茎瘤固氮根瘤菌（Azorhizobium caulinodans ORS571）属于α变形菌纲，作为根瘤菌，A. caulinodans不仅可以在宿主植物毛萼田菁的根部和茎部结瘤，而且在自生状态下也具有固氮能力。趋化是指可运动的细菌感应环境中的化学浓度梯度，从而向有利于自身生长的环境方向运动的应激过程。在细菌的趋化过程中，趋化受体感应外界环境中的变化，并将信号传递至趋化通路中，引导菌体向最适的环境方向运动。本文对A. caulinodans的趋化系统进行了系统进化分析，并对其中的可溶性趋化受体的功能进行了初步探讨，针对可溶性趋化受体IcpB在细菌自生与共生时所发挥的功能进行了深入研究，为揭示其与宿主毛萼田菁的共生机制提供了重要的理论依据。主要的研究内容与结果如下：

（1）A. caulinodans趋化系统的比较基因组学分析

利用生物信息学及比较基因组学的方法对A. caulinodans的趋化基因簇与趋化受体基因进行分析，发现A. caulinodans的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，所有受体的胞内部分序列保守，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。启动子预测显示趋化基因簇的启动子为σ54型。

（2）A. caulinodans可溶性趋化受体功能的初步探讨

 构建了A. caulinodans中六个可溶性趋化受体的基因敲除菌株和回补菌株。利用半固体培养基检测突变株趋化能力发现所有受体缺失菌株的趋化圈均小于野生型，并且在固氮条件下缺失表型更明显。利用绿色荧光蛋白（GFP）对可溶性趋化受体进行亚细胞定位，结果显示所有的受体均定位在细胞的极端，并且在固氮条件下的表达量增强。竞争性结瘤实验表明除含有PiLZ结构域的AZC-3349外，其他五个含有氮端PAS结构域的受体所对应的突变株的竞争性结瘤能力均小于野生型菌株。

（3）可溶性趋化受体IcpB在A. caulinodans自生与共生中的功能研究

利用测定蛋白光吸收峰的方法验证了IcpB氮端的PAS结构域是与血红素相结合，并且位于154位点上的组氨酸（H154）为血红素结合的关键位点。利用氨基酸定点突变证明，H154对IcpB趋化、趋氧功能的发挥起到关键作用。IcpB的缺失使细菌的生物膜及胞外多糖产量增加，并且在固氮条件下二者的增量更为显著。A. caulinodans在自生状态下的固氮酶活均有所下降，icpB突变株所形成的茎瘤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。

 

Azorhizobium caulinodans belongs to the alpha-subdivision of proteobacteria and has the capacity of fixing nitrogen both during free-living growth and in a symbiotic interaction with Sesbania rostrata. Chemotaxis is a stimulated process enabling motile bacterial species to detect chemical gradients and to move in a benefical direction. In bacterial chemotaxis, chemoreceptors sense environmental changes and transmit this information to the chemotactic machinery to guide motile bacteria to preferred niches. In this paper, the chemotaxis system of A. caulinodans was systematically analyzed and the functions of soluble chemoreceptors were preliminarily discussed. Both under free-living and symbiotic states, the role of IcpB was lucubrated, the research contents and results are as follows:

(1) Comparative genome analysis of chemotaxis system of A. caulinodans.

Che clusters and chemoreceptor genes were analyzed via bioinformatic and comparative genomic methods. We found that there is a unique che cluster, a cheY and a cheZ are outside the major che cluster. We identified 43 methyl-accepting chemotaxis protein (MCP) homologs containing conserved methylated domain and core domain in which cytoplasmic domains are composed of 38 heptad repeats. The methylase CheR is pentapeptide-independent. The che cluster is predicted to regulate by σ54 promoter.

(2) Preliminary studies of the soluble chemoreceptors.

We have constructed mutants of six soluble chemoreceptors. The chemotactic rings of all six mutants were smaller than that of wild type strain and this difference was more obvious when bacteria were cultured under nitrogen fixation condition. To visualize the subcellular localization of chemoreceptor in vivo, we expressed the green fluorescent protein (GFP) fused to the C-terminal region of six chemoreceptors. The result showed that All chemoreceptors localized to the cell poles. The fluorescence of polar foci was more stronger under nitrogen fixation condition. In nodulation competition assay, all five PAS containing chemoreceptors could not compete with the wild type strain except the PiLZ containing chemoreceptor (AZC-3349).

(3) The study on function of soluble chemoreceptor IcpB during free-living and symbiotic stage.

This spectrophotometric analysis confirmed that the IcpB N-terminal PAS domain possessed an absorption spectrum typical of oxygen-bound heme proteins. The histidine residue at position 154 is the key site for bind heme. Site-directed mutagenesis confirmed that H154 plays a key role in bacterial chemotaxis and aerotaxis. Mutation of the icpB gene affected the production of biofilm and exopolysaccharides, especially under nitrogen fixation condition. The nodules induced by the icpB mutant lacked sufficient leghemoglobin and were smaller than those induced by the wild type strain.