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 non-legume environments and analyzed them alongside 586 public Bradyrhizobium genomes harboring these genes to reconstruct a robust phylogeny of nif genes. Analysis reveals that the earliest-diverging nif lineages are members capable of free-living nitrogen fixation, establishing this as the ancestral state. The Nod factor-dependent symbiotic lineages are polyphyletic, demonstrating 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 establishes a new framework for the evolution of symbiosis, identifying free-living ancestors as the source from which nitrogen-fixing symbiosis repeatedly and independently evolved in Bradyrhizobium. SignificanceFor over a century, nitrogen-fixing bacteria called rhizobia have been celebrated for their symbiotic partnership with legumes like soybeans and peas. This study overturns the view that this symbiotic lifestyle was the original, advanced state. By discovering and analyzing diverse Bradyrhizobium bacteria from ordinary soils and non-legume plants, we show that the ancestor was capable of free-living nitrogen fixation. Root nodule nitrogen fixation evolved multiple independent times from free-living ancestors. We identify glbO, an oxygen-protective gene located immediately next to the nitrogen fixing genes, as critical for nitrogen fixation in variable soil conditions but dispensable and lost in Bradyrhizobium specialized inside nodules. This repositions free-living Bradyrhizobium as a major potential nitrogen source beyond legumes, with promise for sustainable agriculture.