Doctoral Dissertations

Date of Award

5-2009

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Microbiology

Major Professor

Gary S. Sayler

Abstract

Saxitoxin is a secondary metabolite produced by several species of dinoflagellates and cyanobacteria. The molecular target in mammals and higher eukaryotes is the sodium channel protein in nerve and muscle cells, where it binds with high affinity and effectively blocks the inward flow of sodium ions. The molecular target of saxitoxin in lower eukaryotes, such as those inhabiting the same ecosystems as the toxin-producing algae, is not known. The role of the toxin in the ecology of the algae is also a mystery. This dissertation sought to determine the molecular effects and possible target of saxitoxin on lower eukaryotes as a means to gain insights on the function of the toxin within the ecosystem. Global expression profiling with the yeast Saccharomyces cerevisiae using the Affymetrix GeneChip identified a set of genes commonly associated with copper homeostasis as being significantly differentially expressed upon short-term exposure to saxitoxin.The pattern of regulation of these genes was then compared to the patterns generated upon exposure to excess copper and excess iron using quantitative reverse-transcriptase PCR. The repression of two genes, FET3 and CTR1, suggested intracellular copper levels may be compromised when cells were in the presence of saxitoxin. To further explore the hypothesis of saxitoxin altering internal copper levels, a comparative transcriptomics study was performed with the green alga Chlamydomonas reinhardtii. Expression profiles of a pre-defined set of genes were compared following exposure to saxitoxin and excess copper, with results indicating that saxitoxin was also altering copper homeostasis in C. reinhardtii. The established model of copper transport in S. cerevisiae coupled with the known structural design of the Ctr1 protein suggests that saxitoxin is binding to plasma membrane copper transporters, in a manner analogous to that of sodium channel binding.The final section of this dissertation examined the phylogenetic relationship between two varieties of the dinoflagellate Pyrodinium bahamense. While var. compressum is an established saxitoxin producer, it has been determined that var. bahamense was the source of recent saxitoxin outbreaks in the western Atlantic. Based on small subunit ribosomal RNA gene sequences, the two are 99% identical, suggesting a reclassification based on genetic rather than morphological features.

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