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.