Genome-Wide SNP-genotyping array to study the evolution of the human pathogen Vibrio vulnificus Biotype 3

Vibrio vulnificus is an aquatic bacterium and an important human pathogen. Strains Of V. vulnificus are classified into three different biotypes. The newly emerged biotype 3 has been found to be clonal and restricted to Israel. In the family Vibrionaceae , horizontal gene transfer is the main mechanism responsible for the emergence of new pathogen groups. To better understand the evolution of the bacterium, and in particular to trace the evolution of biotype 3, we performed genome-wide SNP genotyping of 254 clinical and environmental V. vulnificus isolates with worldwide distribution recovered over a 30-year period, representing all phylogeny groups. A custom single-nucleotide polymorphism (SNP) array implemented on the Illumina GoldenGate platform was developed based on 570 SNPs randomly distributed throughout the genome. In general, the genotyping results divided the V. vulnificus species into three main phylogenetic lineages and an additional subgroup, clade B, consisting of environmental and clinical isolates from Israel. Data analysis suggested that 69% of biotype 3 SNPs are similar to SNPs from clade B, indicating that biotype 3 and clade B have a common ancestor. The rest of the biotype 3 SNPs were scattered along the biotype 3 genome, probably representing multiple chromosomal segments that may have been horizontally inserted into the clade B recipient core genome from other phylogroups or bacterial species sharing the same ecological niche. Results emphasize the continuous evolution of V. vulnificus and support the emergence of new pathogenic groups within this species as a recurrent phenomenon. Our findings contribute to a broader understanding of the evolution of this human pathogen

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

Mutator Suppression and Escape from Replication Error–Induced Extinction in Yeast

Cells rely on a network of conserved pathways to govern DNA replication fidelity. Loss of polymerase proofreading or mismatch repair elevates spontaneous mutation and facilitates cellular adaptation. However, double mutants are inviable, suggesting that extreme mutation rates exceed an error threshold. Here we combine alleles that affect DNA polymerase δ (Pol δ) proofreading and mismatch repair to define the maximal error rate in haploid yeast and to characterize genetic suppressors of mutator phenotypes. We show that populations tolerate mutation rates 1,000-fold above wild-type levels but collapse when the rate exceeds 10−3 inactivating mutations per gene per cell division. Variants that escape this error-induced extinction (eex) rapidly emerge from mutator clones. One-third of the escape mutants result from second-site changes in Pol δ that suppress the proofreading-deficient phenotype, while two-thirds are extragenic. The structural locations of the Pol δ changes suggest multiple antimutator mechanisms. Our studies reveal the transient nature of eukaryotic mutators and show that mutator phenotypes are readily suppressed by genetic adaptation. This has implications for the role of mutator phenotypes in cancer

Apparent Loss of Vibrio vulnificus from North Carolina Oysters Coincides with a Drought-Induced Increase in Salinity

Despite years of successful isolation of Vibrio vulnificus from estuarine waters, beginning in 2007, it was extremely difficult to culture V. vulnificus from either North Carolina estuarine water or oyster samples. After employing culture-based methods as well as PCR and quantitative PCR for the detection of V. vulnificus, always with negative results, we concluded that this pathogen had become nearly undetectable in the North Carolina estuarine ecosystem. We ensured that the techniques were sound by seeding North Carolina oysters with V. vulnificus and performing the same tests as those previously conducted on unadulterated oysters. V. vulnificus was readily detected in the seeded oysters using both classes of methods. Furthermore, oysters were obtained from the Gulf of Mexico, and V. vulnificus was easily isolated, confirming that the methodology was sound but that the oysters and waters of North Carolina were lacking the V. vulnificus population studied for decades. Strikingly, the apparent loss of detectable V. vulnificus coincided with the most severe drought in the history of North Carolina. The drought continued until the end of 2009, with an elevated water column salinity being observed throughout this period and with V. vulnificus being nearly nonexistent. When salinities returned to normal after the drought abated in 2010, we were again able to routinely isolate V. vulnificus from the water column, although we were still unable to culture it from oysters. We suggest that the oysters were colonized with a more salt-tolerant bacterium during the drought, which displaced V. vulnificus and may be preventing recolonization

A highly diverse, desert-like microbial biocenosis on solar panels in a Mediterranean city

Microorganisms colonize a wide range of natural and artificial environments although there are hardly any data on the microbial ecology of one the most widespread man-made extreme structures: solar panels. Here we show that solar panels in a Mediterranean city (Valencia, Spain) harbor a highly diverse microbial community with more than 500 different species per panel, most of which belong to drought-, heat- and radiation-adapted bacterial genera, and sun-irradiation adapted epiphytic fungi. The taxonomic and functional profiles of this microbial community and the characterization of selected culturable bacteria reveal the existence of a diverse mesophilic microbial community on the panels’ surface. This biocenosis proved to be more similar to the ones inhabiting deserts than to any human or urban microbial ecosystem. This unique microbial community shows different day/night proteomic profiles; it is dominated by reddish pigment- and sphingolipid-producers, and is adapted to withstand circadian cycles of high temperatures, desiccation and solar radiation