Novel nitrite reductase domain structure suggests a chimeric denitrification repertoire in the phylum Chloroflexi

Abstract

Denitrification plays a central role in the global nitrogen cycle, reducing and removing nitrogen from marine and terrestrial ecosystems. The flux of nitrogen species through this pathway has a widespread impact, affecting ecological carrying capacity, agriculture, and climate. Nitrite reductase (Nir) and nitric oxide reductase (NOR) are the two central enzymes in this pathway. Here we present a previously unreported Nir domain architecture in members of phylum Chloroflexi. Phylogenetic analyses of protein domains within Nir indicate that an ancestral horizontal transfer and fusion event produced this chimeric domain architecture. We also identify an expanded genomic diversity of a rarely reported NOR subtype, eNOR. Together, these results suggest a greater diversity of denitrification enzyme arrangements exist than have been previously reported.

Keywords: Chloroflexi; cytochrome; denitrification; nitric-oxide reductase; nitrite reductase; phylogeny.

Figure 1

Denitrification. Complete denitrification transforms nitrate into dinitrogen gas. Respiratory nitrate reductase gene (nar) and periplasmic nitrate reductase (nap) genes are distributed in non‐denitrifying organisms. Nitrite reductase (Nir) is considered the canonical first enzyme of denitrification (Graf et al., 2014), followed by nitric oxide reductase (NOR) and nitrous oxide reductase (Nos)

Figure 2

Open reading frame domain map. Conserved domain analysis of SURF MAG 42 Chloroflexi nitrite reductase (GenBank RJP53747.1) indicates the presence of two distinct cytochrome superfamily domains and a C‐terminal nitrite reductase domain. MAG, metagenome‐assembled genome; SURF, Sanford Underground Research Facility

Figure 3

NirS gene neighborhood in SURF MAG 42 versus Pseudomonas aeruginosa. Gene neighborhood analyses of the 20,000 base pair region surrounding nirS differ markedly between P. aeruginosa PAO1 (GenBank reference sequence NP_249210.1, top) and two Chloroflexi genomes containing the C1‐C2‐NirS gene: SURF MAG 42 (center) and Anaerolinea thermolimosa (bottom). While the P. aeruginosa nitrite reductase occurs as part of a larger nir operon, and in close proximity to nitric oxide reductase genes, the nirS ORF neighborhoods in SURF MAG 42 and A. thermolimosa do not appear to contain other denitrification‐specific genes. Detailed descriptions of ORF/gene families and functions can be found in Table A1. MAG, metagenome‐assembled genome; ORF, open reading frame; SURF, Sanford Underground Research Facility

Figure 4

Phylogenetic trees for C2 and NirS domains. Phylogenetic analysis of C2 domain homologs (above) places the Chloroflexi within a diverse clade including Epsilonproteobacteria, Aquificae, Bacteroidetes, and Planctomycetes; this clade is sister to a broad radiation of Alpha‐, Beta‐, and Gammaproteobacteria. Analysis of NirS domain homologs (below) places the Chloroflexi within the clade dominated by Alpha‐, Beta‐, and Gammaproteobacteria, which also contains members of the Bacteroidetes and Aquificales. The overall taxonomic representation for the domains is similar. Support values for selected bipartitions are labeled (aLRT/bb). Support for other nodes is indicated with the following color scheme: Strong support with both values ≥90 (black); weak support with both values ≤50 (white); intermediate support with one or both values between 50 and 90 (gray); conflicting support, with one value ≤50 and the other ≥90 (gray)

Figure 5

Phylogenetic tree for C1 domain. A phylogenetic tree for the C1 domain—with no genus‐level filter and inclusion of more distant hits (see Methods)—indicates a limited taxonomic distribution of the domain. The largest group of sequences in Chloroflexi places sister to domains found in Nitrospirae, Nitrospinae, and Deltaproteobacteria. Within this clade, the branch along which C1 is inferred to have fused into nitrite reductase genes in Chloroflexi is labeled. C1 homologs that co‐occur in ORFs with nitrite reductase are indicated with magenta diamonds (NirS) or yellow diamonds (NirK). Support values for selected bipartitions are labeled (aLRT/bb). Support for other nodes is indicated with the following color scheme: Strong support with both values ≥90 (black); weak support with both values ≤50 (white); intermediate support with one or both values between 50 and 90 (gray); conflicting support, with one value ≤50 and the other ≥90 (gray). ORF, open reading frame

Figure 6

eNOR gene tree. A phylogenetic tree of homologs to the nitric oxide reductase from SURF MAG 42 reveals an expanded diversity of putative eNOR subunit I homologs in not only Archaea and Chloroflexi, but also Proteobacteria and other diverse phyla. Putative eNOR sequences (red tips) have the characteristic Gln‐323 in the alignment; outgroup sequences (blue tips) have Tyr‐323 (oxygen reductase superfamily) or other substitutions. Support values for selected bipartitions are labeled (aLRT/bb). Support for other nodes is indicated with the following color scheme: Strong support with both values ≥90 (black); weak support with both values ≤50 (white); intermediate support with one or both values between 50 and 90 (gray); conflicting support, with one value ≤50 and the other ≥90 (gray). MAG, metagenome‐assembled genome; SURF, Sanford Underground Research Facility

Figure A1

C1‐C2‐NirS gene neighborhoods in Chloroflexi. A gene neighborhood showing the 20,000 base pair region adjacent to the putative nitrite reductase gene containing the C1‐C2‐NirS domain architecture. Neighborhoods are shown for homologs in Chloroflexi bacterium 44‐23 (GenBank accession OJX39483.1) and SURF MAG 71 (GenBank accession RJP52528.1). Expanded descriptions of gene names and functions are provided in Table A2

Figure A2

Expanded C2 domain tree. Phylogenetic analysis of C2 domain homologs places the Chloroflexi within a diverse polyphyletic clade including Epsilonproteobacteria, Aquificae, Bacteroidetes, and Planctomycetes; this clade is sister to a broad radiation of Proteobacteria. Support values for selected bipartitions are labeled (aLRT/bb). Support for other nodes is indicated with the following color scheme: Strong support with both values ≥90 (black); weak support with both values ≤50 (white); intermediate support with one or both values between 50 and 90 (gray); conflicting support, with one value ≤50 and the other ≥90 (gray)

Figure A3

Expanded NirS domain tree. Phylogenetic analysis of NirS domain homologs places the Chloroflexi within a polyphyletic clade dominated by Alpha‐, Beta‐, and Gammaproteobacteria. Support values for selected bipartitions are labeled (aLRT/bb). Support for other nodes is indicated with the following color scheme: Strong support with both values ≥90 (black); weak support with both values ≤50 (white); intermediate support with one or both values between 50 and 90 (gray); conflicting support, with one value ≤50 and the other ≥90 (gray)

Figure A4

Subsampled C2 domain tree. Phylogenetic analysis of C2 domain homologs, subsampled to contain only taxa with both C2 and NirS domains in the nitrite reductase ORF, places the largest clade of Chloroflexi as sister to a polyphyletic group including a large group of Alpha‐, Beta‐, and Gammaproteobacteria. Support values for selected bipartitions are labeled (aLRT/bb). Support for other nodes is indicated with the following color scheme: Strong support with both values ≥90 (black); weak support with both values ≤50 (white); intermediate support with one or both values between 50 and 90 (gray); conflicting support, with one value ≤50 and the other ≥90 (gray). ORF, open reading frame

Figure A5

Subsampled NirS domain tree. Phylogenetic analysis of NirS domain homologs, subsampled to contain only taxa with both C2 and NirS domains in the nitrite reductase ORF, places the largest clade of Chloroflexi as sister to a large radiation of Proteobacteria; these groups are nested within a diverse polyphyletic group. Support values for selected bipartitions are labeled (aLRT/bb). Support for other nodes is indicated with the following color scheme: Strong support with both values ≥90 (black); weak support with both values ≤50 (white); intermediate support with one or both values between 50 and 90 (gray); conflicting support, with one value ≤50 and the other ≥90 (gray). ORF, open reading frame

Figure A6

Various nitrite reductase architectures within Chloroflexi. Three different open reading frame types including a C1 cytochrome and a nitrite reductase domain appear in surveyed Chloroflexi. The most commonly seen gene features C1, C2, and cytochrome‐dependent nirS (a); however, C1 is also seen in ORFs with cupredoxin and copper‐dependent nitrite reductase (nirK) domains (b). A limited number of Chloroflexi genomes also contain ORFs placing C1, C2, and nirS together with an N‐terminal cupredoxin, plastocyanin (PetE), or similar copper‐containing domain (c). ORF, open reading frame

Figure A7

eNOR subunit gene neighborhood. A gene neighborhood showing the 20,000 base pair region adjacent to eNOR subunits in SURF MAG 42. Sequence analysis of subunit I (NCBI protein accession RJP50323.1) reflects a conserved glutamine substitution characteristic of the eNOR subfamily. Both proposed subunits of eNOR—the heme‐copper cytochrome‐containing subunit I, and the cupredoxin‐containing subunit II—appear in the MAG. No other denitrification genes appear in the neighborhood. MAG, metagenome‐assembled genome; NCBI, National Center for Biotechnology Information; SURF, Sanford Underground Research Facility

Figure A8

Preliminary C1 domain tree. A preliminary phylogenetic tree for C1, including only sequences with E ≤ 10⁻¹⁰, contains very few overall taxa. The tree contains only members of the Chloroflexi, members of the Nitrospirae, and Nitrospina gracilis; the sampling depth does not recover an outgroup for these sister groups. Support for bipartitions is indicated with the following color scheme: Strong support with both values ≥90 (black); weak support with both values ≤50 (white); intermediate support with one or both values between 50 and 90 (gray); conflicting support, with one value ≤50 and the other ≥90 (gray)