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.

Microbial communities have a central role in ecosystem processes by driving the Earth's biogeochemical cycles. However, the importance of microbial diversity for ecosystem functioning is still debated. Here, we experimentally manipulated the soil microbial community using a dilution approach to analyze the functional consequences of diversity loss. A trait-centered approach was embraced using the denitrifiers as model guild due to their role in nitrogen cycling, a major ecosystem service. How various diversity metrics related to richness, eveness and phylogenetic diversity of the soil denitrifier community were affected by the removal experiment was assessed by 454 sequencing. As expected, the diversity metrics indicated a decrease in diversity in the 1/10 3 and 1/10 5 dilution treatments compared with the undiluted one. However, the extent of dilution and the corresponding reduction in diversity were not commensurate, as a dilution of five orders of magnitude resulted in a 75% decrease in estimated richness. This reduction in denitrifier diversity resulted in a significantly lower potential denitrification activity in soil of up to 4-5 folds. Addition of wheat residues significantly increased differences in potential denitrification between diversity levels, indicating that the resource level can influence the shape of the microbial diversityfunctioning relationship. This study shows that microbial diversity loss can alter terrestrial ecosystem processes, which suggests that the importance of functional redundancy in soil microbial communities has been overstated.