Inter-individual competition for resources and variability of the environment
 
Piotrek Bentkowski
PhD Project Outline

Experimental studies of the biological evolution encounter two major limitations: a long time needed to observe these gradual changes and a large size of the population necessary for beneficial mutations to get fixed. Computational modeling allows us to overcome those limitations for the price of taking some simplifying assumptions. I am investigating can the inter-individual competition for resources and variability of the environment influence the size of the free-living bacteria genomes either by forcing streamlining or expansion of genome's size? The model, an agent-based simulation of individual cell having unique genomes, shows that under stable environmental conditions competition for the resource forces bacteria to do extreme energetic savings including decreasing the number of genes (as their expression cost). When environment is unpredictable the genomes become larger to prepare the individual for a wider span of the environmental conditions.

Gene networks in diatoms that are involved in acclimation to the changing climate
 
Rachel Hipkin
PhD Project Outline

There is an urgent need to understand how important global primary producers such as diatoms perceive environmental changes to make predictions of how global change might influence life in the ocean. I plan to address this challenge using chromatin immunoprecipitation (ChIP) in combination with pyrosequencing (ChIPSeq) to globally identify genomic binding sites of different transcription factors in the sequenced diatom Thalassiosira pseudonana. Over the course of my project I will develop ChIP Seq methods to use with T. Pseudonana, and once established we plan to investigate transcription factors involved in response to biogeochemical stresses. We will establish which genes are upregulated under different environmental stresses. This project will provide an insight into genes which are co regulated and also identify new genes previously unidentified, with important consequences for understanding of important biogeochemical processes. These studies will also allow us to associate “unknown genes” with developmental pathways defined by these regulons and thus begin to infer potential functions for these gene products.

Whole genome expression profiling of the polar diatom Fragilariopsis cylindrus
 
Jan Strauss
PhD Project Outline

A genomic analysis using RNA-Seq to investigate the adaptation of the psychrophilic diatom Fragilariopsis cylindrus to the polar environment.
Diatoms are unicellular photosynthetic eukaryotes with a silicate cell wall. They often dominate polar marine ecosystems, driving the major biogeochemical cycles in these areas. The obligate psychrophilic diatom Fragilariopsis cylindrus is a keystone species in the Southern Ocean. It thrives both in open waters and sea ice and has become a model for studying eukaryotic microalgal adaptations to polar marine conditions. The aim of this thesis was to identify how the genome of F. cylindrus has evolved to cope with marine environmental conditions of the Southern Ocean. To identify key genes, comparative genomics, high-throughput transcriptome sequencing and reverse genetics were applied. Comparative genomics with the sequenced mesophilic diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana was combined with genome-wide RNA-Seq transcriptome analysis, leading to the discovery a new bacteria-like rhodopsin not present in other sequenced diatoms. The characterisation of a bacteria-like rhodopsin in F. cylindrus was conducted by applying reverse genetics tools.


Research Project Outline

Global significance of light-driven proton pumps in eukaryotic marine phytoplankton.
Sunlight is the ultimate source of energy and a major source of environmental information for almost all living organisms. The efficient capture and use of light is a widespread and exquisitely evolved process represented across all kingdoms of life. The metabolic mode in which organisms convert light energy into chemical energy (ATP and NADPH) for growth is known as phototrophy. Two mechanistically distinct processes are known to power phototrophy. While one mechanism employs chlorophyll-containing photochemical reaction centres, the other employs rhodopsins, membrane-embedded photoreceptors composed of opsin proteins and the light-absorbing pigment retinal. Microbial rhodopsins were first discovered in purple membranes of Archaebacteria in the form of the light-driven proton pump bacteriorhodopsin and shown to generate a proton motive force across cell membranes, ultimately powering the production of ATP for energy-requiring processes in the cell. This process is independent from chlorophyll-based photosynthesis. Surprisingly, putative proton-pumping rhodopsins powering phototrophy in prokaryotes were recently also discovered in eukaryotic marine phytoplankton. However, nothing is known about their function in vivo and the role of a light-driven proton-pump in the presence of a proton gradient-generating chlorophyll-based photosynthetic apparatus remains puzzling and speculative. A better understanding of the role of rhodopsins in marine photosynthetic organisms is of global significance, because bacteria-like rhodopsins were shown to be very abundant in metatranscriptome surveys of eukaryotic marine phytoplankton and seem to be of particular significance in iron-limited areas of the ocean, accounting for 35% of the total ocean surface. Furthermore, recent studies suggest that ocean acidification due to increased dissolution of anthropogenic CO2 may decrease iron availability to phytoplankton, which may alter phytoplankton diversity in the oceans, thus, selecting for those phytoplankton species that have a competitive advantage (e.g. by the presence of bacteria-like rhodopsins) under reduced iron concentrations. Therefore we use molecular genetic approaches to elucidate the function and significance of light-driven rhodopsin proton pumps in eukaryotic marine phytoplankton.

The Eukaryotic Meta-Transcriptome of the Upper Ocean
 
Gill Malin, Vincent Moulton, Simon Moxon (University of East Anglia, Norwich, UK)
Ginger Armbrust, Micaela S. Parker, Adrian Marchetti (University of Washington, Seattle, USA)
Andreas Krell, Klaus Valentin (Alfred-Wegener Institute for Polar and Marine Research, Bremerhaven, Germany)
Collaborating Project

The microbial genetic inventory of the ocean is not known yet but we know that marine microbes are present at billions of cells per liter sea water and that they have a long evolutionary history that extends backwards further than other live forms on earth (Irigoien et al. 2004, Nature, 429:863-867).

A phytoplankton bloom off the coast of eastern Scotland
A phytoplankton bloom off the coast of eastern Scotland
 

The genomic diversity that is related to evolutionary dynamics of these organisms influences the flux of energy and matter in the ocean and therefore biogeochemical cycling and global climate (Falkowski et al. 1998, Science, 282:200-206). However, the ultimate link between gene content and fluxes of energy and matter in the ocean is the activity of genes that shape the phenotype. Current metagenome projects have been conducted with genomic DNA from prokaryotic plankton (mostly bacteria) by size-fractionated filtration (pore size between 0.2 – 3μm) (e.g. Rusch et al. 2007, PloS Biol. 5:e77). These projects provided a wealth of novel information about the gene content and thus genomic diversity in the ocean but they excluded most of the eukaryotic microbes because of their expected large genome sizes and potential complications arising from intron/exon structure and intergenic regions. A diverse group of phytoplankton taxa are important components of microbial eukaryotes of the upper ocean. They are responsible for about 25% of global carbon dioxide fixation (Field et al. 1998, Science, 281:237-240) and will be directly affected by warmer, more stratified, and acidic near surface waters caused by an increased burden of carbon dioxide in the atmosphere (Behrenfeld et al. 2006, Nature, 444:752-755). Fundamental knowledge about the expressed metagenome from the phytoplankton community will help to interpret future responses to global change. The expression signature represents the meta-phenotype and thus how these organisms are acclimated to current ambient conditions. We therefore sequence the transcriptome (cDNA) of microbial eukaryotes from biogeochemical hotspots of the upper ocean (productive coastal system, HNLC (high nitrate low chlorophyll) system, and polar ocean) by using the 454/GS-FLX sequencer to provide preliminary data about the expressed blueprint of the upper-ocean transcriptome.

Whole-genome analysis to identify nuclear-encoded plastid proteins in diatoms.
 
Gabrielle Rocap, Ginger Armbrust (University of Washington, Seattle, USA)
Ansgar Gruber, Peter Kroth (University of Konstanz, Germany)
Collaborating Project

Diatoms belong to Stremanopiles, which evolved based on secondary endosymbiosis (see figure). This led to a mixture of several genomes with significant gene transfer into the host nucleus. One consequence of this event includes a complex targeting mechanism to allow nuclear-encoded proteins to be imported into the plastid. The complete genome sequences of the diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum have allowed to identifying these nuclear-encoded plastid proteins. We have taken a two step approach to identify diatom plastid targeted proteins. First, for anabolic pathways known to be localized to the plastids of green algae/higher plants we manually searched upstream of the predicted genes for a signal peptide and plastid transit sequence, both of which are required for plastid localization in diatoms. We identified a set of 118 proteins in both diatoms considered to be highly likely to be plastid targeted. Transit peptides from these 118 proteins were used to determine relative frequency of amino acids and the information content of each position in the transit peptide sequence for each diatom. Based on these “rules” the complete proteome of each diatom was screened to identify proteins with both signal and transit peptide sequences. We have identified over 400 genes in each genome that are putatively targeted to the plastid. This work allows the reconstruction of know plastid pathways and the identification of differences to green algae and higher plants and it furthermore is able to identify new and diatom specific plastid proteins. Current work includes expression analysis of the plastome and transformation experiments to verify predicted targeting.

Endosymbiosis

 
Whole genome sequencing of the polar diatom Fragilariopsis cylindrus
 
Ginger Armbrust, Micaela S. Parker (University of Washington, Seattle, USA)
Chris Bowler (Ecole Normale Supérieure, Paris, France)
Klaus Valentin (Alfred-Wegener Institute for Polar and Marine Research, Bremerhaven, Germany)
Igor V Grigoriev, Alan Kuo, Erica Lindquist, Kerrie Barry (Joint Genome Institute (JGI), Walnut Creek, California, USA)
Collaborating Project

Diatoms are microscopic unicellular algae distributed throughout the world’s marine and freshwater ecosystems, including ice systems. Numerous estimates suggest that, as a group, these organisms are responsible for as much as 20% of global photosynthesis, which is comparable to the amount of primary productivity generated by all the terrestrial rainforests combined. In polar regions, where extensive permafrost and glaciers limit the amount of photosynthesis possible on land, diatoms are responsible for a huge fraction of primary productivity and therefore also for carbon sequestration. Polar diatoms are adapted to cold temperatures and high salinities and they live both within the seawater and the brine channels formed within sea ice. They serve as the base of the entire polar food web. Polar regions are displaying the greatest responses to climate change, with increased melting of sea ice and the potential disruption of this sensitive ecosystem. The whole genome sequencing of a representative polar diatom was proposed in order to gain insights into how these organisms have adapted to the extreme environments in which they thrive.

SEM of two Fragilariopsis cylindrus shells
SEM of two Fragilariopsis cylindrus shells
 
Fragilariopsis cylindrus is regarded as a typical cold-water species found in Arctic and Antarctic sea water and sea ice. It can form large ice-edge blooms and is regularly detected in sea ice investigations. To date, prokaryotes are the only psychrophilic (cold-loving) organisms for which whole genome sequences exist. The whole genome sequencing of F. cylindrus will provide the first insights into a psychrophilic eukaryotic organism with compelling ecological and biotechnological applications. The whole genome sequences of the temperate diatoms Thalassiosira pseudonana, Phaeodactylum tricornutum, and Pseudonitzschia multiseries will provide a basis for comparative analysis with the psychrophilic F. cylindrus. The genome of F. cylindrus will provide insights into the unique physiology of polar-adapted eukaryotes and allow for better predictions of the impact climate change may have on organisms that serve as the basis for all polar ecosystems.
 
From genes to structures: How diatoms form their nano-structured silica shells
 
Amy Kirkham (University of East Anglia, Norwich, UK)
and Angela Falciatore (University Pierre and Marie Curie, Paris, France)
Collaborating Project

Diatoms are nature's nanotechnology masters, able to produce beautifully ornamented shells made of silica (hydrogenated silicon). The beauty of these shells even inspired artists and precise hand drawings of them were already published by Ernst Haeckel in his book: "Artforms in Nature" beginning of the last century. The architecture of these shells differs from species to species but is the consequence of an optimization process that went on for over about 300 million years of evolution. They are light-weight and porous to keep the cells afloat and to ensure exchange of nutrients and gases for growth but on the other side they are very stable to protect the cell. This combination of properties caused a huge interest by engineers because they want to understand how diatoms are able to lay down these complex structures of silica in high order at the sub-nanometer range exceeding any current capabilities of human nano-technology. Diatoms directly build in 3D, which is why they are so attractive for any lithographic approaches. The applications of this knowledge are evident. Silica is related to silicon which is the key element for the semiconductor industry and thus for computer chips. If engineers can genetically control that process of silicon fiber formation as diatoms already do, they would have an entire new way of performing the nanofabrication used to make computer chips. Meanwhile they already have begun to combine diatom shells with semiconductor technology. On application of an electric field, the shells of some diatoms emit light, which is explained by the geometry of shells latticework of pores. They hope that further combinations of semiconductor technology and biologically produced nanostructures may yield novel devices. Diatom shells also influence incoming light by changing its spectral composition. These optical properties on a nano-scale can offer several advantages over current technologies for optical devices. Thus, diatom shells are of broad interests for many different biotechnological application but even more if we understand how these nanostructures are formed. We will then be able to create entirely novel technology with the help of natures knowledge build on million years of evolution. We already have done the first step into this direction by identifying which genes are involved in shell formation. We were able to select about 150 genes out of ca. 14,000 that only responded (turned on or off) by the availability of silicon in a diatom for which the whole genome sequence is available (Thalassiosira pseudonana). Now we know which of the organism's 14,000 genes are most likely to be involved in making the shells. However, the majority of these genes don't have a known function because they display no similarities to known genes from other organisms. We therefore apply modern molecular techniques to shed more light on the function of these unknown genes and also on those genes were we only have vague ideas about their function. We try to elucidate their function by genomic transformations and gene knockdown using RNA silencing technology. These data will significantly improve our knowledge about the genetic underpinnings of diatom silica shells and thus provide a first step towards a genetically controlled process for many different bionanotechnological applications.

SEM and GFP images of Thalassiosira pseudonana
a*) SEM of T. pseudonana silica shell; b*) translational fusion of newly identified silaffin-like protein (ID9558) with GFP; c*) translational fusion of protein with unknown function (ID9619) and GFP; d) extracted nuclear DNA from mutant in c). Arrows indicate girdle bands of silica shell and arrowheads indicate nuclear DNA. Red fluorescence results from chlorophyll a and green fluorescence from GFP. (unpublished data)
* Courtesy of: Nils Kröger, Nicole Poulsen (Georgia Tech, College of Engineering, Atlanta, USA)