根际促生菌SQR9关键趋化受体蛋白McpA识别根系分泌不同结构趋化物的分子机制研究

Bacillus amyloliquefaciens is a relatively important PGPR in rhizosphere, but little research of its molecular mechanism of rhizosphere chemotaxis has been studied. Bacillus amyloliquefaciens SQR9, which was isolated from cucumber rhizosphere by this laboratory and have been widely applied in agricultural production as the microbial fertilizer. The rhizospheric chemotaxis to root exudtaes by SQR9 mediating chemoreceptor McpA is dominantly driven by a handful of strong attractants, but the McpA chemoreceptor from SQR9 was identified as only a few chemoreceptors are known to show chemotactic to more than twenty compounds, which has already been found to bind specifically aspartic acid, citric acid, and galactose. This proposal will illustrate the molecular interaction characteristics between McpA and multiple corresponding chemotactic amino acids, organic acids and sugars attractants. We also attempt to obtain the apo-state structure of McpA-LBD, and the ligand-complex structures by soaking the apo-state crystal with different ligands by X-ray, providing a basis for understanding the mechanism of the binding between chemoreceptor and ligands. This research will provide the theoretical instructions for improving the root colonization and agricultural application of B. amyloliquefaciens SQR9.

解淀粉芽孢杆菌是一类重要的植物根际促生菌，然而对其在根际趋化分子机制的相关研究鲜有报道。解淀粉芽孢杆菌SQR9是本实验室分离自健康黄瓜根际并在农业生产中广泛应用的促生菌，前期研究发现菌株SQR9主要通过趋化受体蛋白McpA识别黄瓜根系分泌物中趋化性较强的物质而实现根际趋化，而且McpA是少数能够同时识别超过20种趋化物的受体，目前已经鉴定到该受体能够特异性结合天冬氨酸、柠檬酸和半乳糖。本项目将在此基础上研究受体蛋白McpA与包括氨基酸、有机酸和糖类等在内的多种小分子趋化物的互作特征，通过X射线衍射解析McpA胞外配体识别结构域的蛋白结构及该蛋白晶体与不同配体共浸泡后的结构，预期研究成果可以为阐明趋化受体蛋白与不同类型配体之间的互作奠定基础，同时也为进一步增强菌株SQR9在根际的定殖能力、更好的发挥其农业应用效果提供理论指导。

为了适应复杂多变的外界环境条件，细菌经过长期的自然进化，已经形成多种信号转导系统。以趋化受体为基础的信号识别系统，一方面在细菌中普遍存在，另外一方面也属于自然界中最复杂的信号系统之一。然而，目前我们对趋化受体识别信号机制的分子模式特征却知之甚少。已知，贝莱斯芽孢杆菌SQR9的趋化受体McpA可以介导对不同类型的小分子配体的趋化表型，在本研究中我们又进一步证明了McpA可以直接结合13种趋化物配体。有机酸和氨基酸等趋化物分别被dCache结构域的膜远端和膜近端所识别，而糖/糖醇类趋化物则只能被完整的dCache结构与所识别。通过结构生物学与点突变等实验的联合证明，我们最终一共鉴定出10个关键氨基酸残基参与感受不同类型的趋化物。其膜远端和膜近端的氨基酸残基对于细菌感受有机酸和氨基酸类趋化物非常重要，而两个模块中的氨基酸残基共同参与了对糖/糖醇趋化物的识别。前期研究发现大多数的趋化受体拥有狭窄而明确的趋化信号范围，而本研究提出了一种新的信号识别机制，即单一趋化受体可以利用其dCache两个结构域调控其对多种结构种类不同趋化信号的识别。