Nitrogen Fixation Mutants of the Actinobacterium Frankia Casuarinae CcI3

Frankia is a representative genus of nitrogen-fixing (N-2-fixing) actinobacteria; however, the molecular mechanisms underlying various phenomena such as the differentiation of a N-2 fixation-specific structure (vesicle) and the regulation of N-2 fixation (nif) genes, have yet to be elucidated in detail. In the present study, we screened hyphal fragments of Frankia casuarinae that were mutagenized by 1-methyl-3-nitro-1-nitrosoguanidine or gamma rays, and isolated 49 candidate N-2 fixation mutants. Twelve of these mutants were selected for further study, and their abilities to grow in NH3-deficient (N-) liquid media and their rates of acetylene reduction activities were evaluated. Eleven mutant strains were confirmed to lack the ability to fix N-2. Five mutant strains formed significantly reduced numbers of vesicles, while some failed to form large mature vesicles. These vesicle mutants also exhibited an aberrant hyphal morphology, suggesting a relationship between vesicle differentiation and hyphal branching. Ten mutants showed significant reductions in the expression of nifE, nifH, and nifV genes under N-conditions. The genome sequencing of eight mutants identified 20 to 400 mutations. Although mutant strains N3H4 and N6F4 shared a large number of mutations (108), most were unique to each strain. Mutant strain N7C9 had 3 mutations in the nifD and nifH genes that may result in the inability to fix N-2. The other mutant strains did not have any mutations in any known N-2 fixation-related genes, indicating that they are novel N-2 fixation mutants.