Biochemical, molecular, and genomic investigations into the response of the diatom Thalassiosira pseudonana to copper stress

Primary producers form the base of aquatic food webs. Diatoms are important primary producers found globally in both freshwater and marine systems and are dominant contributors to processes with global implications, including carbon fixation and biosilicification. Metal pollution is an emerging problem in coastal environments and may be impacting entire marine communities. Although an essential trace element, copper is a potent toxin at elevated concentrations, and attempts to monitor copper pollution through direct measurements have been frustrated by difficulties in measuring the biologically relevant forms of copper. This research explores the possibility of developing a biomarker for copper-stressed environments utilizing the ubiquitous estuarine diatom Thalassiosira pseudonana. Taking advantage of its recently available genome sequence, this work also investigates the mechanisms employed by T. pseudonana to withstand elevated levels of copper.; In laboratory culture experiments, cell-surface proteins induced by copper stress were identified by cell surface labeling techniques and characterized. The proteins were novel and could not be assigned function based on sequence similarity. One of the proteins, termed p150, contained chitin-binding domains and was found to be cell-cycle regulated. While this protein was determined at the molecular and biochemical level to be induced by copper, it was also induced by silicon and iron limitation. Another identified protein, referred to as p100, consists almost entirely of repeats of an unknown domain and belongs to a large family of modular, cell-surface proteins in T. pseudonana .; Antibody probes were generated to p150 and p100, that had been expressed in E. coli, and the specificity and cross reactivity of each antibody was determined. Antibody labeling of cells showed that p150 was induced by elevated copper levels and was localized to the girdle band region of elongated cells. The p100 antibody failed to label whole cells and could not be localized on the cell surface, although it reacted with p100 on westerns of cell-surface protein extracts. The inability to label whole cells limits the utility of p100 as a marker of copper stress. Additionally, the p100 probe may have cross-reacted with other members of a large protein family, as observed by western blotting analysis.; The cellular response of T. pseudonana to copper stress was investigated at the mRNA level through a subtractive cDNA approach. Results indicate that T. pseudonana mounts a rapid and dramatic response to copper that attenuates over time. (Abstract shortened by UMI.)...