Characterization Of RaeE-RaeF-RopN Efflux Pump System And Development Of A Markerless Gene Deletion In Riemerella Anatipestifer

Riemerella anatipestifer(Riemerella anatipestifer,RA)is a gram-negative bacterium belonging to the Flavobacteriaceae,Bacteroides phylum and Riemerella genus.It mainly affects ducklings between 2weeks and 8 weeks of age,causing Serositis in ducklings.At present,antibiotics are commonly used in duck breeding industry to prevent and control infection of RA,which makes multi-drug resistant strains continue to produce and spread,so it is urgent to carry out research on its resistance mechanism.So far,we know very little about the pathogenic mechanism and drug resistance mechanism of Riemerella anatipestifer.A large part of the reason is the lack of efficient genetic manipulation methods.Therefore,the development of new and efficient genetic manipulation methods is one of the key directions in current research.So far,there are few studies on the drug-resistant efflux system of R.anatipestifer at home and abroad.Based on the sequence analysis of the RA-GD strain,it is concluded that the RIA₁117-RIA₁118-RIA₁119 operon in the RA-GD strain encodes a hypothetical three-component RND efflux system,and the RIA₁117,RIA₁118 and RIA₁119 genes encode respectively external member protein(OMP),internal membrane pump protein(pump transport protein)and periplasmic protein(MFP).In this research project,the biological functions of RIA₁117,RIA₁118 and RIA₁119genes were systematically identified.Antibiotic susceptibility tests showed that the deletion of RIA₁117,RIA₁118 and RIA₁119 genes all increased the strain's sensitivity to amikacin,streptomycin and SDS;through the induction of the above-mentioned antibacterial agents,using q RT-PCR detection,the transcription level of the RIA₁117 and RIA₁118 gene was significantly up-regulated,but no significant difference was observed for the transcription level of the RIA₁119 gene.CCCP inhibitor detection test showed that RIA₁117,RIA₁118 and RIA₁119 proteins use proton driving force(PMF)to mediate the efflux of amikacin,streptomycin and SDS.Streptomycin accumulation assay test confirmed that RIA₁117,RIA₁118 and RIA₁119 proteins contribute to the efflux of streptomycin,while CCCP increases the accumulation of streptomycin in the bacteria.In addition,RIA₁117,RIA₁118 and RIA₁119 proteins are also related to the adaptability and virulence of RA-GD strains.These results indicate that the RIA₁117-RIA₁118-RIA₁119 operon encodes an RND efflux system,which has substrate specificity for streptomycin,amikacin and SDS,and is related to the growth and virulence of RA-GD.RIA₁117-RIA₁118-RIA₁119 was named Rae E-Rae F-Rop N efflux system.Based on the above results and structural analysis,RIA₁117,RIA₁118 and RIA₁119 proteins correspond to Rop N(OMP),Rae F(membrane pump transport protein)and Rae E(MFP),respectively.In this study,the Erm F gene was used as a forward marker and sac B as a reverse marker to perform a markerless knockout on the RA-GD genome.The first knockout on the chromosome was carried out by constructing the suicide plasmid p RE112 carrying forward Erm F and reverse marker sac B genes,and using 1?g/m L erythromycin as the selection pressure to screen out gene deletion mutants.Then,a suicide plasmid containing upstream and homologous arms of RIA₁117 gene is constructed to knock out sac B-Erm gene cassette in the second recombination.The unlabeled strain can be selected at 13%(wt/vol)sucrose pressure.A two-step recombination method is used to knock out the target gene without leaving any marks.Many researchers have proven through a lot of practice that this knockout method is operable,and the markerless knockout technique can be used to knock out multiple target genes.Thus,it can be seen that the technology of markerless gene knockout will be more widely used in various scientific research activities.The technology constructed in this article can also provide some new ideas for the research and development of R.anatipestifer vaccine and the in-depth study of drug resistance mechanisms.