Studies on ribonucleotide reductase mutants of Rhizobium meliloti

Optimal reaction concentrations were determined for ribonucleotide reductase of Rhizobium meliloti 3D0al, an ineffective strain in nitrogen fixation. The optimal substrate concentration for GMP, GDP, and GTP was 0.4, 0.1, and 0.4 mM, respectively. Dithio- threitol was the most effective reductant and had an optimal concentration of 20 mM. The optimal B12 coenzyme concentration was 35 uM. Ribonucleotide reductase activity of R. meliloti 3DOal was approximately 2-fold less than in R. meliloti F-28, a strain effective in nitrogen fixation, when both were assayed at their optimal reactant concentrations. Sixteen mutants of R. meliloti F-28 were isolated after treatment with N-methyl-N1-nitro-N-nitrosoguanidine. The growth rates and ribonucleotide reductase activity of most of these organisms were significantly different than that of the prototrophic strain. Two mutants, R. meliloti HP-31 and R. meliloti LZ-26, contained only 10% of the ribonucleotide reductase activity of R. meliloti F-28. These two mutants caused nodule formation on alfalfa plants, and the nodules were capable of acetylene reduction.