Detection, Diversity, and Evolution of Fungal Nitric Oxide Reductases (P450nor)

Nitrous oxide (N₂O) is a gas responsible for significant ozone layer depletion and contributes to greenhouse effects in Earth’s atmosphere. N₂O is primarily generated by denitrification, whereby nitrate (NO_3-) or nitrite (NO_2-) is converted to gaseous N₂O or N2. Teragram quantities of N₂O are emitted annually from agricultural soils treated with nitrogenous fertilizers due to the activity of soil microbiota. Although bacteria and fungi harbor genes permitting denitrification, fungi lack NosZ, an enzyme responsible for reducing N₂O into inert N2 gas. Historically, scientists have linked fungi to soil N₂O production, but uncertainty in fungal contributions remain due to the persistent use of biased, inhibitor-based methodologies used to assess fungal and bacterial contributions to soil N₂O emissions. In the following chapters, the application of PCR- and bioinformaticsbased techniques to detect the nitric oxide reductase gene, p450nor, are presented. N₂O-producing fungi were isolated from microcosms established with two distinct agricultural soils amended with NO_3- or NO_2-. The p450nor PCR primers identified 49 novel p450nor gene amplicons from isolate and environmental DNA. Moreover, the PCR primer’s utility was expanded by their incorporation into Illumina sequencing protocols, enabling analyses of 72 soil samples from two Midwest agricultural soils. Analysis of p450nor sequences (12,675,480 in total) revealed 69.7 and 64.6 % were classified as isolate or environmental sequences previously acquired from these soils. Analysis of fungal internal transcribed spacer (ITS2) sequences revealed 4,591 OTUs, of which 19 OTUs comprised 41.6 % of all fungal sequences. Importantly, ITS2 OTUs assigned to fungal families harboring denitrifying members suggests between 2 and 67 % of the fungal community may be denitrifiers. Finally, a phylogenomics approach was applied to explore denitrification potential across >700 fungal genomes. These analyses demonstrated a low co-occurrence of p450nor and other denitrification genes (narG, napA, nirK). Instead, 11 % of genomes were predicted to contain p450nor in secondary metabolism (SM) gene clusters and 35 % were localized adjacent hallmark SM genes, suggesting an undiscovered role for p450nor in SM. Phylogenetic analyses united p450nor with actinobacterial sequences involved in SM, suggesting recent horizontal gene transfer and challenging the paradigm of p450nor’s primary role in denitrification.