Mechanisms Underlying Vibrio cholerae Biofilm Formation and Dispersion.

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.

The Rvv two-component regulatory system regulates biofilm formation and colonization in Vibrio cholerae.

The facultative human pathogen, Vibrio cholerae, employs two-component signal transduction systems (TCS) to sense and respond to environmental signals encountered during its infection cycle. TCSs consist of a sensor histidine kinase (HK) and a response regulator (RR); the V. cholerae genome encodes 43 HKs and 49 RRs, of which 25 are predicted to be cognate pairs. Using deletion mutants of each HK gene, we analyzed the transcription of vpsL, a biofilm gene required for Vibrio polysaccharide and biofilm formation. We found that a V. cholerae TCS that had not been studied before, now termed Rvv, controls biofilm gene transcription. The Rvv TCS is part of a three-gene operon that is present in 30% of Vibrionales species. The rvv operon encodes RvvA, the HK; RvvB, the cognate RR; and RvvC, a protein of unknown function. Deletion of rvvA increased transcription of biofilm genes and altered biofilm formation, while deletion of rvvB or rvvC lead to no changes in biofilm gene transcription. The phenotypes observed in ΔrvvA depend on RvvB. Mutating RvvB to mimic constitutively active and inactive versions of the RR only impacted phenotypes in the ΔrvvA genetic background. Mutating the conserved residue required for kinase activity in RvvA did not affect phenotypes, whereas mutation of the conserved residue required for phosphatase activity mimicked the phenotype of the rvvA mutant. Furthermore, ΔrvvA displayed a significant colonization defect which was dependent on RvvB and RvvB phosphorylation state, but not on VPS production. We found that RvvA's phosphatase activity regulates biofilm gene transcription, biofilm formation, and colonization phenotypes. This is the first systematic analysis of the role of V. cholerae HKs in biofilm gene transcription and resulted in the identification of a new regulator of biofilm formation and virulence, advancing our understanding of the role TCSs play in regulating these critical cellular processes in V. cholerae.

Nitric oxide stimulates type IV MSHA pilus retraction in Vibrio cholerae via activation of the phosphodiesterase CdpA.

Bacteria use surface appendages called type IV pili to perform diverse activities including DNA uptake, twitching motility, and attachment to surfaces. The dynamic extension and retraction of pili are often required for these activities, but the stimuli that regulate these dynamics remain poorly characterized. To address this question, we study the bacterial pathogen Vibrio cholerae, which uses mannose-sensitive hemagglutinin (MSHA) pili to attach to surfaces in aquatic environments as the first step in biofilm formation. Here, we use a combination of genetic and cell biological approaches to describe a regulatory pathway that allows V. cholerae to rapidly abort biofilm formation. Specifically, we show that V. cholerae cells retract MSHA pili and detach from a surface in a diffusion-limited, enclosed environment. This response is dependent on the phosphodiesterase CdpA, which decreases intracellular levels of cyclic-di-GMP to induce MSHA pilus retraction. CdpA contains a putative nitric oxide (NO)-sensing NosP domain, and we demonstrate that NO is necessary and sufficient to stimulate CdpA-dependent detachment. Thus, we hypothesize that the endogenous production of NO (or an NO-like molecule) in V. cholerae stimulates the retraction of MSHA pili. These results extend our understanding of how environmental cues can be integrated into the complex regulatory pathways that control pilus dynamic activity and attachment in bacterial species.

A Label-Free Approach for Relative Spatial Quantitation of c-di-GMP in Microbial Biofilms.

Microbial biofilms represent an important lifestyle for bacteria and are dynamic three-dimensional structures. Cyclic dimeric guanosine monophosphate (c-di-GMP) is a ubiquitous signaling molecule that is known to be tightly regulated with biofilm processes. While measurements of global levels of c-di-GMP have proven valuable toward understanding the genetic control of c-di-GMP production, there is a need for tools to observe the local changes of c-di-GMP production in biofilm processes. We have developed a label-free method for the direct detection of c-di-GMP in microbial colony biofilms using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). We applied this method to the enteric pathogen Vibrio cholerae, the marine symbiont V. fischeri, and the opportunistic pathogen Pseudomonas aeruginosa PA14 and detected spatial and temporal changes in c-di-GMP signal that accompanied genetic alterations in factors that synthesize and degrade the compound. We further demonstrated how this method can be simultaneously applied to detect additional metabolites of interest from a single sample.

c-di-GMP inhibits LonA-dependent proteolysis of TfoY in Vibrio cholerae.

The LonA (or Lon) protease is a central post-translational regulator in diverse bacterial species. In Vibrio cholerae, LonA regulates a broad range of behaviors including cell division, biofilm formation, flagellar motility, c-di-GMP levels, the type VI secretion system (T6SS), virulence gene expression, and host colonization. Despite LonA's role in cellular processes critical for V. cholerae's aquatic and infectious life cycles, relatively few LonA substrates have been identified. LonA protease substrates were therefore identified through comparison of the proteomes of wild-type and ΔlonA strains following translational inhibition. The most significantly enriched LonA-dependent protein was TfoY, a known regulator of motility and the T6SS in V. cholerae. Experiments showed that TfoY was required for LonA-mediated repression of motility and T6SS-dependent killing. In addition, TfoY was stabilized under high c-di-GMP conditions and biochemical analysis determined direct binding of c-di-GMP to LonA results in inhibition of its protease activity. The work presented here adds to the list of LonA substrates, identifies LonA as a c-di-GMP receptor, demonstrates that c-di-GMP regulates LonA activity and TfoY protein stability, and helps elucidate the mechanisms by which LonA controls important V. cholerae behaviors.

Reciprocal c-di-GMP signaling: Incomplete flagellum biogenesis triggers c-di-GMP signaling pathways that promote biofilm formation.

The assembly status of the V. cholerae flagellum regulates biofilm formation, suggesting that the bacterium senses a lack of movement to commit to a sessile lifestyle. Motility and biofilm formation are inversely regulated by the second messenger molecule cyclic dimeric guanosine monophosphate (c-di-GMP). Therefore, we sought to define the flagellum-associated c-di-GMP-mediated signaling pathways that regulate the transition from a motile to a sessile state. Here we report that elimination of the flagellum, via loss of the FlaA flagellin, results in a flagellum-dependent biofilm regulatory (FDBR) response, which elevates cellular c-di-GMP levels, increases biofilm gene expression, and enhances biofilm formation. The strength of the FDBR response is linked with status of the flagellar stator: it can be reversed by deletion of the T ring component MotX, and reduced by mutations altering either the Na+ binding ability of the stator or the Na+ motive force. Absence of the stator also results in reduction of mannose-sensitive hemagglutinin (MSHA) pilus levels on the cell surface, suggesting interconnectivity of signal transduction pathways involved in biofilm formation. Strains lacking flagellar rotor components similarly launched an FDBR response, however this was independent of the status of assembly of the flagellar stator. We found that the FDBR response requires at least three specific diguanylate cyclases that contribute to increased c-di-GMP levels, and propose that activation of biofilm formation during this response relies on c-di-GMP-dependent activation of positive regulators of biofilm production. Together our results dissect how flagellum assembly activates c-di-GMP signaling circuits, and how V. cholerae utilizes these signals to transition from a motile to a sessile state.

A tyrosine phosphoregulatory system controls exopolysaccharide biosynthesis and biofilm formation in Vibrio cholerae.

Production of an extracellular matrix is essential for biofilm formation, as this matrix both secures and protects the cells it encases. Mechanisms underlying production and assembly of matrices are poorly understood. Vibrio cholerae, relies heavily on biofilm formation for survival, infectivity, and transmission. Biofilm formation requires Vibrio polysaccharide (VPS), which is produced by vps gene-products, yet the function of these products remains unknown. Here, we demonstrate that the vps gene-products vpsO and vpsU encode respectively for a tyrosine kinase and a cognate tyrosine phosphatase. Collectively, VpsO and VpsU act as a tyrosine phosphoregulatory system to modulate VPS production. We present structures of VpsU and the kinase domain of VpsO, and we report observed autocatalytic tyrosine phosphorylation of the VpsO C-terminal tail. The position and amount of tyrosine phosphorylation in the VpsO C-terminal tail represses VPS production and biofilm formation through a mechanism involving the modulation of VpsO oligomerization. We found that tyrosine phosphorylation enhances stability of VpsO. Regulation of VpsO phosphorylation by the phosphatase VpsU is vital for maintaining native VPS levels. This study provides new insights into the mechanism and regulation of VPS production and establishes general principles of biofilm matrix production and its inhibition.

Sensor Domain of Histidine Kinase VxrA of Vibrio cholerae- A Hairpin-swapped Dimer and its Conformational Change.

VxrA and VxrB are cognate histidine kinase (HK) - response regulator (RR) pairs of a two-component signaling system (TCS) found in Vibrio cholerae, a bacterial pathogen that causes cholera. The VxrAB TCS positively regulates virulence, the Type VI Secretion System, biofilm formation, and cell wall homeostasis in V. cholerae, providing protection from environmental stresses and contributing to the transmission and virulence of the pathogen. The VxrA HK has a unique periplasmic sensor domain (SD) and, remarkably, lacks a cytoplasmic linker domain between the second transmembrane helix and the dimerization and histidine phosphotransfer (DHp) domain, indicating that this system may utilize a potentially unique signal sensing and transmission TCS mechanism. In this study, we have determined several crystal structures of VxrA-SD and its mutants. These structures reveal a novel structural fold forming an unusual ß hairpin-swapped dimer. A conformational change caused by relative rotation of the two monomers in a VxrA-SD dimer could potentially change the association of transmembrane helices and, subsequently, the pairing of cytoplasmic DHp domains. Based on the structural observation, we propose a putative scissor-like closing regulation mechanism for the VxrA HK.IMPORTANCE V. cholerae has a dynamic life cycle, which requires rapid adaptation to changing external conditions. Two-component signal transduction (TCS) systems allow V. cholerae to sense and respond to these environmental changes. The VxrAB TCS positively regulates a number of important V. cholerae phenotypes, including virulence, the Type Six Secretion System, biofilm formation, and cell wall homeostasis. Here, we provide the crystal structure of the VxrA sensor histidine kinase sensing domain and propose a mechanism for signal transduction. The cognate signal for VxrAB remains unknown, however, in this work we couple our structural analysis with functional assessments of key residues to further our understanding of this important TCS.

Roadmap on emerging concepts in the physical biology of bacterial biofilms: from surface sensing to community formation.

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.

Synchronous termination of replication of the two chromosomes is an evolutionary selected feature in Vibrionaceae.

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.

Strategies and Approaches for Discovery of Small Molecule Disruptors of Biofilm Physiology.

Biofilms, the predominant growth mode of microorganisms, pose a significant risk to human health. The protective biofilm matrix, typically composed of exopolysaccharides, proteins, nucleic acids, and lipids, combined with biofilm-grown bacteria's heterogenous physiology, leads to enhanced fitness and tolerance to traditional methods for treatment. There is a need to identify biofilm inhibitors using diverse approaches and targeting different stages of biofilm formation. This review discusses discovery strategies that successfully identified a wide range of inhibitors and the processes used to characterize their inhibition mechanism and further improvement. Additionally, we examine the structure-activity relationship (SAR) for some of these inhibitors to optimize inhibitor activity.

NtrC Adds a New Node to the Complex Regulatory Network of Biofilm Formation and vps Expression in Vibrio cholerae.

The biofilm growth mode is important in both the intestinal and environmental phases of the Vibrio cholerae life cycle. Regulation of biofilm formation involves several transcriptional regulators and alternative sigma factors. One such factor is the alternative sigma factor RpoN, which positively regulates biofilm formation. RpoN requires bacterial enhancer-binding proteins (bEBPs) to initiate transcription. The V. cholerae genome encodes seven bEBPs (LuxO, VC1522, VC1926 [DctD-1], FlrC, NtrC, VCA0142 [DctD-2], and PgtA) that belong to the NtrC family of response regulators (RRs) of two-component regulatory systems. The contribution of these regulators to biofilm formation is not well understood. In this study, we analyzed biofilm formation and the regulation of vpsL expression by RpoN activators. Mutants lacking NtrC had increased biofilm formation and vpsL expression. NtrC negatively regulates the expression of core regulators of biofilm formation (vpsR, vpsT, and hapR). NtrC from V. cholerae supported growth and activated glnA expression when nitrogen availability was limited. However, the repressive activity of NtrC toward vpsL expression was not affected by the nitrogen sources present. This study unveils the role of NtrC as a regulator of vps expression and biofilm formation in V. choleraeIMPORTANCE Biofilms play an important role in the Vibrio cholerae life cycle, contributing to both environmental survival and transmission to a human host. Identifying key regulators of V. cholerae biofilm formation is necessary to fully understand how this important growth mode is modulated in response to various signals encountered in the environment and the host. In this study, we characterized the role of RRs that function as coactivators of RpoN in regulating biofilm formation and identified new components in the V. cholerae biofilm regulatory circuitry.

The Two-Component Signal Transduction System VxrAB Positively Regulates Vibrio cholerae Biofilm Formation.

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. choleraevxrB 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.

Vibrio cholerae Response Regulator VxrB Controls Colonization and Regulates the Type VI Secretion System.

Two-component signal transduction systems (TCS) are used by bacteria to sense and respond to their environment. TCS are typically composed of a sensor histidine kinase (HK) and a response regulator (RR). The Vibrio cholerae genome encodes 52 RR, but the role of these RRs in V. cholerae pathogenesis is largely unknown. To identify RRs that control V. cholerae colonization, in-frame deletions of each RR were generated and the resulting mutants analyzed using an infant mouse intestine colonization assay. We found that 12 of the 52 RR were involved in intestinal colonization. Mutants lacking one previously uncharacterized RR, VCA0566 (renamed VxrB), displayed a significant colonization defect. Further experiments showed that VxrB phosphorylation state on the predicted conserved aspartate contributes to intestine colonization. The VxrB regulon was determined using whole genome expression analysis. It consists of several genes, including those genes that create the type VI secretion system (T6SS). We determined that VxrB is required for T6SS expression using several in vitro assays and bacterial killing assays, and furthermore that the T6SS is required for intestinal colonization. vxrB is encoded in a four gene operon and the other vxr operon members also modulate intestinal colonization. Lastly, though ΔvxrB exhibited a defect in single-strain intestinal colonization, the ΔvxrB strain did not show any in vitro growth defect. Overall, our work revealed that a small set of RRs is required for intestinal colonization and one of these regulators, VxrB affects colonization at least in part through its regulation of T6SS genes.

C-di-GMP Regulates Motile to Sessile Transition by Modulating MshA Pili Biogenesis and Near-Surface Motility Behavior in Vibrio cholerae.

In many bacteria, including Vibrio cholerae, cyclic dimeric guanosine monophosphate (c-di-GMP) controls the motile to biofilm life style switch. Yet, little is known about how this occurs. In this study, we report that changes in c-di-GMP concentration impact the biosynthesis of the MshA pili, resulting in altered motility and biofilm phenotypes in V. cholerae. Previously, we reported that cdgJ encodes a c-di-GMP phosphodiesterase and a ΔcdgJ mutant has reduced motility and enhanced biofilm formation. Here we show that loss of the genes required for the mannose-sensitive hemagglutinin (MshA) pilus biogenesis restores motility in the ΔcdgJ mutant. Mutations of the predicted ATPase proteins mshE or pilT, responsible for polymerizing and depolymerizing MshA pili, impair near surface motility behavior and initial surface attachment dynamics. A ΔcdgJ mutant has enhanced surface attachment, while the ΔcdgJmshA mutant phenocopies the high motility and low attachment phenotypes observed in a ΔmshA strain. Elevated concentrations of c-di-GMP enhance surface MshA pilus production. MshE, but not PilT binds c-di-GMP directly, establishing a mechanism for c-di-GMP signaling input in MshA pilus production. Collectively, our results suggest that the dynamic nature of the MshA pilus established by the assembly and disassembly of pilin subunits is essential for transition from the motile to sessile lifestyle and that c-di-GMP affects MshA pilus assembly and function through direct interactions with the MshE ATPase.

Systematic Identification of Cyclic-di-GMP Binding Proteins in Vibrio cholerae Reveals a Novel Class of Cyclic-di-GMP-Binding ATPases Associated with Type II Secretion Systems.

Cyclic-di-GMP (c-di-GMP) is a ubiquitous bacterial signaling molecule that regulates a variety of complex processes through a diverse set of c-di-GMP receptor proteins. We have utilized a systematic approach to identify c-di-GMP receptors from the pathogen Vibrio cholerae using the Differential Radial Capillary Action of Ligand Assay (DRaCALA). The DRaCALA screen identified a majority of known c-di-GMP binding proteins in V. cholerae and revealed a novel c-di-GMP binding protein, MshE (VC0405), an ATPase associated with the mannose sensitive hemagglutinin (MSHA) type IV pilus. The known c-di-GMP binding proteins identified by DRaCALA include diguanylate cyclases, phosphodiesterases, PilZ domain proteins and transcription factors VpsT and VpsR, indicating that the DRaCALA-based screen of open reading frame libraries is a feasible approach to uncover novel receptors of small molecule ligands. Since MshE lacks the canonical c-di-GMP-binding motifs, a truncation analysis was utilized to locate the c-di-GMP binding activity to the N-terminal T2SSE_N domain. Alignment of MshE homologs revealed candidate conserved residues responsible for c-di-GMP binding. Site-directed mutagenesis of these candidate residues revealed that the Arg9 residue is required for c-di-GMP binding. The ability of c-di-GMP binding to MshE to regulate MSHA dependent processes was evaluated. The R9A allele, in contrast to the wild type MshE, was unable to complement the ΔmshE mutant for the production of extracellular MshA to the cell surface, reduction in flagella swimming motility, attachment to surfaces and formation of biofilms. Testing homologs of MshE for binding to c-di-GMP identified the type II secretion ATPase of Pseudomonas aeruginosa (PA14_29490) as a c-di-GMP receptor, indicating that type II secretion and type IV pili are both regulated by c-di-GMP.

The LonA Protease Regulates Biofilm Formation, Motility, Virulence, and the Type VI Secretion System in Vibrio cholerae.

UNLABELLED: The presence of the Lon protease in all three domains of life hints at its biological importance. The prokaryotic Lon protease is responsible not only for degrading abnormal proteins but also for carrying out the proteolytic regulation of specific protein targets. Posttranslational regulation by Lon is known to affect a variety of physiological traits in many bacteria, including biofilm formation, motility, and virulence. Here, we identify the regulatory roles of LonA in the human pathogen Vibrio cholerae. We determined that the absence of LonA adversely affects biofilm formation, increases swimming motility, and influences intracellular levels of cyclic diguanylate. Whole-genome expression analysis revealed that the message abundance of genes involved in biofilm formation was decreased but that the message abundances of those involved in virulence and the type VI secretion system were increased in a lonA mutant compared to the wild type. We further demonstrated that a lonA mutant displays an increase in type VI secretion system activity and is markedly defective in colonization of the infant mouse. These findings suggest that LonA plays a critical role in the environmental survival and virulence of V. cholerae. IMPORTANCE: Bacteria utilize intracellular proteases to degrade damaged proteins and adapt to changing environments. The Lon protease has been shown to be important for environmental adaptation and plays a crucial role in regulating the motility, biofilm formation, and virulence of numerous plant and animal pathogens. We find that LonA of the human pathogen V. cholerae is in line with this trend, as the deletion of LonA leads to hypermotility and defects in both biofilm formation and colonization of the infant mouse. In addition, we show that LonA regulates levels of cyclic diguanylate and the type VI secretion system. Our observations add to the known regulatory repertoire of the Lon protease and the current understanding of V. cholerae physiology.

Phenotypic Analysis Reveals that the 2010 Haiti Cholera Epidemic Is Linked to a Hypervirulent Strain.

Vibrio cholerae O1 El Tor strains have been responsible for pandemic cholera since 1961. These strains have evolved over time, spreading globally in three separate waves. Wave 3 is caused by altered El Tor (AET) variant strains, which include the strain with the signature ctxB7 allele that was introduced in 2010 into Haiti, where it caused a devastating epidemic. In this study, we used phenotypic analysis to compare an early isolate from the Haiti epidemic to wave 1 El Tor isolates commonly used for research. It is demonstrated that the Haiti isolate has increased production of cholera toxin (CT) and hemolysin, increased motility, and a reduced ability to form biofilms. This strain also outcompetes common wave 1 El Tor isolates for colonization of infant mice, indicating that it has increased virulence. Monitoring of CT production and motility in additional wave 3 isolates revealed that this phenotypic variation likely evolved over time rather than in a single genetic event. Analysis of available whole-genome sequences and phylogenetic analyses suggested that increased virulence arose from positive selection for mutations found in known and putative regulatory genes, including hns and vieA, diguanylate cyclase genes, and genes belonging to the lysR and gntR regulatory families. Overall, the studies presented here revealed that V. cholerae virulence potential can evolve and that the currently prevalent wave 3 AET strains are both phenotypically distinct from and more virulent than many El Tor isolates.

CdiA promotes receptor-independent intercellular adhesion.

CdiB/CdiA proteins mediate inter-bacterial competition in a process termed contact-dependent growth inhibition (CDI). Filamentous CdiA exoproteins extend from CDI(+) cells and bind specific receptors to deliver toxins into susceptible target bacteria. CDI has also been implicated in auto-aggregation and biofilm formation in several species, but the contribution of CdiA-receptor interactions to these multi-cellular behaviors has not been examined. Using Escherichia coli isolate EC93 as a model, we show that cdiA and bamA receptor mutants are defective in biofilm formation, suggesting a prominent role for CdiA-BamA mediated cell-cell adhesion. However, CdiA also promotes auto-aggregation in a BamA-independent manner, indicating that the exoprotein possesses an additional adhesin activity. Cells must express CdiA in order to participate in BamA-independent aggregates, suggesting that adhesion could be mediated by homotypic CdiA-CdiA interactions. The BamA-dependent and BamA-independent interaction domains map to distinct regions within the CdiA filament. Thus, CdiA orchestrates a collective behavior that is independent of its growth-inhibition activity. This adhesion should enable 'greenbeard' discrimination, in which genetically unrelated individuals cooperate with one another based on a single shared trait. This kind-selective social behavior could provide immediate fitness benefits to bacteria that acquire the systems through horizontal gene transfer.

Response of Vibrio cholerae to Low-Temperature Shifts: CspV Regulation of Type VI Secretion, Biofilm Formation, and Association with Zooplankton.

UNLABELLED: The ability to sense and adapt to temperature fluctuation is critical to the aquatic survival, transmission, and infectivity of Vibrio cholerae, the causative agent of the disease cholera. Little information is available on the physiological changes that occur when V. cholerae experiences temperature shifts. The genome-wide transcriptional profile of V. cholerae upon a shift in human body temperature (37°C) to lower temperatures, 15°C and 25°C, which mimic those found in the aquatic environment, was determined. Differentially expressed genes included those involved in the cold shock response, biofilm formation, type VI secretion, and virulence. Analysis of a mutant lacking the cold shock gene cspV, which was upregulated >50-fold upon a low-temperature shift, revealed that it regulates genes involved in biofilm formation and type VI secretion. CspV controls biofilm formation through modulation of the second messenger cyclic diguanylate and regulates type VI-mediated interspecies killing in a temperature-dependent manner. Furthermore, a strain lacking cspV had significant defects for attachment and type VI-mediated killing on the surface of the aquatic crustacean Daphnia magna Collectively, these studies reveal that cspV is a major regulator of the temperature downshift response and plays an important role in controlling cellular processes crucial to the infectious cycle of V. cholerae IMPORTANCE: Little is known about how human pathogens respond and adapt to ever-changing parameters of natural habitats outside the human host and how environmental adaptation alters dissemination. Vibrio cholerae, the causative agent of the severe diarrheal disease cholera, experiences fluctuations in temperature in its natural aquatic habitats and during the infection process. Furthermore, temperature is a critical environmental signal governing the occurrence of V. cholerae and cholera outbreaks. In this study, we showed that V. cholerae reprograms its transcriptome in response to fluctuations in temperature, which results in changes to biofilm formation and type VI secretion system activation. These processes in turn impact environmental survival and the virulence potential of this pathogen.