Cloning and map analysis of rifandine resistant E. coli mutant gene digested with restriction endonuclease

In this paper, four rifandine (RFD) resistant strains (MIC 500 μg/ml) were selected from their corresponding sensitive strains by using gradient agar plate method. The RFD resistance of these strains was stable and still well retained after two subcultures in the absence of RFD. The total DNA of the four resistant strains were extracted by three different methods. The agarose gel electrophoresis showed no finding of existance of plasmid DNA. In addition, with two extraction methocls especially for plasmid DNA, there was no plasmid DNA either. Results strongly suggested that the RFD resistance may not be related to plasmid, but mediated by chromosomal mutation. E. coli 2280 RFD resistant strain was chosen to be further studied. Its chromosomal DNA was isolated by using proteinase K - winding up glass rod method and entirely digested with Bgl II. The digested DNA fragments were ligated with BamH I -digested pBR322 (AMP(r), TC(r) was lost, 4.3 kb) prepared by alkaline lysis with T ₄ -DNA ligase. After recombinations these were transformed into E. coli HB101, a pBD802 plasmid carrying the RFD resistant gene has been isolated from the colonies on the selective agar plates containing RFD 50 μg/ml and AMP 60 μg/ml. RFD MIC of pBD802-transformed strain was 512 μg/ml. Agarose gel electrophoresis determined that the size of the RFD resistant DNA fragment was approximately 16 kb. The sites of different restriction endonucleases including EcoR I, BamH I, Hpa I, EcoR V, Sal I, Hind III, Bgl II and Pst I have been defined. These certainly contribute for our further subcloning and determining the detail biological characteristics and DNA sequence of the RFD resistant mutant gene.