The free-living wellspring of symbiotic nitrogen fixation in Bradyrhizobium

The evolutionary origin of nitrogen-fixing symbiosis has been a long-standing question. To address this, we focused on Bradyrhizobium, a globally abundant bacterial genus that includes classic symbiotic lineages, which rely on the common Nod factor signaling pathway to form nodules, and close relatives capable of fixing nitrogen in a free-living state. We isolated 88 strains carrying the key genes for nitrogen fixation (nif) from nonlegume environments and analyzed them alongside 586 public Bradyrhizobium genomes harboring these genes to reconstruct a robust phylogeny of nif genes. Analysis suggests that the earliest-diverging nif lineages are members capable of free-living nitrogen fixation, supporting the interpretation that this lifestyle is ancestral. The Nod factor-dependent symbiotic lineages are polyphyletic, with our data supporting at least three independent origins via horizontal acquisition of symbiosis islands. This evolutionary history is reflected in a genomic dichotomy: lineages capable of free-living nitrogen fixation possess a conserved nif island architecture that consistently includes the oxygen-protective gene glbO, whereas the symbiotic nif-associated regions are highly variable and universally lack glbO. Using both loss-of-function and gain-of-function genetic approaches, we show that glbO contributes significantly to nitrogenase activity under free-living conditions, whereas it is dispensable within the protected nodule environment. This work provides a framework for the evolution of nitrogen-fixing symbiosis, supporting the view that free-living nitrogen-fixing ancestors gave rise repeatedly and independently to symbiotic lineages in Bradyrhizobium.