Abstract:
The mechanism of saxitoxin (STX) biosynthesis in marine dinoflagellates of the genus
Alexandrium is still unknown. The aim of this thesis was to analyze novel aspects of
toxigenesis during the cell cycle in Alexandrium and to apply molecular techniques to gain
new insights on the genetics and regulation of STX biosynthesis.
Synchronized cultures of A. fundyense were studied to determine the dynamics of toxin
production throughout the cell cycle. Toxin production was discontinuous, was induced
by light and always occurred during a period of approximately eight to ten hours in early
G1. Analysis of the cell cycle dynamics suggests the existence of two transition points: one
at the beginning of G1, which is light-dependent and holds the cells in a Go-like period,
and a second one at the end of G1, which is size-dependent and arrests the cells in G1. A
model of the cell cycle of A. fundyense is proposed in which progression through the cell
cycle can be arrested at two different transition points located in G1 and toxin production is
induced by light during G1. The effects of temperature and phosphate limitation on the linkage between changes in
the duration of the cell cycle stages and toxicity were studied in semi-continuous cultures of
A. fundyense. A direct correlation between G1 duration and toxin content was observed,
along with a clear uncoupling of toxin accumulation from the Sand G2 phases of the cell
cycle. In both experiments, toxin production rates remained constant for the respective
range of conditions, implying that the variations in toxin content observed were a result of
increasing periods of biosynthetic activity. Phosphate limitation enhanced toxin production
rates and affected interconversions among STX derivatives in several ways: oxidations to
yield the hydroxy-series of STXs were phosphate-dependent while sulfatation reactions
were not.
Differential Display (DD) analysis was applied to the identification of genes that were
up- or downregulated during toxigenesis in synchronized cultures of A. fundyense. Three
genes were isolated: S-adenosy lhomocysteine hydrolase, methionine aminopeptidase and a
histone-like protein. None could be directly correlated to toxigenesis but instead relate to
general cellular metabolism.
