Comparative And Evolutionary Genomic Analysis Of Vibrio Parahaemolyticus

Backgroud Vibrio parahaemolyticus is an important pathogen which can cause global food poisoning.Outbreak of V.parahaemolyticus infections occurred since 1996 was linked to a proposed clonal complex,the pandemic group.The genomic DNA sequence of a pandemic strain RIMD2210633 of Vibrio parahaemolyticus has been determined in 2003 by Japanese scholars.The purpose of this study is to dissect genome plasticity and phylogeny of the populations of V.parahaemolyticus by microarray technology and microarray-based comparative genomics hybridization of large number representatively global strains.Methods Based on the genomic sequences of Vibrio parahaemolyticus,a total number of 4770 genes was choosen,then they were amplified by PCR and purified, finally they were printed onto glass slides.Two sets of hybridizations were performed by the method of two-fluorescence comparative hybridization to evaluate of the microarray quality and PCR method was carried to verify parts of microarray result. Microarray-based comparative genomics hybridization was performed with a collection of 174 strains of V.parahaemolyticus.Prokaryotic expression system was used to build several important Vibrio parahaemolyticus virulence-related gene expression vector.Results Successfully develop a batch of whole-genome DNA microarray of Vibrio parahaemolyticus.The results of quality evaluation show that microarray hybridization results is completely consistent with theory expectations and PCR verification results and then verify chips can be used in the folllowing comparative genomics hybridization according to the good quality nature of the chips.And we build up a method of microarray-based comparative genomic hybridization of Vibrio parahaemolyticus,and built up a set of microarray data analysis standard method.The six prokaryotic expression vectors of important virulence related genes of Vibrio parahaemolyticus were successfully constructed and it laid the material foundation for the subsequent studies. The phylogenetic analysis of microarray data generated a minimum spanning tree that depicted the population structure of the 174 strains.Strains were assigned into five complexes(C1 to C5),and those in each complex were closely related both genetically and phylogenetically.C3 and C4 represented highly virulent clinical clones,and C5 was closely correlated to non-virulent environment isolates.C2 and C3 constituted two different clonal complexes 'old-O3:K6 clone' and 'pandemic clone',respectively.C3 included all the 39 pandemic strains tested(trh~-,tdh~+ and GS-PCR~+),while C2 contained 12 pre-1996 'old' O3:K6 strains(trh~+,tdh~- and GS-PCR~-).The pandemic clone(post-1996 'new' O3:K6 and its derivates O4:K68, O1:K25,O1:KUT and O6:K18) was emerged from the old-O3:K6 clone,which was promoted by the emerging of toxRS/new sequence and the stepwise acquisition of genomic islands.A phylogenetic intermediate O3:K6 clade(trh-,tdh- and GS-PCR~+) was identified between the pandemic and old-O3:K6 clones.Variably-present genes of the 174 strains accounted for about 22%of the whole gene pool on the genome.Conclusion A comprehensive overview of genomic contents in a large collection of global isolates from the microarray-based comparative genomic hybridization data enabled us to construct a phylogenetic structure of V.parahaemolyticus and an evolutionary history of the pandemic group(clone) of this pathogen.