Diversity of dirammox microorganisms

ABSTRACT Our knowledge of the nitrogen cycle has been advanced by the discovery of anammox bacteria, ammonia-oxidizing archaea, and comammox bacteria, along with their high diversity. Direct ammonia oxidation (dirammox), a recently discovered heterotrophic nitrification pathway, converts ammonium and organic nitrogen directly to N 2 through the DnfABC multienzyme system, further expanding our understanding of the nitrogen cycle. However, the taxonomic distribution, prevalence, and nitrogen metabolic potential of microorganisms encoding dirammox genes remain poorly understood. Here, large-scale genome mining identified complete dnfABC gene clusters in 690 genomes representing 3 phyla and 26 genera. The dnfABC cluster showed a striking bimodal prevalence: it was nearly ubiquitous in Alcaligenes , Delftia , and Microvirgula but occurred sporadically in most other genera. These microorganisms were recovered from diverse environments, particularly organic nitrogen-rich habitats. Comparative analyses of DnfABC phylogeny, gene cluster organization, GC content, and flanking mobile genetic elements indicate that the dnfABC cluster is conserved within several core genera, whereas its sporadic distribution in other lineages suggests that horizontal gene transfer may have contributed to its dissemination. The dnfABC cluster co-occurs with genes associated with assimilatory nitrate/nitrite reduction to ammonium, dissimilatory nitrate/nitrite reduction to ammonium, denitrification, and/or nitrogen fixation, but not with marker genes for nitrification or anammox, indicating diverse nitrogen metabolic potential among potential dirammox microorganisms. Functional assays further showed that diverse dnfABC clusters consistently conferred ammonium- and glutamine-oxidizing activities in Escherichia coli , despite exhibiting low activities in their native hosts. These results suggest that dirammox is a broadly distributed, metabolically flexible, and tightly regulated pathway and may represent an overlooked route of nitrogen transformation in organic-rich habitats. IMPORTANCE Nitrogen is essential for all life, and our understanding of the nitrogen cycle has advanced substantially with the discovery of anammox bacteria, ammonia-oxidizing archaea, and comammox bacteria, which exhibit remarkable taxonomic and metabolic diversity. This study suggests that the recently discovered dirammox pathway may be more widespread in nature than previously recognized. Potential dirammox microorganisms exhibit broad phylogenetic and ecological distributions, high genomic diversity, and notable metabolic versatility. Functional assays indicate that dirammox activity is tightly regulated in native hosts, suggesting that its ecological role depends on specific environmental conditions. Unlike canonical nitrification and anammox, dirammox operates under conditions where other nitrogen transformations are limited, highlighting an important route of nitrogen loss in organic-rich habitats. These findings highlight the previously overlooked potential role of dirammox in nitrogen cycling, expand our understanding of microbial contributions to the global nitrogen cycle, and provide a foundation for future ecological and environmental research.