Publications by authors named "Tatiana Vishnivetskaya"

43 Publications

Comparative Metagenomics of the Active Layer and Permafrost from Low-Carbon Soil in the Canadian High Arctic.

Environ Sci Technol 2021 09 2;55(18):12683-12693. Epub 2021 Sep 2.

Center for Environmental Biotechnology, University of Tennessee, Knoxville, Tennessee 37996, United States.

Approximately 87% of the Arctic consists of low-organic carbon mineral soil, but knowledge of microbial activity in low-carbon permafrost (PF) and active layer soils remains limited. This study investigated the taxonomic composition and genetic potential of microbial communities at contrasting depths of the active layer (5, 35, and 65 cm below surface, bls) and PF (80 cm bls). We showed microbial communities in PF to be taxonomically and functionally different from those in the active layer. 16S rRNA gene sequence analysis revealed higher biodiversity in the active layer than in PF, and biodiversity decreased significantly with depth. The reconstructed 91 metagenome-assembled genomes showed that PF was dominated by heterotrophic, fermenting Bacteroidota using nitrite as their main electron acceptor. Prevalent microbes identified in the active layer belonged to bacterial taxa, gaining energy via aerobic respiration. Gene abundance in metagenomes revealed enrichment of genes encoding the plant-derived polysaccharide degradation and metabolism of nitrate and sulfate in PF, whereas genes encoding methane/ammonia oxidation, cold-shock protein, and two-component systems were generally more abundant in the active layer, particularly at 5 cm bls. The results of this study deepen our understanding of the low-carbon Arctic soil microbiome and improve prediction of the impacts of thawing PF.
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http://dx.doi.org/10.1021/acs.est.1c00802DOI Listing
September 2021

Eight Metagenome-Assembled Genomes Provide Evidence for Microbial Adaptation in 20,000- to 1,000,000-Year-Old Siberian Permafrost.

Appl Environ Microbiol 2021 09 10;87(19):e0097221. Epub 2021 Sep 10.

University of Tennessee, Knoxville, Tennessee, USA.

Permafrost microbes may be metabolically active in microscopic layers of liquid brines, even in ancient soil. Metagenomics can help discern whether permafrost microbes show adaptations to this environment. Thirty-three metagenome-assembled genomes (MAGs) were obtained from six depths (3.5 m to 20 m) of freshly cored permafrost from the Siberian Kolyma-Indigirka Lowland region. These soils have been continuously frozen for ∼20,000 to 1,000,000 years. Eight of these MAGs were ≥80% complete with <10% contamination and were taxonomically identified as , , , and within bacteria and within archaea. MAGs from these taxa have been obtained previously from nonpermafrost environments and have been suggested to show adaptations to long-term energy starvation, but they have never been explored in ancient permafrost. The permafrost MAGs had greater proportions in the Clusters of Orthologous Groups (COGs) categories of energy production and conversion and carbohydrate transport and metabolism than did their nonpermafrost counterparts. They also contained genes for trehalose synthesis, thymine metabolism, mevalonate biosynthesis, and cellulose degradation, which were less prevalent in nonpermafrost genomes. Many of these genes are involved in membrane stabilization and osmotic stress responses, consistent with adaptation to the anoxic, high-ionic-strength, cold environments of permafrost brine films. Our results suggest that this ancient permafrost contains DNA of high enough quality to assemble MAGs from microorganisms with adaptations to survive long-term freezing in this extreme environment. Permafrost around the world is thawing rapidly. Many scientists from a variety of disciplines have shown the importance of understanding what will happen to our ecosystem, commerce, and climate when permafrost thaws. The fate of permafrost microorganisms is connected to these predicted rapid environmental changes. Studying ancient permafrost with culture-independent techniques can give a glimpse into how these microorganisms function under these extreme low-temperature and low-energy conditions. This will facilitate understanding how they will change with the environment. This study presents genomic data from this unique environment ∼20,000 to 1,000,000 years of age.
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http://dx.doi.org/10.1128/AEM.00972-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8432575PMC
September 2021

A living bdelloid rotifer from 24,000-year-old Arctic permafrost.

Curr Biol 2021 06;31(11):R712-R713

Soil Cryology Laboratory, Institute of Physicochemical and Biological Problems in Soil Science, Institutskaya 2, 142290, Pushchino, Russia.

In natural, permanently frozen habitats, some organisms may be preserved for hundreds to tens of thousands of years. For example, stems of Antarctic moss were successfully regrown from an over millennium-old sample covered by ice for about 400 years. Likewise, whole campion plants were regenerated from seed tissue preserved in relict 32,000-year-old permafrost, and nematodes were revived from the permafrost of two localities in northeastern Siberia, with source sediments dated over 30,000 years BP. Bdelloid rotifers, microscopic multicellular animals, are known for their ability to survive extremely low temperatures. Previous reports suggest survival after six to ten years when frozen between -20° to 0°C. Here, we report the survival of an obligate parthenogenetic bdelloid rotifer, recovered from northeastern Siberian permafrost radiocarbon-dated to ∼24,000 years BP. This constitutes the longest reported case of rotifer survival in a frozen state. We confirmed the finding by identifying rotifer actin gene sequences in a metagenome obtained from the same sample. By morphological and molecular markers, the discovered rotifer belongs to the genus Adineta, and aligns with a contemporary Adineta vaga isolate collected in Belgium. Experiments demonstrated that the ancient rotifer withstands slow cooling and freezing (∼1°C min) for at least seven days. We also show that a clonal culture can continuously reproduce in the laboratory by parthenogenesis.
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http://dx.doi.org/10.1016/j.cub.2021.04.077DOI Listing
June 2021

Genomic reconstruction of fossil and living microorganisms in ancient Siberian permafrost.

Microbiome 2021 05 17;9(1):110. Epub 2021 May 17.

Princeton University, B88, Guyot Hall, Princeton, NJ, 08544, USA.

Background: Total DNA (intracellular, iDNA and extracellular, eDNA) from ancient permafrost records the mixed genetic repository of the past and present microbial populations through geological time. Given the exceptional preservation of eDNA under perennial frozen conditions, typical metagenomic sequencing of total DNA precludes the discrimination between fossil and living microorganisms in ancient cryogenic environments. DNA repair protocols were combined with high throughput sequencing (HTS) of separate iDNA and eDNA fraction to reconstruct metagenome-assembled genomes (MAGs) from ancient microbial DNA entrapped in Siberian coastal permafrost.

Results: Despite the severe DNA damage in ancient permafrost, the coupling of DNA repair and HTS resulted in a total of 52 MAGs from sediments across a chronosequence (26-120 kyr). These MAGs were compared with those derived from the same samples but without utilizing DNA repair protocols. The MAGs from the youngest stratum showed minimal DNA damage and thus likely originated from viable, active microbial species. Many MAGs from the older and deeper sediment appear related to past aerobic microbial populations that had died upon freezing. MAGs from anaerobic lineages, including Asgard archaea, however exhibited minimal DNA damage and likely represent extant living microorganisms that have become adapted to the cryogenic and anoxic environments. The integration of aspartic acid racemization modeling and metaproteomics further constrained the metabolic status of the living microbial populations. Collectively, combining DNA repair protocols with HTS unveiled the adaptive strategies of microbes to long-term survivability in ancient permafrost.

Conclusions: Our results indicated that coupling of DNA repair protocols with simultaneous sequencing of iDNA and eDNA fractions enabled the assembly of MAGs from past and living microorganisms in ancient permafrost. The genomic reconstruction from the past and extant microbial populations expanded our understanding about the microbial successions and biogeochemical alterations from the past paleoenvironment to the present-day frozen state. Furthermore, we provided genomic insights into long-term survival mechanisms of microorganisms under cryogenic conditions through geological time. The combined strategies in this study can be extrapolated to examine other ancient non-permafrost environments and constrain the search for past and extant extraterrestrial life in permafrost and ice deposits on Mars. Video abstract.
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http://dx.doi.org/10.1186/s40168-021-01057-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8130349PMC
May 2021

Are permafrost microorganisms as old as permafrost?

FEMS Microbiol Ecol 2021 02;97(2)

Institute of Physicochemical and Biological Problems in Soil Science, Russian Academy of Sciences, Pushchino 142290, Russia.

Permafrost describes the condition of earth material (sand, ground, organic matter, etc.) cemented by ice when its temperature remains at or below 0°C continuously for longer than 2 years. Evidently, permafrost is as old as the time passed from freezing of the earth material. Permafrost is a unique phenomenon and may preserve life forms it encloses. Therefore, in order to talk confidently about the preservation of paleo-objects in permafrost, knowledge about the geological age of sediments, i.e. when the sediments were formed, and permafrost age, when those sediments became permanently frozen, is essential. There are two types of permafrost-syngenetic and epigenetic. The age of syngenetic permafrost corresponds to the geological age of its sediments, whereas the age of epigenetic permafrost is less than the geological age of its sediments. Both of these formations preserve microorganisms and their metabolic products; however, the interpretations of the microbiological and molecular-biological data are inconsistent. This paper reviews the current knowledge of time-temperature history and age of permafrost in relation to available microbiological and metagenomic data.
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http://dx.doi.org/10.1093/femsec/fiaa260DOI Listing
February 2021

Insights into community of photosynthetic microorganisms from permafrost.

FEMS Microbiol Ecol 2020 11;96(12)

Soil Cryology Laboratory, Institute of Physicochemical and Biological Problems in Soil Science, Institutskaya Street, Bldg. 2, Pushchino, Russia.

This work integrates cultivation studies of Siberian permafrost and analyses of metagenomes from different locations in the Arctic with the aim of obtaining insights into the community of photosynthetic microorganisms in perennially frozen deposits. Cyanobacteria and microalgae have been described in Arctic aquatic and surface soil environments, but their diversity and ability to withstand harsh conditions within the permafrost are still largely unknown. Community structure of photosynthetic organisms in permafrost sediments was explored using Arctic metagenomes available through the MG-RAST. Sequences affiliated with cyanobacteria represented from 0.25 to 3.03% of total sequences, followed by sequences affiliated with Streptophyta (algae and vascular plants) 0.01-0.45% and Chlorophyta (green algae) 0.01-0.1%. Enrichment and cultivation approaches revealed that cyanobacteria and green algae survive in permafrost and they could be revived during prolonged incubation at low light intensity. Among photosynthetic microorganisms isolated from permafrost, the filamentous Oscillatoria-like cyanobacteria and unicellular green algae of the genus Chlorella were dominant. Our findings suggest that permafrost cyanobacteria and green algae are expected to be effective members of the re-assembled community after permafrost thawing and soil collapse.
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http://dx.doi.org/10.1093/femsec/fiaa229DOI Listing
November 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

A Microbe Associated with Sleep Revealed by a Novel Systems Genetic Analysis of the Microbiome in Collaborative Cross Mice.

Genetics 2020 03 2;214(3):719-733. Epub 2020 Jan 2.

The Jackson Laboratory, Bar Harbor, Maine 04609

The microbiome influences health and disease through complex networks of host genetics, genomics, microbes, and environment. Identifying the mechanisms of these interactions has remained challenging. Systems genetics in laboratory mice () enables data-driven discovery of biological network components and mechanisms of host-microbial interactions underlying disease phenotypes. To examine the interplay among the whole host genome, transcriptome, and microbiome, we mapped QTL and correlated the abundance of cecal messenger RNA, luminal microflora, physiology, and behavior in a highly diverse Collaborative Cross breeding population. One such relationship, regulated by a variant on chromosome 7, was the association of (Bacteroidales) abundance and sleep phenotypes. In a test of this association in the BKS.Cg- +/+ J mouse model of obesity and diabetes, known to have abnormal sleep and colonization by , treatment with antibiotics altered sleep in a genotype-dependent fashion. The many other relationships extracted from this study can be used to interrogate other diseases, microbes, and mechanisms.
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http://dx.doi.org/10.1534/genetics.119.303013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7054020PMC
March 2020

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

Metagenomes from Late Pleistocene Ice Complex Sediments of the Siberian Arctic.

Microbiol Resour Announc 2019 Oct 24;8(43). Epub 2019 Oct 24.

Institute of Physicochemical and Biological Problems in Soil Science, Russian Academy of Sciences, Pushchino, Russia

The late Pleistocene Ice Complex (also known as Yedoma) encompasses ice-rich permafrost formed when alluvial and/or aeolian sediments accumulated under cold climatic conditions. Three metagenomes obtained from Yedoma deposits continually frozen for periods up to 60,000 years are reported here.
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http://dx.doi.org/10.1128/MRA.01010-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6813392PMC
October 2019

Draft Genome Sequence of Microbacterium sp. Gd 4-13, Isolated from Gydanskiy Peninsula Permafrost Sediments of Marine Origin.

Microbiol Resour Announc 2019 Oct 3;8(40). Epub 2019 Oct 3.

Institute of Physicochemical and Biological Problems in Soil Science, Russian Academy of Sciences, Pushchino, Russia.

Here, we report the draft genome sequence of sp. strain Gd 4-13, isolated from late Pleistocene permafrost of marine origin located on the Gydanskiy Peninsula. Genome sequence analysis was performed to understand strain survivability mechanisms under permafrost conditions and to expand biotechnology applications.
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http://dx.doi.org/10.1128/MRA.00889-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6776773PMC
October 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

Methanogens in the Antarctic Dry Valley permafrost.

FEMS Microbiol Ecol 2018 08;94(8)

Institute of Physicochemical and Biological Problems in Soil Science, Russian Academy of Sciences, Pushchino, 142290, Russia.

Polar permafrost is at the forefront of climate change, yet only a few studies have enriched the native methane-producing microbes that might provide positive feedbacks to climate change. Samples Ant1 and Ant2, collected in Antarctic Miers Valley from permafrost sediments, with and without biogenic methane, respectively, were evaluated for methanogenic activity and presence of methanogens. After a one-year incubation of both samples under anaerobic conditions, methane production was observed only at room temperature in microcosm Ant1 with CO2/H2 (20/80) as carbon and energy sources and was monitored during the subsequent 10 years. The concentration of methane in the headspace of microcosm Ant1 changed from 0.8% to a maximum of 45%. Archaeal 16S rRNA genes from microcosm Ant1 were related to psychrotolerant Methanosarcina lacustris. Repeated efforts at achieving a pure culture of this organism were unsuccessful. Metagenomic reads obtained for the methane-producing microcosm Ant1 were assembled and resulted in a 99.84% complete genome affiliated with the genus Methanosarcina. The metagenome assembled genome contained cold-adapted enzymes and pathways suggesting that the novel uncultured Methanosarcina sp. Ant1 is adapted to sub-freezing conditions in permafrost. This is the first methanogen genome reported from the 15 000 years old permafrost of the Antarctic Dry Valleys.
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http://dx.doi.org/10.1093/femsec/fiy109DOI Listing
August 2018

Microbial community structure with trends in methylation gene diversity and abundance in mercury-contaminated rice paddy soils in Guizhou, China.

Environ Sci Process Impacts 2018 Apr;20(4):673-685

Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831-6036, USA.

Paddy soils from mercury (Hg)-contaminated rice fields in Guizhou, China were studied with respect to total mercury (THg) and methylmercury (MeHg) concentrations as well as Bacterial and Archaeal community composition. Total Hg (0.25-990 μg g-1) and MeHg (1.3-30.5 ng g-1) varied between samples. Pyrosequencing (454 FLX) of the hypervariable v1-v3 regions of the 16S rRNA genes showed that Proteobacteria, Actinobacteria, Chloroflexi, Acidobacteria, Euryarchaeota, and Crenarchaeota were dominant in all samples. The Bacterial α-diversity was higher in samples with relatively Low THg and MeHg and decreased with increasing THg and MeHg concentrations. In contrast, Archaeal α-diversity increased with increasing of MeHg concentrations but did not correlate with changes in THg concentrations. Overall, the methylation gene hgcAB copy number increased with both increasing THg and MeHg concentrations. The microbial communities at High THg and High MeHg appear to be adapted by species that are both Hg resistant and carry hgcAB genes for MeHg production. The relatively high abundance of both sulfate-reducing δ-Proteobacteria and methanogenic Archaea, as well as their positive correlations with increasing THg and MeHg concentrations, suggests that these microorganisms are the primary Hg-methylators in the rice paddy soils in Guizhou, China.
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http://dx.doi.org/10.1039/c7em00558jDOI Listing
April 2018

Draft Genome Sequence of Antarctic Methanogen Enriched from Dry Valley Permafrost.

Genome Announc 2016 Dec 8;4(6). Epub 2016 Dec 8.

Department of Microbiology, University of Tennessee, Knoxville, Tennessee, USA

A genomic reconstruction belonging to the genus Methanosarcina was assembled from metagenomic data from a methane-producing enrichment of Antarctic permafrost. This is the first methanogen genome reported from permafrost of the Dry Valleys and can help shed light on future climate-affected methane dynamics.
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http://dx.doi.org/10.1128/genomeA.01362-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5146446PMC
December 2016

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

Draft genome sequences of 10 strains of the genus exiguobacterium.

Genome Announc 2014 Oct 16;2(5). Epub 2014 Oct 16.

DOE Joint Genome Institute, Walnut Creek, California, USA.

High-quality draft genome sequences were determined for 10 Exiguobacterium strains in order to provide insight into their evolutionary strategies for speciation and environmental adaptation. The selected genomes include psychrotrophic and thermophilic species from a range of habitats, which will allow for a comparison of metabolic pathways and stress response genes.
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http://dx.doi.org/10.1128/genomeA.01058-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4200161PMC
October 2014

Community analysis of plant biomass-degrading microorganisms from Obsidian Pool, Yellowstone National Park.

Microb Ecol 2015 Feb 16;69(2):333-45. Epub 2014 Oct 16.

BioEnergy Science Center, Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN, 37831, USA.

The conversion of lignocellulosic biomass into biofuels can potentially be improved by employing robust microorganisms and enzymes that efficiently deconstruct plant polysaccharides at elevated temperatures. Many of the geothermal features of Yellowstone National Park (YNP) are surrounded by vegetation providing a source of allochthonic material to support heterotrophic microbial communities adapted to utilize plant biomass as a primary carbon and energy source. In this study, a well-known hot spring environment, Obsidian Pool (OBP), was examined for potential biomass-active microorganisms using cultivation-independent and enrichment techniques. Analysis of 33,684 archaeal and 43,784 bacterial quality-filtered 16S rRNA gene pyrosequences revealed that archaeal diversity in the main pool was higher than bacterial; however, in the vegetated area, overall bacterial diversity was significantly higher. Of notable interest was a flooded depression adjacent to OBP supporting a stand of Juncus tweedyi, a heat-tolerant rush commonly found growing near geothermal features in YNP. The microbial community from heated sediments surrounding the plants was enriched in members of the Firmicutes including potentially (hemi)cellulolytic bacteria from the genera Clostridium, Anaerobacter, Caloramator, Caldicellulosiruptor, and Thermoanaerobacter. Enrichment cultures containing model and real biomass substrates were established at a wide range of temperatures (55-85 °C). Microbial activity was observed up to 80 °C on all substrates including Avicel, xylan, switchgrass, and Populus sp. Independent of substrate, Caloramator was enriched at lower (<65 °C) temperatures while highly active cellulolytic bacteria Caldicellulosiruptor were dominant at high (>65 °C) temperatures.
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http://dx.doi.org/10.1007/s00248-014-0500-8DOI Listing
February 2015

Improved yield of high molecular weight DNA coincides with increased microbial diversity access from iron oxide cemented sub-surface clay environments.

PLoS One 2014 17;9(7):e102826. Epub 2014 Jul 17.

Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, United States of America.

Despite over three decades of progress, extraction of high molecular weight (HMW) DNA from high clay soils or iron oxide cemented clay has remained challenging. HMW DNA is desirable for next generation sequencing as it yields the most comprehensive coverage. Several DNA extraction procedures were compared from samples that exhibit strong nucleic acid adsorption. pH manipulation or use of alternative ion solutions offered no improvement in nucleic acid recovery. Lysis by liquid N2 grinding in concentrated guanidine followed by concentrated sodium phosphate extraction supported HMW DNA recovery from clays high in iron oxides. DNA recovered using 1 M sodium phosphate buffer (PB) as a competitive desorptive wash was 15.22±2.33 µg DNA/g clay, with most DNA consisting of >20 Kb fragments, compared to 2.46±0.25 µg DNA/g clay with the Powerlyzer system (MoBio). Increasing PB concentration in the lysis reagent coincided with increasing DNA fragment length during initial extraction. Rarefaction plots of 16S rRNA (V1-V3 region) pyrosequencing from A-horizon and clay soils showed an ∼80% and ∼400% larger accessed diversity compared to the Powerlyzer soil DNA system, respectively. The observed diversity from the Firmicutes showed the strongest increase with >3-fold more operational taxonomic units (OTU) recovered.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0102826PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4102596PMC
November 2015

Diversity and genomic insights into the uncultured Chloroflexi from the human microbiota.

Environ Microbiol 2014 Sep 17;16(9):2635-43. Epub 2014 Apr 17.

Biosciences Division, Oak Ridge National Laboratories, Oak Ridge, TN 37831, USA.

Many microbial phyla that are widely distributed in open environments have few or no representatives within animal-associated microbiota. Among them, the Chloroflexi comprises taxonomically and physiologically diverse lineages adapted to a wide range of aquatic and terrestrial habitats. A distinct group of uncultured chloroflexi related to free-living anaerobic Anaerolineae inhabits the mammalian gastrointestinal tract and includes low-abundance human oral bacteria that appear to proliferate in periodontitis. Using a single-cell genomics approach, we obtained the first draft genomic reconstruction for these organisms and compared their inferred metabolic potential with free-living chloroflexi. Genomic data suggest that oral chloroflexi are anaerobic heterotrophs, encoding abundant carbohydrate transport and metabolism functionalities, similar to those seen in environmental Anaerolineae isolates. The presence of genes for a unique phosphotransferase system and N-acetylglucosamine metabolism suggests an important ecological niche for oral chloroflexi in scavenging material from lysed bacterial cells and the human tissue. The inferred ability to produce sialic acid for cell membrane decoration may enable them to evade the host defence system and colonize the subgingival space. As with other low abundance but persistent members of the microbiota, discerning community and host factors that influence the proliferation of oral chloroflexi may help understand the emergence of oral pathogens and the microbiota dynamics in health and disease states.
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http://dx.doi.org/10.1111/1462-2920.12461DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4149597PMC
September 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

Environmental proteomics reveals early microbial community responses to biostimulation at a uranium- and nitrate-contaminated site.

Proteomics 2013 Oct 22;13(18-19):2921-30. Epub 2013 Aug 22.

Chemical Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN, USA.

High-performance MS instrumentation coupled with improved protein extraction techniques enables metaproteomics to identify active members of soil and groundwater microbial communities. Metaproteomics workflows were applied to study the initial responses (i.e. 4 days post treatment) of the indigenous aquifer microbiota to biostimulation with emulsified vegetable oil (EVO) at a uranium-contaminated site. Members of the Betaproteobacteria (i.e. Dechloromonas, Ralstonia, Rhodoferax, Polaromonas, Delftia, Chromobacterium) and the Firmicutes dominated the biostimulated aquifer community. Proteome characterization revealed distinct differences between the microbial biomass collected from groundwater influenced by biostimulation and groundwater collected upgradient of the EVO injection points. In particular, proteins involved in ammonium assimilation, EVO degradation, and polyhydroxybutyrate granule formation were prominent following biostimulation. Interestingly, the atypical NosZ of Dechloromonas spp. was highly abundant, suggesting active nitrous oxide (N2 O) respiration. c-Type cytochromes were barely detected, as was citrate synthase, a biomarker for hexavalent uranium reduction activity, suggesting that uranium reduction has not commenced 4 days post EVO amendment. Environmental metaproteomics identified microbial community responses to biostimulation and elucidated active pathways demonstrating the value of this technique as a monitoring tool and for complementing nucleic acid-based approaches.
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http://dx.doi.org/10.1002/pmic.201300155DOI Listing
October 2013

Biogeography of the ecosystems of the healthy human body.

Genome Biol 2013 Jan 14;14(1):R1. Epub 2013 Jan 14.

Background: Characterizing the biogeography of the microbiome of healthy humans is essential for understanding microbial associated diseases. Previous studies mainly focused on a single body habitat from a limited set of subjects. Here, we analyzed one of the largest microbiome datasets to date and generated a biogeographical map that annotates the biodiversity, spatial relationships, and temporal stability of 22 habitats from 279 healthy humans.

Results: We identified 929 genera from more than 24 million 16S rRNA gene sequences of 22 habitats, and we provide a baseline of inter-subject variation for healthy adults. The oral habitat has the most stable microbiota with the highest alpha diversity, while the skin and vaginal microbiota are less stable and show lower alpha diversity. The level of biodiversity in one habitat is independent of the biodiversity of other habitats in the same individual. The abundances of a given genus at a body site in which it dominates do not correlate with the abundances at body sites where it is not dominant. Additionally, we observed the human microbiota exhibit both cosmopolitan and endemic features. Finally, comparing datasets of different projects revealed a project-based clustering pattern, emphasizing the significance of standardization of metagenomic studies.

Conclusions: The data presented here extend the definition of the human microbiome by providing a more complete and accurate picture of human microbiome biogeography, addressing questions best answered by a large dataset of subjects and body sites that are deeply sampled by sequencing.
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http://dx.doi.org/10.1186/gb-2013-14-1-r1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4054670PMC
January 2013

Microbial communities involved in biological ammonium removal from coal combustion wastewaters.

Microb Ecol 2013 Jul 13;66(1):49-59. Epub 2013 Jan 13.

Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831-6036, USA.

The efficiency of a novel integrated treatment system for biological removal of ammonium, nitrite, nitrate, and heavy metals from fossil power plant effluent was evaluated. Microbial communities were analyzed using bacterial and archaeal 16S rRNA gene clone libraries (Sanger sequences) and 454 pyrosequencing technology. While seasonal changes in microbial community composition were observed, the significant (P = 0.001) changes in bacterial and archaeal communities were consistent with variations in ammonium concentration. Phylogenetic analysis of 16S rRNA gene sequences revealed an increase of potential ammonium-oxidizing bacteria (AOB), Nitrosomonas, Nitrosococcus, Planctomycetes, and OD1, in samples with elevated ammonium concentration. Other bacteria, such as Nitrospira, Nitrococcus, Nitrobacter, Thiobacillus, ε-Proteobacteria, Firmicutes, and Acidobacteria, which play roles in nitrification and denitrification, were also detected. The AOB oxidized 56 % of the ammonium with the concomitant increase in nitrite and ultimately nitrate in the trickling filters at the beginning of the treatment system. Thermoprotei within the phylum Crenarchaeota thrived in the splitter box and especially in zero-valent iron extraction trenches, where an additional 25 % of the ammonium was removed. The potential ammonium-oxidizing Archaea (AOA) (Candidatus Nitrosocaldus) were detected towards the downstream end of the treatment system. The design of an integrated treatment system consisting of trickling filters, zero-valent iron reaction cells, settling pond, and anaerobic wetlands was efficient for the biological removal of ammonium and several other contaminants from wastewater generated at a coal burning power plant equipped with selective catalytic reducers for nitrogen oxide removal.
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http://dx.doi.org/10.1007/s00248-012-0152-5DOI Listing
July 2013

Anaerobic high-throughput cultivation method for isolation of thermophiles using biomass-derived substrates.

Methods Mol Biol 2012 ;908:153-68

Oak Ridge National Laboratory, BioEnergy Science Center, Oak Ridge, TN, USA.

Flow cytometry (FCM) techniques have been developed for sorting mesophilic organisms, but the difficulty increases if the target microbes are thermophilic anaerobes. We demonstrate a reliable, high-throughput method of screening thermophilic anaerobic organisms using FCM and 96-well plates for growth on biomass-relevant substrates. The method was tested using the cellulolytic thermophiles Clostridium thermocellum (T(opt) = 55 °C), Caldicellulosiruptor obsidiansis (T(opt) = 78 °C) and the fermentative hyperthermophiles, Pyrococcus furiosus (T(opt) = 100 °C) and Thermotoga maritima (T(opt) = 80 °C). Multi-well plates were incubated at various temperatures for approximately 72-120 h and then tested for growth. Positive growth resulting from single cells sorted into individual wells containing an anaerobic medium was verified by OD(600). Depending on the growth substrate, up to 80 % of the wells contained viable cultures, which could be transferred to fresh media. This method was used to isolate thermophilic microbes from Rabbit Creek, Yellowstone National Park (YNP), Wyoming. Substrates for enrichment cultures including crystalline cellulose (Avicel), xylan (from Birchwood), pretreated switchgrass and Populus were used to cultivate organisms that may be of interest to lignocellulosic biofuel production.
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http://dx.doi.org/10.1007/978-1-61779-956-3_15DOI Listing
December 2012

Host genetic and environmental effects on mouse intestinal microbiota.

ISME J 2012 Nov 14;6(11):2033-44. Epub 2012 Jun 14.

Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA.

The mammalian gut harbors complex and variable microbial communities, across both host phylogenetic space and conspecific individuals. A synergy of host genetic and environmental factors shape these communities and account for their variability, but their individual contributions and the selective pressures involved are still not well understood. We employed barcoded pyrosequencing of V1-2 and V4 regions of bacterial small subunit ribosomal RNA genes to characterize the effects of host genetics and environment on cecum assemblages in 10 genetically distinct, inbred mouse strains. Eight of these strains are the foundation of the Collaborative Cross (CC), a panel of mice derived from a genetically diverse set of inbred founder strains, designed specifically for complex trait analysis. Diversity of gut microbiota was characterized by complementing phylogenetic and distance-based, sequence-clustering approaches. Significant correlations were found between the mouse strains and their gut microbiota, reflected by distinct bacterial communities. Cohabitation and litter had a reduced, although detectable effect, and the microbiota response to these factors varied by strain. We identified bacterial phylotypes that appear to be discriminative and strain-specific to each mouse line used. Cohabitation of different strains of mice revealed an interaction of host genetic and environmental factors in shaping gut bacterial consortia, in which bacterial communities became more similar but retained strain specificity. This study provides a baseline analysis of intestinal bacterial communities in the eight CC progenitor strains and will be linked to integrated host genotype, phenotype and microbiota research on the resulting CC panel.
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http://dx.doi.org/10.1038/ismej.2012.54DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3475380PMC
November 2012

Complete genome sequence of the Thermophilic Bacterium Exiguobacterium sp. AT1b.

J Bacteriol 2011 Jun 1;193(11):2880-1. Epub 2011 Apr 1.

The University of Tennessee, Center for Environmental Biotechnology, 676 Dabney-Buehler Hall, Knoxville, TN 37996-1605, USA.

Here we present the genome of strain Exiguobacterium sp. AT1b, a thermophilic member of the genus Exiguobacterium whose representatives were isolated from various environments along a thermal and physicochemical gradient. This genome was sequenced to be a comparative resource for the study of thermal adaptation with a psychroactive representative of the genus, Exiguobacterium sibiricum strain 255-15, that was previously sequenced by the U.S. Department of Energy's (DOE's) Joint Genome Institute (JGI) (http://genome.ornl.gov/microbial/exig/).
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http://dx.doi.org/10.1128/JB.00303-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3133134PMC
June 2011

Enhancement in current density and energy conversion efficiency of 3-dimensional MFC anodes using pre-enriched consortium and continuous supply of electron donors.

Bioresour Technol 2011 Apr 25;102(8):5098-104. Epub 2011 Jan 25.

BioSciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831-6226, USA.

Using a pre-enriched microbial consortium as the inoculum and continuous supply of carbon source, improvement in performance of a three-dimensional, flow-through MFC anode utilizing ferricyanide cathode was investigated. The power density increased from 170 W/m(3) (1800 mW/m(2)) to 580 W/m(3) (6130 mW/m(2)), when the carbon loading increased from 2.5 g/l-day to 50 g/l-day. The coulombic efficiency (CE) decreased from 90% to 23% with increasing carbon loading. The CEs are among the highest reported for glucose and lactate as the substrate with the maximum current density reaching 15.1A/m(2). This suggests establishment of a very high performance exoelectrogenic microbial consortium at the anode. A maximum energy conversion efficiency of 54% was observed at a loading of 2.5 g/l-day. Biological characterization of the consortium showed presence of Burkholderiales and Rhodocyclales as the dominant members. Imaging of the biofilms revealed thinner biofilms compared to the inoculum MFC, but a 1.9-fold higher power density.
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http://dx.doi.org/10.1016/j.biortech.2011.01.045DOI Listing
April 2011

Mercury and other heavy metals influence bacterial community structure in contaminated Tennessee streams.

Appl Environ Microbiol 2011 Jan 5;77(1):302-11. Epub 2010 Nov 5.

Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831-6036, USA.

High concentrations of uranium, inorganic mercury [Hg(II)], and methylmercury (MeHg) have been detected in streams located in the Department of Energy reservation in Oak Ridge, TN. To determine the potential effects of the surface water contamination on the microbial community composition, surface stream sediments were collected 7 times during the year, from 5 contaminated locations and 1 control stream. Fifty-nine samples were analyzed for bacterial community composition and geochemistry. Community characterization was based on GS 454 FLX pyrosequencing with 235 Mb of 16S rRNA gene sequence targeting the V4 region. Sorting and filtering of the raw reads resulted in 588,699 high-quality sequences with lengths of >200 bp. The bacterial community consisted of 23 phyla, including Proteobacteria (ranging from 22.9 to 58.5% per sample), Cyanobacteria (0.2 to 32.0%), Acidobacteria (1.6 to 30.6%), Verrucomicrobia (3.4 to 31.0%), and unclassified bacteria. Redundancy analysis indicated no significant differences in the bacterial community structure between midchannel and near-bank samples. Significant correlations were found between the bacterial community and seasonal as well as geochemical factors. Furthermore, several community members within the Proteobacteria group that includes sulfate-reducing bacteria and within the Verrucomicrobia group appeared to be associated positively with Hg and MeHg. This study is the first to indicate an influence of MeHg on the in situ microbial community and suggests possible roles of these bacteria in the Hg/MeHg cycle.
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http://dx.doi.org/10.1128/AEM.01715-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3019708PMC
January 2011
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