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Discovery of extremely halophilic, methyl-reducing euryarchaea provides insights into the evolutionary origin of methanogenesis

Abstract

Methanogenic archaea are major players in the global carbon cycle and in the biotechnology of anaerobic digestion. The phylum Euryarchaeota includes diverse groups of methanogens that are interspersed with non-methanogenic lineages. So far, methanogens inhabiting hypersaline environments have been identified only within the order Methanosarcinales. We report the discovery of a deep phylogenetic lineage of extremophilic methanogens in hypersaline lakes and present analysis of two nearly complete genomes from this group. Within the phylum Euryarchaeota, these isolates form a separate, class-level lineage ‘Methanonatronarchaeia’ that is most closely related to the class Halobacteria. Similar to the Halobacteria, ‘Methanonatronarchaeia’ are extremely halophilic and do not accumulate organic osmoprotectants. The high intracellular concentration of potassium implies that ‘Methanonatronarchaeia’ employ the ‘salt-in’ osmoprotection strategy. These methanogens are heterotrophic methyl-reducers that use C1-methylated compounds as electron acceptors and formate or hydrogen as electron donors. The genomes contain an incomplete and apparently inactivated set of genes encoding the upper branch of methyl group oxidation to CO2 as well as membrane-bound heterodisulfide reductase and cytochromes. These features differentiate ‘Methanonatronarchaeia’ from all known methyl-reducing methanogens. The discovery of extremely halophilic, methyl-reducing methanogens related to haloarchaea provides insights into the origin of methanogenesis and shows that the strategies employed by methanogens to thrive in salt-saturating conditions are not limited to the classical methylotrophic pathway.

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Figure 1: Cell morphology of methyl-reducing methanogens from hypersaline soda and salt lakes.
Figure 2: Growth and activity of methyl-reducing methanogens from hypersaline soda lakes.
Figure 3: Effect of hydrotroilite (FeS × nH2O) on growth and methanogenic activity of washed and exhausted cells of the AMET1 strain.
Figure 4: Phylogenetic analysis of ‘Methanonatronarchaeia’ (AMET1 and HMET1).
Figure 5: Comparative genomic analysis and reconstruction of gene losses and gains.
Figure 6: Reconstruction of the central metabolic pathways shared by ‘Methanonatronarchaeia’.

References

  1. Ferry, J. G. & Kastead, K. A. in Archaea: Molecular and Cellular Biology (ed. Cavicchioli, R. ) 288–214 (ASM, 2007).

    Book  Google Scholar 

  2. Conrad, R. The global methane cycle: recent advances in understanding the microbial processes involved. Environ. Microbiol. Rep. 1, 285–292 (2009).

    CAS  Article  PubMed  Google Scholar 

  3. Inventory of US Greenhouse Gas Emissions and Sinks: 1990–2014. (US EPA, 2016).

  4. Garrity, G. M. & Holt, J. G. in Bergey's Manual of Systematics of Archaea and Bacteria Vol. 1 http://doi.org/10.1002/9781118960608.pbm00014 (Wiley, 2015).

    Google Scholar 

  5. Iino, T. et al. Candidatus Methanogranum caenicola: a novel methanogen from the anaerobic digested sludge, and proposal of Methanomassiliicoccaceae fam. nov. and Methanomassiliicoccales ord. nov., for a methanogenic lineage of the class Thermoplasmata. Microbes Environ. 28, 244–250 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  6. Borrel, G. et al. Comparative genomics highlights the unique biology of Methanomassiliicoccales, a Thermoplasmatales-related seventh order of methanogenic archaea that encodes pyrrolysine. BMC Genomics 15, 679 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  7. Lang, K. et al. New mode of energy metabolism in the seventh order of methanogens as revealed by comparative genome analysis of ‘Candidatus Methanoplasma termitum’. Appl. Environ. Microbiol. 81, 1338–1352 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  8. Evans, P. N. et al. Methane metabolism in the archaeal phylum Bathyarchaeota revealed by genome-centric metagenomics. Science 350, 434–438 (2015).

    CAS  Article  PubMed  Google Scholar 

  9. Vanwonterghem, I. et al. Methylotrophic methanogenesis discovered in the archaeal phylum Verstraetearchaeota. Nat. Microbiol. 1, 16170 (2016).

    CAS  Article  PubMed  Google Scholar 

  10. Hedderich, R. & Whitman, W. B. in The Prokaryotes—Prokaryotic Physiology and Biochemistry (ed. Rosenberg, E. ) 636–663 (Springer, 2013).

    Google Scholar 

  11. Liu, Y. & Whitman, W. B. Metabolic, phylogenetic, and ecological diversity of the methanogenic archaea. Ann. NY Acad. Sci. 1125, 171–189 (2008).

    CAS  Article  PubMed  Google Scholar 

  12. Thauer, R. K., Kaster, A. K., Seedorf, H., Buckel, W. & Hedderich, R. Methanogenic archaea: ecologically relevant differences in energy conservation. Nat. Rev. Microbiol. 6, 579–591 (2008).

    CAS  Article  PubMed  Google Scholar 

  13. Borrel, G. et al. Phylogenomic data support a seventh order of methylotrophic methanogens and provide insights into the evolution of methanogenesis. Genome Biol. Evol. 5, 1769–1780 (2013).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  14. Dridi, B., Fardeau, M. L., Ollivier, B., Raoult, D. & Drancourt, M. Methanomassiliicoccus luminyensis gen. nov., sp. nov., a methanogenic archaeon isolated from human faeces. Int. J. Syst. Evol. Microbiol. 62, 1902–1907 (2012).

    CAS  Article  PubMed  Google Scholar 

  15. Paul, K., Nonoh, J. O., Mikulski, L. & Brune, A. ‘Methanoplasmatales,’ Thermoplasmatales-related archaea in termite guts and other environments, are the seventh order of methanogens. Appl. Environ. Microbiol. 78, 8245–8253 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  16. Fricke, W. F. et al. The genome sequence of Methanosphaera stadtmanae reveals why this human intestinal archaeon is restricted to methanol and H2 for methane formation and ATP synthesis. J. Bacteriol. 188, 642–658 (2006).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  17. Miller, T. L. & Wolin, M. J. Methanosphaera stadtmaniae gen. nov., sp. nov.: a species that forms methane by reducing methanol with hydrogen. Arch. Microbiol. 141, 116–122 (1985).

    CAS  Article  PubMed  Google Scholar 

  18. Sprenger, W. W., Hackstein, J. H. & Keltjens, J. T. The energy metabolism of Methanomicrococcus blatticola: physiological and biochemical aspects. Antonie van Leeuwenhoek 87, 289–299 (2005).

    CAS  Article  PubMed  Google Scholar 

  19. Sprenger, W. W., Hackstein, J. H. & Keltjens, J. T. The competitive success of Methanomicrococcus blatticola, a dominant methylotrophic methanogen in the cockroach hindgut, is supported by high substrate affinities and favorable thermodynamics. FEMS Microbiol. Ecol. 60, 266–275 (2007).

    CAS  Article  PubMed  Google Scholar 

  20. Sprenger, W. W., van Belzen, M. C., Rosenberg, J., Hackstein, J. H. & Keltjens, J. T. Methanomicrococcus blatticola gen. nov., sp. nov., a methanol- and methylamine-reducing methanogen from the hindgut of the cockroach Periplaneta americana. Int. J. Syst. Evol. Microbiol. 50, 1989–1999 (2000).

    CAS  Article  PubMed  Google Scholar 

  21. Nobu, M. K., Narihiro, T., Kuroda, K., Mei, R. & Liu, W. T. Chasing the elusive Euryarchaeota class WSA2: genomes reveal a uniquely fastidious methyl-reducing methanogen. ISME J. 10, 2478–2487 (2016).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  22. Borrel, G., Adam, P. S. & Gribaldo, S. Methanogenesis and the Wood–Ljungdahl pathway: an ancient, versatile, and fragile association. Genome Biol. Evol. 8, 1706–1711 (2016).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  23. McGenity, T. J. in Handbook of Hydrocarbon and Lipid Microbiology (ed. Timmis, K. N. ) 665–679 (Springer, 2010).

    Book  Google Scholar 

  24. Kelley, C. A., Poole, J. A., Tazaz, A. M., Chanton, J. P. & Bebout, B. M. Substrate limitation for methanogenesis in hypersaline environments. Astrobiology 12, 89–97 (2012).

    CAS  Article  PubMed  Google Scholar 

  25. Oremland, R. S. & King, G. M. in Microbial Mats. Physiological Ecology of Benthic Microbial Communities (eds Cohen, Y. & Rosenberg, E. ) 180–190 (American Society for Microbiology, 1989).

    Google Scholar 

  26. Martin, D. D., Ciulla, R. A. & Roberts, M. F. Osmoadaptation in archaea. Appl. Environ. Microbiol. 65, 1815–1825 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Menaia, J. A. G. F. Osmotics of Halophilic Methanogenic Archaeobacteria. PhD thesis, Oregon Health Sci Univ. (1992).

  28. Sorokin, D. Y . et al. Methanogenesis at extremely haloalkaline conditions in the soda lakes of Kulunda Steppe (Altai, Russia). FEMS Microbiol. Ecol. 91, pii: fiv016 (2015).

    Article  PubMed  Google Scholar 

  29. Ginzburg, M., Sachs, L. & Ginzburg, B. Z. Ion metabolism in a Halobacterium. I. Influence of age of culture on intracellular concentrations. J. Gen. Physiol. 55, 187–207 (1970).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  30. Elevi Bardavid, R. & Oren, A. The amino acid composition of proteins from anaerobic halophilic bacteria of the order Halanaerobiales. Extremophiles 16, 567–572 (2012).

    CAS  Article  PubMed  Google Scholar 

  31. Oren, A. Life at high salt concentrations, intracellular KCl concentrations, and acidic proteomes. Front. Microbiol. 4, 315 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  32. Sorokin, D. Y., Banciu, H. L. & Muyzer, G. Functional microbiology of soda lakes. Curr. Opin. Microbiol. 25, 88–96 (2015).

    CAS  Article  PubMed  Google Scholar 

  33. Abken, H. J. et al. Isolation and characterization of methanophenazine and function of phenazines in membrane-bound electron transport of Methanosarcina mazei Go1. J. Bacteriol. 180, 2027–2032 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Makarova, K. S., Wolf, Y. I. & Koonin, E. V. Archaeal clusters of orthologous genes (arCOGs): an update and application for analysis of shared features between Thermococcales, Methanococcales, and Methanobacteriales. Life (Basel) 5, 818–840 (2015).

    CAS  Google Scholar 

  35. Yutin, N., Puigbo, P., Koonin, E. V. & Wolf, Y. I. Phylogenomics of prokaryotic ribosomal proteins. PLoS ONE 7, e36972 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  36. Eder, W., Schmidt, M., Koch, M., Garbe-Schonberg, D. & Huber, R. Prokaryotic phylogenetic diversity and corresponding geochemical data of the brine–seawater interface of the Shaban Deep, Red Sea. Environ. Microbiol. 4, 758–763 (2002).

    CAS  Article  PubMed  Google Scholar 

  37. Jiang, H. et al. Microbial response to salinity change in Lake Chaka, a hypersaline lake on Tibetan plateau. Environ. Microbiol. 9, 2603–2621 (2007).

    CAS  Article  PubMed  Google Scholar 

  38. Yarza, P. et al. Uniting the classification of cultured and uncultured bacteria and archaea using 16S rRNA gene sequences. Nat. Rev. Microbiol. 12, 635–645 (2014).

    CAS  Article  PubMed  Google Scholar 

  39. Wolf, Y. I., Makarova, K. S., Yutin, N. & Koonin, E. V. Updated clusters of orthologous genes for Archaea: a complex ancestor of the Archaea and the byways of horizontal gene transfer. Biol. Direct 7, 46 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  40. Makarova, K. S., Koonin, E. V. & Albers, S. V. Diversity and evolution of type IV pili systems in archaea. Front. Microbiol. 7, 667 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  41. Zheng, K., Ngo, P. D., Owens, V. L., Yang, X. P. & Mansoorabadi, S. O. The biosynthetic pathway of coenzyme F430 in methanogenic and methanotrophic archaea. Science 354, 339–342 (2016).

    CAS  Article  PubMed  Google Scholar 

  42. Aono, R. et al. Enzymatic characterization of AMP phosphorylase and ribose-1,5-bisphosphate isomerase functioning in an archaeal AMP metabolic pathway. J. Bacteriol. 194, 6847–6855 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  43. Baines, A. J. Evolution of spectrin function in cytoskeletal and membrane networks. Biochem. Soc. Trans. 37, 796–803 (2009).

    CAS  Article  PubMed  Google Scholar 

  44. Hallam, S. J., Girguis, P. R., Preston, C. M., Richardson, P. M. & DeLong, E. F. Identification of methyl coenzyme M reductase A (mcrA) genes associated with methane-oxidizing archaea. Appl. Environ. Microbiol. 69, 5483–5491 (2003).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  45. Sorokin, D. Y. et al. Methanosalsum natronophilum sp. nov., and Methanocalculus alkaliphilus sp. nov., haloalkaliphilic methanogens from hypersaline soda lakes. Int. J. Syst. Evol. Microbiol. 65, 3739–3745 (2015).

    CAS  Article  PubMed  Google Scholar 

  46. Pfennig, N. & Lippert, K. D. Über das vitamin B12-Bedürfnis phototropher schwefelbakterien. Arch. Mikrobiol. 55, 245–256 (1966).

    CAS  Article  Google Scholar 

  47. Plugge, C. M. Anoxic media design, preparation, and considerations. Meth. Enzymol. 397, 3–16 (2005).

    CAS  Article  Google Scholar 

  48. Podar, M. et al. Insights into archaeal evolution and symbiosis from the genomes of a nanoarchaeon and its inferred crenarchaeal host from obsidian pool, Yellowstone national park. Biol. Direct 8, 9 (2013).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  49. Seemann, T. Prokka: rapid prokaryotic genome annotation. Bioinformatics 30, 2068–2069 (2014).

    CAS  Article  PubMed  Google Scholar 

  50. Besemer, J., Lomsadze, A. & Borodovsky, M. GeneMarkS: a self-training method for prediction of gene starts in microbial genomes. Implications for finding sequence motifs in regulatory regions. Nucleic Acids Res. 29, 2607–2618 (2001).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  51. Altschul, S. F. et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402 (1997).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  52. Soding, J., Biegert, A. & Lupas, A. N. The HHpred interactive server for protein homology detection and structure prediction. Nucleic Acids Res. 33, W244–W248 (2005).

    Article  PubMed  PubMed Central  Google Scholar 

  53. Edgar, R. C. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797 (2004).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  54. Price, M. N., Dehal, P. S. & Arkin, A. P. Fasttree 2—approximately maximum-likelihood trees for large alignments. PLoS ONE 5, e9490 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  55. Guindon, S. et al. New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst. Biol. 59, 307–321 (2010).

    CAS  Article  PubMed  Google Scholar 

  56. Darriba, D., Taboada, G. L., Doallo, R. & Posada, D. Prottest 3: fast selection of best-fit models of protein evolution. Bioinformatics 27, 1164–1165 (2011).

    CAS  Article  PubMed  Google Scholar 

  57. Bjellqvist, B. et al. The focusing positions of polypeptides in immobilized pH gradients can be predicted from their amino acid sequences. Electrophoresis 14, 1023–1031 (1993).

    CAS  Article  PubMed  Google Scholar 

  58. Rice, P., Longden, I. & Bleasby, A. EMBOSS: the European Molecular Biology Open Software Suite. Trends Genet. 16, 276–277 (2000).

    CAS  Article  PubMed  Google Scholar 

  59. Parzen, E. On estimation of a probability density function and mode. Ann. Math. Statist. 33, 1065–1076 (1962).

    Article  Google Scholar 

  60. Kullback, S. & Leibler, R. A. On information and sufficiency. Ann. Math. Stat. 22, 79–86 (1951).

    Article  Google Scholar 

  61. Gower, J. C. Some distance properties of latent root and vector methods used in multivariate analysis. Biometrika 53, 325–338 (1966).

    Article  Google Scholar 

  62. Torgeson, W. S. Theory and Methods of Scaling (Wiley, 1958).

    Google Scholar 

  63. R (R Foundation for Statistical Computing, 2013).

  64. Gupta, N. & Pevzner, P. A. False discovery rates of protein identifications: a strike against the two-peptide rule. J. Proteome Res. 8, 4173–4181 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

D.Y.S. was supported by STW (project no. 12226), the Gravitation-SIAM Program (grant no. 24002002 from the Dutch Ministry of Education and Science) and by RFBR (grant no. 16-04-00035). K.S.M., Y.I.W. and E.V.K. are supported by the intramural programme of the US Department of Health and Human Services (to the National Library of Medicine). The proteomic analysis was performed in the Proteomics Facility of The Spanish National Center for Biotechnology (CNB-CSIC), which belongs to ProteoRed (PRB2-ISCIII), supported by grant no. PT13/0001. This project received funding from the European Union's Horizon 2020 research and innovation programme (Blue Growth: Unlocking the potential of Seas and Oceans) under grant agreement no. 634486. This work was further funded by grant no. BIO2014-54494-R from the Spanish Ministry of Economy, Industry and Competitiveness.

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Contributions

D.Y.S. performed the fieldwork, the sediment activity incubations, enrichment and isolation of pure cultures and microbiological investigation of enriched and pure cultures. B.A. and A.Y.M. analysed the mcrA and 16S rRNA genes in sediments and methanogenic cultures. M.F., P.N.G., S.C. and M.C.M.v.L. were responsible for the proteomic analysis. E.A.G. analysed compatible solutes. K.S.M., Y.I.W. and E.V.K. performed genomic analysis and evolutionary reconstructions. D.Y.S., K.S.M. and E.V.K. wrote the paper. M.C.M.v.L. oversaw the project and participated in the data interpretation and discussion.

Corresponding authors

Correspondence to Dimitry Y. Sorokin or Eugene V. Koonin.

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The authors declare no competing financial interests.

Supplementary information

Supplementary information

Supplementary Figures 1–10; Supplementary Tables 1 and 2; Supplementary Data 1–4. (PDF 17605 kb)

Supplementary Table 3

Comparative genomic analysis based on arCOG assignments. (XLSX 6009 kb)

Supplementary Table 4

Reconstruction of gene gain and loss. (XLSX 434 kb)

Supplementary Table 5

Isoelectric point calculation data. (XLSX 25 kb)

Supplementary Table 6

Proteomic analysis for AMET1. (XLSX 121 kb)

Supplementary Table 7

Proteomic analysis for HMET1. (XLSX 179 kb)

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Sorokin, D., Makarova, K., Abbas, B. et al. Discovery of extremely halophilic, methyl-reducing euryarchaea provides insights into the evolutionary origin of methanogenesis. Nat Microbiol 2, 17081 (2017). https://doi.org/10.1038/nmicrobiol.2017.81

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