Research On The Regulation Of (p)ppGpp By The Two-Component System In Extraintestinal Pathogenic Escherichia Coli

Escherichia coli is an important zoonotic pathogen that seriously jeopardizes public safety and animal husbandry.In bacteria,(p)ppGpp and polyP are involved in stress response.It has been reported that there is a direct link between the two-component signaling system and(p)ppGpp and Poly(P)during stress response.In order to study the role of TCS in the regulation of polyphosphorus,we used gel migration assay(EMSA),construction of gene-deficient strains,and real-time PCR to study the regulation mechanism of(p)ppGpp by ArcA,OmpR and PhoB.The specific results are as follows:1.TCS proteins ArcA,OmpR,PhoB can regulate the expression of relA geneIn the EMSA,binding experiments of phosphorylated ArcA,OmpR,PhoB proteins and the PrelA were performed.It was found that all three proteins can block the migration of PrelA on non-denatured protein gel,indicating that ArcA,OmpR and PhoB can bind to PrelA.At the same time,the relative fluorescence quantification experiments of ?arcA,?ompR,?phoB and wild strain PCN033 showed that the expression of relA gene in the deletion strain was significantly down-regulated.Therefore,the TCS proteins ArcA,OmpR,and PhoB can regulate the expression of the relA gene by binding to PrelA.2.TCS proteins ArcA,OmpR,PhoB help bacteria to resist stressful environmentsThe ?arcA,?ompR,?phoB and wild strain PCN033 were treated with low concentration gradient hydrogen peroxide,and the growth curve was determined.It was found that the deletion strain was less tolerant to hydrogen peroxide than the wild strain;At the same time,The above four strains were treated with high concentration of hydrogen peroxide,and the bacterial survival rate was measured.It was found that the survival rate of the deletion strain in the high concentration hydrogen peroxide was significantly lower than that of the wild strain.In the biofilm formation and analysis experiments,it was found that the biofilm formed in the deletion strains ?arcA,?ompR and ?phoB was less than the wild strain PCN033,indicating that the TCS proteins ArcA,OmpR and PhoB could affect the formation of biofilm.In summary,the TCS proteins ArcA,OmpR,and PhoB help bacteria resist hydrogen peroxide stress and can affect biofilm formation.3.Poly(P)can regulate the synthesis of(p)ppGpp by TCS proteins ArcA,OmpR and PhoB.In the(p)ppGpp synthetic assay,it was found that the synthetic amount of(p)ppGpp in the deletion strains ?arcA,?ompR and ?phoB was significantly lower than that in the wild strain.In addition,when overexpression of PPK or PPX caused intracellular accumulation or elimination of poly(P),the expressions of TCS proteins ArcA,OmpR and PhoB were up-regulated or down-regulated accordingly,indicating that poly(P)could regulate the expressions of ArcA,OmpR and PhoB.In combination with the foregoing,the TCS proteins ArcA,OmpR and PhoB can regulate the expression of the relA gene.In summary,Poly(P)regulates the expression of relA by regulating the expression of the TCS proteins ArcA,OmpR and PhoB,thereby realizing the regulation of(p)ppGpp synthesis.