Isolation and global occurrence of nitrogen-fixing <i>Acidobacteriota</i> in soil environments

Abstract Acidobacteriota, one of the most abundant and ubiquitous bacterial phyla in soils, are well recognized for their role in carbon cycling. In contrast, their roles in soil nitrogen cycling remain largely unexplored, although recent metagenome-assembled genome (MAG) analyses suggest that Acidobacteriota may harbor genes involved in nitrogen cycling. Here, we provide culture-based evidence of diazotrophy within this phylum and demonstrate the widespread occurrence of nitrogen-fixing Acidobacteriota across diverse soil types. From grassland and agricultural soils, we isolated five Acidobacteriota strains representing novel taxonomic lineages, four of which harbor functional nitrogenase (nif) gene clusters. These strains were capable of fixing atmospheric nitrogen in vitro and/or in soil microcosms, as evidenced by acetylene reduction, N2-dependent growth, transcription of nif genes, incorporation of 15N into biomass and soil, and inhibition of nitrogenase activity by ammonium. Furthermore, global-scale meta-analysis of soil metagenomes revealed that nif-harboring Acidobacteriota are widely distributed and locally dominant across soil types. These results demonstrate the nitrogen-fixing capability of Acidobacteriota at the organismal level, complementing MAG-based inferences, and underscore their adaptive capacity in nitrogen-limited environments and their potential contribution to terrestrial nitrogen fixation. We also propose novel taxa within the class Terriglobia of the phylum Acidobacteriota, including diazotrophic strains, comprising one novel family, three novel genera, and four novel species: Koromonadaceae fam. nov., Koromonas soli gen. nov., sp. nov., Koromonas humicola sp. nov., Oryzophilus luti gen. nov., sp. nov., and Humiphilus diazotrophicus gen. nov., sp. nov.