茎瘤固氮根瘤菌趋化信号通路中相关蛋白的功能及调控机制

The alphaproteobacterium Azorhizobium caulinodans ORS571 has the capacity to fix nitrogen not only as a free-living organism and an associative-symbiotic bacterium by colonizing the root surface of non-leguminous plants, but also as a symbiotic bacterium by interacting with Sesbania rostrata. Due to its ability to grow and fix nitrogen under three conditions, A.caulinodans employs sophisticated chemotaxis signal transduction systems to transform environmental cues into corresponding behavioural responses. But the mechanism of chemotaxis signal transduction in A.caulinodans is still elusive. Previously it was showed that the chemotaxis protein CheA affected the production of EPS and nodulation capacity. In this study, functions and regulatory mechanisms of seven chemotaxis proteins involved in chemotaxis signal transduction in A.caulinodans will be further determined..The functional characteristic of each chemotaxis protein is studied by constructiog each mutant and analyzing in vitro activity of each protein. A C-terminal translational fusion of the fluorescent protein with each chemotaxis protein, expressed under the control of its native promoter from a broad host range low-copy plasmid, is used to determine the subcellular localization and expression of each protein. The expression level of each chemotaxis protein is examined by real-time quantitative PCR or Western blot when cells are grown under the free-living, symbiotic and non-symbiotic conditions, respectively. The proteins interacted with CheA or CheZ are screened by yeast two-hybrid system, and interactions between two proteins are further identified by Co-IP, ITC, and NMR. It is hoped that this study will provide a theoretical framework for the mechanisms of rhizobia to adapt to the environment.

茎瘤固氮根瘤菌ORS571属于α-变形杆菌纲，既可以与毛萼田菁共生固氮，又可以自生或作为内生菌在其他植物体内固氮。由于其具有三种生活状态及固氮能力，其趋化信号转导系统应当更为复杂多样，但相关功能及其调控机制尚不清楚。本课题组先期研究已获得了趋化蛋白CheA参与田菁结瘤的实验证据。本项目将重点研究趋化信号通路中7个相关蛋白的功能及其调控机制。项目拟通过对各个趋化基因进行缺失突变及蛋白体外功能分析，鉴定各个趋化蛋白的生物学功能；将各个趋化蛋白分别与不同的荧光蛋白融合表达，检测其在活细胞中的表达及定位；采用实时定量PCR及蛋白质印迹法检测各个蛋白分别在共生宿主根际、内生宿主根际及自生状态下的表达差异；利用酵母双杂交系统筛选能够与CheA或CheZ互作的蛋白，通过免疫共沉淀、等温滴定微量热仪、核磁共振等手段进一步揭示蛋白间的互作机制。该项研究将为揭示根瘤菌对于环境的适应机制提供重要的理论依据。

植物可以通过根系分泌物的种类及丰度影响根圈范围内微生物的群体种类及数量，这些分泌物可以作为信号物质协助根瘤菌吸附在豆科植物合适的侵染位点，从而增加被感染的机率。不同于模式菌大肠杆菌，茎瘤固氮根瘤菌ORS571属于α-变形杆菌纲，仅拥有一条趋化基因簇，以及两个孤立趋化基因cheY1及cheZ，趋化基因簇的排序具有独特性，但对其相关功能及其调控机制尚不清楚。. 项目组针对ORS571趋化信号通路中相关蛋白的功能及互作机制进行了研究，主要的研究内容与结果如下：1）鉴定了ORS571中趋化基因cheA和整个趋化基因簇Che的功能，证实了组氨酸激酶CheA及整个趋化基因簇不但调控细胞的运动和趋化行为，而且调节细胞的胞外多糖分泌、生物膜的形成、竞争性结瘤等多重要生理功能，揭示了田菁根系分泌物招募土壤微生物的机理；2）利用生物信息学的手段分析了磷酸酯酶CheZ的功能结构域，利用缺失突变或过量表达的方法鉴定了其功能。CheZ蛋白缺失后，细胞几乎完全丧失了趋化能力，倾向于过快地改变运动方向，进而形成类似“转圈”式的运动轨迹。cheZ缺失突变体更容易粘附到非生物和宿主表面，在早期定植中表现出较强的竞争性定植能力，但在长期定植中失去优势，导致了突变体在结瘤上的缺陷，提出了根际细菌新的调控机制；3）反应调节蛋白CheY1和CheY2均调控细菌的趋化和运动能力、胞外多糖的形成、在宿主根部的定植及竞争性结瘤能力，此结果揭示了ORS571复杂的调控机制。. 综上所述，项目组初步绘制和解析了固氮根瘤菌趋化信号转导的基本框架图，阐明了固氮根瘤菌如何感受细胞外面的刺激物信号来适应其生活环境，进而调节其代谢途径及运动的方向，为阐明细菌与环境及宿主的互作机制提供了重要的理论依据。在趋化信号通路中，CheY1、CheZ和CheY2蛋白均发挥了重要的作用，其磷酸转移机制正在进一步的研究中。