STRUCTURAL DETERMINATION OF THE LPS FROM RHIZOBIUM

这笔赠款的目的是描述 豆类根腐病菌生物群的内毒素(最近重新分类) 根瘤菌等)菌株CE3，并阐明该分子在植物生长发育中的作用。 已知其豆科植物寄主的固氮共生感染 根瘤菌脂多糖是成功感染豆类所必需的 主持人。脂多糖的严重变化，例如O链多糖的丢失， 会导致流产的感染。单抗表明， 在感染过程中，内毒素会发生细微的结构变化。 在感染期间。通过在内毒素生物合成途径中使用已定义的突变体 (马奎特大学的Dale Noel博士提供)，完整的 将确定内毒素的结构，以及这些表位的变化 发生在共生感染期间。突变株的LPSs分析 感染过程的不同阶段将(A)促进 鉴定内毒素生物合成途径中的基因，以及(B)使我们能够 为了确定O链、核心低聚糖和类脂A是如何连接在一起的 完整的内毒素分子。来自这些突变体的内毒素，以及来自 亲本菌株(CE3)将被用来分离携带 在共生感染中很重要的表位。这些表位将是 经酶联免疫吸附抑制试验鉴定。单抗(即 JIM26、JIM27、JIM28和JIM29)由Nick Brewin博士提供 位于英国的约翰·英尼斯研究所。此外，一名研究生将 访问诺埃尔博士的实验室，分离出无法通过 在共生感染期间发生的这些表位变化。这些变种人 将被分析以确定它们的结构(由我们)和共生(由 诺埃尔博士)缺陷。不同LPS和内毒素的结构测定 片段将通过糖基组成和甲基化进行 分析、核磁共振和高分辨率质谱学。我们目前知道 O链重复单元、两个核心低聚糖、 右腿上的脂类A。BV.菜豆CE3LPS。甘草次酸中的脂类A 根瘤菌的独特之处在于它不含磷，它的糖 主干由半乳糖醛酸、氨基葡萄糖和2-氨基葡萄糖酸组成 酸。我们已经证明，根瘤菌提取物含有所有必需的酶。 为了合成肠源性脂类AS常见的脂类A前体， Kdo2IVa.因此，在根瘤菌中，这种常见的前体可能被加工成 形成独特的根瘤菌脂A。一种这样的酶，一种特殊的Kdo2IVa 4‘- 磷酸酶已经被鉴定出来了。这样做的最后一个目标是 建议是对将Kdo2lVa加工成 独一无二的根瘤菌脂A.克里斯·雷茨博士(杜克大学)同意 在这个项目的这方面与我们合作。

The objectives of this grant are to characterize the unique structure of the LPS from R. leguminosarum biovar phaseoli (recently reclassified Rhizobium etli) strain CE3 and to elucidate the role of this molecule in the nitrogen-fixing symbiotic infection of its legume host it is known that Rhizobium LPS are required for a successful infection of the legume host. Gross changes in the LPS, e.g. loss of the O-chain polysaccharide, result in aborted infections. Monoclonal antibodies have shown that during infection there are subtle structural changes that occur in the LPS during infection. By using defined mutants in the LPS biosynthetic pathway (provided by Dr. Dale Noel of Marquette University), the complete structure of the LPS will be determined, as well as those epitope changes that occur during symbiotic infection. Analysis of LPSs from mutants at various stages of the infection process will (a) facilitate the identification of genes in the LPS biosynthetic pathway, and (b) enable us to determine how the O-chain, core oligosaccharides and lipid A linked in the complete LPS molecule. The LPS from these mutants, as well as from the parent strain (CE3) will be used to isolate those LPS fragments that carry the epitopes important in symbiotic infection. These epitopes will be identified by ELISA inhibition assays. The monoclonal antibodies (i.e. JIM26, JIM27, JIM28 and JIM29) have been provided by Dr. Nick Brewin of the John Innes Institute in the UK. Additionally, a graduate student will visit Dr. Noel's laboratory to isolate mutants that are unable to undergo those epitope changes that occur during symbiotic infection. These mutants will be analyzed to determine their structural (by us) and symbiotic (by Dr. Noel) defects. Structural determination of the various LPSs and LPS fragments will be carried out by glycosyl composition and methylation analyses, NMR, and high resolution mass spectrometry. We currently know the structures of the O-chain repeating unit, two core oligosaccharides, and the lipid A from the R. leg. bv. phaseoli CE3 LPS. The lipid A of Rhizobium is unique in that it does not have phosphate, and its sugar backbone consists of galacturonic acid, glucosamine and 2-aminogluconic acid. We have shown that Rhizobium extracts have all the enzymes necessary for the synthesis of a lipid A precursor common to enteric lipid As, Kdo2IVa. Thus, in Rhizobium this common precursor is probably processed to form the unique rhizobial lipid A. One such enzyme, a specific Kdo2IVa 4'- phosphatase has already been identified. The last objective of this proposal is to characterize the other enzymes that process Kdo2lVa into the unique rhizobial lipid A. Dr. Chris Raetz (Duke University) has agreed to collaborate with us in this aspect of the project.