Genetics and biosynthesis of an O antigen essential for symbiosis

说明书(申请人提供)：革兰氏阴性杆菌的脂多糖具有复杂的结构和多种功能。这些细菌表面分子在细菌与环境之间的相互作用中非常重要，包括与动植物宿主的有益和破坏性相互作用。这项拟议的工作涉及到内毒素的外部部分，即O抗原是如何合成的。O-抗原的合成有两种分化良好的机制。ETLI根瘤菌CE3的内毒素是结构和生物学特征较好的例子之一，内毒素的O抗原部分是通过依赖于由WZM和WZT基因编码的ABC转运蛋白的机制合成的。它属于这类LPS中研究相对较少的一个亚群，在这个亚群中，O抗原是杂多糖，而不是同源多糖。它的O-抗原在与菜豆共生中的重要性已经确定，整个脂多糖的结构几乎完全解决，脂类A和分子核心部分的大部分合成步骤已经在体外得到证明，并且已经对一组30kb的基因进行了测序，这些基因是O-抗原生物合成的主要特定域。此外，还确定了整个基因组的核苷酸序列。在拟议的工作中，将确定这种O抗原生物合成的基本特征。它将测试一组假说，这些假说预测转移酶添加糖残留物的顺序，以及哪些基因编码哪些转移酶。大多数拟议的工作都是生化的。一种已经进行得很好的方法是从截短的LPS中推断出阻止不同突变体的步骤。它通过将基因与特定的预测糖基转移酶活性进行匹配来补充了这一假设。另一组方法包括检查体外与不同突变株的细胞提取物的反应，这些突变株被预测在糖基转移酶中有损伤。特别是，这项工作试图确定合成O抗原的第一反应的酶和产物。除了这项生化工作外，还将进行额外的遗传分析，以完成几乎完整的计算这种细菌的O抗原合成所需的基因以及它们可能具有的功能。这些后一种实验将在两个基因位点产生特定的、非极性的突变，这些基因仍然没有突变，我们有强有力的证据表明这些基因致力于O-抗原的合成。此外，两种类型的靶向基因操作将解决两个有趣的问题，这两个问题可能与这种O抗原长度的极端一致性有关。 公共卫生相关性：该项目利用一种不会造成医疗问题的模式细菌，研究有益健康和致病细菌特性的基本基础，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敌意范围内。它在与动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的一种碳水化合物链。这项工作解决的问题是这个链是如何合成的。

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.