Thermophilic archaea activate butane via alkyl-coenzyme M formation


The anaerobic formation and oxidation of methane involve unique enzymatic mechanisms and cofactors, all of which are believed to be specific for C1-compounds. Here we show that an anaerobic thermophilic enrichment culture composed of dense consortia of archaea and bacteria apparently uses partly similar pathways to oxidize the C4 hydrocarbon butane. The archaea, proposed genus ‘Candidatus Syntrophoarchaeum’, show the characteristic autofluorescence of methanogens, and contain highly expressed genes encoding enzymes similar to methyl-coenzyme M reductase. We detect butyl-coenzyme M, indicating archaeal butane activation analogous to the first step in anaerobic methane oxidation. In addition, Ca. Syntrophoarchaeum expresses the genes encoding β-oxidation enzymes, carbon monoxide dehydrogenase and reversible C1 methanogenesis enzymes. This allows for the complete oxidation of butane. Reducing equivalents are seemingly channelled to HotSeep-1, a thermophilic sulfate-reducing partner bacterium known from the anaerobic oxidation of methane. Genes encoding 16S rRNA and methyl-coenzyme M reductase similar to those identifying Ca. Syntrophoarchaeum were repeatedly retrieved from marine subsurface sediments, suggesting that the presented activation mechanism is naturally widespread in the anaerobic oxidation of short-chain hydrocarbons.

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Figure 1: Characterization of the Butane50 culture.
Figure 2: Butyl-CoM as initial metabolic intermediate in butane oxidation
Figure 3: Phylogenetic affiliation of McrA amino acid sequences present in Ca. S. butanivorans and Ca. S. caldarius.
Figure 4: Metabolic scheme proposed for butane oxidation with sulfate based on molecular analyses.
Figure 5: Testing metabolic interaction of Ca. Syntrophoarchaeum and Ca. D. auxilii in Butane50 cultures.


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We thank R. Appel, K. Büttner, I. Kattelmann and S. Menger for assistance in cultivation and molecular work, B. Scheer for proteomic analyses, and M. Sickert, J. Then Bergh and K. Dürkop for support in standard synthesis and LC-MS/MS analyses. We thank as well A. Férnandez-Guerra, H. Gruber-Vodicka and P. Offre for supporting us with bioinformatics and biochemistry. We thank A. Boetius for discussions and financial support provided by her Leibniz Grant of the Deutsche Forschungsgemeinschaft (DFG). Research was further financed by the DFG Research Center and Cluster of Excellence MARUM and the Deep Carbon Observatory (Deep Life grant 11121/6152-2121-2329-9973-CC to G.W.), the Max Planck Society and the Helmholtz Society. We are indebted to A. Teske and the shipboard party, the crew and pilots of research expedition AT15-16 Research Vessel Atlantis and Research Submersible Alvin (NSF Grant OCE-0647633). We acknowledge the Centre for Chemical Microscopy (ProVIS) at the Helmholtz Centre for Environmental Research supported by European regional Development Funds (EFRE – Europe funds Saxony) for using their analytical facilities.

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G.W. and F.M. retrieved the original samples and performed cultivation. R.L.-P., G.W. and F.M. designed research. R.L.-P., K.K., K.J.H. and V.K. designed the CARD-FISH probes and performed microscopy. R.L.-P., G.W. and F.M. performed physiological experiments. H.E.T. prepared and sequenced the DNA and RNA libraries. R.L.-P., V.K., D.V.M. and M.R. performed metagenomic and transcriptomic analyses. R.L.-P., K.K. and K.J.H. performed phylogenetic analysis. D.R. performed thin-sectioning and electron microscopy. H.-H.R., L.A. and F.M. performed proteome analyses. T.R., O.J.L. and F.M. analysed metabolic intermediates. R.L.-P., G.W., F.W. and F.M. developed the metabolic model, and wrote the manuscript with contributions of all co-authors.

Corresponding authors

Correspondence to Gunter Wegener or Florin Musat.

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Nature thanks T. Ettema, M. Jetten, S. Ragsdale and R. Thauer for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 Genetic structure of mcr genes in Ca. Syntrophoarchaeum.

In Ca. S. butanivorans one mcr gene set is separated, with the mcrA subunit in scaffold 1, and mcrB and mcrG in scaffold 4.

Extended Data Figure 2 Experiments validating production of alkyl-CoM compounds in anaerobic cultures.

a, Screening for butyl-CoM in Butane50, in a thermophilic AOM culture supplied with butane (n = 2 with 2 different sampling time points), in BuS5 cultures (n = 3) and in controls. The mass peak of butyl-CoM (m/z = 197.0312) was only found in the Butane50 culture. b, Screening for methyl-CoM (m/z = 154.984; mass accuracy –0.15 p.p.m.) in the thermophilic AOM culture supplied with methane (n = 3) and in the Butane50 culture (n = 2). Methyl-CoM was only found in the thermophilic AOM culture. These analyses (a, b) indicate high specificity for either butane or methane in the corresponding cultures. c, Screening for propyl-CoM (m/z = 183.016; mass accuracy –0.21 p.p.m.) in the propane-degrading culture (n = 1) showing activation of this substrate with CoM.

Extended Data Figure 3 Effect of bromoethanesulfonate (BES, 5 mM final concentration) on Butane50 and Ca. D. auxilii cultures.

a, Upon addition of BES, butane-dependent sulfate reduction in Butane50 cultures (circles, n = 2) was immediately inhibited compared to a control without BES (triangles, n = 1). b, By contrast, BES addition had no influence on hydrogen-dependent sulfate reduction in Ca. D. auxilii cultures (triangles, n = 2).

Extended Data Table 1 Microbial diversity in the AOM enrichment21 used as inoculum and in the Butane50 culture
Extended Data Table 2 Draft genome information and pairwise comparison of whole genome identity of Ca. S. butanivorans and the Ca. S. caldarius
Extended Data Table 3 Genes encoding enzymes for butane activation, candidates for further conversion reactions and butyryl-CoA oxidation in Ca. S. butanivorans
Extended Data Table 4 Genes encoding enzymes of C-1 pathway in Ca. S. butanivorans
Extended Data Table 5 Genes encoding proteins related to electron cycling and energy transfer in Ca. S. butanivorans
Extended Data Table 6 BLASTP search of proteins involved in butyrate oxidation. Best results according to the E-value are shown
Extended Data Table 7 Genes encoding Type IV pili and 10 most expressed cytochromes identified in the genome bin of HotSeep-1 in the Butane50 culture

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Laso-Pérez, R., Wegener, G., Knittel, K. et al. Thermophilic archaea activate butane via alkyl-coenzyme M formation. Nature 539, 396–401 (2016).

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