Extended Data Fig. 3: Analysis of aerobic methane oxidation enzymes and pathways in sampled soils.
From: Trace gas oxidizers are widespread and active members of soil microbial communities
(a) Maximum-likelihood tree of amino acid sequences of particulate methane monooxygenase A subunit (PmoA), a marker for aerobic methane oxidation. The tree shows 2 sequences from soil metagenome-assembled genomes (blue) and 12 sequences from unbinned contigs (red) alongside 93 representative sequences from NCBI reference genomes (black). The tree shows the affiliation of the PmoA from Candidatus Methylotropicum kingii with those of amplicons of the tropical upland soil cluster (TUSC). Also shown are reference sequences of other members of the copper membrane monooxygenase superfamily, namely ammonia monooxygenase (AmoA), hydrocarbon monooxygenase (HmoA), and groups of unknown function (including PxmA). The tree was constructed using the JTT matrix-based model, used all sites, and was bootstrapped with 50 replicates and midpoint-rooted. Source data is provided in Newick (nwk) format. (b) Metabolic reconstruction of the putative novel methanotroph Candidatus Methylotropicum kingii. The core pathways associated with energy conservation and carbon acquisition are shown, with genes detected shown in italics. The bacterium is predicted to use methane, methanol, and acetate as energy and carbon sources. In addition, it can use molecular hydrogen as an electron donor via a group 1f [NiFe]-hydrogenase. The bacterium is predicted to use the electron acceptors oxygen via a cytochrome c oxidase and nitrous oxide via a nitrous oxide reductase. Its particulate methane monooxygenase forms a distinct phylogenetic lineage with amplicons from the Tropical Upland Soil Cluster (TUSC), whereas its methanol dehydrogenase is closely related to those in previously sequenced Gemmatimonadota MAGs inferred to be methylotrophic. The genome encodes key enzymes for the serine cycle for assimilation of one-carbon sources. Abbreviations: H4F = tetrahydrofolate; Hyd = group 1f [NiFe]-hydrogenase; pMMO = particulate methane monooxygenase; MDH = methanol dehydrogenase; PQQ = pyrroloquinoline quinone; I = NADH dehydrogenase (complex I); II = succinate dehydrogenase (complex II); IV = cytochrome aa3 oxidase (complex IV). Dashed black lines indicate diffusion. Dashed gray lines indicate unknown process or undetected genes. (c) Molecular model of the putative particulate methane monooxygenase (Pmo) from Candidatus Methylotropicum kingii and comparison with putative catalytic sites of other experimentally validated Pmos. (i) Molecular model of the functional homotrimer of the putative Pmo complex from Ca. M. kingii, shown as a cartoon representation. PmoA, PmoB, and PmoC subunits from one Pmo complex are colored in light blue, dark blue, and sky blue respectively. The other two Pmo complexes in the trimer are colored in transparent yellow. Cu ions bound in the putative active site of the colour-coded Pmo complex are shown as ochre spheres. TM = transmembrane. (ii) Zoomed view of the Pmo complex from panel A, with the three Pmo subunits labelled and the putative active sites CuB and CuC highlighted. (iii) Stick representation of residues in the CuB and CuC active sites of Pmo molecular models from Ca. M. kingii (Gemmatimonadota) and Methylacidiphilum infernorum (Verrucomicrobiota), and from the crystal structure (PDB ID: 3RGB) of Pmo from the Methylococcus capsulatus (Proteobacteria), showing that both sites are conserved between Pmo from Ca. M. kingii and M. capsulatus. Pmo from M. infernorum lacks the CuB site, suggesting this site is not responsible for methane oxidation. (iv) Sequence alignment of metal binding motifs from the CuB (left) and CuC (right) sites from Pmo from Ca. M. kingii (cMk.), M. capsulatus (Mc.), Candidatus Methylomirabilis limnetica (cMl.; Methylomirabilota), and M. infernorum (Mi.). Amino acids involved in Cu coordination are highlighted.
