Construction Of Transposon Insertion Mutant Library Of Riemerella Anatipestifer And Screening Of Genes Involved In Biofilm Formation

Riemerella anatipestifer (RA) infection is a highly pathogenic and contagious disease of ducks, goose, turkeys and other birds, which has caused giant economic losses, thought to be one of the most serious infectious diseases among duck industry. The coding genes of RA genome are predicted to be over2000, however, few have been known for their functions. Therefore, constructing a mutant library may provide a platform for high-throughput screening the gene functions of RA.In this study, a mutant library of RA was constructed using random insertion mutagenesis by transposon Tn-X, then the genes that influenced the biofilm formation of RA were screened on the genome level. The conjugational transfer was carried out using E.coli BW19851carrying a plasmid pTn-X as donor and RA serotype1strain CH3which could produce biofilm as the recipient. The KanRErmR transconjugants were further selected with Kan (50mg/ml) and Erm (2mg/ml). Finally, a random mutant library which included1500mutants had been constructed. The average conjugal efficience was about4×10-6We screened the mutants which biofilm formation reduced from the mutant library by CV staining assay, and obtained38mutants which biofilm formation were typically reduced with reduced rate ranged between40%-100%. We speculated that these genes involed in different stages of biofilm formation. Among these mutants, there were five mutants that had a reduced rate above99%. Then we identified the insertion times of Tn-X into RA genome by Southern blot, the result showed that the transposon inserted only once in these38mutants. Using Genome walking and some other techniques, we have identified the insertion sites of the transposon in CH3genome. There were four genes which occurred two to four times.In conclusion, in this study, we have constructed transposon insertion mutant library in R. anatipestifer for the first time, and the genes involved in biofilm formation were identified successfully.