STRUCTURAL DETERMINATION OF THE LPS FROM RHIZOBIUM

根瘤菌 LPS 的结构测定

• 批准号: 2179935
• 负责人: RUSSELL W CARLSON
• 金额: $ 12.57万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-06-01 至 1997-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2179935
• 关键词: Leguminoseae; Rhizobiaceae; carbohydrate structure; enzyme linked immunosorbent assay; epitope mapping; lipopolysaccharides; mass spectrometry; methylation; monoclonal antibody; mutant; nuclear magnetic resonance spectroscopy; oligosaccharides; symbiosis

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.

这笔赠款的目标是表征 来自豆科氏菌生物菜菌的LPS（最近重新分类） 根茎Etli）Ce3菌株，并阐明该分子在 其豆类宿主的氮固定共生感染已知 成功感染豆类需要根茎lps 主持人。 LPS的总变化，例如O链多糖的损失， 导致流产的感染。 单克隆抗体表明 在感染期间，LPS中发生了细微的结构变化 在感染期间。通过在LPS生物合成途径中使用定义的突变体 （由Marquette大学的Dale Noel博士提供），完整 LP的结构将被确定，以及这些表位的变化 在共生感染期间发生。分析来自突变体的LPS 感染过程的各个阶段将（a）促进 LPS生物合成途径中基因的识别，（b）使我们能够 确定O链，核心寡糖和脂质A链接的如何 完整的LPS分子。来自这些突变体的LP，以及 父菌株（CE3）将用于隔离那些携带的LPS片段 这些表位在共生感染中很重要。这些表位将是 通过ELISA抑制测定法确定。单克隆抗体（即 JIM26，JIM27，JIM28和JIM29）由Nick Brewin博士提供 英国约翰·英恩斯学院。此外，研究生将 访问Noel博士的实验室，以分离无法进行的突变体 共生感染期间发生的表位变化。这些突变体 将分析以确定其结构（我们）和共生（通过 Noel博士）缺陷。各种LPS和LPS的结构测定 碎片将通过糖基组成和甲基化进行 分析，NMR和高分辨率质谱法。我们目前知道 O链重复单元的结构，两个核心寡糖， 和R.腿的脂质A。 BV。 exepyoli ce3 lps。脂质a 根茎是独一无二的，因为它没有磷酸盐，其糖 骨架由半乳酸，葡萄糖胺和2-氨基葡萄糖组成 酸。我们已经表明，根茎提取物具有所有必要的酶 为了合成脂质，肠脂质的前体 KDO2IVA。因此，在根茎中，这个共同的前体可能被处理为 形成独特的根茎脂质A.一种类似的酶，一种特定的KDO2IVA 4'-- 磷酸酶已经被鉴定出来。最后的目标 建议是将处理KDO2LVA的其他酶表征 独特的根茎脂质A.克里斯·雷兹（Chris Raetz）博士（杜克大学）已同意 在项目的这一方面与我们合作。