The research in this laboratory is broad reaching and presently includes the following projects:
Our most recent cell biology studies have focused on the biogenesis of lipid bodies in unicellular algae. Lipid bodies are structurally complex cellular organelles that are functionally dynamic and indispensable to the maintenance of cellular homeostasis. To date, studies on the genesis of this organelle have focused on mammalian, vascular plant and fungal systems. In contrast, little research has focused on lipid body biology in the Chromalveolata – an enormous crown taxon in the tree of life that includes “golden-brown” photosynthetic algae.
We are developing Chrysochromulina tobin (Cattolico) (Haptophyta) as new model system for the study of lipid body biogenesis in the Chromalveolata. Several attributes make this unicellular organism amenable to lipid body studies: (a) the alga produces copious amounts of fatty acids that are high in PUFA content; (b) the cell is delineated solely by a plasma membrane that facilitates lipid body recovery; (c) unlike most algae that have many lipid bodies that range in size, interphasic Chrysochromulina cells have only two lipid bodies; (d) the alga grows axenically and synchronously on a completely defined medium, thus changes in lipid body size, fatty acid content and composition induced by alterations in cell cycle events, nutrient load or other physiological cues are easily monitored.
We recently sequenced the total genome (nuclear, mitochondrial and chloroplast) of Chrysochromulina tobin with Los Alamos National Laboratory. Notably, this data represents only the second genome to be sequenced for the ~500 species within this large genus of algae. Transcriptome libraries originating from synchronized Chrysochromulina cultures (7 time points over a 24 hour period) when lipid body size is maximum (light phase) and when minimum in volume (dark phase) have been generated. We have also devised a new lipid body isolation technique for Chrysochromulina. Whole cells and isolated lipid bodies were used to compare fatty acid profiles (using our mico-GC/MS method) and proteins (LC/MS/MS at Pacific Northwest Laboratories). Our lipid body isolation method is now being applied to analyze these organelles in several additional Chromalveolate taxa including Synura petersenii, Isochrysis galbana, Emiliania huxleyi, and Ochromonas danica when these algae are subject to cell cycle constraints or physiological stress. Antibodies against heat shock protein 70 (HSP70), and heat shock protein 90 (HSP90) have been used to provide insight into the effects of stress and the hypothesized concurrent increase in lipid production in these cells.
(a) Presently, we are assembling information for a paper that we intend to submit to Science. In this study, phylogenetic analysis of 85 chloroplast genes shared by the stramenopiles, and rhodophytic and haptophyte outgroups, strongly supports the presence of three stramenopile clades and elucidates previously unresolved deep branching among classes—especially those of eustigmatophytes and synurophytes. Newly generated whole genome sequences enabled us to probe conserved relationships between intergenic regulatory domains and specific genes, and to document the frequent occurrence of lateral gene transfer events. Most exciting was the identification of genes that encode proteins novel to existing chloroplast databases (e.g., DNA recombinase, viral-like replication protein, modified RuBisCO, chlorophyll biosynthesis proteins, and unique Clp proteases).
(b) The microalgae Nannochloropsis salina and Nannochloropsis oculata represent two strains of photosynthetic marine Eustigmatophytes. To better understand the genetic complement that drives the metabolic processes of these organisms, we are analyzing their chloroplast and mitochondrial genomes and comparing our newly sequenced genomes with those of published genomes for N. gaditana and N. oceanica (which we have reannotated). We have shown that the chloroplast and mitochondrial genomes of N. salina are 98.4% and 97% identical to their counterparts in N. gaditana. N. oceanica and N. oculata chloroplast and mitochondrial DNA are similarly matched in these two species. Comparison of the Nannochloropsis pangenome to other algae from within and outside of the stramenopiles revealed regions of significant genetic divergence in key genes that encode proteins needed for carbon fixation (RuBisCO), energy conservation (ATP synthase), and protein homeostasis (Clp protease). Many organellar gene modifications in Nannochloropsis are unique and deviate from conserved orthologs found across the tree of life. Implementation of secondary and tertiary structure prediction was crucial to functionally characterize many proteins in this study.
Diatoms are unicellular algae that account for approximately 30% of oceanic primary production, a contribution to world oxygen production and carbon fixation equal to that of the grasses. To maintain efficient photosynthesis these organisms must compensate for changes in irradiance caused by shifts in environmental conditions. These algae modify their photosynthetic capacity by adjusting the composition of their light-harvesting complexes – a process known as photoacclimation. Because chlorophyll serves as the seminal component required for harvesting light energy, changes in the levels of this pigment is required. Chlorophyll synthesis is catalyzed by the light-dependent enzyme, protochlorophyllide oxidoreductase (POR). Importantly, our analyses have revealed the presence of two unique gene homologues (por1 and por2) that encode (POR1 and POR 2) proteins in several diatoms representing divergent taxa. Using a three-pronged approach, our study focuses on understanding how a critical step in the diatom chlorophyll biosynthesis cascade is determined by duplicated por genes and their protein products with respect to their: (a) Function: The enzymatic activity and reaction kinetics of each POR protein homologue are being compared using over expressed proteins; (b) Regulation: The levels of por 1 and 2 transcripts, POR 1 and 2 proteins as well as chlorophyll concentration is being determined as cultures progress through a light:dark photoperiod; (c) Evolution: Phylogenetic methods have been used to determine when in the evolutionary history of the diatoms dual por genes, and to assess which other members of the autotrophic chromoveolate algal taxa also have a dual por gene complement.
Toxic Bloom-forming Algae
(a) Cyst production: Several HAB-forming species exhibit a dual-stage life history, in which they alternate between a pelagic vegetative stage and a benthic resting stage (e.g., cysts, resting spores, and temporary resting cells). Transitions between pelagic and benthic resting stages have potentially important impacts on bloom dynamics. While the physiological conditions required to maintain cell viability in the vegetative stage are generally well understood, relatively little is known metabolic conditions required for survival in the benthic resting stage or that fuel active swimming during benthic emergence. In this research we are analyzing how an autotrophic Heterosigma akashiwo strains can survive in the dark and cold for prolonged time periods by analyzing transcriptome libraries, determining how cells on activation regain their characteristic swimming behavior through analysis of video profiles, and probing what role fatty acid energy reserves play in these processes using GC/MS. H. akashiwo forms toxic, destructive blooms world-wide, including in Puget Sound.
(b) A method for genetic fingerprinting to assess algal strain identity has been devised. We report that mitochondrial gene sequences show better strain resolution, when assessing cryptic species complexes and that this method is especially relevant when monitoring non-chlorophytic algal species. This work shows that Heterosigma is a cryptic species complex, and that virtually all populations of this alga are genetically heterogeneous.
Analytical techniques for assessing cell function
(a) A micro GC/MS method that requires only 250 ug biomass is now available. This method was used in the screening of algal strains of different taxa as they passed through synchronous cell growth, were subject to different physiological cues (e.g., nutrient load, light intensity, temperature). Presented is a fatty acid survey of 29 algal strains representing the stramenopile clade – a huge, complex, marginally studied taxon that produces neutral lipids as a primary photosynthetic product has been accomplished.
(a) The organism Chrysochromulina tobin is introduced as a standard for the algal product industry. This small unicell has no cell covering, a broad spectrum of fatty acids, and a highly defined physiology. Use of such a standard circumvents the challenges of comparing different algal strains that are grown under different environmental conditions and processed via various methods to recover oil. Comparison of fatty acid profiles and productivity for 20 algal strains is presented. Taxa examined include members within the stramenopiles, haptophytes, and dinoflagellates.
(b) Water utilization is a critical factor when discussing effective production of commercial algal species. We show several algal species to be successfully maintained in a fresh water salmon waste-water stream and may require minimal phosphate or nitrogen addition to maintain robust growth. Axenic vs. non-axenic cultures encounter very different physiological cues resulting from the presence or absence of eco-cohorts, which in turn affects lipid profiles and lipid production.