Background: Genetic recombination is a driving force in genome evolution. Among viruses it has a dual role. For genomes with higher fitness, it maintains genome integrity in the face of high mutation rates. Conversely, for genomes with lower fitness, it provides immediate access to sequence space that cannot be reached by mutation alone. Understanding how recombination impacts the cohesion and dissolution of individual whole genomes within viral sequence space is poorly understood across double-stranded DNA bacteriophages (a.k.a phages) due to the challenges of obtaining appropriately scaled genomic datasets. Results: Here we explore the role of recombination in both maintaining and differentiating whole genomes of 142 wild double-stranded DNA marine cyanophages. Phylogenomic analysis across the 51 core genes revealed ten lineages, six of which were well represented. These phylogenomic lineages represent discrete genotypic populations based on comparisons of intra-and inter-lineage shared gene content, genome-wide average nucleotide identity, as well as detected gaps in the distribution of pairwise differences between genomes. McDonald-Kreitman selection tests identified putative niche-differentiating genes under positive selection that differed across the six well-represented genotypic populations and that may have driven initial divergence. Concurrent with patterns of recombination of discrete populations, recombination analyses of both genic and intergenic regions largely revealed decreased genetic exchange across individual genomes between relative to within populations. Conclusions: These findings suggest that discrete double-stranded DNA marine cyanophage populations occur in nature and are maintained by patterns of recombination akin to those observed in bacteria, archaea and in sexual eukaryotes.

There are estimated to be 1030 virus-like particles in the world's oceans. Most are viruses that infect bacteria, called `bacteriophages' or simply `phages'. 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' to which a `tail' 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.

The field of viral ecology is still relatively new and many processes by which viruses influence hosts are still widely unknown. One problem is that there are few standardized techniques in virus ecology, making comparisons of data very difficult. To better understand the methodology, we first set out to make a video showing the process for the viral production assay using the “dilution and reoccurrence” method, which has become the standard to analyze production rates in aquatic ecosystems. Using this method, we also determined the production rates of viruses during a seasonal pelagic phytoplankton bloom during a cruise off the coast of the north island of New Zealand in the subtropical Pacific Ocean. Other biotic and abiotic parameters were also compared throughout the bloom. Production rates were within normal ranges, but showed that viruses were very important for the remobilization of nutrients in the nitrogen-limited system. It is well known that the cyanobacterial genera Synechococcus and Prochlorococcus thrive in the world’s oceans with Synechococcus and other cyanobacterial species also succeeding in freshwater ecosystems. Cyanophages are viruses which infect cyanobacteria and many studies have investigated their diversity using the portal vertex g20 gene in the Cyanomyoviridae family. Although we know that there is significant genetic richness in these phage in marine and freshwater environments, information on their numerical distributions is rare. Using quantitative PCR with the g20 gene, we determined that cyanomyoviruses are ubiquitous and abundant in the Atlantic and Pacific Oceans as well as within Lake Erie. Using statistical analyses we were able to find correlations between cyanomyoviruses and other biotic and abiotic parameters: in the Sargasso Sea, cyanomyovirus abundance correlated well to biology, but in the other systems there was no significant correlation to biological abundances. This suggests that the constraints of this group of viruses may be different in different aquatic realms.

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Part 1: Prophage induction has been demonstrated to be a sensitive indicator for a wide variety of toxic and mutagenic compounds and, as a consequence, has been utilized for biologically based carcinogen screenings. Fourteen marine bacterial isolates were screened for development into the Marine Prophage Induction Assay (MPIA), for marine samples. The selected isolate (P99-4S3) was identified by 16S rDNA sequencing as Pseudomonas aeruginosa. This isolate demonstrated a log-linear response to increasing dose of mutagens, and sensitivity to known environmental contaminants. Field-testing of the assay over two years demonstrated the MPIA would be a useful screening tool for environmental contamination. Part 2: The observed resistance of natural populations of Synechococcus to viral infection may be due to lysogeny with associated homoimmunity. A thirteen-month study of lysogeny in natural populations of Synechococcus demonstrated that lysogeny does occur and exhibits a seasonal pattern. Experiments were performed along a transect of the Mississippi River plume, which provided a variety of ambient nutrient regimes for comparison of lysogeny in Synechococcus. Nutrient amendments did not enable induction and often led to a decrease in viral production. Lysogeny in Synechococcus was primarily correlated with ambient host and cyanophage abundance. Cross-infectivity studies demonstrated cyanophage isolates possess variable virulence. The 35 isolates were examined by transmission electron microscopy (TEM), with 33 identified as myoviruses and two as podoviruses. This dominance of myovirus lytic cyanophage is consistent with prior observations. Twenty-five Synechococcus isolates were screened for prophage induction utilizing the inducing agent Mitomycin C. Eleven isolates demonstrated a statistically significant increase in virus-like particles (VLP’s) in treatment samples. No correlation was observed between their resistance to lytic viral infection and prophage induction. Isolate P99-14, with consistently high levels of prophage induction, was investigated further. In contrast to lytic cyanophage, the induced cyanophage is non-tailed. Differential staining and nuclease digestion experiments indicate that the induced particle contains single-stranded DNA. Environmental conditions potentially leading to prophage induction were investigated with Synechococcus cultures and natural populations. The isolate P99-14 demonstrated that high, continuous light caused prophage induction. Natural populations determined that shifts in salinity, temperature and phosphate are not triggers of prophage induction.

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To initiate algal virus research in the Bay of Quinte, three stations were sampled biweekly throughout 2011. By targeting algal virus DNA polymerase, major capsid protein genes (MCP), and a Microcystis aeruginosa cyanophage (Ma-LMM01) tail sheath protein gene, PCR amplification revealed diverse and unique Phycodnaviruses (viruses of eukaryotic algae) and cyanophage. When analysed statistically, patterns of virus abundance suggested that the seasonality of any one virus cannot be generalised to predict that of other viruses, even among closely related viruses. This study also demonstrated a strong relationship between algal virus abundance and host biomass. It was found that despite the apparent heterogeneity of virus abundance across the Bay, virus abundance patterns clustered by sampling date and geographic location. By providing evidence for diverse algal viruses with complex seasonality, this work highlights significant gaps in the current understanding of Bay of Quinte phytoplankton ecology.

To initiate algal virus research in the Bay of Quinte, three stations were sampled biweekly throughout 2011. By targeting algal virus DNA polymerase, major capsid protein genes (MCP), and a Microcystis aeruginosa cyanophage (Ma-LMM01) tail sheath protein gene, PCR amplification revealed diverse and unique Phycodnaviruses (viruses of eukaryotic algae) and cyanophage. When analysed statistically, patterns of virus abundance suggested that the seasonality of any one virus cannot be generalised to predict that of other viruses, even among closely related viruses. This study also demonstrated a strong relationship between algal virus abundance and host biomass. It was found that despite the apparent heterogeneity of virus abundance across the Bay, virus abundance patterns clustered by sampling date and geographic location. By providing evidence for diverse algal viruses with complex seasonality, this work highlights significant gaps in the current understanding of Bay of Quinte phytoplankton ecology.

No description available.