The Yellowstone caldera contains the most numerous and diverse geothermal systems on Earth, yielding an extensive array of unique high-temperature environments that host a variety of deeply-rooted and understudied Archaea, Bacteria and Eukarya. The combination of extreme temperature and chemical conditions encountered in geothermal environments often results in considerably less microbial diversity than other terrestrial habitats and offers a tremendous opportunity for studying the structure and function of indigenous microbial communities and for establishing linkages between putative metabolisms and element cycling. Metagenome sequence (14–15,000 Sanger reads per site) was obtained for five high-temperature (>65°C) chemotrophic microbial communities sampled from geothermal springs (or pools) in Yellowstone National Park (YNP) that exhibit a wide range in geochemistry including pH, dissolved sulfide, dissolved oxygen and ferrous iron. Metagenome data revealed significant differences in the predominant phyla associated with each of these geochemical environments. Novel members of the Sulfolobales are dominant in low pH environments, while other Crenarchaeota including distantly-related Thermoproteales and Desulfurococcales populations dominate in suboxic sulfidic sediments. Several novel archaeal groups are well represented in an acidic (pH 3) Fe-oxyhydroxide mat, where a higher O2 influx is accompanied with an increase in archaeal diversity. The presence or absence of genes and pathways important in S oxidation-reduction, H2-oxidation, and aerobic respiration (terminal oxidation) provide insight regarding the metabolic strategies of indigenous organisms present in geothermal systems. Multiple-pathway and protein-specific functional analysis of metagenome sequence data corroborated results from phylogenetic analyses and clearly demonstrate major differences in metabolic potential across sites. The distribution of functional genes involved in electron transport is consistent with the hypothesis that geochemical parameters (e.g., pH, sulfide, Fe, O2) control microbial community structure and function in YNP geothermal springs.

Icosahedral nontailed double-stranded DNA (dsDNA) viruses are present in all three domains of life, leading to speculation about a common viral ancestor that predates the divergence of Eukarya, Bacteria, and Archaea. This suggestion is supported by the shared general architecture of this group of viruses and the common fold of their major capsid protein. However, limited information on the diversity and replication of archaeal viruses, in general, has hampered further analysis. Sulfolobus turreted icosahedral virus (STIV), isolated from a hot spring in Yellowstone National Park, was the first icosahedral virus with an archaeal host to be described. Here we present a detailed characterization of the components forming this unusual virus. Using a proteomics-based approach, we identified nine viral and two host proteins from purified STIV particles. Interestingly, one of the viral proteins originates from a reading frame lacking a consensus start site. The major capsid protein (B345) was found to be glycosylated, implying a strong similarity to proteins from other dsDNA viruses. Sequence analysis and structural predication of virion-associated viral proteins suggest that they may have roles in DNA packaging, penton formation, and protein-protein interaction. The presence of an internal lipid layer containing acidic tetraether lipids has also been confirmed. The previously presented structural models in conjunction with the protein, lipid, and carbohydrate information reported here reveal that STIV is strikingly similar to viruses associated with the Bacteria and Eukarya domains of life, further strengthening the hypothesis for a common ancestor of this group of dsDNA viruses from all domains of life.In comparison to viruses with eukaryotic and bacterial hosts, little is known about the viruses that infect Archaea. This is due, in part, to the relatively recent delineation of the archaeal domain of life but, more significantly, to the challenges of isolating and culturing the host organisms (42). The extreme environments favored by many archaeal species and limited knowledge about their biochemistry and biology exacerbate this problem. Often, it is through the study of host-virus interactions that insights to the biology of the host are elucidated. The recent discovery of Sulfolobus turreted icosahedral virus (STIV) presents an opportunity to expand our knowledge of virology, study host biology, and investigate the evolutionary relationship of viruses from all three domains of life. Studies on the structure of STIV have revealed similarities with prokaryotic and eukaryotic viruses that suggest a common ancestry for icosahedral double-stranded DNA (dsDNA) viruses (30, 38).STIV was isolated from Sulfolobus enrichment cultures that were established from a high-temperature acidic hot spring (ϳ80°C, pH ϳ3) in Yellowstone National Park (38). The virus was subsequently shown to infect virus-free isolates of Sulfolobus solfataricus strain P2, for which the complete genome has been sequenced. The electron cryomicroscopy (cry...

Virus enumeration by epifluorescence microscopy (EFM) is routinely done on preserved, refrigerated samples. Concerns about obtaining accurate and reproducible estimates led us to examine procedures for counting viruses by EFM. Our results indicate that aldehyde fixation results in rapid decreases in viral abundance. By 1 h postfixation, the abundance dropped by 16.4% ؎ 5.2% (n ‫؍‬ 6), and by 4 h, the abundance was 20 to 35% lower. The average loss rates for glutaraldehyde-and formaldehyde-fixed samples over the first 2 h were 0.12 and 0.13 h ؊1 , respectively. By 16 days, viral abundance had decreased by 72% (standard deviation, 6%; n ‫؍‬ 6). Aldehyde fixation of samples followed by storage at 4°C, for even a few hours, resulted in large underestimates of viral abundance. The viral loss rates were not constant, and in glutaraldehyde-and formaldehyde-fixed samples they decreased from 0.13 and 0.17 h ؊1 during the first hour to 0.01 h ؊1 between 24 and 48 h. Although decay rates changed over time, the abundance was predicted by using separate models to describe decay over the first 8 h and decay beyond 8 h. Accurate estimates of abundance were easily made with unfixed samples stained with Yo-Pro-1, SYBR Green I, or SYBR Gold, and slides could be stored at ؊20°C for at least 2 weeks or, for Yo-Pro-1, at least 1 year. If essential, samples can be fixed and flash frozen in liquid nitrogen upon collection and stored at ؊86°C. Determinations performed with fixed samples result in large underestimates of abundance unless slides are made immediately or samples are flash frozen. If protocols outlined in this paper are followed, EFM yields accurate estimates of viral abundance.The realization that viruses are abundant in natural waters and have major effects on the mortality of heterotrophic and autotrophic microbial communities (reviewed in references 9, 20, 24, and 25) has provided the impetus to develop procedures to rapidly and accurately enumerate viral particles in cultures and natural samples. Originally, these efforts focused on using electron microscopy (1, 3, 4, 18), but this was soon superseded by more rapid and accurate methods based on epifluorescence microscopy (EFM) (11,12,21,23) and, most recently, flow cytometry (5, 6, 13).Enumeration of viruses by EFM and flow cytometry is based on using highly fluorescent nucleic acid dyes. In the initial methods the approach used for enumerating bacteria in natural samples (17) was modified by staining the virus particles with DAPI (4Ј,6-diamidino-2-phenylindole) and enumerating them on filters by EFM (11,21,23). Subsequently, the approach was modified by replacing DAPI with Yo-Pro-1 (12). Although Yo-Pro-1 has significant advantages over DAPI in that the fluorescence is more stable and the fluorescence yield and the binding coefficient for nucleic acids are higher, it has the disadvantages of relatively long staining times and interference by aldehyde-based fixatives. Hence, Yo-Pro-1-stained slides need to be made with freshly collected samples. Nonetheless, the accura...

Little is known about the replication cycle of archaeal viruses. We have investigated the ultrastructural changes of Sulfolobus solfataricus P2 associated with infection by Sulfolobus turreted icosahedral virus (STIV). A time course of a near synchronous STIV infection was analyzed using both scanning and transmission electron microscopy. Assembly of STIV particles, including particles lacking DNA, was observed within cells, and fully assembled STIV particles were visible by 30 h postinfection (hpi). STIV was determined to be a lytic virus, causing cell disruption beginning at 30 hpi. Prior to cell lysis, virus infection resulted in the formation of pyramid-like projections from the cell surface. These projections, which have not been documented in any other host-virus system, appeared to be caused by the protrusion of the cell membrane beyond the bordering S-layer. These structures are thought to be sites at which progeny virus particles are released from infected cells. Based on these observations of lysis, a plaque assay was developed for STIV. From these studies we propose an overall assembly model for STIV.