Study On The Function And Crystal Structures Of YdiV And YdaM In E.coli Signaling Pathway Transduced By C-di-GMP

The Quorum sensing system (QS) was activated when bacterial cells were clustered. Quorum sensing system, which regulates various physical process of bacteria including pathogenicity, motility and biofilm formation, relys on a kind of signal molecules named Auto Inducer (AI) as messenger in the intercellular communication. AI is a kind of extracellular signaling molecules, which eventually leads to cell's physiological and biochemical reactions through signal transduction by the secondary messenger intracellular. As a secondary messenger, cyclic diguanylate (C-di-GMP) exists widely in many different pathogens, controls various activities such as the pathogenicity of V. cholera, the biofilm formation of E.coli and P.aeruginosa, etc. Concentration of C-di-GMP affects its effect, which was regulated by a pair of catalytically contrary enzymes:Phosphodiesterase (PDE) and Diguanylate cyclase (DGC). Current research on the mechanism of C-di-GMP transduction shows that the concentration level of C-di-GMP directly affects the pathogenicity of V.cholera, P.aeruginosa, S.enterica, etc. Regulation of the concentration and the function of C-di-GMP or relevant proteins has important guiding significance on prevention and treatment of disease caused by these pathogens. Find out relevant proteins exactly and locate most important active site precisely and then clarify the mechanism of whole signaling network were the key points to use C-di-GMP as a pharmic target.Based on the existing conditions of the laboratory, E.coli was used as a carrier to study the structure and function of YdiV and YdaM, as of which various homologous proteins exist widely in V.cholera, P.aeruginosa, and S.enterica. YdiV contains an EAL domain, while YdaM conteins a GGDEF domain. In this thesis, a preliminary crystollographic study of crystalled YdiV was showed for the first time. Through over-expression of YdiV and YdaM and gene knock-out of ydiv and ydam, the intracellular function of YdiV and YdaM were tested, including the influence on biofilm formation. Extracellular enzyme activity of YdiV and YdaM were also tested through reacting system and HPLC. The results that extracellular YdiV and YdaM were inactive while obviously active intracellar indicate a complex regulating network on biofilm fomation.