Lake Erie ice is a repository of organisms.

Organism abundance and diversity were assessed in Lake Erie ice samples using sequences derived from a combined metagenomic and metatranscriptomic analysis. The 68,417 unique sequences were from Bacteria (77.5%), Eukarya (22.3%), and Archaea (0.2%) and indicated diverse species of organisms from 32 bacterial, 8 eukaryotic, and 2 archaeal taxonomic groups.

Ribosomal Intergenic Spacers Are Filled with Transposon Remnants.

Eukaryotic ribosomal DNA (rDNA) comprises tandem units of highly conserved coding genes separated by rapidly evolving spacer DNA. The spacers of all 12 species examined were filled with short direct repeats (DRs) and multiple long tandem repeats (TRs), completing the rDNA maps that previously contained unannotated and inadequately studied sequences. The external transcribed spacers also were filled with DRs and some contained TRs. We infer that the spacers arose from transposon insertion, followed by their imprecise excision, leaving short DRs characteristic of transposon visitation. The spacers provided a favored location for transposon insertion because they occupy loci containing hundreds to thousands of gene repeats. The spacers' primary cellular function may be to link one ribosomal RNA transcription unit to the next, whereas transposons flourish here because they have colonized the most frequently used part of the genome.

Photosynthetic Systems Suggest an Evolutionary Pathway to Diderms.

Bacteria are divided primarily into monoderms (with one cell membrane, and usually Gram-positive, due to a thick peptidoglycan layer) and diderms (with two cell membranes, and mostly Gram-negative, due to a thin peptidoglycan layer sandwiched between the two membranes). Photosynthetic species are spread among the taxonomic groups, some having type I reaction centers (RCI in monoderm phylum Firmicutes; and diderm phyla Acidobacteria and Chlorobi), others with type II reaction centers (RCII in monoderm phylum Chloroflexi; and diderm taxa Gemmatimonadetes, and alpha-, beta-, and gamma-Proteobacteria), and some containing both (RCI and RCII, only in diderm phylum Cyanobacteria). In most bacterial phylograms, photosystem types and diderm taxa are polyphyletic. A more parsimonious arrangement, which is supported by photosystem evolution, as well as additional sets of molecular characters, suggests that endosymbiotic events resulted in the formation of the diderms. In the model presented, monoderms readily form a monophyletic group, while diderms are produced by at least two endosymbiotic events, followed by additional evolutionary changes.

Metatranscriptomic and Metagenomic Analysis of Biological Diversity in Subglacial Lake Vostok (Antarctica).

A combined metatranscriptomic and metagenomic study of Vostok (Antarctica) ice core sections from glacial, basal, and lake water accretion ice yielded sequences that indicated a wide variety of species and possible conditions at the base of the glacier and in subglacial Lake Vostok. Few organisms were in common among the basal ice and accretion ice samples, suggesting little transmission of viable organisms from the basal ice meltwater into the lake water. Additionally, samples of accretion ice, each of which originated from water in several locations of the shallow embayment, exhibit only small amounts of mixing of species. The western-most portion of the embayment had very low numbers of organisms, likely due to biologically challenging conditions. Increasing numbers of organisms were found progressing from west to east, up to approximately 7 km into the embayment. At that point, the numbers of unique sequences and sequence reads from thermophilic, thermotolerant, psychrophilic, and psychrotolerant organisms increased dramatically, as did sequences from alkaliphilic, alkalitolerant, acidophilic, and acidotolerant sequences. The number of unique and total sequences were positively associated with increases in concentrations of Na+, Ca2+, Mg2+, SO42-, Cl-, total amino acids, and non-purgeable organic carbon. The numbers of unique sequences from organisms reported from soil, sediment, ice, aquatic, marine, animal, and plant (probably pollen) sources also peaked in this region, suggesting that this was the most biologically active region. The confluence of the high numbers of organisms, physiologies, and metabolic capabilities suggests the presence of energy and nutrient sources in the eastern half of the embayment. Data from the main basin suggested a cold oligotrophic environment containing fewer organisms. In addition to bacteria, both the basal ice and accretion ice contained sequences from a diverse assemblage of eukaryotes, as well as from bacteria that are known to be associated with multicellular eukaryotes.

Evolution of the genetic code based on conservative changes of codons, amino acids, and aminoacyl tRNA synthetases.

The genetic code, as arranged in the standard tabular form, displays a non-random structure relating to the characteristics of the amino acids. An alternative arrangement can be made by organizing the code according to aminoacyl-tRNA synthetases (aaRSs), codons, and reverse complement codons, which illuminates a coevolutionary process that led to the contemporary genetic code. As amino acids were added to the genetic code, they were recognized by aaRSs that interact with stereochemically similar amino acids. Single nucleotide changes in the codons and anticodons were favored over more extensive changes, such that there was a logical stepwise progression in the evolution of the genetic code. The model presented traces the evolution of the genetic code accounting for these steps. Amino acid frequencies in ancient proteins and the preponderance of GNN codons in mRNAs for ancient proteins indicate that the genetic code began with alanine, aspartate, glutamate, glycine, and valine, with alanine being in the highest proportions. In addition to being consistent in terms of conservative changes in codon nucleotides, the model also is consistent with respect to aaRS classes, aaRS attachment to the tRNA, amino acid stereochemistry, and to a large extent with amino acid physicochemistry, and biochemical pathways.

Integrated evolution of ribosomal RNAs, introns, and intron nurseries.

The initial components of ribosomes first appeared more than 3.8 billion years ago during a time when many types of RNAs were evolving. While modern ribosomes are complex molecular machines consisting of rRNAs and proteins, they were assembled during early evolution by the association and joining of small functional RNA units. Introns may have provided the means to ligate many of these pieces together. All four classes of introns (group I, group II, spliceosomal, and archaeal) are present in many rRNA gene loci over a broad phylogenetic range. A survey of rRNA intron sequences across the three major life domains suggests that some of the classes of introns may have diverged from one another within rRNA gene loci. Analyses of rRNA sequences revealed self-splicing group I and group II introns are present in ancestral regions of the SSU (small subunit) and LSU (large subunit), whereas spliceosomal and archaeal introns appeared in sections of the rRNA that evolved later. Most classes of introns increased in number for approximately 1 billion years. However, their frequencies are low in the most recently evolved regions added to the SSU and LSU rRNAs. Furthermore, many of the introns appear to have been in the same locations for billions of years, suggesting an ancient origin for these sequences. In this Perspectives paper, I reviewed and analyzed rRNA intron sequences, locations, structural characteristics, and splicing mechanisms; and suggest that rRNA gene loci may have served as evolutionary nurseries for intron formation and diversification.

Rapid identification of fungi in culture-negative clinical blood and respiratory samples by DNA sequence analyses.

BACKGROUND: Clinical diagnoses of fungal infections often rely upon culture techniques followed by microscopic examination of positive cultures and histopathological specimens. Culturing of microorganisms is prone to false negatives, while microscopy methods can be complicated by atypical phenotypes and organisms that are morphologically indistinguishable in tissues. Delays in diagnoses (or the lack thereof) and inaccurate identification of infectious organisms contribute to increased morbidity and mortality in patients. METHODS: Two-hundred randomized, heterogeneous patient blood and respiratory samples that were culture-negative were tested using polymerase chain reaction (PCR) amplification of internal transcribed spacer regions of ribosomal RNA genes utilizing panfungal primers. Amplicons were sequenced, subjected to sequence similarity searches, and compared using phylogenetic analyses. RESULTS: Thirteen fungal sequences were detected in three whole-blood samples and nine respiratory samples. Bioinformatic analyses were performed which indicated the presence of multiple pathogens and potential pathogens. CONCLUSIONS: The results from this pilot study demonstrate the utility of PCR assays and sequence analyses in clinical tests for fungi to facilitate rapid diagnosis and appropriate treatments to deal with the false negatives from culture results.

International Space Station environmental microbiome - microbial inventories of ISS filter debris.

Despite an expanding array of molecular approaches for detecting microorganisms in a given sample, rapid and robust means of assessing the differential viability of the microbial cells, as a function of phylogenetic lineage, remain elusive. A propidium monoazide (PMA) treatment coupled with downstream quantitative polymerase chain reaction (qPCR) and pyrosequencing analyses was carried out to better understand the frequency, diversity, and distribution of viable microorganisms associated with debris collected from the crew quarters of the International Space Station (ISS). The cultured bacterial counts were more in the ISS samples than cultured fungal population. The rapid molecular analyses targeted to estimate viable population exhibited 5-fold increase in bacterial (qPCR-PMA assay) and 25-fold increase in microbial (adenosine triphosphate assay) burden than the cultured bacterial population. The ribosomal nucleic acid-based identification of cultivated strains revealed the presence of only four to eight bacterial species in the ISS samples, however, the viable bacterial diversity detected by the PMA-pyrosequencing method was far more diverse (12 to 23 bacterial taxa) with the majority consisting of members of actinobacterial genera (Propionibacterium, Corynebacterium) and Staphylococcus. Sample fractions not treated with PMA (inclusive of both live and dead cells) yielded a great abundance of highly diverse bacterial (94 to 118 taxa) and fungal lineages (41 taxa). Even though deep sequencing capability of the molecular analysis widened the understanding about the microbial diversity, the cultivation assay also proved to be essential since some of the spore-forming microorganisms were detected only by the culture-based method. Presented here are the findings of the first comprehensive effort to assess the viability of microbial cells associated with ISS surfaces, and correlate differential viability with phylogenetic affiliation.

Subglacial Lake Vostok (Antarctica) accretion ice contains a diverse set of sequences from aquatic, marine and sediment-inhabiting bacteria and eukarya.

Lake Vostok, the 7(th) largest (by volume) and 4(th) deepest lake on Earth, is covered by more than 3,700 m of ice, making it the largest subglacial lake known. The combination of cold, heat (from possible hydrothermal activity), pressure (from the overriding glacier), limited nutrients and complete darkness presents extreme challenges to life. Here, we report metagenomic/metatranscriptomic sequence analyses from four accretion ice sections from the Vostok 5G ice core. Two sections accreted in the vicinity of an embayment on the southwestern end of the lake, and the other two represented part of the southern main basin. We obtained 3,507 unique gene sequences from concentrates of 500 ml of 0.22 µm-filtered accretion ice meltwater. Taxonomic classifications (to genus and/or species) were possible for 1,623 of the sequences. Species determinations in combination with mRNA gene sequence results allowed deduction of the metabolic pathways represented in the accretion ice and, by extension, in the lake. Approximately 94% of the sequences were from Bacteria and 6% were from Eukarya. Only two sequences were from Archaea. In general, the taxa were similar to organisms previously described from lakes, brackish water, marine environments, soil, glaciers, ice, lake sediments, deep-sea sediments, deep-sea thermal vents, animals and plants. Sequences from aerobic, anaerobic, psychrophilic, thermophilic, halophilic, alkaliphilic, acidophilic, desiccation-resistant, autotrophic and heterotrophic organisms were present, including a number from multicellular eukaryotes.

Microbial analyses of ancient ice core sections from greenland and antarctica.

Ice deposited in Greenland and Antarctica entraps viable and nonviable microbes, as well as biomolecules, that become temporal atmospheric records. Five sections (estimated to be 500, 10,500, 57,000, 105,000 and 157,000 years before present, ybp) from the GISP2D (Greenland) ice core, three sections (500, 30,000 and 70,000 ybp) from the Byrd ice core, and four sections from the Vostok 5G (Antarctica) ice core (10,500, 57,000, 105,000 and 105,000 ybp) were studied by scanning electron microscopy, cultivation and rRNA gene sequencing. Bacterial and fungal isolates were recovered from 10 of the 12 sections. The highest numbers of isolates were found in ice core sections that were deposited during times of low atmospheric CO2, low global temperatures and low levels of atmospheric dust. Two of the sections (GISP2D at 10,500 and 157,000 ybp) also were examined using metagenomic/metatranscriptomic methods. These results indicated that sequences from microbes common to arid and saline soils were deposited in the ice during a time of low temperature, low atmospheric CO2 and high dust levels. Members of Firmicutes and Cyanobacteria were the most prevalent bacteria, while Rhodotorula species were the most common eukaryotic representatives. Isolates of Bacillus, Rhodotorula, Alternaria and members of the Davidiellaceae were isolated from both Greenland and Antarctica sections of the same age, although the sequences differed between the two polar regions.

Ecology of subglacial lake vostok (antarctica), based on metagenomic/metatranscriptomic analyses of accretion ice.

Lake Vostok is the largest of the nearly 400 subglacial Antarctic lakes and has been continuously buried by glacial ice for 15 million years. Extreme cold, heat (from possible hydrothermal activity), pressure (from the overriding glacier) and dissolved oxygen (delivered by melting meteoric ice), in addition to limited nutrients and complete darkness, combine to produce one of the most extreme environments on Earth. Metagenomic/metatranscriptomic analyses of ice that accreted over a shallow embayment and over the southern main lake basin indicate the presence of thousands of species of organisms (94% Bacteria, 6% Eukarya, and two Archaea). The predominant bacterial sequences were closest to those from species of Firmicutes, Proteobacteria and Actinobacteria, while the predominant eukaryotic sequences were most similar to those from species of ascomycetous and basidiomycetous Fungi. Based on the sequence data, the lake appears to contain a mixture of autotrophs and heterotrophs capable of performing nitrogen fixation, nitrogen cycling, carbon fixation and nutrient recycling. Sequences closest to those of psychrophiles and thermophiles indicate a cold lake with possible hydrothermal activity. Sequences most similar to those from marine and aquatic species suggest the presence of marine and freshwater regions.

Extraction of DNA from plant and fungus tissues in situ.

BACKGROUND: When samples are collected in the field and transported to the lab, degradation of the nucleic acids contained in the samples is frequently observed. Immediate extraction and precipitation of the nucleic acids reduces degradation to a minimum, thus preserving accurate sequence information. An extraction method to obtain high quality DNA in field studies is described. FINDINGS: DNA extracted immediately after sampling was compared to DNA extracted after allowing the sampled tissues to air dry at 21°C for 48 or 72 hours. While DNA extracted from fresh tissues exhibited little degradation, DNA extracted from all tissues exposed to 21°C air for 48 or 72 hours exhibited varying degrees of degradation. Yield was higher for extractions from fresh tissues in most cases. Four microcentrifuges were compared for DNA yield: one standard electric laboratory microcentrifuge (max rcf = 16,000 × g), two battery-operated microcentrifuges (max rcf = 5,000 and 3,000 × g), and one manually-operated microcentrifuge (max rcf = 120 × g). Yields for all centrifuges were similar. DNA extracted under simulated field conditions was similar in yield and quality to DNA extracted in the laboratory using the same equipment. CONCLUSIONS: This CTAB (cetyltrimethylammonium bromide) DNA extraction method employs battery-operated and manually-operated equipment to isolate high quality DNA in the field. The method was tested on plant and fungus tissues, and may be adapted for other types of organisms. The method produced high quality DNA in laboratory tests and under simulated field conditions. The field extraction method should prove useful for working in remote sites, where ice, dry ice, and liquid nitrogen are unavailable; where degradation is likely to occur due to the long distances between the sample site and the laboratory; and in instances where other DNA preservation and transportation methods have been unsuccessful. It may be possible to adapt this method for genomic, metagenomic, transcriptomic and metabolomic projects using samples collected in situ.

Evolution of small putative group I introns in the SSU rRNA gene locus of Phialophora species.

BACKGROUND: Group I introns (specifically subgroup IC1) are common in the nuclear ribosomal RNA genes of fungi. While most range in length from more than 200 to nearly 1800 nucleotides (nt) in length, several small putative (or degenerate) group I introns have been described that are between 56 and 81 nt. Although small, previously we demonstrated that the PaSSU intron in the rRNA small subunit gene of Phialophora americana isolate Wang 1046 is capable of in vitro splicing using a standard group I intron pathway, thus qualifying it as a functional ribozyme. FINDINGS: Here, we describe eight short putative group I introns, ranging in length from 63 to 75 nt, in the rRNA small subunit genes of Phialophora isolates, a fungal genus that ranges from saprobic to pathogenic on plants and animals. All contain putative pairing regions P1, P7, and P10, as well as a pairing region formed between the middle of the intron and part of the 3' exon. The other pairing regions common in the core of standard group I introns are absent. However, parts of the 3' exon may aid in the stabilization of these small introns. Although the eight putative group I introns were from at least three species of Phialophora, phylogenetic analysis indicated that the eight are monophyletic. They are also monophyletic with the small introns of two lichen-forming fungi, Porpidia crustulata and Arthonia lapidicola. CONCLUSIONS: The small putative group I introns in Phialophora have common features that may represent group I introns at their minima. They appear to have a single origin as indicated by their monophyly in phylogenetic analyses.

Isolation of fungi from Lake Vostok accretion ice.

Here we report the characterization of fungi from 10 accretion ice sections (3300-5100 y old) as well as two deep glacial ice sections that are close to the bottom of the glacier (1,000,000-2,000,000 y old) from the Vostok, Antarctica, 5G ice core. Fungi were characterized by fluorescence microscopy culturing and sequence analyses of ribosomal DNA internal transcribed spacers. A total of 270 fungal colonies were cultured from the accretion ice of subglacial Lake Vostok and an additional 14 from the glacial ice immediately above the accretion ice. Mean concentrations were 0-4.42 cells mL(-1) ice meltwater of which 0-100% exhibited viability (as determined by fluorescence microscopy). Thirty-one unique fungal ribosomal DNA sequences (28 from accretion ice and three from glacial ice) were determined and compared to recent taxa. The results, plus tests for growth at low temperatures, indicated that Lake Vostok contains a mixture of heterotrophic psychrotolerant fungal species. This indicates that the lake is not sterile but contains a unique ecosystem.

Splicing and evolution of an unusually small group I intron.

Introns are common in the rRNA gene loci of fungal genomes, but biochemical studies to investigate splicing are rare. Here, self-splicing of a very small (67 nucleotide) group I intron is demonstrated. The PaSSU intron (located within the rRNA small subunit gene of Phialophora americana) splices in vitro under group I intron conditions. Most group I ribozymes contain pairing regions P1-P10, with a conserved G.U pair at the 5' splice site, and a G at the 3' intron border. The PaSSU intron contains only P1, P7, and P10. While it contains the G.U pair at the 5' splice, a U is found at the 3' end of the intron instead of a G. Phylogenetic analysis places it within subgroup IC1, whose members are found in the nuclear rRNA genes of fungi. The structural elements are similar to those in the centermost regions of other group I introns. Its size can be explained by a single large deletion that removed P2 through much of P9. Part of the original P9 region has assumed the function of P7. Its small size and genealogy makes it an excellent model to study RNA catalysis and evolution.

Isolation of microbes from Lake Vostok accretion ice.

Bacteria from seven Lake Vostok accretion and two deep glacial Vostok ice core sections were characterized. The cell concentrations were low, but many of the cells were viable. From the hundreds of cultures, 18 unique bacterial rRNA gene phylotypes were determined. Lake Vostok may contain a complex microbial ecosystem.

Evidence of influenza a virus RNA in siberian lake ice.

Influenza A virus infects a large proportion of the human population annually, sometimes leading to the deaths of millions. The biotic cycles of infection are well characterized in the literature, including in studies of populations of humans, poultry, swine, and migratory waterfowl. However, there are few studies of abiotic reservoirs for this virus. Here, we report the preservation of influenza A virus genes in ice and water from high-latitude lakes that are visited by large numbers of migratory birds. The lakes are along the migratory flight paths of birds flying into Asia, North America, Europe, and Africa. The data suggest that influenza A virus, deposited as the birds begin their autumn migration, can be preserved in lake ice. As birds return in the spring, the ice melts, releasing the viruses. Therefore, temporal gene flow is facilitated between the viruses shed during the previous year and the viruses newly acquired by birds during winter months spent in the south. Above the Arctic Circle, the cycles of entrapment in the ice and release by melting can be variable in length, because some ice persists for several years, decades, or longer. This type of temporal gene flow might be a feature common to viruses that can survive entrapment in environmental ice and snow.

Recycling of pathogenic microbes through survival in ice.

Viable microorganisms (e.g. fungi, bacteria, Archaea and viruses) are distributed by wind over great distances, including globally. Microbes may settle out of the atmosphere or may be incorporated into fog, rain, sleet, hail, or snow. These organisms fall into lakes, streams, oceans, or onto the land or glaciers. When they become incorporated into environmental ice (e.g. glaciers, ice sheets, and snow), those that survive freezing and thawing may persist for years, centuries, millennia, or longer. Once they melt from the ice, they may enter contemporary populations. This mixing of ancient and modern genotypes (i.e. temporal gene flow, or what we term "genome recycling") may lead to a change of allele proportions in the population, which may have effects on mutation rates, fitness, survival, pathogenicity and other characteristics of the organisms. Pathogenic microbes that survive freezing and thawing (e.g. influenza viruses, polioviruses, caliciviruses and tobamoviruses) can remain in these icy reservoirs long enough to avoid resistance mechanisms of the hosts, thereby conveying a selective advantage to these pathogens over those that cannot survive in ice. Ice is an abiotic reservoir of microbes that has been ignored in surveillance activities for human diseases.

Ice as a reservoir for pathogenic human viruses: specifically, caliciviruses, influenza viruses, and enteroviruses.

Hundreds of isolates of viable bacteria and fungi have been recovered from ancient ice and permafrost. Evidence supports the hypothesis that viral pathogens also are preserved in ice repositories, such as glaciers, ice sheets, and lake ice. Proof may depend upon narrowing the search by applying specific criteria, which would target candidate viruses. Such criteria include viral pathogens likely to occur in great abundance, likely to be readily transported into ice, and then participate in ongoing disease cycles suggestive of their having been deposited in and subsequently released from ice. Caliciviruses, influenza A, and some enteroviruses appear to satisfy all three criteria. Environmental ice appears to be an important abiotic reservoir for pathogenic microbes. World health and eradication of specific pathogens could be affected by this huge reservoir.