Gene discovery and expression profilng in the toxin-producing marine diatom, Pseudo-nitzschia multiseries (Hasle) Hasle

Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at the Massachusetts Institute of Technology and the Woods Hole Oceanographic Institution September 2004Toxic algae are a growing concern in the marine environment. One unique marine diatom, Pseudo-nitzschia multiseries (Hasle) Hasle, produces the the neurotoxin domoic acid, which is the cause of amnesic shellfish poisoning. The molecular characterization of this organism has been limited to date. Therefore, the focus of this thesis was to identify and initiate characterization of actively expressed genes that control cell growth and physiology in P. multiseries, with the specific goal of identifying genes that may play a significant role in toxin production. The first step in gene discovery was to establish a complementary DNA (cDNA) library and a database of expressed sequence tags (ESTs) for P. multiseries 2552 cDNAs were sequenced, generating a set of 1955 unique contigs, of which 21 % demonstrated significant similarity with known protein coding sequences. Among the genes identified by sequence similarity were several involved in photosynthetic pathways, including fucoxanthin-chlorophyll a/c light harvesting protein and a C4-specific pyruvate, orthophosphate dikinase. Several genes that may be involved in domoic acid synthesis were also revealed through sequence similarity, for example, glutamate dehydrogenase and 5-oxo-L-prolinase. In addition, the identification of sequences that appear novel to Pseudo-nitzschia may provide insight into unique aspects of Pseudo-nitzschia biology, such as toxin production. Genes whose expression patterns were correlated with toxin production were identified by hybridization to a micro array manufactured from 5376 cDNAs. 121 cDNAs, representing 12 unique cDNA contigs or non-redundant cDNAs, showed significantly increased expression levels in P. multiseries cell populations that were actively producing toxin. The up-regulated transcripts included cDNAs with sequence similarity to 3-carboxymuconate cyclase, phosphoenolpyruvate carboxykinase, an amino acid transporter, a small heat shock protein, a long-chain fatty acid Co-A ligase, and an aldo/keto reductase. These results provide a framework for investigating the control of toxin production in P. multiseries. These transcripts may also be useful in ecological field studies in which they may serve as signatures of toxin production. Prospects for further application of molecular genetic technology to the understanding of the physiology and ecology of P. multiseries is discussed.Financial support for this research was provided by the Woods Hole Academic Programs Offic

Gene discovery and expression profiling in the toxin-producing marine diatom, Pseudo-nitzschia multiseries (Hasle) Hasle

Thesis (Ph. D.)--Joint Program in Biological Oceanography (Massachusetts Institute of Technology, Dept. of Ocean Engineering; and, the Woods Hole Oceanographic Institution), 2004.Includes bibliographical references (leaves 169-180).Toxic algae are a growing concern in the marine environment. One unique marine diatom, Pseudo-nitzschia multiseries (Hasle) Hasle, produces the neurotoxin domoic acid, which is the cause of amnesic shellfish poisoning. The molecular characterization of this organism has been limited to date. Therefore, the focus of this thesis was to identify and initiate characterization of actively expressed genes that control cell growth and physiology in P. multiseries, with the specific goal of identifying genes that may play a significant role in toxin production. The first step in gene discovery was to establish a complementary DNA (cDNA) library and a database of expressed sequence tags (ESTs) for P. multiseries. 2552 cDNAs were sequenced, generating a set of 1955 unique contigs, of which 21% demonstrated significant similarity with known protein coding sequences. Among the genes identified by sequence similarity were several involved in photosynthetic pathways, including fucoxanthin-chlorophyll a/c light harvesting protein and a C4-specific pyruvate, orthophosphate dikinase. Several genes that may be involved in domoic acid synthesis were also revealed through sequence similarity, for example, glutamate dehydrogenase and 5-oxo-L-prolinase. In addition, the identification of sequences that appear novel to Pseudo-nitzschia may provide insight into unique aspects of Pseudo-nitzschia biology, such as toxin production. Genes whose expression patterns were correlated with toxin production were identified by hybridization to a microarray manufactured from 5376 cDNAs. 121 cDNAs, representing 12 unique cDNA contigs or non-redundant cDNAs, showed significantly increased expression levels in P. multiseries cell populations that were actively producing toxin.(cont.) The up-regulated transcripts included cDNAs with sequence similarity to 3-carboxymuconate cyclase, phosphoenolpyruvate carboxykinase, an amino acid transporter, a small heat shock protein, a long- chain fatty acid Co-A ligase, and an aldo/keto reductase. These results provide a framework for investigating the control of toxin production in P. multiseries. These transcripts may also be useful in ecological field studies in which they may serve as signatures of toxin production. Prospects for further application of molecular genetic technology to the understanding of the physiology and ecology of P. multiseries is discussed.by Katie Rose Boissonneault.Ph.D

Microbial food web interactions in two Long Island embayments

Submitted in partial fulfillment of the requirements for the degree of Master of Science at the Massachusetts Institute of Technology and the Woods Hole Oceanographic Institution September 1999Phytoplankton mortality (herbivory) and bacterivory were examined experimentally in West Neck Bay and Coecles Harbor, Long Island, NY from April through September, 1998. Small algae (<5 μm diameter) dominated phytoplankton communities in both ecosystems throughout the summer, and zooplankton were also small (mostly <40 μm). Generally, plankton abundances were indicative of eutrophic ecosystems. Oscillations in standing stocks and mortality of prey indicated tight coupling of growth and grazing mortality in both bays. Phytoplankton mortality rates accounted for the removal of 14% to 65% of total phytoplankton standing stocks daily, while bacterivory accounted for the removal of 14% to 88% of total bacterial standing stocks daily. Estimates of carbon consumption revealed high energy flux through the nano- and microzooplankton assemblages of these estuarine environments.This thesis was supported by a National Science Foundation Graduate Research Traineeship, Grant # DGE-9454129 and New York Sea Grant BTRI Project R/CE-12

Gene expression studies for the analysis of domoic acid production in the marine diatom Pseudo-nitzschia multiseries

Background: Pseudo-nitzschia multiseries Hasle (Hasle) (Ps-n) is distinctive among the ecologically important marine diatoms because it produces the neurotoxin domoic acid. Although the biology of Ps-n has been investigated intensely, the characterization of the genes and biochemical pathways leading to domoic acid biosynthesis has been limited. To identify transcripts whose levels correlate with domoic acid production, we analyzed Ps-n under conditions of high and low domoic acid production by cDNA microarray technology and reverse-transcription quantitative PCR (RT-qPCR) methods. Our goals included identifying and validating robust reference genes for Ps-n RNA expression analysis under these conditions. Results: Through microarray analysis of exponential- and stationary-phase cultures with low and high domoic acid production, respectively, we identified candidate reference genes whose transcripts did not vary across conditions. We tested eleven potential reference genes for stability using RT-qPCR and GeNorm analyses. Our results indicated that transcripts encoding JmjC, dynein, and histone H3 proteins were the most suitable for normalization of expression data under conditions of silicon-limitation, in late-exponential through stationary phase. The microarray studies identified a number of genes that were up- and down-regulated under toxin-producing conditions. RT-qPCR analysis, using the validated controls, confirmed the up-regulation of transcripts predicted to encode a cycloisomerase, an SLC6 transporter, phosphoenolpyruvate carboxykinase, glutamate dehydrogenase, a small heat shock protein, and an aldo-keto reductase, as well as the down-regulation of a transcript encoding a fucoxanthin-chlorophyll a-c binding protein, under these conditions. Conclusion: Our results provide a strong basis for further studies of RNA expression levels in Ps-n, which will contribute to our understanding of genes involved in the production and release of domoic acid, an important neurotoxin that affects human health as well as ecosystem function.Plymouth State University Graduate Programs OfficeWoods Hole Oceanographic Institution Academic Programs OfficeNew Hampshire IDeA Network of Biological Research Excellence (NH-INBRE)National Center for Research Resources (U.S.) (Grant 5P20RR030360-03)National Institute of General Medical Sciences (U.S.) (Grant 8P20GM103506-03