根癌农杆菌化学受体MCP_(1912)调节趋化响应功能的鉴定

Functional identification of Agrobacterium tumefaciens chemoreceptor MCP_(1912) in regulating chemotactic response

【目的】本研究以根癌农杆菌C58为材料,鉴定其甲基趋化受体蛋白(methyl-accepting chemotaxis protein,MCP)MCP_(1912)能够识别的配体,并研究该蛋白在调控根癌农杆菌趋化响应中的具体功能。【方法】通过异源表达MCP_(1912)的配体结合结构域(ligand binding domain,LBD),获得带有His标签的LBD蛋白(LBD_(1912))。利用基于荧光的热位移测定法(fluorescence-based thermal shift assay,TSA)筛选出LBD_(1912)的潜在配体;通过等温滴定量热(isothermal titration calorimetry,ITC),进一步确定筛选出的潜在配体,并测定LBD_(1912)与配体结合之后的解离平衡常数K_(D)。利用基于同源重组的精准DNA片段删除方法,敲除根癌农杆菌C58中编码MCP_(1912)的基因atu1912,获得MCP_(1912)缺失突变体(ΔMCP_(1912));以质粒回补的方法,获得ΔMCP_(1912)回补株(ΔMCP_(1912)C)。利用毛细管趋化测定法,测定根癌农杆菌及其突变体对筛选出来的MCP_(1912)潜在配体的趋化响应,并最终确定MCP_(1912)在调控根癌农杆菌C58趋化响应中的具体功能。【结果】通过TSA,筛选出5种能引起LBD_(1912)的熔解温度(Tm)变化大于2℃的潜在配体(大于2℃意味着可能结合),这5种潜在配体是丙酮酸、L-乳酸、丙酸、乙酸和乙醇酸。ITC实验进一步确定,只有丙酮酸和丙酸能与LBD_(1912)特异性结合。丙酮酸与LBD_(1912)的结合是放热过程,KD为(17.0±0.9)μmol/L,而丙酸与LBD_(1912)的结合是吸热过程,K_(D)为(31.5±5.4)μmol/L。毛细管趋化试验证实,根癌农杆菌C58能够被丙酮酸吸引和躲避丙酸,MCP_(1912)的缺失,完全消除了根癌农杆菌对这两种物质的趋化响应,MCP_(1912)的回补能够恢复其对这两种物质的趋化响应。【结论】化学受体MCP_(1912)识别的配体是丙酸和丙酮酸,MCP_(1912)介导C58对于丙酮酸的吸引趋化响应以及躲避丙酸的趋化响应。

[Objective]To identify the chemoeffectors recognized by Agrobacterium tumefaciens C58 chemoreceptor MCP_(1912)(MCP:methyl-accepting chemotaxis protein),and to verify the function of this MCP in regulating the chemotactic response of A.tumefaciens.[Methods]The ligand binding domain(LBD)of MCP_(1912)was fused with His tag and expressed as an individual recombinant protein(named LBD_(1912))through heterologous expression.We employed fluorescence-based thermal shift assay(TSA)to screen the potential ligands of LBD_(1912),and isothermal titration calorimetry(ITC)to test the binding of the potential ligands to LBD_(1912) and to determine the equilibrium dissociation constant(K_(D))of the ligand-LBD_(1912) complex.We used homologous recombination-based DNA fragment deletion method to construct MCP_(1912)deletion mutant(ΔMCP_(1912)).We introduced the plasmid expressing MCP_(1912)intoΔMCP_(1912)to construct the complemented strain(ΔMCP_(1912)C).Then,we adopted the capillary chemotaxis assay to test the chemotactic response of various A.tumefaciens strains to the potential MCP_(1912)ligands and to confirm the function of MCP_(1912)in regulating the chemotactic response of C58.[Results]Data acquired through TSA showed that 5 chemicals,pyruvate,L-lactate,propionic acid,acetic acid,and glycolate,might be the potential ligands of LBD_(1912).ITC further confirmed that only pyruvate and propionic acid could specifically bind to LBD_(1912).The binding of pyruvate to LBD_(1912) is exothermic with K_(D) of(17.0±0.9)μmol/L,while the binding of propionic acid to LBD_(1912) is endothermic with K_(D) of(31.5±5.4)μmol/L.The capillary chemotaxis assay verified that C58 manifested the attractant response to pyruvate and the repellent response to propionic acid.The deficiency of MCP_(1912)completely eliminated the chemotactic response of A.tumefaciens to pyruvate and propionic acid.The complementation of MCP_(1912)could restore the chemotactic response of MCP_(1912)-deficient strain to pyruvate and propionic acid.[Conclusion]The chemoeffectors recognized by the chemoreceptor MCP_(1912)are propionate and propionic acid.MCP_(1912)mediates the attractant response to pyruvate and the repellent response to propionic acid.