Research Interests
Overview:
Fig. 1: Approximately 75% of our earth is covered by water.
Marine algae contribute almost half of the total carbon processed on earth. These organisms anchor ocean food webs, and form symbiotic associations with a large spectrum of taxa. Coastal ecosystem health is often a reflection of its phytoplankton population.
Chloroplast Evolution Gene Expression Signal Transduction Calvin Cycle Chloroplast replication Motility Fe++ effects, toxicity
Experimental System:
Heterosigma akashiwo is a naturally wall-less, toxic alga that blooms in coastal regions world-wide. In the laboratory, cultures of this alga can be synchronously grown and axenically maintained. In the field, Heterosigma forms massive brown tides that impact the survival of eco-cohorts at every trophic level. This alga has been shown to kill finfish, compromise fish and sea urchin egg development and impact copepod as well as oyster survival.
Fig. 2: Heterosigma cell (insert) and bloom in Puget Sound, Washington
Heterosigma has at least two life history phases. Vegetative cells have been reported to migrate in a circadian manner within the water column - they photosynthesize in the photic zone during the day and then move in the deeper waters at night to glean macronutrients such as nitrate.Heterosigma cells can enter a resting phase in response to specific environmental conditions. When in this state, cells become immobile but do not loose their flagella. Our present studies are focused on understanding how a marine autotroph such as Heterosigma, perceives its environment and then transduces this signal to a metabolic pathway that insures survival.
Fig. 3: Heterosigma life cycle: Vegetative cells are induced by physiological cues to enter a resting phase (or stasis). They can survive in this state for many, many months. When conditions improve, cells regain motility and move to the photic zone.
What genes are present in the chromophytic algal chloroplast genome?
Stramenopiles are a "crown" taxon that evolved about 300 million years ago and radiated after the Cretaceous Period. Photosynthetic members of this taxon vary in morphology from simple unicells to highly complex parenchymous seaweeds with intricate reproductive structures. These autotrophic eukaryotes impact many of the earth's biogeochemical cycles (e.g. sulfur and nitrogen loading) and serve as primary producers that fix a significant portion of the total CO2 processed on earth. The stramenopiles represent a major eukaryotic group, comprised of an estimated 500,000 to one million species that is taxonomically distinct from the chlorophytic or rhodophytic lineages of autotrophs.
To date, of the thousands of species, only two chloroplast genomes are available for the entire stramenopile assemblage (both are from diatoms). In our newly funded work, chloroplast DNA of selected representatives from this taxon are being sequenced. Morphologically diverse organisms that are ecologically contributive and economically valuable have been targeted. We have completed the sequence of the chloroplast genomes from two Heterosigma strains (East Pacific and West Atlantic). Analysis of the chloroplast genomes from 30 additional stramenopiles as part of an NSF Tree of Life project is ongoing. We are using our newly developed Fosmid sequencing approach in these studies. This method completely eliminates the need to isolate chloroplast DNA (an impossible task in 1um-sized algal representatives). I am collaborating with G. Rocap (U of W; Ocean Sciences) and M. Jacobs, (Genome Center) in this endeavor.
Fig. 4: Sequenced Heterosigma akashiwo (strain CCMP 452) chloroplast genome
What chloroplast genes are expressed during Heterosigma's various life history phases?
Heterosigma is an obligate photoauxotroph that not only survives as a free swimming vegetative cell, but also exists for long periods in stasis (in the dark and cold). We have shown that mRNA abundance in synchronously maintained Heterosigma vegetative cells is transcriptionally regulated. This response is determined by photoperiod. Having a large number of chloroplast gene sequences, through our chloroplast genome sequencing project, will allow us to probe selective gene expression profiles at a given life-history phase using quantitative RT PCR and micro-array technologies.
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| Fig. 5: (A) Steady-state analysis of psbA and rbcL, nbcS mRNAs during a 12L:12D cycle shows genes to be expressed in an ocillatory manner |
How do chromophytic algae regulate chloroplast gene expression?
Sequence analysis of the Heterosigma chloroplast genome revealed the presence of a single two-component His-to-Asp (tsg1/Trg1) pair in this golden-brown alga. Our data represent the first documentation of His-to-Asp arrays in chromophytic algae and counter previous reports suggesting that such regulatory proteins are lacking in the stramenopile taxonomic cluster. Molecular modeling of the 27 kd Heterosigma Trg1 regulator protein is consistent with a winged helix-turn-helix identity--a class of proteins that is known to impact gene expression at the level of transcription. Our data support the hypothesis that the response regulators of chromophytes, rhodophytes, and glaucophytes interact with a sigma 70 subunit (encoded by rpoD) of a eubacterial type polymerase. rpoD has now been sequenced in Heterosigma. Both transcriptional and western analysis verify that tsg1 is not a pseudogene. We hypothesize that tsg1 is red/ox regulated. tsg1/trg1 pair is phylogenetically constrained in its distribution. Some chloroplast genomes contain no identifiable tsg1 orthologue,while others contain multiple trg1-like genes.
Fig. 6: Comparison of Heterosigma trg1 DNA and RNA polymerase binding domain to bacterial response regulators
How is carbon metabolized via the Calvin cycle? What are the evolutionary origin(s) of this cycle?
Stramenopiles serve as large-scale bioconverters in CO2 processing via the Calvin-Bensen Cycle. The dogma, present in all text books, that Rubisco small subunit was universally nuclear encoded was disproven by this laboratory (all "red" and "brown" algae have this gene localized to their plastid genome). We have also published some of the only available Km values and enzyme analysis for Rubisco and phosphoribulokinase in these algae. Our present work (in collaboration with Dr. Michael Salvucci; USDA laboratory) is focused on analyzing the molecular biology and biochemistry of a putative Rubisco activase (our target protein is very unlike the Rubisco activase of land plants and green algae). Sequencing of this putative rubisco activase from 24 Heterosigma strains revealed the presence of six naturally occurring varients in the protein. We propose to test the functional significance of these altered proteins.
Fig. 7: Protein model of a putative rubisco activase found in the Heterosigma akashiwo chloroplast genome
How do chloroplasts replicate?
The survival of an autotrophic cell requires that mutiple copies of the plastid genome are reproduced with high fidelity and that these replicated chromosomes are efficiently distributed to the next chloroplast generation. Though Ris and Plaut reported the presence of DNA in chloroplasts over 40 years ago, we still know very little about plastid DNA synthesis. The goal of our study is to analyze the events associated with chloroplast DNA replication and its dispersal to daughter plastids. We are using qPCR to probe the temporal expression of chloroplast-encoded genes that are implicated in DNA replication and division in synchronous Heterosigma cultures. Future studies will use confocal microscopy to document the replication of DNA "beads" on the ring-shaped nucleoid of this organelle and the localization of division proteins.
Fig. 8: Heterosigma chloroplast nucleoids (Courtesy of A. Coleman)
How does motility influence Heterosigma bloom dynamics?
Movement behaviors of phytoplankton within a limited geographic area may significantly affect the intensity and duration of a harmful algal bloom. The ability of one cell type to actively move from the photic zone (where photosynthesis can occur) to deeper waters (for the acquisition of nutrients) gives an alga a competitive advantage over passively moving phytoplankters. Infrared photography coupled with analytical software developed by our collaborators Rachel Bearon and Danny Grumbaum (U of Washington Ocean Sciences), has allowed the 3D tracking of motile Heterosigma cells. Vegetative cell swimming speeds, which range from 65 to 300 um/sec, are also correlated with strain identity. Heterosigma life history phase also impacts swimming capacity. Resting cells are completely immobile. Although cells exiting stasis are mitotically active, resumption of swimming capacity is dependent on induction physiology and strain identity.
Fig. 9: Individual swimming tracks of Heterosigma akashiwo cells
Toxic algae often form high density "slicks" or "patches" on coastal waters. When cell division is coupled with cell aggregation (the concentration of dispersed cells resulting from algal behavior or advective transport), bloom formation is significantly augmented, and the probability of a toxic end point enhanced. Algal blooms are dynamic events that both respond to and create their own environmental signals. These signals drive cellular metabolism and potentially structure the genetic profile of the resident algal population. In the field, the generation of dense concentrations of Heterosigma cells is often correlated with fresh-water stratification of bays and coastal inlets. Similar high-density cell aggregations can be rapidly induced in laboratory culture by layering fresh water over brackish seawater. In this work we are asking how "slick" formation is influenced by Heterosigma strains that swim with different efficiencies, how the "slick" itself impacts cell function (using microarrays; qPCR); drives haplotype selection within a population, and impacts the toxic potential of the composite cells.
Fig. 10: Slicks are when free-swimming cells (A) encounter sea water that has been overlayed with a fresh water lens (B) thus causing cells to be trapped in the low salinity region of the halocline
How does Fe++ impact Heterosigma survival and toxicity?
Estuaries have traditionally been regarded as iron-replete for primary production. However this element has recently been shown to be limiting in some of these semi-enclosed coastal bays. The causes invoked to explain iron limitation in estuarine systems are anthropogenic in origin: a) deforestation may reduce input of low molecular weight humic compounds which bind iron and prevent its precipitation and b) eutrophication via fertilizer and agricultural waste runoff can deliver excess macronutrients, thus driving the limitation further down the nutrient chain to the next-most limiting nutrient, iron. We are researching the potential impacts of this anthropogenic change on populations of the harmful alga Heterosigma akashiwo. Both the responses of individual cell physiology and population-level responses are being examined. We are testing the hypothesis that anthropogenically increased incidences of iron limitation will cause increased reactive oxygen toxicity of Heterosigma blooms. Using laboratory cultures and mathematical models we are also examining the conditions under which an algal bloom may consist of a succession of functionally distinct populations that evolve through a series of selective genetic sweeps.