Figure 2 link
The first cytochrome domain (C1) in the Anaerolineales-type ORF was
identified by NCBI’s Conserved Domains Database (Marchler-Bauer et
al. , 2015; Lu et al. , 2020) as a cytochrome c551/552 (NCBI
COG4654, e-value = 6.4x10-4). The second cytochrome
domain (C2) was predicted with high specificity as a cytochrome C mono-
and diheme variant (NCBI COG2010, e-value=5.31x10-9).
C2 included a region predicted as a cbb3-type cytochrome c oxidase
subunit III (pfam 13442, e-value = 4.06x10-7); such
subunits frequently contain two cytochromes (Bertini, Cavallaro and
Rosato, 2006).
Though the C2 and NirS functional domains frequently co-occur in nitrite
reductases, the inclusion of C1 in the ORF appears extremely rare and
limited to Chloroflexi. A lineage-specific fusion of multiple gene
domains could explain this novel C1-C2-NirS arrangement. Different
evolutionary histories among the domain subunits within Chloroflexi
would provide evidence for an ancestral horizontal acquisition and
fusion event. To determine if the different domains of the enzyme have
different ancestry, maximum-likelihood domain trees were independently
reconstructed (See Methods) for C1, C2, and the NirS-specific domain.
Domain phylogenies indicate a similar overall topology for C2 (Fig. 3a)
and the NirS domains (Fig 3b). These trees both recover a clade
of Chloroflexi closely related to a polyphyletic group containing
diverse NirS sequences from Aquificae, Bacteroidetes,
Epsilonproteobacteria, and Spirochaetia. In both cases, this
polyphyletic group is sister to a large grouping of proteobacterial
sequences from Alpha-, Beta-, and Gammaproteobacteria. The relative
placement of Chloroflexi sequences varies slightly between both domain
trees: For C2, Chloroflexi sequences fall within the polyphyletic group
comprising Aquificae, Bacteroidetes, Epsilonproteobacteria, and
Spirochaetia, with the combined group placing sister to the large
proteobacterial group; for NirS, Chloroflexi are sister to the
polyphyletic grouping.