Genetics and biosynthesis of an O antigen essential for symbiosis

性状（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。拟议的工作涉及如何合成LPS的外部部分，O抗原。O-抗原合成有两种分化良好的机制。Etli根瘤菌CE 3的LPS是较好表征的实例之一（结构和生物学），其中LPS的O-抗原部分通过依赖于由wzm和wzt基因编码的ABC转运蛋白的机制合成。它适合于这种LPS的一个相对研究不足的亚组，其中O抗原是杂多糖，而不是同多糖。其O-抗原在与菜豆（Phaseolus vulgaris）（菜豆）共生中的重要性已经确立，整个LPS的结构几乎完全被解决，脂质A和分子的核心部分的合成中的大部分步骤已经在体外被证明，并且指定大部分O-抗原生物合成的30 kb基因簇已经被测序。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，这些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。大多数拟议的工作是生物化学的。一种已经在进行中的方法是从截短的LPS推断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而增加了这一假设。另一组方法涉及用来自预测具有糖基转移酶损伤的各种突变体的细胞提取物进行体外反应检查。特别是，这项工作旨在确定O抗原合成中第一反应的酶和产物。除了这项生化工作，还将进行额外的遗传分析，以完成对这种细菌中O抗原合成所需的基因以及它们可能具有的功能的几乎完整的说明。这些后面的实验将在基因中产生特定的非极性突变，这些基因在我们有强有力的证据致力于O-抗原合成的两个遗传位点中仍然没有突变。此外，两种类型的有针对性的遗传操作将解决两个有趣的问题，这可能是有关的极端均匀性的长度的O抗原。 公共卫生相关性：该项目利用一种不会引起医学问题的模型细菌，研究了有益健康和致病细菌的一种特性的基本基础，这种特性对它们的生存至关重要，特别是在哺乳动物循环液的恶劣环境中。它在与动物和植物宿主的其他相互作用中也很重要。这种性质是许多细菌表面上的一种碳水化合物链。在这项工作中解决的问题是如何合成这个链。

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.