Evolution and function of nitrogen fixation gene clusters in sugarcane associated Bradyrhizobium strains

Bradyrhizobium spp. are well known to mediate biological nitrogen fixation (BNF) as microsymbionts inhabiting nodules on leguminous plants. However, they may also contribute to plant growth via free-living N₂ fixation (FLNF) in association with non-legumes. Notably, several Bradyrhizobium strains from sugarcane roots display FLNF activity. Among them, Bradyrhizobium sacchari is a legume symbiotic species, whereas strains AG48 and M12 are non-symbiotic. In the present study, a phylogenomic approach was applied to study peculiarities of these and other Bradyrhizobium strains with respect to N fixation (nif) gene content in order to reveal genetic features that enable FNLF in Bradyrhizobium spp. All FLNF strains carry an ancestral 'non-symbiotic' nif-gene cluster (NSC). B. sacchari also contains a second 'symbiotic' nif-gene cluster (SC), a characteristic observed in only three of 156 evaluated genomes. B. sacchari stood out and presented a high level of sequence divergence between individual nif-gene homologues and we discuss scenarios for the evolutionary origin of these clusters. The transcript level of NSC nifH gene increased during FLNF, when compared to symbiotic conditions. The data suggest that sugarcane roots harbor diverse Bradyrhizobium spp. that are genetically adapted to a dynamic environment where leguminous and non-leguminous host plants are alternately available.