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Genetic strategy of gram negative bacteria to acquire the O-antigenic polymorphism

革兰氏阴性菌获得O抗原多态性的遗传策略

基本信息

批准号：
10670253
负责人：
KIDO Nobuo
金额：
$ 2.11万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10670253/
关键词：
lipopolysaccharide Escherichia coli O-antigen mannosyltransferase

项目摘要

O polysaccharides of Gram-negative bacteria are structurally polymorphic, and consequently, they are utilized as the O-antigen for serological typing. The O polysaccharides and covalently binds to lipid A through the core-oligosaccharide portion. Each O polysaccharide consists of many repeating units of an oligosaccaride composed of several sugars with various linkages. The wbィイD1*ィエD1 gene cluster is responsible for the O polysaccharide synthesis. The wbィイD1*ィエD1 cluster of E. coli O9a strain F719 is constituted of eight genes, two for GDP-mannose synthesis, two for the putative ABC-transporter, three for mannosyltransferases, and one whose function is unknown. wbdA is a mannosyltransferase gene that is involved is synthesis of the Escherichia coli O9a polysaccharide, a mannose homopolymer with a repeating unit of 2-αman-1,2-αman-1,3-αman-1,3-αman-1. The equivalent structural O polysaccharide in the polysaccharide in the E. coli O9 and Klebsiella O3 strains is 2-αman-1,2-αman-1,2-αman-1,3-αman-1,3-αman-1, with an excess of one mannose in the 1,2-linkage. We have cloned wbdA genes from the latter strains and shown by genetic and functional studies that wbdA is one only gene determining the O polysaccharide structure of O9 or O9a. Based on functional analysis of chimeric genes and site-directed mutagenesis, we could show that a single amino acid substitution, C55R, in the WbdA of E. coli O9 converts the O9 polysaccharide into O9a. DNA sequencing revealed the substitution to conserved in other E. coli O9a strains. The reverse substitution, R55C, in the WbdA of E. coli O9a results in LPS synthesis showing no ladder profile instead of the conversion of O9a to O9. This suggests that more than one amino acid substitution in the WbdA is required for conversion from O9a to O9.

革兰氏阴性菌的O多糖具有结构多态性，因此可作为O抗原进行血清学分型。O型多糖通过核心低聚糖部分与脂质A共价结合。每一种O型多糖由低聚糖的许多重复单元组成，低聚糖由几种具有不同键的糖组成。wb D1* D1基因簇负责O多糖的合成。世行ィイD1 *ィエD1的大肠杆菌O9a应变F719八个基因的构成,两个GDP-mannose合成,假定的ABC-transporter两,三个mannosyltransferases,和一个未知的函数。wbdA是一种甘露糖基转移酶基因，参与大肠杆菌O9a多糖的合成，这是一种甘露糖均聚物，重复单元为2-α -1,2-α -1,3-α -1,3-α -1。大肠杆菌O9和克雷伯氏菌O3菌株中多糖的等效结构为2-αman-1,2-αman-1,2-αman-1,3-αman-1,3-αman- 1,3-αman-1，在1,2-连锁中多余1个甘露糖。我们克隆了后一种菌株的wbdA基因，通过遗传学和功能研究表明，wbdA是决定O9或O9a O多糖结构的唯一基因。基于嵌合基因的功能分析和定点突变，我们可以发现大肠杆菌O9的WbdA中的单个氨基酸替换C55R将O9多糖转化为O9a。DNA测序结果显示，该基因在其他大肠杆菌O9a菌株中是保守的。在大肠杆菌O9a的WbdA中反向取代R55C，导致LPS合成无阶梯结构，而不是由O9a转化为O9。这表明，从O9a到O9的转化需要在WbdA中进行不止一个氨基酸的取代。

项目成果

期刊论文数量（11）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Sugiyama, T., N. Kido, Y. Kato, N. Koide, T. Yoshida, and T. Yokochi.: "Generation of Escherichia coli O9a serotype, a subtype of E. coli O9, by transfer of the wb ィイD1*ィエD1 gene cluster of Klebsiella O3 into E. coli via recombination"J Bacteriol.. 180. 2

Sugiyama, T.、N. Kido、Y. Kato、N. Koide、T. Yoshida 和 T. Yokochi.：“通过转移 wb D1* 产生大肠杆菌 O9a 血清型，这是大肠杆菌 O9 的一个亚型*克雷伯菌 O3 的 ieD1 基因簇通过重组进入大肠杆菌"J Bacteriol.. 180. 2