Study On The Molecular Mechanism Of The Regulation Of ArgR Transcription Factor On FlrBc Two-component System In Aeromonas Veronii

Aeromonas veronii,as a comorbid pathogen of merman and fish,can cause a variety of diseases and seriously threaten the fish farming industry and human life and health.Previous studies have shown that the global transcriptional regulator ArgR in Aeromonas veronii not only participates in the regulation of arginine metabolism,but also interacts with the trans-translation system to affect bacterial pathogenicity.The purpose of this study is to further explore the function of ArgR in Aeromonas veronii,and to explore its downstream regulatory targets and signal pathways,so as to provide a deeper theoretical basis for the study of the molecular mechanism of the pathogenicity of Aeromonas veronii.Transcriptome analysis was used to compare gene expression differences between ArgR deletion strain and wild-type,and it was found that among 52 genes related to flagella synthesis,a total of 24 genes were down-regulated;at the same time,by analyzing the location of differentially expressed genes on the genome,combined with the analysis of flagellar cascade regulatory network,it is speculated that the downstream target of ArgR is the flrBC two-component system,which affects the expression of flagellar structural genes by regulating the expression of the flrBC two-component system.This study verifies the above hypothesis from the following aspects.Firstly,this study identified that the ArgR protein,as a transcription factor,specifically binds to the promoter of the flrBC two-component system and positively regulates the transcriptional activity of its promoter.On the one hand,bacterial one-hybrid experiments and EMSA experiments proved that ArgR can bind to the P_flrBCpromoter in vivo and in vitro;on the other hand,it was proved by q RT-PCR and fluorescence assay experiments that ArgR knockout resulted in down-regulation of flrBC gene expression,and co-expression of ArgR and P_flrBC-EGFP can promote a significant increase in the green fluorescence intensity downstream of the P_flrBC promoter,indicating that ArgR positively regulates the transcriptional activity of the P_flrBC promoter;the predicted binding site is further mutated and the binding site of ArgR and P_flrBC is detected by EMSA.It is found that the mutant AT-rich region on P_flrBC is the main site for the binding between the two.Secondly,this study started with bacterial phenotype analysis and proved that the flrBC two-component system affects the biofilm formation and motility of Aeromonas veronii.The homologous recombination gene knockout method was used to construct three gene knockout strains ofΔflr B、Δflr C、andΔflrBC and then the biofilm formation and motility of the four strains ofΔflr B、Δflr C、ΔflrBC andΔarg R were detected.The results showed that both the film formation ability and exercise ability were significantly reduced in the recombinant strains ofΔflr B、Δflr C andΔflrBC.Finally,this study preliminarily analyzed the conservation of the regulatory mechanism of ArgR on the flrBC two-component system in other bacterial species.Sequence alignment showed that,in the typical polar flagella bacteria Vibrio cholerae,P_flrBC contains three ArgR conservative binding motifs;in the Flr BC homology system Fle SR promoter in Pseudomonas aeruginosa,two ArgR conservative binding motifs are found.Sequence analysis suggests that the interaction mode of ArgR to the flrBC two-component system may also exist in other polar flagella bacteria,and the interaction relationship is somewhat conservative.This study is the first to explore the downstream regulatory pathways of ArgR in Aeromonas veronii,developing the regulatory network with ArgR as the core,and expanding the functions of ArgR.ArgR positively regulates the transcriptional activity of flrBC,thereby affecting the biofilm formation and locomotor ability of Aeromonas veronii.Both of these abilities are highly related to virulence.The results of this study lays a theoretical foundation for the study of the function of the global transcriptional regulator ArgR and the regulatory network of polar flagellar synthesis,and also provides a new perspective for the study of the pathogenic mechanism of Aeromonas veronii.