Horizontal gene transfer of a unique nif island drives convergent evolution of free-living N2-fixing Bradyrhizobium

The alphaproteobacterial genus Bradyrhizobium has been best known as N2-fixing members that nodulate legumes, supported by the nif and nod gene clusters. Recent environmental surveys show that Bradyrhizobium represents one of the most abundant free-living bacterial lineages in the worlds soils. However, our understanding of Bradyrhizobium comes largely from symbiotic members, biasing the current knowledge of their ecology and evolution. Here, we report the genomes of 88 Bradyrhizobium strains derived from diverse soil samples, including both nif-carrying and non-nif-carrying free-living (nod free) members. Phylogenomic analyses of these and 252 publicly available Bradyrhizobium genomes indicate that nif-carrying free-living members independently evolved from symbiotic ancestors (carrying both nif and nod) multiple times. Intriguingly, the nif phylogeny shows that all nif-carrying free-living members comprise a cluster which branches off earlier than most symbiotic lineages. These results indicate that horizontal gene transfer (HGT) promotes nif expansion among the free-living Bradyrhizobium and that the free-living nif cluster represents a more ancestral version compared to that in symbiotic lineages. Further evidence for this rampant HGT is that the nif in free-living members consistently co-locate with several important genes involved in coping with oxygen tension which are missing from symbiotic members, and that while in free-living Bradyrhizobium nif and the co-locating genes show a highly conserved gene order, they each have distinct genomic context. Given the dominance of Bradyrhizobium in worlds soils, our findings have implications for global nitrogen cycles and agricultural research.