Summary Sulfate‐reducing methanotrophy by anaerobic methanotrophic archaea (ANME) and sulfate‐reducing bacteria (SRB) is a major biological sink of methane in anoxic methane‐enriched marine sediments. The physiology of a microbial community dominated by free‐living ANME‐1 at 14–16 cm below the seafloor in the G11 pockmark at Nyegga was investigated by integrated metagenomic and metaproteomic approaches. Total DNA was subjected to 454‐pyrosequencing (829 527 reads), and 16.6 Mbp of sequence information was assembled into 27352 contigs. Taxonomic analysis supported a high abundance of Euryarchaea (70%) with 66% of the assembled metagenome belonging to ANME‐1. Extracted sediment proteins were separated in two dimensions and subjected to mass spectrometry (LTQ‐Orbitrap XL). Of 356 identified proteins, 245 were expressed by ANME‐1. These included proteins for cold‐adaptation and production of gas vesicles, reflecting both the adaptation of the ANME‐1 community to a permanently cold environment and its potential for positioning in specific sediment depths respectively. In addition, key metabolic enzymes including the enzymes in the reverse methanogenesis pathway (except N5,N10‐methylene‐tetrahydromethanopterin reductase), heterodisulfide reductases and the F420H2:quinone oxidoreductase (Fqo) complex were identified. A complete dissimilatory sulfate reduction pathway was expressed by sulfate‐reducing Deltaproteobacteria. Interestingly, an APS‐reductase comprising Gram‐positive SRB and related sequences were identified in the proteome. Overall, the results demonstrated that our approach was effective in assessing in situ metabolic processes in cold seep sediments.

Energy conservation via the pathway of dissimilatory sulfate reduction is present in a diverse group of prokaryotes, but is most comprehensively studied in Deltaproteobacteria. In this study, whole-genome microarray analyses were used to provide a model of the energy metabolism of the sulfate-reducing archaeon Archaeoglobus fulgidus, based on comparative analysis of litoautotrophic growth with H2/CO2 and thiosulfate, and heterotrophic growth on lactate with sulfate or thiosulfate. Only 72 genes were expressed differentially between the cultures utilizing sulfate or thiosulfate, whereas 269 genes were affected by a shift in energy source. We identified co-located gene cluster encoding putative lactate dehydrogenases (LDHs; lldD, dld, lldEFG), also present in sulfate-reducing bacteria. These enzymes may take part in energy conservation in A. fulgidus by specifically linking lactate oxidation with APS reduction via the Qmo complex. High transcriptional levels of Fqo confirm an important role of F420H2, as well as a menaquinone-mediated electron transport chain, during heterotrophic growth. A putative periplasmic thiosulfate reductase was identified by specific up-regulation. Also, putative genes for transport of sulfate and sulfite are discussed. We present a model for hydrogen metabolism, based on the probable bifurcation reaction of the Mvh:Hdl hydrogenase, which may inhibit the utilization of Fdred for energy conservation. Energy conservation is probably facilitated via menaquinone to multiple membrane-bound heterodisulfide reductase (Hdr) complexes and the DsrC protein—linking periplasmic hydrogenase (Vht) to the cytoplasmic reduction of sulfite. The ambiguous roles of genes corresponding to fatty acid metabolism induced during growth with H2 are discussed. Putative co-assimilation of organic acids is favored over a homologous secondary carbon fixation pathway, although both mechanisms may contribute to conserve the amount of Fdred needed during autotrophic growth with H2.

Methane seepages typically harbor communities of anaerobic methane oxidizers (ANME); however, knowledge about fine-scale vertical variation of ANME in response to geochemical gradients is limited. We investigated microbial communities in sediments below a white microbial mat in the G11 pockmark at Nyegga by 16S rRNA gene tag pyrosequencing and real-time quantitative PCR. A vertical stratification of dominating ANME communities was observed at 4 cmbsf (cm below seafloor) and below in the following order: ANME-2a/b, ANME-1 and ANME-2c. The ANME-1 community was most numerous and comprised single or chains of cells with typical rectangular morphology, accounting up to 89.2% of the retrieved 16S rRNA gene sequences. Detection rates for sulfate-reducing Deltaproteobacteria possibly involved in anaerobic oxidation of methane were low throughout the core. However, a correlation in the abundance of Candidate division JS-1 with ANME-2 was observed, indicating involvement in metabolisms occurring in ANME-2-dominated horizons. The white microbial mat and shallow sediments were dominated by organisms affiliated with Sulfurovum (Epsilonproteobacteria) and Methylococcales (Gammaproteobacteria), suggesting that aerobic oxidation of sulfur and methane is taking place. In intermediate horizons, typical microbial groups associated with methane seeps were recovered. The data are discussed with respect to co-occurring microbial assemblages and interspecies interactions.

Little is known about how lithoautotrophic primary production is connected to microbial organotrophic consumption in hydrothermal systems. Using a multifaceted approach, we analysed the structure and metabolic capabilities within a biofilm growing on the surface of a black smoker chimney in the Loki's Castle vent field. Imaging revealed the presence of rod-shaped Bacteroidetes growing as ectobionts on long, sheathed microbial filaments (> 100 μm) affiliated with the Sulfurovum genus within Epsilonproteobacteria. The filaments were composed of a thick (> 200 nm) stable polysaccharide, representing a substantial fraction of organic carbon produced by primary production. An integrated -omics approach enabled us to assess the metabolic potential and in situ metabolism of individual taxonomic and morphological groups identified by imaging. Specifically, we provide evidence that organotrophic Bacteroidetes attach to and glide along the surface of Sulfurovum filaments utilizing organic polymers produced by the lithoautotrophic Sulfurovum. Furthermore, in situ expression of acetyl-CoA synthetase by Sulfurovum suggested the ability to assimilate acetate, indicating recycling of organic matter in the biofilm. This study expands our understanding of the lifestyles of Epsilonproteobacteria in hydrothermal vents, their metabolic properties and co-operative interactions in deep-sea hydrothermal vent food webs.

The Epsilonproteobacteria, including members of the genus Sulfurovum, are regarded as important primary producers in hydrothermal systems. However, their in situ gene expression in this habitat has so far not been investigated. We report a metatranscriptomic analysis of a Sulfurovum-dominated biofilm from one of the chimneys at the Loki's Castle hydrothermal system, located at the Arctic Mid Ocean Ridge. Transcripts involved in hydrogen oxidation, oxidation of sulfur species, aerobic respiration and denitrification were abundant and mostly assigned to Sulfurovum, indicating that members of this genus utilize multiple chemical energy sources simultaneously for primary production. Sulfurovum also seemed to have a diverse expression of transposases, potentially involved in horizontal gene transfer. Other transcripts were involved in CO₂ fixation by the reverse TCA cycle, the CRISPR-Cas system, heavy metal resistance, and sensing and responding to changing environmental conditions. Through pyrosequencing of PCR amplified 16S rRNA genes, the Sulfurovum-dominated biofilm was compared with another biofilm from the same chimney, revealing a large shift in the community structure of Epsilonproteobacteria-dominated biofilms over a few metres.