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Analysis of transfer genes of incompatibility group I plasmids

不相容I组质粒的转移基因分析

基本信息

批准号：
11640622
负责人：
KOMANO Teruya
金额：
$ 2.24万
依托单位：
Tokyo Metropolitan University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11640622/
关键词：
IncI plasmids Conjugal transfer Type IV pilus Shufflon oriT operon

项目摘要

The 54-kb transfer region of IncI 1 plasmid R64 contains 49 genes. E.coli cells harboring R64 form two types of sex pili, a thin pilus and a thick pilus. Multiple DNA inversions of R64 shufflon select one of seven PilV adhesins, components of thin pili, with different C-terminal segments. The purpose of the present study is to elucidate the functions of various transfer genes in plasmids R64, ColIb-P9, R721 and R621a. 1. The products of several R64 pil and tra genes were overproduced and purified Antibodies against the pil and tra products were raised. The pilN product functions as a secretin forming a ring structure within the outer membrane. The pilQ product forms a multimer and exhibits ATPase activity responsible for thin pilus biogenesis. Intracellular localization of pilKLMOPRT and traMN products were determined. 2. DNA sequence of nonconserved arm of asymmetric shufflon recombination sites greatly affected their inversion frequencies. Deletion of an artificial shufflon segment containing symmetric recombination site was observed. Specific binding of lipopolysaccharide of recipient cell envelope to various PilV adhesins was demonstrated by blotting experiments. 3. Many pilS and pilV mutants were isolated to reveal relationship between structure and function of their products and also interaction of their products. 4. The R64 oriT operon consists of oriT sequence and nikAB genes. NikAB proteins were purified and their interaction with oriT sequence was analyzed. 5. Transfer region of IncIγ plasmid R621a was found to be similar to that of R64, although the structue of their traY and exc genes are different each other. R621a and R64 were found to belong to different exclusion groups.

IncI 1质粒R64的54-kb转移区含有49个基因。携带R64的大肠杆菌细胞形成两种类型的性皮利，薄菌毛和厚菌毛。R64 shufflon的多个DNA倒位选择具有不同C末端区段的七种PilV粘附素（薄皮利的组分）中的一种。本研究的目的是阐明质粒R64、ColIb-P9、R721和R621 a中各种转移基因的功能。1.几个R64 β和tra基因的产物过量产生，并产生了针对β和tra产物的纯化抗体。pilN产物作为外膜内形成环状结构的胰泌素起作用。pilQ产物形成多聚体并表现出负责薄菌毛生物发生的ATP酶活性。测定pilKLMOPRT和traMN产物的细胞内定位。2.不对称shufflon重组位点的非保守臂序列对其倒位频率有很大影响。观察到含有对称重组位点的人工shufflon片段的缺失。通过印迹实验证明受体细胞包膜的脂多糖与各种PilV粘附素的特异性结合。3.许多pilS和pilV突变体被分离出来，以揭示其产物的结构和功能之间的关系，以及它们的产物之间的相互作用。4. R64 oriT操纵子由oriT序列和nikAB基因组成。纯化NikAB蛋白并分析其与oriT序列的相互作用。5. IncIγ质粒R621 a的转移区与R64的转移区相似，但traY和exc基因的结构不同。R621 a和R64属于不同的排斥群。