Functional Identification Of Type ? Secretion System Effector YPK₀405 In Yersinia Pseudotuberculosis

Bacterial Type?secretion system(T6SS)is a recently characterized secretion system appears to constitute a phage-tail-spike-like contractile machine that has the potential to introduce effector proteins to surroundings or directly into the cytoplasm of host cells.More than a quarter of sequenced gram-negative bacterial genomes contain genes for T6SS components,mostly within the proteobacteria,but also within the planctomycetes and acidobacteria.T6SS is required for several crucial physiological processes,such as pathogenicity,competition and stresses combatting.Here,we take an unknown gene ypk?0405 in T6SS-1 gene cluster of Yersinia pseudotuberculosis YP?as a research object,exploring the new features of T6SS by studying the physical activity biochemical function and biology function of its expression product.The results of the experiments are as follows:1.The Bacterial two hybrid assay suggests that there are protein-protein interaction between YPK?0405 and YPK?0406 which indicated that YPK?0405 and YPK?0406 is probably a pair of effector and immune protein.2.DNA hydrolysis activity assay suggests that YPK?0405 possesses DNA hydrolysis activity,which need assistance of 2m M Mg²⁺,and the supernatant hydrolysis activity assay of WT,mutant of YPK?0405 and complement strain has the same results.3.Secretion assay suggest that YPK?0405 is a secretion protein,and the secretion depends onT6SS-1 of Yersinia pseudotuberculosis.4.When expressed in E.coli,YPK?0405 is toxic,and genomic DNA extraction assay suggests that the toxicity may be caused by DNA hydrolysis activity.5.The detection of physiological property suggest that YPK?0405 could form dimers after treated by GA for 12 hours.In addition,PAR competition assay illustrate that YPK?0405could bind Zn²⁺.6.Biofilm formation assay suggest that YPK?0405 could not only influence the biofilm formation on abiotic surface,but also be related to the activity of biofilm formation biological surface.To sum up,YPK?0405 is a T6SS-1 effector that possesses DNA hydrolysis activity.It could bind Zn²⁺and form dimers when treated with GA after 12hours.In addition,when expressed in E.coli,YPK?0405 could lead bacteria to death.Finally,YPK?0405 is related to Y.pseudotuberculosis biofilm formation ability on abiotic surface or biological surface by an unknown mechanism.