| Bacterial chemotaxis refers to the ability of microorganisms with flagella structure to perceive the concentration gradient of chemical signals in the surrounding environment and make effective responses autonomously,such as migrating to favorable substances or escaping from harmful substances.Chemotaxis is crucial for bacteria to survive in harsh and nutrientpoor environments,and the chemotactic stage is also one of the important links in the infection process of pathogenic microorganisms.Chemotaxis widely exists in the vast majority of bacteria that have been sequenced,among which Escherichia coli is the most thoroughly studied.The model bacteria E.coli has a complete chemotaxis system,including seven extremely important components:methyl-accepting chemotaxis protein MCP,methyltransferase CheR,coupling protein CheW,sensor kinase CheA,methylesterase CheB,chemotaxis response regulator CheY and phosphatase CheZ.When methyl-accepting chemotaxis proteins MCPs recognize chemotactic signals in the environment,the ternary core complex formed by these receptor proteins,coupling protein CheW and sensor kinase CheA,changes subtly in structure,that is,it can directly affect the phosphorylation degree of response regulator CheY via controling the output of phosphorated signal.Thus,the amount of phosphorylated CheY(CheY-P)bound to the two components of motor switch complex(FliM and FliN)at the bottom of flagellum is reduced,and the rotary mode of motor is adjusted to enable the bacteria to respond accordingly.Agrobacterium fabrum(tumefaciens)is a kind of Gram-negative bacteria distributed on the surface of plant roots and surrounding soil.Because of its capability to transfer T-DNA gene and integrate it into the host genome,A.fabrum has become the most important tool in current plant transgenic engineering.Like E.coli,A.fabrum has the flagellar structure and chemotaxis,but there are many differences between the two strains.The chemotaxis system of A.fabrum encodes 20 methyl-accepting chemotaxis proteins(MCPs),two coupling proteins(CheWs)and two chemotaxis response regulators(CheYs,encoded by atu0516 and atu0520).At present,why the chemotaxis system of A.fabrum needs to encode two regulators CheYs but only one kinase CheA and the specific biological functions of two CheYs are not clear.Bioinformatics analysis showed that the two CheYs of A.fabrum were similar to E.coli CheY(EcCheY)in gross structure,but the root mean square deviation of skeleton atomic displacement between the homologous modeling structures of the two CheY proteins is only 2.507 A.It is suggested that they may play different regulatory efficiency or even different roles in chemotaxis signal transduction.Based on this,this study investigated the biology function of A.fabrum CheYs,analyzed the different regulatory efficiency and molecular mechanism of the differences in regulating chemotaxis signal,and expanded the research of two CheYs in adhesion,secretion of extracellular polysaccharide,biofilm formation and virulence through three chapters content.These results provided reference for recognizing the overall biological function of chemotaxis response regulators.The main results are summarized as follows:1.atu0516(cheY1)and atu0520(cheY2)genes are regulated by the same chemotactic promoter,and the deletion of cheY genes does not affect the growth and flagellar synthesis of A.fabrum.Firstly,this study used unmarked counterselectable homologous recombination method to construct two cheY genes deletion mutants(single gene deletion mutants and cheY1-cheY2 double-deletion mutant)in which open reading frame was deleted precisely.Then this study determined the growth curve of these mutants.Results showed that under the condition with shaking flask culture in laboratory,the deletion of che Y1 and cheY2 genes does not affect the normal growth of A.fabrum.The results also showed that the deletion of two che Y genes dose not affect the synthesis and assembly of flagellum.The chemotaxis of A.fabrum was completely eliminated after the promoter of the chemotactic gene cluster was knocked out.Furthermore,RT-PCR showed transcriptional levels of cheY1 and cheY2 in A.fabrum chemotaxis promoter-deletion strain were significantly decreased,indicating that cheY1 and che Y2 genes were regulated by the same chemotactic promoter.Total RNA was extracted from A.fabrum in liquid culture and solid plate culture,and the result showed that the transcription level of chemotactic operon of A.fabrum in solid plate culture was similar with that in liquid culture,indicating that the short-term different growth modes(liquid free growth and solid attached growth)had no effect on the expression of chemotactic gene cluster.2.cheY1 and cheY2 genes are both involved in regulating chemotaxis in A.fabrum,in which CheY2 is the main regulator of chemotaxis signals transduction,and CheY1 assists CheY2 in conducting chemotactic signals.The difference in regulatory efficiency between CheY1 and CheY2 is mainly due to the difference in affinity with FliM.The difference of key residues on the motor binding surface is important molecular mechanism leading to the functional differentiation of the two CheYs.Firstly,the comprehensive chemotactic response of two cheY gene deletion mutants to nutrients was investigated via a swim plate.The results showed that the deletion of cheY2 completely disrupted chemotaxis signal transduction,while the deletion of cheY1 slightly affected chemotaxis,and che Y1 could not regulate chemotaxis alone in the absence of che Y2.Furthermore,the swimming behavior of cheY mutant single cell showed that cheY2 was mainly responsible for regulating the rotatory rate and mode of the flagellar motor in A.fabrum,and the complement with cheY1 gene did not significantly affect the swimming behavior of A.fabrum.A split fluorescence method further demonstrated that both CheY1 and CheY2 could bind to CheA in A.fabrum cells to form a complex,which was located at one polar point of the cell.CheY1 was able to form more intracellular polar fluorescence with Che A compared with CheY2 regardless of the presence or absence of CheS.Then,bacterial two-hybrid system and western blot were used to explore the interaction between CheY 1(CheY2)and CheA.The results showed that the affinity of CheY1 with CheA and the affinity of CheY2 with CheA were in the same level.This also suggested that the functional differences between CheY1 and CheY2 did not result from affinity differences with CheA.However,the affinity of CheY1 with FliM and CheY2 with FliM was significant different.The affinity between CheY2-P and FliM is 5 folds that between CheY1-P and FliM,suggesting that the functional difference between CheY1 and CheY2 was due to the different reactive mechanism between CheYs and FliM.Subsequently,via substituting the key residues at the C-terminal of CheY1 and CheY2 proteins,this study further verified the hypothesis that the difference of key residues in the a4?5-?5 domain results in a significant difference in the regulatory efficiency of two CheYs in chemotaxis signal tranduction.Unexpectedly,two CheY2 protein variants(CheY2R93A and CheY2A109V)were obtained to significantly enhance chemotactic activity.Their responses to plant secretion signals,sucrose and acetY1eugenone(AS)demonstrated that CheY2R93A and CheY2A109V could significantly increase chemotaxis activity of native CheY2 in the absence of CheYl,suggesting that it is possible to enhance the biological function of the original protein by substituting key residues from homologous proteins.The results of substituting of C-terminal structures also indicated that ?4-?5-?5 domain is an important but not the only determinant of distinguishing CheY's function.Finally,the interactions between two CheYs with VirA provided the evidence that VirA is directly involved in the chemotaxis of A.fabrum to respone to AS.VirA may be involved in responsing to AS via directly interacting with CheY2 or CheY2-P,although the phosphate transfer between VirA and CheY2 has not been confirmed.3.The deletion of cheY2 gene significantly increases the adhesion,synthesis of extracellular polysaccharide,SSA-biofilm formation and virulence in A.fabrum.It was found that the cheY2 gene deletion significantly improved the adhesion of A.fabrum cells to hydrophilic biological and abiotic surfaces via testing the adhesion capabilities of key chemotactic components mutants(cheA deletion,che Y deletion)and mobile component mutant(motA deletion).Then,the adhesion of A.fabrum cheY deletion mutant cells to hydrophobic surface was determined,and result showed that the deletion of cheY2 significantly increased the adhesion of A.fabrum cell to hydrophobic hydrocarbon while the defect of cheA and motA had no significant effect on adhesion,suggesting that cheY2 may influence biological adhesion through a unique pathway.Subsequently,Congo Red binding assay showed that three che Y2-deficiency strains(?y2,?y and ?y+y1 strains)combined with Congo red reagent to produce the deeper red reaction than the other groups,suggesting that cheY2 deletion significantly increased the synthesis of extracellular polysaccharide in A.fabrum.This conclusion was further proved quantitatively by a sulfuric acid-anthrone assay.The solid-surface-associated biofilm(SSA-Biofilm)formation of each strain was verified via a typical glass tube biofilm formation assay.The result showed that che Y2-deficiency strains were the first to switch from liquid free growth to solid attached growth and accumulated the most biofilm products.The mutant CheY2D58A in which the phosphorylated active site D58 was replaced by alanine(A),the C-terminal domain heterozygote CheY2(NY2CY1),and the CheY of E.coli(EcCheY)could not effectively restore the biofilm phenotype like native CheY2 dose in che Y2-deletion strain ?y2.These results suggested that the effect of CheY2 on A.fabrum biofilm formation depends on its own specific and active spatial conformation.Later,virulence infection assay also showed that the deletion of cheY2 gene significantly increased the tumorgenesis of A.fabrum on the three plant hosts represented by root,stem and leave.RT-PCR result showed that deletion of cheY2 gene significantly affected the transcriptional levels of several DGCs and PDEs enzymes.Among them,the overexpression of Atu1114,Atu3207 and Atu4490 significantly reduced the biofilm formation in ?y2 strain.This suggests that deletion of the cheY2 gene affects the expression levels of at least three important phosphodiesterases(Atu1114,Atu3207,and Atu4490)with strong activity of decompose c-diGMP,leading to the excess synthesis of extracellular polysaccharide and the accumulation of biofilm.Finally,the interaction between CheY2 and 30 DGCs(or PDEs)proteins of A.fabrum was detected via Bacterial Two-hybrid System.The result showed that CheY2 significantly interacted with Atu0701,Atu1119 and Atu1257 in E.coli,which may be directly involved in the regulation of c-di-GMP. |
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