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Microorganisms play an essential role in nitrogen cycling and greenhouse gas emissions in soils and sediments. The recently discovered oxygenic denitrifiers are proposed to reduce nitrate and nitrite via nitric oxide dismutation directly to N 2 and O 2 . So far, the ecological role of these microbes is not well understood. The only available tool for a targeted study of oxygenic denitrifiers is their respective maker gene, nitric oxide dismutase (nod). Here, we established the use of PacBio long-read sequencing of nod gene amplicons to study the diversity and community structure of oxygenic denitrifiers. Two distinct sets of environmental samples, agricultural soil and lake sediment, were investigated as examples. The circular consensus sequences (ca 1.0 kb) obtained covered most substitution characteristic of NO dismutase and allowed for reliable classification of oxygenic denitrifiers. Distinct nod gene pools and community structure were revealed for the different habitats, with most sequence types affiliated to yet unidentified environmental nod lineages. The abundance of nod genes ranged 2.2 × 10 6 -3.2 × 10 7 gene copies g -1 soil or sediment, accounting for up to 3% of total bacterial 16S rRNA gene counts. This study indicates that nod-gene-targeted long-read sequencing can be a powerful tool for studying the ecology of these novel microbes, and the results also suggest that oxygenic denitrifiers are prevalent and abundant in different terrestrial samples, where they could play an important, but yet overlooked role in nitrogen transformations.

Long-Read Amplicon Sequencing of Nitric Oxide Dismutase (nod) Genes Reveal Diverse Oxygenic Denitrifiers in Agricultural Soils and Lake Sediments