Molecular insights into the niche of harmful brown tides

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 2011Recurrent brown tide blooms caused by the harmful alga Aureococcus anophagefferens have decimated coastal ecosystems and shellfisheries along the Eastern U.S. and South Africa. The exact mechanisms controlling bloom formation, sustenance, and decline are unclear, however bottom-up factors such as nutrient type and supply are thought to be critical. Traditional assays for studying algal nutrient physiology require bulk community measurements or in situ nutrient perturbations. Although useful, these techniques lack the ability to target individual species in complex, mixed microbial assemblages. The motivation for this thesis is to examine the metabolic strategies utilized by A. anophagefferens for meeting its nitrogen (N) and phosphorus (P) demand at the cellular level using molecular tools that, even in the presence of complex microbial assemblages, can be used to track how nutrients influence the bloom dynamics of A. anophagefferens in the environment. Chapter two examines the global transcriptional responses of A. anophagefferens to N and P deficiency. Results demonstrate that A. anophagefferens has the capacity to utilize multiple forms of organic N and P when inorganic forms become unavailable. Chapter three analyzed the global protein changes in response to P deficiency and P re-supply. Consistent with transcript patterns, A. anophagefferens increases protein abundance for a number of genes involved in inorganic and organic P metabolism when inorganic P is deficient. Furthermore, increases in a sulfolipid biosynthesis protein combined with lipid data suggest A. anophagefferens can adjust its P requirement by switching from phospholipids to sulfolipids when inorganic P is unavailable. Analysis of protein abundances from P-deficient cells that were re-fed inorganic P demonstrates variations in the timing of turnover among various proteins upon release from phosphate deficiency. Chapter four tests the expression patterns of candidate gene markers of nutrient physiology under controlled culture experiments. Results show that expression patterns of a phosphate transporter and xanthine/uracil/vitamin C permease are indicators of P and N deficiency, respectively. Taken together, these findings provide insight into the fundamental and ecological niche space of this harmful algal species with respect to N and P and provide a platform for assaying nutrient controls on natural brown tide blooms.Throughout my career as a graduate student I have was supported by the Woods Hole Oceanographic Institution Academic Programs Office, an EPA STAR graduate fellowship (#FP916901). I also utilized funds from a student research grant from the Woods Hole Coastal Ocean Institute, a National Oceanic and Atmospheric Administration ECOHAB grant (#NA09NOS4780206), and National Science Foundation grant (#OCE-0723667)

Molecular insights into the niche of harmful brown tides

Thesis (Ph. D.)--Joint Program in Oceanography/Applied Ocean Science and Engineering (Massachusetts Institute of Technology, Dept. of Biology; and the Woods Hole Oceanographic Institution), 2011.Cataloged from PDF version of thesis.Includes bibliographical references.Recurrent brown tide blooms caused by the harmful alga Alureococcus anophagefferens have decimated coastal ecosystems and shellfisheries along the Eastern U.S and South Africa. The exact mechanisms controlling bloom formation, sustenance, and decline are unclear, however bottom-up factors such as nutrient type and supply are thought to be critical. Traditional assays for studying algal nutrient physiology require bulk community measurements or in situ nutrient perturbations. Although useful, these techniques lack the ability to target individual species in complex, mixed microbial assemblages. The motivation for this thesis is to examine the metabolic strategies utilized by A. anophagefferens for meeting its nitrogen (N) and phosphorus (P) demand at the cellular level using molecular tools that, even in the presence of complex microbial assemblages, can be used to track how nutrients influence the bloom dynamics of A. anophageferens in the environment. Chapter two examines the global transcriptional responses of A. anophagefferens to N and P deficiency. Results demonstrate that A. anophagefferens has the capacity to utilize multiple forms of organic N and P when inorganic forms become unavailable. Chapter three analyzed the global protein changes in response to P deficiency and P re-supply. Consistent with transcript patterns, A. anophagefferens increases protein abundance for a number of genes involved in inorganic and organic P metabolism when inorganic P is deficient. Furthermore, increases in a sulfolipid biosynthesis protein combined with lipid data suggest A. anophagefferens can adjust its P requirement by switching from phospholipids to sulfolipids when inorganic P is unavailable. Analysis of protein abundances from Pdeficient cells that were re-fed inorganic P demonstrates variations in the timing of turnover among various proteins upon release from phosphate deficiency. Chapter four tests the expression patterns of candidate gene markers of nutrient physiology under controlled culture experiments. Results show that expression patterns of a phosphate transporter and xanthine/uracil/vitamin C permease are indicators of P and N deficiency, respectively. Taken together, these findings provide insight into the fundamental and ecological niche space of this harnful algal species with respect to N and P and provide a platform for assaying nutrient controls on natural brown tide blooms.by Louie L. Wurch.Ph.D

Virus-host relationships of marine single-celled eukaryotes resolved from metatranscriptomics

Establishing virus–host relationships has historically relied on culture-dependent approaches. Here we report on the use of marine metatranscriptomics to probe virus–host relationships. Statistical co-occurrence analyses of dsDNA, ssRNA and dsRNA viral markers of polyadenylation-selected RNA sequences from microbial communities dominated by Aureococcus anophagefferens (Quantuck Bay, NY), and diatoms (Narragansett Bay, RI) show active infections by diverse giant viruses (NCLDVs) associated with algal and nonalgal hosts. Ongoing infections of A. anophagefferens by a known Mimiviridae (AaV) occur during bloom peak and decline. Bloom decline is also accompanied by increased activity of viruses other than AaV, including (+) ssRNA viruses. In Narragansett Bay, increased temporal resolution reveals active NCLDVs with both ‘boom-and-bust’ and ‘steady-state infection’-like ecologies that include known as well as novel virus–host interactions. Our approach offers a method for screening active viral infections and develops links between viruses and their potential hosts in situ. Our observations further demonstrate that previously unknown virus–host relationships in marine systems are abundant

Long serial analysis of gene expression for gene discovery and transcriptome profiling in the widespread marine coccolithophore Emiliania huxleyi

Author Posting. © American Society for Microbiology, 2006. This article is posted here by permission of American Society for Microbiology for personal use, not for redistribution. The definitive version was published in Applied and Environmental Microbiology 72 (2006): 252-260, doi:10.1128/AEM.72.1.252-260.2006.The abundant and widespread coccolithophore Emiliania huxleyi plays an important role in mediating CO2 exchange between the ocean and the atmosphere through its impact on marine photosynthesis and calcification. Here, we use long serial analysis of gene expression (SAGE) to identify E. huxleyi genes responsive to nitrogen (N) or phosphorus (P) starvation. Long SAGE is an elegant approach for examining quantitative and comprehensive gene expression patterns without a priori knowledge of gene sequences via the detection of 21-bp nucleotide sequence tags. E. huxleyi appears to have a robust transcriptional-level response to macronutrient deficiency, with 42 tags uniquely present or up-regulated twofold or greater in the N-starved library and 128 tags uniquely present or up-regulated twofold or greater in the P-starved library. The expression patterns of several tags were validated with reverse transcriptase PCR. Roughly 48% of these differentially expressed tags could be mapped to publicly available genomic or expressed sequence tag (EST) sequence data. For example, in the P-starved library a number of the tags mapped to genes with a role in P scavenging, including a putative phosphate-repressible permease and a putative polyphosphate synthetase. In short, the long SAGE analyses have (i) identified many new differentially regulated gene sequences, (ii) assigned regulation data to EST sequences with no database homology and unknown function, and (iii) highlighted previously uncharacterized aspects of E. huxleyi N and P physiology. To this end, our long SAGE libraries provide a new public resource for gene discovery and transcriptional analysis in this biogeochemically important marine organism.This work was supported by the Woods Hole Oceanographic Institution Ocean Life Institute, the J. Lamar Worzel Assistant Scientist Fund, and the Frank and Lisina Hoch Endowed Fund. A.G.M., S.R.B., and M.J.C. were supported in part by the Marine Biological Laboratory's Program in Global Infectious Diseases, funded by the Ellison Medical Foundation. Computational resources were provided by the Josephine Bay Paul Center for Comparative Molecular Biology and Evolution (Marine Biological Laboratory) through funds provided by the W. M. Keck Foundation and the G. Unger Vetlesen Foundation

Characterization of Fatty Acids in Crenarchaeota by GC-MS and NMR

Lipids composed of condensed isoprenyl units connected to glycerol backbones by ether linkages are a distinguishing feature of Archaea. Data suggesting that fatty acids with linear hydrocarbon chains are present in some Archaea have been available for decades. However, lack of genomic and biochemical evidence for the metabolic machinery required to synthesize and degrade fatty acids has left the field unclear on this potentially significant biochemical aspect. Because lipids are energy currency and cell signaling molecules, their presence in Archaea is significant for understanding archaeal biology. A recent large-scale bioinformatics analysis reignited the debate as to the importance of fatty acids in Archaea by presenting genetic evidence for the presence of enzymes required for anabolic and catabolic fatty acid metabolism across the archaeal domain. Here, we present direct biochemical evidence from gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) spectroscopy for the presence of fatty acids in two members of the Crenarchaeota, Sulfolobus solfataricus and Ignicoccus hospitalis. This is the first report providing biochemical data for the existence of fatty acids in these Crenarchaeota, opening new discussions on energy balance and the potential for the discovery of new thermostable enzymes for industry

Transcriptional Shifts Highlight the Role of Nutrients in Harmful Brown Tide Dynamics

Harmful algal blooms (HABs) threaten ecosystems and human health worldwide. Controlling nitrogen inputs to coastal waters is a common HAB management strategy, as nutrient concentrations often suggest coastal blooms are nitrogen-limited. However, defining best nutrient management practices is a long-standing challenge: in part, because of difficulties in directly tracking the nutritional physiology of harmful species in mixed communities. Using metatranscriptome sequencing and incubation experiments, we addressed this challenge by assaying the in situ physiological ecology of the ecosystem destructive alga, Aureococcus anophagefferens. Here we show that gene markers of phosphorus deficiency were expressed in situ, and modulated by the enrichment of phosphorus, which was consistent with the observed growth rate responses. These data demonstrate the importance of phosphorus in controlling brown-tide dynamics, suggesting that phosphorus, in addition to nitrogen, should be evaluated in the management and mitigation of these blooms. Given that nutrient concentrations alone were suggestive of a nitrogen-limited ecosystem, this study underscores the value of directly assaying harmful algae in situ for the development of management strategies

Niche of harmful alga Aureococcus anophagefferens revealed through ecogenomics

Author Posting. © The Author(s), 2011. This is the author's version of the work. It is posted here by permission of National Academy of Sciences for personal use, not for redistribution. The definitive version was published in Proceedings of the National Academy of Sciences 108 (2011): 4352-4357, doi:10.1073/pnas.1016106108.Harmful algal blooms (HABs) cause significant economic and ecological damage worldwide. Despite considerable efforts, a comprehensive understanding of the factors that promote these blooms has been lacking because the biochemical pathways that facilitate their dominance relative to other phytoplankton within specific environments have not been identified. Here, biogeochemical measurements demonstrated that the harmful 43 Aureococcus anophagefferens outcompeted co-occurring phytoplankton in estuaries with elevated levels of dissolved organic matter and turbidity and low levels of dissolved inorganic nitrogen. We subsequently sequenced the first HAB genome (A. anophagefferens) and compared its gene complement to those of six competing phytoplankton species identified via metaproteomics. Using an ecogenomic approach, we specifically focused on the gene sets that may facilitate dominance within the environmental conditions present during blooms. A. anophagefferens possesses a larger genome (56 mbp) and more genes involved in light harvesting, organic carbon and nitrogen utilization, and encoding selenium- and metal-requiring enzymes than competing phytoplankton. Genes for the synthesis of microbial deterrents likely permit the proliferation of this species with reduced mortality losses during blooms. Collectively, these findings suggest that anthropogenic activities resulting in elevated levels of turbidity, organic matter, and metals have opened a niche within coastal ecosystems that ideally suits the unique genetic capacity of A. anophagefferens and thus has facilitated the proliferation of this and potentially other HABs.Joint Genome Institute is supported by the Office of Science of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231. Efforts were also supported by awards from New York Sea Grant to Stony Brook University, National Oceanic and Atmospheric Administration Center for Sponsored Coastal Ocean Research award #NA09NOS4780206 to Woods Hole Oceanographic Institution, NIH grant GM061603 to Harvard University, and NSF award IOS-0841918 to The University of Tennessee

Adhiron: a stable and versatile peptide display scaffold for molecular recognition applications

We have designed a novel non-antibody scaffold protein, termed Adhiron, based on a phytocystatin consensus sequence. The Adhiron scaffold shows high thermal stability (Tm ca. 101°C), and is expressed well in Escherichia coli. We have determined the X-ray crystal structure of the Adhiron scaffold to 1.75 Å resolution revealing a compact cystatin-like fold. We have constructed a phage-display library in this scaffold by insertion of two variable peptide regions. The library is of high quality and complexity comprising 1.3 × 10(10) clones. To demonstrate library efficacy, we screened against the yeast Small Ubiquitin-like Modifier (SUMO). In selected clones, variable region 1 often contained sequences homologous to the known SUMO interactive motif (V/I-X-V/I-V/I). Four Adhirons were further characterised and displayed low nanomolar affinities and high specificity for yeast SUMO with essentially no cross-reactivity to human SUMO protein isoforms. We have identified binders against >100 target molecules to date including as examples, a fibroblast growth factor (FGF1), platelet endothelial cell adhesion molecule (PECAM-1; CD31), the SH2 domain Grb2 and a 12-aa peptide. Adhirons are highly stable and well expressed allowing highly specific binding reagents to be selected for use in molecular recognition applications

Proteome changes driven by phosphorus deficiency and recovery in the brown tide-forming alga Aureococcus anophagefferens

© The Author(s), 2011. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in PLoS One 6 (2011): e28949, doi:10.1371/journal.pone.0028949.Shotgun mass spectrometry was used to detect proteins in the harmful alga, Aureococcus anophagefferens, and monitor their relative abundance across nutrient replete (control), phosphate-deficient (−P) and −P refed with phosphate (P-refed) conditions. Spectral counting techniques identified differentially abundant proteins and demonstrated that under phosphate deficiency, A. anophagefferens increases proteins involved in both inorganic and organic phosphorus (P) scavenging, including a phosphate transporter, 5′-nucleotidase, and alkaline phosphatase. Additionally, an increase in abundance of a sulfolipid biosynthesis protein was detected in −P and P-refed conditions. Analysis of the polar membrane lipids showed that cellular concentrations of the sulfolipid sulphoquinovosyldiacylglycerol (SQDG) were nearly two-fold greater in the −P condition versus the control condition, while cellular phospholipids were approximately 8-fold less. Transcript and protein abundances were more tightly coupled for gene products involved in P metabolism compared to those involved in a range of other metabolic functions. Comparison of protein abundances between the −P and P-refed conditions identified differences in the timing of protein degradation and turnover. This suggests that culture studies examining nutrient starvation responses will be valuable in interpreting protein abundance patterns for cellular nutritional status and history in metaproteomic datasets.Research for this work was supported by a National Oceanic and Atmospheric Administration ECOHAB grant (#NA09NOS4780206) and National Science Foundation grant (#OCE-0723667) and a STAR Research Assistance Agreement No. R-83041501-0 awarded by the U.S. Environmental Protection Agency. Further support came from the Woods Hole Coastal Ocean Institute. LLW was supported by a Environmental Protection Agency STAR Fellowship (#FP916901). EMB was supported by a National Science Foundation (NSF) Graduate Research Fellowship (#2007037200) and an Environmental Protection Agency STAR Fellowship (#F6E20324)