Phylogenetic diversity of NO reductases, new tools for nor monitoring, and insights into N₂O production in natural and engineered environments

Highlights

● 548 representative nor genes were collected to create complete phylogenetic trees.

● The distribution of nor and nod genes were detected in 18 different phyla.

● The most conserved amino acids in NOR were located adjacent to the active site.

● nor-universal and Clade-specific primers were designed, suggested, and tested.

Abstract

Nitric oxide reductases (NORs) have a central role in denitrification, detoxification of nitric oxide (NO) in host-pathogen interactions, and NO-mediated cell-cell signaling. In this study, we focus on the phylogeny and detection of qNOR and cNOR genes because of their nucleotide sequence similarity and evolutionary relatedness to cytochrome oxidases, their key role in denitrification, and their abundance in natural, agricultural, and wastewater ecosystems. We also include nitric oxide dismutase (NOD) due to its similarity to qNOR. Using 548 nor sequences from publicly accessible databases and sequenced isolates from N₂O-producing bioreactors, we constructed phylogenetic trees for 289 qnor/nod genes and 259 cnorB genes. These trees contain evidence of horizontal gene transfer and gene duplication, with 13.4% of the sequenced strains containing two or more nor genes. By aligning amino acid sequences for qnor + cnor, qnor, and cnor, we identified four highly conserved regions for NOR and NOD, including two highly conserved histidine residues at the active site for qNOR and cNOR. Extending this approach, we identified conserved sequences for: 1) all nor (nor-universal); 2) all qnor (qnor-universal) and all cnor (cnor-universal); 3) qnor of Comamonadaceae; 4) Clade-specific sequences; and 5) nod of Candidatus Methylomirabilis oxyfera. Examples of primer performance were confirmed experimentally.

Graphical abstract

Keywords

N₂O / Greenhouse gas / NO reductase / NO dismutase / Primer / Crystal structure

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Acknowledgements

This research was supported in part by a grant from the US National Science Foundation Engineering Research Center Reinventing the Nation’s Urban Water Infrastructure (ReNUWIt) (Award No. EEC-1028968) and in part by a grant from the NASA Center (USA) for the Utilization of Biological Engineering in Space (CUBES) (Award No. NNX17AJ31G). We thank Dr. Jizhong Zhou and the Institute of Environmental Genomics at the University of Oklahoma, Norman (OK, USA), for sequencing of Alicycliphilus sp. CD02.

Competing Interests

The authors declare no conflict of interests.

Electronic Supplementary Material

Supplementary material is available in the online version of this article at https://doi.org/10.1007/s11783-022-1562-3 and is accessible for authorized users.

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