Isolation and Characterization of High-Temperature-Tolerant Mutants of Bradyrhizobium diazoefficiens USDA110 by Carbon-Ion Beam Irradiation

Biofertilizers are promising technologies for achieving sustainable agriculture. However, high-temperature tolerance is a constraint that limits the function of microbial inoculants. To characterize the genetic changes responsible for the high-temperature tolerance of rhizobia, mutant screening was performed using <i>Bradyrhizobium diazoefficiens</i> USDA110. The wild-type cells were mutagenized with carbon-ion irradiation, and two mutant strains, designated M10 and M14, were obtained after a three-day heat-shock treatment at 43 °C. In particular, M14 showed superior growth at 36 °C, at which temperature growth of the wild type was extremely slow, whereas M14 grew more slowly than the wild type at 32 °C. Whole-genome sequencing revealed that M10 had seven point mutations, whereas M14 had eight point mutations together with a 1.27 Mb inversion. RNA sequencing showed that the number of differentially expressed genes greatly exceeded the actual number of induced mutations. In M14, a gene cluster associated with pyruvate metabolism was markedly downregulated, probably because of disjunction with the promoter region after inversion, and was considered to be the cause of the slow growth rate of M14 at 32 °C. Notably, transmembrane proteins, including porins, were enriched among the genes upregulated in both M10 and M14. M14 was confirmed to retain symbiotic functions with soybeans. These results indicate that high-temperature tolerance was conferred by random mutagenesis while the symbiotic functions of rhizobia was maintained.