Functional Study Of Aeromonas Hydrophila AhyI Gene And K7 Acetylation

Aeromonas hydrophila(A.hydrophila)is a Gram-negative bacterium that is widely distributed in various water bodies,which is a typically conditional-pathogenic microorganism.In recent years,the problem of economic loss caused by A.hydrophila infection of fishes has been becoming more and more serious with the development of fisheries.Quorum sensing(QS)is a cell communication mechanism by which bacteria secrete and recognize some signal molecules,thereby regulating group behaviors such as gene level transfer,virulence factor secretion,spore production and biofilm formation.These functions play an important role in the pathogenesis of bacterial invasion,colonization and proliferation.AhyI is a synthetase that catalyzes the production of autoinducer molecules in the LuxI family of A.hydrophila QS system.The ahyI gene regulates the production of A.hydrophila biofilm,serine protease and metalloproteinase activity.This protein plays an important role in the maintenance of basic physiological functions of the bacteria.Additionally,protein post-translational modifications(PTM)reversibly modifies the protein of interest at the protein level,which make the spatial structure of the protein more complex,more functional,more precise,and more specific.Therefore,it has become a hot spot in the research of current epigenetic.Therefore,understanding the PTM information of target proteins will be helpful to better understand our aging and disease states.A previous study by the research team showed that a site of acetylation modification exsits at the K7 site of A.hydrophila AhyI protein,but its biological function still remains unclear.To study the effect of AhyI protein and its acetylation modification on the physiological traits of A.hydrophila,the strain with a deletion of ?ahyI was firstly constructed by homologous recombination.Additionally,?ahyI::Vector strain,mutant strains and the repair strain were also constructed in this study and used for further researches.The mutant strain was used to simulate the K7 acetylation modification and the biological characteristics of the ahyI strain were analyzed.The signal molecules of the strain were also extracted to study the effect of AhyI protein disruption and acetylation modification on quorum sensing.The results showed in thepaper are as follows:1.The ahyI gene was successfully deleted in A.hydrophila strain by homologous recombination,and the repair strain of ?ahyI::ahyI,vacant strain of ?ahyI::Vector were made in the study.According to the analysis of previous mass spectrometry data in our laboratory,site-directed mutations on the sites K7 Q and K7 R ?ahyI::ahyI-K7 Q,?ahyI::ahyI-K7 R were made.The results showed that K7 Q and K7 R mutations in the AhyI protein significantly reduced the acetylation modification on the proteins encoded by A.hydrophila.Compared with K7 R mutation,less proteins were modified by acetylation in the K7 Q mutant strain.2.The signal molecules from A.hydrophila ATCC7966,ahyI gene knockout strain,repair strain,K7 Q and K7 R point mutant strain were extracted to study the effect of ahyI gene on the production of A.hydrophila signal molecules by thin film chromatography(TCL)and E.coli pSB536 luminescence experiment.The results showed that the point mutation of K7 Q in A.hydrophila AhyI reduced the secretion of AI-1 signaling molecules,suggesting that acetylation modifications on the AhyI protein has a regulatory role in the production of signaling molecules.3.The strain with a deletion of ahyI affect the secretion of A.hydrophila virulence factors to a certain extent by extracellular protease activity and hemolytic analysis.The results showed that the ?ahyI strain of A.hydrophila gene significantly enhanced the ability of bacterial hemolysis and inhibited extracellular protease activity.Compared with the repair type,there was no significant difference between the K7 Q and K7 R mutations in the AhyI protein,indicating that K7 acetylation modification had no significant effect on the physiological activity of A.hydrophila.4.The growth curve and biofilm formation revealed that there are no significant difference among the abiotic strain,the repair strain,the point mutants and the wild type of A.hydrophila AhyI.These results indicated that the ahyI gene was not essential for the growth of A.hydrophila,but may play a regulatory role in propagation of the bacteria.As mentioned above,it was found that AhyI protein may regulate the secretion ofAI-1 signaling molecules through acetylation on the K7 site,but did not affect other physiological functions by studying the physiological phenotype of A.hydrophila ahyI deletion and a series of point mutation strains.Meanwhile,the above studies also provide a theoretical basis for further understanding the role of acetylation of AhyI protein in maintaining biological functions,and lay a foundation to systematically study the regulatory mechanism of quorum sensing.