Diversity and Distribution of a Novel Genus of Hyperthermophilic Aquificae Viruses Encoding a Proof-Reading Family-A DNA Polymerase.

Despite the high abundance of Aquificae in many geothermal systems, these bacteria are difficult to culture and no viruses infecting members of this phylum have been isolated. Here, we describe the complete, circular dsDNA Uncultivated Virus Genome (UViG) of Thermocrinis Octopus Spring virus (TOSV), derived from metagenomic data, along with eight related UViGs representing three additional viral species. Despite low overall similarity among viruses from different hot springs, the genomes shared a high degree of synteny, and encoded numerous genes for nucleotide metabolism, including a PolA-type DNA polymerase polyprotein with likely accessory functions, a DNA Pol III sliding clamp, a thymidylate kinase, a DNA gyrase, a helicase, and a DNA methylase. Also present were conserved genes predicted to code for phage capsid, large and small subunits of terminase, portal protein, holin, and lytic transglycosylase, all consistent with a distant relatedness to cultivated Caudovirales. These viruses are predicted to infect Aquificae, as multiple CRISPR spacers matching the viral genomes were identified within the genomes and metagenomic contigs from these bacteria. Based on the predicted atypical bi-directional replication strategy, low sequence similarity to known viral genomes, and unique position in gene-sharing networks, we propose a new putative genus, "Pyrovirus," in the order Caudovirales.

Engineering of a thermostable viral polymerase using metagenome-derived diversity for highly sensitive and specific RT-PCR.

Reverse transcription is an essential initial step in the analysis of RNA for most PCR-based amplification and detection methods. Despite advancements in these technologies, efficient conversion of RNAs that form stable secondary structures and double-stranded RNA targets remains challenging as retroviral-derived reverse transcriptases are often not sufficiently thermostable to catalyze synthesis at temperatures high enough to completely relax these structures. Here we describe the engineering and improvement of a thermostable viral family A polymerase with inherent reverse transcriptase activity for use in RT-PCR. Using the 3173 PyroPhage polymerase, previously identified from hot spring metagenomic sampling, and additional thermostable orthologs as a source of natural diversity, we used gene shuffling for library generation and screened for novel variants that retain high thermostability and display elevated reverse transcriptase activity. We then created a fusion enzyme between a high-performing variant polymerase and the 5'→3' nuclease domain of Taq DNA polymerase that provided compatibility with probe-based detection chemistries and enabled highly sensitive detection of structured RNA targets. This technology enables a flexible single-enzyme RT-PCR system that has several advantages compared with standard heat-labile reverse transcription methods.

A Multifunctional Reactor with Dry-Stored Reagents for Enzymatic Amplification of Nucleic Acids.

To enable inexpensive molecular detection at the point-of-care and at home with minimal or no instrumentation, it is necessary to streamline unit operations and store reagents refrigeration-free. To address this need, a multifunctional enzymatic amplification reactor that combines solid-phase nucleic acid extraction, concentration, and purification; refrigeration-free storage of reagents with just-in-time release; and enzymatic amplification is designed, prototyped, and tested. A nucleic acid isolation membrane is placed at the reactor's inlet, and paraffin-encapsulated reagents are prestored within the reactor. When a sample mixed with chaotropic agents is filtered through the nucleic acid isolation membrane, the membrane binds nucleic acids from the sample. Importantly, the sample volume is decoupled from the reaction volume, enabling the use of relatively large sample volumes for high sensitivity. When the amplification reactor's temperature increases to its operating level, the paraffin encapsulating the reagents melts and moves out of the way. The reagents are hydrated, just-in-time, and the polymerase reaction proceeds. The amplification process can be monitored, in real-time. We demonstrate our reactors' ability to amplify both DNA and RNA targets using polymerase with both reverse-transcriptase and strand displacement activities to obtain sensitivities on-par with benchtop equipment and a shelf life exceeding 6 months.

Complete Genome Sequence of Thermus aquaticus Y51MC23.

Thermus aquaticus Y51MC23 was isolated from a boiling spring in the Lower Geyser Basin of Yellowstone National Park. Remarkably, this T. aquaticus strain is able to grow anaerobically and produces multiple morphological forms. Y51MC23 is a Gram-negative, rod-shaped organism that grows well between 50°C and 80°C with maximum growth rate at 65°C to 70°C. Growth studies suggest that Y51MC23 primarily scavenges protein from the environment, supported by the high number of secreted and intracellular proteases and peptidases as well as transporter systems for amino acids and peptides. The genome was assembled de novo using a 350 bp fragment library (paired end sequencing) and an 8 kb long span mate pair library. A closed and finished genome was obtained consisting of a single chromosome of 2.15 Mb and four plasmids of 11, 14, 70, and 79 kb. Unlike other Thermus species, functions usually found on megaplasmids were identified on the chromosome. The Y51MC23 genome contains two full and two partial prophage as well as numerous CRISPR loci. The high identity and synteny between Y51MC23 prophage 2 and that of Thermus sp. 2.9 is interesting, given the 8,800 km separation of the two hot springs from which they were isolated. The anaerobic lifestyle of Y51MC23 is complex, with multiple morphologies present in cultures. The use of fluorescence microscopy reveals new details about these unusual morphological features, including the presence of multiple types of large and small spheres, often forming a confluent layer of spheres. Many of the spheres appear to be formed not from cell envelope or outer membrane components as previously believed, but from a remodeled peptidoglycan cell wall. These complex morphological forms may serve multiple functions in the survival of the organism, including food and nucleic acid storage as well as colony attachment and organization.

A novel thermostable polymerase for RNA and DNA loop-mediated isothermal amplification (LAMP).

Meeting the goal of providing point of care (POC) tests for molecular detection of pathogens in low resource settings places stringent demands on all aspects of the technology. OmniAmp DNA polymerase (Pol) is a thermostable viral enzyme that enables true POC use in clinics or in the field by overcoming important barriers to isothermal amplification. In this paper, we describe the multiple advantages of OmniAmp Pol as an isothermal amplification enzyme and provide examples of its use in loop-mediated isothermal amplification (LAMP) for pathogen detection. The inherent reverse transcriptase activity of OmniAmp Pol allows single enzyme detection of RNA targets in RT-LAMP. Common methods of nucleic acid amplification are highly susceptible to sample contaminants, necessitating elaborate nucleic acid purification protocols that are incompatible with POC or field use. OmniAmp Pol was found to be less inhibited by whole blood components typical in certain crude sample preparations. Moreover, the thermostability of the enzyme compared to alternative DNA polymerases (Bst) and reverse transcriptases allows pretreatment of complete reaction mixes immediately prior to amplification, which facilitates amplification of highly structured genome regions. Compared to Bst, OmniAmp Pol has a faster time to result, particularly with more dilute templates. Molecular diagnostics in field settings can be challenging due to the lack of refrigeration. The stability of OmniAmp Pol is compatible with a dry format that enables long term storage at ambient temperatures. A final requirement for field operability is compatibility with either commonly available instruments or, in other cases, a simple, inexpensive, portable detection mode requiring minimal training or power. Detection of amplification products is shown using lateral flow strips and analysis on a real-time PCR instrument. Results of this study show that OmniAmp Pol is ideally suited for low resource molecular detection of pathogens.

Lateral gene transfer of family A DNA polymerases between thermophilic viruses, aquificae, and apicomplexa.

Bioinformatics and functional screens identified a group of Family A-type DNA Polymerase (polA) genes encoded by viruses inhabiting circumneutral and alkaline hot springs in Yellowstone National Park and the US Great Basin. The proteins encoded by these viral polA genes (PolAs) shared no significant sequence similarity with any known viral proteins but were remarkably similar to PolAs encoded by two of three families of the bacterial phylum Aquificae and by several apicoplast-targeted PolA-like proteins found in the eukaryotic phylum Apicomplexa, which includes the obligate parasites Plasmodium, Babesia, and Toxoplasma. The viral gene products share signature elements previously associated only with Aquificae and Apicomplexa PolA-like proteins and were similar to proteins encoded by prophage elements of a variety of otherwise unrelated Bacteria, each of which additionally encoded a prototypical bacterial PolA. Unique among known viral DNA polymerases, the viral PolA proteins of this study share with the Apicomplexa proteins large amino-terminal domains with putative helicase/primase elements but low primary sequence similarity. The genomic context and distribution, phylogeny, and biochemistry of these PolA proteins suggest that thermophilic viruses transferred polA genes to the Apicomplexa, likely through secondary endosymbiosis of a virus-infected proto-apicoplast, and to the common ancestor of two of three Aquificae families, where they displaced the orthologous cellular polA gene. On the basis of biochemical activity, gene structure, and sequence similarity, we speculate that the xenologous viral-type polA genes may have functions associated with diversity-generating recombination in both Bacteria and Apicomplexa.

Evaluation of the specificity and sensitivity of a potential rapid influenza screening system.

Influenza remains a serious worldwide health threat with numerous deaths attributed to influenza-related complications. It is likely that transmission of influenza and both the morbidity and mortality of influenza could be reduced if inexpensive but reliable influenza screening assays were more available to the general public or local medical treatment facilities. This report provides the initial evaluation of a pilot system designed by Lucigen Corp. (Middleton, WI, USA) as a potential rapid near point-of-care screening system for influenza A and influenza B. The evaluation of specificity and sensitivity was conducted on stored nasal swab samples collected from emergency department patients presenting with influenza-like symptoms at a large military academic hospital and on de-identified nasal swabs and isolated RNA from a local epidemiology laboratory. The gold standard for assessment of specificity and sensitivity was the Luminex® Respiratory Viral Panel.

Thermostable DNA polymerase from a viral metagenome is a potent RT-PCR enzyme.

Viral metagenomic libraries are a promising but previously untapped source of new reagent enzymes. Deep sequencing and functional screening of viral metagenomic DNA from a near-boiling thermal pool identified clones expressing thermostable DNA polymerase (Pol) activity. Among these, 3173 Pol demonstrated both high thermostability and innate reverse transcriptase (RT) activity. We describe the biochemistry of 3173 Pol and report its use in single-enzyme reverse transcription PCR (RT-PCR). Wild-type 3173 Pol contains a proofreading 3'-5' exonuclease domain that confers high fidelity in PCR. An easier-to-use exonuclease-deficient derivative was incorporated into a PyroScript RT-PCR master mix and compared to one-enzyme (Tth) and two-enzyme (MMLV RT/Taq) RT-PCR systems for quantitative detection of MS2 RNA, influenza A RNA, and mRNA targets. Specificity and sensitivity of 3173 Pol-based RT-PCR were higher than Tth Pol and comparable to three common two-enzyme systems. The performance and simplified set-up make this enzyme a potential alternative for research and molecular diagnostics.

Complete Genome Sequence of Paenibacillus strain Y4.12MC10, a Novel Paenibacillus lautus strain Isolated from Obsidian Hot Spring in Yellowstone National Park.

Paenibacillus sp.Y412MC10 was one of a number of organisms isolated from Obsidian Hot Spring, Yellowstone National Park, Montana, USA under permit from the National Park Service. The isolate was initially classified as a Geobacillus sp. Y412MC10 based on its isolation conditions and similarity to other organisms isolated from hot springs at Yellowstone National Park. Comparison of 16 S rRNA sequences within the Bacillales indicated that Geobacillus sp.Y412MC10 clustered with Paenibacillus species, and the organism was most closely related to Paenibacillus lautus. Lucigen Corp. prepared genomic DNA and the genome was sequenced, assembled, and annotated by the DOE Joint Genome Institute. The genome sequence was deposited at the NCBI in October 2009 (NC_013406). The genome of Paenibacillus sp. Y412MC10 consists of one circular chromosome of 7,121,665 bp with an average G+C content of 51.2%. Comparison to other Paenibacillus species shows the organism lacks nitrogen fixation, antibiotic production and social interaction genes reported in other paenibacilli. The Y412MC10 genome shows a high level of synteny and homology to the draft sequence of Paenibacillus sp. HGF5, an organism from the Human Microbiome Project (HMP) Reference Genomes. This, combined with genomic CAZyme analysis, suggests an intestinal, rather than environmental origin for Y412MC10.

Functional viral metagenomics and the next generation of molecular tools.

The enzymes of bacteriophages and other viruses have been essential research tools since the first days of molecular biology. However, the current repertoire of viral enzymes only hints at their overall potential. The most commonly used enzymes are derived from a surprisingly small number of cultivated viruses, which is remarkable considering the extreme abundance and diversity of viruses revealed over the past decade by metagenomic analysis. To access the treasure trove of enzymes hidden in the global virosphere and develop them for research, therapeutic and diagnostic uses, improvements are needed in our ability to rapidly and efficiently discover, express and characterize viral genes to produce useful proteins. In this paper, we discuss improvements to sampling and cloning methods, functional and genomics-based screens, and expression systems, which should accelerate discovery of new enzymes and other viral proteins for use in research and medicine.

Germ warfare in a microbial mat community: CRISPRs provide insights into the co-evolution of host and viral genomes.

CRISPR arrays and associated cas genes are widespread in bacteria and archaea and confer acquired resistance to viruses. To examine viral immunity in the context of naturally evolving microbial populations we analyzed genomic data from two thermophilic Synechococcus isolates (Syn OS-A and Syn OS-B') as well as a prokaryotic metagenome and viral metagenome derived from microbial mats in hotsprings at Yellowstone National Park. Two distinct CRISPR types, distinguished by the repeat sequence, are found in both the Syn OS-A and Syn OS-B' genomes. The genome of Syn OS-A contains a third CRISPR type with a distinct repeat sequence, which is not found in Syn OS-B', but appears to be shared with other microorganisms that inhabit the mat. The CRISPR repeats identified in the microbial metagenome are highly conserved, while the spacer sequences (hereafter referred to as "viritopes" to emphasize their critical role in viral immunity) were mostly unique and had no high identity matches when searched against GenBank. Searching the viritopes against the viral metagenome, however, yielded several matches with high similarity some of which were within a gene identified as a likely viral lysozyme/lysin protein. Analysis of viral metagenome sequences corresponding to this lysozyme/lysin protein revealed several mutations all of which translate into silent or conservative mutations which are unlikely to affect protein function, but may help the virus evade the host CRISPR resistance mechanism. These results demonstrate the varied challenges presented by a natural virus population, and support the notion that the CRISPR/viritope system must be able to adapt quickly to provide host immunity. The ability of metagenomics to track population-level variation in viritope sequences allows for a culture-independent method for evaluating the fast co-evolution of host and viral genomes and its consequence on the structuring of complex microbial communities.

Genome signature analysis of thermal virus metagenomes reveals Archaea and thermophilic signatures.

BACKGROUND: Metagenomic analysis provides a rich source of biological information for otherwise intractable viral communities. However, study of viral metagenomes has been hampered by its nearly complete reliance on BLAST algorithms for identification of DNA sequences. We sought to develop algorithms for examination of viral metagenomes to identify the origin of sequences independent of BLAST algorithms. We chose viral metagenomes obtained from two hot springs, Bear Paw and Octopus, in Yellowstone National Park, as they represent simple microbial populations where comparatively large contigs were obtained. Thermal spring metagenomes have high proportions of sequences without significant Genbank homology, which has hampered identification of viruses and their linkage with hosts. To analyze each metagenome, we developed a method to classify DNA fragments using genome signature-based phylogenetic classification (GSPC), where metagenomic fragments are compared to a database of oligonucleotide signatures for all previously sequenced Bacteria, Archaea, and viruses. RESULTS: From both Bear Paw and Octopus hot springs, each assembled contig had more similarity to other metagenome contigs than to any sequenced microbial genome based on GSPC analysis, suggesting a genome signature common to each of these extreme environments. While viral metagenomes from Bear Paw and Octopus share some similarity, the genome signatures from each locale are largely unique. GSPC using a microbial database predicts most of the Octopus metagenome has archaeal signatures, while bacterial signatures predominate in Bear Paw; a finding consistent with those of Genbank BLAST. When using a viral database, the majority of the Octopus metagenome is predicted to belong to archaeal virus Families Globuloviridae and Fuselloviridae, while none of the Bear Paw metagenome is predicted to belong to archaeal viruses. As expected, when microbial and viral databases are combined, each of the Octopus and Bear Paw metagenomic contigs are predicted to belong to viruses rather than to any Bacteria or Archaea, consistent with the apparent viral origin of both metagenomes. CONCLUSION: That BLAST searches identify no significant homologs for most metagenome contigs, while GSPC suggests their origin as archaeal viruses or bacteriophages, indicates GSPC provides a complementary approach in viral metagenomic analysis.

Assembly of viral metagenomes from yellowstone hot springs.

Thermophilic viruses were reported decades ago; however, knowledge of their diversity, biology, and ecological impact is limited. Previous research on thermophilic viruses focused on cultivated strains. This study examined metagenomic profiles of viruses directly isolated from two mildly alkaline hot springs, Bear Paw (74 degrees C) and Octopus (93 degrees C). Using a new method for constructing libraries from picograms of DNA, nearly 30 Mb of viral DNA sequence was determined. In contrast to previous studies, sequences were assembled at 50% and 95% identity, creating composite contigs up to 35 kb and facilitating analysis of the inherent heterogeneity in the populations. Lowering the assembly identity reduced the estimated number of viral types from 1,440 and 1,310 to 548 and 283, respectively. Surprisingly, the diversity of viral species in these springs approaches that in moderate-temperature environments. While most known thermophilic viruses have a chronic, nonlytic infection lifestyle, analysis of coding sequences suggests lytic viruses are more common in geothermal environments than previously thought. The 50% assembly included one contig with high similarity and perfect synteny to nine genes from Pyrobaculum spherical virus (PSV). In fact, nearly all the genes of the 28-kb genome of PSV have apparent homologs in the metagenomes. Similarities to thermoacidophilic viruses isolated on other continents were limited to specific open reading frames but were equally strong. Nearly 25% of the reads showed significant similarity between the hot springs, suggesting a common subterranean source. To our knowledge, this is the first application of metagenomics to viruses of geothermal origin.

Anatomical contributions to odorant sampling and representation in rodents: zoning in on sniffing behavior.

Odorant sampling behaviors such as sniffing bring odorant molecules into contact with olfactory receptor neurons (ORNs) to initiate the sensory mechanisms of olfaction. In rodents, inspiratory airflow through the nose is structured and laminar; consequently, the spatial distribution of adsorbed odorant molecules during inspiration is predictable. Physicochemical properties such as water solubility and volatility, collectively called sorptiveness, interact with behaviorally regulable variables such as inspiratory flow rate to determine the pattern of odorant deposition along the inspiratory path. Populations of ORNs expressing the same odorant receptor are distributed in strictly delimited regions along this inspiratory path, enabling different deposition patterns of the same odorant to evoke different patterns of neuronal activation across the olfactory epithelium and in the olfactory bulb. We propose that both odorant sorptive properties and the regulation of sniffing behavior may contribute to rodents' olfactory capacities by this mechanism. In particular, we suggest that the motor regulation of sniffing behavior is substantially utilized for purposes of "zonation" or the direction of odorant molecules to defined intranasal regions and hence toward distinct populations of receptor neurons, pursuant to animals' sensory goals.

The anatomical logic of smell.

Olfactory receptor neurons (ORNs) expressing the same odorant receptor gene share ligand-receptor affinity profiles and converge onto common glomerular targets in the brain. The activation patterns of different ORN populations, evoked by differential binding of odorant molecular moieties, constitute the primary odor representation. However, odorants possess properties other than receptor-binding sites that can contribute to odorant discrimination. Among terrestrial vertebrates, odorant sorptiveness--volatility and water solubility--imposes physicochemical constraints on migration through the nose during inspiration. The non-uniform distributions of ORN populations along the inspiratory axis enable sorptiveness to modify odor representations by affecting the number of molecules reaching different receptors during a sniff. Animals can then modify and analyze odor representation further by the dynamic regulation of sniffing.

Evidence for the disproportionate mapping of olfactory airspace onto the main olfactory bulb of the hamster.

Olfactory receptor neurons (ORNs) project to the rodent main olfactory bulb (MOB) from spatially distinct air channels in the olfactory recesses of the nose. The relatively smooth central channels of the dorsal meatus map onto the dorsal MOB, whereas the highly convoluted peripheral channels of the ethmoid turbinates project to the ventral MOB. Medial and lateral components of each projection stream innervate the medial and lateral MOB, respectively. To ascertain whether such topography entails the disproportionate representation seen in other sensory maps, we used disector-based stereological techniques in hamsters to estimate the number of ORNs associated with each channel in the nose and the number of their targets (glomeruli and mitral and tufted cells) in corresponding divisions of the MOB. Each circumferential half of the MOB (dorsal/ventral, medial/lateral) contained about 50% of the 3,100 glomeruli and about 50% of the 160,000 mitral and tufted cells per bulb. We found equivalent numbers of ORNs with dendritic knobs in the medial and lateral channels (4.5 million each). However, the central channels had only 2 million knobbed ORNs, whereas the peripheral channels had 7 million. Thus, there is a disproportionate mapping of the central-peripheral axis of olfactory airspace onto the dorsal-ventral axis of the MOB, encompassing a greater than threefold variation in the average convergence of ORNs onto MOB secondary neurons. We hypothesize that the disproportionate projections help to optimize chemospecific processing by compensating, with differing sensitivity, for significant variation in the distribution and concentration of odorant molecules along the olfactory air channels during sniffing.

NADPH diaphorase activity in olfactory receptor neurons and their axons conforms to a rhinotopically-distinct dorsal zone of the hamster nasal cavity and main olfactory bulb.

NADPH diaphorase histochemical protocols were optimized for the histochemical labeling of olfactory receptor neurons (ORNs) in the nasal cavity and their axon terminals in glomeruli of the main olfactory bulb (MOB) in the Syrian hamster. This labeling was then used to map and quantify the spatial distribution of ORNs and their central projections. Diaphorase-positive ORNs were found to be rhinotopically restricted to dorsal-medially situated segments of sensory mucosa associated with central air channels in the nose, together constituting about 25% of the total receptor sheet. This topography closely resembles the zonal expression patterns of putative odorant receptor genes and cell surface glycoconjugates in the nose. Moreover, the projections of ORNs in the diaphorase-positive dorsal/central zone were found to expand onto the entire dorsal half of the MOB, consistent with spatial patterns discerned in retrograde tract-tracing studies. These boundaries indicate that dorsal/central zone ORNs project to a disproportionately larger region of the MOB than do those in the more ventral/peripheral zones. The demonstration of NADPH diaphorase activity in ORNs is inconsistent with the expression of the best-known NADPH-dependent enzymes, such as nitric oxide synthase (neuronal and endothelial isoforms) and NADPH cytochrome P450 oxidoreductase. Understanding the spatial patterning of histochemical labeling in ORNs should facilitate the biochemical identification of this diaphorase.