Publications by authors named "Tullis Onstott"

37 Publications

Ancestral Absence of Electron Transport Chains in Patescibacteria and DPANN.

Front Microbiol 2020 17;11:1848. Epub 2020 Aug 17.

Bigelow Laboratory for Ocean Sciences, East Boothbay, ME, United States.

Recent discoveries suggest that the candidate superphyla Patescibacteria and DPANN constitute a large fraction of the phylogenetic diversity of Bacteria and Archaea. Their small genomes and limited coding potential have been hypothesized to be ancestral adaptations to obligate symbiotic lifestyles. To test this hypothesis, we performed cell-cell association, genomic, and phylogenetic analyses on 4,829 individual cells of Bacteria and Archaea from 46 globally distributed surface and subsurface field samples. This confirmed the ubiquity and abundance of Patescibacteria and DPANN in subsurface environments, the small size of their genomes and cells, and the divergence of their gene content from other Bacteria and Archaea. Our analyses suggest that most Patescibacteria and DPANN in the studied subsurface environments do not form specific physical associations with other microorganisms. These data also suggest that their unusual genomic features and prevalent auxotrophies may be a result of ancestral, minimal cellular energy transduction mechanisms that lack respiration, thus relying solely on fermentation for energy conservation.
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http://dx.doi.org/10.3389/fmicb.2020.01848DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7507113PMC
August 2020

Thaumarchaea Genome Sequences from a High Arctic Active Layer.

Microbiol Resour Announc 2020 May 21;9(21). Epub 2020 May 21.

Department of Geosciences, Princeton University, Princeton, New Jersey, USA

The role of archaeal ammonia oxidizers often exceeds that of bacterial ammonia oxidizers in marine and terrestrial environments but has been understudied in permafrost, where thawing has the potential to release ammonia. Here, three thaumarchaea genomes were assembled and annotated from metagenomic data sets from carbon-poor Canadian High Arctic active-layer cryosols.
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http://dx.doi.org/10.1128/MRA.00326-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7242670PMC
May 2020

The genome of a subterrestrial nematode reveals adaptations to heat.

Nat Commun 2019 11 21;10(1):5268. Epub 2019 Nov 21.

Biology Department, American University, Washington, DC, 20016, USA.

The nematode Halicephalobus mephisto was originally discovered inhabiting a deep terrestrial aquifer 1.3 km underground. H. mephisto can thrive under conditions of abiotic stress including heat and minimal oxygen, where it feeds on a community of both chemolithotrophic and heterotrophic prokaryotes in an unusual ecosystem isolated from the surface biosphere. Here we report the comprehensive genome and transcriptome of this organism, identifying a signature of adaptation: an expanded repertoire of 70 kilodalton heat-shock proteins (Hsp70) and avrRpt2 induced gene 1 (AIG1) proteins. The expanded Hsp70 genes are transcriptionally induced upon growth under heat stress, and we find that positive selection is detectable in several members of this family. We further show that AIG1 may have been acquired by horizontal gene transfer (HGT) from a rhizobial fungus. Over one-third of the genes of H. mephisto are novel, highlighting the divergence of this nematode from other sequenced organisms. This work sheds light on the genomic basis of heat tolerance in a complete subterrestrial eukaryotic genome.
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http://dx.doi.org/10.1038/s41467-019-13245-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6872716PMC
November 2019

Metagenome-Assembled Genome of USCα AHI, a Potential High-Affinity Methanotroph from Axel Heiberg Island, Canadian High Arctic.

Microbiol Resour Announc 2019 Nov 14;8(46). Epub 2019 Nov 14.

Department of Geosciences, Princeton University, Princeton, New Jersey, USA

Metagenomic sequencing of active-layer cryosols from the Canadian High Arctic has yielded a nearly complete genome for an atmospheric CH-oxidizing bacterium belonging to upland soil cluster α (USCα). This genome contains genes involved in CH metabolism, H metabolism, and multiple carbon assimilation pathways.
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http://dx.doi.org/10.1128/MRA.01178-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6856278PMC
November 2019

gen. nov. sp. nov., a Novel Fumarate-Fermenting Microorganism From a Deep Fractured Carbonate Aquifer of the US Great Basin.

Front Microbiol 2019 27;10:2224. Epub 2019 Sep 27.

Division of Earth and Ecosystems Sciences, Desert Research Institute, Las Vegas, NV, United States.

Deep fractured rock ecosystems across most of North America have not been studied extensively. However, the US Great Basin, in particular the Nevada National Security Site (NNSS, formerly the Nevada Test Site), has hosted a number of influential subsurface investigations over the years. This investigation focuses on resident microbiota recovered from a hydrogeologically confined aquifer in fractured Paleozoic carbonate rocks at 863 - 923 meters below land surface. Analysis of the microorganisms living in this oligotrophic environment provides a perspective into microbial metabolic strategies required to endure prolonged hydrogeological isolation deep underground. Here we present a microbiological and physicochemical characterization of a deep continental carbonate ecosystem and describe a bacterial genus isolated from the ecosystem. Strain DRI-13 is a strictly anaerobic, moderately thermophilic, fumarate-respiring member of the phylum . This bacterium grows optimally at 55°C and pH 8.0, can tolerate a concentration of 100 mM NaCl, and appears to obligately metabolize fumarate to acetate and succinate. Culture-independent 16S rRNA gene sequencing indicates a global subsurface distribution, while the closest cultured relatives of DRI-13 are (90.0% similarity) and (88.0% similarity). The predominant fatty acid profile is iso-C, C, C and C. The percentage of the straight-chain fatty acid C is a defining characteristic not present in the other closely related species. The genome is estimated to be 3,649,665 bp, composed of 87.3% coding regions with an overall average of 45.1% G + C content. Strain DRI-13 represents a novel genus of subsurface bacterium isolated from a previously uncharacterized rock-hosted geothermal habitat. The characterization of the bacterium combined with the sequenced genome provides insights into metabolism strategies of the deep subsurface biosphere. Based on our characterization analysis we propose the name (DRI-13 = DSM 100382 = ATCC TSD-12).
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http://dx.doi.org/10.3389/fmicb.2019.02224DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6776889PMC
September 2019

Aspartic acid racemization constrains long-term viability and longevity of endospores.

FEMS Microbiol Ecol 2019 10;95(10)

Department of Geosciences, Princeton University, Princeton, NJ, 08544, USA.

Certain microorganisms survive long periods of time as endospores to cope with adverse conditions. Since endospores are metabolically inactive, the extent of aspartic acid (Asp) racemization will increase over time and might kill the spores by preventing their germination. Therefore, understanding the relationship between endospore survivability and Asp racemization is important for constraining the long-term survivability and global dispersion of spore-forming bacteria in nature. Geobacillus stearothermophilus was selected as a model organism to investigate racemization kinetics and survivability of its endospores at 65°C, 75°C and 98°C. This study found that the Asp racemization rates of spores and autoclaved spores were similar at all temperatures. The Asp racemization rate of spores was not significantly different from that of vegetative cells at 65°C. The Asp racemization rate of G. stearothermophilus spores was not significantly different from that of Bacillus subtilis spores at 98°C. The viability of spores and vegetative cells decreased dramatically over time, and the mortality of spores correlated exponentially with the degree of racemization (R2 = 0.9). This latter correlation predicts spore half-lives on the order of hundreds of years for temperatures typical of shallow marine sediments, a result consistent with studies about the survivability of thermophilic spores found in these environments.
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http://dx.doi.org/10.1093/femsec/fiz132DOI Listing
October 2019

Cretaceous dinosaur bone contains recent organic material and provides an environment conducive to microbial communities.

Elife 2019 06 18;8. Epub 2019 Jun 18.

Department of Geosciences, Princeton University, Princeton, United States.

Fossils were thought to lack original organic molecules, but chemical analyses show that some can survive. Dinosaur bone has been proposed to preserve collagen, osteocytes, and blood vessels. However, proteins and labile lipids are diagenetically unstable, and bone is a porous open system, allowing microbial/molecular flux. These 'soft tissues' have been reinterpreted as biofilms. Organic preservation versus contamination of dinosaur bone was examined by freshly excavating, with aseptic protocols, fossils and sedimentary matrix, and chemically/biologically analyzing them. Fossil 'soft tissues' differed from collagen chemically and structurally; while degradation would be expected, the patterns observed did not support this. 16S rRNA amplicon sequencing revealed that dinosaur bone hosted an abundant microbial community different from lesser abundant communities of surrounding sediment. Subsurface dinosaur bone is a relatively fertile habitat, attracting microbes that likely utilize inorganic nutrients and complicate identification of original organic material. There exists potential post-burial taphonomic roles for subsurface microorganisms.
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http://dx.doi.org/10.7554/eLife.46205DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6581507PMC
June 2019

Predominance of Anaerobic, Spore-Forming Bacteria in Metabolically Active Microbial Communities from Ancient Siberian Permafrost.

Appl Environ Microbiol 2019 08 18;85(15). Epub 2019 Jul 18.

Princeton University, Princeton, New Jersey, USA.

The prevalence of microbial life in permafrost up to several million years (Ma) old has been well documented. However, the long-term survivability, evolution, and metabolic activity of the entombed microbes over this time span remain underexplored. We integrated aspartic acid (Asp) racemization assays with metagenomic sequencing to characterize the microbial activity, phylogenetic diversity, and metabolic functions of indigenous microbial communities across a ∼0.01- to 1.1-Ma chronosequence of continuously frozen permafrost from northeastern Siberia. Although Asp in the older bulk sediments (0.8 to 1.1 Ma) underwent severe racemization relative to that in the youngest sediment (∼0.01 Ma), the much lower d-Asp/l-Asp ratio (0.05 to 0.14) in the separated cells from all samples suggested that indigenous microbial communities were viable and metabolically active in ancient permafrost up to 1.1 Ma. The microbial community in the youngest sediment was the most diverse and was dominated by the phyla and In contrast, microbial diversity decreased dramatically in the older sediments, and anaerobic, spore-forming bacteria within became overwhelmingly dominant. In addition to the enrichment of sporulation-related genes, functional genes involved in anaerobic metabolic pathways such as fermentation, sulfate reduction, and methanogenesis were more abundant in the older sediments. Taken together, the predominance of spore-forming bacteria and associated anaerobic metabolism in the older sediments suggest that a subset of the original indigenous microbial community entrapped in the permafrost survived burial over geological time. Understanding the long-term survivability and associated metabolic traits of microorganisms in ancient permafrost frozen millions of years ago provides a unique window into the burial and preservation processes experienced in general by subsurface microorganisms in sedimentary deposits because of permafrost's hydrological isolation and exceptional DNA preservation. We employed aspartic acid racemization modeling and metagenomics to determine which microbial communities were metabolically active in the 1.1-Ma permafrost from northeastern Siberia. The simultaneous sequencing of extracellular and intracellular genomic DNA provided insight into the metabolic potential distinguishing extinct from extant microorganisms under frozen conditions over this time interval. This in-depth metagenomic sequencing advances our understanding of the microbial diversity and metabolic functions of extant microbiomes from early Pleistocene permafrost. Therefore, these findings extend our knowledge of the survivability of microbes in permafrost from 33,000 years to 1.1 Ma.
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http://dx.doi.org/10.1128/AEM.00560-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6643238PMC
August 2019

Denitrifiers, nitrogen-fixing bacteria and N2O soil gas flux in high Arctic ice-wedge polygon cryosols.

FEMS Microbiol Ecol 2019 05;95(5)

Department of Natural Resource Sciences, Faculty of Agricultural and Environmental Sciences, Macdonald Campus, McGill University, 21,111 Lakeshore Rd, Ste Anne-de-Bellevue, QC, H9X 3V9, Canada.

Climate warming and subsequent permafrost thaw may result in organic carbon and nutrient stores being metabolized by microbial communities, resulting in a positive feedback loop of greenhouse gas (GHG) soil emissions. As the third most important GHG, understanding nitrous oxide (N2O) flux in Arctic mineral ice-wedge polygon cryosols and its relationship to the active microbial community is potentially a key parameter for understanding future GHG emissions and climatic warming potential. In the present study, metatranscriptomic analyses of active layer Arctic cryosols, at a representative ice-wedge polygon site, identified active nitrogen-fixing and denitrifying bacteria that included members of Rhizobiaceae, Nostocaceae, Cyanothecaceae, Rhodobacteraceae, Burkholderiaceae, Chloroflexaceae, Azotobacteraceae and Ectothiorhodospiraceae. Unique microbial assemblages with higher proportion of Rhodobacteriales and Rhocyclales were identified by targeted functional gene sequencing at locations with higher (P = 0.053) N2O emissions in the wetter trough soils compared with the dryer polygon interior soils. This coincided with a higher relative abundance of the denitrification nirS gene and higher nitrate/nitrite concentrations in trough soils. The elevated N2O flux observed from wetter trough soils compared with drier polygon interior soils is concerning from a climate warming perspective, since the Arctic is predicted to become warmer and wetter.
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http://dx.doi.org/10.1093/femsec/fiz049DOI Listing
May 2019

Draft Genome Sequence of " Bathyarchaeota" Archaeon BE326-BA-RLH, an Uncultured Denitrifier and Putative Anaerobic Methanotroph from South Africa's Deep Continental Biosphere.

Microbiol Resour Announc 2018 Nov 21;7(20). Epub 2018 Nov 21.

Department of Geosciences, Princeton University, Princeton, New Jersey, USA.

Metagenomic sequencing of fracture fluid from South Africa recovered a nearly complete " Bathyarchaeota" archaeon genome. The metagenome-assembled genome of BE326-BA-RLH contains genes involved in methane metabolism and dissimilatory nitrate reduction. This study presents the first genomic evidence for potential anaerobic methane oxidation in the phylum " Bathyarchaeota."
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http://dx.doi.org/10.1128/MRA.01295-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6256629PMC
November 2018

Valuing Life-Detection Missions.

Astrobiology 2018 07;18(7):834-840

3 Princeton University , Princeton, New Jersey.

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http://dx.doi.org/10.1089/ast.2017.1813DOI Listing
July 2018

Taxonomic and Functional Compositions Impacted by the Quality of Metatranscriptomic Assemblies.

Front Microbiol 2018 20;9:1235. Epub 2018 Jun 20.

Department of Geosciences, Princeton University, Princeton, NJ, United States.

Metatranscriptomics has recently been applied to investigate the active biogeochemical processes and elemental cycles, and responses of microbiomes to environmental stimuli and stress factors. assembly of RNA-Sequencing (RNA-Seq) data can reveal a more detailed description of the metabolic interactions amongst the active microbial communities. However, the quality of the assemblies and the depiction of the metabolic network provided by various assemblers have not yet been thoroughly assessed. In this study, we compared 15 metatranscriptomic assemblies for a fracture fluid sample collected from a borehole located at 1.34 km below land surface in a South African gold mine. These assemblies were constructed from total, non-coding, and coding reads using five transcriptomic assemblers (Trans-ABySS, Trinity, Oases, IDBA-tran, and Rockhopper). They were evaluated based on the number of transcripts, transcript length, range of transcript coverage, continuity, percentage of transcripts with confident annotation assignments, as well as taxonomic and functional diversity patterns. The results showed that these parameters varied considerably among the assemblies, with Trans-ABySS and Trinity generating the best assemblies for non-coding and coding RNA reads, respectively, because the high number of transcripts assembled covered a wide expression range, and captured extensively the taxonomic and metabolic gene diversity, respectively. We concluded that the choice of transcriptomic assemblers impacts substantially the taxonomic and functional compositions. Care should be taken to obtain high-quality assemblies for informing the metabolic landscape.
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http://dx.doi.org/10.3389/fmicb.2018.01235DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6019464PMC
June 2018

Rokubacteria: Genomic Giants among the Uncultured Bacterial Phyla.

Front Microbiol 2017 28;8:2264. Epub 2017 Nov 28.

Bigelow Laboratory for Ocean Sciences, East Boothbay, ME, United States.

Recent advances in single-cell genomic and metagenomic techniques have facilitated the discovery of numerous previously unknown, deep branches of the tree of life that lack cultured representatives. Many of these candidate phyla are composed of microorganisms with minimalistic, streamlined genomes lacking some core metabolic pathways, which may contribute to their resistance to growth in pure culture. Here we analyzed single-cell genomes and metagenome bins to show that the "Candidate phylum Rokubacteria," formerly known as SPAM, represents an interesting exception, by having large genomes (6-8 Mbps), high GC content (66-71%), and the potential for a versatile, mixotrophic metabolism. We also observed an unusually high genomic heterogeneity among individual Rokubacteria cells in the studied samples. These features may have contributed to the limited recovery of sequences of this candidate phylum in prior cultivation and metagenomic studies. Our analyses suggest that Rokubacteria are distributed globally in diverse terrestrial ecosystems, including soils, the rhizosphere, volcanic mud, oil wells, aquifers, and the deep subsurface, with no reports from marine environments to date.
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http://dx.doi.org/10.3389/fmicb.2017.02264DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5712423PMC
November 2017

Draft Genome Sequence of Uncultured Upland Soil Cluster Gives Molecular Insights into High-Affinity Methanotrophy.

Genome Announc 2017 Apr 27;5(17). Epub 2017 Apr 27.

Department of Geosciences, Princeton University, Princeton, New Jersey, USA

Aerated soils form the second largest sink for atmospheric CH A near-complete genome of uncultured upland soil cluster that oxidize CH at <2.5 ppmv was obtained from incubated Antarctic mineral cryosols. This first genome of high-affinity methanotrophs can help resolve the mysteries about their phylogenetic affiliation and metabolic potential.
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http://dx.doi.org/10.1128/genomeA.00047-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5408097PMC
April 2017

An oligotrophic deep-subsurface community dependent on syntrophy is dominated by sulfur-driven autotrophic denitrifiers.

Proc Natl Acad Sci U S A 2016 12 21;113(49):E7927-E7936. Epub 2016 Nov 21.

Department of Geosciences, Princeton University, Princeton, NJ 08544.

Subsurface lithoautotrophic microbial ecosystems (SLiMEs) under oligotrophic conditions are typically supported by H Methanogens and sulfate reducers, and the respective energy processes, are thought to be the dominant players and have been the research foci. Recent investigations showed that, in some deep, fluid-filled fractures in the Witwatersrand Basin, South Africa, methanogens contribute <5% of the total DNA and appear to produce sufficient CH to support the rest of the diverse community. This paradoxical situation reflects our lack of knowledge about the in situ metabolic diversity and the overall ecological trophic structure of SLiMEs. Here, we show the active metabolic processes and interactions in one of these communities by combining metatranscriptomic assemblies, metaproteomic and stable isotopic data, and thermodynamic modeling. Dominating the active community are four autotrophic β-proteobacterial genera that are capable of oxidizing sulfur by denitrification, a process that was previously unnoticed in the deep subsurface. They co-occur with sulfate reducers, anaerobic methane oxidizers, and methanogens, which each comprise <5% of the total community. Syntrophic interactions between these microbial groups remove thermodynamic bottlenecks and enable diverse metabolic reactions to occur under the oligotrophic conditions that dominate in the subsurface. The dominance of sulfur oxidizers is explained by the availability of electron donors and acceptors to these microorganisms and the ability of sulfur-oxidizing denitrifiers to gain energy through concomitant S and H oxidation. We demonstrate that SLiMEs support taxonomically and metabolically diverse microorganisms, which, through developing syntrophic partnerships, overcome thermodynamic barriers imposed by the environmental conditions in the deep subsurface.
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http://dx.doi.org/10.1073/pnas.1612244113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5150411PMC
December 2016

Deep subsurface mine stalactites trap endemic fissure fluid Archaea, Bacteria, and Nematoda possibly originating from ancient seas.

Front Microbiol 2015 11;6:833. Epub 2015 Aug 11.

Department of Biotechnology, University of the Free State Bloemfontein, South Africa.

Stalactites (CaCO3 and salt) from water seeps are frequently encountered in ceilings of mine tunnels whenever they intersect water-bearing faults or fractures. To determine whether stalactites could be mineralized traps for indigenous fracture water microorganisms, we analyzed stalactites collected from three different mines ranging in depth from 1.3 to 3.1 km. During sampling in Beatrix gold mine (1.4 km beneath the surface), central South Africa, CaCO3 stalactites growing on the mine tunnel ceiling were collected and observed, in two cases, to contain a living obligate brackish water/marine nematode species, Monhystrella parvella. After sterilization of the outer surface, mineral layers were physically removed from the outside to the interior, and DNA extracted. Based upon 16S and 18S rRNA gene sequencing, Archaea, Bacteria, and Eukarya in different combinations were detected for each layer. Using CT scan and electron microscopy the inner structure of CaCO3 and salt stalactites were analyzed. CaCO3 stalactites show a complex pattern of lamellae carrying bacterially precipitated mineral structures. Nematoda were clearly identified between these layers confirming that bacteria and nematodes live inside the stalactites and not only in the central straw. Salt stalactites exhibit a more uniform internal structure. Surprisingly, several Bacteria showing highest sequence identities to marine species were identified. This, together with the observation that the nematode M. parvella recovered from Beatrix gold mine stalactite can only survive in a salty environment makes the origin of the deep subsurface colonization enigmatic. The possibility of a Permian origin of fracture fluids is discussed. Our results indicate stalactites are suitable for biodiversity recovery and act as natural traps for microorganisms in the fissure water long after the water that formed the stalactite stopped flowing.
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http://dx.doi.org/10.3389/fmicb.2015.00833DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4585192PMC
October 2015

A metagenomic window into carbon metabolism at 3 km depth in Precambrian continental crust.

ISME J 2016 Mar 1;10(3):730-41. Epub 2015 Sep 1.

Department of Geosciences, Guyot Hall, Princeton University, Princeton, NJ, USA.

Subsurface microbial communities comprise a significant fraction of the global prokaryotic biomass; however, the carbon metabolisms that support the deep biosphere have been relatively unexplored. In order to determine the predominant carbon metabolisms within a 3-km deep fracture fluid system accessed via the Tau Tona gold mine (Witwatersrand Basin, South Africa), metagenomic and thermodynamic analyses were combined. Within our system of study, the energy-conserving reductive acetyl-CoA (Wood-Ljungdahl) pathway was found to be the most abundant carbon fixation pathway identified in the metagenome. Carbon monoxide dehydrogenase genes that have the potential to participate in (1) both autotrophic and heterotrophic metabolisms through the reversible oxidization of CO and subsequent transfer of electrons for sulfate reduction, (2) direct utilization of H2 and (3) methanogenesis were identified. The most abundant members of the metagenome belonged to Euryarchaeota (22%) and Firmicutes (57%)-by far, the highest relative abundance of Euryarchaeota yet reported from deep fracture fluids in South Africa and one of only five Firmicutes-dominated deep fracture fluids identified in the region. Importantly, by combining the metagenomics data and thermodynamic modeling of this study with previously published isotopic and community composition data from the South African subsurface, we are able to demonstrate that Firmicutes-dominated communities are associated with a particular hydrogeologic environment, specifically the older, more saline and more reducing waters.
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http://dx.doi.org/10.1038/ismej.2015.150DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4817676PMC
March 2016

Single cell genomics indicates horizontal gene transfer and viral infections in a deep subsurface Firmicutes population.

Front Microbiol 2015 22;6:349. Epub 2015 Apr 22.

Bigelow Laboratory for Ocean Sciences East Boothbay, ME, USA.

A major fraction of Earth's prokaryotic biomass dwells in the deep subsurface, where cellular abundances per volume of sample are lower, metabolism is slower, and generation times are longer than those in surface terrestrial and marine environments. How these conditions impact biotic interactions and evolutionary processes is largely unknown. Here we employed single cell genomics to analyze cell-to-cell genome content variability and signatures of horizontal gene transfer (HGT) and viral infections in five cells of Candidatus Desulforudis audaxviator, which were collected from a 3 km-deep fracture water in the 2.9 Ga-old Witwatersrand Basin of South Africa. Between 0 and 32% of genes recovered from single cells were not present in the original, metagenomic assembly of Desulforudis, which was obtained from a neighboring subsurface fracture. We found a transposable prophage, a retron, multiple clustered regularly interspaced short palindromic repeats (CRISPRs) and restriction-modification systems, and an unusually high frequency of transposases in the analyzed single cell genomes. This indicates that recombination, HGT and viral infections are prevalent evolutionary events in the studied population of microorganisms inhabiting a highly stable deep subsurface environment.
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http://dx.doi.org/10.3389/fmicb.2015.00349DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4406082PMC
May 2015

Comparisons of the composition and biogeographic distribution of the bacterial communities occupying South African thermal springs with those inhabiting deep subsurface fracture water.

Front Microbiol 2014 17;5:679. Epub 2014 Dec 17.

Department of Geosciences, Princeton University Princeton, NJ, USA.

South Africa has numerous thermal springs that represent topographically driven meteoric water migrating along major fracture zones. The temperature (40-70°C) and pH (8-9) of the thermal springs in the Limpopo Province are very similar to those of the low salinity fracture water encountered in the South African mines at depths ranging from 1.0 to 3.1 km. The major cation and anion composition of these thermal springs are very similar to that of the deep fracture water with the exception of the dissolved inorganic carbon and dissolved O2, both of which are typically higher in the springs than in the deep fracture water. The in situ biological relatedness of such thermal springs and the subsurface fracture fluids that feed them has not previously been evaluated. In this study, we evaluated the microbial diversity of six thermal spring and six subsurface sites in South Africa using high-throughput sequencing of 16S rRNA gene hypervariable regions. Proteobacteria were identified as the dominant phylum within both subsurface and thermal spring environments, but only one genera, Rheinheimera, was identified among all samples. Using Morisita similarity indices as a metric for pairwise comparisons between sites, we found that the communities of thermal springs are highly distinct from subsurface datasets. Although the Limpopo thermal springs do not appear to provide a new window for viewing subsurface bacterial communities, we report that the taxonomic compositions of the subsurface sites studied are more similar than previous results would indicate and provide evidence that the microbial communities sampled at depth are more correlated to subsurface conditions than geographical distance.
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http://dx.doi.org/10.3389/fmicb.2014.00679DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4269199PMC
January 2015

Direct measurements of methane emissions from abandoned oil and gas wells in Pennsylvania.

Proc Natl Acad Sci U S A 2014 Dec 8;111(51):18173-7. Epub 2014 Dec 8.

Geosciences Department, Princeton University, Princeton, NJ 08544.

Abandoned oil and gas wells provide a potential pathway for subsurface migration and emissions of methane and other fluids to the atmosphere. Little is known about methane fluxes from the millions of abandoned wells that exist in the United States. Here, we report direct measurements of methane fluxes from abandoned oil and gas wells in Pennsylvania, using static flux chambers. A total of 42 and 52 direct measurements were made at wells and at locations near the wells ("controls") in forested, wetland, grassland, and river areas in July, August, October 2013 and January 2014, respectively. The mean methane flow rates at these well locations were 0.27 kg/d/well, and the mean methane flow rate at the control locations was 4.5 × 10(-6) kg/d/location. Three out of the 19 measured wells were high emitters that had methane flow rates that were three orders of magnitude larger than the median flow rate of 1.3 × 10(-3) kg/d/well. Assuming the mean flow rate found here is representative of all abandoned wells in Pennsylvania, we scaled the methane emissions to be 4-7% of estimated total anthropogenic methane emissions in Pennsylvania. The presence of ethane, propane, and n-butane, along with the methane isotopic composition, indicate that the emitted methane is predominantly of thermogenic origin. These measurements show that methane emissions from abandoned oil and gas wells can be significant. The research required to quantify these emissions nationally should be undertaken so they can be accurately described and included in greenhouse gas emissions inventories.
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http://dx.doi.org/10.1073/pnas.1408315111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4280601PMC
December 2014

Metagenomes from thawing low-soil-organic-carbon mineral cryosols and permafrost of the canadian high arctic.

Genome Announc 2014 Nov 20;2(6). Epub 2014 Nov 20.

Microbial release of greenhouse gases from thawing permafrost is a global concern. Seventy-six metagenomes were generated from low-soil-organic-carbon mineral cryosols from Axel Heiberg Island, Nunavut, Canada, during a controlled thawing experiment. Permafrost thawing resulted in an increase in anaerobic fermenters and sulfate-reducing bacteria but not methanogens.
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http://dx.doi.org/10.1128/genomeA.01217-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4239366PMC
November 2014

Phylogeny and phylogeography of functional genes shared among seven terrestrial subsurface metagenomes reveal N-cycling and microbial evolutionary relationships.

Front Microbiol 2014 31;5:531. Epub 2014 Oct 31.

Department of Geosciences, Princeton University Princeton, NJ, USA.

Comparative studies on community phylogenetics and phylogeography of microorganisms living in extreme environments are rare. Terrestrial subsurface habitats are valuable for studying microbial biogeographical patterns due to their isolation and the restricted dispersal mechanisms. Since the taxonomic identity of a microorganism does not always correspond well with its functional role in a particular community, the use of taxonomic assignments or patterns may give limited inference on how microbial functions are affected by historical, geographical and environmental factors. With seven metagenomic libraries generated from fracture water samples collected from five South African mines, this study was carried out to (1) screen for ubiquitous functions or pathways of biogeochemical cycling of CH4, S, and N; (2) to characterize the biodiversity represented by the common functional genes; (3) to investigate the subsurface biogeography as revealed by this subset of genes; and (4) to explore the possibility of using metagenomic data for evolutionary study. The ubiquitous functional genes are NarV, NPD, PAPS reductase, NifH, NifD, NifK, NifE, and NifN genes. Although these eight common functional genes were taxonomically and phylogenetically diverse and distinct from each other, the dissimilarity between samples did not correlate strongly with geographical or environmental parameters or residence time of the water. Por genes homologous to those of Thermodesulfovibrio yellowstonii detected in all metagenomes were deep lineages of Nitrospirae, suggesting that subsurface habitats have preserved ancestral genetic signatures that inform the study of the origin and evolution of prokaryotes.
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http://dx.doi.org/10.3389/fmicb.2014.00531DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4215791PMC
November 2014

Trends and future challenges in sampling the deep terrestrial biosphere.

Front Microbiol 2014 12;5:481. Epub 2014 Sep 12.

Department of Geological Sciences, Michigan State University East Lansing, MI, USA.

Research in the deep terrestrial biosphere is driven by interest in novel biodiversity and metabolisms, biogeochemical cycling, and the impact of human activities on this ecosystem. As this interest continues to grow, it is important to ensure that when subsurface investigations are proposed, materials recovered from the subsurface are sampled and preserved in an appropriate manner to limit contamination and ensure preservation of accurate microbial, geochemical, and mineralogical signatures. On February 20th, 2014, a workshop on "Trends and Future Challenges in Sampling The Deep Subsurface" was coordinated in Columbus, Ohio by The Ohio State University and West Virginia University faculty, and sponsored by The Ohio State University and the Sloan Foundation's Deep Carbon Observatory. The workshop aims were to identify and develop best practices for the collection, preservation, and analysis of terrestrial deep rock samples. This document summarizes the information shared during this workshop.
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http://dx.doi.org/10.3389/fmicb.2014.00481DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4162470PMC
October 2014

Commercial DNA extraction kits impact observed microbial community composition in permafrost samples.

FEMS Microbiol Ecol 2014 Jan 17;87(1):217-30. Epub 2013 Oct 17.

University of Tennessee, Knoxville, TN, USA.

The total community genomic DNA (gDNA) from permafrost was extracted using four commercial DNA extraction kits. The gDNAs were compared using quantitative real-time PCR (qPCR) targeting 16S rRNA genes and bacterial diversity analyses obtained via 454 pyrosequencing of the 16S rRNA (V3 region) amplified in single or nested PCR. The FastDNA(®) SPIN (FDS) Kit provided the highest gDNA yields and 16S rRNA gene concentrations, followed by MoBio PowerSoil(®) (PS) and MoBio PowerLyzer™ (PL) kits. The lowest gDNA yields and 16S rRNA gene concentrations were from the Meta-G-Nome™ (MGN) DNA Isolation Kit. Bacterial phyla identified in all DNA extracts were similar to that found in other soils and were dominated by Actinobacteria, Firmicutes, Gemmatimonadetes, Proteobacteria, and Acidobacteria. Weighted UniFrac and statistical analyses indicated that bacterial community compositions derived from FDS, PS, and PL extracts were similar to each other. However, the bacterial community structure from the MGN extracts differed from other kits exhibiting higher proportions of easily lysed β- and γ-Proteobacteria and lower proportions of Actinobacteria and Methylocystaceae important in carbon cycling. These results indicate that gDNA yields differ between the extraction kits, but reproducible bacterial community structure analysis may be accomplished using gDNAs from the three bead-beating lysis extraction kits.
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http://dx.doi.org/10.1111/1574-6941.12219DOI Listing
January 2014

A carbon free filter for collection of large volume samples of cellular biomass from oligotrophic waters.

J Microbiol Methods 2012 Sep 3;90(3):145-51. Epub 2012 May 3.

Department of Environmental Science, Barnard College, New York, NY 10025-6598, USA.

Isotopic analysis of cellular biomass has greatly improved our understanding of carbon cycling in the environment. Compound specific radiocarbon analysis (CSRA) of cellular biomass is being increasingly applied in a number of fields. However, it is often difficult to collect sufficient cellular biomass for analysis from oligotrophic waters because easy-to-use filtering methods that are free of carbon contaminants do not exist. The goal of this work was to develop a new column based filter to autonomously collect high volume samples of biomass from oligotrophic waters for CSRA using material that can be baked at 450°C to remove potential organic contaminants. A series of filter materials were tested, including uncoated sand, ferrihydrite-coated sand, goethite-coated sand, aluminum-coated sand, uncoated glass wool, ferrihydrite-coated glass wool, and aluminum-coated glass wool, in the lab with 0.1 and 1.0 μm microspheres and Escherichia coli. Results indicated that aluminum-coated glass wool was the most efficient filter and that the retention capacity of the filter far exceeded the biomass requirements for CSRA. Results from laboratory tests indicate that for oligotrophic waters with 1×10(5) cells ml(-1), 117l of water would need to be filtered to collect 100 μg of PLFA for bulk PLFA analysis and 2000 l for analysis of individual PLFAs. For field sampling, filtration tests on South African mine water indicated that after filtering 5955l, 450 μg of total PLFAs were present, ample biomass for radiocarbon analysis. In summary, we have developed a filter that is easy to use and deploy for collection of biomass for CSRA including total and individual PLFAs.
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http://dx.doi.org/10.1016/j.mimet.2012.04.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5926177PMC
September 2012

Lessons learned from bacterial transport research at the South Oyster Site.

Ground Water 2011 Sep-Oct;49(5):745-63. Epub 2011 Jun 15.

Pacific Northwest National Laboratory, P.O. Box 999, MS K9-36, Richland, WA 99352, USA.

This paper provides a review of bacterial transport experiments conducted by a multiinvestigator, multiinstitution, multidisciplinary team of researchers under the auspices of the U.S. Department of Energy (DOE). The experiments were conducted during the time period 1999-2001 at a field site near the town of Oyster, Virginia known as the South Oyster Site, and included four major experimental campaigns aimed at understanding and quantifying bacterial transport in the subsurface environment. Several key elements of the research are discussed here: (1) quantification of bacterial transport in physically, chemically, and biologically heterogeneous aquifers, (2) evaluation of the efficacy of conventional colloid filtration theory, (3) scale effects in bacterial transport, (4) development of new methods for microbial enumeration and screening for low adhesion strains, (5) application of novel hydrogeophysical techniques for aquifer characterization, and (6) experiences regarding management of a large field research effort. Lessons learned are summarized in each of these areas. The body of literature resulting from South Oyster Site research has been widely cited and continues to influence research into the controls exerted by aquifer heterogeneity on reactive transport (including microbial transport). It also served as a model (and provided valuable experience) for subsequent and ongoing highly-instrumented field research efforts conducted by DOE-sponsored investigators.
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http://dx.doi.org/10.1111/j.1745-6584.2011.00831.xDOI Listing
December 2011

Hydrogeology, chemical and microbial activity measurement through deep permafrost.

Ground Water 2011 May-Jun;49(3):348-64

Department of Earth Sciences, University of Waterloo, Waterloo, Ontario, Canada.

Little is known about hydrogeochemical conditions beneath thick permafrost, particularly in fractured crystalline rock, due to difficulty in accessing this environment. The purpose of this investigation was to develop methods to obtain physical, chemical, and microbial information about the subpermafrost environment from a surface-drilled borehole. Using a U-tube, gas and water samples were collected, along with temperature, pressure, and hydraulic conductivity measurements, 420 m below ground surface, within a 535 m long, angled borehole at High Lake, Nunavut, Canada, in an area with 460-m-thick permafrost. Piezometric head was well above the base of the permafrost, near land surface. Initial water samples were contaminated with drill fluid, with later samples <40% drill fluid. The salinity of the non-drill fluid component was <20,000 mg/L, had a Ca/Na ratio above 1, with δ(18) O values ∼5‰ lower than the local surface water. The fluid isotopic composition was affected by the permafrost-formation process. Nonbacteriogenic CH(4) was present and the sample location was within methane hydrate stability field. Sampling lines froze before uncontaminated samples from the subpermafrost environment could be obtained, yet the available time to obtain water samples was extended compared to previous studies. Temperature measurements collected from a distributed temperature sensor indicated that this issue can be overcome easily in the future. The lack of methanogenic CH(4) is consistent with the high sulfate concentrations observed in cores. The combined surface-drilled borehole/U-tube approach can provide a large amount of physical, chemical, and microbial data from the subpermafrost environment with few, controllable, sources of contamination.
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http://dx.doi.org/10.1111/j.1745-6584.2010.00724.xDOI Listing
August 2011

Sulfur isotope enrichment during maintenance metabolism in the thermophilic sulfate-reducing bacterium Desulfotomaculum putei.

Appl Environ Microbiol 2009 Sep 26;75(17):5621-30. Epub 2009 Jun 26.

Department of Geosciences, Princeton University, Princeton, NJ 08544, USA.

Values of Delta(34)S (=delta(34)S(HS)-delta(34)S(SO(4)), where delta(34)S(HS) and delta(34)S(SO(4)) indicate the differences in the isotopic compositions of the HS(-) and SO(4)(2-) in the eluent, respectively) for many modern marine sediments are in the range of -55 to -75 per thousand, much greater than the -2 to -46 per thousand epsilon(34)S (kinetic isotope enrichment) values commonly observed for microbial sulfate reduction in laboratory batch culture and chemostat experiments. It has been proposed that at extremely low sulfate reduction rates under hypersulfidic conditions with a nonlimited supply of sulfate, isotopic enrichment in laboratory culture experiments should increase to the levels recorded in nature. We examined the effect of extremely low sulfate reduction rates and electron donor limitation on S isotope fractionation by culturing a thermophilic, sulfate-reducing bacterium, Desulfotomaculum putei, in a biomass-recycling culture vessel, or "retentostat." The cell-specific rate of sulfate reduction and the specific growth rate decreased progressively from the exponential phase to the maintenance phase, yielding average maintenance coefficients of 10(-16) to 10(-18) mol of SO(4) cell(-1) h(-1) toward the end of the experiments. Overall S mass and isotopic balance were conserved during the experiment. The differences in the delta(34)S values of the sulfate and sulfide eluting from the retentostat were significantly larger, attaining a maximum Delta(34)S of -20.9 per thousand, than the -9.7 per thousand observed during the batch culture experiment, but differences did not attain the values observed in marine sediments.
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http://dx.doi.org/10.1128/AEM.02948-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2737900PMC
September 2009