Structural And Functional Studies Of The C-di-GMP Receptors XC3703 And Smlt4090 Of YajQ Family And Adaptor Protein Atu4349 Of T6SS From Agrobacterium Tumefaciens

Xanthomonas campestris pv.Campestris is the pathogen of the plant disease black rot.It can infect all crucifers worldwide and cause significant economic losses.Stenotrophomonas maltophilia is a rare gram negative opportunistic pathogen.It can infect immunocompromised individuals and cause pulmonary infection.C-di-GMP is a well known intracellular second messenger,which is involved in many important physiological functions,such as biofilm synthesis,bacterial virulence,cell motility and so on.In the bacteria,there are many kinds of c-di-GMP receptors which can interact with c-di-GMP to cause the changes of physiological and biochemical functions.C-di-GMP is involved in a variety of important physiological activities of bacteria.Up to now,the number of receptors which are found is significantly lower than the actual number.In recent research,a new family of c-di-GMP receptor family called YajQ was found.The secondary structure of YajQ family proteins is relatively conserved in bacteria,and the protein potentially has the ability to bind to nucleic acids.In Xcc,a member of the YajQ family XC3703 has been shown to interact with the transcription factor XC2801 and the interaction can be reversed by the combination of c-di-GMP.XC3703 can influence biofilm formation,virulence and other biological functions.Smlt4090 in S.Maltophilia which also belongs to YajQ family was proved to have the ability to bind to c-di-GMP,too.The amount of biofilm and the virulence were significantly reduced in the mutant.In the first part of this thesis,I will introduce the structural and functional research of XC3703 and Smlt4090.According to the alignment of their sequences,the identity of XC3703 and Smlt4090 is up to 83%.The structures of these two proteins were resolved successfully.However,it is a pity that the structures of complex with c-di-GMP have not been obtained.According to the analysis of the structure,XC3703 contains two molecules in an asymmetric unit,but the subsequent result showed that it exists as a monomer in solution state.In the structure of Smlt4090,each asymmetric unit contains three molecules.The result of size exclusion chromatography also proved that it exists as a monomer in solution state.Agrobacterium tumefaciens is the pathogen of crown gall disease.The plants infected will form tumours.A.tumefaciens has important application in plant genetic manipulation.Type six secretion system is a kind of bacterial secretion system which is well concerned in recent years.The secretion apparatus is similar to the phage tail structurally and functionally.Bacteria gain competitive or survival advantage by injecting a variety of effectors into adjacent prokaryotic or eukaryotic cells.Recent research showed that the effective output of effectors needs cognate adapter proteins.The adaptor proteins can form a complex with VgrG,which is part of injection apparatus,and the corresponding effectors.In the second part of this thesis,the main research content is conditions of purification and crystallization of Tap-1 from A.tumefaciens.Tap-1 can be overexpressed in E.coli BL21 cells,but protein obtained could not grow into crystals.Considering that there are three cysteines in Tap-1,the growth of crystals may be affected by formation of the disulfide bond.So I used three methods to try to obtain the crystals.And finally crystals were obtained successfully.But the quality of crystals could not satisfy the requirement of structure analysis.