Nitrous oxide (N2O) is a potent greenhouse gas and the predominant ozone depleting substance. The only enzyme known to reduce N2O is the nitrous oxide reductase, encoded by the nosZ gene, which is present among bacteria and archaea capable of either complete denitrification or only N2O reduction to di-nitrogen gas. To determine whether the occurrence of nosZ, being a proxy for the trait N2O reduction, differed among taxonomic groups, preferred habitats or organisms having either NirK or NirS nitrite reductases encoded by the nirK and nirS genes, respectively, 652 microbial genomes across 18 phyla were compared. Furthermore, the association of different co-occurrence patterns with enzymes reducing nitric oxide to N2O encoded by nor genes was examined. We observed that co-occurrence patterns of denitrification genes were not randomly distributed across taxa, as specific patterns were found to be more dominant or absent than expected within different taxonomic groups. The nosZ gene had a significantly higher frequency of co-occurrence with nirS than with nirK and the presence or absence of a nor gene largely explained this pattern, as nirS almost always co-occurred with nor. This suggests that nirS type denitrifiers are more likely to be capable of complete denitrification and thus contribute less to N2O emissions than nirK type denitrifiers under favorable environmental conditions. Comparative phylogenetic analysis indicated a greater degree of shared evolutionary history between nosZ and nirS. However 30% of the organisms with nosZ did not possess either nir gene, with several of these also lacking nor, suggesting a potentially important role in N2O reduction. Co-occurrence patterns were also non-randomly distributed amongst preferred habitat categories, with several habitats showing significant differences in the frequencies of nirS and nirK type denitrifiers. These results demonstrate that the denitrification pathway is highly modular, thus underpinning the importance of community structure for N2O emissions.

Nitrous oxide (N 2 O) is a major radiative forcing and stratospheric ozone-depleting gas emitted from terrestrial and aquatic ecosystems. It can be transformed to nitrogen gas (N 2 ) by bacteria and archaea harboring the N 2 O reductase (N 2 OR), which is the only known N 2 O sink in the biosphere. Despite its crucial role in mitigating N 2 O emissions, knowledge of the N 2 OR in the environment remains limited. Here, we report a comprehensive phylogenetic analysis of the nosZ gene coding the N 2 OR in genomes retrieved from public databases. The resulting phylogeny revealed two distinct clades of nosZ, with one unaccounted for in studies investigating N 2 O-reducing communities. Examination of N 2 OR structural elements not considered in the phylogeny revealed that the two clades differ in their signal peptides, indicating differences in the translocation pathway of the N 2 OR across the membrane. Sequencing of environmental clones of the previously undetected nosZ lineage in various environments showed that it is widespread and diverse. Using quantitative PCR, we demonstrate that this clade was most often at least as abundant as the other, thereby more than doubling the known extent of the overall N 2 O-reducing community in the environment. Furthermore, we observed that the relative abundance of nosZ from either clade varied among habitat types and environmental conditions. Our results indicate a physiological dichotomy in the diversity of N 2 Oreducing microorganisms, which might be of importance for understanding the relationship between the diversity of N 2 O-reducing microorganisms and N 2 O reduction in different ecosystems.

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The rhizosphere is a hotspot for denitrification. The nitrous oxide (N2O) reductase among denitrifiers and non-denitrifying N2O reducers is the only known N2O sink in the biosphere. We hypothesized that the composition of root-associated N2O-reducing communities when establishing on annual crops depend on soil type and plant species, but that assembly processes are independent of these factors and differ between nosZ clade I and II. Using a pot experiment with barley and sunflower and two soils, we analyzed the abundance, composition and diversity of soil and root-associated N2O reducing communities by qPCR and amplicon sequencing of nosZ. Clade I was more abundant on roots compared to soil, while clade II showed the opposite. In barley, this pattern coincided with N2O availability, determined as potential N2O production rates, but for sunflower no N2O production was detected in the root compartment. Root and soil nosZ communities differed in composition and phylogeny-based community analyses indicated that assembly of root-associated N2O reducers was driven by the interaction between plant and soil type, with inferred competition being more influential than habitat selection. Selection between clades I and II in the root/soil interface is suggested, which may have functional consequences since most clade I microorganisms can produce N2O.

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