根癌农杆菌化学受体MCP1912调节趋化响应功能的鉴定

Functional identification of Agrobacterium tumefaciens chemoreceptor MCP1912 in regulating chemotactic response

摘要:

【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) MCP₁₉₁₂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MCP₁₉₁₂的配体结合结构域(ligand binding domain，LBD)，获得带有His标签的LBD蛋白(LBD₁₉₁₂)。利用基于荧光的热位移测定法(fluorescence-based thermal shift assay，TSA)筛选出LBD₁₉₁₂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LBD₁₉₁₂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MCP₁₉₁₂的基因atu1912，获得MCP₁₉₁₂缺失突变体(ΔMCP₁₉₁₂)；以质粒回补的方法，获得ΔMCP₁₉₁₂回补株(ΔMCP₁₉₁₂C)。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MCP₁₉₁₂潜在配体的趋化响应，并最终确定MCP₁₉₁₂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LBD₁₉₁₂的熔解温度(T_m)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LBD₁₉₁₂特异性结合。丙酮酸与LBD₁₉₁₂的结合是放热过程，K_D为(17.0±0.9)μmol/L，而丙酸与LBD₁₉₁₂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，MCP₁₉₁₂的缺失，完全消除了根癌农杆菌对这两种物质的趋化响应，MCP₁₉₁₂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MCP₁₉₁₂识别的配体是丙酸和丙酮酸，MCP₁₉₁₂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。

关键词:根癌农杆菌;甲基趋化受体蛋白;趋化响应;吸引物;驱避剂;配体结合结构域

Abstract:

[Objective]To identify the chemoeffectors recognized by Agrobacterium tumefaciens C58 chemoreceptor MCP₁₉₁₂ (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 MCP₁₉₁₂ was fused with His tag and expressed as an individual recombinant protein (named LBD₁₉₁₂) through heterologous expression. We employed fluorescence-based thermal shift assay (TSA) to screen the potential ligands of LBD₁₉₁₂, and isothermal titration calorimetry (ITC) to test the binding of the potential ligands to LBD₁₉₁₂ and to determine the equilibrium dissociation constant (K_D) of the ligand-LBD₁₉₁₂ complex. We used homologous recombination-based DNA fragment deletion method to construct MCP₁₉₁₂ deletion mutant (ΔMCP₁₉₁₂). We introduced the plasmid expressing MCP₁₉₁₂ into ΔMCP₁₉₁₂ to construct the complemented strain (ΔMCP₁₉₁₂C). Then, we adopted the capillary chemotaxis assay to test the chemotactic response of various A.tumefaciens strains to the potential MCP₁₉₁₂ ligands and to confirm the function of MCP₁₉₁₂ 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 LBD₁₉₁₂. ITC further confirmed that only pyruvate and propionic acid could specifically bind to LBD₁₉₁₂. The binding of pyruvate to LBD₁₉₁₂ is exothermic with K_D of (17.0±0.9) μmol/L, while the binding of propionic acid to LBD₁₉₁₂ 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 MCP₁₉₁₂ completely eliminated the chemotactic response of A.tumefaciens to pyruvate and propionic acid. The complementation of MCP₁₉₁₂ could restore the chemotactic response of MCP₁₉₁₂-deficient strain to pyruvate and propionic acid. [Conclusion] The chemoeffectors recognized by the chemoreceptor MCP₁₉₁₂ are propionate and propionic acid. MCP₁₉₁₂ mediates the attractant response to pyruvate and the repellent response to propionic acid.

Key words:Agrobacterium tumefaciens;methyl-accepting chemotaxis protein;chemotaxis;attractant;repellent;ligand binding domain