Unexpected novel clade III type nitrous oxide-reducing bacteria from incubated lake sediments

Nitrous oxide-reducing bacteria (N₂ORB) play a pivotal role in regulating N₂O emissions in aquatic ecosystems, with clade I and clade II nosZ-harboring microorganisms representing well-recognized contributors to microbial N₂O consumption. Beyond conventional N₂ORB, the recently identified clade III nosZ from soil may represent a previously overlooked potential N₂O sink, yet the distribution and characterization remain largely unexplored in aquatic ecosystems. Here we established microcosm systems using sediments from five lakes and subjected them to warming temperature gradients to investigate the diversity and genomic characteristics of N₂ORB. Hidden Markov model (HMM)-based analyses identified a total of 45 nonredundant nosZ sequences, including 12 affiliated with clade III nosZ. Clade III nosZ accounted for 10.2%-40.6% of total nosZ genes, indicating that clade III nosZ-harboring N₂ORB is widespread and non-negligible. Reconstruction of metagenome-assembled genomes (MAGs) identified four phylogenetically novel clade III nosZ-harboring N₂ORB, with these MAGs showing low average amino acid identity to their closest known reference genomes. These MAGs showed different denitrification gene inventories, with MAG33 lacking identifiable genes for upstream N₂O-producing steps, suggesting a potential non-denitrifying N₂O reducer. They also encoded oxygen-related stress-response genes, suggesting a potential ability to perform N₂O respiration in the presence of oxygen. Unlike canonical clade I/II nosZ clusters, clade III nosZ-harboring MAGs lacked typical accessory genes and instead exhibited distinct neighboring transporter- and cytochrome-related genes. Together, our results provide evidence for the occurrence of clade III nosZ-harboring N₂ORB in non-soil ecosystems and expand current understanding of their genomic traits.