Phage Communities in Hot Springs

Boiling thermal pools are unique ecosystems to study microbial ecology, biogeochemistry and evolution, because they are physically isolated from other ecosystems, they are relatively biologically simple, and they are among the most hostile environments known. The water column of boiling hot springs represents an extreme environment that has not been previously explored for viruses or microbes. Planktonic organisms found in the water column potentially originate from significantly higher temperatures and pressures than those found in surface sediments. For example, water temperatures as high as 238°C have been measured in shallow drill holes (< 330 m) in Yellowstone National Park. Bacteriophage and host cells emanating from this formidable environment have important implications for a number of disciplines. Constructing genomic libraries from extreme environmental niches is challenging due to the low abundance of microbes and bacteriophage; for example, as little as 10,000 cells per ml are found in thermal aquifers, yielding only picogram amounts of DNA. Lucigen has developed methods to make complex gene libraries from anonymous DNA sequences, starting with less than a picogram of purified material. Improved sampling methods and our NanoClone, Single Cell Genomics and CloneSmart technologies have allowed construction of complex community genomic libraries from very limited samples of directly isolated microbial and viral DNA. Limited 16S rRNA sequencing of NanoClone libraries made from Bath hot spring has revealed at least 20 distinct Bacteria and Archaea, many without significant similarity to cultivated microbes. Sequence analysis of approximately 5,000 reads from a bacteriophage metagenomic library derived from Little Hot Creek also shows limited similarity to the NCBI database and a surprising degree of diversity. Comparative data analysis of phage DNA from four different thermal pools will provide a unique glimpse into the diversity and community makeup of a unique set of environments. The goal of this field trip is to collect additional bacteriophage and microbial samples from new hot springs to construct metagenomic libraries for sequence analysis and expression studies. Analysis of the diversity of bacteriophage genes in different thermal pools from geographically similar and isolated sites will begin to answer important questions about the ecology of hot springs. We will use methods similar to those employed by Breitbart et al. (Genomic analysis of an uncultured marine viral community. Proceedings of the National Academy USA 99:14250-14255, 2002). D Mead will collect samples on this trip and they will be processed at Lucigen for phage and microbial expression analysis (T Schoenfeld, R Godiska, P Brumm). Data analysis will be performed by F Rohwer. Applicants: Grieg F Steward, Asst Professor; Jennifer R. Brum, Graduate Student Grieg Steward Department of Oceanography School of Ocean & Earth Science & Technology University of Hawaii at Manoa 1000 Pope Road Honolulu, HI 96822 808-956-6775 (Office) 808-956-8629 (Lab) 808-956-9516 (Fax) ABSTRACT Morphological and biochemical characterization of viral communities in extreme environments.Morphological and biochemical characterization of viral communities in extreme environments. Viruses are the most diverse and numerous microbes in aquatic systems, but their ecology in extreme environments remains poorly studied compared to oceans and freshwater lakes. Extreme environments are likely to harbor many novel viruses. Although differences in viral assemblages among diverse environments is proximally controlled by the host presence and diversity, lytic viruses must also be adapted to survive as free virions in the chemical and physical conditions of their particular habitat. Comparisons among diverse environments may thus reveal themes in the evolution, adaptation, and diversity of viruses. We propose to obtain an overview of the characteristics of the free virions within hot springs and /or Mono Lake to seek novel morphologies and relate these to physical properties and biochemical composition. Viruses would be harvested by filtration and ultrafiltration in the field. In the lab the viruses in the concentrate would be subjected to 2-dimensional physical fractionation to determine the range of biophysical and biochemical properties present in the assemblages. The viral concentrates will be fractionated in a CsCl equilibrium density gradient. Each CsCl fraction will then be separated in a second dimension by ion exchange chromatography. Fractions will be examined by transmission electron microscopy to document morphologies and analyzed to determine the associated nucleic acid size and type (RNA vs DNA). Given sufficient starting material, additional analyses could also be performed on the archived viral fractions (biochemical composition, genome sequence, proteome analysis). Applicants: Grieg F Steward, Asst Professor; Jennifer R. Brum, Graduate Student