Analysis of transfer genes of incompatibility group I plasmids

点击翻译按钮获取中文摘要

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.