STRUCTURAL DETERMINATION OF THE LPS FROM RHIZOBIUM

根瘤菌与豆科植物寄主之间的固氮共生体 涉及识别信号分子的复杂过程 共生体和寄主豆科植物的高度协调 基因在植物和共生体中的表达。其结果是 过程是一种根瘤，其中含有固氮菌，称为 类杆菌。最近的许多报告表明，主要的外膜 根瘤菌的组成成分--脂多糖(LPS)在这方面是必不可少的 共生感染过程。缺乏已知的部分内毒素的突变体 由于O-抗原多糖(O-链)是缺陷感染 在细菌释放到皮质根的过程中形成细丝或 细胞。此外，使用单抗还表明， 在共生感染和感染期间，内毒素发生了微妙的结构变化 这些变化很可能是形成固氮体系的关键 结节。这些结构变化也被观察到，当细菌 在某种程度上模仿根部内部的条件下生长 结节，例如低pH值(4.8)。这项建议的主要目的是 描述所需的这些重要的脂多糖结构变化 用于共生感染。具体地说，关于LPSR。 豆科植物豆科植物菜豆共生菌CE3和BV.维克泰(共生体) 豌豆)，目的是：确定O-抗原的结构 多糖；测定内毒素核心低聚糖、脂类A和O- 抗原结构区域连接形成完整的内毒素分子；以及 确定在共生过程中发生的内毒素结构变化。O- 将使用甲基化、GC-MS、FAB-MS和 核磁共振分析技术。带和不带O链的LPS将是 其特征是确定脂类A、核心低聚糖和O- 链条连接在一起。在pH 4.8的条件下生长的细菌的LPS将是 以确定正在发生的结构变化为特征的 在结瘤期间。根据这项拟议工作的结果，它应该 (在未来)能够识别这些内毒素所需的基因 并确定它们的表达如何受到宿主的调控 种。

Nitrogen fixing symbioss between a Rhizobium and its legume host is a complex process involving the recognition of signal molecules from both the symbiont and the host legume which results in a highly coordinated expression of genes in both the plant and symbiont. The result of this process is a root nodule which contains nitrogen-fixing bacteria, called bacteroids. Many recent reports have shown that the major outer membrane component of rhizobia, the lipopolysaccharide (LPS), is essential in this symbiotic infection process. Mutants which lack a portion of the LPS known as the O-antigen polysaccharide (O-chain) are defective either infection thread formation or in the release of the bacteria into the cortical root cells. Additionally it has been shown, using monoclonal antibodies, that subtle structural changes occur in the LPS during symbiotic infection and that these changes are likely to be crucial in forming a nitrogen-fixing nodule. These structural alterations are also observed when the bacteria are grown under conditions which, in part, mimic those inside the root nodule, e.g. low pH (4.8). The main objective of this proposal is to characterize these important LPS structural alterations that are required for symbiotic infection. Specifically, with regard to the LPSs R. leguminosarum bv. phaseoli CE3 (symbiont of bean) and bv. victae (symbiont of pea), the objectives are to: determine the structure of the O-antigen polysaccharides; determine how the LPS core oligosaccharide, lipid A and O- antigen structural regions are joined to form a complete LPS molecule; and determine the LPS structural changes that occur during symbiosis. The O- antigen structures will be determined using methylation, GC-MS, FAB-MS and NMR analytical techniques. The LPSs with and without the O-chain will be characterized to determine how the lipid A, core oligosaccharide and O- chain are joined together. The LPSs from bacteria grown at pH 4.8 will be characterized to determine the structural alterations that are occuring during nodulation. Based on the results of this proposed work, it should (in the future) be able to identify the genes required for these LPS alterations and to determined how their expression is regulated by the host plant.