The Research On ClpV Type ATPase, The Key Component Of Type Ⅵ Secretion System In Agrobacterium Tumefaciens C58

Type VI secretion system (T6SS) is a new discovered secretion system which widely distributed in many Gram-negative bacterial pathogens. T6SS is involved in a broad variety of functions:host-bacteria interaction mediation, bacteria-bacteria interaction mediation and stress sensing. Therefore, investigation of the secretion mechanism of T6SS has a great meaning in understanding how plant, animal, and human pathogens cause diseases, and how to diagnose those diseases.Furthermore, as T6SS has a bacteria-killing function, it can be potentially used for novel bacteria resistance strategy and medicine screen. Hence, it has been given highlight on T6SS in the field of bacteria, whose function and role are still little known.Therefore, we studied T6SS in Agrohacterium tumefaciens C58, which is a model bacteria. Through bio-information analysis, we found that ClpV, one of the key components of T6SS containing a conserved AAA+domain (ATPase associated with diverse cellular activities), has been presumed to be the ATPase which drives effector protein secretion through hydrolyzing ATP. However, the functional and biochemical roles of ClpV remain largely uncharacterized.In this study, we carried out primary investigation about the structure and function of ClpV ATPase:1) In this study, the clpV gene (atu4344) of Agrobacterium tumefaciens C58 was cloned and heterologously expressed in E. coli. By using the coupled-enzyme assay and site-directed mutagenesis, it was confirmed that the wild-type ClpV exhibited ATPase activity, but neither the K232/608 nor E299/675 mutants were active, which demonstrated that both the Walker A and Walker B motif are essential for ClpV activity in vitro. And the optimum pH for it is 6.5, as Vmax=42.8nmol min-1 mg-1, Km=0.68mM, and its activity decreases along with the increase of Ac-ion.2) The structure of ClpV:based on native PAGE, we found ClpV protein form a polymer and further investigation via gel filtration and electric microscopy confirmed its hexamer structure, which is in the same situation as InvC, an ATPase providing energy in T3SS.3) Polymerization and depolymerization of ClpV:based on pull-down test, we found ClpV can interact with Atu4341and Atu4342 and form a complex. However, ClpV can not form complex with either of those two proteins. Besides, ClpV can depolymerize Atu 4341-Atu4342 when ATP is present.4) Primary study about the function ClpV ATPase:based on our results, unlike wild-type strain, ClpV mutant strain can not secret HCP to the supernatant. In contrast, in ClpV complemented strain, the secretion of HCP were recovered in some extent.5) We studied protein-protein interaction between 23 components of T6SS in Agrobacterium tumefaciens C58 based on yeast two-hybrid system and constructed a interaction net.