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.