Characterization of the Vibrio cholerae extracellular matrix: a top-down solid-state NMR approach.

Bacterial biofilms are communities of bacterial cells surrounded by a self-secreted extracellular matrix. Biofilm formation by Vibrio cholerae, the human pathogen responsible for cholera, contributes to its environmental survival and infectivity. Important genetic and molecular requirements have been identified for V. cholerae biofilm formation, yet a compositional accounting of these parts in the intact biofilm or extracellular matrix has not been described. As insoluble and non-crystalline assemblies, determinations of biofilm composition pose a challenge to conventional biochemical and biophysical analyses. The V. cholerae extracellular matrix composition is particularly complex with several proteins, complex polysaccharides, and other biomolecules having been identified as matrix parts. We developed a new top-down solid-state NMR approach to spectroscopically assign and quantify the carbon pools of the intact V. cholerae extracellular matrix using ¹³C CPMAS and ¹³C{(¹5N}, ¹5N{³¹P}, and ¹³C{³¹P}REDOR. General sugar, lipid, and amino acid pools were first profiled and then further annotated and quantified as specific carbon types, including carbonyls, amides, glycyl carbons, and anomerics. In addition, ¹5N profiling revealed a large amine pool relative to amide contributions, reflecting the prevalence of molecular modifications with free amine groups. Our top-down approach could be implemented immediately to examine the extracellular matrix from mutant strains that might alter polysaccharide production or lipid release beyond the cell surface; or to monitor changes that may accompany environmental variations and stressors such as altered nutrient composition, oxidative stress or antibiotics. More generally, our analysis has demonstrated that solid-state NMR is a valuable tool to characterize complex biofilm systems.

Identification and characterization of VpsR and VpsT binding sites in Vibrio cholerae.

UNLABELLED: The ability to form biofilms is critical for environmental survival and transmission of Vibrio cholerae, a facultative human pathogen responsible for the disease cholera. Biofilm formation is controlled by several transcriptional regulators and alternative sigma factors. In this study, we report that the two main positive regulators of biofilm formation, VpsR and VpsT, bind to nonoverlapping target sequences in the regulatory region of vpsL in vitro. VpsR binds to a proximal site (the R1 box) as well as a distal site (the R2 box) with respect to the transcriptional start site identified upstream of vpsL. The VpsT binding site (the T box) is located between the R1 and R2 boxes. While mutations in the T and R boxes resulted in a decrease in vpsL expression, deletion of the T and R2 boxes resulted in an increase in vpsL expression. Analysis of the role of H-NS in vpsL expression revealed that deletion of hns resulted in enhanced vpsL expression. The level of vpsL expression was higher in an hns vpsT double mutant than in the parental strain but lower than that in an hns mutant. In silico analysis of the regulatory regions of the VpsR and VpsT targets resulted in the identification of conserved recognition motifs for VpsR and VpsT and revealed that operons involved in biofilm formation and vpsT are coregulated by VpsR and VpsT. Furthermore, a comparative genomics analysis revealed substantial variability in the promoter region of the vpsT and vpsL genes among extant V. cholerae isolates, suggesting that regulation of biofilm formation is under active selection. IMPORTANCE: Vibrio cholerae causes cholera and is a natural inhabitant of aquatic environments. One critical factor that is important for environmental survival and transmission of V. cholerae is the microbe's ability to form biofilms, which are surface-associated communities encased in a matrix composed of the exopolysaccharide VPS (Vibrio polysaccharide), proteins, and nucleic acids. Two proteins, VpsR and VpsT, positively regulate VPS production and biofilm formation. We characterized the structural features of the promoter of the vpsL gene, determined the target sequences recognized by VpsT and VpsR, and analyzed their distribution and conservation patterns in multiple V. cholerae isolates. This work fills a fundamental gap in our understanding of the regulatory mechanisms employed by the master regulators VpsR and VpsT in controlling biofilm matrix production.

Polymyxin B resistance and biofilm formation in Vibrio cholerae are controlled by the response regulator CarR.

Two-component systems play important roles in the physiology of many bacterial pathogens. Vibrio cholerae's CarRS two-component regulatory system negatively regulates expression of vps (Vibrio polysaccharide) genes and biofilm formation. In this study, we report that CarR confers polymyxin B resistance by positively regulating expression of the almEFG genes, whose products are required for glycine and diglycine modification of lipid A. We determined that CarR directly binds to the regulatory region of the almEFG operon. Similarly to a carR mutant, strains lacking almE, almF, and almG exhibited enhanced polymyxin B sensitivity. We also observed that strains lacking almE or the almEFG operon have enhanced biofilm formation. Our results reveal that CarR regulates biofilm formation and antimicrobial peptide resistance in V. cholerae.

Molecular determinants of mechanical properties of V. cholerae biofilms at the air-liquid interface.

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.

Cellular levels and binding of c-di-GMP control subcellular localization and activity of the Vibrio cholerae transcriptional regulator VpsT.

The second messenger, cyclic diguanylate (c-di-GMP), regulates diverse cellular processes in bacteria. C-di-GMP is produced by diguanylate cyclases (DGCs), degraded by phosphodiesterases (PDEs), and receptors couple c-di-GMP production to cellular responses. In many bacteria, including Vibrio cholerae, multiple DGCs and PDEs contribute to c-di-GMP signaling, and it is currently unclear whether the compartmentalization of c-di-GMP signaling components is required to mediate c-di-GMP signal transduction. In this study we show that the transcriptional regulator, VpsT, requires c-di-GMP binding for subcellular localization and activity. Only the additive deletion of five DGCs markedly decreases the localization of VpsT, while single deletions of each DGC do not impact VpsT localization. Moreover, mutations in residues required for c-di-GMP binding, c-di-GMP-stabilized dimerization and DNA binding of VpsT abrogate wild type localization and activity. VpsT does not co-localize or interact with DGCs suggesting that c-di-GMP from these DGCs diffuses to VpsT, supporting a model in which c-di-GMP acts at a distance. Furthermore, VpsT localization in a heterologous host, Escherichia coli, requires a catalytically active DGC and is enhanced by the presence of VpsT-target sequences. Our data show that c-di-GMP signaling can be executed through an additive cellular c-di-GMP level from multiple DGCs affecting the localization and activity of a c-di-GMP receptor and furthers our understanding of the mechanisms of second messenger signaling.

Quantitative image analysis of microbial communities with BiofilmQ.

Biofilms are microbial communities that represent a highly abundant form of microbial life on Earth. Inside biofilms, phenotypic and genotypic variations occur in three-dimensional space and time; microscopy and quantitative image analysis are therefore crucial for elucidating their functions. Here, we present BiofilmQ-a comprehensive image cytometry software tool for the automated and high-throughput quantification, analysis and visualization of numerous biofilm-internal and whole-biofilm properties in three-dimensional space and time.

Role of Vibrio polysaccharide (vps) genes in VPS production, biofilm formation and Vibrio cholerae pathogenesis.

Biofilm formation enhances the survival and persistence of the facultative human pathogen Vibrio cholerae in natural ecosystems and its transmission during seasonal cholera outbreaks. A major component of the V. cholerae biofilm matrix is the Vibrio polysaccharide (VPS), which is essential for development of three-dimensional biofilm structures. The vps genes are clustered in two regions, the vps-I cluster (vpsU, vpsA-K, VC0916-27) and the vps-II cluster (vpsL-Q, VC0934-39), separated by an intergenic region containing the rbm gene cluster that encodes biofilm matrix proteins. In-frame deletions of the vps clusters and genes encoding matrix proteins drastically altered biofilm formation phenotypes. To determine which genes within the vps gene clusters are required for biofilm formation and VPS synthesis, we generated in-frame deletion mutants for all the vps genes. Many of these mutants exhibited reduced capacity to produce VPS and biofilms. Infant mouse colonization assays revealed that mutants lacking either vps clusters or rbmA (encoding secreted matrix protein RbmA) exhibited a defect in intestinal colonization compared to the wild-type. Understanding the roles of the various vps gene products will aid in the biochemical characterization of the VPS biosynthetic pathway and elucidate how vps gene products contribute to VPS biosynthesis, biofilm formation and virulence in V. cholerae.

Overexpression of VpsS, a hybrid sensor kinase, enhances biofilm formation in Vibrio cholerae.

Vibrio cholerae causes the disease cholera and inhabits aquatic environments. One key factor in the environmental survival of V. cholerae is its ability to form matrix-enclosed, surface-associated microbial communities known as biofilms. Mature biofilms rely on Vibrio polysaccharide to connect cells to each other and to a surface. We previously described a core regulatory network, which consists of two positive transcriptional regulators, VpsR and VpsT, and a negative transcriptional regulator HapR, that controls biofilm formation by regulating the expression of vps genes. In this study, we report the identification of a sensor histidine kinase, VpsS, which can control biofilm formation and activates the expression of vps genes. VpsS required the response regulator VpsR to activate vps expression. VpsS is a hybrid sensor histidine kinase that is predicted to contain both histidine kinase and response regulator domains, but it lacks a histidine phosphotransferase (HPT) domain. We determined that VpsS acts through the HPT protein LuxU, which is involved in a quorum-sensing signal transduction network in V. cholerae. In vitro analysis of phosphotransfer relationships revealed that LuxU can specifically reverse phosphotransfer to CqsS, LuxQ, and VpsS. Furthermore, mutational and phenotypic analyses revealed that VpsS requires the response regulator LuxO to activate vps expression, and LuxO positively regulates the transcription of vpsR and vpsT. The induction of vps expression via VpsS was also shown to occur independent of HapR. Thus, VpsS utilizes components of the quorum-sensing pathway to modulate biofilm formation in V. cholerae.

Interplay between cyclic AMP-cyclic AMP receptor protein and cyclic di-GMP signaling in Vibrio cholerae biofilm formation.

Vibrio cholerae is a facultative human pathogen. The ability of V. cholerae to form biofilms is crucial for its survival in aquatic habitats between epidemics and is advantageous for host-to-host transmission during epidemics. Formation of mature biofilms requires the production of extracellular matrix components, including Vibrio polysaccharide (VPS) and matrix proteins. Biofilm formation is positively controlled by the transcriptional regulators VpsR and VpsT and is negatively regulated by the quorum-sensing transcriptional regulator HapR, as well as the cyclic AMP (cAMP)-cAMP receptor protein (CRP) regulatory complex. Transcriptome analysis of cyaA (encoding adenylate cyclase) and crp (encoding cAMP receptor protein) deletion mutants revealed that cAMP-CRP negatively regulates transcription of both VPS biosynthesis genes and genes encoding biofilm matrix proteins. Further mutational and expression analysis revealed that cAMP-CRP negatively regulates transcription of vps genes indirectly through its action on vpsR transcription. However, negative regulation of the genes encoding biofilm matrix proteins by cAMP-CRP can also occur independent of VpsR. Transcriptome analysis also revealed that cAMP-CRP regulates the expression of a set of genes encoding diguanylate cyclases (DGCs) and phosphodiesterases. Mutational and phenotypic analysis of the differentially regulated DGCs revealed that a DGC, CdgA, is responsible for the increase in biofilm formation in the Deltacrp mutant, showing the connection between of cyclic di-GMP and cAMP signaling in V. cholerae.

Heterologous expression of the alcohol dehydrogenase (adhI) gene from Geobacillus thermoglucosidasius strain M10EXG.

A thermostable alcohol dehydrogenase (ADH-I) isolated from the potential thermophilic ethanologen Geobacillus thermoglucosidasius strain M10EXG has been characterised. Inverse PCR showed that the gene (adhI) was localised with 3-hexulose-6-phosphate synthase (HPS) and 6-phospho-3 hexuloisomerase (PHI) on its genome. The deduced peptide sequence of the 1020-bp M10EXG adhI, which corresponds to 340 amino acids, shows 96% and 89% similarity to ADH-hT and ADH-T from Geobacillus stearothermophilus strains LLD-R and NCA 1503, respectively. Over-expression of M10EXG ADH-I in Escherichia coli DH5alpha (pNF303) was confirmed using an ADH activity assay and SDS-PAGE analysis. The specific ADH activity in the extract from this recombinant strain was 9.7(+/-0.3) U mg(-1) protein, compared to 0.1(+/-0.01) U mg(-1) protein in the control strain. The recombinant E. coli showed enzymatic activity towards ethanol, 1-butanol, 1-pentanol, 1-heptanol, 1-hexanol, 1-octanol and 2-propanol, but not methanol. In silico analysis, including phylogenetic reconstruction and protein modeling, confirmed that the thermostable enzyme from G. thermoglucosidasius is likely to belong to the NAD-Zn-dependent family of alcohol dehydrogenases.

Vibrio cholerae use pili and flagella synergistically to effect motility switching and conditional surface attachment.

We show that Vibrio cholerae, the causative agent of cholera, use their flagella and mannose-sensitive hemagglutinin (MSHA) type IV pili synergistically to switch between two complementary motility states that together facilitate surface selection and attachment. Flagellar rotation counter-rotates the cell body, causing MSHA pili to have periodic mechanical contact with the surface for surface-skimming cells. Using tracking algorithms at 5 ms resolution we observe two motility behaviours: 'roaming', characterized by meandering trajectories, and 'orbiting', characterized by repetitive high-curvature orbits. We develop a hydrodynamic model showing that these phenotypes result from a nonlinear relationship between trajectory shape and frictional forces between pili and the surface: strong pili-surface interactions generate orbiting motion, increasing the local bacterial loiter time. Time-lapse imaging reveals how only orbiting mode cells can attach irreversibly and form microcolonies. These observations suggest that MSHA pili are crucial for surface selection, irreversible attachment, and ultimately microcolony formation.

Development of quinoline-based disruptors of biofilm formation against Vibrio cholerae.

Biofilm formation is a major cause of bacterial persistence in nosocomial infections, leading to extended treatment times and increased rates of morbidity and mortality. Despite this, there are currently no biofilm inhibitors approved for clinical use. The synthesis and biological evaluation of a library of amino alcohol quinolines as lead compounds for the disruption of biofilm formation in Vibrio cholerae is now reported. Application of selective metal-halogen exchange chemistry installed both stereocenters in one step, to afford a simpler scaffold than the initial lead molecule, with an EC50 < 10 µM.

Molecular architecture and assembly principles of Vibrio cholerae biofilms.

In their natural environment, microbes organize into communities held together by an extracellular matrix composed of polysaccharides and proteins. We developed an in vivo labeling strategy to allow the extracellular matrix of developing biofilms to be visualized with conventional and superresolution light microscopy. Vibrio cholerae biofilms displayed three distinct levels of spatial organization: cells, clusters of cells, and collections of clusters. Multiresolution imaging of living V. cholerae biofilms revealed the complementary architectural roles of the four essential matrix constituents: RbmA provided cell-cell adhesion; Bap1 allowed the developing biofilm to adhere to surfaces; and heterogeneous mixtures of Vibrio polysaccharide, RbmC, and Bap1 formed dynamic, flexible, and ordered envelopes that encased the cell clusters.

Vibrio cholerae VpsT regulates matrix production and motility by directly sensing cyclic di-GMP.

Microorganisms can switch from a planktonic, free-swimming life-style to a sessile, colonial state, called a biofilm, which confers resistance to environmental stress. Conversion between the motile and biofilm life-styles has been attributed to increased levels of the prokaryotic second messenger cyclic di-guanosine monophosphate (c-di-GMP), yet the signaling mechanisms mediating such a global switch are poorly understood. Here we show that the transcriptional regulator VpsT from Vibrio cholerae directly senses c-di-GMP to inversely control extracellular matrix production and motility, which identifies VpsT as a master regulator for biofilm formation. Rather than being regulated by phosphorylation, VpsT undergoes a change in oligomerization on c-di-GMP binding.

The rbmBCDEF gene cluster modulates development of rugose colony morphology and biofilm formation in Vibrio cholerae.

Vibrio cholerae, the causative agent of cholera, can undergo phenotypic variation generating rugose and smooth variants. The rugose variant forms corrugated colonies and well-developed biofilms and exhibits increased levels of resistance to several environmental stresses. Many of these phenotypes are mediated in part by increased expression of the vps genes, which are organized into vps-I and vps-II coding regions, separated by an intergenic region. In this study, we generated in-frame deletions of the five genes located in the vps intergenic region, termed rbmB to -F (rugosity and biofilm structure modulators B to F) in the rugose genetic background, and characterized the mutants for rugose colony development and biofilm formation. Deletion of rbmB, which encodes a protein with low sequence similarity to polysaccharide hydrolases, resulted in an increase in colony corrugation and accumulation of exopolysaccharides relative to the rugose variant. RbmC and its homolog Bap1 are predicted to encode proteins with carbohydrate-binding domains. The colonies of the rbmC bap1 double deletion mutant and bap1 single deletion mutant exhibited a decrease in colony corrugation. Furthermore, the rbmC bap1 double deletion mutant was unable to form biofilms at the air-liquid interface after 2 days, while the biofilms formed on solid surfaces detached readily. Although the colony morphology of rbmDEF mutants was similar to that of the rugose variant, their biofilm structure and cell aggregation phenotypes were different than those of the rugose variant. Taken together, these results indicate that vps intergenic region genes encode proteins that are involved in biofilm matrix production and maintenance of biofilm structure and stability.

Identification and characterization of RbmA, a novel protein required for the development of rugose colony morphology and biofilm structure in Vibrio cholerae.

Phase variation between smooth and rugose colony variants of Vibrio cholerae is predicted to be important for the pathogen's survival in its natural aquatic ecosystems. The rugose variant forms corrugated colonies, exhibits increased levels of resistance to osmotic, acid, and oxidative stresses, and has an enhanced capacity to form biofilms. Many of these phenotypes are mediated in part by increased production of an exopolysaccharide termed VPS. In this study, we compared total protein profiles of the smooth and rugose variants using two-dimensional gel electrophoresis and identified one protein that is present at a higher level in the rugose variant. A mutation in the gene encoding this protein, which does not have any known homologs in the protein databases, causes cells to form biofilms that are more fragile and sensitive to sodium dodecyl sulfate than wild-type biofilms. The results indicate that the gene, termed rbmA (rugosity and biofilm structure modulator A), is required for rugose colony formation and biofilm structure integrity in V. cholerae. Transcription of rbmA is positively regulated by the response regulator VpsR but not VpsT.

Isolation and characterization of two novel ethanol-tolerant facultative-anaerobic thermophilic bacteria strains from waste compost.

In a search for potential ethanologens, waste compost was screened for ethanol-tolerant thermophilic microorganisms. Two thermophilic bacterial strains, M5EXG and M10EXG, with tolerance of 5 and 10% (v/v) ethanol, respectively, were isolated. Both isolates are facultative anaerobic, non-spore forming, non-motile, catalase-positive, oxidase-negative, Gram-negative rods that are capable of utilizing a range of carbon sources including arabinose, galactose, mannose, glucose and xylose and produce low amounts of ethanol, acetate and lactate. Growth of both isolates was observed in fully defined minimal media within the temperature range 50-80 degrees C and pH 6.0-8.0. Phylogenetic analysis of the 16S rDNA sequences revealed that both isolates clustered with members of subgroup 5 of the genus Bacillus. G+C contents and DNA-DNA relatedness of M5EXG and M10EXG revealed that they are strains belonging to Geobacillus thermoglucosidasius. However, physiological and biochemical differences were evident when isolates M5EXG and M10EXG were compared with G. thermoglucosidasius type strain (DSM 2542(T)). The new thermophilic, ethanol-tolerant strains of G. thermoglucosidasius may be candidates for ethanol production at elevated temperatures.