The Tolerance To Heavy Metal Ions Study Of RaeX-RaeY-RopM Efflux Pump In Riemerella Anatipestifer

Riemerella anatipestifer is one of the important bacterial diseases that seriously endanger poultry industry.It mainly infects ducks,geese,turkeys and other birds.There are multiple serotypes of Riemerella anatipestifer,and 21 serotypes have been identified.In China,the main prevalent serotypes are type 1,type 2 and type 10.The lack of effective cross-protection among different serotypes makes it more difficult to effectively prevent vaccines.Up to now,the disease is mainly treated by antibiotics,but with the wide use of antibiotics,the prevalence of multi-drug resistant Riemerella anatipestifer strains has appeared,which greatly reduces the therapeutic effect of antibiotics.Therefore,it is of great theoretical and practical significance to analyze the drug resistance mechanism of Riemerella anatipestifer and control the drug resistance of bacteria at the molecular level.The multiple drug transporter of the resistance-nodulation-cell division(RND)is one of the main causes of multiple drugs in Gram-negative bacteria,and it also exists in some Gram-positive bacteria.Generally,a triple efflux complex traverses the cell membrane,directly expelling the antibacterial drugs that enter the cell,so that the bacteria have a very broad antibacterial spectrum.In this study,the genome of Riemerella anatipestifer RA-LZ01 was used to construct gene deletion strains LZ01?1445 and LZ01?1450,complement strains LZ01::c?1445andLZ01::c?1450andexpressionstrains C43-p ET32a::1445-1450-1455,Thefunctionsof GE296?RS01445-GE296?RS01450-GE296?RS01455 efflux pump in Riemerella anatipestifer were systematically identified from three aspects:strain growth,antibiotic resistance and heavy metal ion tolerance.At first,the erythromycin resistance marker was used as a forward screening method to construct the targeted fragment by fusion PCR method,which was ligated with the suicide plasmid p RE112,and transformed into E.coli X7213 to construct the donor bacteria,and then the donor bacteria and wild strain RA-LZ01 Co-cultivation,using a combination of transfer and homologous recombination methods to construct gene-deleted strains(LZ01?1445and LZ01?1450);Afterwards,using the E.coli-Riemerella anatipestifer shuttle plasmid p CPRA,the GE296?RS01445 and GE296?RS01450 genes were reintroduced into the deleted strain to construct complement strains(LZ01::c?1445 and LZ01::c?1450);Atlast,insertthethreecomponents GE296?RS01445-GE296?RS01450-GE296?RS01455 into the plasmid p ET32a,and transform the recombinant plasmid into E.coli C43 to construct the expression strain C43-p ET32a::1445-1450-1455;The dynamic graph of growth curve of wild strains and deletion strain was drawn by measuring OD₆₀₀value within 12 hours;the minimum inhibitory amount(MIC)of the wild strains and the deletion strains was determined by the micro broth dilution method;The tolerance of wild strains,deleted strains and complemented strains to heavy metal ions was determined by dot plate experiment.The growth curve results showed that after knocking out the GE296?RS01445 and GE296?RS01450 genes,the growth of the deleted strain did not change significantly compared with the wild strain;The results of the determination of the MIC values of antibiotics showed that compared with the wild strains,the MIC values of the 11 types of antibiotics did not change significantly;The results of heavy metal ion efflux experiment showed that the growth ability of the deleted strain was significantly lower than that of the wild strain and the restorer strain,and the growth of C43-p ET32a::1445-1450-1455 was better than that of Escherichia coli C43 and C43-p ET32a.The above research results indicate that the R.anatipestifer gene locus GE296?RS01445-GE296?RS01450-GE296?RS01455 has no obvious correlation with the growth of the bacteria and the efflux of antibiotics,and it plays an important role in the efflux of metallic nickel,cobalt and manganese ions.We named the GE296?RS01445-GE296?RS01450-GE296?RS01455effluxpump Rae X-Rae Y-Rop M,Where GE296?RS01445,GE296?RS01450 and GE296?RS01455correspond to the efflux pump Rae X,outer membrane protein Rop M and periplasmic protein Rae Y,respectively.This study laid the foundation for the study of the mechanism of heavy metal efflux by Riemerella anatipestifer.