Analysis of transfer genes of incompatibility group I plasmids

INCI-1质粒R64的54kb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毛，一种是细的菌毛，另一种是厚的菌毛。R64 Shufflon的多重DNA倒置选择了具有不同C-末端片段的七个PilV粘附素中的一个，PilV粘附素是细菌毛的组成部分。本研究的目的是阐明R64、ColIb-P9、R721和R621a载体中各种转移基因的功能。1.对多个R64 pIL和trA基因的产物进行了大量生产，并获得了针对pIL和trA产物的纯化抗体。Piln产物的功能是在外膜内形成环状结构的分泌素。PilQ产物形成一个多聚体，并显示出与细菌毛生物发生有关的ATPase活性。通过细胞内定位实验，确定了PilKLMOPRT和traMN产物的细胞内定位。2.非对称洗牌子重组位点非保守臂DNA序列对其倒置频率有很大影响。观察到含有对称重组位点的人工改组片段的缺失。通过印迹实验，证实了受体细胞囊膜内毒素与多种PilV粘附素的特异性结合。3.筛选到了多个突变体Pils和PilV，以揭示其产物的结构与功能之间的关系及其相互作用。4.R64 ORIT操纵子由ORIT序列和nikab基因组成。对Nikab蛋白进行纯化，并分析其与ORIT序列的相互作用。5.INCIγ质粒R621a的转移区与R64相似，但它们的托盘和exc基因的结构不同。R621a和R64属于不同的排除群。

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.