Function Study Of CheW₁ And CheW₂ Genes In The Chemotaxis System Of Agrobacterium Tumefaciens

Chemotaxis allows bacterial cells to modulate their swimming patterns in response to chemoeffectors and keeps them moving forward to an optimal environment.In the chemotaxis model bacterium Escherichia coli,the minimal functional unit that senses chemoeffectors contains six chemoreceptor molecules,organized as trimers of dimers,two CheW molecules,and one dimer CheA molecule.CheW bridges the histidine kinase CheA and the chemoreceptors to form the chemotaxis core signaling complex and plays a crucial role in the assembly and function of the large chemosensory array at the cell poles.The gram-negative bacterium Agrobacterium tumefaciens can cause the crown gall tumor disease by transferring a DNA fragment(called T-DNA)from its Ti plasmid to host plant cell and genetically transforming the host.Chemotaxis of A.tumefaciens toward the wound sites of the host plant is the first step to recognize the host.Unlike all the previously reported chemotaxis systems in other bacteria or archaea,A.tumefaciens has only one major che operon,but two coupling protein encoded genes(atu2075 as cheWi and atu2617 as cheW2)on unlinked loci.Sequence aligment showed that CheWi shares 47.17%sequence identity with CheW2.The homology models of two A.tumefaciens CheW proteins manifested the structures of both A.tumefaciens CheW proteins are very similar to the structure of E.coli CheW,with root-mean-square deviations of backbone atoms less than 0.148 A.These analyses indicated the function of CheW1 and CheW2 might be similar to that of E.coli CheW.So two che W genes were precisely deleted by homologous recombination,and their roles in chemotaxis were investigated by a series of experiments.The main results are as follows:1.Deletion of cheWi and cheW2 genes affects A.tumefaciens chemotaxis and tumorgensisFirstly,the expressions of two cheW genes in different strains were verified by Western blot.Immunoblotting results showed that two cheW genes are expressed simultaneously in the same cell;the deletion of either cheW gene does not affect the expression of another cheW gene.Secondly,swimming in swim agar plate and attraction toward plant leaf disc were used to evaluate the effects of cheW deletions on A.tumefaciens chemotaxis.The results indicated that the in-frame deletion of either cheW gene significantly impaired A.tumefaciens chemotaxis,but does not abolish the chemotaxis,unless both two cheWs were deleted.The effect of cheW2-deletion on the chemotaxis is more severe than that of cheW1-deletion.In the capillary assay,the chemotactic responses of the cheW deletion mutants to the single chemoattractant acetosyringone(AS)were the same as above two results.Bacteria use different MCP to sense different chemoeffectors.The chemotactic response of the ceW2-deletion mutant was always weaker than that of the cheW1-deletion mutant no matter what chemoeffectors they responded to,implying that the effects of the two CheW proteins on the chemotaxis signaling transduction do not depend on the MCP.They can recognize the same MCP and transfer the signal from the same MCP to CheA.2.CheW1 and CheW2 are involved in the formation of the ternary signaling complexes in A.tumefaciensIn order to investgate the role of CheWi and CheW2 on the chemotaxis of A.tumefaciens at a molecular level,bacterial two-hybrid system and in vitro pull-down assay were conducted to vertify the interaction between two CheW proteins and CheA.Both experiements showed that CheW1 and CheW2 could interact with CheA respectively,which suggested that CheW1 and CheW2 might be involved in the formation of the ternary signaling complexes at the cell pole.To verify this hypothesis,eGFP-CheA was expressed in different che W-deletion mutants,in which cheA was deleted.The observation of the fluorescence of these mutant strains showed that the eGFP-CheA fusion protein is located at the cell poles in all of the cheA-deletion mutant strains with either or both of the cheW genes but uniformly distributed throughout whole cell in the cheA-deletion mutant with neither of the cheW genes.These results manifested that CheWi and CheW2 are incorporated into one chemosensory pathway in a redundant manner;they can couple CheA to MCP and form stable ternary signaling complexes respectively.3.The difference of promoter activity and encoded protein itself of two cheW genes lead to the different efficiencies of chemotaxis signal transduction in A.tumefaciensThe expression level of cheW directly affects bacterial chemotaxis.To determine the promoter activities of two cheW genes,an rfp gene was used as a reporter gene for the in situ substitute for cheWi and cheW2.The new strains were named C58RS1(cheW1 was substituted for rfp)and C58RS2(cheW2 was substituted for rfp).The results indicated that the promoter activity of cheW2 is always higher than that of cheWi under all the tested conditions.This means that the concentration of CheW2 is always higher than that of CheWi in A.tumefaciens cells.In the protein function analysis,CheWi and CheW2 had different effects in restoring the chemotactic response of cheW double deletion-mutant even if both cheWi and cheW2 were under the control of the identical promoter.This implies that the expression products of the two cheW genes,CheWi and CheW2 proteins,have different effects on A.tumefaciens chemotaxis in the same concentration.In addition,we also mutated some amino acid residues of CheW1 and CheW2 that are involoved in the interaction between CheWs and CheA.The results showed that the key amino acid residues in two CheWs are different,implying that the affinities of two CheWs with CheA are different.Therefore,we envision that both the different molecular ratio of two CheWs in cell and the different affinities of two CheWs with CheA and chemoreceptors result in the efficiency difference of two CheWs in functioning in the large chemosensory array.From the above,two A.tumefaciens coupling proteins encoded by cheWi and cheW2 are incorporated into one chemotaxis signaling pathway with different efficiencies in a redundant manner.The different efficiencies of cheWi and cheW2 in chemotaxis signaling transduction arise from both the promoter activity and the protein itself.Under the tested condition,the difference of the promoter activity of two cheW genes is the main reason.