Graduation Year

2005

Document Type

Dissertation

Degree

Ph.D.

Degree Granting Department

Marine Science

Major Professor

John H. Paul, Ph.D.

Committee Member

Gabriel A. Vargo, Ph.D.

Committee Member

Pamela Hallock-Muller, Ph.D.

Committee Member

Andrew Cannons, Ph.D.

Committee Member

Joel Thompson, Ph.D.

Keywords

Viruses, Cyanophage, Lysogeny, Picoplankton, phytoplankton, Synechococcus, environmental mutagenesis, MPIA, prophage induction

Abstract

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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