The free-living wellspring of symbiotic nitrogen fixation in <i>Bradyrhizobium</i>.

Abstract

The evolutionary origin of nitrogen-fixing symbiosis has been a long-standing question. To address this, we focused on <i>Bradyrhizobium</i>, 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 (<i>nif</i>) from nonlegume environments and analyzed them alongside 586 public <i>Bradyrhizobium</i> genomes harboring these genes to reconstruct a robust phylogeny of <i>nif</i> genes. Analysis suggests that the earliest-diverging <i>nif</i> 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 <i>nif</i> island architecture that consistently includes the oxygen-protective gene <i>glbO</i>, whereas the symbiotic <i>nif</i>-associated regions are highly variable and universally lack <i>glbO</i>. Using both loss-of-function and gain-of-function genetic approaches, we show that <i>glbO</i> 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 <i>Bradyrhizobium</i>.