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Methanotrophy by a Mycobacterium species that dominates a cave microbial ecosystem


So far, only members of the bacterial phyla Proteobacteria and Verrucomicrobia are known to grow methanotrophically under aerobic conditions. Here we report that this metabolic trait is also observed within the Actinobacteria. We enriched and cultivated a methanotrophic Mycobacterium from an extremely acidic biofilm growing on a cave wall at a gaseous chemocline interface between volcanic gases and the Earth’s atmosphere. This Mycobacterium, for which we propose the name Candidatus Mycobacterium methanotrophicum, is closely related to well-known obligate pathogens such as M. tuberculosis and M. leprae. Genomic and proteomic analyses revealed that Candidatus M. methanotrophicum expresses a full suite of enzymes required for aerobic growth on methane, including a soluble methane monooxygenase that catalyses the hydroxylation of methane to methanol and enzymes involved in formaldehyde fixation via the ribulose monophosphate pathway. Growth experiments combined with stable isotope probing using 13C-labelled methane confirmed that Candidatus M. methanotrophicum can grow on methane as a sole carbon and energy source. A broader survey based on 16S metabarcoding suggests that species closely related to Candidatus M. methanotrophicum may be abundant in low-pH, high-methane environments.

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Fig. 1: Phylogenetic position and unique genetic make-up of the Sulfur Cave mycobacteria.
Fig. 2: Metabolic reconstruction of methane utilization in Candidatus M. methanotrophicum.
Fig. 3: Growth curves, methane assimilation and morphology of Candidatus M. methanotrophicum.
Fig. 4: Correlative imaging analysis of cells from a Candidatus M. methanotrophicum enrichment culture grown on 13C-labelled methane.
Fig. 5: Microbial community characteristics based on amplicon-based sequencing of 16S rRNA genes in samples from Puturosu Mountain.

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Data availability

Raw data from the proteomic analysis of the Sulfur Cave biofilm, annotation files and supplementary data can be found in a Zenodo repository72. Illumina 16S rRNA gene amplicon data from the Sulfur Cave biofilm are associated with the NCBI BioProject PRJNA675490. Illumina reads from the sequencing of the two metagenomes from the cave and one from the culture as well as the MAGs M. MAGs 1, 2 and 3 are deposited at ENA (PRJEB45004) with the accession numbers ERR10036468, ERR10036469, ERR10036470, ERS6581338, ERS6581340 and ERS6581341, respectively and accession CAJUXY010000000. The 16S rRNA gene sequence obtained from the Candidatus M. methanotrophicum culture is available at GenBank (MW243585). The Illumina and Nanopore reads from this culture as well as the resulting whole-genome assembly are available at NCBI under BioProject PRJNA837300.


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The authors thank C. Murrell for valuable discussions, C. P. Antony for assistance in metagenomics, A. Grootemaat for TEM imaging, M. Kienhuis for technical support with nanoSIMS analysis, I. Grigoriev for support during fluorescence microsopy, and Z. Para and B. Hegyeli, the Romanian custodians, for facilitating the experiments in Sulfur Cave. Also, we thank Utrecht Sequencing Facility ( for providing sequencing service and data. The nanoSIMS facility at Utrecht University was financed through a large infrastructure grant by the Netherlands Organisation for Scientific Research (NWO, grant no. 175.010.2009.011). C.M. was supported by the Dutch Research Council, as part of the MiCRop Consortium (NWO/OCW grant no. 024.004.014). A.P. and Q.G. are supported by a faculty baseline grant (BAS/1/1020-01-01) from KAUST to A.P. The authors also thank members of the Bioscience Core Laboratory in KAUST for providing assistance with the generation of raw genome sequence datasets. PIE research was funded by FONDECYT-CONCYTEC (216-2015-FONDECYT). This is publication number 7467 of the Netherlands Institute of Ecology (NIOO-KNAW).

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Authors and Affiliations



R.J.M.v.S., organizing ideas, writing, sampling, pathway reconstructions and physiology. Q.G., R.U., J.G., C.M.B. and A.P., metagenomics and proteomics. C.M., writing, pathway reconstructions, phylogenomics and bioinformatic analyses. L.P., writing, 13C-methane experiments, fluorescence and nanoSIMS imaging, data analysis and interpretation. J.-F.F., whole-genome assembly and annotation. E.J.F., fruitful discussions. B.W.B., Illumina sequencing and data processing. J.W.A., sampling and fruitful discussions. M.B. and R.U., technical support. P.I.E., DNA extractions. M.M.M.-F. and P.L.E.B., culturing and 13C-methane experiments. S.R.P. and R.U., mass spectrometry. C.M.B., mycobacterial physiology. N.N.v.d.W., TEM imaging. V.D.G., sampling and culturing. S.M.S., sampling, culturing and fruitful discussions. W.B., organizing ideas, writing, sampling and mycobacterial genetics.

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Correspondence to Rob J. M. van Spanning or Wilbert Bitter.

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Extended data

Extended Data Fig. 1 Sulfur Cave on the Puturosu Mountain (Stinky Mountain), Romania, from which Candidatus M. methanotrophicum was enriched.

(a) General overview of the cave. (b) Detailed images of the cave biofilms. The dashed line in panel a marks the stable gaseous chemocline between the volcanic gases (below the chemocline) and atmospheric air (above the chemocline). The cave walls below the chemocline are yellow due to sulphur deposition (marks 2, 5, 8), whereas no such depositions are present above the chemocline (marks 1, 3, 6). Biofilms on the cave walls are only present at the chemocline interface (mark 4). Mark 7 shows the bare cave wall after biofilm sampling.

Extended Data Fig. 2 Maps of sMMO gene clusters in known methanotrophs and selected members of the order Mycobacteriales.

(a) Gene clusters encoding sMMO in Methylosinu strichosporium OB3b, Methylococcus capsulatus Bath, which are known methanotrophs, and Candidatus M. methanotrophicum from this study. Abbreviations: hyp, gene encoding a hypothetical protein; B, mmoB; D, mmoD; Z, mmoZ; hyd, hydrogenase gene cluster; empty arrow, unknown ORF; r, hypothetical transcriptional regulator; PEP-ck, gene encoding phosphoenolpyruvate carboxykinase; SS, signal sensor of a two-component regulatory system; RR, response regulator of a two-component regulatory system. Each structural mmo gene is shown in distinct color, regulatory mmoR and groEL homologue mmoG genes are in red and orange, respectively. Candidatus M. methanotrophicum does not have mmoG in close proximity, but does have groEL genes elsewhere (see Extended Data Fig. 3). (b) Gene maps of genomic regions containing one or more of the mmoRXYBDCZ genes. The maps are shown for the species in Cluster 1 of the phylogenetic tree shown in Supplementary Fig. 4. Note that, in this figure, the direction of the arrows does not reflect the direction of gene transcription.

Extended Data Fig. 3 Maps of mft gene clusters in selected Mycobacterium species.

Abbreviations: M. MAG 1, Candidatus M. methanotrophicum; R, mftR; A, mftA; B, mftB; dac, D-aminocyclase gene; adh, alcohol dehydrogenase gene; hpr, hydroxypyruvate reductase gene; fdr, Ferredoxin-NAD(P)+ reductase gene; gmc, GMC-type oxidoreductase gene. adh from M. MAG 2 (ORF 0656) is closely related to the one from M. tuberculosis H37Rv (see also Supplementary Fig. 8).

Extended Data Fig. 4 Metabolic graph of the pentose phosphate pathway based on a corresponding KEGG map (map00030).

Shown are reactions of enzymes potentially encoded by the genes of Candidatus M. methanotrophicum (M. MAG 1, green), M. MAG 2 (red) and M. MAG 3 (purple) with the corresponding KEGG reaction IDs. Reactions with no color codes are present in all three M. MAGs. Orange circles represent intermediate compounds (corresponding KEGG compound IDs are shown in blue). KEGG reaction names: R05338: D-arabino-hex-3-ulose-6-phosphate formaldehyde-lyase (D-ribulose-5-phosphate-forming); R09780: D-arabino-hex-3-ulose-6-phosphate isomerase; R01741: D-Gluconate:(acceptor) 2-oxidoreductase; R02750: ATP:2-deoxy-D-ribose 5-phosphotransferase; R01051: ATP:D-ribose 5-phosphotransferase.

Extended Data Fig. 5 Number of unique proteins detected by LFQ intensity in the cave biofilm and in the enrichment culture of Candidatus M. methanotrophicum.

Only proteins from four prokaryotic species that remain in the enrichment culture are shown (see legend). Note the increasing relative contribution of proteins from Candidatus M. methanotrophicum in the enrichment culture compared to the cave biofilm.

Extended Data Fig. 6 Morphology and CO2 production of Candidatus M. methanotrophicum.

(a–b) Electron micrographs of Candidatus M. methanotrophicum (a) and M. smegmatis (a). Inset in panel a shows vesicles in the periplasm of Candidatus M. methanotrophicum at a higher magnification. Schematic representations of the micrographs in panels a’ and b’ show the cytosol (grey), the capsular layer (green), the plasma membrane (black line), the periplasm (yellow), the DNA (blue), the elucent areas (white), and the vesicles (red). (c–d) Evolution of CO2 during incubations of Candidatus M. methanotrophicum enrichment cultures with CH4. Incubations were conducted under aerobic (c) or anaerobic (d) conditions using either live cultures (green symbols) or a sterile NMS medium (black symbols). Different symbols correspond to replicate cultures (rep 1–3), and the values correspond to the total amount of CO2 per incubation bottle. Green lines show fits of the experimental data within the time interval of 4–22 days (c) and 0–22 days (d) with a linear model. For the replicate culture 1 incubated under aerobic conditions (rep 1), the slope of this linear model is significantly different from the corresponding negative slope of the linear model characterizing the removal of CH4 during the incubation (two-sided ANOVA, F = 6.79, p = 0.03; CH4 data shown in Fig. 3b). In contrast, the corresponding slopes are not significantly different for replicate cultures 2 (F = 0.0118, p = 0.92) and 3 (F = 3.38, p = 0.10). For both the aerobic and anaerobic incubations, the abiotic controls showed no significant variation in the CO2 amounts over time in comparison to the live cultures.

Extended Data Fig. 7 Additional nanoSIMS images of cells from Candidatus M. methanotrophicum enrichment cultures and cell biomass dynamics.

(a–d) Images of the 13C atom fraction, which is a measure of carbon assimilation from methane provided during the incubation. (e–h) Images of an overlay between the 12C14N ion counts intensity (blue), which is a proxy for biomass, and the 13C atom fraction (green). Cells shown in panels a–c and e–g were grown on 13C-labelled methane for 110 days, while cells in panels d and h were grown on unlabelled methane (control cells). Some cells in panels e–g appear blue because their 13C labeling is significantly lower compared to cells that appear cyan, although it was still significant compared to the control cells (see Fig. 4d). Note the filament in panels c and g, which belongs to a fungus from the genus Acidomyces. (i) Cell biomass as a function of time in two parallel subcultures of Candidatus M. methanotrophicum grown on CH4 as the sole carbon and energy source. One culture used 13C-labelled CH4 (13C atom fraction of 0.5), the other one used unlabelled CH4. Lines show the modelled biomass assuming an exponential growth with a doubling time of 94 days (solid line) and 98 days (dashed line) and a lag phase of 21 days. The decrease in the doubling time from 150–200 in the original culture to 94–98 days in these subcultures was possibly due to small changes in the culturing conditions combined with improved growth properties of Candidatus M. methanotrophicum. (j)13C atom fractions in the cells of Candidatus M. methanotrophicum grown on 13C-labelled CH4. Symbol shows the mean value, error bar corresponds to the standard deviation (calculated based on the measurement of N = 110 cells, where the cells were not treated by any post-incubation chemical procedure prior to the nanoSIMS analysis). Solid line shows the 13C atom fraction modelled based on the assumption that the 13C-labelled CH4 was the sole carbon and energy source and the growth characteristics were as shown in panel i.

Supplementary information

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Supplemental materials, Tables 1–5, Figs. 1–12 and references.

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van Spanning, R.J.M., Guan, Q., Melkonian, C. et al. Methanotrophy by a Mycobacterium species that dominates a cave microbial ecosystem. Nat Microbiol 7, 2089–2100 (2022).

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