Fu C, Maier R J
Department of Biology, McCollum-Pratt Institute, Johns Hopkins University, Baltimore, Maryland 21218.
FEMS Microbiol Lett. 1993 May 1;109(1):33-8. doi: 10.1111/j.1574-6968.1993.tb06139.x.
Due to the high incidence of spontaneous antibiotic resistance and slow growth of Bradyrhizobium japonicum strains, screening for site-directed mutants is cumbersome and time-consuming. A rapid method for selection of recombinant site-directed mutants of B. japonicum was developed. A kanamycin (Km) and a spectinomycin (Sp) cassette were each used to replace DNA fragments in the chromosome by homologous recombination. The primary new features of this method involve a simple plate selection for the antibiotic (Km or Sp) resistant mutants, then colony streaking, and lysis for DNA hybridization on a nitrocellulose filter enabling direct identification of the recombinant site-directed mutants. This method has permitted us to quickly and easily identify a large number of positive recombinant mutants from a large number of individual colonies. The procedure eliminates the need to first isolate genomic DNA from each mutant for Southern hybridization. All of the tested site-directed mutants from this method were confirmed to exhibit the expected mutant phenotype.
