Development And Application Of Multiple Markerless Gene Deletion Strategies In Riemerella Anatipestifer

Riemerella anatipestifer(R.anatipestifer,RA)belongs to the family of Flavobacterium,and it is a Gram-negative bacterium.Although a variety of genetic manipulation strategies have been used to identify virulence factors of R.anatipestifer,these established methods have various defective for application.In this study,we successfully established multiple methods for markerless gene deletion in R.anatipestifer using rps L or sac B as counterselection marker.Using this method to identify the functions of related genes.The main results are as follows:1.Development of a markerless gene deletion method in ATCC11845s using rps L as a counterselection markerStreptomycin-sensitive ATCC11845 strain was cultured on a streptomycin-containing medium,and then we successfully obtained spontaneously mutated rps L mutants,ATCC11845s,which are resistant to streptomycin.The wild-type rpsL gene was cloned into the shuttle plasmid pLMF02 and transferred into ATCC11845s.The ATCC11845s strains expressing the rps L gene were sensitive to streptomycin again.This proved that rps L can be used as a counterselection marker for R.anatipestifer ATCC11845s.Subsequently,the suicide vector pORS expressing the rps L gene and the plasmid pBAD24-ermR-rpsL were constructed.The gene deletion method based on the suicide vector or natural transformation was established,respectively.Both of them were successfully applied to the deletion of RA0C?1534 gene in ATCC11845s.2.Development of a markerless gene deletion method in R.anatipestifer using sacB as a counterselection markerThe sucrose lethal gene sac B gene was cloned into the shuttle plasmid pLMF02 and transferred intoR.anatipestifer CH-1 strain.The strain expressing sacB gene had limited growth in sucrose-containing medium,suggesting that sacB can be used as a counterselection marker for R.anatipestifer CH-1.Subsequently,the suicide plasmid pOBS expressing the sacB gene and the plasmid pBAD24-cfx-sac B were constructed.Based on this,a pOBS-mediated and a natural transformation-mediated gene deletion methods were established.The dps mutant strain of R.anatipestifer CH-1was then successfully constructed.3.Study on the function of Dps in R.anatipestifer CH-1Compared with R.anatipestifer CH-1,the dps mutant strain was more sensitive to H₂O₂during the stationary phase of R.anatipestifer CH-1.Meanwhile,it was found that the antioxidant stress ability of Dps depends on the intracellular iron level,and it only works under the iron-rich conditions.The results of the electrophoretic mobility shift assay further indicate that Dps protects bacterial DNA from oxidative damage by binding with DNA.Consistent with the phenotype,transcriptional regulation results indicate that R.anatipestifer dps transcription was higher in the stationary phase than in the other gowth phases and was activated by OxyR in the presence of H₂O₂.Ducklings were infected with the same amount of CH-1 or CH-1?dps by injection.At 24,48 and 60 h post-infection,bacteria were isolated from multiple organs and then counted.The colonization ability of the dps mutant strain is significantly lower than that of the wild-type strain.Thus it is speculated that dps of R.anatipestifer CH-1 plays a role in colonization,but not in host invasion.