Collaborative Research: Development of Molecular and Biochemical Signatures for the Detection of Toxin Production in Pseudo-nitzchia spp. Under Nutrient Stress

San Jose State University Foundation, San Jose CA

Investigators

Abstract

The two intertwined goals of this project are to determine the suite of genes expressed by Pseudo-nitzschia under toxin-producing conditions, and to acquire a better understanding of the connections between environmental conditions and physiological responses leading to toxin production. A set of physiological experiments will permit evaluation of molecular probes generated from gene expression studies. In turn, the molecular probes will be used to interrogate natural populations and help determine the physiological status of Pseudo-nitzschia in the field. The ultimate goal is to find a specific gene transcript or a pattern of gene expression that is correlated with toxin production in the field. The following hypotheses will be tested: H1: There are genes or a suite of genes whose expression pattern is highly correlated with toxin production in Pseudo-nitzschia. H2: A primary trigger for toxin production in Monterey Bay is silicate limitation, so that certain oceanographic conditions permit bloom development. H3: Silicate limitation may sensitize cells to trace-metal (e.g. copper) stress and the toxin (domoic acid) can function as a metal ion buffer. Batch and continuous cultures will be stressed with silicate, copper, and iron. Growth, substrate utilization, and physiological parameters (variable fluorescence, nutrient quotas, amino acid pools, including domoic acid) will be assessed. Cells will be harvested for development of cDNA subtraction libraries under different stressors. Gene arrays developed from these libraries will provide molecular probes for field testing. Identification of genes related to toxin production, but not general metabolism, will be facilitated by information generated by the physiology experiments. The laboratory work will be combined with a limited field program for assessment of environmental triggers (e.g. copper, silicate, iron stress) and for testing of the molecular probes. Results from the molecular expression and physiological assays will permit an initial description of the cellular pathways mediating environmental triggers (e.g silicate and metals) for production of toxin.