STRENGTHS AND LIMITATIONS OF PROPOSED RESEARCH 



The strengths of the approach are that we can begin to look at the physiological 

 characteristics and state of specific phytoplankton cells within a population and the different 

 factors limiting their growth. Ecosystems are complex and the factors limiting one group of 

 organisms might not be limiting others so that the single species/single cell approach will be 

 extremely valuable and will eventually provide a more mechanistic understanding of the whole 

 ecosystem. On the negative side identifying candidate cell surface proteins as markers, purifying 

 them, identifying their function, and making antibodies to them is extremely time consuming and 

 these kinds of proteins may not always be present in all organisms. 



STATUS OF RESEARCH 



The first goal of the project was to work out the best conditions for labeling 

 phytoplankton cell surfaces with biotinylating reagents in order to detect cell surface proteins. 

 The method has been essentially perfected. We have since used the method to look for 

 nitrogen-limitation specific cell surface proteins in Emiliania huxleyi, one of the most commonly 

 found marine phytoplankton, and in two diatom (Thalassiosira) species. We have found such a 

 protein in Emiliania but not in the diatoms. The diatoms, however, have proteins found in 

 healthy, but not stressed, cells suggesting that antibodies to these proteins could also be used as 

 indicators of cell state. The candidate N-imitation protein in Emiliania is found in the 

 membrane/cell wall fraction of the cells and is about 80 kDa in size based on SDS-PAGE. It is 

 found in three strains of Emiliania from widely different locations including a strain from the 

 Gulf of Maine, the site of yearly blooms of this organism. We have also found a candidate for 

 a phosphorus limitation induced protein in Emiliania so that antibodies to the two proteins could 

 be used to look at P vs N limitation of this organism in the field. We are continuing to screen 

 other coastal phytoplankton for marker proteins and we are beginning the purification of the 80 

 kDa protein for antibody production. The second goal of the project was to purify a cell surface 

 L-amino acid oxidase from a coccolithophorid. This protein allows certain phytoplankton to grow 

 on amino acids as a nitrogen source and it is induced in some organisms under N limitation. The 

 protein seemed like a good candidate for a test of the approach of using cell surface antibodies 

 to look at environmental stress. We purchased and set up a Gradifrac system from Pharmacia and 

 have used it to purify the amino acid oxidase using gel filtration and ion exchange 

 chromatography. Antibodies are currently being made. These antibodies will be fluorescently 

 labeled and used to work out the best conditions for cell surface protein detection in seawater and 

 will also be used on field samples in the future. Some additional work is needed to complete the 

 characterization of this protein. 



Keywords: phytoplankton, antibody, cell surface proteins, physiological state 



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