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Author(s): Rafael Laso-Prez [1, 2]; Gunter Wegener (corresponding author) [1, 2, 3]; Katrin Knittel [1]; Friedrich Widdel [1]; Katie J. Harding [1]; Viola Krukenberg [1, 2]; Dimitri V. Meier [1]; [...], The anaerobic formation and oxidation of methane involve unique enzymatic mechanisms and cofactors, all of which are believed to be specific for C[sub.1]-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 C[sub.4] 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 -oxidation enzymes, carbon monoxide dehydrogenase and reversible C[sub.1] 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.

As a cause of genomic innovations and a catalyst of diversification, close interactions between eukaryotes and prokaryotes are driving forces of evolution (7). The importance of eukaryote-prokaryote interactions is clearly [...], Breviatea form a lineage of free living, unicellular protists, distantly related to animals and fungi (1,2). This lineage emerged almost one billion years ago, when the oceanic oxygen content was low, and extant Breviatea have evolved or retained an anaerobic lifestyle (3,4). Here we report the cultivation of Lenisia limosa, gen. et sp. nov., a newly discovered breviate colonized by relatives of animal-associated Arcobacter. Physiological experiments show that the association of L. limosa with Arcobacter is driven by the transfer of hydrogen and is mutualistic, providing benefits to both partners. With whole-genome sequencing and differential proteomics, we show that an experimentally observed fitness gain of L. limosa could be explained by the activity of a so far unknown type of NAD(P)H-accepting hydrogenase, which is expressed in the presence, but not in the absence, of Arcobacter. Differential proteomics further reveal that the presence of Lenisia stimulates expression of known 'virulence' factors by Arcobacter. These proteins typically enable colonization of animal cells during infection (5), but may in the present case act for mutual benefit. Finally, re-investigation of two currently available transcriptomic data sets of other Breviatea (4) reveals the presence and activity of related hydrogen-consuming Arcobacter, indicating that mutualistic interaction between these two groups of microbes might be pervasive. Our results support the notion that molecular mechanisms involved in virulence can also support mutualism (6), as shown here for Arcobacter and Breviatea.

Author(s): Gunter Wegener [sup.1] [sup.2] , Viola Krukenberg [sup.1] , Dietmar Riedel [sup.3] , Halina E. Tegetmeyer [sup.4] [sup.5] , Antje Boetius [sup.1] [sup.2] [sup.4] Author Affiliations: (1) Max-Planck Institute [...], Marine anaerobic methanotrophic archaea and sulfate-reducing bacteria connect by pili-like nanowires, suggesting that direct interspecies exchange of electrons could be a fundamental mechanism in the anaerobic oxidation of methane. A novel mechanism for microbial cooperation Anaerobic oxidation of methane in marine sediments, of central importance for the global methane cycle, is a collaborative process performed by consortia of methane-oxidizing archaea and sulfate-reducing bacteria. The biochemical basis of this syntrophic relationship is not fully understood. It has been suggested that exchange of a diffusible metabolite between the cooperating microbes is essential, but two groups reporting in this issue of Nature challenge this idea. Victoria Orphan and colleagues examined the biosynthetic activity at the single-cell level in microbial consortia prepared from sediment sampled from an active methane seep at Hydrate Ridge North in the Northwest Pacific. They find that cell activities are independent of the distance between syntrophic partners, which is inconsistent with a model involving the diffusion of intermediates over short distances. Instead, direct electron transfer between archaea and bacteria, mediated by large multi-haem cytochromes produced by ANME-2 archaea, is a central mechanism of their interaction. Gunter Wegener et al. show that interspecies exchange of electrons in microbial samples derived from hydrothermal vent sediments from Guaymas Basin in the Gulf of California is most probably through direct transfer of electrons by means of 'nanowires' connecting the two partners. These authors propose that electron transfer is mediated by pili-like structures and outer-membrane multi-haem cytochromes. The anaerobic oxidation of methane (AOM) with sulfate controls the emission of the greenhouse gas methane from the ocean floor.sup.1,2. In marine sediments, AOM is performed by dual-species consortia of anaerobic methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB) inhabiting the methane-sulfate transition zone.sup.3,4,5. The biochemical pathways and biological adaptations enabling this globally relevant process are not fully understood. Here we study the syntrophic interaction in thermophilic AOM (TAOM) between ANME-1 archaea and their consortium partner SRB HotSeep-1 (ref. 6) at 60 °C to test the hypothesis of a direct interspecies exchange of electrons.sup.7,8. The activity of TAOM consortia was compared to the first ANME-free culture of an AOM partner bacterium that grows using hydrogen as the sole electron donor. The thermophilic ANME-1 do not produce sufficient hydrogen to sustain the observed growth of the HotSeep-1 partner. Enhancing the growth of the HotSeep-1 partner by hydrogen addition represses methane oxidation and the metabolic activity of ANME-1. Further supporting the hypothesis of direct electron transfer between the partners, we observe that under TAOM conditions, both ANME and the HotSeep-1 bacteria overexpress genes for extracellular cytochrome production and form cell-to-cell connections that resemble the nanowire structures responsible for interspecies electron transfer between syntrophic consortia of Geobacter.sup.9,10. HotSeep-1 highly expresses genes for pili production only during consortial growth using methane, and the nanowire-like structures are absent in HotSeep-1 cells isolated with hydrogen. These observations suggest that direct electron transfer is a principal mechanism in TAOM, which may also explain the enigmatic functioning and specificity of other methanotrophic ANME-SRB consortia.

The anaerobic oxidation of methane with sulfate (AOM) controls the emission of methane from the seabed (1,3,4). At environmental conditions the net reaction C[H.sub.4](aq) + S[O.sub.4.sup.2] → H[S.sup.-] + HC[o.sub.3.sup.-] [...], The anaerobic oxidation of methane (AOM) with sulfate controls the emission of the greenhouse gas methane from the ocean floor (1,2). In marine sediments, AOM is performed by dual-species consortia of anaerobic methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB) inhabiting the methane-sulfate transition zone (3-5). The biochemical pathways and biological adaptations enabling this globally relevant process are not fully understood. Here we study the syntrophic interaction in thermophilic AOM (TAOM) between ANME-1 archaea and their consortium partner SRB HotSeep-1 (ref. 6) at 60°C to test the hypothesis of a direct interspecies exchange of electrons (7,8). The activity of TAOM consortia was compared to the first ANME-free culture of an AOM partner bacterium that grows using hydrogen as the sole electron donor. The thermophilic ANME-1 do not produce sufficient hydrogen to sustain the observed growth of the HotSeep-1 partner. Enhancing the growth of the HotSeep-1 partner by hydrogen addition represses methane oxidation and the metabolic activity of ANME-1. Further supporting the hypothesis of direct electron transfer between the partners, we observe that under TAOM conditions, both ANME and the HotSeep-1 bacteria overexpress genes for extracellular cytochrome production and form cell-to-cell connections that resemble the nanowire structures responsible for interspecies electron transfer between syntrophic consortia of Geobacter (9,10). HotSeep-1 highly expresses genes for pili production only during consortial growth using methane, and the nanowire-like structures are absent in HotSeep-1 cells isolated with hydrogen. These observations suggest that direct electron transfer is a principal mechanism in TAOM, which may also explain the enigmatic functioning and specificity of other methanotrophic ANME-SRB consortia.