The ecology of C-genotype and E-genotype strains of the bacterium Vibrio vulnificus and their interactions with the American oyster Crassostrea virginica

Vibrio vulnificus is a pathogenic bacterium, routinely found in waters of estuarine environments as part of the normal microflora. This organism can be divided into two genotypes, a C-type associated with clinical isolation, and an E-type associated with environmental isolation. While it was previously known that C- and E-type cells were genetically distinct, a further distinction was found among C-type strains that has the potential to predict pathogenicity using simple PCR. It was also found that C- types cells are more rapidly taken up by oyster hosts than E-type cells in some cases, but that depuration was just as rapid for both types. These studies revealed that addition of laboratory grown bacterial strains can cause endogenous bacteria in oyster to resuscitate from the viable-but-non-culturable state. Inefficiencies in bacterial uptake in oysters led to a study that concludes that E-type strains are more readily integrated into marine aggregates that C-type strains. Most notably, V. vulnificus was observed to decline in North Carolina estuaries in response to a prolonged and severe drought

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

Key differential genes found in <i>V. vulnificus</i> E-genotypes but not in C-genotypes.

*<p>indicates there are more than 1 GO term at the lowest level for this gene. +indicates that no significant GO term was associated with gene. Significance adjusted-p value <.005. Box highlights differential genes which aligned to locally conserved blocks in Chromosome 1 of <i>V. vulnificus</i> CMCP6, suggesting a possible location on Chromosome 1 in the E-genotype genomes.</p

<i>Vibrio vulnificus</i> genomic content differential Venn diagram.

<p>A 6-way Venn diagram representing the differential and shared gene counts between the <i>V. vulnificus</i> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037553#pone.0037553-Li2" target="_blank">[74]</a>, using as a clustering criterion shared sequence similarity of 70% or greater.</p

Circular maps of the sequence contigs of <i>V. vulnificus</i> JY1305, JY1701, and E64MW.

<p>From the outside in, the first circle (red) represents <i>V. vulnificus</i> JY1305 genomic contigs, the second circle (green) represents <i>V. vulnificus</i> JY1701 genomic contigs, and third circle (blue) represents <i>V. vulnificus</i> E64MW genomic contigs. The circles represent BLAST alignment of contigs against the <i>V. vulnificus</i> CMCP6 reference genome. Circle 4 shows GC content. Figure generated using CGView <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037553#pone.0037553-Stothard1" target="_blank">[21]</a>.</p

Phylogenetic relationships among sequenced <i>Vibrio</i> genomes.

<p>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 <i>Vibrio</i> 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 <i>V. vulnificus</i>.</p

Key differential genes between found in <i>V. vulnificus</i> C-genotypes that are NOT present in the E-genotypes.

*<p>indicates there are more than 1 GO term at the lowest level for this gene. +indicates that no significant GO term was associated with gene. Significance adjusted-p value <.005. Box highlights genes that are found on Chromosome 1 of <i>V. vulnificus</i> CMCP6. All other differential genes are found on Chromosome 2.</p

Summary of assembly and annotation characteristics for the <i>V. vulnificus</i> JY1305, E64MW, and JY1701 genomes.

<p>Summary of assembly and annotation characteristics for the <i>V. vulnificus</i> JY1305, E64MW, and JY1701 genomes.</p