RbmC adhesins secreted by existing pellicle microcolonies. Planktonic V. cholerae cells readily attach to the interface by VPS-adhesin binding when they exist in the low-cell-density quorum-sensing state because this is the mode that promotes VPS production. Single-molecule FISH of V. cholerae pellicles reveals that regions near pellicle microcolonies, where secreted Bap1 and RbmC adhesin levels are the highest, recruit a higher proportion of VPS-producing planktonic cells than do more distant regions. Thus, existing pellicle microcolony inhabitants in an advanced phase of sessile growth prime the surface for new colonizer cell attachment, driving a spatial pattern of gene expression within the pellicle community.

microbiology↗

bioRxiv2026

Phage VP882 possesses quorum-sensing-driven lytic induction and stress-mediated host growth suppression mechanisms

Vibriophage VP882 launches its lytic cascade upon detection of a quorum-sensing autoinducer produced by its bacterial host. This capability enables the phage to maximize transmission by transitioning from lysogeny to lysis only at high host cell density, when abundant host cells are present to infect. Here, we show that two different pathways can be triggered upon prophage induction. When the prophage is induced via quorum sensing, Qtip, an anti-repressor, inhibits the cI lysogeny maintenance protein, driving expression of the lysis genes. The phage can also launch a cascade that causes host growth arrest. In this case, cI derepresses two genes, one encoding a DksA homolog, TraRVP882, and one encoding a protein that we name QisA. TraRVP882 and QisA form a complex that causes host growth arrest, relying on RNAP-binding by TraRVP882. Phage VP882 quorum sensing also activates production of a protein we call QtiQ, which inactivates the QisA-TraRVP882 complex, reestablishing host cell growth. Thus, in the presence of QtiQ, the phage lysis program is enacted. To our knowledge, QtiQ is the first protein inactivator of a TraR or DksA-like homolog. By inducing growth arrest, phage VP882 may enable its host to survive under stress-inducing conditions, or the phage may delay host lysis until optimal conditions are met. In both cases, the phage thereby enhances its own prospects for spread.

microbiology↗

bioRxiv2026

A pectin-based formulation protects milk fat globule membranes in stored human milk

During household storage, expressed human milk can develop odor and flavor changes that trigger infant refusal and lead caregivers to discard their saved milk supply. We show that typical refrigeration and freezing conditions disrupt the milk fat globule membrane (MFGM), exposing milk lipids to lipases that catalyze hydrolysis and oxidation. A pectin-based formulation (PBF) maintains MFGM integrity during storage and following lipase challenge, suppressing production of glycerol, free fatty acid, and oxylipin byproducts without broadly affecting milk macronutrients, the proteome, and culturable microbial burden. Across an independent cohort of lactating individuals, lipase activity varied but tracked with maternal milk lipase gene expression, implicating endogenous lipolysis in stored-milk deterioration. In a blinded olfactory panel, PBF-treated, lipase-challenged milk smelled more like fresh milk than untreated controls. Together, these findings show that stabilizing the MFGM can protect stored human milk from lipase-driven deterioration, preserve sensory quality, and support use for infant feeding.

biochemistry↗

bioRxiv2026

Competing Inter-Domain Quorum-Sensing Systems Control Prophage Lysis-Lysogeny Decisions

Phaeobacter inhibens T5T harbors three LuxI/LuxR quorum-sensing systems: one encoded by the host and one in each of two prophages. Each prophage quorum-sensing autoinducer activates its own lysogeny program and that of the co-resident prophage. By driving competitors toward lysogeny, prophages deny their rivals access to susceptible hosts. One prophage engages in a bidirectional interaction with the host: the prophage activates host quorum sensing, while the host represses prophage quorum sensing. Because the host and prophage autoinducers exert opposing effects on the same prophage promoter, the prophages lysis-lysogeny decision depends on the ratio of the two autoinducers rather than their absolute concentrations. Ratiometric sensing allows the prophage to infer the relative abundances of infected and uninfected hosts and to commit to lysis only when uninfected hosts predominate. Phage quorum-sensing modules are widespread and undergo diversification and exchange. These findings reveal how prophages surveil and manipulate competitors that share chemical environments.

microbiology↗

bioRxiv2026

Molecular basis of quorum-sensing signal transduction by CqsS and its inhibition by CqsA the autoinducer synthase

Vibrio bacteria possess multiple quorum-sensing systems, each of which conveys information into the cell concerning the cell density and species composition of the vicinal community. Germane to the present work is the CqsA-CqsS quorum-sensing pathway that vibrios use for intra-genus communication. CqsA is the synthase for the autoinducer (S)-3-hydroxytridecan-4-one (CAI-1), and CqsS, a bi-functional two-component kinase-phosphatase, is the CAI-1 receptor. Here, we determine the cryo-EM structures of the full-length Vibrio harveyi CqsS dimer in the apo- and ATP{gamma}S-bound states. Our structures reveal the molecular basis for the initiation of the phosphorylation cascade, which we support through biochemical and genetic analyses. Autophosphorylation of CqsS occurs in cis at the conserved histidine residue (H1), whereas phospho-transfer to the conserved aspartate residue (D1) in the C-terminal receiver domain occurs in trans. By co-expressing CqsS and CqsA to capture CqsS bound to CAI-1, we unexpectedly discover a tight interaction between the CqsS intracellular domain and CqsA. In the complex, the CqsS H1 residues are positioned away from the catalytic cavity, and CqsS kinase activity is inhibited. Cryo-EM analysis, focused on the transmembrane domain of CqsS in this complex, reveals the presence of two CAI-1 molecules. CAI-1 binding induces an obvious movement of transmembrane helix 6, disrupting its extension into the cytosol, providing a structural basis for how CAI-1 binding in the periplasm is coupled to signal transduction into the cytoplasm. Together, our findings advance the understanding of CqsS-CAI-1-mediated quorum-sensing signal transduction, an activity that underpins the coordination of group behaviors in vibrios.

microbiology↗

bioRxiv2026

Vibrio parahaemolyticus quorum sensing controls phage VP882 transmission

Quorum sensing is a cell-to-cell communication process bacteria use to orchestrate collective behaviors. Quorum sensing involves the production, release, and detection of extracellular signal molecules called autoinducers. Some temperate phages can monitor bacterial autoinducers enabling them to track the abundance of potential host cells in the vicinity. Quorum-sensing-responsive phages can preferentially launch the transition from lysogeny to lytic replication at high cell density, presumably maximizing transmission. Once the phage lytic program is enacted, if nearby host cells are already lysogens, infections initiated by released virions could be nonproductive due to homoimmunity or superinfection exclusion mechanisms, posing a conundrum for temperate phages, including those that surveil quorum-sensing autoinducers. Here, we define host and phage components that influence transmission of the first discovered quorum-sensing-responsive phage, phage VP882, in populations of its host, Vibrio parahaemolyticus. We show that phage VP882 uses the K-antigen of serotype O3:K6 as its receptor. We demonstrate that host cells can prevent phage access to the O3:K6 K-antigen via quorum-sensing-control of the export of polysaccharides that shield the K-antigen from the phage at high cell density. We discover that phage VP882 can superinfect and superlysogenize V. parahaemolyticus, overcoming the challenge of detecting whether or not potential hosts are lysogens. Following superlysogenization, recombination of the resident and newly infecting phage genomes can occur possibly promoting phage VP882 genome diversification. ImportanceA longstanding mystery is how temperate phages optimally time the launch of their lytic cascades to maximize spread. Quorum-sensing-responsive phages can preferentially execute their lytic replication programs at high host cell density, which in principle should foster transmission. However, if nearby host cells are already lysogens, infections initiated by released virions could be nonproductive due to homoimmunity or superinfection exclusion. We define host and phage components influencing transmission of the quorum-sensing-responsive phage VP882 in Vibrio parahaemolyticus populations. Phage VP882 uses the O3:K6 K-antigen as its receptor. Host cells prevent phage infection via quorum-sensing-controlled export of polysaccharides that shield the K-antigen at high cell density. We discover that phage VP882 can superinfect and superlysogenize V. parahaemolyticus, overcoming the challenge of detecting whether or not potential hosts are lysogens. These findings reveal how phages can capitalize on interception of host quorum-sensing cues to maximize their reproductive success.

microbiology↗

bioRxiv2026

Discovery of a Human Metabolite that Mimics the Bacterial Quorum-Sensing Autoinducer AI-2

Bacteria use small molecules to orchestrate collective behaviors in a process called quorum sensing (QS), which relies on the production, release, and group-wide detection of extracellular signal molecules referred to as autoinducers. One QS autoinducer, termed AI-2, is broadly used for inter-species bacterial communication, including in the mammalian gut. AI-2 consists of a family of interconverting compounds and adducts originating from 4,5-hydroxy-2,3-pentanedione. This complex speciation, coupled with the inherent instability of AI-2 congeners, have complicated isolation efforts. It has been known that mammalian epithelial cells produce an AI-2 mimic to which bacteria respond. However, the identity of the AI-2 mimic has remained elusive, presumably due to its instability, similar to that of known AI-2 compounds. Here, we developed a reactivity-based metabolomics approach to capture and identify a mammalian AI-2 mimic. Using a chemical strategy targeted at the -diketone moiety of known AI-2s, we identify the unusual sugar L-xylosone, as well as the related metabolite L-xylulose, as AI-2 mimics. While L-xylulose is a common and naturally occurring sugar known in human metabolism, L-xylosone is a rare and highly reactive oxidation product. We established a facile synthetic route to access pure enantiomers of xylosone and confirmed that, like AI-2, the L-configuration is required for recognition by the bacterial AI-2 receptor, LuxP, whereas D-xylosone is inactive. L-xylosone is new to the human metabolome, suggesting that other chemically reactive small molecules that mediate host-microbe interactions await discovery. The identification of L-xylosone expands the AI-2 family of molecules and adds a new word to the lexicon of host-bacterial interactions.

biochemistry↗

bioRxiv2025

Inhibitors of the PqsR Quorum-Sensing Receptor Reveal Differential Roles for PqsE and RhlI in Control of Phenazine Production

Pseudomonas aeruginosa is a leading cause of hospital-acquired infections and it is resistant to many current antibiotic therapies, making development of new anti-microbial treatments imperative. The cell-to-cell communication process called quorum sensing controls P. aeruginosa pathogenicity. Quorum sensing relies on the production, release, and group-wide detection of extracellular signal molecules called autoinducers. Quorum sensing enables bacteria to synchronize group behaviors. P. aeruginosa possesses multiple quorum-sensing systems that control overlapping regulons, including those required for virulence and biofilm formation. Interventions that target P. aeruginosa quorum-sensing receptors are considered a fruitful avenue to pursue for new therapeutic advances. Here, we developed a P. aeruginosa strain that carries a bioluminescent reporter fused to a target promoter that is controlled by two P. aeruginosa quorum-sensing receptors. The receptors are PqsR, which binds and responds to the autoinducer called PQS (2-heptyl-3-hydroxy-4(1H)-quinolone) and RhlR, which binds and responds to the autoinducer called C4-HSL (C4-homoserine lactone). We used this reporter strain to screen >100,000 compounds with the aim of identifying inhibitors of either or both the PqsR and RhlR quorum-sensing receptors. We report results for 30 PqsR inhibitors from this screen. All of the identified compounds inhibit PqsR with IC50 values in the nanomolar to low micromolar range and they are readily docked into the autoinducer binding site of the PqsR crystal structure, suggesting they function competitively. The majority of hits identified are not structurally related to previously reported PqsR inhibitors. Recently, RhlR was shown to rely on the accessory protein PqsE for full function. Specifically, RhlR controls different subsets of genes depending on whether it is bound to PqsE or to C4-HSL, however, the consequences of differential regulation on the quorum-sensing output response have not been defined. PqsR regulates pqsE. That feature of the system enabled us to exploit our new set of PqsR inhibitors to show that RhlR requires PqsE to activate the biosynthetic genes for pyocyanin, a key P. aeruginosa virulence factor, while C4-HSL is dispensable. These results highlight the promise of inhibition of PqsR as a possible P. aeruginosa therapeutic to suppress production of factors under RhlR-PqsE control.

microbiology↗

bioRxiv2024

Food-Derived Compounds Extend the Shelf-Life of Frozen Human Milk

Breastmilk is known to provide optimal nutrition for infant growth and development. A cross-sectional analysis of nationally representative US data from 2016 to 2021 revealed that >90% of lactating mothers reported using breast pumps to express milk.1 We conducted a survey of n = 1,049 lactating or recently lactating individuals from a US nationally representative population to explore breastmilk storage practices among this group. The data revealed that 83% of respondents store breastmilk in their homes, with 68% using freezers to do so for >1 month. The lowest available temperature in most household freezers is -20 {degrees}C, a temperature that is inadequate to maintain human milks emulsified structure, leading to separation, degradation of fats, loss of key vitamins, and changes in palatability. We developed a first-of-its-kind high-throughput screening platform to identify food-derived compounds and combinations of compounds that, when added to human breastmilk, preserve fat content, retain antioxidant capacity, and reduce production of rancid-associated free fatty acids during extended freezer storage. These formulations represent leads for the development of safe and affordable frozen breastmilk shelf-life extenders for easy at-home use to increase the longevity of stored breastmilk.

molecular biology↗

bioRxiv2024

Quorum sensing orchestrates parallel cell death pathways in Vibrio cholerae via Type 6 secretion dependent and independent mechanisms

Quorum sensing (QS) is a cell-to-cell communication process that enables bacteria to coordinate group behaviors. In Vibrio cholerae colonies, a program of spatial-temporal cell death is among the QS-controlled traits. Cell death occurs in two phases, first along the colony rim, and subsequently, at the colony center. Both cell death phases are driven by the type VI secretion system (T6SS). Here, we show that HapR, the master QS regulator, does not control t6ss gene expression nor T6SS-mediated killing activity. Nonetheless, a {Delta}hapR strain displays no cell death at the colony rim. RNA-Seq analyses reveal that HapR activates expression of an operon containing four genes of unknown function, vca0646-0649. Epistasis and overexpression studies show that two of the genes, vca0646 and vca0647, are required to drive cell death in both a {Delta}hapR and a {Delta}hapR {Delta}t6ss strain. Thus, vca0646-0649 are regulated by HapR but act independently of the T6SS machinery to cause cell death, suggesting that a second, parallel pathway to cell death exists in V. cholerae. SignificanceCell death is a fundamental biological process. In mammals, cell death sculpts tissues during development, enables injury recovery, and regulates immunity. In bacteria, cell death mechanisms remain little explored. Recently, colonies formed by the pathogen Vibrio cholerae were demonstrated to undergo a spatio-temporal program of cell death. The program is controlled by quorum sensing (QS) and driven by the Type VI secretion system. Here, we discover QS-controlled genes, called vca0646-0649, that cause cell death in V. cholerae colonies independently of the Type VI secretion system. These findings indicate that a second cell death pathway exists in V. cholerae. The results expand our understanding of bacterial cell death mechanisms and provide insight into how cell death shapes bacterial community structure.

microbiology↗

bioRxiv2024

Single-cell gene-expression measurements in Vibrio cholerae biofilms reveal spatiotemporal patterns underlying development

Bacteria commonly exist in multicellular, surface-attached communities called biofilms. Biofilms are central to ecology, medicine, and industry. The Vibrio cholerae pathogen forms biofilms from single founder cells that, via cell division, mature into three-dimensional structures with distinct, yet reproducible, regional architectures. To define mechanisms underlying biofilm developmental transitions, we establish a single-molecule fluorescence in situ hybridization (smFISH) approach that enables accurate quantitation of spatiotemporal gene-expression patterns in biofilms at cell-scale resolution. smFISH analyses of V. cholerae biofilm regulatory and structural genes demonstrate that, as biofilms mature, overall matrix gene expression decreases, and simultaneously, a pattern emerges in which matrix gene expression becomes largely confined to peripheral biofilm cells. Both quorum sensing and c-di-GMP-signaling are required to generate the proper temporal pattern of matrix gene expression. Quorum sensing autoinducer levels are uniform across the biofilm, and thus, c-di-GMP-signaling alone sets the regional matrix gene expression pattern. The smFISH strategy provides insight into mechanisms conferring particular fates to individual biofilm cells.

microbiology↗

bioRxiv2024

The LuxO-OpaR quorum-sensing cascade differentially controls Vibriophage VP882 lysis-lysogeny decision making in liquid and on surfaces

Quorum sensing (QS) is a process of cell-to-cell communication that bacteria use to synchronize collective behaviors. QS relies on the production, release, and group-wide detection of extracellular signaling molecules called autoinducers (AIs). Vibrios use two QS systems: the LuxO-OpaR circuit and the VqmA-VqmR circuit. Both QS circuits control group behaviors including biofilm formation and surface motility. The Vibrio parahaemolyticus temperate phage {varphi}VP882 encodes a VqmA homolog (called VqmA{varphi}). When VqmA{varphi} is produced by {varphi}VP882 lysogens, it binds to the host-produced AI called DPO and launches the {varphi}VP882 lytic cascade. This activity times induction of lysis with high host cell density and presumably promotes maximal phage transmission to new cells. Here, we explore whether, in addition to induction from lysogeny, QS controls the initial establishment of lysogeny by {varphi}VP882 in naive host cells. Using mutagenesis, phage infection assays, and phenotypic analyses, we show that {varphi}VP882 connects its initial lysis-lysogeny decision to both host cell density and whether the host resides in liquid or on a surface. Only host cells in the low-density QS state undergo lysogenic conversion. The QS regulator LuxO[~]P promotes {varphi}VP882 lysogenic conversion of low cell density planktonic host cells. By contrast, the ScrABC surface-sensing system regulates lysogenic conversion of low cell density surface-associated host cells. ScrABC controls the abundance of the second messenger molecule cyclic diguanylate, which in turn, modulates motility. The scrABC operon is only expressed when its QS repressor, OpaR, is absent. Thus, at low cell density, QS-dependent derepression of scrABC drives lysogenic conversion in surface-associated host cells. These results demonstrate that {varphi}VP882 integrates cues from multiple sensory pathways into its lifestyle decision making upon infection of a new host cell. AUTHOR SUMMARYBacteria in nature often exist in surface-associated communities including sessile biofilms and highly motile swarms. Thus, bacteriophages can encounter their hosts in structured communities. Much bacteriophage research is performed in homogenous, planktonic cultures containing cells that neither display the gene expression patterns nor the behaviors that occur in surface communities. The Vibrio parahaemolyticus temperate phage {varphi}VP882, after lysogenizing its host, can monitor the vicinal cell density and time lytic induction with high host cell density. Here, we show that, upon infection of a new host cell, {varphi}VP882 assesses host cell density to make the decision whether to lyse or lysogenize. Only host cells at low density undergo lysogenic conversion, and the components driving the phage decision-making process vary depending on whether the host cell is in liquid or associated with a solid surface. We propose that by tuning its lysis-lysogeny decision making to both host cell density and the physical environment of the host, {varphi}VP882 can maximize transmission to new host cells and dispersal to new environments.

microbiology↗

bioRxiv2023

Natural Silencing of Quorum-Sensing Activity Protects Vibrio parahaemolyticus from Lysis by an Autoinducer-Detecting Phage

Quorum sensing (QS) is a chemical communication process that bacteria use to track population density and orchestrate collective behaviors. QS relies on the production, accumulation, and group-wide detection of extracellular signal molecules called autoinducers. Vibriophage 882 (phage VP882), a bacterial virus, encodes a homolog of the Vibrio QS receptor-transcription factor, called VqmA, that monitors the Vibrio QS autoinducer DPO. Phage VqmA binds DPO at high host-cell density and activates transcription of the phage gene qtip. Qtip, an antirepressor, launches the phage lysis program. Phage-encoded VqmA when bound to DPO also manipulates host QS by activating transcription of the host gene vqmR. VqmR is a small RNA that controls downstream QS target genes. Here, we sequence Vibrio parahaemolyticus strain O3:K6 882, the strain from which phage VP882 was initially isolated. The chromosomal region normally encoding vqmR and vqmA harbors a deletion encompassing vqmR and a portion of the vqmA promoter, inactivating that QS system. We discover that V. parahaemolyticus strain O3:K6 882 is also defective in its other QS systems, due to a mutation in luxO, encoding the central QS transcriptional regulator LuxO. Both the vqmR-vqmA and luxO mutations lock V. parahaemolyticus strain O3:K6 882 into the low-cell density QS state. Reparation of the QS defects in V. parahaemolyticus strain O3:K6 882 promotes activation of phage VP882 lytic gene expression and LuxO is primarily responsible for this effect. Phage VP882-infected QS-competent V. parahaemolyticus strain O3:K6 882 cells lyse more rapidly and produce more viral particles than the QS-deficient parent strain. We propose that, in V. parahaemolyticus strain O3:K6 882, constitutive maintenance of the low-cell density QS state suppresses the launch of the phage VP882 lytic cascade, thereby protecting the bacterial host from phage-mediated lysis.

microbiology↗

bioRxiv2022

Natural and synthetic inhibitors of a phage-encoded quorum-sensing receptor affect phage-host dynamics in mixed bacterial communities

Viruses that infect bacteria, called phages, shape the composition of bacterial communities and are important drivers of bacterial evolution. We recently showed that temperate phages, when residing in bacteria (i.e., prophages), are capable of manipulating the bacterial cell-to-cell communication process called quorum sensing (QS). QS relies on the production, release, and population-wide detection of signaling molecules called autoinducers (AI). Gram-negative bacteria commonly employ N-acyl homoserine lactones (HSL) as AIs that are detected by LuxR-type QS receptors. Phage ARM81ld is a prophage of the aquatic bacterium Aeromonas sp. ARM81, and it encodes a homolog of a bacterial LuxR, called LuxRARM81ld. LuxRARM81ld detects host Aeromonas-produced C4-HSL, and in response, activates the phage lytic program, triggering death of its host and release of viral particles. Here, we show that phage LuxRARM81ld activity is modulated by non-cognate HSL ligands and by a synthetic small molecule inhibitor. We determine that HSLs with acyl chain lengths equal to or longer than C8 antagonize LuxRARM81ld. For example, the C8-HSL AI produced by Vibrio fischeri that co-exists with Aeromonads in marine environments, binds to and inhibits LuxRARM81ld, and consequently, protects the host from lysis. Co-culture of V. fischeri with the Aeromonas sp. ARM81 lysogen suppresses phage ARM81ld virion production. We propose that the cell density and species composition of the bacterial community could determine outcomes in bacterial-phage partnerships. SIGNIFICANCEBacteria use the cell-to-cell communication process called quorum sensing to orchestrate group behaviors. Quorum sensing relies on extracellular molecules called autoinducers. Bacteria-infecting viruses (phages) can possess homologs of bacterial quorum-sensing receptors that detect autoinducers to control lysis-lysogeny transitions. We show that a phage LuxR-type quorum-sensing receptor is activated by the autoinducer produced by its host bacterium and is inhibited by non-cognate autoinducers made by bacteria that naturally co-exist with the phages host and by a synthetic quorum-sensing inhibitor. Our findings demonstrate that microbial community composition, mediated through quorum-sensing-communication, influences phage lysis-lysogeny transitions. These results deepen the understanding of host-phage interactions in communities and could inspire new phage-specific, quorum-sensing interventions.

microbiology↗

bioRxiv2022

Small protein modules dictate prophage fates during polylysogeny

DNA-damaging agents are the pervasive inducers of temperate phages in model bacteria. However, most bacteria in the biosphere are predicted to carry multiple prophages, a state called polylysogeny, making it unclear how co-residing prophages compete for host cell resources if they all respond to the identical trigger. We discover regulatory modules encoded on phage genomes that control prophage induction independently of the DNA damage cue. Genes specifying these pathways exist in linear plasmid-like phages at sites essential for phage propagation. The modules lack sequence similarity but display a shared regulatory logic of a transcription factor that activates expression of a neighboring gene encoding a small protein. The small protein inactivates the master repressor of lysis, leading to prophage induction. In some phages, the regulatory unit detects sensory information including quorum-sensing autoinducers, making lysis host-cell-density dependent. Exposure of the polylysogens studied here to different induction scenarios reveals that mixed phage populations emerge following DNA damage, however, induction through the SOS-independent module drives near-exclusive production of the phage sensitive to that specific cue. Considering the lack of potent DNA-damaging agents in natural habitats, we propose that additional phage-encoded sensory pathways that drive lysis play fundamental roles in phage-host biology and inter-prophage competition.

microbiology↗

bioRxiv2022

Quorum-sensing control of matrix protein production drives fractal wrinkling and interfacial localization of Vibrio cholerae pellicles

Bacterial cells at fluid interfaces can self-assemble into collective communities with stunning macroscopic morphologies. Within these soft, living materials, called pellicles, constituent cells gain group-level survival advantages including increased antibiotic resistance. However, the regulatory and structural components that drive pellicle self-patterning are not well defined. Here, using Vibrio cholerae as our model system, we report that two sets of matrix proteins and a key quorum-sensing regulator jointly orchestrate the sequential mechanical instabilities underlying pellicle morphogenesis, culminating in fractal patterning. A pair of matrix proteins, RbmC and Bap1, maintain pellicle localization at the interface and prevent self-peeling. A single matrix protein, RbmA, drives a morphogenesis program marked by a cascade of ever finer wrinkles with fractal scaling in wavelength. Artificial expression of rbmA restores fractal wrinkling to a {Delta}rbmA mutant and enables precise tuning of fractal dimensions. The quorum-sensing regulatory small RNAs Qrr1-4 first activate matrix synthesis to launch pellicle primary wrinkling and ridge instabilities. Subsequently, via a distinct mechanism, Qrr1-4 suppress fractal wrinkling to promote fine modulation of pellicle morphology. Our results connect cell-cell signaling and architectural components to morphogenic patterning and suggest that manipulation of quorum-sensing regulators or synthetic control of rbmA expression could underpin strategies to engineer soft biomaterial morphologies on demand.

microbiology↗

bioRxiv2022

Quorum-sensing- and type VI secretion-mediated spatio-temporal cell death drives genetic diversity in Vibrio cholerae

Bacterial colonies composed of genetically identical individuals can diversify to yield variant cells with distinct genotypes. Variant outgrowth manifests as sectors. Here, we show that Type 6 secretion system (T6SS)-driven cell death in Vibrio cholerae colonies imposes a selective pressure for the emergence of variant strains that can evade T6SS-mediated killing. T6SS-mediated cell death occurs in two distinct spatio-temporal phases, and each phase is driven by a particular T6SS toxin. The first phase is regulated by quorum sensing and drives sectoring. The second phase does not require the T6SS-injection machinery. Variant V. cholerae strains isolated from colony sectors encode mutated quorum-sensing components that confer growth advantages by suppressing T6SS-killing activity while simultaneously boosting T6SS-killing defenses. Our findings show that the T6SS can eliminate sibling cells suggesting a role in intra-specific antagonism. We propose that quorum-sensing-controlled T6SS-driven killing promotes V. cholerae genetic diversity, including in natural habitats and during disease.

microbiology↗

bioRxiv2022

The PqsE active site as a target for small molecule antimicrobial agents against Pseudomonas aeruginosa

The opportunistic pathogen Pseudomonas aeruginosa causes antibiotic resistant, nosocomial infections in immuno-compromised individuals, and is a high priority for antimicrobial development. Key to pathogenicity in P. aeruginosa are biofilm formation and virulence factor production. Both traits are controlled by the cell-to-cell communication process called quorum sensing (QS). QS involves the synthesis, release, and population-wide detection of signal molecules called autoinducers. We previously reported that activity of the RhlR QS transcription factor depends on a protein-protein interaction with the hydrolase, PqsE, and PqsE catalytic activity is dispensable for this interaction. Nonetheless, the PqsE-RhlR interaction could be disrupted by substitution of an active site glutamate residue with tryptophan (PqsE(E182W)). Here, we show that disruption of the PqsE-RhlR interaction via either the E182W change or alteration of PqsE surface residues that are essential for the interaction with RhlR, attenuates P. aeruginosa infection in a murine host. We use crystallography to characterize the conformational changes induced by the PqsE(E182W) substitution to define the mechanism underlying disruption of the PqsE-RhlR interaction. A loop rearrangement that repositions the E280 residue in PqsE(E182W) is responsible for the loss of interaction. We verify the implications garnered from the PqsE(E182W) structure using mutagenic, biochemical, and additional structural analyses. We present the next generation of molecules targeting the PqsE active site, including a structure of the tightest binding of these compounds, BB584, in complex with PqsE. The findings presented here provide insight for drug discovery against P. aeruginosa with PqsE as the target. Author SummaryThe human pathogen Pseudomonas aeruginosa is resistant to many currently used antibiotics, making it a burden of urgent clinical importance. P. aeruginosa pathogenicity is controlled by the bacterial cell-to-cell communication process called quorum sensing (QS). The function of one protein that controls P. aeruginosa QS-directed virulence, RhlR, requires a protein-protein interaction with an enzyme called PqsE. When PqsE is blocked from interacting with RhlR, P. aeruginosa is avirulent and incapable of infecting an animal host. Here, we validate the PqsE-RhlR interaction as a target for antibiotic development, and we present a mechanism for how such antibiotics could disrupt the PqsE-RhlR interaction. Discovery of new antibiotics would fulfill an unmet healthcare need by providing treatments to combat P. aeruginosa infections.

microbiology↗