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In growing active matter systems, a large collection of engineered or living autonomous units metabolize free energy and create order at different length scales as they proliferate and migrate collectively. One such example is bacterial biofilms, surface-attached aggregates of bacterial cells embedded in an extracellular matrix that can exhibit community-scale orientational order. However, how bacterial growth coordinates with cell-surface interactions to create distinctive, long-range order during biofilm development remains elusive. Here we report a collective cell reorientation cascade in growing Vibrio cholerae biofilms that leads to a differentially ordered, spatiotemporally coupled core-rim structure reminiscent of a blooming aster. Cell verticalization in the core leads to a pattern of differential growth that drives radial alignment of the cells in the rim, while the growing rim generates compressive stresses that expand the verticalized core. Such self-patterning disappears in nonadherent mutants but can be restored through opto-manipulation of growth. Agent-based simulations and two-phase active nematic modeling jointly reveal the strong interdependence of the driving forces underlying the differential ordering. Our findings offer insight into the developmental processes that shape bacterial communities and provide ways to engineer phenotypes and functions in living active matter., (© 2021. The Author(s).)

Bacterial species have diverse cell shapes that enable motility, colonization and virulence. The cell wall defines bacterial shape and is primarily built by two cytoskeleton-guided synthesis machines, the elongasome and the divisome. However, the mechanisms producing complex shapes, like the curved-rod shape of Vibrio cholerae, are incompletely defined. Previous studies have reported that species-specific regulation of cytoskeleton-guided machines enables formation of complex bacterial shapes such as cell curvature and cellular appendages. In contrast, we report that CrvA and CrvB are sufficient to induce complex cell shape autonomously of the cytoskeleton in V. cholerae. The autonomy of the CrvAB module also enables it to induce curvature in the Gram-negative species Escherichia coli, Pseudomonas aeruginosa, Caulobacter crescentus and Agrobacterium tumefaciens. Using inducible gene expression, quantitative microscopy and biochemistry, we show that CrvA and CrvB circumvent the need for patterning via cytoskeletal elements by regulating each other to form an asymmetrically localized, periplasmic structure that binds directly to the cell wall. The assembly and disassembly of this periplasmic structure enables dynamic changes in cell shape. Bioinformatics indicate that CrvA and CrvB may have diverged from a single ancestral hybrid protein. Using fusion experiments in V. cholerae, we find that a synthetic CrvA/B hybrid protein is sufficient to induce curvature on its own, but that expression of two distinct proteins, CrvA and CrvB, promotes more rapid curvature induction. We conclude that morphological complexity can arise independently of cell-shape specification by the core cytoskeleton-guided synthesis machines.

Vibrio cholerae, the cause of cholera disease, exhibits a characteristic curved rod morphology, which promotes infectivity and motility in dense hydrogels. Periplasmic protein CrvA determines cell curvature in V. cholerae, yet the regulatory factors controlling CrvA are unknown. Here, we discover the VadR small RNA (sRNA) as a post-transcriptional inhibitor of the crvA mRNA. Mutation of vadR increases cell curvature, whereas overexpression has the inverse effect. We show that vadR transcription is activated by the VxrAB two-component system and triggered by cell-wall-targeting antibiotics. V. cholerae cells failing to repress crvA by VadR display decreased survival upon challenge with penicillin G indicating that cell shape maintenance by the sRNA is critical for antibiotic resistance. VadR also blocks the expression of various key biofilm genes and thereby inhibits biofilm formation in V. cholerae. Thus, VadR is an important regulator for synchronizing peptidoglycan integrity, cell shape, and biofilm formation in V. cholerae.

Motility is required for many bacterial pathogens to reach and colonize target sites. Vibrio cholerae traverses a thick mucus barrier coating the small intestine to reach the underlying epithelium. We screened a transposon library in motility medium containing mucin to identify factors that influence mucus transit. Lesions in structural genes of the type VI secretion system (T6SS) were among those recovered. Two-dimensional (2D) and 3D single-cell tracking was used to compare the motility behaviors of wild-type cells and a mutant that collectively lacked three essential T6SS structural genes (T6SS - ). In the absence of mucin, wild-type and T6SS - cells exhibited similar speeds and run-reverse-flick (RRF) swimming patterns, in which forward-moving cells briefly backtrack before stochastically reorienting (flicking) in a new direction upon resuming forward movement. We show that mucin induced T6SS expression and activity in wild-type bacteria but significantly decreased their swimming speed and flicking, yielding curvilinear or near-surface circular traces for many cells. Conversely, mucin slowed T6SS - cells to a lesser extent, and many continued to flick and produce RRF-like traces. Δ cheY3 cells, which exclusively swim in the forward direction and thus cannot flick, also produced curvilinear traces with or without mucin present and, on occasion, near-surface circular traces in the presence of mucin. The dependence of flicking on swimming speed suggested that mucin-induced T6SS activity further decreased V. cholerae motility and thereby reduced flicking probability during reverse-to-forward transitions. We propose that this encourages cells to continue on their current trajectory rather than reorienting, which may benefit those tracking toward the epithelial surface. IMPORTANCE V. cholerae deploys an arsenal of virulence factors as it attempts to traverse a protective mucus layer and reach the epithelial surface of the distal small intestine. The T6SS used to cull bacterial competition during infection is induced by mucus. We show that this activity may serve an additional purpose by further decreasing motility in the presence of mucin, thereby reducing the probability of speed-dependent, near-perpendicular directional changes. We posit that this encourages cells to maintain course rather than change direction, which may aid those attempting to reach and colonize the epithelial surface., (Copyright © 2020 American Society for Microbiology.)

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.)

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.

The phenotypic heterogeneity in a large population arises because of fluctuation in microenvironments and stochastic gene expressions. In this report, we isolated two types of persistent sub-populations of Vibrio cholerae, one triggered by starvation and another by antibiotics. We characterised starvation-induced (E-cells) and antibiotic-induced (P-cell) persister cells for stress tolerance, colony morphology and toxin gene expressions. Both the sub-populations differ with respect to morphology, temperature tolerance and oxidative stress tolerance. The E-cells were smaller than the P-cells and formed tiny colonies (1-2 mm). The E-cells were more sensitive to heat and oxidative stress compared with P-cells. The up-regulated genes of P-cells include, genes of antioxidant enzymes (>5 fold), cholera toxin (>26 fold) and toxin: antitoxin protein hipA (>100 fold). Upon nutrient up-shift, the E-cells recovered after lag time of 6 h. However, such lag extension was not visible during P-cell recovery, suggesting that P-cell physiology is more akin to normal cells than E-cells. This is the first comparative report on the two different persister sub-populations of V. cholerae. The E-cells and P-cells are similar regarding antibiotic tolerance. However, the sub-populations differ significantly in stress tolerance and other phenotypes studied.

BolA family proteins are conserved in Gram-negative bacteria and many eukaryotes. While diverse cellular phenotypes have been linked to this protein family, the molecular pathways through which these proteins mediate their effects are not well described. Here, we investigated the roles of BolA family proteins in Vibrio cholerae , the cholera pathogen. Like Escherichia coli , V. cholerae encodes two BolA proteins, BolA and IbaG. However, in marked contrast to E. coli , where bolA is linked to cell shape and ibaG is not, in V. cholerae , bolA mutants lack morphological defects, whereas ibaG proved critical for the generation and/or maintenance of the pathogen's morphology. Notably, the bizarre-shaped, multipolar, elongated, and wide cells that predominated in exponential-phase Δ ibaG V. cholerae cultures were not observed in stationary-phase cultures. The V. cholerae Δ ibaG mutant exhibited increased sensitivity to cell envelope stressors, including cell wall-acting antibiotics and bile, and was defective in intestinal colonization. Δ ibaG V. cholerae had reduced peptidoglycan and lipid II and altered outer membrane lipids, likely contributing to the mutant's morphological defects and sensitivity to envelope stressors. Transposon insertion sequencing analysis of ibaG 's genetic interactions suggested that ibaG is involved in several processes involved in the generation and homeostasis of the cell envelope. Furthermore, copurification studies revealed that IbaG interacts with proteins containing iron-sulfur clusters or involved in their assembly. Collectively, our findings suggest that V. cholerae IbaG controls cell morphology and cell envelope integrity through its role in biogenesis or trafficking of iron-sulfur cluster proteins. IMPORTANCE BolA-like proteins are conserved across prokaryotes and eukaryotes. These proteins have been linked to a variety of phenotypes, but the pathways and mechanisms through which they act have not been extensively characterized. Here, we unraveled the role of the BolA-like protein IbaG in the cholera pathogen Vibrio cholerae The absence of IbaG was associated with dramatic changes in cell morphology, sensitivity to envelope stressors, and intestinal colonization defects. IbaG was found to be required for biogenesis of several components of the V. cholerae cell envelope and to interact with numerous iron-sulfur cluster-containing proteins and factors involved in their assembly. Thus, our findings suggest that IbaG governs V. cholerae cell shape and cell envelope homeostasis through its effects on iron-sulfur proteins and associated pathways. The diversity of processes involving iron-sulfur-containing proteins is likely a factor underlying the range of phenotypes associated with BolA family proteins., (Copyright © 2019 Fleurie et al.)

Vibrio cholerae is an aquatic bacterium with the potential to infect humans and cause the cholera disease. While most bacteria have single chromosomes, the V. cholerae genome is encoded on two replicons of different size. This study focuses on the DNA replication and cell division of this bi-chromosomal bacterium during the stringent response induced by starvation stress. V. cholerae cells were found to initially shut DNA replication initiation down upon stringent response induction by the serine analog serine hydroxamate. Surprisingly, cells temporarily restart their DNA replication before finally reaching a state with fully replicated single chromosome sets. This division-replication pattern is very different to that of the related single chromosome model bacterium Escherichia coli. Within the replication restart phase, both chromosomes of V. cholerae maintained their known order of replication timing to achieve termination synchrony. Using flow cytometry combined with mathematical modeling, we established that a phase of cellular regrowth be the reason for the observed restart of DNA replication after the initial shutdown. Our study shows that although the stringent response induction itself is widely conserved, bacteria developed different ways of how to react to the sensed nutrient limitation, potentially reflecting their individual lifestyle requirements., (© 2019 John Wiley & Sons Ltd.)

Acute Vibrio cholerae infection triggers significant inflammatory response and immense fluid secretion in the intestine. In the present study, methyl gallate (MG) isolated from Terminalia chebula was evaluated to determine the in vivo fluid accumulation-inhibitory, anticolonization and anti-inflammatory and in vitro biofilm-inhibitory activities against multi-drug resistant (MDR) V. cholerae. Bacterial membrane-damaging and biofilm-inhibitory activities were determined by membrane perturbation and transmission electron microscopy (TEM); and microdilution assays, respectively. Fluid accumulation-inhibitory and anticolonization activities of MG (23.80-95.23 mg/kg body weight) were determined in 4-5 days old BALB/c mice with an incubation time of 18 h. The effect of MG (1, 50 and 500 mg/kg body weight) on intestinal inflammatory reaction induced by V. cholerae was studied by performing histology in Swiss albino mice. MIC and MBC of MG against the test strains were 32-64 and 64-256 μg/ml, respectively. MG showed the fluid accumulation-inhibitory activity with inhibition values of 42.86-89.08% at doses between 23.80 and 95.23 mg/kg body weight and significant anticolonization activity (p < 0.0001) against V. choleare in the suckling mouse intestine. MG (500 mg/kg body weight) significantly inhibited the inflammatory reactions induced by V. cholerae compared to the vehicle control. MG exhibited 70% minimum biofilm inhibition concentration of 64 μg/ml and bacterial membrane damaging activity at 1 × MBC. The results obtained in the present study suggest that MG has potential as an effective agent for the treatment of severe secretory and inflammatory diarrheal disease caused by MDR V. cholerae., (Copyright © 2018. Published by Elsevier Ltd.)

Gram-negative bacteria are surrounded by an inner cytoplasmic membrane and by an outer membrane, which serves as a protective barrier to limit entry of many antibiotics. The distinctive properties of the outer membrane are due to the presence of lipopolysaccharide 1 . This large glycolipid, which contains numerous sugars, is made in the cytoplasm; a complex of proteins forms a membrane-to-membrane bridge that mediates transport of lipopolysaccharide from the inner membrane to the cell surface 1 . The inner-membrane components of the protein bridge comprise an ATP-binding cassette transporter that powers transport, but how this transporter ensures unidirectional lipopolysaccharide movement across the bridge to the outer membrane is unknown 2 . Here we describe two crystal structures of a five-component inner-membrane complex that contains all the proteins required to extract lipopolysaccharide from the membrane and pass it to the protein bridge. Analysis of these structures, combined with biochemical and genetic experiments, identifies the path of lipopolysaccharide entry into the cavity of the transporter and up to the bridge. We also identify a protein gate that must open to allow movement of substrate from the cavity onto the bridge. Lipopolysaccharide entry into the cavity is ATP-independent, but ATP is required for lipopolysaccharide movement past the gate and onto the bridge. Our findings explain how the inner-membrane transport complex controls efficient unidirectional transport of lipopolysaccharide against its concentration gradient.

Quorum sensing (QS) and nucleotide-based second messengers are vital signaling systems that regulate bacterial physiology in response to changing environments. Disrupting bacterial signal transduction is a promising direction to combat infectious diseases, and QS and the second messengers are undoubtedly potential targets. In Vibrio cholerae, both QS and the second messenger 3', 5'-cyclic diguanylate (c-di-GMP) play a central role in controlling motility, motile-to-sessile life transition, and virulence. In this study, we found that water-soluble extract from the North American cranberry could significantly inhibit V. cholerae biofilm formation during the development/maturation stage by reducing the biofilm matrix production and secretion. The anti-biofilm effect by water-soluble cranberry extract was possibly through modulating the intracellular c-di-GMP level and was independent of QS and the QS master regulator HapR. Our results suggest an opportunity to explore more functional foods to fight stubborn infections through interference with the bacterial signaling systems., Competing Interests: The authors have declared that no competing interests exist.

Vibrio cholerae O1, the etiological agent of cholera, is a natural inhabitant of aquatic ecosystems. Motility is a critical element for the colonization of both the human host and its environmental reservoirs. In this study, we investigated the molecular mechanisms underlying the chemotactic response of V. cholerae in the presence of some of its environmental reservoirs. We found that, from the several oligosaccharides found in mucin, two specifically triggered motility of V. cholerae O1: N-acetylneuraminic acid (Neu5Ac) and N-acetylglucosamine (GlcNAc). We determined that the compounds need to be internally catabolized in order to trigger motility of V. cholerae. Interestingly, the catabolism of Neu5Ac and GlcNAc converges and the production of one molecule common to both pathways, glucosamine-6-phosphate (GlcN-6P), is essential to induce motility in the presence of both compounds. Mutants unable to produce GlcN-6P show greatly reduced motility towards mucin. Furthermore, we determined that the production of GlcN-6P is necessary to induce motility of V. cholerae in the presence of some of its environmental reservoirs such as crustaceans or cyanobacteria, revealing a molecular link between the two distinct modes of the complex life cycle of V. cholerae. Finally, cross-species comparisons revealed varied chemotactic responses towards mucin, GlcNAc, and Neu5Ac for environmental (non-pathogenic) strains of V. cholerae, clinical and environmental isolates of the human pathogens Vibrio vulnificus and Vibrio parahaemolyticus, and fish and squid isolates of the symbiotic bacterium Vibrio fischeri. The data presented here suggest nuance in convergent strategies across species of the same bacterial family for motility towards suitable substrates for colonization., Competing Interests: The authors have declared that no competing interests exist.

Sensitive and specific detection of pathogens via nucleic acid amplification is currently constrained to laboratory settings and portable equipment with costly fluorescent detectors. Nucleic acid-detecting lateral flow immunoassay strips (LFIAs) offer a low-cost visual transduction strategy at points of need. Unfortunately, these LFIAs frequently detect amplification byproducts that can yield spurious results which can only be deciphered through statistical analysis. We integrated customizable strand displacement probes into standard loop mediated isothermal amplification (LAMP) assays to prevent byproduct capture on commercial LFIAs. We find that combining strand displacement with LAMP (SD-LAMP) yields LFIA test band intensities that can be unequivocally interpreted by human subjects without additional instrumentation, thereby alleviating the need for a portable reader's analysis. Using SD-LAMP, we capture target amplicons on commercially available LFIAs from as few as 3.5 Vibrio cholerae and 2 750 Escherichia coli bacteria without false positive or false negative interpretation. Moreover, we demonstrate that LFIA capture of SD-LAMP products remain specific even in the presence of complex sample matrixes, providing a significant step toward reliable instrument-free pathogen detection outside of laboratories.

Interactions between bacteria and colon cancer cells influence the transcription of the host cell. Yet is it undetermined whether the bacteria itself or the communication between the host and bacteria is responsible for the genomic changes in the eukaryotic cell. Now, we have investigated the genomic and epigenetic consequences of co-culturing colorectal carcinoma cells with membrane vesicles from pathogenic bacteria Vibrio cholerae and non-pathogenic commensal bacteria Escherichia coli. Our study reveals that membrane vesicles from pathogenic and commensal bacteria have a global impact on the gene expression of colon-carcinoma cells. The changes in gene expression correlate positively with both epigenetic changes and chromatin accessibility of promoters at transcription start sites of genes induced by both types of membrane vesicles. Moreover, we have demonstrated that membrane vesicles obtained only from V. cholerae induced the expression of genes associated with epithelial cell differentiation. Altogether, our study suggests that the observed genomic changes in host cells might be due to specific components of membrane vesicles and do not require communication by direct contact with the bacteria.

The bacterial Type VI secretion system (T6SS) assembles from three major parts: a membrane complex that spans inner and outer membranes, a baseplate, and a sheath-tube polymer. The baseplate assembles around a tip complex with associated effectors and connects to the membrane complex by TssK. The baseplate assembly initiates sheath-tube polymerization, which in some organisms requires TssA. Here, we analyzed both ends of isolated non-contractile Vibrio cholerae sheaths by cryo-electron microscopy. Our analysis suggests that the baseplate, solved to an average 8.0 Å resolution, is composed of six subunits of TssE/F 2 /G and the baseplate periphery is decorated by six TssK trimers. The VgrG/PAAR tip complex in the center of the baseplate is surrounded by a cavity, which may accommodate up to ~450 kDa of effector proteins. The distal end of the sheath, resolved to an average 7.5 Å resolution, shows sixfold symmetry; however, its protein composition is unclear. Our structures provide an important step toward an atomic model of the complete T6SS assembly., (© 2017 The Authors. Published under the terms of the CC BY 4.0 license.)

Bacteria display a highly flexible cell cycle in which cell division can be temporally disconnected from the replication/segregation cycle of their genome. The accuracy of genetic transmission is enforced by restricting the assembly of the cell division apparatus to the low DNA-density zones that develop between the regularly spaced nucleoids originating from the concurrent replication and segregation of genomic DNA. In most bacteria, the process is simplified because the genome is encoded on a single chromosome. This is notably the case in Escherichia coli, the most well studied bacterial model organism. However, ~10% of bacteria have domesticated horizontally acquired mega-plasmids into extra-numerous chromosomes. Most of our current knowledge on the cell cycle regulation of multi-chromosomal species derives from the study of replication, segregation and cell division in Vibrio cholerae, the agent of the deadly epidemic human diarrheal disease cholera. A nicety of this model is that it is closely related to E. coli in the phylogenetic tree of bacteria. Here, we review recent findings on the V. cholerae cell cycle in the context of what was previously known on the E. coli cell cycle., (Copyright© by the Spanish Society for Microbiology and Institute for Catalan Studies.)

The CrbS/R two-component signal transduction system is a conserved regulatory mechanism through which specific Gram-negative bacteria control acetate flux into primary metabolic pathways. CrbS/R governs expression of acetyl-CoA synthase (acsA), an enzyme that converts acetate to acetyl-CoA, a metabolite at the nexus of the cell's most important energy-harvesting and biosynthetic reactions. During infection, bacteria can utilize this system to hijack host acetate metabolism and alter the course of colonization and pathogenesis. In toxigenic strains of Vibrio cholerae, CrbS/R-dependent expression of acsA is required for virulence in an arthropod model. Here, we investigate the function of the CrbS/R system in Pseudomonas aeruginosa, Pseudomonas entomophila, and non-toxigenic V. cholerae strains. We demonstrate that its role in acetate metabolism is conserved; this system regulates expression of the acsA gene and is required for growth on acetate as a sole carbon source. As a first step towards describing the mechanism of signaling through this pathway, we identify residues and domains that may be critical for phosphotransfer. We further demonstrate that although CrbS, the putative hybrid sensor kinase, carries both a histidine kinase domain and a receiver domain, the latter is not required for acsA transcription. In order to determine whether our findings are relevant to pathogenesis, we tested our strains in a Drosophila model of oral infection previously employed for the study of acetate-dependent virulence by V. cholerae. We show that non-toxigenic V. cholerae strains lacking CrbS or CrbR are significantly less virulent than are wild-type strains, while P. aeruginosa and P. entomophila lacking CrbS or CrbR are fully pathogenic. Together, the data suggest that the CrbS/R system plays a central role in acetate metabolism in V. cholerae, P. aeruginosa, and P. entomophila. However, each microbe's unique environmental adaptations and pathogenesis strategies may dictate conditions under which CrbS/R-mediated acs expression is most critical.

The Na+-translocating NADH:quinone oxidoreductase (NQR) is the entry site for electrons into the respiratory chain of Vibrio cholerae, the causative agent of cholera disease. NQR couples the electron transfer from NADH to ubiquinone to the translocation of sodium ions across the membrane. We investigated the pH dependence of electron transfer and generation of a transmembrane voltage (ΔΨ) by NQR reconstituted in liposomes with Na+ or Li+ as coupling cation. ΔΨ formation was followed with the voltage-sensitive dye oxonol. With Na+, ΔΨ was barely influenced by pH (6.5-8.5), while Q reduction activity exhibited a maximum at pH 7.5-8.0. With Li+, ΔΨ was generally lower, and the pH profile of electron transfer activity did not reveal a pronounced maximum. We conclude that the coupling efficiency of NQR is influenced by the nature of the transported cation, and by the concentration of protons. The 3D structure of NQR reveals a transmembrane channel in subunit NqrB. It is proposed that partial uncoupling of the NQR observed with the smaller Li+, or with Na+ at pH 7.5-8.0, is caused by the backflow of the coupling cation through the channel in NqrB.

Pathogenic Vibrio cholerae remains a major human health concern. V. cholerae has a characteristic curved rod morphology, with a longer outer face and a shorter inner face. The mechanism and function of this curvature were previously unknown. Here, we identify and characterize CrvA, the first curvature determinant in V. cholerae. CrvA self-assembles into filaments at the inner face of cell curvature. Unlike traditional cytoskeletons, CrvA localizes to the periplasm and thus can be considered a periskeletal element. To quantify how curvature forms, we developed QuASAR (quantitative analysis of sacculus architecture remodeling), which measures subcellular peptidoglycan dynamics. QuASAR reveals that CrvA asymmetrically patterns peptidoglycan insertion rather than removal, causing more material insertions into the outer face than the inner face. Furthermore, crvA is quorum regulated, and CrvA-dependent curvature increases at high cell density. Finally, we demonstrate that CrvA promotes motility in hydrogels and confers an advantage in host colonization and pathogenesis., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Cell division must be coordinated with chromosome replication and segregation to ensure the faithful transmission of genetic information during proliferation. In most bacteria, assembly of the division apparatus, the divisome, starts with the polymerization of a tubulin homologue, FtsZ, into a ring-like structure at mid-cell, the Z-ring(1). It typically occurs at half of the cell cycle when most of the replication and segregation cycle of the unique chromosome they generally harbour is achieved(2). The chromosome itself participates in the regulation of cell division, at least in part because it serves as a scaffold to position FtsZ polymerization antagonists(3). However, about 10% of bacteria have more than one chromosome(4), which raises questions about the way they license cell division(3). For instance, the genome of Vibrio cholerae, the agent of cholera, is divided between a 3 Mbp replicon that originates from the chromosome of its mono-chromosomal ancestor, Chr1, and a 1 Mbp plasmid-derived replicon, Chr2 (ref. 5). Here, we show that Chr2 harbours binding motifs for an inhibitor of Z-ring formation, which helps accurately position the V. cholerae divisome at mid-cell and postpones its assembly to the very end of the cell cycle.

Background: Outer membrane vesicles (OMVs) are known to release from almost all Gram-negative bacteria during normal growth. OMVs carry different biologically active toxins and enzymes into the surrounding environment. We suggest that OMVs may therefore be able to transport bacterial proteases into the target host cells. We present here an analysis of the Vibrio cholerae OMV-associated protease PrtV., Methodology/principal Findings: In this study, we demonstrated that PrtV was secreted from the wild type V. cholerae strain C6706 via the type II secretion system in association with OMVs. By immunoblotting and electron microscopic analysis using immunogold labeling, the association of PrtV with OMVs was examined. We demonstrated that OMV-associated PrtV was biologically active by showing altered morphology and detachment of cells when the human ileocecum carcinoma (HCT8) cells were treated with OMVs from the wild type V. cholerae strain C6706 whereas cells treated with OMVs from the prtV isogenic mutant showed no morphological changes. Furthermore, OMV-associated PrtV protease showed a contribution to bacterial resistance towards the antimicrobial peptide LL-37., Conclusion/significance: Our findings suggest that OMVs released from V. cholerae can deliver a processed, biologically active form of PrtV that contributes to bacterial interactions with target host cells.

Proteinaceous components of the biofilm matrix include secreted extracellular proteins, cell surface adhesins, and protein subunits of cell appendages such as flagella and pili. Biofilm matrix proteins play diverse roles in biofilm formation and dissolution. They are involved in attaching cells to surfaces, stabilizing the biofilm matrix via interactions with exopolysaccharide and nucleic acid components, developing three-dimensional biofilm architectures, and dissolving biofilm matrix via enzymatic degradation of polysaccharides, proteins, and nucleic acids. In this article, we will review functions of matrix proteins in a selected set of microorganisms, studies of the matrix proteomes of Vibrio cholerae and Pseudomonas aeruginosa, and roles of outer membrane vesicles and of nucleoid-binding proteins in biofilm formation.

Bacteria use rapid contraction of a long sheath of the type VI secretion system (T6SS) to deliver effectors into a target cell. Here, we present an atomic-resolution structure of a native contracted Vibrio cholerae sheath determined by cryo-electron microscopy. The sheath subunits, composed of tightly interacting proteins VipA and VipB, assemble into a six-start helix. The helix is stabilized by a core domain assembled from four β strands donated by one VipA and two VipB molecules. The fold of inner and middle layers is conserved between T6SS and phage sheaths. However, the structure of the outer layer is distinct and suggests a mechanism of interaction of the bacterial sheath with an accessory ATPase, ClpV, that facilitates multiple rounds of effector delivery. Our results provide a mechanistic insight into assembly of contractile nanomachines that bacteria and phages use to translocate macromolecules across membranes., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Biofilm formation increases both the survival and infectivity of Vibrio cholerae, the causative agent of cholera. V. cholerae is capable of forming biofilms on solid surfaces and at the air-liquid interface, termed pellicles. Known components of the extracellular matrix include the matrix proteins Bap1, RbmA, and RbmC, an exopolysaccharide termed Vibrio polysaccharide, and DNA. In this work, we examined a rugose strain of V. cholerae and its mutants unable to produce matrix proteins by interfacial rheology to compare the evolution of pellicle elasticity in real time to understand the molecular basis of matrix protein contributions to pellicle integrity and elasticity. Together with electron micrographs, visual inspection, and contact angle measurements of the pellicles, we defined distinct contributions of the matrix proteins to pellicle morphology, microscale architecture, and mechanical properties. Furthermore, we discovered that Bap1 is uniquely required for the maintenance of the mechanical strength of the pellicle over time and contributes to the hydrophobicity of the pellicle. Thus, Bap1 presents an important matrix component to target in the prevention and dispersal of V. cholerae biofilms.

Synthesis and hydrolysis of septal peptidoglycan (PG) are critical processes at the conclusion of cell division that enable separation of daughter cells. Cleavage of septal PG is mediated by PG amidases, hydrolytic enzymes that release peptide side chains from the glycan strand. Most gammaproteobacteria, including Escherichia coli, encode several functionally redundant periplasmic amidases. However, members of the Vibrio genus, including the enteric pathogen Vibrio cholerae, encode only a single PG amidase, AmiB. Here, we show that V. cholerae AmiB is crucial for cell division and growth. Genetic and biochemical analyses indicated that AmiB is regulated by two activators, EnvC and NlpD, at least one of which is required for AmiB's localization to the cell division site. Localization of the activators (and thus of AmiB) is dependent upon the cell division protein FtsN. These factors mediate septal PG cleavage in E. coli as well; however, their precise roles vary between the two organisms in a number of ways. Notably, even though V. cholerae EnvC and NlpD appear to be functionally redundant under most growth conditions tested, NlpD is specifically required for intestinal colonization in the infant mouse model of cholera and for V. cholerae resistance against bile salts, perhaps due to environmental regulation of AmiB or its activators. Collectively, our findings reveal that although the cellular components that enable cleavage of septal PG appear to be generally conserved between E. coli and V. cholerae, they can be combined into diverse functional regulatory networks., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

In rod shaped bacteria, numerous cellular components are targeted to the cell poles, and such localization is often important for optimal function. In particular, recognition of poles is often linked to division site selection, chromosome segregation, chemotactic signaling, and motility. Recent advances in understanding polarity include identification of a Vibrio cholerae protein that mediates polar localization of a chromosome origin and chemotaxis clusters, as well as a downstream protein that contributes solely to localization of chemotaxis proteins. In Caulobacter crescentus, the molecular mechanisms by which polar determinants and effectors are localized, and the key roles for nucleotide-dependent switches, have been defined. Finally, roles for, and interactions between, factors that mediate environmentally determined polarity in Myxococcus xanthus have recently been characterized., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has become the method of choice for introducing DNA into prokaryotes that are not naturally competent. Electroporation is a rapid, efficient, and streamlined transformation method that, in addition to purified DNA and competent bacteria, requires commercially available gene pulse controller and cuvettes. In contrast to the pulsing step, preparation of electrocompetent cells is time consuming and labor intensive involving repeated rounds of centrifugation and washes in decreasing volumes of sterile, cold water, or non-ionic buffers of large volumes of cultures grown to mid-logarithmic phase of growth. Time and effort can be saved by purchasing electrocompetent cells from commercial sources, but the selection is limited to commonly employed E. coli laboratory strains. We are hereby disseminating a rapid and efficient method for preparing electrocompetent E. coli, which has been in use by bacteriology laboratories for some time, can be adapted to V. cholerae and other prokaryotes. While we cannot ascertain whom to credit for developing the original technique, we are hereby making it available to the scientific community.

A unique event in bacterial epidemiology was the emergence of the El Tor biotype of Vibrio cholerae O1 and the subsequent rapid displacement of the existing classical biotype as the predominant cause of epidemic cholera. We demonstrate that when the El Tor and classical biotypes were cocultured in standard laboratory medium a precipitous decline in colony forming units (CFU) of the classical biotype occurred in a contact dependent manner. Several lines of evidence including DNA release, microscopy and flow cytometric analysis indicated that the drastic reduction in CFU of the classical biotype in cocultures was not accompanied by lysis, although when the classical biotype was grown individually in monocultures, lysis of the cells occurred concomitant with decrease in CFU starting from late stationary phase. Furthermore, uptake of a membrane potential sensitive dye and protection of genomic DNA from extracellular DNase strongly suggested that the classical biotype cells in cocultures retained viability in spite of loss of culturability. These results suggest that coculturing the classical biotype with the El Tor biotype protects the former from lysis allowing the cells to remain viable in spite of the loss of culturability. The stationary phase sigma factor RpoS may have a role in the loss of culturability of the classical biotype in cocultures. Although competitive exclusion of closely related strains has been reported for several bacterial species, conversion of the target bacterial population to the viable non-culturable state has not been demonstrated previously and may have important implications in the evolution of bacterial strains.

The chromosomal origin, chemotaxis arrays and flagellum of Vibrio cholerae congregate at the same pole of the cell. How? A recent study identifies a new pole-organizing protein, HubP, that recruits members of the ParA family of spatial regulators of subcellular structures to the pole., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Unlabelled: Vibrio cholerae has two chromosomes (chrI and chrII) whose replication and segregation are under different genetic controls. The region covering the replication origin of chrI resembles that of the Escherichia coli chromosome, and both origins are under control of the highly conserved initiator, DnaA. The origin region of chrII resembles that of plasmids that have iterated initiator-binding sites (iterons) and is under control of the chrII-specific initiator, RctB. Both chrI and chrII encode chromosome-specific orthologs of plasmid partitioning proteins, ParA and ParB. Here, we have interfered with chrII replication, segregation, or both, using extra copies of sites that titrate RctB or ParB. Under these conditions, replication and segregation of chrI remain unaffected for at least 1 cell cycle. In this respect, chrI behaves similarly to the E. coli chromosome when plasmid maintenance is disturbed in the same cell. Apparently, no checkpoint exists to block cell division before the crippled chromosome is lost by a failure to replicate or to segregate. Whether blocking chrI replication can affect chrII replication remains to be tested., Importance: Chromosome replication, chromosome segregation, and cell division are the three main events of the cell cycle. They occur in an orderly fashion once per cell cycle. How the sequence of events is controlled is only beginning to be answered in bacteria. The finding of bacteria that possess more than one chromosome raises the important question: how are different chromosomes coordinated in their replication and segregation? It appears that in the evolution of the two-chromosome genome of V. cholerae, either the secondary chromosome adapted to the main chromosome to ensure its maintenance or it is maintained independently, as are bacterial plasmids. An understanding of chromosome coordination is expected to bear on the evolutionary process of chromosome acquisition and on the efficacy of possible strategies for selective elimination of a pathogen by targeting a specific chromosome.

Type VI secretion systems are bacterial virulence-associated nanomachines composed of proteins that are evolutionarily related to components of bacteriophage tails. Here we show that protein secretion by the type VI secretion system of Vibrio cholerae requires the action of a dynamic intracellular tubular structure that is structurally and functionally homologous to contractile phage tail sheath. Time-lapse fluorescence light microscopy reveals that sheaths of the type VI secretion system cycle between assembly, quick contraction, disassembly and re-assembly. Whole-cell electron cryotomography further shows that the sheaths appear as long tubular structures in either extended or contracted conformations that are connected to the inner membrane by a distinct basal structure. These data support a model in which the contraction of the type VI secretion system sheath provides the energy needed to translocate proteins out of effector cells and into adjacent target cells.

Surface associated microbes have historically been difficult to accurately and effectively enumerate. In the current study, we propose a rapid and simple method for estimating abundance of surface associated microbial cells by fluorescence of SYBRGreen stained bacteria and in vivo chlorophyll a fluorescence of benthic diatoms in 24 and 48-well microtiter plates. The effectiveness of this high-throughput technique is demonstrated by assessing sensitivity of a clinical strain of Vibrio cholerae, a benthic bacterial isolate and the benthic microalgae Cylindrotheca closterium to three antibiotics--tylosin, lincomycin and ciproflaxacin. We report on the significant linear relationships between spectral chl a fluorescence and cell abundance and between microalgal growth rates derived from cell counts and fluorescence. Additionally, we provide a simplified and improved method for preparation of a silica gel matrix (SGM), which is an ideal plating media for fluorescence applications. These findings indicate that spectrofluorometry is an inexpensive tool for rapidly estimating abundance of surface associated microbiota and can be employed for assessing antibiotic sensitivity., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The resistance-nodulation-division (RND) efflux systems are ubiquitous transporters that function in antimicrobial resistance. Recent studies showed that RND systems were required for virulence factor production in Vibrio cholerae. The V. cholerae genome encodes six RND efflux systems. Three of the RND systems (VexB, VexD, and VexK) were previously shown to be redundant for in vitro resistance to bile acids and detergents. A mutant lacking the VexB, VexD, and VexK RND pumps produced wild-type levels of cholera toxin (CT) and the toxin co-regulated pilus (TCP) and was moderately attenuated for intestinal colonization. In contrast, a RND negative mutant produced significantly reduced amounts of CT and TCP and displayed a severe colonization defect. This suggested that one or more of the three uncharacterized RND efflux systems (i.e. VexF, VexH, and VexM) were required for pathogenesis. In this study, a genetic approach was used to generate a panel of V. cholerae RND efflux pump mutants in order to determine the function of VexH in antimicrobial resistance, virulence factor production, and intestinal colonization. VexH contributed to in vitro antimicrobial resistance and exhibited a broad substrate specificity that was redundant with the VexB, VexD, and VexK RND efflux pumps. These four efflux pumps were responsible for in vitro antimicrobial resistance and were required for virulence factor production and intestinal colonization. Mutation of the VexF and/or VexM efflux pumps did not affect in vitro antimicrobial resistance, but did negatively affect CT and TCP production. Collectively, our results demonstrate that the V. cholerae RND efflux pumps have redundant functions in antimicrobial resistance and virulence factor production. This suggests that the RND efflux systems contribute to V. cholerae pathogenesis by providing the bacterium with protection against antimicrobial compounds that are present in the host and by contributing to the regulated expression of virulence factors.

Based on modification of separate structural features of the Vibrio cholerae quorum sensing signal, (S)-3-hydroxytridecan-4-one (CAI-1), three focused compound libraries have been synthesized and evaluated for biological activity. Modifications to the acyl tail and α-hydroxy ketone typically provided agonists with activities correlated to tail length and conservative changes to the hydroxy ketone. Among the molecules identified within this collection of agonists is Am-CAI-1 (B11), which is among the most potent agonists reported to date with an EC(50) of 0.21 μM. Modifications to the ethyl side chain delivered molecules with both agonist and antagonist activity, including m-OH-Ph-CAI-1 (C13) which is the most potent antagonist reported to date with an IC(50) of 36 μM. The molecules described in this manuscript are anticipated to serve as valuable tools in the study of quorum sensing in Vibrio cholerae and provide new leads in the development of an antivirulence therapy against this human pathogen., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The acute diarrhoeal disease cholera is caused by the aquatic pathogen Vibrio cholerae upon ingestion of contaminated food or water by the human host. The mechanisms by which V. cholerae is able to persist and survive in the host and aquatic environments have been studied for years; however, little is known about the factors involved in the adaptation or response of V. cholerae transitioning between these two environments. The transition from bacillary to coccoid morphology is thought to be one mechanism of survival that V. cholerae uses in response to environmental stress. Coccoid morphology has been observed for V. cholerae while in a viable but non-culturable (VBNC) state, during times of nutrient limitation, and in the water-diluted stool of cholera-infected patients. In this study we sought conditions to study the coccoid morphology of V. cholerae, and found that coccoid-shaped cells can express and produce the virulence factor toxin co-regulated pilus (TCP) and are able to colonize the infant mouse to the same extent as bacillus-shaped cells. This study suggests that TCP may be one factor that V. cholerae utilizes for adaptation and survival during the transition between the host and the aquatic environment.

Bacteria commonly grow in densely populated surface-bound communities, termed biofilms, where they gain benefits including superior access to nutrients and resistance to environmental insults. The secretion of extracellular polymeric substances (EPS), which bind bacterial collectives together, is ubiquitously associated with biofilm formation. It is generally assumed that EPS secretion is a cooperative phenotype that benefits all neighboring cells, but in fact little is known about the competitive and evolutionary dynamics of EPS production. By studying Vibrio cholerae biofilms in microfluidic devices, we show that EPS-producing cells selectively benefit their clonemates and gain a dramatic advantage in competition against an isogenic EPS-deficient strain. However, this advantage carries an ecological cost beyond the energetic requirement for EPS production: EPS-producing cells are impaired for dispersal to new locations. Our study establishes that a fundamental tradeoff between local competition and dispersal exists among bacteria. Furthermore, this tradeoff can be governed by a single phenotype.

Stochastic processes are thought to mediate localization of membrane-associated chemotaxis signaling clusters in peritrichous bacteria. Here, we identified a new family of ParA-like ATPases (designated ParC [for partitioning chemotaxis]) encoded within chemotaxis operons of many polar-flagellated γ-proteobacteria that actively promote polar localization of chemotaxis proteins. In Vibrio cholerae, a single ParC focus is found at the flagellated old pole in newborn cells, and later bipolar ParC foci develop as the cell matures. The cell cycle-dependent redistribution of ParC occurs by its release from the old pole and subsequent relocalization at the new pole, consistent with a "diffusion and capture" model for ParC dynamics. Chemotaxis proteins encoded in the same cluster as ParC have a similar unipolar-to-bipolar transition; however, they reach the new pole after the arrival of ParC. Cells lacking ParC exhibit aberrantly localized foci of chemotaxis proteins, reduced chemotaxis, and altered motility, which likely accounts for their enhanced colonization of the proximal small intestine in an animal model of cholera. Collectively, our findings indicate that ParC promotes the efficiency of chemotactic signaling processes. In particular, ParC-facilitated development of a functional chemotaxis apparatus at the new pole readies this site for its development into a functional old pole after cell division.

Plasmid origins of replication are rare in bacterial chromosomes, except in multichromosome bacteria. The replication origin of Vibrio cholerae chromosome II (chrII) closely resembles iteron-bearing plasmid origins. Iterons are repeated initiator binding sites in plasmid origins and participate both in replication initiation and its control. The control is mediated primarily by coupling of iterons via the bound initiators ("handcuffing"), which causes steric hindrance to the origin. The control in chrII must be different, since the timing of its replication is cell cycle-specific, whereas in plasmids it is random. Here we show that chrII uses, in addition to iterons, another kind of initiator binding site, named 39-mers. The 39-mers confer stringent control by increasing handcuffing of iterons, presumably via initiator remodeling. Iterons, although potential inhibitors of replication themselves, restrain the 39-mer-mediated inhibition, possibly by direct coupling ("heterohandcuffing"). We propose that the presumptive transition of a plasmid to a chromosomal mode of control requires additional regulators to increase the stringency of control, and as will be discussed, to gain the capacity to modulate the effectiveness of the regulators at different stages of the cell cycle.

Vibrio cholerae carries homologs of plasmid-borne parA and parB genes on both of its chromosomes. The par genes help to segregate many plasmids and chromosomes. Here we have studied the par genes of V. cholerae chromosome I. Earlier studies suggested that ParBI binds to the centromeric site parSI near the origin of replication (oriI), and parSI-ParBI complexes are placed at the cell poles by ParAI. Deletion of parAI and parSI caused the origin-proximal DNA to be less polar. Here we found that deletion of parBI also resulted in a less polar localization of oriI. However, unlike the deletion of parAI, the deletion of parBI increased the oriI number. Replication was normal when both parAI and parBI were deleted, suggesting that ParBI mediates its action through ParAI. Overexpression of ParAI in a parABI-deleted strain also increased the DNA content. The results are similar to those found for Bacillus subtilis, where ParA (Soj) stimulates replication and this activity is repressed by ParB (SpoOJ). As in B. subtilis, the stimulation of replication most likely involves the replication initiator DnaA. Our results indicate that control of chromosomal DNA replication is an additional function of chromosomal par genes conserved across the Gram-positive/Gram-negative divide.

The present strategy proposes a simple and single step aqueous route for synthesizing stable, fluorescent ZnTe/dendrimer nanocomposites with varying dendrimer terminal groups. In these hybrid materials, the fluorescence of the semiconductor combines with the biomimetic properties of the dendrimer making them suitable for various biomedical applications. The ZnTe nanocomposites thus obtained demonstrate bactericidal activity against enteropathogenic bacteria without having toxic effects on the human erythrocytes. The average size of the ZnTe nanoparticles within the dendrimer matrix was in the range of 2.9-6.0 nm, and they have a good degree of crystallinity with a hexagonal crystal phase. The antibacterial activities of the ZnTe/dendrimer nanocomposites (ZnTe DNCs) as well other semiconductor nanocomposites were evaluated against enteropathogenic bacteria including multi-drug resistant Vibrio cholerae serogroup O1 and enterotoxigenic Escherichia coli (ETEC). ZnTe DNCs had significant antibacterial activity against strains of V. cholerae and ETEC with minimum inhibitory concentrations ranging from 64 to 512 μg ml(-1) and minimum bactericidal concentrations ranging from 128 to 1000 μg ml(-1). Thus, the observed results suggest that these water-soluble active nanocomposites have potential for the treatment of enteric diseases like diarrhoea and cholera.

The Gram-negative bacteria Vibrio cholerae poses significant public health concerns by causing an acute intestinal infection afflicting millions of people each year. V. cholerae motility, as well as virulence factor expression and outer membrane protein production, has been shown to be affected by bile. The current study examines the effects of bile on V. cholerae phospholipids. Bile exposure caused significant alterations to the phospholipid profile of V. cholerae but not of other enteric pathogens. These changes consisted of a quantitative increase and migratory difference in cardiolipin, decreases in phosphatidylglycerol and phosphatidylethanolamine, and the dramatic appearance of an unknown phospholipid determined to be lyso-phosphatidylethanolamine. Major components of bile were not responsible for the observed changes, but long-chain polyunsaturated fatty acids, which are minor components of bile, were shown to be incorporated into phospholipids of V. cholerae. Although the bile-induced phospholipid profile was independent of the V. cholerae virulence cascade, we identified another relevant environment in which V. cholerae assimilates unique fatty acids into its membrane phospholipids - marine sediment. Our results suggest that Vibrio species possess unique machinery conferring the ability to take up a wider range of exogenous fatty acids than other enteric bacteria., (© 2010 Blackwell Publishing Ltd.)

The two-component quorum sensing (QS) system, first described in the marine bacterium Vibrio harveyi and evolutionarily conserved among members of the genus Vibrio, has been best studied in the human pathogen Vibrio cholerae (1, 2). In the V. cholerae QS system, the response to the accumulation of extracellular autoinducers triggers a signaling cascade resulting in the transcription of four small regulatory RNAs (sRNAs). Our results support the model that the QS sRNAs bind to the 5' untranslated region of multiple mRNAs and alter the fate of one in a positive manner and several others in a negative manner. This mechanism ensures the proper timing of the QS response, which includes the expression of traits critical for virulence and for the formation of biofilms (2-6).

This work presents the first demonstration of a cantilever based cholerae sensor. Dynamic force microscopy within atomic force microscope (AFM) is applied to measure the cantilever's resonance frequency shift due to mass of cell bound on microcantilever surface. The Vibrio cholerae O1, a food and waterborne pathogen that caused cholera disease in human, is a target bacterium cell of interest. Commercial gold-coated AFM microcantilevers are immobilized with monoclonal antibody (anti-V. cholerae O1) by self-assembled monolayer method. V. cholerae O1 detection experiment is then conducted in concentrations ranging from 1×10(3) to 1×10(7) CFU/ml. The microcantilever-based sensor has a detection limit of ∼1×10(3) CFU/ml and a mass sensitivity, Δm/ΔF, of ∼146.5 pg/Hz, which is at least two orders of magnitude lower than other reported techniques and sufficient for V. cholerae detection in food products without pre-enrichment steps. In addition, V. cholerae O1 antigen-antibody binding on microcanilever is confirmed by scanning electron microscopy. The results demonstrate that the new biosensor is promising for high sensitivity, uncomplicated and rapid detection of V. cholerae O1., (Copyright © 2010 Elsevier B.V. All rights reserved.)

VBNC Vibrio cholerae O139 VC-280 obtained by incubation in 1% solution of artificial sea water IO at 4°C for 74 days converted to the culturable state when co-cultured with CHO cells. Other eukaryotic cell lines, including HT-29, Caco-2, T84, HeLa, and Intestine 407, also supported conversion of VBNC cells to the culturable state. Conversion of VBNC V. cholerae O1 N16961 and V. cholerae O139 VC-280/pG13 to the culturable state, under the same conditions, was also confirmed. When VBNC V. cholerae O139 VC-280 was incubated in 1% IO at 4°C for up to 91 days, the number of cells converted by co-culture with CHO cells declined with each additional day of incubation and after 91 days conversion was not observed.

DNA adenine methylation is widely used to control many DNA transactions, including replication. In Escherichia coli, methylation serves to silence newly synthesized (hemimethylated) sister origins. SeqA, a protein that binds to hemimethylated DNA, mediates the silencing, and this is necessary to restrict replication to once per cell cycle. The methylation, however, is not essential for replication initiation per se but appeared so when the origins (oriI and oriII) of the two Vibrio cholerae chromosomes were used to drive plasmid replication in E. coli. Here we show that, as in the case of E. coli, methylation is not essential for oriI when it drives chromosomal replication and is needed for once-per-cell-cycle replication in a SeqA-dependent fashion. We found that oriII also needs SeqA for once-per-cell-cycle replication and, additionally, full methylation for efficient initiator binding. The requirement for initiator binding might suffice to make methylation an essential function in V. cholerae. The structure of oriII suggests that it originated from a plasmid, but unlike plasmids, oriII makes use of methylation for once-per-cell-cycle replication, the norm for chromosomal but not plasmid replication., Competing Interests: The authors have declared that no competing interests exist.

The factors that enhance the waterborne spread of bacterial epidemics and sustain the pathogens in nature are unclear. The epidemic diarrheal disease cholera caused by Vibrio cholerae spreads through water contaminated with the pathogen. However, the bacteria exist in water mostly as clumps of cells, which resist cultivation by standard techniques but revive into fully virulent form in the intestinal milieu. These conditionally viable environmental cells (CVEC), alternatively called viable but nonculturable cells, presumably play a crucial role in cholera epidemiology. However, the precise mechanism causing the transition of V. cholerae to the CVEC form and this form's significance in the biology of the pathogen are unknown. Here we show that this process involves biofilm formation that is dependent on quorum sensing, a regulatory response that is controlled by cell density. V. cholerae strains carrying mutations in genes required for quorum sensing and biofilm formation displayed altered CVEC formation in environmental water following intestinal infections. Analysis of naturally occurring V. cholerae CVEC showed that organisms that adopt this quiescent physiological state typically exist as clumps of cells that comprise a single clone closely related to isolates causing the most recent local cholera epidemic. These results support a model of cholera transmission in which in vivo-formed biofilms convert to CVEC upon the introduction of cholera stools into environmental water. Our data further suggest that a temporary loss of quorum sensing due to dilution of extracellular autoinducers confers a selective advantage to communities of V. cholerae by blocking quorum-mediated regulatory responses that would break down biofilms and thus interfere with CVEC formation.

Signaling through the second messenger cyclic di-GMP (c-di-GMP) is central to the life cycle of Vibrio cholerae. However, relatively little is known about the signaling mechanism, including the specific external stimuli that regulate c-di-GMP concentration. Here, we show that the phosphate responsive regulator PhoB regulates an operon, acgAB, which encodes c-di-GMP metabolic enzymes. We show that induction of acgAB by PhoB positively regulates V. cholerae motility in vitro and that PhoB regulates expression of acgAB at late stages during V. cholerae infection in the infant mouse small intestine. These data support a model whereby PhoB becomes activated at a late stage of infection in preparation for dissemination of V. cholerae to the aquatic environment and suggest that the concentration of exogenous phosphate may become limited at late stages of infection.

The flagellar motor consists of a rotor and a stator and couples the flux of cations (H(+) or Na(+)) to the generation of the torque necessary to drive flagellum rotation. The inner membrane proteins PomA and PomB are stator components of the Na(+)-driven flagellar motor from Vibrio cholerae. Affinity-tagged variants of PomA and PomB were co-expressed in trans in the non-motile V. cholerae pomAB deletion strain to study the role of the conserved D23 in the transmembrane helix of PomB. At pH 9, the D23E variant restored motility to 100% of that observed with wild type PomB, whereas the D23N variant resulted in a non-motile phenotype, indicating that a carboxylic group at position 23 in PomB is important for flagellum rotation. Motility tests at decreasing pH revealed a pronounced decline of flagellar function with a motor complex containing the PomB-D23E variant. It is suggested that the protonation state of the glutamate residue at position 23 determines the performance of the flagellar motor by altering the affinity of Na(+) to PomB. The conserved aspartate residue in the transmembrane helix of PomB and its H(+)-dependent homologs might act as a ligand for the coupling cation in the flagellar motor.

The formation of biofilm communities enhances the persistence of Vibrio cholerae in aquatic environments. Biofilm production is repressed by the quorum-sensing regulator HapR in response to the accumulation of CAI-1 and AI-2. CAI-1 is the strongest input signal activating HapR, whereas the role of AI-2 remains ill-defined. In the present study, we show that a V. cholerae luxS (AI-2-defective) mutant made increased biofilm. Interestingly, cells in the biofilm were more responsive to AI-2 deficiency than cells from the planktonic population.