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Recent single-gene-based surveys of deep continental aquifers demonstrated the widespread occurrence of archaea related to Candidatus Methanoperedens nitroreducens (ANME-2d) known to mediate anaerobic oxidation of methane (AOM). However, it is unclear whether ANME-2d mediates AOM in the deep continental biosphere. In this study, we found the dominance of ANME-2d in groundwater enriched in sulfate and methane from a 300-m deep underground borehole in granitic rock. A near-complete genome of one representative species of the ANME-2d obtained from the underground borehole has most of functional genes required for AOM and assimilatory sulfate reduction. The genome of the subsurface ANME-2d is different from those of other members of ANME-2d by lacking functional genes encoding nitrate and nitrite reductases and multiheme cytochromes. In addition, the subsurface ANME-2d genome contains a membrane-bound NiFe hydrogenase gene putatively involved in respiratory H2 oxidation, which is different from those of other methanotrophic archaea. Short-term incubation of microbial cells collected from the granitic groundwater with 13C-labeled methane also demonstrates that AOM is linked to microbial sulfate reduction. Given the prominence of granitic continental crust and sulfate and methane in terrestrial subsurface fluids, we conclude that AOM may be widespread in the deep continental biosphere.

Geological sequestration in deep underground repositories is the prevailing proposed route for radioactive waste disposal. After the disposal of radioactive waste in the subsurface, H2 may be produced by corrosion of steel and, ultimately, radionuclides will be exposed to the surrounding environment. To evaluate the potential for microbial activities to impact disposal systems, we explored the microbial community structure and metabolic functions of a sediment-hosted ecosystem at the Horonobe Underground Research Laboratory, Hokkaido, Japan. Overall, we found that the ecosystem hosted organisms from diverse lineages, including many from the phyla that lack isolated representatives. The majority of organisms can metabolize H2, often via oxidative [NiFe] hydrogenases or electron-bifurcating [FeFe] hydrogenases that enable ferredoxin-based pathways, including the ion motive Rnf complex. Many organisms implicated in H2 metabolism are also predicted to catalyze carbon, nitrogen, iron and sulfur transformations. Notably, iron-based metabolism is predicted in a novel lineage of Actinobacteria and in a putative methane-oxidizing ANME-2d archaeon. We infer an ecological model that links microorganisms to sediment-derived resources and predict potential impacts of microbial activity on H2 consumption and retardation of radionuclide migration.

The tree of life is one of the most important organizing principles in biology(1). Gene surveys suggest the existence of an enormous number of branches(2), but even an approximation of the full scale of the tree has remained elusive. Recent depictions of the tree of life have focused either on the nature of deep evolutionary relationships(3-5) or on the known, well-classified diversity of life with an emphasis on eukaryotes(6). These approaches overlook the dramatic change in our understanding of life's diversity resulting from genomic sampling of previously unexamined environments. New methods to generate genome sequences illuminate the identity of organisms and their metabolic capacities, placing them in community and ecosystem contexts(7,8). Here, we use new genomic data from over 1,000 uncultivated and little known organisms, together with published sequences, to infer a dramatically expanded version of the tree of life, with Bacteria, Archaea and Eukarya included. The depiction is both a global overview and a snapshot of the diversity within each major lineage. The results reveal the dominance of bacterial diversification and underline the importance of organisms lacking isolated representatives, with substantial evolution concentrated in a major radiation of such organisms. This tree highlights major lineages currently underrepresented in biogeochemical models and identifies radiations that are probably important for future evolutionary analyses.

The bacterium Caulobacter crescentus and related stalk bacterial species are known for their distinctive ability to live in low nutrient environments, a characteristic of most heavy metal contaminated sites. Caulobacter crescentus is a model organism for studying cell cycle regulation with well developed genetics. We have identified the pathways responding to heavy metal toxicity in C. crescentus to provide insights for possible application of Caulobacter to environmental restoration. We exposed C. crescentus cells to four heavy metals (chromium, cadmium, selenium and uranium) and analyzed genome wide transcriptional activities post exposure using a Affymetrix GeneChip microarray. C. crescentus showed surprisingly high tolerance to uranium, a possible mechanism for which may be formation of extracellular calcium-uranium-phosphate precipitates. The principal response to these metals was protection against oxidative stress (up-regulation of manganese-dependent superoxide dismutase, sodA). Glutathione S-transferase, thioredoxin, glutaredoxins and DNA repair enzymes responded most strongly to cadmium and chromate. The cadmium and chromium stress response also focused on reducing the intracellular metal concentration, with multiple efflux pumps employed to remove cadmium while a sulfate transporter was down-regulated to reduce non-specific uptake of chromium. Membrane proteins were also up-regulated in response to most of the metals tested. A two-component signal transduction system involved in the uranium response was identified. Several differentially regulated transcripts from regions previously not known to encode proteins were identified, demonstrating the advantage of evaluating the transcriptome using whole genome microarrays.

The family Ca. Methanoperedenaceae archaea mediates the anaerobic oxidation of methane (AOM) in different terrestrial environments. Using a newly developed high‐pressure laboratory incubation system, we investigated 214‐ and 249‐m deep groundwater samples at Horonobe Underground Research Laboratory, Japan, where the high and low abundances of Ca. Methanoperedenaceae archaea have been shown by genome‐resolved metagenomics, respectively. The groundwater samples amended with 13C‐labelled methane and amorphous Fe(III) were incubated at a pressure of 1.6 MPa. After 3–7 days of incubation, the AOM rate was 45.8 ± 19.8 nM/day in 214‐m groundwater. However, almost no activity was detected from 249‐m groundwater. Based on the results from 16S rRNA gene analysis, the abundance of Ca. Methanoperedenaceae archaea was high in the 214‐m deep groundwater sample, whereas Ca. Methanoperedenaceae archaea was undetected in the 249‐m deep groundwater sample. These results support the in situ AOM activity of Ca. Methanoperedenaceae archaea in the 214‐m deep subsurface borehole interval. Although the presence of Fe‐bearing phyllosilicates was demonstrated in the 214‐m deep groundwater, it needs to be determined whether Ca. Methanoperedenaceae archaea use the Fe‐bearing phyllosilicates as in situ electron acceptors by high‐pressure incubation amended with the Fe‐bearing phyllosilicates. [ABSTRACT FROM AUTHOR]

The microbial community in the black rust from a circulation obviation retrofit kit (CORK) observatory was investigated by using both culture-dependent and independent methods to understand the microbial ecosystem within ridge flank. It provided important insights into the phylogenetic, physiological and trophic characteristics of subseafloor microbial ecosystems in warm dridge flank crusts.