DISCUSSION
The phylogenetic analyses of nirS and eNOR ORFs in Chloroflexi suggest that subsurface ecosystems may harbor an under-described diversity of denitrification enzymes, which may reflect adaptations to the unique challenges of nutrient cycling within these environments. More broadly, a deeper understanding of the ecological extent of microbial denitrification has important implications for basic and applied microbial ecology. The reduction of fixed nitrogen species plays a crucial role in global nitrogen cycling and is also an essential component of smaller-scale systems, such as those associated with agricultural or waste treatment (Butterbach-Bahl and Dannenmann, 2011; Lu, Chandran and Stensel, 2014). The discovery and characterization of novel variants of genes such as nirS and eNOR may therefore pave the way for future biotechnological applications.
Although the C2 and NirS domains do not have identical evolutionary histories or distributions, the taxonomic representation of these groups is very similar, and the presence of the paired C2-NirS domains in cytochrome-type nitrite reductases appears broadly throughout the Proteobacteria. In contrast, the taxonomic distribution and phylogeny of the C1 domain tree is strikingly different than that of the other domains in the nitrite reductase ORF. Combined with the apparent absence of a full C1-C2-NirS ORF in any taxonomic group other than Chloroflexi, these data suggest that the C1 cytochrome was likely incorporated intonirS in a gene fusion event within Chloroflexi, following HGT. As there is no evidence of the C2-NirS ORF in Chloroflexi without the fused C1 domain present, the fusion probably occurred very soon after the acquisition of the C2-NirS region and may be necessary for the function of the gene in Chloroflexi.
Interestingly, putative homologs of C1 cytochrome domains were found in some Chloroflexi genomes in ORFs containing nirK , not nirS(Fig. 4, Fig. S1). Though NirS and NirK are functionally equivalent, the two enzymes do not show a shared evolutionary origin, and are often—though not always—mutually exclusive among known denitrifier genomes (Jones et al. , 2008; Graf, Jones and Hallin, 2014). Unlike the cytochrome-containing NirS, NirK is a copper-type enzyme. The co-occurrence of cytochrome c domains in ORFs with the copper-type nirK has been identified in rare instances in Proteobacteria, and noted as surprising, given the cupredoxin-like fold of the NirK enzyme (Bertini, Cavallaro and Rosato, 2006). Similarly surprising is the inverse relationship revealed in the C1 domain tree: Several Chloroflexi ORFs contain a cupredoxin or similar copper-containing domain N-terminal to the C1-C2-NirS architecture (Fig. 4, Fig. S1). The co-occurrence of C1 with both cytochrome- and copper-dependent Nir domains suggests a general evolutionary trend within Chloroflexi to incorporate this cytochrome into denitrification ORFs. This distribution pattern raises the possibility that the C1-type cytochrome may serve an important but generalized role in nitrite reduction—regardless of the evolutionary history or genetic profile of the nitrite reduction domain itself.
The apparent absence of a nor homolog in the majority of genomes with the C1-nirS fusion is unexpected. Beyond providing downstream redox capacity, nitric oxide reductase provides an efficient means of reducing and detoxifying nitric oxide, the highly cytotoxic product of NirS. It is not unprecedented for bacterial genomes to harbor a nir gene without a nor gene, particularly for organisms with nirK (Heylen et al. , 2007; Graf, Jones and Hallin, 2014). This nir-nor mismatch is much rarer for putative denitrifiers with nirS , representing fewer than 4% of genomes in a recent survey—but a small number of surveyed bacteria do, interestingly, appear to harbor nirS without also harboringcNOR or qNOR (Heylen et al. , 2007; Graf, Jones and Ha llin, 2014). To our knowledge, however, eNOR has not been included in such analyses of the genomic correlation between nitrite reductases and nitric oxide reductases. The phylogenetic evidence for diverse eNOR homologs suggests likely undocumented or underexplored diversity for divergent nitric oxide reductases. Diversity and function of cytochrome-dependent (cNOR) and quinol-dependent nitric oxide reductases (qNOR) are fairly well-established. However, divergent enzymes such as eNOR and sNOR are less-extensively documented and may not be accurately distinguished from broader oxygen reductase superfamily members in genomic or metagenomic analyses.
Cytochrome c proteins function as electron transfer proteins in anaerobic respiration and are often fused to redox enzymes to allow electron passage (Bertini, Cavallaro and Rosato, 2006). It is not surprising, therefore, to find cytochrome c-containing subunits in frame with nitrite reductase. NirS itself is cytochrome-dependent (Bertini, Cavallaro and Rosato, 2006). However, the unusual addition of the upstream cytochrome domain (C1) may reflect additional redox requirement or capacity. It is also possible that the inclusion of this construct could be linked to the conspicuous absence of nitric oxide reductase enzymes in several metagenome-assembled genomes containing a NirS ORF with the C1 fusion. Nitric oxide reduction can be cytochrome-dependent; the well-studied cNOR nitric oxide reductases contain a membrane-anchored cytochrome c (Hemp and Gennis, 2008). Further, the C1 domain tree recovers ORFs in the Nitrospirae that contain C1 homologs and are annotated as nitric oxide reductases, with detectable similarity to Proteobacteria nitric oxide reductase subunits. It is therefore possible that the inclusion of a C1 domain in nir genes within genomes lacking eNOR reflects some generalized NOR-like role in detoxification of the cytotoxic product of NirS. Additionally, while the presence of NirS suggests an active denitrification pathway, and the NirS domain tree reflects the homology between this domain and NirS from known denitrifying groups, the possibility remains that this group of Chloroflexi do not perform denitrification, and instead use this gene product for a different metabolic function, potentially enabled or constrained by the C1 domain. Experimental validation would be necessary to determine if the novel Chloroflexi-associated NirS performs differently than canonical NirS in vivo ; this work, therefore, suggests a promising direction for future investigation.
The divergent denitrification enzymes described above may or may not reflect different metabolic strategies in situ . But the identification of both a novel nirS ORF and an expanded diversity of eNOR enzymes suggests that the existing understanding of denitrification may underestimate the genetic diversity and ecological distribution of constituent enzymes. This may be especially true in deep subsurface biomes, such as those from which several Chloroflexi analyzed in this study were isolated. These systems have garnered increasing attention in recent years; extensive evidence supports the existence of dynamic, diverse microbial subsurface ecosystems with the metabolic potential to influence global biogeochemical cycles (Hug et al. , 2013; Osburn et al. , 2014, 2019; Momper et al. , 2017). Chloroflexi are frequently cited as well-represented members of deep sediment and aquifer systems, where they play key roles in carbon cycling dynamics (Hug et al. , 2013; Momper et al. , 2017; Kadnikov et al. , 2020). But Chloroflexi are known to also harbor diverse nitrogen metabolisms (Hemp et al. , 2015; Denef et al. , 2016; Spieck et al. , 2020), and previous studies have linked subsurface Chloroflexi to denitrification pathway genes such as nitrous oxide reductase (nos ) (Sanford et al. , 2012; Huget al. , 2016; Momper et al. , 2017). The role of Chloroflexi in subsurface nitrogen cycling—as well as the scope of subsurface microbial nitrogen dynamics at large—requires further investigation.