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.

The anaerobic formation and oxidation of methane involve unique enzymatic mechanisms and cofactors, all of which are believed to be specific for C-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 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 C 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.

In the biogeochemical nitrogen cycle, microbial respiration processes compete for nitrate as an electron acceptor. Denitrification converts nitrate into nitrogenous gas and thus removes fixed nitrogen from the biosphere, whereas ammonification converts nitrate into ammonium, which is directly reusable by primary producers. We combined multiple parallel long-term incubations of marine microbial nitrate-respiring communities with isotope labeling and metagenomics to unravel how specific environmental conditions select for either process. Microbial generation time, supply of nitrite relative to nitrate, and the carbon/nitrogen ratio were identified as key environmental controls that determine whether nitrite will be reduced to nitrogenous gas or ammonium. Our results define the microbial ecophysiology of a biogeochemical feedback loop that is key to global change, eutrophication, and wastewater treatment.

No abstract

SummaryThe 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.

So far, microbial physiology has dedicated itself mainly to pure cultures. In nature, cross feeding and competition are important aspects of microbial physiology and these can only be addressed by studying complete communities such as enrichment cultures. Metagenomic sequencing is a powerful tool to characterize such mixed cultures. In the analysis of metagenomic data, well established algorithms exist for the assembly of short reads into contigs and for the annotation of predicted genes. However, the binning of the assembled contigs or unassembled reads is still a major bottleneck and required to understand how the overall metabolism is partitioned over different community members. Binning consists of the clustering of contigs or reads that apparently originate from the same source population. In the present study eight metagenomic samples from the same habitat, a laboratory enrichment culture, were sequenced. Each sample contained 13–23 Mb of assembled contigs and up to eight abundant populations. Binning was attempted with existing methods but they were found to produce poor results, were slow, dependent on non-standard platforms or produced errors. A new binning procedure was developed based on multivariate statistics of tetranucleotide frequencies combined with the use of interpolated Markov models. Its performance was evaluated by comparison of the results between samples with BLAST and in comparison to existing algorithms for four publicly available metagenomes and one previously published artificial metagenome. The accuracy of the new approach was comparable or higher than existing methods. Further, it was up to a 100 times faster. It was implemented in Java Swing as a complete open source graphical binning application available for download and further development ().

SummaryThe 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 508C and 708C with an activity optimum at 608C 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.

Genetic diversity of closely-related free-living microbes is widespread and underpins ecosystem functioning, but most evolutionary theories predict that it destabilizes intimate mutualisms. Indeed, symbiont strain diversity has long assumed to be restricted in intracellular bacteria associated with animals. Here, we sequenced the metagenomes and metatranscriptomes of 18 Bathymodiolus mussel individuals from four species, covering their known distribution range at deep-sea hydrothermal vents in the Atlantic. We show that as many as 16 strains of intracellular, sulfuroxidizing symbionts coexist in individual Bathymodiolus mussels. Co-occurring symbiont strains differed extensively in key metabolic functions, such as the use of energy and nutrient sources, electron acceptors and viral defense mechanisms. Most strain-specific genes were expressed, highlighting their adaptive potential. We show that fine-scale diversity is pervasive in Bathymodiolus symbionts, and hypothesize that it may be widespread in low-cost symbioses where the environment, not the host, feeds the symbionts.