Thermophilic viruses (phages) were first reported decades ago; however, our knowledge of their ecological significance is still very limited. In contrast to previous research on thermophilic viruses that focused on cultured viruses, this study examined the metagenomic profiles of uncultured viruses directly isolated from two alkaline hot springs in Yellowstone National Park, one at 74? and the other at 93?C. New methods of sampling and library construction were required to determine nearly 30 Mb of viral DNA sequence from these extreme environments. We are currently analyzing the metagenomes of the corresponding microbial populations, as well. This sequence data allows inferences about diversity, distribution and ecological impact of viruses in extreme thermal environments and the development of otherwise inaccessible analytical and industrial reagent enzymes.
Octopus Hot Spring
To date, researchers have used enrichment cultivation to identify hundreds of thermophilic bacteriophages and dozens of thermophilic crenarchaeal viruses, all from terrestrial hot springs, as well as one euryarchaeal virus from a marine vent. Morphologies and genomic analysis both suggest crenarchaeal viruses are unrelated to viruses of Euryarchaeota, Bacteria or Eukarya. Little is known about the roles of viruses in the ecology of hydrothermal environments, although they appear to play a role in host mortality and carbon cycling and are probably the only predators. In better studied low temperature environments, viruses are responsible for a significant proportion of microbial mortality and thus have a profound influence on carbon and other nutrient cycles. Marine viruses are also thought to be important vehicles for lateral gene transfer via lysogeny and transduction and probably promote diversity by preferentially lysing the most abundant species. Several lines of evidence suggest viruses in high temperature aquifers may play a unique role in global diversity and evolution. While enrichment cultures have been invaluable, important contextual information such as relative abundance, diversity and distribution is lost, and all information about the majority of viruses and cells that are not readily cultivated is lost.
The two mildly alkaline siliceous hot springs that were the sources of the viral metagenomic libraries, Octopus and Bear Paw, are 5 km apart in the Yellowstone Lower Geyser Basin. Both are direct outflows of a much larger thermal aquifer and not secondarily heated surface water. In this respect they are distinct from the acidic springs, mudpots, and other thermal features that have provided most of the published thermophilic virus samples. It is likely that the viruses proliferate deep in the vent, where increased pressures allow much higher temperatures than at the surface. Water temperatures of 150-200?C at 200-300 psi are found at depths of 100-300 meters throughout the caldera of YNP. If the viruses proliferate in the subsurface aquifer, hot springs separated by kilometer distances may share a common viral population. The pH value of both springs is 8; however, the temperatures and apparent microflora vary widely. Bear Paw (74?C) hot spring is characterized by orange sedentary microbial growth in the pool. Octopus hot spring water emerges at 93?C, the boiling point at the local elevation of 2500 meters and its bottom surface is grey.
Bear Paw Hot Spring
Viral abundances in the hot springs ranged from about 10e5 to 10e6 viruses per ml, within the range of 10e4 to 10e9 reported for thermal springs in California and moderate temperature aquatic environments (Schoenfeld et al., submitted). Metagenomic analysis suggests between 400 and 1400 different viral types in each hot spring. Although most sequences are unrelated to known viral or cellular genomic sequences, hundreds of BLASTx similarities to genes associated with replication, recombination, lysis, transcription, lysogeny, and viral structural were identified. 28% of the viral metagenome was shared between the two hot springs, suggesting important overlaps between the populations. Certain cultured viral genomes had remarkable similarity to the metagenomic sequences. For example, the metagenomic libraries have apparent homology to almost the entire 28 kb genome of Pyrobacculum spherical virus, a creanarchaeal virus that was isolated from an acidic hot spring some 30 km away from the sample sites of the current project. Similarities to bacteriophages and acidophilic archaeal viruses isolated on other continents were limited to specific open reading frames, but were equally strong. This research has also resulted in the development of DNA polymerases as research reagents and carbohydrases as industrial processing tools.
