MEES Theses and Dissertations
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Item INSIGHTS INTO DINOFLAGELLATE NATURAL PRODUCT SYNTHESIS VIA CATALYTIC DOMAIN INTERACTIONS(2022) Williams, Ernest Patrick; Place, Allen R; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Dinoflagellates are protists that can be split into two evolutionary groups, the parasitic syndinians and the largely photosynthetic “core” dinoflagellates. They represent a major portion of aquatic biomass which means that they are responsible for large portions of carbon that are both fixed and released. Other than biomass, the fixed carbon can be made into natural products such as polyunsaturated fatty acids that support the biota of many ecosystems or toxins that are harmful to aquatic life and humans. DNA and RNA analyses have been used to discover the putative genes that may make these compounds, but their non-colinear arrangement in the genome is very different from model organisms and their gene copy number is very high, making it nearly impossible to determine the exact biosynthetic pathways. The goal of my studies was to develop methods to differentiate biosynthetic pathways such as lipid and toxin synthesis by comparing the ability of domains to interact with each other with the assumption that domains that preferentially interact are more likely to participate in the same pathway. Initially, a survey was performed on available dinoflagellate transcriptomes to enumerate domains potentially involved in natural product synthesis and bin them based on sequence similarity to identify genes that could be used in biochemical assays. An interesting integration of analogous genes involved in lipid synthesis with those involved in natural product synthesis was observed as well as trends in domain expansion and contraction during core dinoflagellate evolution. Ultimately, the domain that scaffolds natural product synthesis, the thiolation domain, was chosen for further study because it exhibited two clear functional bins and is acted on directly by another enzyme, a phosphopantetheinyl transferase (PPTase). The PPTase activates the thiolation domain by transferring the phosphopantetheinate group from Coenzyme A to the thiolation domain, creating a free thiol group upon which the natural products are synthesized. These PPTases were then enumerated in dinoflagellates and characterized by looking for sequence motifs and observing expression patterns over a diel cycle as well as during growth in the model species Amphidinium carterae, a basal toxic dinoflagellate. Amphidinium carterae appears to have three PPTases, two of which (PPTase 1 and 2) are very similar, except that PPTase 2 does not appear to have a stop codon and has never been observed as a full-size protein. The remaining two PPTases (PPTase 1 and 3) had alternating expression patterns that did not appear to directly correlate to the acyl carrier protein, the thiolation domain required specifically for lipid biosynthesis. This carrier protein, like other enzymes for natural product synthesis in dinoflagellates, had a chloroplast targeting sequence while the three PPTases did not. To investigate the ability of these three PPTases to activate various thiolation domains, a total of 8 domains from A. carterae were substituted into the blue pigment synthesizing gene BpsA from Streptomyces lavendulae. These recombinant constructs were used for coexpression in E. coli as well as in vitro to reduce as many artifacts as possible and assess the interactions of each PPTase with the thiolation domains. Some of the recombinant BpsA genes were able to make blue dye with all three PPTases, while others never made blue dye both in E. coli as well as in vitro. In vitro quantification of free thiol added by the PPTase showed that all the thiolation domains, as well as the acyl carrier protein could be phosphopantetheinated by all the PPTases. This generalist substrate recognition, along with the alternating expression patterns and lack of chloroplast signaling peptide, indicate that the two active PPTases are performing the same function on all available thiolation domains, probably before export to the chloroplast. This lack of pathway segregation by PPTases is a completely novel way of synthesizing natural products compared to bacteria and fungi, likely due to the acquisition of both photosynthesis and natural product/lipid biosynthesis during dinoflagellate evolution that was not present in the common ancestor. Additionally, the techniques to identify genes of interest and perform biochemical characterization developed here are useful for future experiments annotating the function of dinoflagellate genes.Item EXAMINING HIBERNATION IN THE BIG BROWN BAT THROUGH DNA METHYLATION(2021) Sullivan, Isabel; Wilkinson, Gerald S; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Hibernation allows individuals to conserve energy during seasonal low temperatures. As the physiological regulation of hibernation is inadequately understood, I examine hibernation using DNA methylation (DNAm). DNAm is the addition of a methyl group to cytosine at cytosine guanine dinucleotide (CpG) sites in the genome. DNAm in promoters can repress gene expression and be influenced by histone modifications. Using the big brown bat, Eptesicus fuscus, I examined how hibernation influences DNAm, independent of age, through comparing DNAm from bats that differed in hibernation history and comparing DNAm from the same individual between hibernating and active seasons. Both comparisons found evidence of differential enrichment of genes near significant CpG sites resulting from hibernation. The latter analysis found evidence consistent with a histone mark, associated with active transcription, is likely enriched in hibernating bats. These results suggest that DNAm and histone modifications associated with transcription factor binding regulate gene expression during hibernation.Item TOXIN-ANTITOXIN SYSTEMS AND OTHER STRESS RESPONSE ELEMENTS IN PICOCYANOBACTERIA AND THEIR ECOLOGICAL IMPLICATIONS.(2020) Fucich, Daniel Christopher Ehlers; Chen, Feng; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Picocyanobacteria (mainly Synechococcus and Prochlorococcus) contribute significantly to oceanic primary production. Unlike Prochlorococcus, which is mainly constrained to the warm and oligotrophic ocean, Synechococcus has a ubiquitous distribution. Synechococcus is present in freshwater, estuarine, coastal, and open ocean habitats. They have also been found in polar regions and hot springs. Endemic to the hot and the cold, the saline and the fresh, and every condition in between, Synechococcus appears to have the capability to adapt and tolerate nearly any environment and climate. This ability to adapt to any aquatic environment is possible through their genome plasticity, a character that is not present in the Prochlorococcus.Due to the differential distribution of the genera, Synechococcus is considered a generalist and Prochlorococcus is considered a specialist in ecological theory. More than 400 picocyanobacterial genomes have now been sequenced, and this large genomic resource enables comprehensive genome mining and comparison. One possibility is to study the prevalence of Toxin-Antitoxin (TA) systems in picocyanobacterial genomes. TA systems are present in nearly all bacteria and archaea and are involved in cell growth regulation in response to environmental stresses. However, little is known about the presence and complexity of TA systems in picocyanobacteria. By querying 77 complete genomes of freshwater, estuarine, coastal and ocean picocyanobacteria, Type II TA systems (the most well studied TA family) were predicted in 27 of 33 (81%) Synechococcus strains, but no type II TA genes were predicted in any of the 38 Prochlorococcus strains. The number of TA pairs varies from 0 to 80 in Synechococcus strains, with a trend for more type II TA systems being predicted in larger genomes. A linear correlation between the genome size and the number of putative TA systems in both coastal and freshwater Synechococcus was established. In general, open ocean Synechococcus contain no or few TA systems, while coastal and freshwater Synechococcus contain more TA systems. The type II TA systems inhibit microbial translation via ribonucleases and allow cells to enter the “dormant” stage in adverse environments. Inheritance of more TA genes in freshwater and coastal Synechococcus could confer a recoverable persister state which would be an important mechanism to survive in variable environments. Different genotypes of Synechococcus are present in the Chesapeake Bay in winter and summer. Winter isolates of Synechococcus have shown high tolerance to cold conditions and other stressors. To explore their potential genetic capability, complete genomes of five representative winter Synechococcus strains CBW1002, CBW1004, CBW1006, CBW1107, and CBW1108 were fully sequenced. These five winter strains share many homologs that are unique to them and not shared with pelagic Synechococcus. Winter Synechococcus genomes are enriched with particular desaturases, chaperones, and transposases. Similar amino acid sequences and annotated features were not found in distantly related Synechococcus from Subcluster 5.1. These shared genomic features between the winter strains imply that maintaining membrane fluidity, protein stability, and genomic plasticity are important to cold adaption of Synechococcus. The winter strains also contain genes that are not traditionally considered with the canonical bacterial cold shock response. They contain a particularly high abundance of Type II TA pairs with complex association networks. They feature promiscuous toxins, like VapC, that pair with multiple antitoxins, which support the mix and match hypothesis. Winter strains also contain more monogamous toxins, such as BrnT, which tend to pair with their traditionally named antitoxin, BrnA. Expression of certain TA transcripts in response to environmental stress has been observed in the model strain CB0101, and the activity of one TA pair in CB0101 for growth arrest has been experimentally confirmed via heterologous expression in E. coli. My thesis work has identified interesting genetic systems related to niche partitioning of picocyanobacteria, particularly among the Chesapeake Bay Synechococcus. Future studies are paramount to understand the functional role of TA systems, desaturases, chaperons, and transposases of picocyanobacteria under various environmental stressors.Item POPULATION GENETICS OF EASTERN OYSTER Crassostrea virginica RESTORATION IN THE CHESPEAKE BAY(2020) Hornick, Katherine; Plough, Louis V; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The strategic release of captive-bred organisms is one of the most popular methods to restore species, but concerns exist regarding genetic impacts on natural populations over the long-term. Slow recovery of depleted eastern oyster C. virginica populations in the Chesapeake Bay prompted a large-scale hatchery-based restoration program consisting of the mass-release of hatchery-produced juveniles from local, wild broodstock. This dissertation characterized the genetic impact of this program, with the overall goal of understanding how characteristics of species life-history interact with hatchery practices to shape genetic variation in populations over short and long-time scales. In Chapter 2, analysis of genetic diversity changes resulting from hatchery production under two spawning designs (mass- and controlled-spawns) revealed substantial reductions in diversity and the number of breeders from parents to offspring, due primarily to high variance in reproductive success among adults in hatchery culture. In Chapter 3, high-resolution genomic data was used in a population genetic analysis comparing diversity of restored reefs in Harris Creek with variable planting histories and husbandry practices to ‘wild’ Chesapeake Bay oyster reefs. While restored reefs showed similar levels of diversity as wild reefs, strong positive relationships between planting frequency or broodstock numbers and genetic diversity were found, suggesting that hatchery practices can significantly impact diversity in natural populations. These genomic data also permitted the investigation of local adaptation and genotype by environment associations which revealed that salinity was correlated with loci putatively under selection, suggesting potential fitness tradeoffs for sourcing non-local broodstock. In Chapter 4, an individual-based model was created using biological and demographic data from Chesapeake Bay oysters to simultaneously evaluate the impact of multiple hatchery practices on natural population genetic diversity over time scales not possible with empirical methods. Overall, hatchery practices had a large effect on genetic diversity in most scenarios, but spawning practices (mass or controlled) and broodstock rotation were more important than broodstock number, suggesting that broodstock-limited programs may have other options to maintain diversity. In summary, these studies advance our understanding of how marine supplementation impacts both neutral and adaptive variation and will provide critical information for future oyster restoration efforts.Item Adaptive Mechanisms of an Estuarine Synechococcus based on Genomics, Transcriptomics, and Proteomics(2016) Marsan, David Wilfred; Chen, Feng; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Picocyanobacteria are important phytoplankton and primary producers in the ocean. Although extensive work has been conducted for picocyanobacteria (i.e. Synechococcus and Prochlorococcus) in coastal and oceanic waters, little is known about those found in estuaries like the Chesapeake Bay. Synechococcus CB0101, an estuarine isolate, is more tolerant to shifts in temperature, salinity, and metal toxicity than coastal and oceanic Synechococcus strains, WH7803 and WH7805. Further, CB0101 has a greater sensitivity to high light intensity, likely due to its adaptation to low light environments. A complete and annotated genome sequence of CB0101 was completed to explore its genetic capacity and to serve as a basis for further molecular analysis. Comparative genomics between CB0101, WH7803, and WH7805 show that CB0101 contains more genes involved in regulation, sensing, and stress response. At the transcript and protein level, CB0101 regulates its metabolic pathways, transport systems, and sensing mechanisms when nitrate and phosphate are limited. Zinc toxicity led to oxidative stress and a global down regulation of photosystems and the translation machinery. From the stress response studies seven chromosomal toxin-antitoxin (TA) genes, were identified in CB0101, which led to the discovery of TA genes in several marine Synechococcus strains. The activation of the relB2/relE1 TA system allows CB0101 to arrest its growth under stressful conditions, but the growth arrest is reversible, once the stressful environment dissipates. The genome of CB0101 contains a relatively large number of genomic island (GI) genes compared to known marine Synechococcus genomes. Interestingly, a massive shutdown (255 out of 343) of GI genes occurred after CB0101 was infected by a lytic phage. On the other hand, phage-encoded host-like proteins (hli, psbA, ThyX) were highly expressed upon phage infection. This research provides new evidence that estuarine Synechococcus like CB0101 have inherited unique genetic machinery, which allows them to be versatile in the estuarine environment.Item THE VIRAL GENOMICS REVOLUTION: BIG DATA APPROACHES TO BASIC VIRAL RESEARCH, SURVEILLANCE, AND VACCINE DEVELOPMENT(2015) Schobel, Seth A.; Cummings, Michael P; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Since the decoding of the first RNA virus in 1976, the field of viral genomics has exploded, first through the use of Sanger sequencing technologies and later with the use next-generation sequencing approaches. With the development of these sequencing technologies, viral genomics has entered an era of big data. New challenges for analyzing these data are now apparent. Here, we describe novel methods to extend the current capabilities of viral comparative genomics. Through the use of antigenic distancing techniques, we have examined the relationship between the antigenic phenotype and the genetic content of influenza virus to establish a more systematic approach to viral surveillance and vaccine selection. Distancing of Antigenicity by Sequence-based Hierarchical Clustering (DASH) was developed and used to perform a retrospective analysis of 22 influenza seasons. Our methods produced vaccine candidates identical to or with a high concordance of antigenic similarity with those selected by the WHO. In a second effort, we have developed VirComp and OrionPlot: two independent yet related tools. These tools first generate gene-based genome constellations, or genotypes, of viral genomes, and second create visualizations of the resultant genome constellations. VirComp utilizes sequence-clustering techniques to infer genome constellations and prepares genome constellation data matrices for visualization with OrionPlot. OrionPlot is a java application for tailoring genome constellation figures for publication. OrionPlot allows for color selection of gene cluster assignments, customized box sizes to enable the visualization of gene comparisons based on sequence length, and label coloring. We have provided five analyses designed as vignettes to illustrate the utility of our tools for performing viral comparative genomic analyses. Study three focused on the analysis of respiratory syncytial virus (RSV) genomes circulating during the 2012- 2013 RSV season. We discovered a correlation between a recent tandem duplication within the G gene of RSV-A and a decrease in severity of infection. Our data suggests that this duplication is associated with a higher infection rate in female infants than is generally observed. Through these studies, we have extended the state of the art of genotype analysis, phenotype/genotype studies and established correlations between clinical metadata and RSV sequence data.Item CHARACTERIZATION OF THE BACTERIAL COMMUNITIES ASSOCIATED WITH THE TROPICAL SACOGLOSSAN MOLLUSKS ELYSIA RUFESCENS AND ELYSIA CRISPATA.(2014) Davis, Jeanette; Hill, Russell T; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Mollusks are the largest group of marine invertebrates and are known to harbor bacterial communities; however, the characterization and metabolic roles of these communities to the biology of mollusks are unknown. Sacoglossan sea slugs are herbivorous mollusks well known for their unique ability among metazoans to sequester functional chloroplasts from their algal food through a process called kleptoplasty, enabling a few species of these slugs to photosynthesize. Sacoglossan mollusks are also known to sequester chemical compounds from their algal diets through a process called kleptochemistry and use these compounds as defense molecules. These defense molecules often display medicinal properties. The mechanisms for such phenomena are unknown. I characterized the bacterial communities associated with the Hawaiian sea slug Elysia rufescens and its algal diet Bryopsis sp., in which the promising anticancer compound, Kahalalide F (KF) was extracted, through both culture-based and molecular analysis. I cultured a total of 460 bacteria from the mollusk and Bryopsis and screened them for KF production. I found a diverse assemblage of bacteria associated with this sacoglossan comprising 16 different bacterial phyla. In addition, a photosynthetic sacoglossan slug, Elysia crispata from two Caribbean locations and their associated alga bacterial communities were characterized. I discovered less bacterial diversity associated with this sacoglossan and found that the bacterial communities associated with E. crispata from different locations are more similar to each other than the bacterial communities of the associated alga. This work forms the basis for describing the bacterial community of the sacoglossans E. rufescens and E. crispata and furthers our understanding of the potential roles bacteria may play in the unusual sacoglossan niche.