Investigating the Diversity of Single-Stranded DNA Bacteriophages in Marine Environments

There are estimated to be 1030 virus-like particles in the world\u27s oceans. Most are viruses that infect bacteria, called `bacteriophages\u27 or simply `phages\u27. Phages exert tremendous influence on marine biogeochemical cycling because they are responsible for about half of all bacterial death in the oceans, causing nutrient release into the dissolved and particulate organic matter pools. Traditional paradigms of phage biology held that most of these ocean phages belonged to the Caudovirales group: phages that contain a double-stranded DNA genome within a geometric capsid `head\u27 to which a `tail\u27 is joined, in one of several morphological variants, that is the main structure allowing the phage to interact and infect a host bacterium. Compared to tailed phages, small, non- tailed, single-stranded DNA-containing phages have been an historical afterthought; believed to exist only in specialized, niche environments. However, recent studies harnessing advances in technology have revealed that single-stranded DNA phages are ubiquitous to nearly every marine environment yet tested. Small, icosahedral, single-stranded DNA bacteriophages of the subfamily Gokushovirinae (family Microviridae) exemplify the difficulty that viruses can present as study subjects. They are difficult to visualize by epifluorescence microscopy and contain a paucity of genetic and protein material. As a result, recognition of their importance in marine environments has lagged behind that of tailed, double-stranded DNA bacteriophages. This thesis seeks to redress this knowledge gap. The first chapter expands knowledge of gokushovirus diversity in the environment by developing a degenerate PCR assay to amplify a portion of the major capsid protein (MCP) gene of gokushoviruses. Over 500 amplicons were sequenced from ten diverse environmental samples (sediments, sewage, seawater and freshwater), revealing the ubiquity and high diversity of this understudied phage group. The data was aggregated in several informative ways. Multiple alignments were combined with a predicted 3D-structure to reveal regions of both high and low conservation. Viewed in a phylogenetic framework, many gokushovirus MCP clades contained samples from multiple environments, although distinct clades dominated the different sample types. Some environments, particularly pelagic sediments, appear as hotbeds of gokushovirus diversity, while freshwater springs were the least diverse. The second chapter used the same primer set to detect gokushovirus communities at 0 m and 100 m depth in two seasons from three years at the Bermuda Atlantic Time- series Study (BATS) site. As a result of twenty-six years of constant sampling, the annual hydrodynamic cycling of BATS is very well understood. This wealth of knowledge allows us to hypothesize that the winter deep mixing layer will act to connect the viral communities between 0 m and 100 m. Conversely, in summer when stratification occurs, viral communities at the two depths will become divergent. We find compelling evidence to support this hypothesis. The final chapter of this thesis details continuing efforts to characterize the first non-tailed, single-stranded DNA, temperate phage to infect a member of the globally important genus of marine autotroph, Synechococcus. Efforts undertaken have spanned genomic, metagenomic and proteomic methodologies. The lack of culturable, phage-host model systems for small, single-stranded DNA phages is today one of the most glaring impediments to increased understanding of these viruses. In combination with the data presented on environmental diversity, steps taken towards establishing this Synechococcus phage as a culturable model system makes this thesis a major contribution to the understanding of environmental ssDNA phages