Genetics and biosynthesis of an O antigen essential for symbiosis

共生所必需的 O 抗原的遗传学和生物合成

基本信息

批准号：
7779181
负责人：
KENNETH D NOEL
金额：
$ 21.78万
依托单位：
MARQUETTE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-01-15 至 2014-12-31
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): The lipopolysaccharides (LPS) of Gram-negative bacteria have complex structures and multiple functions. These bacterial surface molecules are very important in interactions between bacteria and their environment, including beneficial and destructive interactions with animal and plant hosts. The proposed work deals with how the outer portion, the O antigen, of the LPS is synthesized. There are two well-differentiated mechanisms of O-antigen synthesis. The LPS of Rhizobium etli CE3 is one of the better characterized examples (structurally and biologically) in which the O-antigen portion of the LPS is synthesized by a mechanism dependent on an ABC transporter encoded by wzm and wzt genes. It fits into a relatively under-studied subgroup of such LPSs in which the O antigen is a heteropolysaccharide, rather than a homopolysaccharide. The importance of its O-antigen in the symbiosis with Phaseolus vulgaris (bean) has been established, the structure of the entire LPS is almost completely solved, most of the steps in the synthesis of the lipid A and core portion of the molecule have been demonstrated in vitro, and a 30 kb cluster of genes that specify most of O-antigen biosynthesis has been sequenced. Moreover, the nucleotide sequence of the entire genome has been determined. In the proposed work, basic features of the biosynthesis of this O antigen would be determined. It would test a set of hypotheses that predict the order in which the transferases add the sugar residues and which genes encode which transferases. Most of the proposed work is biochemical. One approach that already is well-underway is to to deduce from truncated LPSs the steps at which different mutants are blocked. It has added to the hypothesis by matching genes with specific predicted glycosyltransferase activities. Another set of approaches involve examination of reactions in vitro with cell extracts from the various mutants predicted to have lesions in glycosyltransferases. In particular the work seeks to define the enzymes and products of the first reaction in the synthesis of the O antigen. In addition to this biochemical work, there will be additional genetic analysis to finish the nearly complete accounting of which genes are required in O-antigen synthesis in this bacterium and which functions they might have. These latter experiments will generate specific, nonpolar mutations in genes that still have not been mutated in two genetic loci that we have strong evidence are devoted to O-antigen synthesis. In addition two types of targeted genetic manipulations will address two interesting questions that may be related to the extreme uniformity of length of this O antigen. PUBLIC HEALTH RELEVANCE: With a model bacterium that does not cause medical problems, this project examines the fundamental basis of a property of health-beneficial and pathogenic bacteria that is crucial to their survival, especially in the hostile confines of the mammalian circulatory fluids. It also is important in other interactions with animal and plant hosts. This property is a type of carbohydrate chain on the surfaces of many bacteria. The question addressed in this work is how this chain is synthesized.

描述（申请人提供）：革兰氏阴性菌的脂多糖（LPS）结构复杂，功能多样。这些细菌表面分子对于细菌与其环境之间的相互作用非常重要，包括与动植物宿主的有益和破坏性相互作用。拟议的工作涉及 LPS 的外部部分（O 抗原）是如何合成的。 O-抗原合成有两种截然不同的机制。根瘤菌 CE3 的 LPS 是特征较好的例子之一（结构上和生物学上），其中 LPS 的 O 抗原部分是通过依赖于 wzm 和 wzt 基因编码的 ABC 转运蛋白的机制合成的。它属于此类 LPS 中研究相对较少的亚组，其中 O 抗原是杂多糖，而不是同多糖。其O-抗原在与菜豆（菜豆）共生中的重要性已经确定，整个LPS的结构几乎完全解析，脂质A和分子核心部分合成的大部分步骤已经被解决。已在体外得到证实，并且已对指定大部分 O 抗原生物合成的 30 kb 基因簇进行了测序。此外，整个基因组的核苷酸序列已经确定。在拟议的工作中，将确定这种 O 抗原生物合成的基本特征。它将测试一组假设，预测转移酶添加糖残基的顺序以及哪些基因编码哪些转移酶。大部分拟议的工作是生物化学的。一种已经在顺利进行的方法是从截短的 LPS 中推断出不同突变体被阻断的步骤。它通过将基因与特定的预测糖基转移酶活性相匹配来补充这一假设。另一组方法涉及用预测糖基转移酶损伤的各种突变体的细胞提取物检查体外反应。特别是，这项工作旨在定义 O 抗原合成中第一个反应的酶和产物。除了这项生化工作之外，还将进行额外的遗传分析，以完成该细菌中 O 抗原合成所需的基因以及它们可能具有哪些功能的几乎完整的计算。后面的这些实验将在基因中产生特异性的非极性突变，而这些基因在我们有强有力的证据致力于 O 抗原合成的两个基因位点中尚未突变。此外，两种类型的靶向基因操作将解决两个有趣的问题，这些问题可能与这种 O 抗原长度的极端均匀性有关。公共健康相关性：该项目利用不会引起医疗问题的模型细菌，研究了对健康有益和致病细菌的生存至关重要的特性的根本基础，特别是在哺乳动物循环液的恶劣环境中。它在与动植物宿主的其他相互作用中也很重要。这种特性是许多细菌表面的一种碳水化合物链。这项工作解决的问题是如何合成这条链。