Study On The Regulation Mechanism Of FleQ And C-di-GMP In Pseudomonas Syringae Pv.tomato DC3000

Pseudomonas syringae is an important plant pathogen,which is widespread in the natural environments and infects many plant cultivars.P.syringae pv.tomato DC3000(Pst DC3000)can cause bacterial spots on tomatoes and Arabidopsis.As an important type strain,it was widely used in the study of molecular plant pathology.since it carries a large repertoire of potential virulence factors,it is also often used in the study of some effector proteins secreted through the type III secretory system.Cyclic dimeric guanosine monophosphate(c-di-GMP)is a ubiquitous bacterial second messenger that regulates cell functions in response to diverse environmental chemical and physical signals.The previous study reported that c-di-GMP plays an important role to modulate biological functions including regulating flagellar motility and exopolysaccharide secretion to secure a planktonic to sessile life-form transition.c-di-GMP also could regulate virulence,especially the expression of some virulence factors.The study on the regulation mechanism and biological function could provide guidances for the molecular mechanisms of infection and pathogenesis.In Pseudomonas aeruginosa,FleQ,as the master regulator of flagella and receptor of c-di-GMP,not only participates in the regulation of motility,but also participates in the regulation of bacterial biofilm formation.Whether FleQ in Pst DC3000 also would affects biological phenotype and the relationship with c-di-GMP are still unclear.This study focused on exploring the regulatory mechanism of c-di-GMP and FleQ in Pst DC3000,and the main results were as follows:C-di-GMP inhabits the binding of FleQ to fliC promoter in Pst DC3000.In this study,fleQ deletion mutant could cause the losing of swimming and swarming motility.It was also found that the ability of biofilm formation reduced.Considering that fleQ deletion mutant in P.aeruginosa PAO1 also showed the same phenotype about losing motility,we compare the amino acid sequence alignment and analyse the homology of FleQ in Pst DC3000 and PAO1.The homology was up to 83%.Based on the experiment that FleQ in PA01 could compensate for the motility function caused by FleQ deficiency in Pst DC3000,we speculated that FleQ in Pst DC3000 might have some similar functions to which in PA01.Previous studies have found that FleQ is a multi-domain transcriptional regulator which dependents on σ54.FleQ regulates many flagellum genes and operons.Here we show that FleQ,the flagellar master regulator,is a c-di-GMP effector in Pst DC3000.According to the β-galactose experiment,we found that the expression of flagellum gene fliC was significantly decreased in fleQ deletion mutant.It was also found that FleQ could directly bind c-di-GMP in vitro by isothermal titration calorimetry(ITC)experiment.The EMSA experiment verified that FleQ controls the target gene expression by direct binding to the DNA promoters,whereas the binding is reversed by both c-di-GMP.It indicates that FleQ regulates them directly.We also analyzed the influence of FleQ on the synthesis and degradation of c-di-GMP in cells by the enzyme activity experiment.It was showed that in the reaction system of c-di-GMP synthesis,FleQ could not synthesize c-di-GMP,but could hydrolyze GTP to GDP.The binding of FleQ protein to GTP in vitro was further analyzed by the ITC experiment.The results showed that FleQ could bind to GTP in vitro.Considering that physiological activities are often regulated by protein-protein interaction in bacteria,we constructed a two-hybrid library of FleQ in Pst DC3000,and found that the 4654 protein which regulating methylation,as well as the antagonist Fle N interacting with FleQ.2757 belonging to c-di-GMP degradation enzyme was also found interaction with FleQ by bacterial double hybridization.The interaction in vitro need to be further verified.The enzyme activity experiment showed that FleQ could not degrade c-diGMP,but could prevent 2757 from degrading c-di-GMP.By constructing fle N and 2757+deletion strains,it was found that the motility was decreased compared with the wild type.We will do further exploration in the following study.The results of this study determined the influence of c-di-GMP on bacterial motility and biofilm formation in Pst DC3000,and revealed the role and expression regulation of FleQ,laying a foundation for further studies on c-di-GMP metabolism in Pst DC3000.