茎瘤固氮根瘤菌中参与趋化作用的受体蛋白Mcp5的功能机理研究

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. Previously it was showed that the methyl-accepting chemotaxis protein Mcp5 affected the chemotatic response to proline in A.caulinodans. In this study, the functions and regulatory mechanisms of the Mcp5 protein involved in chemotaxis signal transduction in A.caulinodans ORS571 will be further determined. The functional characteristics of the Mcp5 protein are studied by the construction of mutants, and the chemotactic defect could be complemented by expressing the wild-type gene from its own promoter in trans from a plasmid. Then key functional sites are identified by site-specific mutagenesis of the Mcp5 protein. A C-terminal translational fusion of the yellow fluorescent protein (Yfp) with the Mcp5 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 the Mcp5 protein. The expression level of the Mcp5 gene or the Mcp5 protein was examined by real-time quantitative PCR or Western blot when cells were grown under the free-living, symbiotic and non-symbiotic conditions, respectively. Interactions between the Mcp5 protein and chemotactic attractants or repllents are identified by yeast two-hybrid system, pull-down and ITC. Our findings further highlight the mechanisms of chemotaxis signal transduction in nodule bacteria and legume-rhizobium interactions. It is hoped that this study will provide a theoretical framework for this system that may lead to more productive hypotheses.

茎瘤固氮根瘤菌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，其感受外界信号的受体系统应当更为复杂多样。本项目将在前期工作基础上，重点研究茎瘤固氮根瘤菌信号受体蛋白Mcp5感应脯氨酸等信号物质过程中的功能机理。项目拟通过缺失突变及表型分析鉴定Mcp5的生物学功能，定点突变鉴定其功能位点；将Mcp5与绿色荧光蛋白融合表达，检测Mcp5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Mcp5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、pull-down、等温滴定微量热仪等手段研究Mcp5与信号物质之间的互作。该项研究将为揭示根瘤菌的趋化信号转导机制及其与宿主植物的互作机理提供重要理论依据。

茎瘤固氮根瘤菌不仅可以与毛萼田菁形成根瘤和茎瘤共生固氮，还能够侵入到植物侧根皮层内进行固氮。趋化性被认为是根瘤菌侵染植物根部的第一步。本项目针对茎瘤固氮根瘤菌ORS571趋化受体蛋白Mcp5的功能、调控机理及其对根际微生物的影响进行了初步探讨，并对细菌在自生或与宿主共生时所发挥的功能进行了深入研究，主要的研究内容与结果如下：（1）利用生物信息学及比较基因组学的方法对茎瘤固氮根瘤菌ORS571的趋化基因簇与趋化受体进行分析，发现ORS571具有一套独特的趋化系统。不同于其它的固氮菌，该基因组中只含有一条趋化基因簇，还存在一个CheY基因与CheZ基因游离于趋化基因簇之外。基因组中共含有43条受体基因，均属于38H型，并存在保守的甲基化位点与核心区域，甲基化酶CheR是非五肽依赖型。（2）首次报道了可溶性趋化受体Mcp5影响到根瘤菌与宿主的共生能力。Mcp5蛋白定位在细胞的极端，氮端的PAS结构域以血红素为辅基，感应外界氧信号。Mcp5保守的组氨酸位点为血红素结合的关键位点，对Mcp5趋化、趋氧功能的发挥起到关键作用。Mcp5的缺失使细菌的生物膜及胞外多糖产量增加，自生状态下的固氮酶活下降，结瘤能力减弱，瘤中豆血红蛋白含量明显减少，瘤的体积与固氮酶活也明显小于野生型。（3）Mcp5的定位受到TlpA1蛋白的影响。TlpA1位于趋化基因簇上游，是一个N端包括两个的跨膜区甲基化受体，缺失突变后细胞在自生状态下对多种碳源的趋化性以及生物被膜的形成能力均减弱。与宿主共生时，tlpA1基因缺失突变体的竞争性定殖和结瘤能力也都下降。这些结果显示受体蛋白可能共同通过感应环境信号，调节其生理活性及与宿主的共生过程。（4）受体蛋白缺失后，导致植物根系根瘤菌种群结构发生改变。该研究结果为揭示宿主植物共生信号转导和根瘤共生体形成的机制提供重要理论依据。