The aims of this proposal are twofold. First, the investigators propose to identify and characterize new toxins, produced by algae and cyanobacteria, found in Florida?s aquatic environments. Florida waters harbor more than sixty toxic or potentially toxic microorganisms. These toxic microorganisms have significant impacts on public health, recreation, tourism, wildlife, and local economies. Gymnodinium breve blooms annually on the West Coast of Florida causing closure of shellfish beds to prevent the occurrence of neurotoxic shellfish poisoning. Cyanobacterial blooms are becoming increasingly frequent in freshwater lakes, making recreational use hazardous. In addition to these well-known and well-characterized toxin-producing organisms, other toxic microorganisms are present for which toxins have yet to be identified. This proposal will focus on the identification of these as yet uncharacterized toxins. Prior to mass culturing, small-scale cultures and assemblages will be screened by RT-PCR for the expression of biosynthetic capability. Specifically, flagellates will be screened for polyketide synthase gene expression, while cyanobacteria will be screened for non-ribosomal peptide biosynthesis. New toxins will be isolated by bioassay-guided fractionation using a variety of screening methods. Second, the role of associated bacteria in dinoflagellate toxin biosynthesis will be examined. Since many dinoflagellate cultures have not been successfully maintained axenically, the ultimate origins of the toxins remains a topic of ongoing debate. The existence of toxic and nontoxic strains of dinoflagellates, and the sudden unexplained loss of toxicity of dinoflagellates in culture, has led to speculation that associated bacteria are responsible for toxin biosynthesis. The investigators have isolated bacteria from dinoflagellate cultures that carry polyketide synthase encoding genes, but express them only in the presence of dinoflagellates. The role of these bacteria in the biosynthesis of dinoflagellate-derived polyketides will be examined.
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