Irene Roalkvam

Integrated metagenomic and metaproteomic analyses of an ANME-1-dominated community in marine cold seep sediments

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 N(5) ,N(10) -methylene-tetrahydromethanopterin reductase), heterodisulfide reductases and the F(420) H(2) :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.

New insight into stratification of anaerobic methanotrophs in cold seep sediments

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.

Physiological and genomic characterization of Arcobacter anaerophilus IR-1 reveals new metabolic features in Epsilonproteobacteria.

In this study we characterized and sequenced the genome of Arcobacter anaerophilus strain IR-1 isolated from enrichment cultures used in nitrate-amended corrosion experiments. A. anaerophilus IR-1 could grow lithoautotrophically on hydrogen and hydrogen sulfide and lithoheterothrophically on thiosulfate and elemental sulfur. In addition, the strain grew organoheterotrophically on yeast extract, peptone, and various organic acids. We show for the first time that Arcobacter could grow on the complex organic substrate tryptone and oxidize acetate with elemental sulfur as electron acceptor. Electron acceptors utilized by most Epsilonproteobacteria, such as oxygen, nitrate, and sulfur, were also used by A. anaerophilus IR-1. Strain IR-1 was also uniquely able to use iron citrate as electron acceptor. Comparative genomics of the Arcobacter strains A. butzleri RM4018, A. nitrofigilis CI and A. anaerophilus IR-1 revealed that the free-living strains had a wider metabolic range and more genes in common compared to the pathogen strain. The presence of genes for NAD+-reducing hydrogenase (hox) and dissimilatory iron reduction (fre) were unique for A. anaerophilus IR-1 among Epsilonproteobacteria. Finally, the new strain had an incomplete denitrification pathway where the end product was nitrite, which is different from other Arcobacter strains where the end product is ammonia. Altogether, our study shows that traditional characterization in combination with a modern genomics approach can expand our knowledge on free-living Arcobacter, and that this complementary approach could also provide invaluable knowledge about the physiology and metabolic pathways in other Epsilonproteobacteria from various environments.

Functional interactions among filamentous Epsilonproteobacteria and Bacteroidetes in a deep-sea hydrothermal vent biofilm

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 Lokis 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.

Modeling of Heavy Nitrate Corrosion in Anaerobe Aquifer Injection Water Biofilm: A Case Study in a Flow Rig

Heavy carbon steel corrosion developed during nitrate mitigation of a flow rig connected to a water injection pipeline flowing anaerobe saline aquifer water. Genera-specific QPCR primers quantified 74% of the microbial biofilm community, and further 87% of the community of the nonamended parallel rig. The nonamended biofilm hosted 6.3 × 10(6) SRB cells/cm(2) and the S(35)-sulfate-reduction rate was 1.1 μmol SO4(2-)/cm(2)/day, being congruent with the estimated SRB biomass formation and the sulfate areal flux. Nitrate amendment caused an 18-fold smaller SRB population, but up to 44 times higher sulfate reduction rates. This H2S formation was insufficient to form the observed Fe3S4 layer. Additional H2S was provided by microbial disproportionation of sulfur, also explaining the increased accessibility of sulfate. The reduced nitrate specie nitrite inhibited the dominating H2-scavenging Desulfovibrio population, and sustained the formation of polysulfide and Fe3S4, herby also dissolved sulfur. This terminated the availability of acetate in the inner biofilm and caused cell starvation that initiated growth upon metallic electrons, probably by the sulfur-reducing Desulfuromonas population. On the basis of these observations we propose a model of heavy nitrate corrosion where three microbiological processes of nitrate reduction, disproportionation of sulfur, and metallic electron growth are nicely woven into each other.

The versatile in situ gene expression of an Epsilonproteobacteria-dominated biofilm from a hydrothermal chimney

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 Lokis 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.

Identification of key components in the energy metabolism of the hyperthermophilic sulfate-reducing archaeon Archaeoglobus fulgidus by transcriptome analyses

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.

Fine-Scale Community Structure Analysis of ANME in Nyegga Sediments with High and Low Methane Flux

To obtain knowledge on how regional variations in methane seepage rates influence the stratification, abundance, and diversity of anaerobic methanotrophs (ANME), we analyzed the vertical microbial stratification in a gravity core from a methane micro-seeping area at Nyegga by using 454-pyrosequencing of 16S rRNA gene tagged amplicons and quantitative PCR. These data were compared with previously obtained data from the more active G11 pockmark, characterized by higher methane flux. A down core stratification and high relative abundance of ANME were observed in both cores, with transition from an ANME-2a/b dominated community in low-sulfide and low methane horizons to ANME-1 dominance in horizons near the sulfate-methane transition zone. The stratification was over a wider spatial region and at greater depth in the core with lower methane flux, and the total 16S rRNA copy numbers were two orders of magnitude lower than in the sediments at G11 pockmark. A fine-scale view into the ANME communities at each location was achieved through operational taxonomical units (OTU) clustering of ANME-affiliated sequences. The majority of ANME-1 sequences from both sampling sites clustered within one OTU, while ANME-2a/b sequences were represented in unique OTUs. We suggest that free-living ANME-1 is the most abundant taxon in Nyegga cold seeps, and also the main consumer of methane. The observation of specific ANME-2a/b OTUs at each location could reflect that organisms within this clade are adapted to different geochemical settings, perhaps due to differences in methane affinity. Given that the ANME-2a/b population could be sustained in less active seepage areas, this subgroup could be potential seed populations in newly developed methane-enriched environments.

Lutibacter profundi sp. nov., isolated from a deep-sea hydrothermal system on the Arctic Mid-Ocean Ridge and emended description of the genus Lutibacter.

A bacterial strain designated LP1T was isolated from a microbial mat growing on the surface of a black smoker chimney at the Lokis Castle hydrothermal system, which is located on the Arctic Mid-Ocean Ridge. Phylogenetic analyses based on 16S rRNA gene sequences positioned strain LP1T within the family Flavobacteriaceae with Lutibacterholmesii as the closest relative (97.5 % 16S rRNA gene sequence similarity). Strain LP1T was rod-shaped, Gram-reaction-negative and non-motile. It grew in a modified artificial seawater medium supplemented with tryptone and vitamins at pH 5.5-7.5 (optimum pH 6.0-6.5), within a temperature range of 13-34 °C (optimum 23 °C), and under microaerobic conditions. The most abundant fatty acids (>10 %) were iso-C15 : 0 (25.2 %) and iso-C15 : 0 3-OH (14.5 %). The genome of strain LP1T has a DNA G+C content of 29.8 mol%. Based on the results of the polyphasic characterization presented here, strain LP1T is considered to represent a novel species of the genus Lutibacter, for which the name Lutibacter profundi sp. nov. is proposed. The type strain is LP1T (=DSM 100437T =JCM 30585T). An emended description of the genus Lutibacter is also provided to fit the description of strain LP1T.

Novel Barite Chimneys at the Loki's Castle Vent Field Shed Light on Key Factors Shaping Microbial Communities and Functions in Hydrothermal Systems

In order to fully understand the cycling of elements in hydrothermal systems it is critical to understand intra-field variations in geochemical and microbiological processes in both focused, high-temperature and diffuse, low-temperature areas. To reveal important causes and effects of this variation, we performed an extensive chemical and microbiological characterization of a low-temperature venting area in the Lokis Castle Vent Field (LCVF). This area, located at the flank of the large sulfide mound, is characterized by numerous chimney-like barite (BaSO4) structures (≤ 1 m high) covered with white cotton-like microbial mats. Results from geochemical analyses, microscopy (FISH, SEM), 16S rRNA gene amplicon-sequencing and metatranscriptomics were compared to results from previous analyses of biofilms growing on black smoker chimneys at LCVF. Based on our results, we constructed a conceptual model involving the geochemistry and microbiology in the LCVF. The model suggests that CH4 and H2S are important electron donors for microorganisms in both high-temperature and low-temperature areas, whereas the utilization of H2 seems restricted to high-temperature areas. This further implies that sub-seafloor processes can affect energy-landscapes, elemental cycling, and the metabolic activity of primary producers on the seafloor. In the cotton-like microbial mats on top of the active barite chimneys, a unique network of single cells of Epsilonproteobacteria interconnected by threads of extracellular polymeric substances (EPS) was seen, differing significantly from the long filamentous Sulfurovum filaments observed in biofilms on the black smokers. This network also induced nucleation of barite crystals and is suggested to play an essential role in the formation of the microbial mats and the chimneys. Furthermore, it illustrates variations in how different genera of Epsilonproteobacteria colonize and position cells in different vent fluid mixing zones within a vent field. This may be related to niche-specific physical characteristics. Altogether, the model provides a reference for future studies and illustrates the importance of systematic comparative studies of spatially closely connected niches in order to fully understand the geomicrobiology of hydrothermal systems.