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 ß-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.

Intercellular wiring enables electron transfer between methanotrophic archaea and bacteria.

The anaerobic oxidation of methane (AOM) with sulfate controls the emission of the greenhouse gas methane from the ocean floor. 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. 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. 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. 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.

Gene expression and ultrastructure of meso- and thermophilic methanotrophic consortia.

The sulfate-dependent, anaerobic oxidation of methane (AOM) is an important sink for methane in marine environments. It is carried out between anaerobic methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB) living in syntrophic partnership. In this study, we compared the genomes, gene expression patterns and ultrastructures of three phylogenetically different microbial consortia found in hydrocarbon-rich environments under different temperature regimes: ANME-1a/HotSeep-1 (60°C), ANME-1a/Seep-SRB2 (37°C) and ANME-2c/Seep-SRB2 (20°C). All three ANME encode a reverse methanogenesis pathway: ANME-2c encodes all enzymes, while ANME-1a lacks the gene for N5,N10-methylene tetrahydromethanopterin reductase (mer) and encodes a methylenetetrahydrofolate reductase (Met). The bacterial partners contain the genes encoding the canonical dissimilatory sulfate reduction pathway. During AOM, all three consortia types highly expressed genes encoding for the formation of flagella or type IV pili and/or c-type cytochromes, some predicted to be extracellular. ANME-2c expressed potentially extracellular cytochromes with up to 32 hemes, whereas ANME-1a and SRB expressed less complex cytochromes (≤ 8 and ≤ 12 heme respectively). The intercellular space of all consortia showed nanowire-like structures and heme-rich areas. These features are proposed to enable interspecies electron exchange, hence suggesting that direct electron transfer is a common mechanism to sulfate-dependent AOM, and that both partners synthesize molecules to enable it.

Candidatus Desulfofervidus auxilii, a hydrogenotrophic sulfate-reducing bacterium involved in the thermophilic anaerobic oxidation of methane.

The anaerobic oxidation of methane (AOM) is mediated by consortia of anaerobic methane-oxidizing archaea (ANME) and their specific partner bacteria. In thermophilic AOM consortia enriched from Guaymas Basin, members of the ANME-1 clade are associated with bacteria of the HotSeep-1 cluster, which likely perform direct electron exchange via nanowires. The partner bacterium was enriched with hydrogen as sole electron donor and sulfate as electron acceptor. Based on phylogenetic, genomic and metabolic characteristics we propose to name this chemolithoautotrophic sulfate reducer Candidatus Desulfofervidus auxilii. Ca. D. auxilii grows on hydrogen at temperatures between 50°C and 70°C with an activity optimum at 60°C and doubling time of 4-6 days. Its genome draft encodes for canonical sulfate reduction, periplasmic and soluble hydrogenases and autotrophic carbon fixation via the reductive tricarboxylic acid cycle. The presence of genes for pili formation and cytochromes, and their similarity to genes of Geobacter spp., indicate a potential for syntrophic growth via direct interspecies electron transfer when the organism grows in consortia with ANME. This first ANME-free enrichment of an AOM partner bacterium and its characterization opens the perspective for a deeper understanding of syntrophy in anaerobic methane oxidation.

Diversity and function of methyl-coenzyme M reductase-encoding archaea in Yellowstone hot springs revealed by metagenomics and mesocosm experiments.

Metagenomic studies on geothermal environments have been central in recent discoveries on the diversity of archaeal methane and alkane metabolism. Here, we investigated methanogenic populations inhabiting terrestrial geothermal features in Yellowstone National Park (YNP) by combining amplicon sequencing with metagenomics and mesocosm experiments. Detection of methyl-coenzyme M reductase subunit A (mcrA) gene amplicons demonstrated a wide diversity of Mcr-encoding archaea inhabit geothermal features with differing physicochemical regimes across YNP. From three selected hot springs we recovered twelve Mcr-encoding metagenome assembled genomes (MAGs) affiliated with lineages of cultured methanogens as well as Candidatus (Ca.) Methanomethylicia, Ca. Hadesarchaeia, and Archaeoglobi. These MAGs encoded the potential for hydrogenotrophic, aceticlastic, hydrogen-dependent methylotrophic methanogenesis, or anaerobic short-chain alkane oxidation. While Mcr-encoding archaea represent minor fractions of the microbial community of hot springs, mesocosm experiments with methanogenic precursors resulted in the stimulation of methanogenic activity and the enrichment of lineages affiliated with Methanosaeta and Methanothermobacter as well as with uncultured Mcr-encoding archaea including Ca. Korarchaeia, Ca. Nezhaarchaeia, and Archaeoglobi. We revealed that diverse Mcr-encoding archaea with the metabolic potential to produce methane from different precursors persist in the geothermal environments of YNP and can be enriched under methanogenic conditions. This study highlights the importance of combining environmental metagenomics with laboratory-based experiments to expand our understanding of uncultured Mcr-encoding archaea and their potential impact on microbial carbon transformations in geothermal environments and beyond.

Culexarchaeia, a novel archaeal class of anaerobic generalists inhabiting geothermal environments.

Geothermal environments, including terrestrial hot springs and deep-sea hydrothermal sediments, often contain many poorly understood lineages of archaea. Here, we recovered ten metagenome-assembled genomes (MAGs) from geothermal sediments and propose that they constitute a new archaeal class within the TACK superphylum, "Candidatus Culexarchaeia", named after the Culex Basin in Yellowstone National Park. Culexarchaeia harbor distinct sets of proteins involved in key cellular processes that are either phylogenetically divergent or are absent from other closely related TACK lineages, with a particular divergence in cell division and cytoskeletal proteins. Metabolic reconstruction revealed that Culexarchaeia have the capacity to metabolize a wide variety of organic and inorganic substrates. Notably, Culexarchaeia encode a unique modular, membrane associated, and energy conserving [NiFe]-hydrogenase complex that potentially interacts with heterodisulfide reductase (Hdr) subunits. Comparison of this [NiFe]-hydrogenase complex with similar complexes from other archaea suggests that interactions between membrane associated [NiFe]-hydrogenases and Hdr may be more widespread than previously appreciated in both methanogenic and non-methanogenic lifestyles. The analysis of Culexarchaeia further expands our understanding of the phylogenetic and functional diversity of lineages within the TACK superphylum and the ecology, physiology, and evolution of these organisms in extreme environments.

Microbial Community Response to Polysaccharide Amendment in Anoxic Hydrothermal Sediments of the Guaymas Basin.

Organic-rich, hydrothermal sediments of the Guaymas Basin are inhabited by diverse microbial communities including many uncultured lineages with unknown metabolic potential. Here we investigated the short-term effect of polysaccharide amendment on a sediment microbial community to identify taxa involved in the initial stage of macromolecule degradation. We incubated anoxic sediment with cellulose, chitin, laminarin, and starch and analyzed the total and active microbial communities using bioorthogonal non-canonical amino acid tagging (BONCAT) combined with fluorescence-activated cell sorting (FACS) and 16S rRNA gene amplicon sequencing. Our results show a response of an initially minor but diverse population of Clostridia particularly after amendment with the lower molecular weight polymers starch and laminarin. Thus, Clostridia may readily become key contributors to the heterotrophic community in Guaymas Basin sediments when substrate availability and temperature range permit their metabolic activity and growth, which expands our appreciation of the potential diversity and niche differentiation of heterotrophs in hydrothermally influenced sediments. BONCAT-FACS, although challenging in its application to complex samples, detected metabolic responses prior to growth and thus can provide complementary insight into a microbial community's metabolic potential and succession pattern. As a primary application of BONCAT-FACS on a diverse deep-sea sediment community, our study highlights important considerations and demonstrates inherent limitations associated with this experimental approach.

Conspicuous Smooth and White Egg-Shaped Sulfur Structures on a Deep-Sea Hydrothermal Vent Formed by Sulfide-Oxidizing Bacteria.

Conspicuous egg-shaped, white, and smooth structures were observed at a hydrothermal vent site in the Guaymas Basin, Gulf of California. The gelatinous structures decomposed within hours after sampling. Scanning electron microscopy (SEM) and light microscopy showed that the structure consisted of filaments of less than 0.1 µm thickness, similar to those observed for "Candidatus Arcobacter sulfidicus." SEM-energy-dispersive X-ray spectroscopy (EDS) showed that the filaments were sulfur rich. According to 16S rRNA gene amplicon and fluorescence in situ hybridization (FISH) analyses, Arcobacter, a sulfide oxidizer that is known to produce filamentous elemental sulfur, was among the dominant species in the structure and was likely responsible for its formation. Arcobacter normally produces woolly snowflake like structures in opposed gradients of sulfide and oxygen. In the laboratory, we observed sulfide consumption in the anoxic zone of the structure, suggesting an anaerobic conversion. The sulfide oxidation and decomposition of the structure in the laboratory may be explained by dissolution of the sulfur filaments by reaction with sulfide under formation of polysulfides. IMPORTANCE At the deep-sea Guaymas Basin hydrothermal vent system, sulfide-rich hydrothermal fluids mix with oxygenated seawater, thereby providing a habitat for microbial sulfur oxidation. Microbial sulfur oxidation in the deep sea involves a variety of organisms and processes and can result in the excretion of elemental sulfur. Here, we report on conspicuous white and smooth gelatinous structures found on hot vents. These strange egg-shaped structures were often observed on previous occasions in the Guaymas Basin, but their composition and formation process were unknown. Our data suggest that the notable and highly ephemeral structure was likely formed by the well-known sulfide-oxidizing Arcobacter. While normally Arcobacter produces loose flocs or woolly layers, here smooth gel-like structures were found.

Next-generation physiology approaches to study microbiome function at single cell level.

The function of cells in their native habitat often cannot be reliably predicted from genomic data or from physiology studies of isolates. Traditional experimental approaches to study the function of taxonomically and metabolically diverse microbiomes are limited by their destructive nature, low spatial resolution or low throughput. Recently developed technologies can offer new insights into cellular function in natural and human-made systems and how microorganisms interact with and shape the environments that they inhabit. In this Review, we provide an overview of these next-generation physiology approaches and discuss how the non-destructive analysis of cellular phenotypes, in combination with the separation of the target cells for downstream analyses, provide powerful new, complementary ways to study microbiome function. We anticipate that the widespread application of next-generation physiology approaches will transform the field of microbial ecology and dramatically improve our understanding of how microorganisms function in their native environment.

Activity-based cell sorting reveals responses of uncultured archaea and bacteria to substrate amendment.

Metagenomic studies have revolutionized our understanding of the metabolic potential of uncultured microorganisms in various ecosystems. However, many of these genomic predictions have yet to be experimentally tested, and the functional expression of genomic potential often remains unaddressed. In order to obtain a more thorough understanding of cell physiology, novel techniques capable of testing microbial metabolism under close to in situ conditions must be developed. Here, we provide a benchmark study to demonstrate that bioorthogonal non-canonical amino acid tagging (BONCAT) in combination with fluorescence-activated cell sorting (FACS) and 16S rRNA gene sequencing can be used to identify anabolically active members of a microbial community incubated in the presence of various growth substrates or under changing physicochemical conditions. We applied this approach to a hot spring sediment microbiome from Yellowstone National Park (Wyoming, USA) and identified several microbes that changed their activity levels in response to substrate addition, including uncultured members of the phyla Thaumarchaeota, Acidobacteria, and Fervidibacteria. Because shifts in activity in response to substrate amendment or headspace changes are indicative of microbial preferences for particular growth conditions, results from this and future BONCAT-FACS studies could inform the development of cultivation media to specifically enrich uncultured microbes. Most importantly, BONCAT-FACS is capable of providing information on the physiology of uncultured organisms at as close to in situ conditions as experimentally possible.

Establishing anaerobic hydrocarbon-degrading enrichment cultures of microorganisms under strictly anoxic conditions.

Traditionally, the description of microorganisms starts with their isolation from an environmental sample. Many environmentally relevant anaerobic microorganisms grow very slowly, and often they rely on syntrophic interactions with other microorganisms. This impedes their isolation and characterization by classic microbiological techniques. We developed and applied an approach for the successive enrichment of syntrophic hydrocarbon-degrading microorganisms from environmental samples. We collected samples from microbial mat-covered hydrothermally heated hydrocarbon-rich sediments of the Guaymas Basin and mixed them with synthetic mineral medium to obtain sediment slurries. Supplementation with defined substrates (i.e., methane or butane), incubation at specific temperatures, and a regular maintenance procedure that included the measurement of metabolic products and stepwise dilutions enabled us to establish highly active, virtually sediment-free enrichment cultures of actively hydrocarbon-degrading communities in a 6-months to several-years' effort. Using methane as sole electron donor shifted the originally highly diverse microbial communities toward defined mixed cultures dominated by syntrophic consortia consisting of anaerobic methane-oxidizing archaea (ANME) and different sulfate-reducing bacteria. Cultivation with butane at 50 °C yielded consortia of archaea belonging to Candidatus Syntrophoarchaeum and Candidatus Desulfofervidus auxilii partner bacteria. This protocol also describes sampling for further molecular characterization of enrichment cultures by fluorescence in situ hybridization (FISH), and transcriptomics and metabolite analyses, which can provide insights into the functioning of hydrocarbon metabolism in archaea and resolve important mechanisms that enable electron transfer to their sulfate-reducing partner bacteria.

Metabolic Capabilities of Microorganisms Involved in and Associated with the Anaerobic Oxidation of Methane.

In marine sediments the anaerobic oxidation of methane with sulfate as electron acceptor (AOM) is responsible for the removal of a major part of the greenhouse gas methane. AOM is performed by consortia of anaerobic methane-oxidizing archaea (ANME) and their specific partner bacteria. The physiology of these organisms is poorly understood, which is due to their slow growth with doubling times in the order of months and the phylogenetic diversity in natural and in vitro AOM enrichments. Here we study sediment-free long-term AOM enrichments that were cultivated from seep sediments sampled off the Italian Island Elba (20°C; hereon called E20) and from hot vents of the Guaymas Basin, Gulf of California, cultivated at 37°C (G37) or at 50°C (G50). These enrichments were dominated by consortia of ANME-2 archaea and Seep-SRB2 partner bacteria (E20) or by ANME-1, forming consortia with Seep-SRB2 bacteria (G37) or with bacteria of the HotSeep-1 cluster (G50). We investigate lipid membrane compositions as possible factors for the different temperature affinities of the different ANME clades and show autotrophy as characteristic feature for both ANME clades and their partner bacteria. Although in the absence of additional substrates methane formation was not observed, methanogenesis from methylated substrates (methanol and methylamine) could be quickly stimulated in the E20 and the G37 enrichment. Responsible for methanogenesis are archaea from the genus Methanohalophilus and Methanococcoides, which are minor community members during AOM (1-7‰ of archaeal 16S rRNA gene amplicons). In the same two cultures also sulfur disproportionation could be quickly stimulated by addition of zero-valent colloidal sulfur. The isolated partner bacteria are likewise minor community members (1-9‰ of bacterial 16S rRNA gene amplicons), whereas the dominant partner bacteria (Seep-SRB1a, Seep-SRB2, or HotSeep-1) did not grow on elemental sulfur. Our results support a functioning of AOM as syntrophic interaction of obligate methanotrophic archaea that transfer non-molecular reducing equivalents (i.e., via direct interspecies electron transfer) to obligate sulfate-reducing partner bacteria. Additional katabolic processes in these enrichments but also in sulfate methane interfaces are likely performed by minor community members.