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Cultivation and visualization of a methanogen of the phylum Thermoproteota

Nature volume 632, pages 1118–1123 (2024)Cite this article

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Abstract

Methane is the second most abundant climate-active gas, and understanding its sources and sinks is an important endeavour in microbiology, biogeochemistry, and climate sciences1,2. For decades, it was thought that methanogenesis, the ability to conserve energy coupled to methane production, was taxonomically restricted to a metabolically specialized group of archaea, the Euryarchaeota1. The discovery of marker genes for anaerobic alkane cycling in metagenome-assembled genomes obtained from diverse habitats has led to the hypothesis that archaeal lineages outside the Euryarchaeota are also involved in methanogenesis3,4,5,6. Here we cultured Candidatus Methanosuratincola verstraetei strain LCB70, a member of the archaeal class Methanomethylicia (formerly Verstraetearchaeota) within the phylum Thermoproteota, from a terrestrial hot spring. Growth experiments combined with activity assays, stable isotope tracing, and genomic and transcriptomic analyses demonstrated that this thermophilic archaeon grows by means of methyl-reducing hydrogenotrophic methanogenesis. Cryo-electron tomography revealed that Ca. M. verstraetei are coccoid cells with archaella and chemoreceptor arrays, and that they can form intercellular bridges connecting two to three cells with continuous cytoplasm and S-layer. The wide environmental distribution of Ca. M. verstraetei suggests that they might play important and hitherto overlooked roles in carbon cycling within diverse anoxic habitats.

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Fig. 1: Characterization of the methanogenic enrichment culture.
Fig. 2: Translational activity and conversion of stable isotope tracers.
Fig. 3: Proposed methane and hydrogen metabolism in Ca. M. verstraetei strain LCB70.
Fig. 4: Cryoelectron tomography of LCB70 cells.

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Data availability

The LCB70 enrichment culture metagenomic, metatranscriptomic, and amplicon data are deposited under NCBI BioProject ID PRJNA916083. The LCB070 hot spring metagenome is publicly available on the IMG database under the taxon ID 3300043544 and the strain LCB70 genome under taxon ID 8012933652. Cryo-tomograms are deposited at EMDB/EMPIAR under accession numbers EMD-50298 to EMD-50305, EMD-50308 to EMD-50313 and EMD-50315. Raman spectra are available in the MicrobioRaman database (https://www.ebi.ac.uk/biostudies/MicrobioRaman/studies) with accession number S-MBRS7.  Source data are provided with this paper.

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Acknowledgements

This study was funded through awards by the NASA Exobiology programme (80NSSC19K1633 to R.H.) and the Simons and the Gordon and Betty Moore Foundations (award 737750 to R.H.). M.P. was supported by the European Research Council (CoG 101000232) and the NOMIS foundation. A.J.K. was supported in part by the Thermal Biology Institute and MSU’s Vice President’s Office of Research, Economic Development and Graduate Education. V.K. was supported in part by a grant from the NSF (MCB-1817428 to R.H.). A portion of this research was performed under the Community Sciences Program (proposal https://doi.org/10.46936/10.25585/60008108) and used resources at the DOE Joint Genome Institute (https://ror.org/04xm1d337), which is a DOE Office of Science User Facility operated under contract no. DE-AC02-05CH11231. Raman imaging was made possible by The Center for Biofilm Engineering Imaging Facility at MSU, which is supported by funding from the NSF MRI program (2018562), the M. J. Murdock Charitable Trust (202016116), the US Department of Defense (77369LSRIP) and the Montana Nanotechnology Facility (an NNCI member supported by NSF grant ECCS-2025391). MSU’s Confocal Raman microscope was acquired with support by the National Science Foundation (DBI-1726561) and the M. J. Murdock Charitable Trust (SR-2017331). We thank the US National Park Service for permitting work in YNP under permit number YELL-SCI-8010 and ScopeM for instrument access at ETH Zürich. We thank G. Chadwick for discussions on archaeal cell biology and physiology; L. Baranek for assistance with culture maintenance; C. Gauvin, A. Charbonneau and A. Kokhanovska for help with cryo-ET; M. Lynes for discussions that informed field sampling efforts; and M. Palmer for input on taxonomy. We thank W. Verstraete for allowing us to name this newly cultured methanogen after him.

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Author notes

  1. These authors contributed equally: Nikolai Petrosian, Viola Krukenberg, Zackary J. Jay

Authors and Affiliations

  1. Department of Chemistry and Biochemistry, Center for Biofilm Engineering, and Thermal Biology Institute, Montana State University, Bozeman, MT, USA

    Anthony J. Kohtz, Viola Krukenberg, Zackary J. Jay & Roland Hatzenpichler

  2. Institute of Molecular Biology and Biophysics, ETH Zürich, Zürich, Switzerland

    Nikolai Petrosian & Martin Pilhofer

  3. Department of Microbiology and Cell Biology, Montana State University, Bozeman, MT, USA

    Roland Hatzenpichler

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  1. Anthony J. Kohtz
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Contributions

A.J.K. and R.H. developed the research project. A.J.K., V.K., N.P., M.P. and R.H. designed the experiments. A.J.K., V.K. and Z.J.J. conducted the field sampling. A.J.K. conducted cultivation, BONCAT, SIP, FISH, Raman and transcriptomic experiments, performed culture DNA and RNA extractions and phylogenetic analysis of hydrogenases. V.K. extracted DNA from LCB070 sediment, advised on cultivation and performed phylogenetic analysis of MCR sequences. Z.J.J. and A.J.K. analysed metagenomic data and performed phylogenetic analysis of genomes. A.J.K. performed the metabolic reconstruction with input from Z.J.J. and V.K. N.P. collected cryo-ET data. N.P. analysed cryo-ET data, with input from A.J.K., M.P. and R.H. R.H. and M.P. were responsible for funding and supervision of the project. A.J.K. and R.H. wrote the manuscript, which was then edited by all the authors.

Corresponding author

Correspondence to Roland Hatzenpichler.

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Extended data figures and tables

Extended Data Fig. 1 Image of hot spring LCB070 and diversity of Methanomethylicia MAGs recovered from the hot spring sediment metagenome.

A. Hot spring LCB070 in Yellowstone National Park. The arrow indicates the location where sediment samples were taken. B. Phylogenomic tree of Methanomethylicia, rooted with MAGs from the closely related lineage, Culexarchaeia31. MAGs recovered from the sediment metagenome are highlighted in green.

Extended Data Fig. 2 Pairwise comparison of 16S rRNA sequence identity and phylogeny and pairwise comparison of Methanomethylicia MAGs.

A. Pairwise percent sequence identity was calculated with BLASTn. B. Maximum likelihood phylogenetic tree of full length and partial 16S rRNA genes encoded in strain LCB70 and representative Methanomethylicia MAGs. Numbers at the nodes indicate bootstrap support, only values above 90 are shown. C. Pairwise average amino acid identity of MAGs (AAI, top) and average nucleotide identity (ANI, bottom) values (in %) were calculated by compareM and FastANI, respectively. Two MAGs from the closely related class Culexarchaeia31, were used as an outgroup. The two Verstraetearchaeota-affiliated MAGs reconstructed from the original hot spring (LCB070 bin14 and bin 23) have less than 75% ANI similarity to the genome of Ca. M. verstraetei strain LCB70.

Extended Data Fig. 3 Habitat distribution of Ca. M. verstraetei LCB70 and related species.

Sequences were identified by searching the NCBI SRA database using the IMNGS webserver with default settings. Graph is based on the presence of 16S rRNA gene sequences with >97% sequence identity to strain LCB70 across 664 public samples.

Extended Data Fig. 4 Growth of culture LCB70 on different carbon substrates and genomic context of putative lactate dehydrogenase and heterodisulfide reductase (HdrD) subunits.

A. Growth of culture LCB70 on 50 mM methanol (MeOH) and 50% hydrogen (H2) in the presence or absence of L-lactate (10 mM). Data symbols for methane are the mean (n = 3 biological replicates) ± s.d. Where not visible, the error bars are smaller than the symbol. B. Growth of culture LCB70 on 50 mM MeOH and 50% H2 with 10 mM of either lactate, pyruvate, or acetate. Data symbols for methane are the mean (n = 3 biological replicates) and error bars are one standard deviation. C. Transcriptional activity of LCB70 during growth on methanol and hydrogen with lactate, pyruvate, or acetate. Plotted are the mean ± s.d. (n = 3 biological replicates). Where not visible, the error bar is smaller than the symbol. Dashed lines represents the average TPM of all genes. D. Organization of putative lactate dehydrogenase genes, double slashes indicate the GlcD and LldG clusters are in different sections of the strain LCB70 genome. Sequences are indicated by arrows and distinct domains within the protein coding sequence are indicated by black bars. LUD, Lactate Utilization Domain.

Source Data

Extended Data Fig. 5 Diversity of Group 4 [NiFe] hydrogenases encoded by strain LCB70.

Maximum likelihood phylogenetic tree of group 4 [NiFe]-hydrogenases. The colors for the Ehb, Ehd, and Ehi clades match those in Fig. 3a. Circles at nodes indicate ultrafast bootstrap values higher than 95.

Extended Data Fig. 6 Genomic organization, expression, and occurrence of archaella and chemoreceptors within tomograms.

A. Organization of archaella (yellow) and chemotaxis gene clusters (blue). Hypothetical proteins (Hyp.) are shown in gray. MCP, Methyl-accepting chemotaxis proteins. PAS domain, Per-Arnt-Sim domain. B. Expression profile of archaella and chemotaxis genes for cultures grown on methanol and hydrogen and either lactate, pyruvate, or acetate as the carbon source (n = 3 biological replicates). Plotted are the mean values +/− SD. Where not visible, the error bar is smaller than the symbol. Dashed line represents the average TPM of all genes. C. Occurrence of archaella and chemoreceptor arrays in tomograms of strain LCB70. Note that due to differences in contrast between the tomograms, estimates of chemoreceptor arrays may be slightly underestimated.

Source Data

Extended Data Fig. 7 Cell biology of strain LCB70.

A, B. Two tomographic slices of the same archaellated LCB70 cell. C. A LCB70 cell with semi-ordered unidentified filamentous structures. A zoomed-in view is shown in the inset. D. A tomographic slice of a cell with putative storage granules. E,F. Examples of potentially dividing LCB70 cells. G. Variation in LCB70 cell diameters (n = 55 cells). The center line represents the mean cell diameter and whiskers represent +/− SD. For all tomographic slices the thickness is 18.04 nm. Scale bars, 200 nm; 25 nm for the inset. H. Raman spectra of the fingerprint region (400–2,000 cm−1) of 18 LCB70 cells. Black line shows the average and pink shaded area is one standard deviation. Gray shaded regions are marker regions for glycogen (478–484, and 840–860 cm−1)94. I. Transcriptional activity of genes involved in glycogen metabolism for cells grown on methanol and hydrogen with either lactate, pyruvate, or acetate as carbon sources (n = 3 biological replicates each). Data are presented as mean values +/− SD. Where not visible, the error bar is smaller than the symbol. Dashed lines indicate the average TPM for each treatment. For full gene names see Supplementary Table 4.

Source Data

Extended Data Fig. 8 Representative tomograms of LCB70 cells grown with different carbon sources.

A. Tomographic slices of LCB70 cells grown on 50 mM methanol and 50% hydrogen with 10 mM lactate; B. 10 mM lactate with BES treatment; C. 10 mM pyruvate; D. 10 mM acetate. Scale bar, 200 nm. The thickness of tomographic slices is 18.04 nm.

Extended Data Fig. 9 Examples of cell aggregates in culture LCB70.

A,B. Representative epifluorescence images of the LCB70 culture showing large aggregates. Cells are stained with the general nucleic acid stain DAPI (blue), and the Methanomethylicaceae-specific FISH probe Msur657 (green), and the general archaeal probe Arch915 (red). LCB70 cells bind to both the Msur657 and Arch915 probes, appearing orange. Scale bars, 5 µm. Shown are representative micrographs from 3 independent biological replicates.

Extended Data Fig. 10 Cell-cell connections between LCB70 cells.

A, B. Two examples of interactions between LCB70 cells. Cell-cell bridges without (panel A) or with S-layer (panel B) formed from the cytoplasmic membrane connecting two cells. C,D. Tomographic slice (C) and corresponding model (D) of a cell-cell bridge without a surrounding S-layer. Structures in black are interpreted to either represent virus-like particles or protrusions of the cell membrane. E. Barplot represents the variation in length (n = 4 tomograms), diameter (n = 4 tomograms), and S-layer diameter of the tube connection between cells (n = 3 tomograms), from which only one tube is fully covered by S-layer, two are covered only in area of the cell contact, and one is not covered at all in S-layer. The number in the bar is the mean and error bars represent the standard deviation. For all tomographic slices, the scale bar is 200 nm and the thickness is 18.04 nm. F. A zoomed out view of the model of a junction between the three cells shown in Fig. 4c. S-layer colored in yellow, cytoplasmic membrane in blue, putative ribosomes in red, and thin filaments in green. Scale bars, 200 nm.

Source Data

Supplementary information

Supplementary Information

Supplementary Results and Discussion, including the complete etymology of Methanosuratincola verstraetei strain LCB70 and supplemental discussion on lactate metabolism.

Reporting Summary

Supplementary Tables

This file contains Supplementary Tables 1–6.

Supplementary Video 1

The video shows tomographic slices through a Ca. M. verstraetei LCB70 cell followed by the corresponding 3D model representation (same dataset as shown in Fig. 4a,b). In the 3D model the colours refer to: S-layer (yellow), cytoplasmic membrane (blue), putative ribosomes (red), archaella (dark pink), putative virus-like particles (cyan), chemotaxis receptor arrays (pink), and unidentified filamentous structures (grey).

Supplementary Video 2

The video shows tomographic slices through a Ca. M. verstraetei LCB70 cell (same dataset as shown in Fig. 4c).

Source data

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Kohtz, A.J., Petrosian, N., Krukenberg, V. et al. Cultivation and visualization of a methanogen of the phylum Thermoproteota. Nature 632, 1118–1123 (2024). https://doi.org/10.1038/s41586-024-07631-6

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