Nitrate reductase sequence analysis and expression in the raphidophytes, Heterosigma akashiwo and Chattonella subsalsa

The research presented in this dissertation focuses on the nitrate reductase (NR) enzyme and nitrogen metabolism in two harmful algal bloom species in the class Raphidophyceae, Heterosigma akashiwo and Chattonella subsalsa. Raphidophytes are a group of harmful algae well known for forming dense, toxic and destructive blooms worldwide. Nitrate is often the dominant source of nitrogen in coastal waters, and the capacity to compete for nitrate contributes to species dominance within phytoplankton communities. Therefore, increasing anthropogenic nitrogen inputs may stimulate raphidophyte blooms. In order to utilize NO3-, an organism must invest energy in the form of reducing equivalents to convert NO3- to NH4+ in a two-enzyme process involving nitrate reductase and nitrite reductase. The analysis presented here shows that H. akashiwo and C. subsalsa NR is unique and may contribute to the success of raphidophyte blooms in coastal waters.;In chapter 2, a novel NR containing a truncated hemoglobin domain ( NR2-trHbN) is described for the raphidophytes, H. akashiwo and C. subsalsa. To date, this is the first example of a truncated hemoglobin domain found in a multi-domain protein in eukaryotes. In the present study, mRNA transcript and genomic sequences for NR2-trHbN in H. akashiwo and C. subsalsa are reported and the potential mechanisms involved in the evolution and function of this hybrid protein are discussed. Since phylogenetic and sequence analyses suggest that the trHbN domain of NR2-trHbN may possess NO reactivity, the ability of H. akashiwo cultures to metabolize NO and the expression of NR2-trHbN after addition of the chemical NO donor, sodium pentacyanonitrosylferrate (II) (aka sodium nitroprusside; SNP), was evaluated. Here, the hemoglobin domain may be involved in the detoxification of nitric oxide that is produced in high concentrations during raphidophyte blooms. Future studies of this novel enzyme may provide insights into the involvement of nitric oxide in raphidophyte toxicity, as well as characterizing a novel method of nitric oxide detoxification by microalgae.;In chapter 3, a novel assay for detecting species-specific nitrate reductase activity using the fluorescent molecular probe, 6-chloro-9-nitro-5-oxo-5H-benzo[a]phenoxazine (CNOB), was evaluated and found to be ineffective at determining NR activity in live cells visualized by flow cytometry. Interestingly, the 6 e - reduction of CNOB to a fluorescent aminosubstituted product was visualized in the membranes of raphidophytes and not any other species tested. Although the origin of this fluorescence was not determined, these results indicate that raphidophytes posses a unique mechanism for the reduction of aromatic nitro compounds and/or nitrate that warrants further study.;Finally in chapter 4, NR transcript expression, in concert with expression of genes in the carbon and lipid metabolic pathways, is studied in response to increasing temperature and CO2 supply in two strains of H. akashiwo. Here, a cold water strain isolated from Washington and a warm water strain isolated from Delaware showed very different responses in gene expression for three key enzymes involved in carbon, lipid, and nitrogen metabolism to increases in temperature and CO2. Transcript levels were affected by CO2 concentration in the Washington strain of H. akashiwo, whereas temperature affected transcript levels in the Delaware strain. In the absence of increased cell growth, gene expression data suggest that carbon was being allocated to carbon skeletons for lipid and/or protein synthesis. Although treatments did not stimulate growth and had little effect on photosynthetic parameters for these strains, the gene expression data presented here suggest an effect on cellular metabolism in response to changes in temperature and/or CO2 concentrations. Overall, these results imply that studies on individual species or strains cannot be used to make generalizations on the effects of climate change on phytoplankton.;In conclusion, raphidophytes possess a unique nitrate reductase and future work is needed to define the role of NR2-trHbN in nitrate and/or nitric oxide metabolism. The results presented in this dissertation contribute to the advancement of knowledge in a variety of fields and are of interest to a diverse audience that includes the study of protein evolution, hemoglobin function, algal physiology and ecology, nitric oxide metabolism, and nitrate reductase structure and function.