Genetic analysis and prevalence studies of the brp exopolysaccharide locus of Vibrio vulnificus

Phase variation in the Gram-negative human pathogen Vibrio vulnificus involves three colonial morphotypes- smooth opaque colonies due to production of capsular polysaccharide (CPS), smooth translucent colonies as the result of little or no CPS expression, and rugose colonies due to production of a separate extracellular polysaccharide (EPS), which greatly enhances biofilm formation. Previously, it was shown that the brp locus, which consists of nine genes arranged as an operon, is up-regulated in rugose strains in a c-di-GMP-dependent manner, and that plasmid insertions into the locus resulted in loss of rugosity and efficient biofilm production. Here, we have used non-polar mutagenesis to assess the involvement of individual brp genes in production of EPS and related phenotypes. Inactivation of genes predicted to be involved in various stages of EPS biosynthesis eliminated both the rugose colonial appearance and production of EPS, while knockout of a predicted flippase function involved in EPS transport resulted in a dry, lightly striated phenotype, which was associated with a reduction of brp-encoded EPS on the cell surface. All brp mutants retained the reduced motility characteristic of rugose strains. Lastly, we provide evidence that the brp locus is highly prevalent among strains of V. vulnificus. © 2014 Garrison-Schilling et al

Capsular polysaccharide production and serum survival of Vibrio vulnificus are dependent on antitermination control by RfaH

© 2016 Federation of European Biochemical Societies The human pathogen Vibrio vulnificus undergoes phase variation among colonial morphotypes, including a virulent opaque form which produces capsular polysaccharide (CPS) and a translucent phenotype that produces little or no CPS and is attenuated. Here, we found that a V. vulnificus mutant defective for RfaH antitermination control showed a diminished capacity to undergo phase variation and displayed significantly reduced distal gene expression within the Group I CPS operon. Moreover, the rfaH mutant produced negligible CPS and was highly sensitive to killing by normal human serum, results which indicate that RfaH is likely essential for virulence in this bacterium

No interaction between serotonin transporter gene (5-HTTLPR) polymorphism and adversity on depression among Japanese children and adolescents

Background: Identification of gene × environment interactions (G × E) for depression is a crucial step in ascertaining the mechanisms underpinning the disorder. Earlier studies have indicated strong genetic influences and numerous environmental risk factors. In relation to childhood and adolescent depression, evidence is accumulating that the quality of the parental environment is associated with serotonin biology in children. We hypothesized that maternal depression is a crucial environmental risk factor associated with serotonin-regulating genes.Methods: This study was designed to ascertain the G × E interaction for diagnosis of depression in a Japanese pediatric sample. DNA samples from 55 pediatric patients with depression and 58 healthy schoolchildren were genotyped for the 5-HTT (2 short (S) alleles at the 5-HTT locus) promoter serotonin-transporter-linked polymorphic region (5-HTTLPR) polymorphism. We examined whether an adverse parental environment, operationalized as the mother\u27s history of recurrent major depressive disorder, interacts with 5-HTTLPR polymorphism to predict patients\u27 depression symptoms.Results: Binary logistic regression analyses revealed that maternal depression (adversity), gender, and FSIQ significantly affect the diagnosis of depression among children and adolescents. However, no main effect was found for adversity or genotype. Results of multivariable logistic regression analyses using stepwise procedure have elicited some models with a good fit index, which also suggests no interaction between 5-HTTLPR and adversity on depression.Conclusions: To assess G × E interaction, data obtained from children and adolescents who had been carefully diagnosed categorically and data from age-matched controls were analyzed using logistic regression. Despite an equivocal interaction effect, adversity and gender showed significant main effects

Identification of genetic and environmental factors that control exopolysaccharide expression and phase variation in the human pathogen Vibrio vulnificus

Vibrio vulnificus is a gram-negative bacterium found in estuaries and coastal waters and is associated with human disease caused by the ingestion of raw shellfish. Pathogenesis is directly related to the presence of capsular polysaccharide (CPS). Encapsulated virulent strains exhibit an opaque colony phenotype (OpS), while unencapsulated attenuated strains appear translucent (TrS). A third colony type, rugose (R), is caused by expression of rugose extracellular polysaccharide (rEPS) and forms robust biofilms. V. vulnificus undergoes spontaneous phase variation associated with altered levels of CPS and rEPS, and the work presented here identifies genetic and environmental parameters that control this process. A cluster of nine genes (brpABCDFHIJK) was found to be up-regulated in R isolates when compared to OpS or TrS isolates. We assessed the role of the brp gene cluster in CPS and rEPS production by creating non-polar mutants and characterizing their colony morphotypes, rEPS production, biofilm formation, and motility phenotypes. We demonstrate that the brp genes are required for rEPS production and robust biofilm formation, and that the decreased motility of R isolates is subject to regulation by the second messenger cyclic-di-GMP. Approximately 130-135bp upstream of brpA, we identify a promoter, which is activated at significantly higher levels in R variants than in TrS or OpS variants. In addition to characterizing the genetic components of rEPS phase variation, we show that environmental factors, such as media composition and temperature, influence the rate of polysaccharide phase variation. Specifically, calcium (Ca2+) significantly increases the rate of CPS and rEPS phase variation in V. vulnificus. Multiple phenotypic responses to increased [Ca2+] were observed among strains, which suggests the existence of underlying cognate genetic or epigenetic differences. Certain TrS isolates contained deletions at the group I CPS operon, inferring increased [Ca2+] up-regulates existing phase variation mechanisms. Expanding on a previous observation, increased [Ca2+] also enhanced biofilm formation for all phase variants. Our results show that [Ca2+] increases polysaccharide phase variation and contributes to biofilm formation, thereby likely playing a dual role in the persistence of V. vulnificus in the environment

Biofilm formation by individual <i>brp</i> gene mutants.

<p>Biofilm formation was assessed qualitatively and quantitatively for opaque, rugose, and translucent control variants, the 4 <i>brp</i> mutant strains, and the complemented mutants. <b>A</b>. Following ON growth with shaking, 3 cultures per strain were assessed for pellicle formation and a representative is pictured. Pellicle thickness was scored qualitatively as — (no pellicle), + (thin pellicle), ++ (pellicle), or +++ (thick pellicle). <b>B</b>. Biofilm assays were performed on at least 6 independent culture replicates of each statically grown strain and OD₅₇₀ values, which correspond to the amount of crystal violet staining of biofilm material, were averaged. Averages ± standard deviations (SD) are pictured here. Asterisk denotes <i>p</i><0.001 versus KG3(R).</p

Phenotypes of individual <i>brp</i> gene mutants.

<p><b>A</b>. Diagram of mutant construction for 4 <i>brp</i> genes. Open rectangles indicate the non-polar Kan^R cassette, while the filled rectangle indicates the mini-Tn<i>10</i> insertion in strain TDB3(T) (41). Shading of arrows for the <i>brp</i> cluster indicates putative function encoded: black, flippase involved in EPS transport (for <i>brpJ</i>) or EPS export-related protein (for <i>brpC</i>); light grey, tyrosine autokinase involved in EPS biosynthesis; dark grey, glycosyltransferase involved in EPS biosynthesis; white, unknown function. <b>B</b>. Colony morphology of opaque, rugose, and translucent control variants, the 4 <i>brp</i> mutant strains derived from strain KG3(R), and the complemented mutants. Strains were streaked for isolation on HI agar (containing kanamycin, chloramphenicol and arabinose, as appropriate) and incubated at 30°C ON. <b>C</b>. Streak plate of opaque, rugose, and translucent control variants and the 4 <i>brp</i> mutant strains. Strains were inoculated into HI broth (with kanamycin, where appropriate), shaken ON at 30°C, streaked onto HI (with no antibiotics), and incubated ON at 30°C. <b>D</b>. Colony morphology of the YJ016-derived <i>brpJ</i> mutant YJ-10. The strain was streaked for isolation on HI agar containing kanamycin and incubated at 30°C ON.</p

Evidence of EPS production for <i>brpJ</i> mutant strain KG3-03.

<p>An EPS extraction procedure (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100890#s2" target="_blank">Materials and Methods</a> for details) was performed on KG3(R) and the 4 <i>brp</i> mutant strains, and the results were analyzed by SDS-PAGE using 4% stacking/10% resolving gels and subsequent staining with Stains-All.</p