Bullet-shaped magnetosomes and metagenomic-based magnetosome gene profiles in a deep-sea hydrothermal vent chimney.

Magnetosome-producing microorganisms can sense and move toward the redox gradient and have been extensively studied in terrestrial and shallow marine sediment environments. However, given the difficulty of sampling, magnetotactic bacteria (MTB) are poorly explored in deep-sea hydrothermal fields. In this study, a deep-sea hydrothermal vent chimney from the Southern Mariana Trough was collected using a remotely operated submersible. The mineralogical and geochemical characterization of the vent chimney sample showed an internal iron redox gradient. Additionally, the electron microscopy of particles collected by magnetic separation from the chimney sample revealed MTB cells with bullet-shaped magnetosomes, and there were minor occurrences of cuboctahedral and hexagonal prismatic magnetosomes. Genome-resolved metagenomic analysis was performed to identify microorganisms that formed magnetosomes. A metagenome-assembled genome (MAG) affiliated with Nitrospinae had magnetosome genes such as mamA, mamI, mamM, mamP, and mamQ. Furthermore, a diagnostic feature of MTB genomes, such as magnetosome gene clusters (MGCs), including mamA, mamP, and mamQ, was also confirmed in the Nitrospinae-affiliated MAG. Two lines of evidence support the occurrence of MTB in a deep-sea, inactive hydrothermal vent environment.

Anaerobic methane-oxidizing activity in a deep underground borehole dominantly colonized by Ca. Methanoperedenaceae.

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 13 C-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.

Copper-Nanocoated Ultra-Small Cells in Grain Boundaries Inside an Extinct Vent Chimney.

Chemosynthetic organisms flourish around deep-sea hydrothermal vents where energy-rich fluids are emitted from metal sulfide chimneys. However, microbial life hosted in mineral assemblages in extinct chimneys lacking fluid venting remains largely unknown. The interior of extinct chimneys remains anoxic where the percolation of oxygenated seawater is limited within tightly packed metal sulfide grains. Given the scarcity of photosynthetic organics in deep seawater, anaerobic microbes might inhabit the grain boundaries energetically depending on substrates derived from rock-water interactions. In this study, we reported ultra-small cells directly visualized in grain boundaries of CuFeS2 inside an extinct metal sulfide chimney from the southern Mariana Trough. Nanoscale solid analyses reveal that ultra-small cells are coated with Cu2O nanocrystals in grain boundaries enriched with C, N, and P. In situ spectroscopic and spectrometric characterizations demonstrate the distribution of organics with amide groups and a large molecular organic compound in the grain boundaries. We inferred that the ultra-small cells are anaerobes because of the fast dissolution of Cu2O nanocrystals in oxygenated solution. This Cu2O property also excludes the possibility of microbial contamination from ambient seawater during sampling. It is shown by 16S rRNA gene sequence analysis that the chimney interior is dominated by Pacearchaeota known to have anaerobic metabolisms and ultra-small cells. Our results support the potential existence of photosynthesis-independent microbial ecosystems in grain boundaries in submarine metal sulfides deposits on the early Earth.

The Deep Rocky Biosphere: New Geomicrobiological Insights and Prospects.

Rocks that react with liquid water are widespread but spatiotemporally limited throughout the solar system, except for Earth. Rock-forming minerals with high iron content and accessory minerals with high amounts of radioactive elements are essential to support rock-hosted microbial life by supplying organics, molecular hydrogen, and/or oxidants. Recent technological advances have broadened our understanding of the rocky biosphere, where microbial inhabitation appears to be difficult without nutrient and energy inputs from minerals. In particular, microbial proliferation in igneous rock basements has been revealed using innovative geomicrobiological techniques. These recent findings have dramatically changed our perspective on the nature and the extent of microbial life in the rocky biosphere, microbial interactions with minerals, and the influence of external factors on habitability. This study aimed to gather information from scientific and/or technological innovations, such as omics-based and single-cell level characterizations, targeting deep rocky habitats of organisms with minimal dependence on photosynthesis. By synthesizing pieces of rock-hosted life, we can explore the evo-phylogeny and ecophysiology of microbial life on Earth and the life's potential on other planetary bodies.

Deep microbial proliferation at the basalt interface in 33.5-104 million-year-old oceanic crust.

The upper oceanic crust is mainly composed of basaltic lava that constitutes one of the largest habitable zones on Earth. However, the nature of deep microbial life in oceanic crust remains poorly understood, especially where old cold basaltic rock interacts with seawater beneath sediment. Here we show that microbial cells are densely concentrated in Fe-rich smectite on fracture surfaces and veins in 33.5- and 104-million-year-old (Ma) subseafloor basaltic rock. The Fe-rich smectite is locally enriched in organic carbon. Nanoscale solid characterizations reveal the organic carbon to be microbial cells within the Fe-rich smectite, with cell densities locally exceeding 1010 cells/cm3. Dominance of heterotrophic bacteria indicated by analyses of DNA sequences and lipids supports the importance of organic matter as carbon and energy sources in subseafloor basalt. Given the prominence of basaltic lava on Earth and Mars, microbial life could be habitable where subsurface basaltic rocks interact with liquid water.

Evidence in the Japan Sea of microdolomite mineralization within gas hydrate microbiomes.

Over the past 15 years, massive gas hydrate deposits have been studied extensively in Joetsu Basin, Japan Sea, where they are associated primarily with active gas chimney structures. Our research documents the discovery of spheroidal microdolomite aggregates found in association with other impurities inside of these massive gas hydrates. The microdolomites are often conjoined and show dark internal cores occasionally hosting saline fluid inclusions. Bacteroidetes sp. are concentrated on the inner rims of microdolomite grains, where they degrade complex petroleum-macromolecules present as an impurity within yellow methane hydrate. These oils show increasing biodegradation with depth which is consistent with the microbial activity of Bacteroidetes. Further investigation of these microdolomites and their contents can potentially yield insight into the dynamics and microbial ecology of other hydrate localities. If microdolomites are indeed found to be ubiquitous in both present and fossil hydrate settings, the materials preserved within may provide valuable insights into an unusual microhabitat which could have once fostered ancient life.

Deep Microbial Colonization in Saponite-Bearing Fractures in Aged Basaltic Crust: Implications for Subsurface Life on Mars.

One of the most promising planetary bodies that might harbor extraterrestrial life is Mars, given the presence of liquid water in the deep subsurface. The upper crust of Mars is mainly composed of >3.7-billion-year-old basaltic lava where heat-driven fluid circulation is negligible. The analogous crustal environment to the Martian subsurface is found in the Earth's oceanic crust composed of basaltic lava. The basaltic crust tends to cool down for 10-20-million-years after formation. However, microbial life in old cold basaltic lava is largely unknown even in the Earth's oceanic crust, because the lack of vigorous circulation prevents sampling of pristine crustal fluid from boreholes. Alternatively, it is important to investigate deep microbial life using pristine drill cores obtained from basaltic lava. We investigated a basaltic rock core sample with mineral-filled fractures drilled during Integral Ocean Drilling Project Expedition 329 that targeted 104-million-year-old oceanic crust. Mineralogical characterizations of fracture-infilling minerals revealed that fractures/veins were filled with Mg-rich smectite called saponite and calcium carbonate. The organic carbon content from the saponite-rich clay fraction in the core sample was 23 times higher than that from the bulk counterpart, which appears to be sufficient to supply energy and carbon sources to saponite-hosted life. Furthermore, a newly developed method to detect microbial cells in a thin-section of the saponite-bearing fracture revealed the dense colonization of SYBR-Green-I stained microbial cells spatially associated with saponite. These results suggest that the presence of saponite in old cold basaltic crust is favorable for microbial life. In addition to carbonaceous chondrite, saponite is a common product of low-temperature reactions between water and mafic minerals on Earth and Mars. It is therefore expected that deep saponite-bearing fractures could host extant life and/or the past life on Mars.

Metabolic Potential of As-yet-uncultured Archaeal Lineages of Candidatus Hydrothermarchaeota Thriving in Deep-sea Metal Sulfide Deposits.

Candidatus Hydrothermarchaeota, formally called Marine Benthic Group E, has often been detected in iron- and sulfur-rich marine environments, such as hydrothermal vents and cold seeps. However, their ecology and physiology remain unclear. Cultivated representatives of this group are still lacking and only several metagenome-assembled genomes (MAGs) and single-amplified genomes (SAGs) are available from two deep-sea hydrothermal areas, the Juan de Fuca Ridge (JdFR) and Guaymas Basin (GB), in the north-east Pacific. We herein report four MAGs of Ca. Hydrothermarchaeota recovered from hydrothermally-inactive metal sulfide deposits at the Southern Mariana Trough (SMT) in the north-west Pacific. A phylogenetic analysis indicated that the MAGs of the SMT were distinct from those of the JdFR and GB at the genus or potentially family level. Ca. Hydrothermarchaeota MAGs from the SMT commonly possessed putative genes for carboxydotrophic and hydrogenotrophic respiration using oxidized chemical species of sulfur as electron acceptors and also for carbon fixation, as reported previously in MAGs/SAGs from the JdFR and GB. This result strongly supports Ca. Hydrothermarchaeota containing anaerobic chemolithoautotrophs using carbon monoxide and/or hydrogen as electron donors. A comparative genome analysis highlighted differences in the capability of nitrogen fixation between MAGs from the SMT and the other fields, which are consistent with environmental differences in the availability of nitrogen sources for assimilation between the fields. Based on the wide distribution in various areas, abundance, and metabolic potential of Ca. Hydrothermarchaeota, they may play a role in the biogeochemical cycling of carbon, nitrogen, sulfur, and iron in marine environments, particularly in deep-sea hydrothermal fields.

Ecological and genomic profiling of anaerobic methane-oxidizing archaea in a deep granitic environment.

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.

Deep microbial life in high-quality granitic groundwater from geochemically and geographically distinct underground boreholes.

Deep granitic aquifer is one of the largest, but least understood, microbial habitats. To avoid contamination from the surface biosphere, underground drilling was conducted for 300 m deep granitic rocks at the Mizunami underground research laboratory (URL), Japan. Slightly alkaline groundwater was characterized by low concentrations of dissolved organic matter and sulfate and the presence of > 100 nM H2 . The initial biomass was the highest (∼10(5) cells ml(-1) ) with the dominance of Hydrogenophaga spp., whereas the phylum Nitrospirae became predominant after 3 years with decreasing biomass (∼10(4) cells ml(-1) ). One week incubation of groundwater microbes after 3 years with (13) C-labelled bicarbonate and 1% H2 and subsequent single-cell imaging with nanometer-scale secondary ion mass spectrometry demonstrated that microbial cells were metabolically active. Pyrosequencing of microbial communities in groundwater retrieved at 3-4 years after drilling at the Mizunami URL and at 14 and 25 years after the drilling at the Grimsel Test Site, Switzerland, revealed the occurrence of common Nitrospirae lineages at the geographically distinct sites. As the close relatives of the Nitrospirae lineages were exclusively detected from deep groundwaters and terrestrial hot springs, it suggests that these bacteria are indigenous and potentially adapted to the deep terrestrial subsurface.

Novel microbial populations in deep granitic groundwater from Grimsel Test Site, Switzerland.

Freshwater aquifers in granitic rocks are widespread microbial habitats in the terrestrial subsurface. Microbial populations in deep granitic groundwater from two recently drilled (1 and 2 years) and two old boreholes (14 and 25 years) were compared. The 16S rRNA gene sequences related to "Candidatus Magnetobacterium bavaricum", Thermodesulfovibrio spp. of Nitrospirae (90.5-93.1 % similarity) and a novel candidate division with <90 % similarity to known cultivated species were dominant in all boreholes. Most of the environmental clones closely related to the novel lineages in Nitrospirae, which have been detected exclusively in deep groundwater samples. In contrast, betaproteobacterial sequences related to the family Rhodocyclaceae were obtained only from the recently drilled boreholes, which had higher total cell numbers. Catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) analysis supported the result from clone library analysis; betaproteobacterial cells were dominantly detected in recently drilled boreholes. These results suggest that while indigenous microbial populations represented by the novel phylotypes persisted in the boreholes for 25 years, betaproteobacterial species disappeared after 2 years owing to the change of substrate availability.

A new DNA extraction method by controlled alkaline treatments from consolidated subsurface sediments.

Microbial communities that thrive in subterranean consolidated sediments are largely unknown owing to the difficulty of extracting DNA. As this difficulty is often attributed to DNA binding onto the silica-bearing sediment matrix, we developed a DNA extraction method for consolidated sediment from the deep subsurface in which silica minerals were dissolved by being heated under alkaline conditions. NaOH concentrations (0.07 and 0.33 N), incubation temperatures (65 and 94 °C) and incubation times (30-90 min) before neutralization were evaluated based on the copy number of extracted prokaryotic DNA. Prokaryotic DNA was detected by quantitative PCR analysis after heating the sediment sample at 94 °C in 0.33 N NaOH solution for 50-80 min. Results of 16S rRNA gene sequence analysis of the extracted DNA were all consistent with regard to the dominant occurrence of the metallophilic bacterium, Cupriavidus metallidurans, and Pseudomonas spp. Mineralogical analysis revealed that the dissolution of a silica mineral (opal-CT) during alkaline treatment was maximized at 94 °C in 0.33 N NaOH solution for 50 min, which may have resulted in the release of DNA into solution. Because the optimized protocol for DNA extraction is applicable to subterranean consolidated sediments from a different locality, the method developed here has the potential to expand our understanding of the microbial community structure of the deep biosphere.

Geomicrobiological properties of ultra-deep granitic groundwater from the Mizunami Underground Research Laboratory (MIU), central Japan.

Although deep subterranean crystalline rocks are known to harbor microbial ecosystems, geochemical factors that constrain the biomass, diversity, and metabolic activities of microorganisms remain to be clearly defined. To better understand the geochemical and microbiological relationships, we characterized granitic groundwater collected from a 1,148- to 1,169-m-deep borehole interval at the Mizunami Underground Research Laboratory site, Japan, in 2005 and 2008. Geochemical analyses of the groundwater samples indicated that major electron acceptors, such as NO(3)(-) and SO(4)(2-), were not abundant, while dissolved organic carbon (not including organic acids), CH(4) and H(2), was moderately rich in the groundwater sample collected in 2008. The total number of acridine orange-stained cells in groundwater samples collected in 2005 and 2008 were 1.1 x 10(4) and 5.2 x 10(4) cells/mL, respectively. In 2005 and 2008, the most common phylotypes determined by 16S rRNA gene sequence analysis were both related to Thauera spp., the cultivated members of which can utilize minor electron donors, such as aromatic and aliphatic hydrocarbons. After a 3-5-week incubation period with potential electron donors (organic acids or CH(4) + H(2)) and with/without electron acceptors (O(2) or NO(3)(-)), dominant microbial populations shifted to Brevundimonas spp. These geomicrobiological results suggest that deep granitic groundwater has been stably colonized by Thauera spp. probably owing to the limitation of O(2), NO(3)(-), and organic acids.

A solution for universal classification of species based on genomic DNA.

Traditionally, organisms have been classified on the basis of their phenotype. Recently, genotype-based classification has become possible through the development of sequencing technology. However, it is still difficult to apply sequencing approaches to the analysis of a large number of species due to the cost and labor. In most biological fields, the analysis of complex systems comprising various species has become an important theme, demanding an effective method for handling a vast number of species. In this paper, we have demonstrated, using plants, fish, and insects, that genome profiling, a compact technology for genome analysis, can classify organisms universally. Surprisingly, in all three of the domains of organisms tested, the phylogenetic trees generated from the phenotype topologically matched completely those generated from the genotype. Furthermore, a single probe was sufficient for the genome profiling, thereby demonstrating that this methodology is universal and compact.

Acquisition of genome information from single-celled unculturable organisms (radiolaria) by exploiting genome profiling (GP).

BACKGROUND: There is no effective method to obtain genome information from single-celled unculturable organisms such as radiolarians. Even worse, such organisms are often very difficult to collect. Sequence analysis of 18S rDNA has been carried out, but obtaining the data has been difficult and it has provided a rather limited amount of genome information. In this paper, we have developed a method which provides a sufficient amount of data from an unculturable organism. The effectiveness of this method was demonstrated by applying it to the provisional classification of a set of unculturable organisms (radiolarians). RESULTS: Dendrogram was drawn regarding the single-celled unculturable species based on the similarity score termed PaSS, offering a consistent result with the conventional taxonomy of them built up based on phenotypes. This fact has shown that genome profiling-based technology developed here can obtain genome information being sufficient for identifying and classifying species from a single-celled organism. CONCLUSION: Since this method is so simple, general, and yet powerful, it can be applied to various organisms and cells, especially single-celled, uncluturable ones, for their genome analysis.