Pyrosequencing-based comparative genome analysis of Vibrio vulnificus environmental isolates

Abstract

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.

Figure 1. Circular maps of the sequence contigs of V. vulnificus JY1305, JY1701, and E64MW.

From the outside in, the first circle (red) represents V. vulnificus JY1305 genomic contigs, the second circle (green) represents V. vulnificus JY1701 genomic contigs, and third circle (blue) represents V. vulnificus E64MW genomic contigs. The circles represent BLAST alignment of contigs against the V. vulnificus CMCP6 reference genome. Circle 4 shows GC content. Figure generated using CGView .

Figure 2. Genomic alignment of Vibrio vulnificus Biotype 1 strains CMCP6, YJ016, MO6-24/O, JY1305, E64MW, and JY1701.

Locally conserved block based alignment between the reference genome CMCP6 and the newly sequenced genomes of JY1305, E64MW, and JY1701 as locally collinear blocks (LCB). Figure generated using Mauve .

Figure 3. Vibrio vulnificus genomic content differential Venn diagram.

A 6-way Venn diagram representing the differential and shared gene counts between the V. vulnificus YJ016, CMCP6, MO6-24/O, JY1305, E64MW, and JY1701. The main Venn diagram represents the overlap between C- and E- genotype groups, while the nested Venn diagrams represent the content relationships among the individual C-genotype or E-genotype strains. Gene counts are based on presence or absence of orthologs, where orthology is defined by OrthoMCL , using as a clustering criterion shared sequence similarity of 70% or greater.

Figure 4. Phylogenetic relationships among sequenced Vibrio genomes.

Phylogenetic relationships computed using maximum likelihood estimation, from a random sampling of 175 single copy gene ortholog sequences common among the newly sequenced E-genotype genomes and other sequenced Vibrio species. Three randomly sampled replicates produce trees with highly similar topologies. Purple box indicates strains classified as C-genotypes and green box indicates strains classified as E-genotypes for V. vulnificus.

Figure 5. Gene Ontology (GO) functional differences between C- and E- genotypes.

Figure shows GO functional categories which are enriched in C-genotypes of V. vulnificus relative to E-genotypes (blue) or E-genotypes relative to C-genotypes (red). Percentages represent percent of genes under each category that are differential between the genotypes. Percentages of less than 20% are not depicted.