Kenneth Stedman

Date of Award

Winter 3-4-2014

Document Type


Degree Name

Doctor of Philosophy (Ph.D.) in Biology



Physical Description

1 online resource (xi, 137 pages)


Metagenomics, Thermophilic microorganisms -- California -- Lassen Volcanic National Park, Viruses -- Evolution -- California -- Lassen Volcanic National Park, Viruses -- Genome mapping, Viral genomes




Viruses are the most abundant organisms on Earth, yet their collective evolutionary history, biodiversity and functional capacity is not well understood. Viral metagenomics offers a potential means of establishing a more comprehensive view of virus diversity and evolution, as vast amounts of new sequence data becomes available for comparative analysis.
Metagenomic DNA from virus-sized particles (smaller than 0.2 microns in diameter) was isolated from approximately 20 liters of sediment obtained from Boiling Springs Lake (BSL) and sequenced. BSL is a large, acidic hot-spring (with a pH of 2.2, and temperatures ranging from 50°C to 96°C) located in Lassen Volcanic National Park, USA. BSL supports a purely microbial ecosystem comprised largely of Archaea and Bacteria, however, the lower temperature regions permit the growth of acid- and thermo-tolerant Eukarya. This distinctive feature of the BSL microbial ecosystem ensures that virus types infecting all domains of life will be present. The metagenomic sequence data was used to characterize the types of viruses present within the microbial ecosystem, to ascertain the extent of genetic diversity and novelty comprising the BSL virus assemblage, and to explore the genomic and structural modalities of virus evolution.
Metagenomic surveys of natural virus assemblages, including the survey of BSL, have revealed that the diversity within the virosphere far exceeds what has currently been determined through the detailed study of viruses that are relevant to human health and agriculture. The number of as-yet-uncharacterized virus protein families present in the BSL assemblage was estimated by clustering analysis. Genomic context analysis of the predicted viral protein sequences in the BSL dataset indicates that most of the putative uncharacterized proteins are endemic or unique to BSL, and are largely harbored by known virus types. A comparative metagenomic analysis approach identified a set of conserved, yet uncharacterized BSL protein sequences that are commonly found in other similar and dissimilar environments.
New sequence data from metagenomic surveys of natural virus assemblages was also used to better characterize and define known virus protein families, as some of the viruses found in the BSL environment represent distant relatives of well-characterized isolates. By comparing viral genes and protein sequences from these highly divergent species, it is possible to better understand the dynamics of adaptation and evolution in the virosphere. Additionally, as structures of virus proteins continue to be experimentally determined by X-ray crystallography and cryo-electron microscopy, a merger of structural and metagenomic sequence data allows the opportunity to observe the structural dynamics underlying virus protein evolution.
Capsid (structural) proteins from two distinct Microviridae strains; a globally ubiquitous and highly sequence-diverse virus family, were identified in, and isolated from the BSL metagenomic DNA sample. These BSL capsid protein sequences, along with several other homologous sequences derived from metagenomic surveys and laboratory isolates, were mapped to the solved structure of a closely related capsid protein from the Spiroplasma phage-4 microvirus. Patterns of amino acid sequence conservation, unveiled by structure-based homology modeling analysis, revealed that the protein sequences within this family exhibit a remarkable level of plasticity, while remaining structurally and functionally congruent.
Lateral gene transfer is thought to have had a significant impact on the genomic evolution and adaptation of virus families. Genomic context analysis was also utilized to identify interviral gene transfer within the BSL virus assemblage. An ostensibly rare interviral gene transfer event, having transpired between single-stranded RNA and DNA virus types, was detected in the BSL metagenome. Similar genomes were subsequently detected in other ecosystems around the globe. The discovery of this new virus genome dramatically underscores the scope and importance of genetic mobility and genomic mosaicism as major forces driving the evolution of viruses.
The analyses conducted herein demonstrate the many ways in which viral metagenomic sequence data may be utilized to not only evaluate the composition of a natural virus assemblage, but to discover new viral genes, and to better understand the dynamics of both genomic and structural evolution within the virosphere.

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