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（由申请人提供）：革兰氏阴性细菌的脂多糖（LPS）具有复杂的结构和多个功能。这些细菌表面分子在细菌与其环境之间的相互作用中非常重要，包括与动物和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）如何合成。 O-抗原合成有两种差异化的机制。根瘤菌ETLI CE3的LPS是一个更好的特征示例之一（结构和生物学上），其中LPS的O-抗原部分由依赖于WZM和WZT基因编码的ABC转运蛋白的机制合成。它适合此类LPS的相对研究的亚组，其中O抗原是杂多糖，而不是均质糖。已经确定了其O-抗原在与阶段果素（Bean）的共生中的重要性，整个LP的结构几乎完全解决，在脂质A和分子的合成中的大多数步骤已在体外证明，并且在体外证明了30 kB的基因群，该基因量表大多数e-o-antigennebenthe pepeciped execiped execiped execiped execiped execiped execiped。此外，已经确定了整个基因组的核苷酸序列。在拟议的工作中，将确定该O抗原的生物合成的基本特征。它将测试一组假设，以预测转移酶添加糖残基以及哪些基因编码哪个转移酶的顺序。大多数拟议的工作都是生化的。一种已经出色的方法是从截短的LPS中推断出不同的突变体被阻断的步骤。它通过将基因与特定预测的糖基转移酶活性匹配来增加了假设。另一种方法涉及在体外检查反应与预测在糖基转移酶中有病变的各种突变体的细胞提取物。特别是，该作品旨在定义O抗原合成中第一反应的酶和产物。除了这项生化工作外，还将进行其他遗传分析，以完成该细菌中O-抗原合成中需要哪些基因以及它们可能具有哪些功能的基因。这些后一个实验将在基因中产生特定的非极性突变，这些突变仍未在两个遗传基因座中突变，即我们有强有力的证据专门用于O-抗原的合成。此外，有两种类型的靶向遗传操作将解决两个有趣的问题，这些问题可能与该O抗原长度的极端均匀性有关。公共卫生相关性：使用不会引起医学问题的模型细菌，该项目研究了健康剥皮和致病细菌特性的基本基础，这对于它们的生存至关重要，尤其是在哺乳动物循环液的敌对范围内。它在与动物和植物宿主的其他相互作用中也很重要。该特性是许多细菌表面上的碳水化合物链。这项工作中解决的问题是如何合成该链。