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.