Active agricultural soil denitrifiers possess extensive nitrite reductase gene diversity. Active agricultural soil denitrifiers

Denitrification in agricultural fields transforms nitrogen applied as fertilizer, reduces crop production, and emits N2O, which is a potent greenhouse gas. Existing primers and probes used for detecting denitrifiers are biased due to their development based primarily on sequence information from cultured denitrifiers; very little is known about the identities of active denitrifiers in agricultural soils. Using targeted metagenomics, we explored active and uncultivated denitrifiers from two agricultural sites in Ottawa, Ontario. The DNA stable-isotope probing (DNA-SIP) method recovered labelled nucleic acids from active soil denitrifiers by exposing intact replicate soil cores to nitrate and 13C6-glucose under anoxic conditions using flow-through reactors. Multivariate statistics of assembled 16S rRNA genes confirmed unique taxonomic representation in heavy fractions from flow-through reactors fed 13C6-glucose, which was consistent across sites and time points. Labelled taxa affiliated with the Betaproteobacteria (71%; Janthinobacterium, Acidovorax, Azoarcus, Dechloromonas), Alphaproteobacteria (8%; Rhizobium), Gammaproteobacteria (4%; Pseudomonas), and Actinobacteria (4%; Streptomycetaceae). High-throughput sequencing of metagenomic DNA from the original soil and DNA-SIP heavy fractions confirmed amplicon sequence data by demonstrating enrichment of denitrification genes affiliated with Janthinobacterium, Ralstonia, and Azoarcus in heavy fractions of flow-through reactors. The vast majority of heavy-DNA-associated nitrite-reductase reads annotated to the copper-containing form (nirK), rather than the heme-containing enzyme (nirS). Analysis of recovered nirK genes demonstrated low sequence identity across common primer-binding sites used for the detection and quantification of soil denitrifiers, indicating that these active denitrifiers would not have been detected in molecular surveys of these same soils and suggesting that no single primer set can be used for detecting the nirK genes of all known soil denitrifiers.