Quantification and diversity of a ubiquitous ssDNA phage group (Gokushovirinae) in the Red Sea through the polony technique

Graduation Year

2024

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Marine Science

Major Professor

Mya Breitbart, Ph.D.

Committee Member

Debbie Lindell, Ph.D.

Committee Member

Karyna Rosario Cora, Ph.D.

Committee Member

Larry Dishaw, Ph.D.

Committee Member

Kristen N. Buck, Ph.D.

Keywords

Marine Viruses, Microviridae, Solid phase PCR, Viral Metagenomics, Gokushoviruses

Abstract

With average concentrations of 107 virus-like particles per milliliter of seawater, viruses comprise the second largest biomass in the ocean, where they are a driving force of microbial evolution and constitute important roles in Earth’s biogeochemical cycles. Bacteriophage (phage), viruses that infect bacteria, are of particular importance in the marine ecosystem as they impact the efficiency of the microbial loop, which recycles carbon in surface waters, counteracting vertical export through the biological pump. Although double-stranded DNA phage have been researched extensively, single-stranded DNA (ssDNA) phage remain understudied, mostly due to methodological limitations.

The incorporation of rolling circle amplification (RCA) into metagenomic studies revealed the ubiquity of ssDNA phage, particularly those belonging to the Gokushovirinae subfamily of the Microviridae family, in marine environments. RCA is now known to selectively amplify ssDNA circular genomes, which is advantageous for studying their diversity; however, quantifying the abundance of ssDNA phage with current molecular techniques remains difficult. This dissertation addresses this limitation by optimizing the polony method to determine ssDNA gokushovirus abundance in the Red Sea (Appendix A,B). The polony method is a solid phase polymerase chain reaction (PCR) that immobilizes the template DNA in an acrylamide gel, then hybridizes the PCR products with a fluorescent probe to enable determination of the abundance of targeted phage groups. The polony approach is compatible with degenerate primer sets, enabling the first quantification of this diverse gokushovirus group in any environment.

Due to its deep basin and oligotrophic ecosystem, the Red Sea is analogous to an open ocean system despite its proximity to land. This ecosystem experiences seasonal water column stratification that predictably affects the phytoplankton community structure, making it an ideal system to quantify the variation in gokushovirus abundance across depths and seasons. This dissertation achieved the following two objectives in the Red Sea: 1) Adapt and optimize the polony method for gokushovirus quantification and 2) quantify the abundance of ssDNA gokushoviruses as well as assess their diversity. Completion of these objectives tested the hypothesis that gokushovirus abundance and diversity vary seasonally in response to water column stratification. To test this hypothesis, the polony method was applied to samples collected across three years (2015-2018) in February (mixed water column) and September (stratified water column).

The results of this dissertation showed that gokushovirus abundance changes drastically with the seasons. Gokushoviruses were up to 80-fold more abundant in September, when the water column is stratified, than in February, when the water column is mixed. Additionally, in September gokushovirus abundance also changed with depth, with a subsurface peak that often occurred around 40 m. Though initial amplicon sequencing revealed sequences with distinct depth distributions within the month of September, this pattern was not detected when looking at the wider diversity of gokushoviruses using viral metagenomics. Additionally, there was no clear shift in gokushovirus diversity between the two seasons, despite their distinct differences in abundance.

The molecular markers designed for this polony assay were compared to gokushovirus sequences recovered by viral metagenomics in the Red Sea. This analysis revealed that the current polony assay captures about half of the gokushovirus diversity in the Red Sea, yet recovers gokushoviruses across both months and all depths, emphasizing this assay has minimal spatiotemporal biases and the differences in gokushovirus abundance by season and depth reflect their ecology in the Red Sea. Discovering these clear spatiotemporal shifts in abundance has expanded our knowledge of gokushovirus ecology in the Red Sea and lays the groundwork for future studies targeting their host range and ecological roles in the marine environment.

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