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This study highlights the enhanced motility of filamentous Vibrio cholerae in viscous environments, an adaptation that may provide a survival advantage in the human gastrointestinal tract. By demonstrating increased reversal behavior at mucin interfaces, filamentous V. cholerae cells exhibit a superior ability to penetrate the mucus layer, which is crucial for effective colonization and infection. Filamentous cells in bile-supplemented media further underscores their potential role in disease pathogenesis. These findings offer critical insights into the morphological flexibility of V. cholerae and its potential implications for infection dynamics, paving the way for more effective strategies in managing and preventing cholera outbreaks.

The mosaic-structured Vibrio cholerae genome points to the importance of horizontal gene transfer (HGT) in the evolution of this human pathogen. We showed that V. cholerae can acquire new genetic material by natural transformation during growth on chitin, a biopolymer that is abundant in aquatic habitats (e.g., from crustacean exoskeletons), where it lives as an autochthonous microbe. Transformation competence was found to require a type IV pilus assembly complex, a putative DNA binding protein, and three convergent regulatory cascades, which are activated by chitin, increasing cell density, and nutrient limitation, a decline in growth rate, or stress.

Recovery from cholera is characterized by a pattern of accumulation of bacterial taxa that shows similarities to the pattern of maturation of the gut microbiota in healthy children, raising the possibility that some of these taxa may be useful for ‘repair’ of the gut microbiota in individuals whose gut communities have been ‘wounded’ through a variety of insults. Gut microbes aid recovery from cholera Cholera and other diarrhoeal diseases caused by bacterial pathogens affect millions of people worldwide each year. Understanding how the gut microbiota affects diarrhoeal disease, in particular that associated with Vibrio cholera infection, is therefore an important goal. Jeffrey Gordon and colleagues carried out a time-series metagnomic analysis of the gut microbiota during acute and recovery phases of the disease in a cohort of Bangladeshi adults. They find that the recovery phase is characterized by a pattern of accumulation of bacterial taxa that mirrors the assembly pattern of normal microbiota of healthy children. In a mouse model they show that the abundance of one species, Ruminococcus obeum increased upon infection by V. cholerae and that R. obeum restricts colonization by V. cholerae in a quorum sensing dependent manner. These findings suggest that mining the gut microbiota of suitable populations for isolates that use autoinducers or other mechanisms to limit V. cholera colonization could provide a means of restoring the gut microbiota in cholera sufferers. Given the global burden of diarrhoeal diseases 1 , it is important to understand how members of the gut microbiota affect the risk for, course of, and recovery from disease in children and adults. The acute, voluminous diarrhoea caused by Vibrio cholerae represents a dramatic example of enteropathogen invasion and gut microbial community disruption. Here we conduct a detailed time-series metagenomic study of faecal microbiota collected during the acute diarrhoeal and recovery phases of cholera in a cohort of Bangladeshi adults living in an area with a high burden of disease 2 . We find that recovery is characterized by a pattern of accumulation of bacterial taxa that shows similarities to the pattern of assembly/maturation of the gut microbiota in healthy Bangladeshi children 3 . To define the underlying mechanisms, we introduce into gnotobiotic mice an artificial community composed of human gut bacterial species that directly correlate with recovery from cholera in adults and are indicative of normal microbiota maturation in healthy Bangladeshi children 3 . One of the species, Ruminococcus obeum , exhibits consistent increases in its relative abundance upon V. cholerae infection of the mice. Follow-up analyses, including mono- and co-colonization studies, establish that R. obeum restricts V. cholerae colonization, that R. obeum luxS (autoinducer-2 (AI-2) synthase) expression and AI-2 production increase significantly with V. cholerae invasion, and that R. obeum AI-2 causes quorum-sensing-mediated repression of several V. cholerae colonization factors. Co-colonization with V. cholerae mutants discloses that R. obeum AI-2 reduces Vibrio colonization/pathogenicity through a novel pathway that does not depend on the V. cholerae AI-2 sensor, LuxP. The approach described can be used to mine the gut microbiota of Bangladeshi or other populations for members that use autoinducers and/or other mechanisms to limit colonization with V. cholerae , or conceivably other enteropathogens.

The bacterial type VI secretion system (T6SS) is a nanomachine that delivers toxic effector proteins into target cells, killing them. In mice, we found that the T6SS attacks members of the host commensal microbiota in vivo, facilitating the pathogen's colonization of the gut. This microbial antagonistic interaction drives measurable changes in the pathogenicity of through enhanced intestinal colonization, expression of bacterial virulence genes, and activation of host innate immune genes. Because ablation of mouse commensals by this enteric pathogen correlated with more severe diarrheal symptoms, we conclude that antagonism toward the gut microbiota could improve the fitness of as a pathogen by elevating its transmission to new susceptible hosts.

Natural competence for transformation is a common mode of horizontal gene transfer and contributes to bacterial evolution. Transformation occurs through the uptake of external DNA and its integration into the genome. Here we show that the type VI secretion system (T6SS), which serves as a predatory killing device, is part of the competence regulon in the naturally transformable pathogen Vibrio cholerae. The T6SS-encoding gene cluster is under the positive control of the competence regulators TfoX and QstR and is induced by growth on chitinous surfaces. Live-cell imaging revealed that deliberate killing of nonimmune cells via competence-mediated induction of T6SS releases DNA and makes it accessible for horizontal gene transfer in V. cholerae.

Biofilms, surface-attached communities of bacteria encased in an extracellular matrix, are a major mode of bacterial life. How the material properties of the matrix contribute to biofilm growth and robustness is largely unexplored, in particular in response to environmental perturbations such as changes in osmotic pressure. Here, using Vibrio cholerae as our model organism, we show that during active cell growth, matrix production enables biofilm-dwelling bacterial cells to establish an osmotic pressure difference between the biofilm and the external environment. This pressure difference promotes biofilm expansion on nutritious surfaces by physically swelling the colony, which enhances nutrient uptake, and enables matrix-producing cells to outcompete non-matrix-producing cheaters via physical exclusion. Osmotic pressure together with crosslinking of the matrix also controls the growth of submerged biofilms and their susceptibility to invasion by planktonic cells. As the basic physicochemical principles of matrix crosslinking and osmotic swelling are universal, our findings may have implications for other biofilm-forming bacterial species. Most bacteria live in biofilms, surface-attached communities encased in an extracellular matrix. Here, Yan et al. show that matrix production in Vibrio cholerae increases the osmotic pressure within the biofilm, promoting biofilm expansion and physical exclusion of non-matrix producing cheaters.

Many bacterial species colonize surfaces and form dense 3D structures, known as biofilms, which are highly tolerant to antibiotics and constitute one of the major forms of bacterial biomass on Earth. Bacterial biofilms display remarkable changes during their development from initial attachment to maturity, yet the cellular architecture that gives rise to collective biofilm morphology during growth is largely unknown. Here, we use high-resolution optical microscopy to image all individual cells in Vibrio cholerae biofilms at different stages of development, including colonies that range in size from 2 to 4,500 cells. From these data, we extracted the precise 3D cellular arrangements, cell shapes, sizes, and global morphological features during biofilm growth on submerged glass substrates under flow. We discovered several critical transitions of the internal and external biofilm architectures that separate the major phases of V. cholerae biofilm growth. Optical imaging of biofilms with single-cell resolution provides a new window into biofilm formation that will prove invaluable to understanding the mechanics underlying biofilm development.

Key Points Biofilms of Vibrio cholerae , the causative agent of cholera, have an important role during the aquatic and intestinal phases of the bacterial life cycle, conferring greater resistance to environmental stresses and increasing infectivity. V. cholerae biofilm formation is a multistep process that begins with initial attachment via the bacterial mannose-sensitive haemagglutinin (MSHA) pili. The key components of a V. cholerae biofilm are secreted by the cell at various times during biofilm formation and include Vibrio polysaccharide (VPS), the biofilm proteins rugosity and biofilm structure modulator A (RbmA), Bap1 and RbmC, and extracellular DNA, all of which are critical for the formation of mature biofilms. Biofilm formation in V. cholerae is controlled by an integrated network of transcriptional regulators. The major transcriptional activators include VpsR, VpsT and AphA, and major transcriptional repressors include HapR and H-NS; alternative RNA polymerase sigma factors, regulatory small RNAs and signalling molecules also function as regulators of this complex process. Nucleotide-based signals play an important part in controlling biofilm formation and include cyclic di-GMP (c-di-GMP), which positively regulates biofilm formation and negatively regulates motility to influence the planktonic-to-biofilm transition. Additionally, cyclic AMP represses biofilm formation, and guanosine tetraphosphate and guanosine pentaphosphate (collectively called (p)ppGpp) enhance biofilm formation. V. cholerae biofilm formation is influenced by a number of fluctuating environmental factors, including nutritional status, shifts in salinity and osmolarity, phosphate limitation, the presence of polyamines, variations in calcium levels, and exposure to indole and bile. The ability of V. cholerae to activate or repress biofilm formation in response to external signals probably contributes to the environmental survival and persistence of the bacterium and demonstrates the complexity of the biofilm regulation programme. New small-molecule therapeutics have emerged that target and disrupt V. cholerae biofilm formation. Such therapeutics include quorum sensing inhibitors, disruptors of c-di-GMP signalling and compounds with unknown molecular targets. Whole-cell phenotypic imaging coupled with cellular-viability measurements have been used to differentiate bactericidal agents and compounds that selectively disrupt biofilm formation without affecting cell survival. These studies have led to the discovery of several compounds that show promise for biofilm inhibition and for the treatment of cholera. Bacteria form biofilms as a strategy for survival and persistence. In this Review, Yildiz and colleagues discuss Vibrio cholerae surface attachment and the biofilm matrix components. They also review the regulatory network that governs V. cholerae biofilm formation, including the transcriptional regulators of key genes involved in this process, as well as the roles of small nucleotides and small RNAs. Nearly all bacteria form biofilms as a strategy for survival and persistence. Biofilms are associated with biotic and abiotic surfaces and are composed of aggregates of cells that are encased by a self-produced or acquired extracellular matrix. Vibrio cholerae has been studied as a model organism for understanding biofilm formation in environmental pathogens, as it spends much of its life cycle outside of the human host in the aquatic environment. Given the important role of biofilm formation in the V. cholerae life cycle, the molecular mechanisms underlying this process and the signals that trigger biofilm assembly or dispersal have been areas of intense investigation over the past 20 years. In this Review, we discuss V. cholerae surface attachment, various matrix components and the regulatory networks controlling biofilm formation.

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.

Biofilm formation by Vibrio cholerae facilitates environmental persistence, and hyperinfectivity within the host. Biofilm formation is regulated by 3’,5’-cyclic diguanylate (c-di-GMP) and requires production of the type IV mannose-sensitive hemagglutinin (MSHA) pilus. Here, we show that the MSHA pilus is a dynamic extendable and retractable system, and its activity is directly controlled by c-di-GMP. The interaction between c-di-GMP and the ATPase MshE promotes pilus extension, whereas low levels of c-di-GMP correlate with enhanced retraction. Loss of retraction facilitated by the ATPase PilT increases near-surface roaming motility, and impairs initial surface attachment. However, prolonged retraction upon surface attachment results in reduced MSHA-mediated surface anchoring and increased levels of detachment. Our results indicate that c-di-GMP directly controls MshE activity, thus regulating MSHA pilus extension and retraction dynamics, and modulating V. cholerae surface attachment and colonization. Biofilm formation by Vibrio cholerae is regulated by c-di-GMP and requires the type IV MSHA pilus. Here, Floyd et al. show that the MSHA pilus is a dynamic system, and that both extension and retraction are directly controlled by c-di-GMP via regulation of activity of the extension ATPase MshE.