Tn5 mutants of Bradyrhizobium japonicum Is-1 with altered compatibility with Rj₂-soybean cultivars

Bradyrhizobium japonicum Is-1 is incompatible with soybean (Glycine max L. Merr.) cv. CNS (Rj₂Rj₃) and compatible with cv. Hill (Rj₄) and is, therefore, classified into nodulation-type B. Tn5 mutants (1C1, 1C2, 5C1, 6C1, 7C1, 7C2, 10C1 and 10C2) were isolated from eight nodules produced on CNS that had been inoculated with the kanamycin-resistant (Km^r) transconjugants of strain Is-1. To investigate the shift of host range, Tn5 mutants were inoculated to soybean cvs. CNS, D-51 (Rj ₃), Hardee (Rj₂Rj₃), Hill and IAC-2 (Rj ₂Rj₃). They acquired the ability to nodulate Rj ₂Rj₃ soybeans without losing the ability to nodulate both Rj₃ and Rj₄. These results suggest that the Tn5 mutation in B. japonicum strain Is-1 is only related to the ability to nodulate Rj ₂ soybean. Although the host ranges were common to Tn5 mutants and all sections of nodules produced by Tn5 mutants indicate a red color, the nodule numbers and nitrogen fixation activities were different for each mutant. These results suggest that Tn5 insertion sites are different from each other. Tn5-flanking sequences in these mutants were specifically amplified. The electrophoresis analyses of the polymerase chain reaction (PCR) products showed that Tn5 mutants contained a single copy of Tn5, except for 10C1. These PCR products were cloned and sequenced. The sequence analysis of Tn5-flanking sequences revealed that these Tn5 insertion sites were different from each other and all homologous sequences to them were found in the complete genome sequence of B. japonicum USDA 110. These results are in good accordance with our expectation that Tn5 insertion sites are different for each mutant. Most Tn5-inserted gene products relate to the cell membrane structure. This suggests that the change in the cell surface structure in Tn5 mutants may overcome nodulation restriction conditioned by Rj₂ soybean.