2004 Progress Report: The Development of a Single-cell Field Diagnostic for Nitrogen Limitation in Harmful AlgaeEPA Grant Number: R830415
Title: The Development of a Single-cell Field Diagnostic for Nitrogen Limitation in Harmful Algae
Investigators: Dyhrman, Sonya , Anderson, Donald M.
Institution: Woods Hole Oceanographic Institution
EPA Project Officer: Hiscock, Michael
Project Period: November 18, 2002 through November 17, 2005 (Extended to August 17, 2007)
Project Period Covered by this Report: November 18, 2003 through November 17, 2004
Project Amount: $451,953
RFA: Ecology and Oceanography of Harmful Algal Blooms (2002) RFA Text | Recipients Lists
Research Category: Aquatic Ecosystems , Water Quality , Ecosystems , Water
The overall objective of this research project is to test and further refine an assay for the N’-acetylglucosaminidase (NAG) activity using the enzyme labeled fluorescence (ELF)-NAG substrate. The specific objectives of this research project are to:
- test for ELF-NAG labeling in a suite of harmful algal bloom (HAB) species;
- pursue a method for quantifying ELF-NAG labeling on a single-cell basis in a model Alexandrium species;
- relate the regulation of the ELF-NAG labeling to the physiological condition of the Alexandrium cells;
- further characterize the physiological function of the NAG enzyme;
- develop the assay for use on field populations of Alexandrium fundyense;
- and test the assay on field populations of the toxic dinoflagellate A. fundyense during nutrient addition experiments in limno-corrals.
Nutrients play a significant regulatory role in phytoplankton growth and bloom dynamics. Nitrogen, a critical macronutrient, exists in inorganic and organic forms in the ocean. Dissolved organic forms of nitrogen (DON) comprise a nutrient pool that often requires enzymatic hydrolysis before they can be exploited by many species, including harmful ones, for cell growth. Potential DON utilization by harmful algal species can be assessed through measures of enzymatic activity. Specifically, ELF technology provides a cell-specific method for diagnosing the nitrogen status of various HAB species. To date, 13 HAB species have been tested with the ELF-NAG labeling. More than one-half of these species have been found to express NAG activity when grown under nitrogen deplete conditions, while those grown under nitrogen-replete conditions did not express NAG activity. With this data in hand, we sought to further satisfy our overall objective and focus our work on assessing the regulation of ELF-NAG labeling to the physiological condition and characterizing the function of the NAG enzyme. Dr. Da-Zhi Wang, a visiting scientist from Xiamen University in China, directed his sabbatical work on these questions. Dr. Wang augmented our initial protein expression data with 2-D polyacrylamide gel electrophoresis (2DE) to analyze the proteome of the HAB species, Karenia brevis, grown with inorganic and organic nitrogen sources. K. brevis grew in the presence of low nitrate, ammonium, and urea. The cell density of K. brevis when grown with ammonium or urea was lower than that in the low nitrate condition. Proteomes of K. brevis grown in nitrate-replete, low nitrate, and urea also were compared. Significant protein differences were noted between the three treatments and corresponding protein spots were cut from each gel and stored for later identification and characterization by mass spectroscopy. Ultimately, our investigation into the proteomics of K. brevis not only provided valuable protein regulation data, but it also furnished our laboratory with a new tool, 2DE, which can be easily adapted for other species of harmful algae.
In conjunction with the 2DE work, we also began preliminary work to examine gene expression within K. brevis to identify and characterize the function of the NAG enzyme. To date, we have designed primers to successfully amplify both glutamine synthetase and NAG from a replete culture. Additional work is currently underway to investigate the regulation of these particular genes in nitrogen deplete K. brevis cultures.
We do not anticipate any major changes from the tasks outlined in the proposal. A particular focus will be work on specific objectives 1and 2 and field work for specific objective 6. A graduate student, Liz Orchard, has already begun collecting additional data for specific objective 1. Furthermore, we intend to follow up on our 2DE work by analyzing several of the most interesting excised spots by mass spectroscopy. Also, we will continue our investigation of K. brevis gene expression (specific objective 4). Specifically, we plan on growing K. brevis under nitrogen-replete and nitrogen-deplete conditions and investigating the regulation of glutamine synthetase and NAG.
Journal Articles on this Report : 1 Displayed | Download in RIS Format
|Other project views:||All 7 publications||3 publications in selected types||All 2 journal articles|
||Dyhrman S. Ectoenzymes in Prorocentrum minimum. Harmful Algae 2005;4(3):619-627.||