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

Genetic analysis of the capsule polysaccharide (K antigen) and exopolysaccharide genes in pandemic Vibrio parahaemolyticus O3:K6

<p>Abstract</p> <p>Background</p> <p>Pandemic <it>Vibrio parahaemolyticus </it>has undergone rapid changes in both K- and O-antigens, making detection of outbreaks more difficult. In order to understand these rapid changes, the genetic regions encoding these antigens must be examined. In <it>Vibrio cholerae </it>and <it>Vibrio vulnificus</it>, both O-antigen and capsular polysaccharides are encoded in a single region on the large chromosome; a similar arrangement in pandemic <it>V. parahaemolyticus </it>would help explain the rapid serotype changes. However, previous reports on "capsule" genes are controversial. Therefore, we set out to clarify and characterize these regions in pandemic <it>V. parahaemolyticus </it>O3:K6 by gene deletion using a chitin based transformation strategy.</p> <p>Results</p> <p>We generated different deletion mutants of putative polysaccharide genes and examined the mutants by immuno-blots with O and K specific antisera. Our results showed that O- and K-antigen genes are separated in <it>V. parahaemolyticus </it>O3:K6; the region encoding both O-antigen and capsule biosynthesis in other vibrios, i.e. genes between <it>gmhD </it>and <it>rjg</it>, determines the K6-antigen but not the O3-antigen in <it>V. parahaemolyticus</it>. The previously identified "capsule genes" on the smaller chromosome were related to exopolysaccharide synthesis, not K-antigen.</p> <p>Conclusion</p> <p>Understanding of the genetic basis of O- and K-antigens is critical to understanding the rapid changes in these polysaccharides seen in pandemic <it>V. parahaemolyticus. </it>This report confirms the genetic location of K-antigen synthesis in <it>V. parahaemolyticus </it>O3:K6 allowing us to focus future studies of the evolution of serotypes to this region.</p

Over-expression of the IGI1 leading to altered shoot-branching development related to MAX pathway in Arabidopsis

Shoot branching and growth are controlled by phytohormones such as auxin and other components in Arabidopsis. We identified a mutant (igi1) showing decreased height and bunchy branching patterns. The phenotypes reverted to the wild type in response to RNA interference with the IGI1 gene. Histochemical analysis by GUS assay revealed tissue-specific gene expression in the anther and showed that the expression levels of the IGI1 gene in apical parts, including flowers, were higher than in other parts of the plants. The auxin biosynthesis component gene, CYP79B2, was up-regulated in igi1 mutants and the IGI1 gene was down-regulated by IAA treatment. These results indicated that there is an interplay regulation between IGI1 and phytohormone auxin. Moreover, the expression of the auxin-related shoot branching regulation genes, MAX3 and MAX4, was down-regulated in igi1 mutants. Taken together, these results indicate that the overexpression of the IGI1 influenced MAX pathway in the shoot branching regulation

Protein Domain of Unknown Function 3233 is a Translocation Domain of Autotransporter Secretory Mechanism in Gamma proteobacteria

Vibrio cholerae, the enteropathogenic gram negative bacteria is one of the main causative agents of waterborne diseases like cholera. About 1/3rd of the organism's genome is uncharacterised with many protein coding genes lacking structure and functional information. These proteins form significant fraction of the genome and are crucial in understanding the organism's complete functional makeup. In this study we report the general structure and function of a family of hypothetical proteins, Domain of Unknown Function 3233 (DUF3233), which are conserved across gram negative gammaproteobacteria (especially in Vibrio sp. and similar bacteria). Profile and HMM based sequence search methods were used to screen homologues of DUF3233. The I-TASSER fold recognition method was used to build a three dimensional structural model of the domain. The structure resembles the transmembrane beta-barrel with an axial N-terminal helix and twelve antiparallel beta-strands. Using a combination of amphipathy and discrimination analysis we analysed the potential transmembrane beta-barrel forming properties of DUF3233. Sequence, structure and phylogenetic analysis of DUF3233 indicates that this gram negative bacterial hypothetical protein resembles the beta-barrel translocation unit of autotransporter Va secretory mechanism with a gene organisation that differs from the conventional Va system

Pyrosequencing-Based Comparative Genome Analysis of Vibrio vulnificus Environmental Isolates

Between 1996 and 2006, the US Centers for Disease Control reported that the only category of food-borne infections increasing in frequency were those caused by members of the genus Vibrio. The Gram-negative bacterium Vibrio vulnificus is a ubiquitous inhabitant of estuarine waters, and is the number one cause of seafood-related deaths in the US. Many V. vulnificus isolates have been studied, and it has been shown that two genetically distinct subtypes, distinguished by 16S rDNA and other gene polymorphisms, are associated predominantly with either environmental or clinical isolation. While local genetic differences between the subtypes have been probed, only the genomes of clinical isolates have so far been completely sequenced. In order to better understand V. vulnificus as an agent of disease and to identify the molecular components of its virulence mechanisms, we have completed whole genome shotgun sequencing of three diverse environmental genotypes using a pyrosequencing approach. V. vulnificus strain JY1305 was sequenced to a depth of 33×, and strains E64MW and JY1701 were sequenced to lesser depth, covering approximately 99.9% of each genome. We have performed a comparative analysis of these sequences against the previously published sequences of three V. vulnificus clinical isolates. We find that the genome of V. vulnificus is dynamic, with 1.27% of genes in the C-genotype genomes not found in the E- genotype genomes. We identified key genes that differentiate between the genomes of the clinical and environmental genotypes. 167 genes were found to be specifically associated with environmental genotypes and 278 genes with clinical genotypes. Genes specific to the clinical strains include components of sialic acid catabolism, mannitol fermentation, and a component of a Type IV secretory pathway VirB4, as well as several other genes with potential significance for human virulence. Genes specific to environmental strains included several that may have implications for the balance between self-preservation under stress and nutritional competence

Characterization of Polysaccharide Biosynthesis, Structure and Regulation in Vibrio vulnificus

Vibrio vulnificus are marine bacteria causing fatal septicemia through wound infections or consumption of contaminated seafood. V. vulnificus is an excellent model for the study of surface polysaccharides, as it is capable of synthesizing capsular polysaccharide (CPS), lipopolysaccharide (LPS) and exopolysaccharide (EPS). V. vulnificus strains exhibit a multitude of carbotypes that evolve through unknown mechanisms. CPS is a confirmed virulence factor, but the genetics of its biosynthesis are unknown. The main objective of these experiments was to gain insight into the biosynthesis, regulation and evolution of ATCC 27562 outer surface polysaccharides. A miniTn10 transposon (Tn) system was used for mutagenesis and single insertions were confirmed through Southern analysis. A novel 25 kb CPS biosynthesis locus was identified through sequencing of regions surrounding Tn insertions; a region encoding putative LPS core biosynthetic functions was identified adjacent to the CPS cluster. The CPS locus contained features of O-antigen biosynthetic loci and was unusual in carrying characteristics of both group I and IV capsular biosynthetic loci. Mutations in this region resulted in elimination of CPS and LPS, and both were shown to be dependent on the activity of the polymerase Wzy. Evidence is presented here supporting horizontal transfer (HT) as a contributor to V. vulnificus CPS evolution. CPS regions of V. vulnificus 27562, YJ016 and CMCP6 contain strain specific genes surrounded by conserved regions, suggestive of HT. Moreover, a CPS locus virtually identical to that of 27562 was discovered in Shewanella putrefaciens strain 200. 27562 CPS is distinctive as it contains N-acetylmuramic acid. Genes encoding murA and murB activities were identified within the cluster and shown to be functionally redundant, supporting HT acquisition of this region. A screen of V. vulnificus gDNA library using CPS biosynthesis and transport mutants identified a cyclic diguanylate cyclase, dcpA. dcpA-mediated increase in cyclic diguanylate lead to EPS production, rugosity phenotypes and enhanced biofilm formation. Interestingly, virulence and motility were not affected suggesting complexity of cyclic diguanylate regulation in V. vulnificus, supported by the large number of cyclic diguanylate related proteins in Vulnificus strains.Ph

Cyclic-di-GMP Regulates Extracellular Polysaccharide Production, Biofilm Formation, and Rugose Colony Development by Vibrio vulnificus▿

Vibrio vulnificus is a human and animal pathogen that carries the highest death rate of any food-borne disease agent. It colonizes shellfish and forms biofilms on the surfaces of plankton, algae, fish, and eels. Greater understanding of biofilm formation by the organism could provide insight into approaches to decrease its load in filter feeders and on biotic surfaces and control the occurrence of invasive disease. The capsular polysaccharide (CPS), although essential for virulence, is not required for biofilm formation under the conditions used here. In other bacteria, increased biofilm formation often correlates with increased exopolysaccharide (EPS) production. We exploited the translucent phenotype of acapsular mutants to screen a V. vulnificus genomic library and identify genes that imparted an opaque phenotype to both CPS biosynthesis and transport mutants. One of these encoded a diguanylate cyclase (DGC), an enzyme that synthesizes bis-(3′-5′)-cyclic-di-GMP (c-di-GMP). This prompted us to use this DGC, DcpA, to examine the effect of elevated c-di-GMP levels on several developmental pathways in V. vulnificus. Increased c-di-GMP levels induced the production of an EPS that was distinct from the CPS and dramatically enhanced biofilm formation and rugosity in a CPS-independent manner. However, the EPS could not compensate for the loss of CPS production that is required for virulence. In contrast to V. cholerae, motility and virulence appeared unaffected by elevated levels of c-di-GMP