Functional genomics of a non-toxic Alexandrium lusitanicum culture

Submitted to the department of Biology in partial fulfillment of the requirements for the degree of Doctor of Philosophy at the Massachusetts Institute of Technology and the Woods Hole Oceanographic Institution February 2007Paralytic shellfish poisoning (PSP) is a human intoxication associated with the consumption of shellfish contaminated with a family of neurotoxins called saxitoxins. Many species in the dinoflagellate genus Alexandrium have been shown to produce these toxins. Here I report the first case of a culture of Alexandrium that has completely lost the ability to produce saxitoxins. The loss of toxicity was accompanied by a reduction in growth capability. A subculture of this isolate maintains the ability to produce toxins and to grow at rates and to cell abundances that were characteristic of the original Alexandrium culture. The growth and toxicity differences in the two isolates were demonstrated to be a property of the dinoflagellate itself and were not dependent on the different bacterial symbionts associated with each culture. The pair of subcultures is a novel experimental system to study gene expression related to toxin production and growth in dinoflagellates. The products of gene expression were analyzed in the two subcultures of Alexandrium grown under the same conditions, but where toxicity and growth differ. At the metabolome level, compounds were identified that were unique to the non-toxic isolate; their emergence may be correlated to a disruption of the biosynthetic pathway for PSP toxins. These compounds share some characteristics and potential structural similarities with saxitoxins, though they are not any of the known toxin derivatives. Difference gel electrophoresis (DIGE) identified proteins differentially expressed between the two subcultures. Identification of some of these proteins was possible by searching the expressed sequence tag (EST) database for dinoflagellates. Proteins shown to be down-regulated in the non-toxic, slower growing subculture are all enzymes from the Calvin cycle, which may explain the limited growth of the non-toxic isolate. Other unknown, differentially expressed proteins may relate to the loss of toxicity, but their identity and function remain unresolved.This work was supported by a graduate fellowship from the Fundação para a Ciência e Tecnologia, Portugal