Preface 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.

Microbial species often exist in complex communities where they must avoid predation and compete for favorable niches. The type VI secretion system (T6SS) is a contact-dependent bacterial weapon that allows for direct killing of competitors through the translocation of proteinaceous toxins. Vibrio cholerae is a Gram-negative pathogen that can use its T6SS during antagonistic interactions with neighboring prokaryotic and eukaryotic competitors. The T6SS not only promotes V. cholerae's survival during its aquatic and host life cycles, but also influences its evolution by facilitating horizontal gene transfer. This review details the recent insights regarding the structure and function of the T6SS as well as the diverse signals and regulatory pathways that control its activation in V. cholerae.

Infectious diseases kill nearly 9 million people annually. Bacterial pathogens are responsible for a large proportion of these diseases and the bacterial agents of pneumonia, diarrhea, and tuberculosis are leading causes of death and disability worldwide (1). Increasingly, the crucial role of non-host environments in the life cycle of bacterial pathogens is being recognized. Heightened scrutiny has been given to the biological processes impacting pathogen dissemination and survival in the natural environment, as these processes are essential for the transmission of pathogenic bacteria to new hosts. This chapter focuses on the model environmental pathogen, Vibrio cholerae, to describe recent advances in our understanding of how pathogens survive between hosts and highlight the processes necessary to support the cycle of environmental survival, transmission, and dissemination. We describe the physiological and molecular responses of V. cholerae to changing environmental conditions, focusing on its survival in aquatic reservoirs between hosts and its entry and exit from human hosts.

Vibrio cholerae, the causative agent of the cholera disease, is commonly used as a model organism for the study of bacteria with multipartite genomes. Its two chromosomes of different sizes initiate their DNA replication at distinct time points in the cell cycle and terminate in synchrony. In this study, the time-delayed start of Chr2 was verified in a synchronized cell population. This replication pattern suggests two possible regulation mechanisms for other Vibrio species with different sized secondary chromosomes: Either all Chr2 start DNA replication with a fixed delay after Chr1 initiation, or the timepoint at which Chr2 initiates varies such that termination of chromosomal replication occurs in synchrony. We investigated these two models and revealed that the two chromosomes of various Vibrionaceae species terminate in synchrony while Chr2-initiation timing relative to Chr1 is variable. Moreover, the sequence and function of the Chr2-triggering crtS site recently discovered in V. cholerae were found to be conserved, explaining the observed timing mechanism. Our results suggest that it is beneficial for bacterial cells with multiple chromosomes to synchronize their replication termination, potentially to optimize chromosome related processes as dimer resolution or segregation.

Two-component signal transduction systems (TCSs), typically composed of a sensor histidine kinase (HK) and a response regulator (RR), are the primary mechanism by which pathogenic bacteria sense and respond to extracellular signals. The pathogenic bacterium Vibrio cholerae is no exception and harbors 52 RR genes. Using in-frame deletion mutants of each RR gene, we performed a systematic analysis of their role in V. cholerae biofilm formation. We determined that 7 RRs impacted the expression of an essential biofilm gene and found that the recently characterized RR, VxrB, regulates the expression of key structural and regulatory biofilm genes in V. cholerae. vxrB is part of a 5-gene operon, which contains the cognate HK vxrA and three genes of unknown function. Strains carrying ΔvxrA and ΔvxrB mutations are deficient in biofilm formation, while the ΔvxrC mutation enhances biofilm formation. The overexpression of VxrB led to a decrease in motility. We also observed a small but reproducible effect of the absence of VxrB on the levels of cyclic di-GMP (c-di-GMP). Our work reveals a new function for the Vxr TCS as a regulator of biofilm formation and suggests that this regulation may act through key biofilm regulators and the modulation of cellular c-di-GMP levels.IMPORTANCE Biofilms play an important role in the Vibrio cholerae life cycle, providing protection from environmental stresses and contributing to the transmission of V. cholerae to the human host. V. cholerae can utilize two-component systems (TCS), composed of a histidine kinase (HK) and a response regulator (RR), to regulate biofilm formation in response to external cues. We performed a systematic analysis of V. cholerae RRs and identified a new regulator of biofilm formation, VxrB. We demonstrated that the VxrAB TCS is essential for robust biofilm formation and that this system may regulate biofilm formation via its regulation of key biofilm regulators and cyclic di-GMP levels. This research furthers our understanding of the role that TCSs play in the regulation of V. cholerae biofilm formation.KEYWORDS Vibrio cholerae, VxrAB, biofilms, c-di-GMP, motility V ibrio cholerae is the causative agent of the gastrointestinal disease cholera, responsible for approximately 3 to 5 million cases of severe diarrhea and 120,000 deaths annually (1, 2). A resident of aquatic reservoirs, V. cholerae can be found as freeswimming planktonic cells or in matrix-protected cellular aggregates, known as biofilms (2-4). Evidence suggests that biofilms form during the aquatic and intestinal phases of the V. cholerae life cycle and play an important role in environmental survival, as well as in the intestinal and transmission stages of infection (5-9). V. cholerae biofilm formation requires the production and secretion of an extracellular matrix composed of matrix proteins, nucleic acids, and Vibrio polysaccharide (VPS), a glycoconjugate that is essential for the formation of three-dimensional biofilm structures (10-16). A complex regulatory network governs this process,...

Vibrio cholerae remains a major global health threat, disproportionately impacting parts of the world without adequate infrastructure and sanitation resources. In aquatic environments, V. cholerae exists both as planktonic cells and as biofilms, which are held together by an extracellular matrix. V. cholerae biofilms have been shown to be hyperinfective, but the mechanism of hyperinfectivity is unclear. Here we show that biofilm-grown cells, irrespective of the surfaces on which they are formed, are able to markedly outcompete planktonic-grown cells in the infant mouse. Using an imaging technique designed to render intestinal tissue optically transparent and preserve the spatial integrity of infected intestines, we reveal and compare three-dimensional V. cholerae colonization patterns of planktonic-grown and biofilm-grown cells. Quantitative image analyses show that V. cholerae colonizes mainly the medial portion of the small intestine and that both the abundance and localization patterns of biofilm-grown cells differ from that of planktonic-grown cells. In vitro biofilm-grown cells activate expression of the virulence cascade, including the toxin coregulated pilus (TCP), and are able to acquire the cholera toxin-carrying CTXФ phage. Overall, virulence factor gene expression is also higher in vivo when infected with biofilm-grown cells, and modulation of their regulation is sufficient to cause the biofilm hyperinfectivity phenotype. Together, these results indicate that the altered biogeography of biofilm-grown cells and their enhanced production of virulence factors in the intestine underpin the biofilm hyperinfectivity phenotype.

Biofilms are a widely observed growth mode in which microbial communities are spatially structured and embedded in a polymeric extracellular matrix. Here, we focus on the model bacterium Vibrio cholerae and summarize the current understanding of biofilm formation, including initial attachment, matrix components, community dynamics, social interactions, molecular regulation, and dispersal. The regulatory network that orchestrates the decision to form and disperse from biofilms coordinates various environmental inputs. These cues are integrated by several transcription factors, regulatory RNAs, and second-messenger molecules, including bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP). Through complex mechanisms, V. cholerae weighs the energetic cost of forming biofilms against the benefits of protection and social interaction that biofilms provide. Expected final online publication date for the Annual Review of Microbiology, Volume 76 is September 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

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