Figure 4 link
The majority of ORFs represented in the C1 domain tree contain the C1 domain homolog either as a free cytochrome, or as one of multiple cytochrome-type or cytochrome superfamily domains. In rare or isolated cases, C1 homologs co-occur in ORFs with membrane or structural protein domains (Table S1). The ORF containing the C1 homolog was annotated as a nitric oxide reductase in several members of the Nitrospirae and one Geobacteraceae genome (Table S1); domain analysis of these genes yielded limited additional data, but representative sequences showed detectable sequence similarity to Pseudomonas norC genes.
The occurrence of C1 domain homologs within predicted nitrite reductase genes is restricted to the Chloroflexi. The majority of these C1-containing nir genes have the cytochrome-type NirS domain; however, a small number of Chloroflexi MAGs contain an ORF pairing the C1 cytochrome with the copper-type NirK domain instead. This NirK ORF also included an N-terminal cupredoxin/plastocyanin domain. As this fusion is only apparent within a small number of MAGs, which are identical across the length of the analyzed ORF, this may represent an assembly artifact. However, several of the nir genes that contained a C1 homolog and a cytochrome-type NirS (not copper-type NirK) also contained cupredoxins or other copper-containing domains (Fig. S1).
The distinct phylogeny and taxonomic distribution of C1, as compared with C2 and NirS domains, strongly suggest that the C1-C2-NirS domain structure observed in Chloroflexi is the result of fusion of the C1 domain with a horizontally-acquired Proteobacterial nir gene. Though topology and extant taxon sampling of these gene trees does not allow us to reliably infer the donor lineage, the C2-NirS architecture—or similar arrangements of functional domains—is widespread among members of the Alpha-, Beta-, and Gammaproteobacteria. Additionally, it appears that Chloroflexi may have been the source for an independent transfer of the free C1 domain into Nitrospirae and Nitrospinae.