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Methanotrophic Methanoperedens archaea host diverse and interacting extrachromosomal elements

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Abstract

Methane emissions are mitigated by anaerobic methane-oxidizing archaea, including Methanoperedens. Some Methanoperedens host huge extrachromosomal genetic elements (ECEs) called Borgs that may modulate their activity, yet the broader diversity of Methanoperedens ECEs is understudied. Here we report small enigmatic linear ECEs, circular viruses and unclassified ECEs that are predicted to replicate within Methanoperedens. Linear ECEs have inverted terminal repeats, tandem repeats and coding patterns that are strongly reminiscent of Borgs, but they are only 52–145 kb in length. As they share proteins with Borgs and Methanoperedens, we refer to them as mini-Borgs. Mini-Borgs are genetically diverse and can be assigned to at least five family-level groups. We identify eight families of Methanoperedens viruses, some of which encode multi-haem cytochromes, and circular ECEs encoding transposon-associated TnpB genes with proximal population-heterogeneous CRISPR arrays. These ECEs exchange genetic information with each other and with Methanoperedens, probably impacting their archaeal host activity and evolution.

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Fig. 1: Comparison of genomic features of Methanoperedens and associated ECEs.
Fig. 2: Evidence linking mini-Borgs to Methanoperedens.
Fig. 3: Genomic comparison and phylogeny of Methanoperedens viruses.
Fig. 4: TnpB and associated CRISPR array in an unclassified circular ECE.
Fig. 5: Evidence for lateral gene transfer among Methanoperedens and associated ECEs based on gene phylogenies.

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Data availability

Metagenomic sequencing reads related to Methanoperedens ECEs are available under NCBI BioProject PRJNA999944. The genomes described in this study can be accessed at https://ggkbase.berkeley.edu/project_groups/methanoperedens_ece. Please note that it is necessary to sign up as a user (simply provide an email address) to download the data. We also submit genomes to figshare (https://doi.org/10.6084/m9.figshare.24481222)90. Reference protein sequences used in this study can be found in NCBI (https://www.ncbi.nlm.nih.gov/), KEGG (https://www.genome.jp/kegg/) and UniProt databases (https://www.uniprot.org/). Protein structures (for example, 6R10) can be accessed at RCSB PDB (https://www.rcsb.org/). Source data are provided with this paper.

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Acknowledgements

We thank J. A. Doudna and B. Adler for helpful discussion of the TnpB-associated CRISPR complex. We also thank Adam Abate’s lab (University of California, San Francisco) for their efforts to obtain cell concentrates from soil samples, and the Oxford Nanopore research team (New York) for the efforts to perform Hi-C sequencing. Funding for this research was provided by the Bill and Melinda Gates Foundation (grant number INV-037174 to J.F.B.). The findings and conclusions are those of the authors and do not necessarily reflect positions or policies of the Bill and Melinda Gates Foundation. Funding was also provided via the Innovative Genomics Institute Climate fund philanthropic donation to J.F.B., a DFG postdoctoral fellowship to M.C.S. (project number 447383558 to M.C.S.) and The Ministry of Economy, Trade and Industry of Japan as ‘The project for validating near-field system assessment methodology in geological disposal system’ (2022 FY, grant number JPJ007597).

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Author notes

  1. Marie C. Schoelmerich

    Present address: Department of Environmental Systems Sciences, ETH Zurich, Zurich, Switzerland

Authors and Affiliations

  1. Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA

    Ling-Dong Shi, Marie C. Schoelmerich, Petar I. Penev, LinXing Chen, Rohan Sachdeva & Jillian F. Banfield

  2. Environmental Science, Policy and Management, University of California, Berkeley, Berkeley, CA, USA

    Jacob West-Roberts & Jillian F. Banfield

  3. Sector of Decommissioning and Radioactive Wastes Management, Japan Atomic Energy Agency, Ibaraki, Japan

    Yuki Amano

  4. Earth and Planetary Science, University of California, Berkeley, Berkeley, CA, USA

    Shufei Lei & Jillian F. Banfield

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Contributions

The study was designed by L.-D.S. and J.F.B. Binning and genome curation and analysis were performed by L.-D.S. and J.F.B. Proteome and phylogenetic analyses were carried out by L.-D.S. Correlation analyses were performed by L.-D.S. J.W.-R. provided the Corona Mine sequences and computational support. M.C.S. contributed to proteome analyses, and L.-D.S. and P.I.P. carried out the protein structural analyses. L.C. contributed to TnpB and CRISPR array analyses. Y.A. provided the Horonobe metagenomic sequences, and S.L. and R.S. contributed to the data handling and bioinformatic analyses. L.-D.S. and J.F.B. wrote the paper with input from all the authors.

Corresponding author

Correspondence to Jillian F. Banfield.

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Competing interests

J.F.B. is a co-founder of Metagenomi. The other authors declare no competing interests.

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Nature Microbiology thanks Marc Strous and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Genomic architectures and predicted replichores of representative complete mini-Borgs.

Yellow blocks indicate predicted genes while red and blue lines indicate predicted replication origin and terminus, respectively. Gray dots and green lines show GC skew and cumulative GC skew across genomes.

Extended Data Fig. 2 Coverage profile of representative complete and near-complete mini-Borgs.

Yellow blocks indicate predicted genes. Red arrows in the Venus_106kb_34_23_complete denote inverted terminal repeats (ITR). Mapped reads are depicted below the genomes.

Extended Data Fig. 3 Examples of tandem repeats in the Jupiter_54kb_36_306_complete genome.

Yellow blocks indicate predicted genes; red and blue blocks denote, respectively, left inverted terminal repeat and perfect tandem repeats distributed in various regions.

Extended Data Fig. 4 Structural comparison of ATPase subunit K.

a. Representative proteins from mini-Borgs, Methanoperedens, and a reference Thermus thermophilus (PDB: 6R10) were superimposed and highlighted in red, blue, and cyan, respectively. b, c. Two copies of the T. thermophilus ATPase subunit K ring were substituted by that from mini-Borg and Methanoperedens, as shown in the surface form. The surface was colored based on calculated electrostatic potential, where kb, T, e in the unit indicate the Boltzmann’s constant, temperature in Kelvin, and the charge of an electron.

Source data

Extended Data Fig. 5 Phylogeny of Borgs and mini-Borgs inferred from a concatenation of two marker proteins.

Blastp of markers against the NCBI database recruited no significant hits, therefore no other sequences are included in the tree. Support values were calculated based on 1000 replicates.

Source data

Extended Data Fig. 6 Genomic comparison of Methanoperedens-associated ECEs to other archaeal viruses and plasmids.

References were downloaded from the NCBI genome database accessed on October 30, 2023. Colored blocks indicate dissimilarity between pairs of genomes, in which the redder color is, the more similar genomes are. The original vector figure can be found in figshare under the DOI of 10.6084/m9.figshare.24481222.

Source data

Extended Data Fig. 7 An example of recombination in the Jupiter_54kb_36_306_complete genome.

The obvious dip in the coverage profile (blue area in the upper panel) suggests a region for which the more divergent reads were not recruited. Sequence blocks surrounded by colored rectangles indicate alleles that are linked in a variety of configurations, consistent with recombination involving mini-Borg variants.

Extended Data Fig. 8 Modeled structure and genomic context of mini-Borg capsid-like proteins.

a. The reference is the icosahedral asymmetric unit of the major capsid from Haloarcula californiae icosahedral virus (PDB: 6H9C). Detailed superimposition is shown for the predicted structure of a representative mini-Borg capsid-like protein from Jupiter_54kb_36_306_complete and the cryo-EM structure of a capsid VP7 protein. b. The genes encoding capsid-like proteins are highlighted and identified in all 12 complete or near-complete mini-Borgs. Coordinates listed next to the names indicate the positions within aligned regions. Typically, these regions are located in the middle of corresponding genomes (note the genome length in mini-Borg names). Genes belonging to the same group are connected if pairwise amino acid identity is > 30%, as shown by shading.

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Extended Data Fig. 9 Phylogeny of TnpB transposase shared by Methanoperedens and associated ECEs.

Homologous references were recruited from the NCBI nr database. Arc colors indicate different taxonomic clades. Proteins found in Methanoperedens and associated ECEs are marked using colored dots. The tree was mid-point rerooted and support values were calculated based on 1000 replicates (only show those at the boundary of different taxonomic branches).

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Supplementary information

Supplementary Information

Supplementary Figs. 1–23.

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Supplementary Tables 1–6.

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Original protein sequences.

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Original protein sequences.

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Original protein sequences.

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Original protein sequences.

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Original protein sequences.

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Original protein sequences.

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Shi, LD., West-Roberts, J., Schoelmerich, M.C. et al. Methanotrophic Methanoperedens archaea host diverse and interacting extrachromosomal elements. Nat Microbiol 9, 2422–2433 (2024). https://doi.org/10.1038/s41564-024-01740-8

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