Study On The Enzymatic Function Of Type ? Secretion System Effector Protein YPK?2589 Of Yersinia Pseudotuberculosis(YP?)

Bacterial type VI secretory system(Type VI secretion system,T6SS)is common in Gram-negative bacteria,accounting for about 1/4 in the whole genome of Gram-negative bacteria.And it is highly conserved.T6 SS plays important roles in physiological and biochemical processes like competetion between and within species,quorum sensing,and stress responsion by secreting some effector proteins and other substances.Up to now,the main structural components of T6 SS and their functions are a more in-depth part of research.However,there are still a large number of genes that can encode T6 SS effectors with unknown functions on the T6 SS gene cluster,ypk?2589 is one of these genes.In the previous work in our team,the effector protein gene ypk?2589 from Yersinia pseudotuberculosis(YP?)was isolated and part of its physiological function was explored.In order to further explore its biological significance,the effector protein YPK?2589 was taken as the main object of study.On the basis of previous experimental data,this paper focuses on the in-depth study on the function and functional mechanism of YPK?2589.In this study,by expressing the target gene in Escherichia coli BL21(DE3),it was preliminarily confirmed that the gene had a effect of delaying growth of E.coli BL21(DE3).In order to further reveal the toxic mechanism of YPK?2589,the secondary structure and function of were analyzed by protein structure prediction and other bioinformatical analysis.The recombinant YPK?2589 protein was overexpressed by E.coli strain BL21(DE3)and purified by affinity chromatography.The RNA hydrolysis experiment was carried out and showed that YPK?2589 protein was a non-specific RNase with both r RNA and t RNA hydrolysis activities.By setting variant reaction gradients on reaction temperature,p H and cation concentration,the effects of the above conditions on the hydrolysis activity of YPK?2589 protein were explored.The optimal reaction condition for YPK?2589 protein as a RNase was preliminarily determined.Then,in the study,the key domain of RNase activity of YPK?2589 was verified in E.coli BL21(DE3)by constructing ypk?2589 gene interceptor and point mutation,and the key active sites of RNA hydrolysis were preliminarily explored.YPK?2589 was proved to be a new type of RNase in this study.It hydrolysis RNAs in the cytosol of host cell or bacteria to suppress their development.This work promoted the research on development and pathogenic mechanism of Yersinia pseudotuberculosis(YP?).