Gamma Ray-induced Mutations in pyrEF Genes in Frankia casuarinae Strain CcI3

Frankia spp. are multicellular actinobacteria with the ability to fix atmospheric dinitrogen (N₂). Frankia fixes N₂ not only in the free-living state, but also in root-nodule symbioses with more than 200 plant species called actinorhizal plants. In the present study, we isolated mutants of the pyrE (orotate phosphoribosyltransferase) and pyrF (orotidine-5'-phosphate decarboxylase) genes in Frankia casuarinae strain CcI3 using gamma rays as a mutagen and systematically identified the types of mutations that occurred in these genes. pyrEF mutants were isolated as uracil auxotrophs using the antimetabolite 5-fluoroorotic acid. We elucidated the nucleotide sequences of the pyrEF genes in 32 uracil auxotrophs, and detected eight substitutions, 17 single-nucleotide deletions, and seven large insertions. Large insertions were insertion sequences (IS elements); four belonged to the IS4 family, two to the IS66 family, and one to the IS110 family. This is the first study to demonstrate the mobilization of IS elements in the Frankia genome.