Rapid and efficient selection of recombinant site-directed mutants of Bradyrhizobium japonicum by colony hybridization.
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.
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Department of Biology, McCollum-Pratt Institute, Johns Hopkins University, Baltimore, Maryland 21218.
Maier
R J
RJ
D004269:DNA, Bacterial / Q000235:genetics; D004352:Drug Resistance, Microbial / Q000235:genetics; D007612:Kanamycin / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D009693:Nucleic Acid Hybridization; D010957:Plasmids / Q000235:genetics; D011995:Recombination, Genetic; D012230:Rhizobiaceae / Q000187:drug effects / Q000235:genetics*; D000198:Spectinomycin / Q000494:pharmacology
D004269:DNA, Bacterial; D007612:Kanamycin; D000198:Spectinomycin