Rugosity in Grimontia hollisae

Grimontia hollisae, formerly Vibrio hollisae, produces both smooth and rugose colonial variants. The rugose colony phenotype is characterized by wrinkled colonies producing copious amounts of exopolysaccharide. Cells from a rugose colony grown at 30 degrees C form rugose colonies, while the same cells grown at 37 degrees C form smooth colonies, which are characterized by a nonwrinkled, uncrannied appearance. Stress response studies revealed that after exposure to bleach for 30 min, rugose survivors outnumbered smooth survivors. Light scatter information obtained by flow cytometry indicated that rugose cells clumped into clusters of three or more cells (average, five cells) and formed two major clusters, while smooth cells formed only one cluster of single cells or doublets. Fluorescent lectin-binding flow cytometry studies revealed that the percentages of rugose cells that bound either wheat germ agglutinin (WGA) or Galanthus nivalis lectin (GNL) were greater than the percentages of smooth cells that bound the same lectins (WGA, 35% versus 3.5%; GNL, 67% versus 0.21%). These results indicate that the rugose exopolysaccharide consists partially of N-acetylglucosamine and mannose. Rugose colonies produced significantly more biofilm material than did smooth colonies, and rugose colonies grown at 30 degrees C produced more biofilm material than rugose colonies grown at 37 degrees C. Ultrastructurally, rugose colonies show regional cellular differentiation, with apical and lateral colonial regions containing cells embedded in a matrix stained by Alcian Blue. The cells touching the agar surface are packed tightly together in a palisade-like manner. The central region of the colony contains irregularly arranged, fluid-filled spaces and loosely packed chains or arrays of coccoid and vibrioid cells. Smooth colonies, in contrast, are flattened, composed of vibrioid cells, and lack distinct regional cellular differences. Results from suckling mouse studies showed that both orally fed rugose and smooth variants elicited significant, but similar, amounts of fluid accumulated in the stomach and intestines. These observations comprise the first report of expression and characterization of rugosity by G. hollisae and raise the possibility that expression of rugose exopolysaccharide in this organism is regulated at least in part by growth temperature.     

Rugosity in Grimontia hollisae

Grimontia hollisae, formerly Vibrio hollisae, produces both smooth and rugose colonial variants. The rugose colony phenotype is characterized by wrinkled colonies producing copious amounts of exopolysaccharide. Cells from a rugose colony grown at 30°C form rugose colonies, while the same cells grown at 37°C form smooth colonies, which are characterized by a nonwrinkled, uncrannied appearance. Stress response studies revealed that after exposure to bleach for 30 min, rugose survivors outnumbered smooth survivors. Light scatter information obtained by flow cytometry indicated that rugose cells clumped into clusters of three or more cells (average, five cells) and formed two major clusters, while smooth cells formed only one cluster of single cells or doublets. Fluorescent lectin-binding flow cytometry studies revealed that the percentages of rugose cells that bound either wheat germ agglutinin (WGA) or Galanthus nivalis lectin (GNL) were greater than the percentages of smooth cells that bound the same lectins (WGA, 35% versus 3.5%; GNL, 67% versus 0.21%). These results indicate that the rugose exopolysaccharide consists partially of N-acetylglucosamine and mannose. Rugose colonies produced significantly more biofilm material than did smooth colonies, and rugose colonies grown at 30°C produced more biofilm material than rugose colonies grown at 37°C. Ultrastructurally, rugose colonies show regional cellular differentiation, with apical and lateral colonial regions containing cells embedded in a matrix stained by Alcian Blue. The cells touching the agar surface are packed tightly together in a palisade-like manner. The central region of the colony contains irregularly arranged, fluid-filled spaces and loosely packed chains or arrays of coccoid and vibrioid cells. Smooth colonies, in contrast, are flattened, composed of vibrioid cells, and lack distinct regional cellular differences. Results from suckling mouse studies showed that both orally fed rugose and smooth variants elicited significant, but similar, amounts of fluid accumulated in the stomach and intestines. These observations comprise the first report of expression and characterization of rugosity by G. hollisae and raise the possibility that expression of rugose exopolysaccharide in this organism is regulated at least in part by growth temperature.     

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.     

Cyclic-diGMP signal transduction systems in Vibrio cholerae: modulation of rugosity and biofilm formation

Cyclic di-guanylic acid (c-diGMP) is a second messenger that modulates the cell surface properties of several microorganisms. Concentrations of c-diGMP in the cell are controlled by the opposing activities of diguanylate cyclases and phosphodiesterases, which are carried out by proteins harbouring GGDEF and EAL domains respectively. In this study, we report that the cellular levels of c-diGMP are higher in the Vibrio cholerae rugose variant compared with the smooth variant. Modulation of cellular c-diGMP levels by overexpressing proteins with GGDEF or EAL domains increased or decreased colony rugosity respectively. Several genes encoding proteins with either GGDEF or EAL domains are differentially expressed between the two V. cholerae variants. The generation and characterization of null mutants of these genes (cdgA-E, rocS and mbaA) revealed that rugose colony formation, exopolysaccharide production, motility and biofilm formation are controlled by their action. Furthermore, epistasis analysis suggested that cdgC, rocS and mbaA act in convergent pathways to regulate the phenotypic properties of the rugose and smooth variants, and are part of the VpsR, VpsT and HapR signal transduction pathway.     

Phenotypic and genetic differences between opaque and translucent colonies of Vibrio alginolyticus

Many pathogens undergo phase variation between rugose and smooth colony morphology or between opaque and translucent colony morphology, which is mainly due to the variation in the surface polysaccharides. In this study, Vibrio alginolyticus ZJ-51 displayed phase variation between opaque, rugose colonies (Op) and translucent, smooth colonies (Tr). Unlike the vibrios reported previously, Tr cells of ZJ-51 enhanced biofilm formation and motility, but they did not differ from Op cells in the quantity of surface polysaccharides produced. Real time PCR was used to analyze the expression of the genes involved in polysaccharide biosynthesis, flagellar synthesis, and the AI-2 quorum-sensing system. The results revealed that the K-antigen capsule gene cluster (which consists of homologs to the cpsA-K in Vibrio parahaemolyticus) and O-antigen polysaccharide gene cluster (which contains homologs to the wza-wzb-wzc) were significantly more transcribed in Tr cells. The AI-2 quorum-sensing genes showed enhanced expression in the Tr variant which also exhibited greater expression of genes associated with polar flagellar biosynthesis. These results suggest that colony phase variation might affect the virulence and survival ability in the stressful environment inhabited by V. alginolyticus.     

Phase variation, capsular polysaccharide, pilus and flagella contribute to uptake of Vibrio vulnificus by the Eastern oyster (Crassostrea virginica)

Vibrio vulnificus infections are associated with raw oyster consumption, and disease reservoirs are determined by the ability of this bacterium to infect and persist in oysters. Surface structures, such as capsular polysaccharide (CPS), pili and flagella, function as virulence factors in mouse infection models. Furthermore, virulence is related to phase variation in colony morphology, which reflects CPS expression and includes opaque (encapsulated, virulent), translucent (reduced encapsulation, avirulent) and rugose (wrinkled, biofilm-enhanced) colony types. The role of these factors in environmental survival is unknown; therefore, mutational analysis and phase variation of V. vulnificus were examined in an oyster infection model. Oysters ( Crassostrea virginica ) were pre-treated with tetracycline to reduce background bacteria and subsequently inoculated via filter feeding with 10⁶ colony-forming units (cfu) ml⁻¹ of V. vulnificus wild-type strains and phase variants, as well as strains with deletion mutations in genes related to CPS (Δ wza ), pili (Δ pilA ), flagella (Δ flaCDE/ Δ flaFBA ) and motility (Δ motAB ). All mutants were significantly reduced in their dissemination to oyster haemolymph as compared with wild type; however, recovery of mutants from gills and intestinal tissue was generally similar to wild type. Translucent and rugose inocula showed induction of high-frequency phase variation to the opaque encapsulated phenotype (100% and 72% respectively) during oyster infections that did not occur in strains recovered from seawater. Thus, multiple bacterial factors determine uptake of V. vulnificus in oysters, and phase variation during oyster infection is a likely mechanism for environmental survival and for induction of the more virulent phenotype.     

A gene co-expression network in whole blood of schizophrenia patients is independent of antipsychotic-use and enriched for brain-expressed genes

Despite large-scale genome-wide association studies (GWAS), the underlying genes for schizophrenia are largely unknown. Additional approaches are therefore required to identify the genetic background of this disorder. Here we report findings from a large gene expression study in peripheral blood of schizophrenia patients and controls. We applied a systems biology approach to genome-wide expression data from whole blood of 92 medicated and 29 antipsychotic-free schizophrenia patients and 118 healthy controls. We show that gene expression profiling in whole blood can identify twelve large gene co-expression modules associated with schizophrenia. Several of these disease related modules are likely to reflect expression changes due to antipsychotic medication. However, two of the disease modules could be replicated in an independent second data set involving antipsychotic-free patients and controls. One of these robustly defined disease modules is significantly enriched with brain-expressed genes and with genetic variants that were implicated in a GWAS study, which could imply a causal role in schizophrenia etiology. The most highly connected intramodular hub gene in this module (ABCF1), is located in, and regulated by the major histocompatibility (MHC) complex, which is intriguing in light of the fact that common allelic variants from the MHC region have been implicated in schizophrenia. This suggests that the MHC increases schizophrenia susceptibility via altered gene expression of regulatory genes in this network.     

Smooth to rugose phase variation in Vibrio cholerae can be mediated by a single nucleotide change that targets c-di-GMP signalling pathway

Microorganisms use phase variation to increase population diversity to maximize evolutionary success. One such variation is the smooth to rugose phenotype change in Vibrio cholerae. We determined that the variation between smooth and rugose phenotypes can be controlled by a single nucleotide change in a gene (vpvC) predicted to encode a diguanylate cyclase. The vpvC allele found in the rugose genetic background is more active at producing c-di-GMP while that in smooth genetic background is less active. In support of this finding, disruption of vpvC in the rugose genetic variant decreases cellular c-di-GMP levels, diminishes rugose-associated phenotypes and yields a smooth variant. Furthermore, the frequency of phase variation decreases dramatically when the vpvC locus is deleted in the smooth genetic background. Evidence is presented that the rugose variant is less susceptible to phage infection than the smooth variant. As phage infection is known to control populations of V. cholerae and thus outbreaks of cholera, phase variation may increase the evolutionary success of the pathogen.     

Rare variants regulate expression of nearby individual genes in multiple tissues

The rapid decrease in sequencing cost has enabled genetic studies to discover rare variants associated with complex diseases and traits. Once this association is identified, the next step is to understand the genetic mechanism of rare variants on how the variants influence diseases. Similar to the hypothesis of common variants, rare variants may affect diseases by regulating gene expression, and recently, several studies have identified the effects of rare variants on gene expression using heritability and expression outlier analyses. However, identifying individual genes whose expression is regulated by rare variants has been challenging due to the relatively small sample size of expression quantitative trait loci studies and statistical approaches not optimized to detect the effects of rare variants. In this study, we analyze whole-genome sequencing and RNA-seq data of 681 European individuals collected for the Genotype-Tissue Expression (GTEx) project (v8) to identify individual genes in 49 human tissues whose expression is regulated by rare variants. To improve statistical power, we develop an approach based on a likelihood ratio test that combines effects of multiple rare variants in a nonlinear manner and has higher power than previous approaches. Using GTEx data, we identify many genes regulated by rare variants, and some of them are only regulated by rare variants and not by common variants. We also find that genes regulated by rare variants are enriched for expression outliers and disease-causing genes. These results suggest the regulatory effects of rare variants, which would be important in interpreting associations of rare variants with complex traits.     

VpsR, a Member of the Response Regulators of the Two-Component Regulatory Systems, Is Required for Expression of vps Biosynthesis Genes and EPS(ETr)-Associated Phenotypes in Vibrio cholerae O1 El Tor

The rugose colonial variant of Vibrio cholerae O1 El Tor produces an exopolysaccharide (EPS(ETr)) that enables the organism to form a biofilm and to resist oxidative stress and the bactericidal action of chlorine. Transposon mutagenesis of the rugose variant led to the identification of vpsR, which codes for a homologue of the NtrC subclass of response regulators. Targeted disruption of vpsR in the rugose colony genetic background yielded a nonreverting smooth-colony morphotype that produced no detectable EPS(ETr) and did not form an architecturally mature biofilm. Analysis of two genes, vpsA and vpsL, within the vps cluster of EPS(ETr) biosynthesis genes revealed that their expression is induced above basal levels in the rugose variant, compared to the smooth colonial variant, and requires vpsR. These results show that VpsR functions as a positive regulator of vpsA and vpsL and thus acts to positively regulate EPS(ETr) production and biofilm formation.