茎瘤固氮根瘤菌ORS571受体TlpA1对琥珀酸的感应机理

趋化作用在根瘤菌与宿主建立共生关系的起始阶段起着至关重要的作用。TlpA1是茎瘤固氮根瘤菌ORS571重要的跨膜趋化受体，但其感应机理尚未清楚。考察茎瘤固氮根瘤菌ORS571跨膜趋化受体TlpA1对趋化物的感应机理及生物学功能。通过平末端连接的方法构建N端表达菌株进行原核表达蛋白及纯化，利用等温滴定量热法（isothermal titration calorimetry）检测蛋白与趋化物的相互作用，测定野生型与突变株的生物膜及絮凝等表型。结果显示，跨膜趋化受体TlpA1通过胞外部分第29～183位氨基酸与琥珀酸结合。固氮条件下，突变株ΔTlpA1生物膜形成量相较于野生型下降约38%。絮凝量与野生型无显著性差异。跨膜趋化受体TlpA1通过感应琥珀酸调控茎瘤固氮根瘤菌ORS571生物膜的形成，对细胞絮凝无调控作用。

Sensing Mechanism of Receptor TlpA1 to Succinic Acid in Azorhizobium caulinodans ORS571

Chemotaxis plays an important role in the initial stage of symbiosis between rhizobium and host. TlpA1 is an important transmembrane chemoreceptor in A. caulinodans ORS571, but its sensing mechanism is not clear. To study the mechanism of the interaction between receptor protein TlpA1 and chemotaxin, and the biological function of TlpA1 in A.caulinodans ORS571. The N-terminal expression plasmid was constructed by the blunt end ligation, and the target protein was expressed and purified in Escherichia coli. The interaction between protein and chemotaxin was detected by isothermal titration calorimetry. The phenotypes, such as biofilm formation and cell flocculation, were investigated. The results showed that the receptor protein TlpA1 bound to succinic acid through its extracellular part (29～183 amino acids). Under the condition of nitrogen fixation, the biofilm formation of ΔTlpA1 mutant decreased about 38% compared with wild type. And there was no significant difference in flocculation between the ΔTlpA1 mutant and wild type. The transmembrane chemoreceptors TlpA1 could regulate the biofilm formation of A. caulinodans ORS571 by sensing of succinic acid, but could not regulate the flocculation of A. caulinodans ORS571.