大腸桿菌素 colicin Ib (Col Ib)的定性

Abstract

Bacteriocins are proteins that are produced by bacteria and toxic to bacteria with the same or related species. Bacteriocins produced by E. coli specifically are called colicins. E. coli strains producing colicins also produce the immunity proteins specific to the colicins. It is generally believed that inside the bacteria the immunity proteins interact with the colicins so that colicins would not kill the bacteria that produce them. Some E. coli stains carry plasmid ColIb-P9 which contains genes for colicin Ib (cib) and its immunity protein (cbi). In the environment, free colicin Ib would bind the Cir receptor on the outer membrane of E. coli and reach the inner membrane through the Ton translocation system, where channels or pores are formed and bacteria would die due to ion leakage. Our laboratory previously isolated a fosmid clone from a Shigella flexneri genomic library that contains cib and cbi. Comparing to the sequences of cib and cbi in ColIb-P9, the clone only contained one transversion mutation in cib (A to T at bp 1860; glutamic acid to aspartic acid at amino acid residue 620) in the region including cib and cbi. The purpose of this study is to characterize this specific colicin Ib in E. coli. First, 18 out of 59 E. coli strains isolated from cattle, chicken and swine were found to have something to do with colicin Ib, either they were killed by colicin Ib, carried cib gene and/or cbi gene, or carried the colicin Ib killing activity. Particularly, five strains that carry cib and the colicin Ib killing activity also contain cbi. A high-copied plasmid only carrying cib was transformed successfully into cir+ and cir- laboratory E. coli strains. The transformants expressed the killing activity but could not be killed by exogenous colicin Ib. Comparing to the transformants that contain cib and cbi, separately in the high-copied plasmid and a low-copied plasmid, transformants that only contain cib in the high-copied plasmid showed lower killing activity of the cell-free medium and higher killing activity of the bacterial cell extract, indicating that the immunity protein is likely to help the bacteria to release colicin Ib into medium. By cloning egfp into the 5’ or 3’ end of cib in the high-copied plasmid and examining the colony morphology of the transformants under fluorescence microscopy, it was found only the transformants that carry the construct with egfp in the 5’ end of cib showed green fluorescence. Similarly, the β-lactamase gene (bla) was cloned into the 5’ or 3’ end of cib in the high-copied plasmid, and only the transformants that carry the construct with bla in the 3’ end of cib showed growth on the ampicillin-containing plates. Proteins of the transformants that only carry cib in the high-copied plasmid were fractionated and analyzed by western hybridization, the rersults showed that colicin Ib was located in the cytoplasmic, inner membrane, and periplasmic fractions. These results indicate that colicin Ib, when existed solely in E. coli, was located in the inner membrane with N terminus in the cytoplasm and C terminus in the periplasm.