Construction And Biological Characterizations Of Ndk And IlvI Mutant In Aeromonas Veronii TH0426

Aeromonas veronii(A.veronii)is a Gram-negative pathogen bacterium that can infect human-animal-aquatic animals.It is widely distributed and has strong pathogenicity,which has brought huge economic losses to aquaculture industry.Recent research on A.veronii is on the rise,and reports on its increasing pathogenicity are growing year by year.Nucleoside diphosphate kinase(NDK)is a highly conserved multifunctional protein encoded by NDK gene.Its main function is to maintain the balance of ATP and NTP concentration in cells through phosphoryl transfer reaction and participate in UTP and GTP biosynthesis.Previous research showed that ndk can regulate cell proliferation,development,apoptosis and bacterial virulence.The acetolactate synthase(ALS),encoded by the ilvI gene,is a key enzyme in the biosynthesis of branched chain amino acids Val,Leu and Ile in plants,fungi and bacteria,and TPP,FAD and divalent metal ions are needed for their activity.Current research on the enzyme focused on plants,especially herbicides.Herbicide compounds block the synthesis of branched-chain amino acids by inactivating the ALS.As a result,the synthesis of branched chain amino acids is blocked,thus affecting the growth of organisms.However,there is no study on nucleotide diphosphate kinase and acetolactate synthase in A.veronii.Therefore,we studied the effects of ndk and ilvI genes on A.veronii virulence,biofilm and gene functions.In this study,we selected the ndk and ilvI genes closely related to biofilm formation and virulence obtained by differential protein analysis in our early findings.The recombinant suicide plasmid pRE112 vector was constructed by recombinant technology,and the strains which were knocked ndk and ilvI genes were screened by antibiotic selection method.Meanwhile the same construction method was used in the complemented strains.We found that the growth ability of ndk and ilvI mutant strain was significantly lower than that of the wild strain and complemented strain.The plate motility experiment showed that the swimming motility of ndk and ilvI mutant strain was decreased,while there was no change in swarming.The biofilm formation test of bacteria was detected by 96-well plate and scanning electron microscope.It was found that the biofilm formation ability of ndk mutant strain was significantly decreased(P<0.001).Meantime the biofilm formation ability of ilvI mutant strain was decreased(P<0.05),indicating that ndk and ilvI genes were associated with biofilm formation.Cell experiments showed that the adhesion and invasion of ndk mutant to EPC cells were decreased significantly(P < 0.001);the cytotoxicity decreased significantly after 3,6 and 12 h of treatment;and flow cytometry detected that the apoptotic activity of EPC cells also decreased.However,there was no difference in the adhesion and invasion of ilvI gene to EPC cells;after 3 and 6h of cyclization,the cytotoxicity was decreased significantly,but there was no difference at 12 h.The LD50 test found that the LD50 of ndk mutant increased by 5.5 times and ilvI mutant increased by 1.7 times compared with the wild strain,and the virulence was significantly reduced.Drug sensitivity test of ilvI mutant strain found that the drugs were highly sensitive to carbenicillin,cefradine and tetracycline.In summary,we successfully constructed ndk and ilvI deletion strains and complemented strain,and analyzed the biological characteristics of wild strains,deletion strains and complemented strain.It was found that after gene deletion,the motility ability,biofilm formation ability and pathogenicity were decreased significantly.The research lays a foundation for subsequent research.