Phylogenomics reveals dynamic evolution of fungal nitric oxide reductases and their relationship to secondary metabolism

Abstract

Fungi expressing P450nor, an unconventional nitric oxide (NO) reducing cytochrome P450, are thought to be significant contributors to soil nitrous oxide (N2O) emissions. However, fungal contributions to N2O emissions remain uncertain due to inconsistencies in measurements of N2O formation by fungi. Much of the N2O emitted from antibiotic-amended soil microcosms is attributed to fungal activity, yet fungal isolates examined in pure culture are poor N2O producers. To assist in reconciling these conflicting observations and produce a benchmark genomic analysis of fungal denitrifiers, genes underlying fungal denitrification were examined in >700 fungal genomes. Of 167 p450nor–containing genomes identified, 0, 30, and 48 also harbored the denitrification genes narG, napA or nirK, respectively. Compared to napA and nirK, p450nor was twice as abundant and exhibited two to five-fold more gene duplications, losses, and transfers, indicating a disconnect between p450nor presence and denitrification potential. Furthermore, co-occurrence of p450nor with genes encoding NO-detoxifying flavohemoglobins (Spearman r = 0.87, p = 1.6e−10) confounds hypotheses regarding P450nor’s primary role in NO detoxification. Instead, ancestral state reconstruction united P450nor with actinobacterial cytochrome P450s (CYP105) involved in secondary metabolism (SM) and 19 (11 %) p450nor-containing genomic regions were predicted to be SM clusters. Another 40 (24 %) genomes harbored genes nearby p450nor predicted to encode hallmark SM functions, providing additional contextual evidence linking p450nor to SM. These findings underscore the potential physiological implications of widespread p450nor gene transfer, support the novel affiliation of p450nor with fungal SM, and challenge the hypothesis of p450nor’s primary role in denitrification.

Nitrous oxide production and isotopomer composition by fungi isolated from salt marsh sediments

“…The ~10-fold increase in both N 2 O and CO 2 production by P. lilacinum grown with 100 mM NO − 2 (compared to 10 mM ) further supports the notion that respiratory reduction of NO − 2 was the primary mechanism for N 2 O production, although we cannot rule out the possibility that secondary metabolisms also contribute to N 2 O production (Higgins et al, 2018). Such a response by P. lilacinum implies that fungal N 2 O production from salt marsh sediments will scale linearly with nutrient inputs from anthropogenic sources.…”

“…The ability to produce N 2 O was widespread among the hundreds of strains tested by Maeda and colleagues (Maeda et al, 2015), but only one (Mucorales) of the 70 N 2 O-producing strains in their study was not from the phylum Ascomycota. A survey of P450nor in over 700 fungal genomes also showed that this diagnostic gene for fungal denitrification was primarily found in Ascomycota (Higgins et al, 2018). While Ascomycota seem to be the main lineage capable of N 2 O production, fungi from other phyla, particularly early diverging lineages, are poorly represented in these studies, partially due to their low abundance in terrestrial environments (Berbee et al, 2017).…”

“…All filamentous species (P. lilacinum, T. virens, and T. harzianum) we isolated from salt marsh sediments have been isolated from soil and shown to produce N 2 O (Lavrent'ev et al, 2008;Jirout, 2015;Maeda et al, 2015). A comparative genomics studies showed that all these three species possess the diagnostic gene for fungal denitrification, the cytochrome P450 nitric oxide reductase (P450nor) (Higgins et al, 2018). The positive correlations between N 2 O and CO 2 production by filamentous fungi indicate respiratory denitrification is responsible for N 2 O production.…”

“…fungus isolated anaerobically was the basidiomycetous yeast R. glutinis, which is not known to possess P450nor in its genome (Higgins et al, 2018), but it does possess a fungal nitrite reductase (fungal nirK). The decoupling between N 2 O and CO 2 production in R. glutinis cultures (Figure 3) suggests that N 2 O production by R. glutinis may not be from respiratory denitrification.…”

“…Fungi differ from bacteria and archaea in that they lack nosZ (Shoun et al, 1992;Hayatsu et al, 2008). Fungal N 2 O is thereby released into the environment where it can be consumed by N 2 O-reducing bacteria or emitted to the atmosphere (Higgins et al, 2018).…”