Screening The Survive In Vivo-related Genes Of Riemerella Anatipestifer By Signature-tagged Transposon Mutagenesis

Riemerella anatipestifer infection,also called duck infectious serositis,is a kind of contagious disease infecting ducks,geese,turkeys and other birds.It can cause huge economic losses to the duck industry worldwide especially China.The pathogen,R.anatipestifer,is the type species of genus Riemerella in the family Flavobacteriaceae.Currently,at least 21 serotypies has been identified,but no or little cross protection was found among different serotypes,which has cause great difficulties for the disease control and prevention.So far,little is known about the molecular mechanism of pathogenesis.In this study,the transposon random mutation libraries with different singal tags of serotype 1 R.anatipestifer strainWJ4 were established to screen the survive in vivo-related genes using signature-tagged transposon mutagenesis.In this study,17 different tagged transposon based random mutation libraries of R.anatipestifer strain WJ4 containing 5100 mutants were constructed.In the first screen,sixty-three mutants which cannot be detected from recovered pools were screened out based on the infection experiment of inoculated ducklings,and then after several screening,20 mutants,which cannot be detected from single mutant infected ducklings,were screened out.The transposon insertion sites on the chrosome in 17 out of 20 mutants were determined by inverse-PCR.The mutants 7-295 and 16-284 were selected to construct the corresponding complemented strains.The related genes were amplificated from the wild type WJ4 by PCR and cloned into E.coli-R.anatipestifer shuttle express vector pRES1 respectively.The complemented plasmids were introducted into the mutants 7-295 and 16-284 by biparental mating respectively,to generate the complemented strains c7-295 and c16-284.Then,the growth curves and the blood bacterial loads from the wild strain WJ4,the mutants 7-295,16-284,and the complemented strains c7-295 and c16-284 infected ducklings were compared.The results showed that there was no significantly difference on the growth curves among the wild strain WJ4,the mutants and the complemented strains.In addition,the blood bacterial loads of the mutant 7-295 or 16-284 infected duckings at 24 h after inoculation were zero,while that of the complemented strain c16-284 was partily restored,but not that of the complemented strain c7-295.In conclusion,in this study,17 putative survive in vivo-related genes were screen out based on signature-tagged transposon mutagenesis using serotype 1 R.anatipestifer strain WJ4,and partial biological characteristics two mutants 7-295 and 16-284 were analyzed.This study has laid a foundation for the further studying the molecular mechanisms of pathogenesis of R.anatipestifer infection.