Construction And Biological Characterization Of Bfr And LsrB Gene Deletion Strains Of Aeromonas Veronii TH0426

Aeromonas veronii is a ubiquitous Gram-negative opportunistic pathogen that infects mammals,amphibians and aquatic animals.While bringing economic losses to the aquaculture industry,it also seriously threatens food health.With the increase in the infection rate of A.veronii,related research reports have gradually increased,but the mining of its virulence factors needs to be supplemented.Lsr B is a high-affinity substrate-binding protein encoded by the lsrB gene.As a substrate-binding protein of the ABC transport system,it is mainly responsible for the internalization of extracellular AI-2.Lsr B receptors bind to AI-2 when extracellular AI-2 accumulates to a threshold concentration.The cells were then introduced through the Lsr ABC transmission system,resulting in changes in gene expression.Relevant studies have shown that the lsrB gene can affect the ability of bacteria to produce biofilms,virulence factors and pathogenicity.To maintain iron balance in the body,bacteria have evolved an efficient iron uptake and storage protein:Bacterioferritin（Bfr）.In this study,the bfr and lsrB genes,which may be closely related to virulence,obtained from our previous study in differential protein analysis of the research group,were selected.The gene deletion mutants were constructed by using the suicide plasmid p RE112 and the wide host expression plasmid p BBR1-MCS,and the biological characteristics of the deletion strain,the complement strain and the wild strain were compared,It was found that there was no significant difference in drug sensitivity and bacterial growth ability after bfr and lsrB gene deletions,but they both weakened bacterial swimming ability.And after the bfr gene was deleted,the strain was positive for glycine-AMC（GLYB）metabolism.Under the transmission electron microscope,the structure of the flagella of the two gene deletion strains could be observed to be damaged.The expression levels of 15pairs of flagella formation-related genes in the deletion strain were further detected by RT-q PCR,and it was found that flgL,flgK,flgE,flgF,flgG,mot A,mot B,fli E,fli G,flgB,flgC,fli M,flh B,fla A and fla B genes.All the expressions were up-regulated,and no down-regulated genes were found.It is speculated that bfr and lsrB genes affect the motility of bacteria by regulating the expression of flagellar genes.The results of 96 well plate detection of bacterial biofilm formation showed that the biofilm forming ability ofΔbfr andΔlsrB decreased,and the difference was extremely significant compared with the wild strain TH0426（p<0.01）,while the biofilm forming ability of the complementary strain recovered.,indicating that the gene is related to the formation of biofilm,and it can be clearly observed by scanning electron microscope that the cells of the deletion strain are loosely connected and have different shapes.The adhesion and invasion of EPC cells were significantly decreased after lsrB gene deletion（p<0.01）.After 2 h and 2 h,the cytotoxicity decreased significantly;the LD₅₀ of zebrafish was increased by 2.06-fold after the deletion of the bfr gene,and the LD₅₀ increased by 1.74-fold after the deletion of the lsrB gene,and the virulence decreased significantly.To sum up,the bfr and lsrB gene deletion strains and complemented strains were successfully constructed in this experiment,and the comparison of wild strains,deletion strains and complementary strains found that after gene deletion,the motility,biofilm formation ability,and EPC were significantly reduced.Cell adhesion and pathogenicity were significantly reduced.This experiment lays a foundation for subsequent research.