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Phages have wide influence on bacterial physiology, and likewise, bacterial processes impinge on phage biology. Key to these interactions are phage small proteins (<100 aa). Long underappreciated, recent work has revealed millions of phage small proteins, and increasingly, mechanisms by which they function to dictate phage and/or bacterial behavior and evolution. Here, we describe select phage small proteins that mediate phage-bacterial interactions by modulating phage lifestyle decision-making components or by altering host gene expression., Competing Interests: Declaration of Competing Interest The authors declare they have no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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. 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 φVP882 encodes a VqmA homolog (called VqmAφ). When VqmAφ is produced by φVP882 lysogens, it binds to the host-produced autoinducer called DPO and launches the φVP882 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 φVP882 in naïve host cells. Using mutagenesis, phage infection assays, and phenotypic analyses, we show that φVP882 connects its initial lysis-lysogeny decision to both host cell density and whether the host resides in liquid or on a surface. Host cells in the low-cell-density QS state primarily undergo lysogenic conversion. The QS regulator LuxO~P promotes φVP882 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 φVP882 integrates cues from multiple sensory pathways into its lifestyle decision making upon infection of a new host cell., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Santoriello, Bassler. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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 individual-cell resolution. smFISH analyses of V. cholerae biofilm regulatory and structural genes demonstrate that, as biofilms mature, matrix gene expression decreases, and simultaneously, a pattern emerges in which matrix gene expression is 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, while c-di-GMP-signaling sets the regional expression pattern without input from quorum sensing. The smFISH strategy provides insight into mechanisms conferring particular fates to individual biofilm cells., Competing Interests: Declaration of interests The authors have no competing interests to declare.

Aggregation behavior provides bacteria protection from harsh environments and threats to survival. Two uncharacterized proteases, LapX and Lap, are important for Vibrio cholerae liquid-based aggregation. Here, we determined that LapX is a serine protease with a preference for cleavage after glutamate and glutamine residues in the P1 position, which processes a physiologically based peptide substrate with a catalytic efficiency of 180 ± 80 M -1 s -1 . The activity with a LapX substrate identified by a multiplex substrate profiling by mass spectrometry screen was 590 ± 20 M -1 s -1 . Lap shares high sequence identity with an aminopeptidase (termed VpAP) from Vibrio proteolyticus and contains an inhibitory bacterial prepeptidase C-terminal domain that, when eliminated, increases catalytic efficiency on leucine p-nitroanilide nearly four-fold from 5.4 ± 4.1 × 10 4  M -1 s -1 to 20.3 ± 4.3 × 10 4  M -1 s -1 . We demonstrate that LapX processes Lap to its mature form and thus amplifies Lap activity. The increase is approximately eighteen-fold for full-length Lap (95.7 ± 5.6 × 10 4  M -1 s -1 ) and six-fold for Lap lacking the prepeptidase C-terminal domain (11.3 ± 1.9 × 10 5  M -1 s -1 ). In addition, substrate profiling reveals preferences for these two proteases that could inform in vivo function. Furthermore, purified LapX and Lap restore the timing of the V. cholerae aggregation program to a mutant lacking the lapX and lap genes. Both proteases must be present to restore WT timing, and thus they appear to act sequentially: LapX acts on Lap, and Lap acts on the substrate involved in aggregation., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Most bacteria in the biosphere are predicted to be polylysogens harbouring multiple prophages 1-5 . In studied systems, prophage induction from lysogeny to lysis is near-universally driven by DNA-damaging agents 6 . Thus, how co-residing prophages compete for cell resources if they respond to an identical trigger is unknown. Here we discover regulatory modules that control prophage induction independently of the DNA-damage cue. The modules bear little resemblance at the sequence level but share a regulatory logic by having a transcription factor that activates the expression of a neighbouring gene that encodes a small protein. The small protein inactivates the master repressor of lysis, which leads to induction. Polylysogens that harbour two prophages exposed to DNA damage release mixed populations of phages. Single-cell analyses reveal that this blend is a consequence of discrete subsets of cells producing one, the other or both phages. By contrast, induction through the DNA-damage-independent module results in cells producing only the phage sensitive to that specific cue. Thus, in the polylysogens tested, the stimulus used to induce lysis determines phage productivity. Considering the lack of potent DNA-damaging agents in natural habitats, additional phage-encoded sensory pathways to lysis likely have fundamental roles in phage-host biology and inter-prophage competition., (© 2023. The Author(s).)

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Duddy et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Bacterial biofilms are communities of cells adhered to surfaces. These communities represent a predominant form of bacterial life on Earth. A defining feature of a biofilm is the three-dimensional extracellular polymer matrix that protects resident cells by acting as a mechanical barrier to the penetration of chemicals, such as antimicrobials. Beyond being recalcitrant to antibiotic treatment, biofilms are notoriously difficult to remove from surfaces. A promising, but relatively underexplored, approach to biofilm control is to disrupt the extracellular polymer matrix by enabling penetration of particles to increase the susceptibility of biofilms to antimicrobials. In this work, we investigate externally imposed chemical gradients as a mechanism to transport polystyrene particles into bacterial biofilms. We show that preconditioning the biofilm with a prewash step using deionized (DI) water is essential for altering the biofilm so it takes up the micro- and nanoparticles by the application of a further chemical gradient created by an electrolyte. Using different particles and chemicals, we document the transport behavior that leads to particle motion into the biofilm and its further reversal out of the biofilm. Our results demonstrate the importance of chemical gradients in disrupting the biofilm matrix and regulating particle transport in crowded macromolecular environments, and suggest potential applications of particle transport and delivery in other physiological systems.

Competing Interests: The authors have declared that no competing interests exist.

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 LuxR ARM81ld . LuxR ARM81ld 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 LuxR ARM81ld activity is modulated by noncognate HSL ligands and by a synthetic small molecule inhibitor. We determine that HSLs with acyl chain lengths equal to or longer than C8 antagonize LuxR ARM81ld . For example, the C8-HSL AI produced by Vibrio fischeri that coexists with Aeromonads in aquatic environments, binds to and inhibits LuxR ARM81ld , and consequently, protects the host from lysis. Coculture 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.

Pseudomonas aeruginosa is a human pathogen that relies on quorum sensing to establish infections. The PqsE quorum-sensing protein is required for P. aeruginosa virulence factor production and infection. PqsE has a reported enzymatic function in the biosynthesis of the quorum-sensing autoinducer called PQS. However, this activity is redundant because, in the absence of PqsE, this role is fulfilled by alternative thioesterases. Rather, PqsE drives P. aeruginosa pathogenic traits via a protein-protein interaction with the quorum-sensing receptor/transcription factor RhlR, an interaction that enhances the affinity of RhlR for target DNA sequences. PqsE catalytic activity is dispensable for interaction with RhlR. Thus, the virulence function of PqsE can be decoupled from its catalytic function. Here, we present an immunoprecipitation-mass spectrometry method employing enhanced green fluorescent protein-PqsE fusions to define the protein interactomes of wild-type PqsE and the catalytically inactive PqsE(D73A) variant in P. aeruginosa and their dependence on RhlR. Several proteins were identified to have specific interactions with wild-type PqsE while not forming associations with PqsE(D73A). In the Δ rhlR strain, an increased number of specific PqsE interactors were identified, including the partner autoinducer synthase for RhlR, called RhlI. Collectively, these results suggest that specific protein-protein interactions depend on PqsE catalytic activity and that RhlR may prevent proteins from interacting with PqsE, possibly due to competition between RhlR and other proteins for PqsE binding. Our results provide a foundation for the identification of the in vivo PqsE catalytic function and, potentially, new proteins involved in P. aeruginosa quorum sensing. IMPORTANCE Pseudomonas aeruginosa causes hospital-borne infections in vulnerable patients, including immunocompromised individuals, burn victims, and cancer patients undergoing chemotherapy. There are no effective treatments for P. aeruginosa infections, which are usually broadly resistant to antibiotics. Animal models show that, to establish infection and to cause illness, P. aeruginosa relies on an interaction between two proteins, namely, PqsE and RhlR. There could be additional protein-protein interactions involving PqsE, which, if defined, could be exploited for the design of new therapeutic strategies to combat P. aeruginosa. Here, we reveal previously unknown protein interactions in which PqsE participates, which will be investigated for potential roles in pathogenesis.

Transitions between individual and communal lifestyles allow bacteria to adapt to changing environments. Bacteria must integrate information encoded in multiple sensory cues to appropriately undertake these transitions. Here, we investigate how two prevalent sensory inputs converge on biofilm morphogenesis: quorum sensing, which endows bacteria with the ability to communicate and coordinate group behaviors, and second messenger c-di-GMP signaling, which allows bacteria to detect and respond to environmental stimuli. We use Vibrio cholerae as our model system, the autoinducer AI-2 to modulate quorum sensing, and the polyamine norspermidine to modulate NspS-MbaA-mediated c-di-GMP production. Individually, AI-2 and norspermidine drive opposing biofilm phenotypes, with AI-2 repressing and norspermidine inducing biofilm formation. Surprisingly, however, when AI-2 and norspermidine are simultaneously detected, they act synergistically to increase biofilm biomass and biofilm cell density. We show that this effect is caused by quorum-sensing-mediated activation of nspS - mbaA expression, which increases the levels of NspS and MbaA, and in turn, c-di-GMP biosynthesis, in response to norspermidine. Increased MbaA-synthesized c-di-GMP activates the VpsR transcription factor, driving elevated expression of genes encoding key biofilm matrix components. Thus, in the context of biofilm morphogenesis in V. cholerae, quorum-sensing regulation of c-di-GMP-metabolizing receptor levels connects changes in cell population density to detection of environmental stimuli. IMPORTANCE The development of multicellular communities, known as biofilms, facilitates beneficial functions of gut microbiome bacteria and makes bacterial pathogens recalcitrant to treatment. Understanding how bacteria regulate the biofilm life cycle is fundamental to biofilm control in industrial processes and in medicine. Here, we demonstrate how two major sensory inputs-quorum-sensing communication and second messenger c-di-GMP signaling-jointly regulate biofilm morphogenesis in the global pathogen Vibrio cholerae. We characterize the mechanism underlying a surprising synergy between quorum-sensing and c-di-GMP signaling in controlling biofilm development. Thus, the work connects changes in cell population density to detection of environmental stimuli in a pathogen of clinical significance.

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 ΔrbmA 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., (© 2022. The Author(s).)

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 VI 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 spatiotemporal 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., Competing Interests: Declaration of interests The authors have no competing interests to declare., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Bacteria orchestrate collective behaviors and accomplish feats that would be unsuccessful if carried out by a lone bacterium. Processes undertaken by groups of bacteria include bioluminescence, biofilm formation, virulence factor production, and release of public goods that are shared by the community. Collective behaviors are controlled by signal transduction networks that integrate sensory information and transduce the information internally. Here, we discuss network features and mechanisms that, even in the face of dramatically changing environments, drive precise execution of bacterial group behaviors. We focus on representative quorum-sensing and second-messenger cyclic dimeric GMP (c-di-GMP) signal relays. We highlight ligand specificity versus sensitivity, how small-molecule ligands drive discrimination of kin versus nonkin, signal integration mechanisms, single-input sensory systems versus coincidence detectors, and tuning of input-output dynamics via feedback regulation. We summarize how different features of signal transduction systems allow groups of bacteria to successfully interpret and collectively react to dynamically changing environments.

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 the 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 the 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 insights into drug discovery against P. aeruginosa with PqsE as the target.

Chemical communication between bacteria and between bacteria and the bacteriophage (phage) viruses that prey on them can shape the outcomes of phage-bacterial encounters. Quorum sensing (QS) is a bacterial cell-to-cell communication process that promotes collective undertaking of group behaviors. QS relies on the production, release, accumulation, and detection of signal molecules called autoinducers. Phages can exploit QS-mediated communication to manipulate their hosts and maximize their own survival. In the opportunistic pathogen Pseudomonas aeruginosa, the LasI/R QS system induces the RhlI/R QS system, and in opposing manners, these two systems control the QS system that relies on the autoinducer called PQS. A P. aeruginosa Δ lasI mutant is impaired in PQS synthesis, leading to accumulation of the precursor molecule HHQ, and HHQ suppresses growth of the P. aeruginosa Δ lasI strain. We show that, in response to a phage infection, the P. aeruginosa Δ lasI mutant reactivates QS, which, in turn, restores pqsH expression, enabling conversion of HHQ into PQS. Moreover, downstream QS target genes encoding virulence factors are induced. Additionally, phage-infected P. aeruginosa Δ lasI cells transiently exhibit superior growth compared to uninfected cells. IMPORTANCE Clinical isolates of P. aeruginosa frequently harbor mutations in particular QS genes. Here, we show that infection by select temperate phages restores QS, a cell-to-cell communication mechanism in a P. aeruginosa QS mutant. Restoration of QS increases expression of genes encoding virulence factors. Thus, phage infection of select P. aeruginosa strains may increase bacterial pathogenicity, underscoring the importance of characterizing phage-host interactions in the context of bacterial mutants that are relevant in clinical settings.

Bacterial biofilms are multicellular communities that collectively overcome environmental threats and clinical treatments. To regulate the biofilm lifecycle, bacteria commonly transduce sensory information via the second messenger molecule cyclic diguanylate (c-di-GMP). Using experimental and modeling approaches, we quantitatively capture c-di-GMP signal transmission via the bifunctional polyamine receptor NspS-MbaA, from ligand binding to output, in the pathogen Vibrio cholerae. Upon binding of norspermidine or spermidine, NspS-MbaA synthesizes or degrades c-di-GMP, respectively, which, in turn, drives alterations specifically to biofilm gene expression. A long-standing question is how output specificity is achieved via c-di-GMP, a diffusible molecule that regulates dozens of effectors. We show that NspS-MbaA signals locally to specific effectors, sensitizing V. cholerae to polyamines. However, local signaling is not required for specificity, as changes to global cytoplasmic c-di-GMP levels can selectively regulate biofilm genes. This work establishes the input-output dynamics underlying c-di-GMP signaling, which could be useful for developing bacterial manipulation strategies., Competing Interests: The authors have declared that no competing interests exist.

Pseudomonas aeruginosa is an opportunistic pathogen that causes disease in immunocompromised individuals and individuals with underlying pulmonary disorders. P. aeruginosa virulence is controlled by quorum sensing (QS), a bacterial cell-cell communication mechanism that underpins transitions between individual and group behaviors. In P. aeruginosa, the PqsE enzyme and the QS receptor RhlR directly interact to control the expression of genes involved in virulence. Here, we show that three surface-exposed arginine residues on PqsE comprise the site required for interaction with RhlR. We show that a noninteracting PqsE variant [PqsE(NI)] possesses catalytic activity, but is incapable of promoting virulence phenotypes, indicating that interaction with RhlR, and not catalysis, drives these PqsE-dependent behaviors. Biochemical characterization of the PqsE-RhlR interaction coupled with RNA-seq analyses demonstrates that the PqsE-RhlR complex increases the affinity of RhlR for DNA, enabling enhanced expression of genes encoding key virulence factors. These findings provide the mechanism for PqsE-dependent regulation of RhlR and identify a unique regulatory feature of P. aeruginosa QS and its connection to virulence. IMPORTANCE Bacteria use a cell-cell communication process called quorum sensing (QS) to orchestrate collective behaviors. QS relies on the group-wide detection of molecules called autoinducers (AI). QS is required for virulence in the human pathogen Pseudomonas aeruginosa, which can cause fatal infections in patients with underlying pulmonary disorders. In this study, we determine the molecular basis for the physical interaction between two virulence-driving QS components, PqsE and RhlR. We find that the ability of PqsE to bind RhlR correlates with virulence factor production. Since current antimicrobial therapies exacerbate the growing antibiotic resistance problem because they target bacterial growth, we suggest that the PqsE-RhlR interface discovered here represents a new candidate for targeting with small molecule inhibition. Therapeutics that disrupt the PqsE-RhlR interaction should suppress virulence. Targeting bacterial behaviors such as QS, rather than bacterial growth, represents an attractive alternative for exploration because such therapies could potentially minimize the development of resistance.

Quorum sensing (QS) is a chemical communication process in which bacteria produce, release, and detect extracellular signaling molecules called autoinducers. Via combined transcriptional and posttranscriptional regulatory mechanisms, QS allows bacteria to collectively alter gene expression on a population-wide scale. Recently, the TetR family transcriptional regulator LuxT was shown to control Vibrio harveyi qrr 1, encoding the Qrr1 small RNA that functions at the core of the QS regulatory cascade. Here, we use RNA sequencing to reveal that, beyond the control of qrr 1, LuxT is a global regulator of 414 V. harveyi genes, including those involved in type III secretion, siderophore production, and aerolysin toxin biosynthesis. Importantly, LuxT directly represses swrZ , encoding a GntR family transcriptional regulator, and LuxT control of type III secretion, siderophore, and aerolysin genes occurs by two mechanisms, one that is SwrZ dependent and one that is SwrZ independent. All of these target genes specify QS-controlled behaviors that are enacted when V. harveyi is at low cell density. Thus, LuxT and SwrZ function in parallel with QS to drive particular low-cell-density behaviors. Phylogenetic analyses reveal that luxT is highly conserved among Vibrionaceae , but swrZ is less well conserved. In a test case, we find that in Aliivibrio fischeri, LuxT also represses swrZ . SwrZ is a repressor of A. fischeri siderophore production genes. Thus, LuxT repression of swrZ drives the activation of A. fischeri siderophore gene expression. Our results indicate that LuxT is a major regulator among Vibrionaceae , and in the species that also possess swrZ , LuxT functions with SwrZ to control gene expression. IMPORTANCE Bacteria precisely tune gene expression patterns to successfully react to changes that occur in the environment. Defining the mechanisms that enable bacteria to thrive in diverse and fluctuating habitats, including in host organisms, is crucial for a deep understanding of the microbial world and also for the development of effective applications to promote or combat particular bacteria. In this study, we show that a regulator called LuxT controls over 400 genes in the marine bacterium Vibrio harveyi and that LuxT is highly conserved among Vibrionaceae species, ubiquitous marine bacteria that often cause disease. We characterize the mechanisms by which LuxT controls genes involved in virulence and nutrient acquisition. We show that LuxT functions in parallel with a set of regulators of the bacterial cell-to-cell communication process called quorum sensing to promote V. harveyi behaviors at low cell density.

Bacteria orchestrate collective behaviors using the cell-cell communication process called quorum sensing (QS). QS relies on the synthesis, release, and group-wide detection of small molecules called autoinducers. In Vibrio cholerae, a multicellular community aggregation program occurs in liquid, during the stationary phase, and in the high-cell-density QS state. Here, we demonstrate that this aggregation program consists of two subprograms. In one subprogram, which we call void formation, structures form that contain few cells but provide a scaffold within which cells can embed. The other subprogram relies on flagellar machinery and enables cells to enter voids. A genetic screen for factors contributing to void formation, coupled with companion molecular analyses, showed that four extracellular proteases, Vca0812, Vca0813, HapA, and PrtV, control the onset timing of both void formation and aggregation; moreover, proteolytic activity is required. These proteases, or their downstream products, can be shared between void-producing and non-void-forming cells and can elicit aggregation in a normally nonaggregating V. cholerae strain. Employing multiple proteases to control void formation and aggregation timing could provide a redundant and irreversible path to commitment to this community lifestyle. IMPORTANCE Bacteria can work as collectives to form multicellular communities. Vibrio cholerae, the bacterium that causes the disease cholera in humans, forms aggregated communities in liquid. Aggregate formation relies on a chemical communication process called quorum sensing. Here, we show that, beyond overarching control by quorum sensing, there are two aggregation subprograms. One subprogram, which we call void formation, creates a scaffold within which cells can embed. The second subprogram, which allows bacteria to enter the scaffold, requires motility. We discovered that four extracellular proteases control the timing of both void formation and aggregation. We argue that, by using redundant proteases, V. cholerae ensures the reliable execution of this community formation process. These findings may provide insight into how V. cholerae persists in the marine environment or colonizes the human host, as both lifestyles are central to the spread of the disease cholera.

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., Competing Interests: The authors declare no competing interest.

Quorum sensing is a chemical communication process that bacteria use to coordinate group behaviors. In the global pathogen Vibrio cholerae, one quorum-sensing receptor and transcription factor, called VqmA (VqmAVc), activates expression of the vqmR gene encoding the small regulatory RNA VqmR, which represses genes involved in virulence and biofilm formation. Vibriophage VP882 encodes a VqmA homolog called VqmAPhage that activates transcription of the phage gene qtip, and Qtip launches the phage lytic program. Curiously, VqmAPhage can activate vqmR expression but VqmAVc cannot activate expression of qtip. Here, we investigate the mechanism underlying this asymmetry. We find that promoter selectivity is driven by each VqmA DNA-binding domain and key DNA sequences in the vqmR and qtip promoters are required to maintain specificity. A protein sequence-guided mutagenesis approach revealed that the residue E194 of VqmAPhage and A192, the equivalent residue in VqmAVc, in the helix-turn-helix motifs contribute to promoter-binding specificity. A genetic screen to identify VqmAPhage mutants that are incapable of binding the qtip promoter but maintain binding to the vqmR promoter delivered additional VqmAPhage residues located immediately C-terminal to the helix-turn-helix motif as required for binding the qtip promoter. Surprisingly, these residues are conserved between VqmAPhage and VqmAVc. A second, targeted genetic screen revealed a region located in the VqmAVc DNA-binding domain that is necessary to prevent VqmAVc from binding the qtip promoter, thus restricting DNA binding to the vqmR promoter. We propose that the VqmAVc helix-turn-helix motif and the C-terminal flanking residues function together to prohibit VqmAVc from binding the qtip promoter., Competing Interests: The authors have declared that no competing interests exist.

Bacterial biofilms are communities of bacteria that exist as aggregates that can adhere to surfaces or be free-standing. This complex, social mode of cellular organization is fundamental to the physiology of microbes and often exhibits surprising behavior. Bacterial biofilms are more than the sum of their parts: single-cell behavior has a complex relation to collective community behavior, in a manner perhaps cognate to the complex relation between atomic physics and condensed matter physics. Biofilm microbiology is a relatively young field by biology standards, but it has already attracted intense attention from physicists. Sometimes, this attention takes the form of seeing biofilms as inspiration for new physics. In this roadmap, we highlight the work of those who have taken the opposite strategy: we highlight the work of physicists and physical scientists who use physics to engage fundamental concepts in bacterial biofilm microbiology, including adhesion, sensing, motility, signaling, memory, energy flow, community formation and cooperativity. These contributions are juxtaposed with microbiologists who have made recent important discoveries on bacterial biofilms using state-of-the-art physical methods. The contributions to this roadmap exemplify how well physics and biology can be combined to achieve a new synthesis, rather than just a division of labor., (Creative Commons Attribution license.)

Bacterial cells can self-organize into structured communities at fluid-fluid interfaces. These soft, living materials composed of cells and extracellular matrix are called pellicles. Cells residing in pellicles garner group-level survival advantages such as increased antibiotic resistance. The dynamics of pellicle formation and, more generally, how complex morphologies arise from active biomaterials confined at interfaces are not well understood. Here, using Vibrio cholerae as our model organism, a custom-built adaptive stereo microscope, fluorescence imaging, mechanical theory, and simulations, we report a fractal wrinkling morphogenesis program that differs radically from the well-known coalescence of wrinkles into folds that occurs in passive thin films at fluid-fluid interfaces. Four stages occur: growth of founding colonies, onset of primary wrinkles, development of secondary curved ridge instabilities, and finally the emergence of a cascade of finer structures with fractal-like scaling in wavelength. The time evolution of pellicle formation depends on the initial heterogeneity of the film microstructure. Changing the starting bacterial seeding density produces three variations in the sequence of morphogenic stages, which we term the bypass, crystalline, and incomplete modes. Despite these global architectural transitions, individual microcolonies remain spatially segregated, and thus, the community maintains spatial and genetic heterogeneity. Our results suggest that the memory of the original microstructure is critical in setting the morphogenic dynamics of a pellicle as an active biomaterial., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Pseudomonas aeruginosa is an opportunistic human pathogen that causes fatal infections. There exists an urgent need for new antimicrobial agents to combat P. aeruginosa . We conducted a screen for molecules that bind the virulence-controlling protein PqsE and characterized hit compounds for inhibition of PqsE enzymatic activity. The binding conformations of two inhibitory molecules, BB391 and BB393, were identified by crystallography, and inhibitor binding was mimicked by the substitution of PqsE residues E182 and S285 with tryptophan. Comparison of the inhibitor-mimetic mutations to the catalytically inactive PqsE D73A protein demonstrated that catalysis is not responsible for the role PqsE plays in driving virulence factor production. Rather, the PqsE E182W protein fails to interact with the quorum-sensing receptor, RhlR, and our results suggest that it is this interaction that is responsible for promoting virulence factor production in P. aeruginosa . These findings provide a new route for drug discovery efforts targeting PqsE.

The global pathogen Vibrio cholerae undergoes cycles of biofilm formation and dispersal in the environment and the human host. Little is understood about biofilm dispersal. Here, we show that MbaA, a periplasmic polyamine sensor, and PotD1, a polyamine importer, regulate V. cholerae biofilm dispersal. Spermidine, a commonly produced polyamine, drives V. cholerae dispersal, whereas norspermidine, an uncommon polyamine produced by vibrios, inhibits dispersal. Spermidine and norspermidine differ by one methylene group. Both polyamines control dispersal via MbaA detection in the periplasm and subsequent signal relay. Our results suggest that dispersal fails in the absence of PotD1 because endogenously produced norspermidine is not reimported, periplasmic norspermidine accumulates, and it stimulates MbaA signaling. These results suggest that V. cholerae uses MbaA to monitor environmental polyamines, blends of which potentially provide information about numbers of 'self' and 'other'. This information is used to dictate whether or not to disperse from biofilms., Competing Interests: AB, BB No competing interests declared, (© 2021, Bridges and Bassler.)

Quorum sensing (QS) is a process of chemical communication bacteria use to transition between individual and collective behaviors. QS depends on the production, release, and synchronous response to signaling molecules called autoinducers (AIs). The marine bacterium Vibrio harveyi monitors AIs using a signal transduction pathway that relies on five small regulatory RNAs (called Qrr1-5) that post-transcriptionally control target genes. Curiously, the small RNAs largely function redundantly making it difficult to understand the necessity for five of them. Here, we identify LuxT as a transcriptional repressor of qrr1. LuxT does not regulate qrr2-5, demonstrating that qrr genes can be independently controlled to drive unique downstream QS gene expression patterns. LuxT reinforces its control over the same genes it regulates indirectly via repression of qrr1, through a second transcriptional control mechanism. Genes dually regulated by LuxT specify public goods including an aerolysin-type pore-forming toxin. Phylogenetic analyses reveal that LuxT is conserved among Vibrionaceae and sequence comparisons predict that LuxT represses qrr1 in additional species. The present findings reveal that the QS regulatory RNAs can carry out both shared and unique functions to endow bacteria with plasticity in their output behaviors., Competing Interests: The authors have declared that no competing interests exist.

Quorum sensing is a process of cell-to-cell communication that bacteria use to orchestrate collective behaviors. Quorum sensing depends on the production, release, and detection of extracellular signal molecules called autoinducers (AIs) that accumulate with increasing cell density. While most AIs are species specific, the AI called AI-2 is produced and detected by diverse bacterial species, and it mediates interspecies communication. We recently reported that mammalian cells produce an AI-2 mimic that can be detected by bacteria through the AI-2 receptor LuxP, potentially expanding the role of the AI-2 system to interdomain communication. Here, we describe a second molecule capable of interdomain signaling through LuxP, 4-hydroxy-5-methylfuran-3(2H)-one (MHF), that is produced by the yeast Saccharomyces cerevisiae Screening the S. cerevisiae deletion collection revealed Cff1p, a protein with no known role, to be required for MHF production. Cff1p is proposed to be an enzyme, with structural similarity to sugar isomerases and epimerases, and substitution at the putative catalytic residue eliminated MHF production in S. cerevisiae Sequence analysis uncovered Cff1p homologs in many species, primarily bacterial and fungal, but also viral, archaeal, and higher eukaryotic. Cff1p homologs from organisms from all domains can complement a cff1Δ S. cerevisiae mutant and restore MHF production. In all cases tested, the identified catalytic residue is conserved and required for MHF to be produced. These findings increase the scope of possibilities for interdomain interactions via AI-2 and AI-2 mimics, highlighting the breadth of molecules and organisms that could participate in quorum sensing. IMPORTANCE Quorum sensing is a cell-to-cell communication process that bacteria use to monitor local population density. Quorum sensing relies on extracellular signal molecules called autoinducers (AIs). One AI called AI-2 is broadly made by bacteria and used for interspecies communication. Here, we describe a eukaryotic AI-2 mimic, 4-hydroxy-5-methylfuran-3(2H)-one, (MHF), that is made by the yeast Saccharomyces cerevisiae , and we identify the Cff1p protein as essential for MHF production. Hundreds of viral, archaeal, bacterial, and eukaryotic organisms possess Cff1p homologs. This finding, combined with our results showing that homologs from all domains can replace S. cerevisiae Cff1p, suggests that like AI-2, MHF is widely produced. Our results expand the breadth of organisms that may participate in quorum-sensing-mediated interactions., (Copyright © 2021 Valastyan et al.)

Competing Interests: The authors have declared that no competing interests exist.

Bacteria alternate between being free-swimming and existing as members of sessile multicellular communities called biofilms. The biofilm lifecycle occurs in three stages: cell attachment, biofilm maturation, and biofilm dispersal. Vibrio cholerae biofilms are hyperinfectious, and biofilm formation and dispersal are considered central to disease transmission. While biofilm formation is well studied, almost nothing is known about biofilm dispersal. Here, we conducted an imaging screen for V. cholerae mutants that fail to disperse, revealing three classes of dispersal components: signal transduction proteins, matrix-degradation enzymes, and motility factors. Signaling proteins dominated the screen and among them, we focused on an uncharacterized two-component sensory system that we term DbfS/DbfR for dispersal of biofilm sensor/regulator. Phospho-DbfR represses biofilm dispersal. DbfS dephosphorylates and thereby inactivates DbfR, which permits dispersal. Matrix degradation requires two enzymes: LapG, which cleaves adhesins, and RbmB, which digests matrix polysaccharides. Reorientation in swimming direction, mediated by CheY3, is necessary for cells to escape from the porous biofilm matrix. We suggest that these components act sequentially: signaling launches dispersal by terminating matrix production and triggering matrix digestion, and subsequent cell motility permits escape from biofilms. This study lays the groundwork for interventions aimed at modulating V. cholerae biofilm dispersal to ameliorate disease., Competing Interests: Competing interest statement: B.L.B. and S.G. are co-authors on a 2017 comment article concerning gender discrimination in science. The piece has a total of 37 co-authors., (Copyright © 2020 the Author(s). Published by PNAS.)

When two or more bacterial species inhabit a shared niche, often, they must compete for limited nutrients. Iron is an essential nutrient that is especially scarce in the marine environment. Bacteria can use the production, release, and re-uptake of siderophores, small molecule iron chelators, to scavenge iron. Siderophores provide fitness advantages to species that employ them by enhancing iron acquisition, and moreover, by denying iron to competitors incapable of using the siderophore-iron complex. Here, we show that cell-free culture fluids from the marine bacterium Vibrio fischeri ES114 prevent the growth of other vibrio species. Mutagenesis reveals the aerobactin siderophore as the inhibitor. Our analysis reveals a gene, that we name aerE, encodes the aerobactin exporter, and LuxT is a transcriptional activator of aerobactin production. In co-culture, under iron-limiting conditions, aerobactin production allows V. fischeri ES114 to competitively exclude Vibrio harveyi, which does not possess aerobactin production and uptake genes. In contrast, V. fischeri ES114 mutants incapable of aerobactin production lose in competition with V. harveyi. Introduction of iutA, encoding the aerobactin receptor, together with fhuCDB, encoding the aerobactin importer are sufficient to convert V. harveyi into an "aerobactin cheater.", (© 2020 John Wiley & Sons Ltd.)

Quorum sensing is a chemical communication process in which bacteria use the production, release, and detection of signal molecules called autoinducers to orchestrate collective behaviors. The human pathogen Vibrio cholerae requires quorum sensing to infect the small intestine. There, V. cholerae encounters the absence of oxygen and the presence of bile salts. We show that these two stimuli differentially affect quorum-sensing function and, in turn, V. cholerae pathogenicity. First, during anaerobic growth, V. cholerae does not produce the CAI-1 autoinducer, while it continues to produce the DPO autoinducer, suggesting that CAI-1 may encode information specific to the aerobic lifestyle of V. cholerae Second, the quorum-sensing receptor-transcription factor called VqmA, which detects the DPO autoinducer, also detects the lack of oxygen and the presence of bile salts. Detection occurs via oxygen-, bile salt-, and redox-responsive disulfide bonds that alter VqmA DNA binding activity. We propose that VqmA serves as an information processing hub that integrates quorum-sensing information, redox status, the presence or absence of oxygen, and host cues. In response to the information acquired through this mechanism, V. cholerae appropriately modulates its virulence output. IMPORTANCE Quorum sensing (QS) is a process of chemical communication that bacteria use to orchestrate collective behaviors. QS communication relies on chemical signal molecules called autoinducers. QS regulates virulence in Vibrio cholerae , the causative agent of the disease cholera. Transit into the human small intestine, the site of cholera infection, exposes V. cholerae to the host environment. In this study, we show that the combination of two stimuli encountered in the small intestine, the absence of oxygen and the presence of host-produced bile salts, impinge on V. cholerae QS function and, in turn, pathogenicity. We suggest that possessing a QS system that is responsive to multiple environmental, host, and cell density cues enables V. cholerae to fine-tune its virulence capacity in the human intestine., (Copyright © 2020 Mashruwala and Bassler.)

Bacterial biofilms represent a basic form of multicellular organization that confers survival advantages to constituent cells. The sequential stages of cell ordering during biofilm development have been studied in the pathogen and model biofilm-former Vibrio cholerae It is unknown how spatial trajectories of individual cells and the collective motions of many cells drive biofilm expansion. We developed dual-view light-sheet microscopy to investigate the dynamics of biofilm development from a founder cell to a mature three-dimensional community. Tracking of individual cells revealed two distinct fates: one set of biofilm cells expanded ballistically outward, while the other became trapped at the substrate. A collective fountain-like flow transported cells to the biofilm front, bypassing members trapped at the substrate and facilitating lateral biofilm expansion. This collective flow pattern was quantitatively captured by a continuum model of biofilm growth against substrate friction. Coordinated cell movement required the matrix protein RbmA, without which cells expanded erratically. Thus, tracking cell lineages and trajectories in space and time revealed how multicellular structures form from a single founder cell., (Copyright © 2020, American Association for the Advancement of Science.)

The Genetics Society of America (GSA) Medal recognizes researchers who have made outstanding contributions to the field of genetics in the past 15 years. The 2019 GSA Medal is awarded to Bonnie L. Bassler of Princeton University and the Howard Hughes Medical Institute in recognition of her groundbreaking studies of bacterial chemical communication and regulation of group behaviors., (Copyright © 2020 by the Genetics Society of America.)

Quorum sensing is a process of chemical communication that bacteria use to track cell density and coordinate gene expression across a population. Bacteria-infecting viruses, called phages, can encode quorum-sensing components that enable them to integrate host cell density information into the lysis-lysogeny decision. Vibriophage VP882 is one such phage, and activation of its quorum-sensing pathway leads to the production of an antirepressor called Qtip. Qtip interferes with the prophage repressor (cI VP882 ), leading to host-cell lysis. Here, we show that Qtip interacts with the N terminus of cI VP882 , inhibiting both cI VP882 DNA binding and cI VP882 autoproteolysis. Qtip also sequesters cI VP882 , localizing it to the poles. Qtip can localize to the poles independently of cI VP882 . Alanine-scanning mutagenesis of Qtip shows that its localization and interference with cI VP882 activities are separable. Comparison of Qtip to a canonical phage antirepressor reveals that despite both proteins interacting with their partner repressors, only Qtip drives polar localization., Competing Interests: Declaration of Interests The authors declare no competing financial interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

During development, organisms acquire three-dimensional (3D) shapes with important physiological consequences. While basic mechanisms underlying morphogenesis are known in eukaryotes, it is often difficult to manipulate them in vivo. To circumvent this issue, here we present a study of developing Vibrio cholerae biofilms grown on agar substrates in which the spatiotemporal morphological patterns were altered by varying the agar concentration. Expanding biofilms are initially flat but later undergo a mechanical instability and become wrinkled. To gain mechanistic insights into this dynamic pattern-formation process, we developed a model that considers diffusion of nutrients and their uptake by bacteria, bacterial growth/biofilm matrix production, mechanical deformation of both the biofilm and the substrate, and the friction between them. Our model shows quantitative agreement with experimental measurements of biofilm expansion dynamics, and it accurately predicts two distinct spatiotemporal patterns observed in the experiments-the wrinkles initially appear either in the peripheral region and propagate inward (soft substrate/low friction) or in the central region and propagate outward (stiff substrate/high friction). Our results, which establish that nonuniform growth and friction are fundamental determinants of stress anisotropy and hence biofilm morphology, are broadly applicable to bacterial biofilms with similar morphologies and also provide insight into how other bacterial biofilms form distinct wrinkle patterns. We discuss the implications of forming undulated biofilm morphologies, which may enhance the availability of nutrients and signaling molecules and serve as a "bet hedging" strategy., Competing Interests: The authors declare no competing interest.

Quorum sensing is a bacterial communication process whereby bacteria produce, release, and detect extracellular signaling molecules called autoinducers to coordinate collective behaviors. In the pathogen Vibrio cholerae , the quorum-sensing autoinducer 3,5-dimethyl-pyrazin-2-ol (DPO) binds the receptor and transcription factor VqmA. The DPO-VqmA complex activates transcription of vqmR , encoding the VqmR small RNA, which represses genes required for biofilm formation and virulence factor production. Here, we show that VqmA is soluble and properly folded and activates basal-level transcription of its target vqmR in the absence of DPO. VqmA transcriptional activity is increased in response to increasing concentrations of DPO, allowing VqmA to drive the V. cholerae quorum-sensing transition at high cell densities. We solved the DPO-VqmA crystal structure to 2.0 Å resolution and compared it with existing structures to understand the conformational changes VqmA undergoes upon DNA binding. Analysis of DPO analogs showed that a hydroxyl or carbonyl group at the 2'-position is critical for binding to VqmA. The proposed DPO precursor, a linear molecule, N -alanyl-aminoacetone (Ala-AA), also bound and activated VqmA. Results from site-directed mutagenesis and competitive ligand-binding analyses revealed that DPO and Ala-AA occupy the same binding site. In summary, our structure-function analysis identifies key features required for VqmA activation and DNA binding and establishes that, whereas VqmA binds two different ligands, VqmA does not require a bound ligand for folding or basal transcriptional activity. However, bound ligand is required for maximal activity., (© 2020 Huang et al.)

Pseudomonas aeruginosa is a leading cause of hospital-acquired infections in the United States. PqsE, a thioesterase enzyme, is vital for virulence of P. aeruginosa , making PqsE an attractive target for inhibition. Neither the substrate nor the product of PqsE catalysis has been identified. A library of 550 million DNA-encoded drug-like small molecules was screened for those that bind to the purified PqsE protein. The structures of the bound molecules were identified by high throughput sequencing of the attached DNA barcodes. Putative PqsE binders with the strongest affinity features were examined for inhibition of PqsE thioesterase activity in vitro . The most potent inhibitors were resynthesized off DNA and examined for the ability to alter PqsE thermal melting and for PqsE thioesterase inhibition. Here, we report the synthesis, biological activity, mechanism of action, and early structure-activity relationships of a series of 2-(phenylcarbamoyl)benzoic acids that noncompetitively inhibit PqsE. A small set of analogs designed to probe initial structure-activity relationships showed increases in potency relative to the original hits, the best of which has an IC 50 = 5 μM. Compound refinement is required to assess their in vivo activities as the current compounds do not accumulate in the P. aeruginosa cytosol. Our strategy validates DNA-encoded compound library screening as a rapid and effective method to identify catalytic inhibitors of the PqsE protein, and more generally, for discovering binders to bacterial proteins revealed by genetic screening to have crucial in vivo activities but whose biological functions have not been well-defined.

Bacteria convert changes in sensory inputs into alterations in gene expression, behavior, and lifestyles. A common lifestyle choice that bacteria make is whether to exhibit individual behavior and exist in the free-living planktonic state or to engage in collective behavior and form sessile communities called biofilms. Transitions between individual and collective behaviors are controlled by the chemical cell-to-cell communication process called quorum sensing. Here, we show that quorum sensing represses Pseudomonas aeruginosa biofilm formation and virulence by activating expression of genes encoding the KinB-AlgB two-component system (TCS). Phospho-AlgB represses biofilm and virulence genes, while KinB dephosphorylates and thereby inactivates AlgB. We discover that the photoreceptor BphP is the kinase that, in response to light, phosphorylates and activates AlgB. Indeed, exposing P. aeruginosa to light represses biofilm formation and virulence gene expression. To our knowledge, P. aeruginosa was not previously known to detect and respond to light. The KinB-AlgB-BphP module is present in all pseudomonads, and we demonstrate that AlgB is the partner response regulator for BphP in diverse bacterial phyla. We propose that in the KinB-AlgB-BphP system, AlgB functions as the node at which varied sensory information is integrated. This network architecture provides a mechanism enabling bacteria to integrate at least two different sensory inputs, quorum sensing (via RhlR-driven activation of algB) and light (via BphP-AlgB), into the control of collective behaviors. This study sets the stage for light-mediated control of P. aeruginosa infectivity., Competing Interests: The authors have declared that no competing interests exist.

Vibrio cholerae possesses multiple quorum-sensing (QS) systems that control virulence and biofilm formation among other traits. At low cell densities, when QS autoinducers are absent, V. cholerae forms biofilms. At high cell densities, when autoinducers have accumulated, biofilm formation is repressed, and dispersal occurs. Here, we focus on the roles of two well-characterized QS autoinducers that function in parallel. One autoinducer, called cholerae autoinducer-1 (CAI-1), is used to measure Vibrio abundance, and the other autoinducer, called autoinducer-2 (AI-2), is widely produced by different bacterial species and presumed to enable V. cholerae to assess the total bacterial cell density of the vicinal community. The two V. cholerae autoinducers funnel information into a shared signal relay pathway. This feature of the QS system architecture has made it difficult to understand how specific information can be extracted from each autoinducer, how the autoinducers might drive distinct output behaviors, and, in turn, how the bacteria use QS to distinguish kin from nonkin in bacterial communities. We develop a live-cell biofilm formation and dispersal assay that allows examination of the individual and combined roles of the two autoinducers in controlling V. cholerae behavior. We show that the QS system works as a coincidence detector in which both autoinducers must be present simultaneously for repression of biofilm formation to occur. Within that context, the CAI-1 QS pathway is activated when only a few V. cholerae cells are present, whereas the AI-2 pathway is activated only at much higher cell density. The consequence of this asymmetry is that exogenous sources of AI-2, but not CAI-1, contribute to satisfying the coincidence detector to repress biofilm formation and promote dispersal. We propose that V. cholerae uses CAI-1 to verify that some of its kin are present before committing to the high-cell-density QS mode, but it is, in fact, the broadly made autoinducer AI-2 that sets the pace of the V. cholerae QS program. This first report of unique roles for the different V. cholerae autoinducers suggests that detection of kin fosters a distinct outcome from detection of nonkin., Competing Interests: The authors have declared that no competing interests exist.

Biofilms are surface-associated bacterial communities that play both beneficial and harmful roles in nature, medicine, and industry. Tolerant and persister cells are thought to underlie biofilm-related bacterial recurrence in medical and industrial contexts. Here, we review recent progress aimed at understanding the mechanical features that drive biofilm resilience and the biofilm formation process at single-cell resolution. We discuss findings regarding mechanisms underlying bacterial tolerance and persistence in biofilms and how these phenotypes are linked to antibiotic resistance. New strategies for combatting tolerance and persistence in biofilms and possible methods for biofilm eradication are highlighted to inspire future development., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Quorum sensing is a chemical communication process that bacteria use to coordinate group behaviors. Pseudomonas aeruginosa, an opportunistic pathogen, employs multiple quorum-sensing systems to control behaviors including virulence factor production and biofilm formation. One P. aeruginosa quorum-sensing receptor, called RhlR, binds the cognate autoinducer N-butryl-homoserine lactone (C4HSL), and the RhlR:C4HSL complex activates transcription of target quorum-sensing genes. Here, we use a genetic screen to identify RhlR mutants that function independently of the autoinducer. The RhlR Y64F W68F V133F triple mutant, which we call RhlR*, exhibits ligand-independent activity in vitro and in vivo. RhlR* can drive wildtype biofilm formation and infection in a nematode animal model. The ability of RhlR* to properly regulate quorum-sensing-controlled genes in vivo depends on the quorum-sensing regulator RsaL keeping RhlR* activity in check. RhlR is known to function together with PqsE to control production of the virulence factor called pyocyanin. Likewise, RhlR* requires PqsE for pyocyanin production in planktonic cultures, however, PqsE is dispensable for RhlR*-driven pyocyanin production on surfaces. Finally, wildtype RhlR protein is not sufficiently stabilized by C4HSL to allow purification. However, wildtype RhlR can be stabilized by the synthetic ligand mBTL (meta-bromo-thiolactone) and RhlR* is stable without a ligand. These features enabled purification of the RhlR:mBTL complex and of RhlR* for in vitro examination of their biochemical activities. To our knowledge, this work reports the first RhlR protein purification., Competing Interests: No. The authors have declared that no competing interests exist.

Quorum sensing is a process of bacterial cell-to-cell chemical communication that relies on the production, detection and response to extracellular signalling molecules called autoinducers. Quorum sensing allows groups of bacteria to synchronously alter behaviour in response to changes in the population density and species composition of the vicinal community. Quorum-sensing-mediated communication is now understood to be the norm in the bacterial world. Elegant research has defined quorum-sensing components and their interactions, for the most part, under ideal and highly controlled conditions. Indeed, these seminal studies laid the foundations for the field. In this Review, we highlight new findings concerning how bacteria deploy quorum sensing in realistic scenarios that mimic nature. We focus on how quorums are detected and how quorum sensing controls group behaviours in complex and dynamically changing environments such as multi-species bacterial communities, in the presence of flow, in 3D non-uniform biofilms and in hosts during infection.

Virulence induction in the Staphylococcus aureus is under the control of a quorum sensing (QS) circuit encoded by the accessory gene regulator (agr) locus. Allelic variation within agr produces four QS specificity groups, each producing a unique secreted autoinducer peptide (AIP) and receptor histidine kinase (RHK), AgrC. Cognate AIP-AgrC interactions activate virulence through a two-component signaling cascade, whereas non-cognate pairs are generally inhibitory. Here we pinpoint a key hydrogen-bonding interaction within AgrC that acts as a switch to convert helical motions propagating from the receptor sensor domain into changes in inter-domain association within the kinase module. AgrC mutants lacking this interaction are constitutively active in vitro and in vivo, the latter leading to a pronounced attenuation of S. aureus biofilm formation. Thus, our work sheds light on the regulation of this biomedically important RHK., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Quorum sensing (QS) is a process of cell-to-cell communication that bacteria use to orchestrate collective behaviors. QS relies on the cell-density-dependent production, accumulation, and receptor-mediated detection of extracellular signaling molecules called autoinducers (AIs). Gram-negative bacteria commonly use N -acyl homoserine lactones (AHLs) as their AIs, and they are detected by LuxR-type receptors. Often, LuxR-type receptors are insoluble when not bound to a cognate AI. In this report, we show that LuxR-type receptors are encoded on phage genomes, and in the cases we tested, the phage LuxR-type receptors bind to and are solubilized specifically by the AHL AI produced by the host bacterium. We do not yet know the viral activities that are controlled by these phage QS receptors; however, our observations, coupled with recent reports, suggest that their occurrence is more widespread than previously appreciated. Using receptor-mediated detection of QS AIs could enable phages to garner information concerning the population density status of their bacterial hosts. We speculate that such information can be exploited by phages to optimize the timing of execution of particular steps in viral infection. IMPORTANCE Bacteria communicate with chemical signal molecules to regulate group behaviors in a process called quorum sensing (QS). In this report, we find that genes encoding receptors for Gram-negative bacterial QS communication molecules are present on genomes of viruses that infect these bacteria. These viruses are called phages. We show that two phage-encoded receptors, like their bacterial counterparts, bind to the communication molecule produced by the host bacterium, suggesting that phages can "listen in" on their bacterial hosts. Interfering with bacterial QS and using phages to kill pathogenic bacteria represent attractive possibilities for development of new antimicrobials to combat pathogens that are resistant to traditional antibiotics. Our findings of interactions between phages and QS bacteria need consideration as new antimicrobial therapies are developed., (Copyright © 2019 Silpe and Bassler.)

Bacteria use a cell-cell communication process called quorum sensing to coordinate collective behaviors. Quorum sensing relies on production and group-wide detection of extracellular signal molecules called autoinducers. Here, we probe the activity of the Pseudomonas aeruginosa LasR quorum-sensing receptor using synthetic agonists based on the structure of the native homoserine lactone autoinducer. The synthetic compounds range from low to high potency, and agonist activity tracks with the ability of the agonist to stabilize the LasR protein. Structural analyses of the LasR ligand binding domain complexed with representative synthetic agonists reveal two modes of ligand binding, one mimicking the canonical autoinducer binding arrangement, and the other with the lactone head group rotated approximately 150°. Iterative mutagenesis combined with chemical synthesis reveals the amino acid residues and the chemical moieties, respectively, that are key to enabling each mode of binding. Simultaneous alteration of LasR residues Thr75, Tyr93, and Ala127 converts low-potency compounds into high-potency compounds and converts ligands that are nearly inactive into low-potency compounds. These results show that the LasR binding pocket displays significant flexibility in accommodating different ligands. The ability of LasR to bind ligands in different conformations, and in so doing, alter their potency as agonists, could explain the difficulties that have been encountered in the development of competitive LasR inhibitors.

Surface-attached bacterial communities called biofilms display a diversity of morphologies. Although structural and regulatory components required for biofilm formation are known, it is not understood how these essential constituents promote biofilm surface morphology. Here, using Vibrio cholerae as our model system, we combine mechanical measurements, theory and simulation, quantitative image analyses, surface energy characterizations, and mutagenesis to show that mechanical instabilities, including wrinkling and delamination, underlie the morphogenesis program of growing biofilms. We also identify interfacial energy as a key driving force for mechanomorphogenesis because it dictates the generation of new and the annihilation of existing interfaces. Finally, we discover feedback between mechanomorphogenesis and biofilm expansion, which shapes the overall biofilm contour. The morphogenesis principles that we discover in bacterial biofilms, which rely on mechanical instabilities and interfacial energies, should be generally applicable to morphogenesis processes in tissues in higher organisms., Competing Interests: JY, CF, SM, AM, NW, AK, HS, BB No competing interests declared, (© 2019, Yan et al.)

Vibrio cholerae uses a quorum-sensing (QS) system composed of the autoinducer 3,5-dimethylpyrazin-2-ol (DPO) and receptor VqmA (VqmA Vc ), which together repress genes for virulence and biofilm formation. vqmA genes exist in Vibrio and in one vibriophage, VP882. Phage-encoded VqmA (VqmA Phage ) binds to host-produced DPO, launching the phage lysis program via an antirepressor that inactivates the phage repressor by sequestration. The antirepressor interferes with repressors from related phages. Like phage VP882, these phages encode DNA-binding proteins and partner antirepressors, suggesting that they, too, integrate host-derived information into their lysis-lysogeny decisions. VqmA Phage activates the host VqmA Vc regulon, whereas VqmA Vc cannot induce phage-mediated lysis, suggesting an asymmetry whereby the phage influences host QS while enacting its own lytic-lysogeny program without interference. We reprogram phages to activate lysis in response to user-defined cues. Our work shows that a phage, causing bacterial infections, and V. cholerae, causing human infections, rely on the same signal molecule for pathogenesis., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Quorum sensing is a cell-cell communication process that bacteria use to orchestrate group behaviors. Quorum sensing is mediated by signal molecules called autoinducers. Autoinducers are often structurally similar, raising questions concerning how bacteria distinguish among them. Here, we use the Pseudomonas aeruginosa LasR quorum-sensing receptor to explore signal discrimination. The cognate autoinducer, 3OC 12 homoserine lactone (3OC 12 HSL), is a more potent activator of LasR than other homoserine lactones. However, other homoserine lactones can elicit LasR-dependent quorum-sensing responses, showing that LasR displays ligand promiscuity. We identify mutants that alter which homoserine lactones LasR detects. Substitution at residue S129 decreases the LasR response to 3OC 12 HSL, while enhancing discrimination against noncognate autoinducers. Conversely, the LasR L130F mutation increases the potency of 3OC 12 HSL and other homoserine lactones. We solve crystal structures of LasR ligand-binding domains complexed with noncognate autoinducers. Comparison with existing structures reveals that ligand selectivity/sensitivity is mediated by a flexible loop near the ligand-binding site. We show that LasR variants with modified ligand preferences exhibit altered quorum-sensing responses to autoinducers in vivo. We suggest that possessing some ligand promiscuity endows LasR with the ability to optimally regulate quorum-sensing traits., Competing Interests: The authors declare no conflict of interest.