The Ectocarpus genome and the independent evolution of multicellularity in brown algae.

Brown algae (Phaeophyceae) are complex photosynthetic organisms with a very different evolutionary history to green plants, to which they are only distantly related. These seaweeds are the dominant species in rocky coastal ecosystems and they exhibit many interesting adaptations to these, often harsh, environments. Brown algae are also one of only a small number of eukaryotic lineages that have evolved complex multicellularity (Fig. 1). We report the 214 million base pair (Mbp) genome sequence of the filamentous seaweed Ectocarpus siliculosus (Dillwyn) Lyngbye, a model organism for brown algae, closely related to the kelps (Fig. 1). Genome features such as the presence of an extended set of light-harvesting and pigment biosynthesis genes and new metabolic processes such as halide metabolism help explain the ability of this organism to cope with the highly variable tidal environment. The evolution of multicellularity in this lineage is correlated with the presence of a rich array of signal transduction genes. Of particular interest is the presence of a family of receptor kinases, as the independent evolution of related molecules has been linked with the emergence of multicellularity in both the animal and green plant lineages. The Ectocarpus genome sequence represents an important step towards developing this organism as a model species, providing the possibility to combine genomic and genetic approaches to explore these and other aspects of brown algal biology further.

Genome structure and metabolic features in the red seaweed Chondrus crispus shed light on evolution of the Archaeplastida.

Red seaweeds are key components of coastal ecosystems and are economically important as food and as a source of gelling agents, but their genes and genomes have received little attention. Here we report the sequencing of the 105-Mbp genome of the florideophyte Chondrus crispus (Irish moss) and the annotation of the 9,606 genes. The genome features an unusual structure characterized by gene-dense regions surrounded by repeat-rich regions dominated by transposable elements. Despite its fairly large size, this genome shows features typical of compact genomes, e.g., on average only 0.3 introns per gene, short introns, low median distance between genes, small gene families, and no indication of large-scale genome duplication. The genome also gives insights into the metabolism of marine red algae and adaptations to the marine environment, including genes related to halogen metabolism, oxylipins, and multicellularity (microRNA processing and transcription factors). Particularly interesting are features related to carbohydrate metabolism, which include a minimalistic gene set for starch biosynthesis, the presence of cellulose synthases acquired before the primary endosymbiosis showing the polyphyly of cellulose synthesis in Archaeplastida, and cellulases absent in terrestrial plants as well as the occurrence of a mannosylglycerate synthase potentially originating from a marine bacterium. To explain the observations on genome structure and gene content, we propose an evolutionary scenario involving an ancestral red alga that was driven by early ecological forces to lose genes, introns, and intergenetic DNA; this loss was followed by an expansion of genome size as a consequence of activity of transposable elements.

The Phaeodactylum genome reveals the evolutionary history of diatom genomes.

Diatoms are photosynthetic secondary endosymbionts found throughout marine and freshwater environments, and are believed to be responsible for around one-fifth of the primary productivity on Earth. The genome sequence of the marine centric diatom Thalassiosira pseudonana was recently reported, revealing a wealth of information about diatom biology. Here we report the complete genome sequence of the pennate diatom Phaeodactylum tricornutum and compare it with that of T. pseudonana to clarify evolutionary origins, functional significance and ubiquity of these features throughout diatoms. In spite of the fact that the pennate and centric lineages have only been diverging for 90 million years, their genome structures are dramatically different and a substantial fraction of genes ( approximately 40%) are not shared by these representatives of the two lineages. Analysis of molecular divergence compared with yeasts and metazoans reveals rapid rates of gene diversification in diatoms. Contributing factors include selective gene family expansions, differential losses and gains of genes and introns, and differential mobilization of transposable elements. Most significantly, we document the presence of hundreds of genes from bacteria. More than 300 of these gene transfers are found in both diatoms, attesting to their ancient origins, and many are likely to provide novel possibilities for metabolite management and for perception of environmental signals. These findings go a long way towards explaining the incredible diversity and success of the diatoms in contemporary oceans.

Diatoms: self assembled silica nanostructures, and templates for bio/chemical sensors and biomimetic membranes.

In this review we highlight recent advances in the understanding of biosilica production, biomodification of diatom frustules and their subsequent applications in bio/chemical sensors, and as a model membrane for filtration and separation.

Whole-cell response of the pennate diatom Phaeodactylum tricornutum to iron starvation.

Marine primary productivity is iron (Fe)-limited in vast regions of the contemporary oceans, most notably the high nutrient low chlorophyll (HNLC) regions. Diatoms often form large blooms upon the relief of Fe limitation in HNLC regions despite their prebloom low cell density. Although Fe plays an important role in controlling diatom distribution, the mechanisms of Fe uptake and adaptation to low iron availability are largely unknown. Through a combination of nontargeted transcriptomic and metabolomic approaches, we have explored the biochemical strategies preferred by Phaeo dactylum tricornutum at growth-limiting levels of dissolved Fe. Processes carried out by components rich in Fe, such as photosynthesis, mitochondrial electron transport, and nitrate assimilation, were down-regulated. Our results show that this retrenchment is compensated by nitrogen (N) and carbon (C) reallocation from protein and carbohydrate degradation, adaptations to chlorophyll biosynthesis and pigment metabolism, removal of excess electrons by mitochondrial alternative oxidase (AOX) and non-photochemical quenching (NPQ), and augmented Fe-independent oxidative stress responses. Iron limitation leads to the elevated expression of at least three gene clusters absent from the Thalassiosira pseudonana genome that encode for components of iron capture and uptake mechanisms.

Plasticity and robustness of pattern formation in the model diatom Phaeodactylum tricornutum.

Understanding the morphogenesis of mineralized structures found in shells, bones, teeth, spicules and plant cell walls is difficult because of the complexities underlying biomineralization, and the requirement of accurate models for pattern formation. Here, we investigated the spatial and temporal development of siliceous structures found in a model diatom species, Phaeodactylum tricornutum, for which the entire genome has been sequenced and transformation is routine. Analyses of pattern formation revealed that the process of silicification starts from a 'pi-like' structure that controls the spatial organization of a sternum upon which regular instabilities are initiated and developed. Detailed analyses also demonstrate that morphogenesis of silica is nonuniform. We also tested the sensitivity of pattern formation to perturbation of proton pumps, and found that selective inhibitors of H(+)-V-ATPases affect silica biomineralization both quantitatively and qualitatively. Morphometric analyses of valves purified from isogenic populations of cells show that the morphometric noise of several traits is under exquisite regulation, explaining why the overall valve pattern is reproducibly maintained. Altogether our analyses demonstrate that silica morphogenesis is a robust but nonuniform process, and allow us to propose a model for the dynamic growth of materials within a spatially controlled geometry.

Biomimetic dual templating of silica by polysaccharide/protein assemblies.

The control of silica growth by living organisms such as diatoms is known to involve the templating effect of several biomolecules working concomitantly. However, until now, biomimetic studies involving model molecules have mainly been performed with single templates. We show here that the addition of two biopolymers, gelatin and alginic acid, to silicate solutions allows the formation of complex structures resulting from the combined templating effect of both components at different scales. Gelatin is able to activate silica formation resulting in hybrid aggregates at the nanoscale. Alginic acid does not interfere with silica condensation but is able to control silica morphology through the assembly of these gelatin-silica aggregates at the microscale. For all materials, calcination up to 700 degrees C degrades the polymer component of the hybrid material and opens macroporosity in the silica network. In parallel, the high thermal stability of gelatin allows a good preservation of initial silica nanoparticle size upon heating whereas a coarsening process is observed in the sole presence of alginate. These results correlate well with previous models of biosilicification and suggest that the use of multiple templates is a suitable approach to elaborate more complex silica architectures.

Influence of poly-L-lysine on the biomimetic growth of silica tubes in confined media.

Pore channels of polycarbonate membranes were recently used as biomimetic models to study the effect of confinement on silicate condensation, leading to the formation of silica tubes exhibiting a core-shell structure. In this work, we preimmobilized poly-L-lysine on the membrane pores, leading to modification of the tube shell formation process and variation in core particle size. These data strengthen previous assumptions on the role of confinement on silica growth, i.e., interfacial interactions and perturbation of the diffusion coefficient. They also suggest that this approach is suitable to investigate in more detail the contribution of confinement effects on silica biomineralization.

Sol-gel encapsulation extends diatom viability and reveals their silica dissolution capability.

Several strains of diatom exhibit a long-term viability in silica gels and demonstrate the ability to dissolve the silica in their surroundings.

Prospects in diatom research.

Diatoms are unicellular photosynthetic eukaryotes that play a major role in the global cycling of carbon and silicon. They are believed to have arisen from a secondary endosymbiotic event between two eukaryotes, a red alga and a flagellated heterotroph. Recent analysis of a diatom genome indeed reveals a 'mosaic' nature, with genes derived from plant, animal and bacterial lineages. Advances in molecular genomics are facilitating the use of diatom-specific genes or pathways for biotechnology. Another interest is in understanding the artistry of the amorphous silica shell and the underlying biomineralization process. Materials scientists and chemists are now exploiting diatoms to develop new biomimetic approaches and to create silicon-based microdevices with specific features.

Diatomics: toward diatom functional genomics.

For diatom biologists one of the most interesting research areas over the next years will be in linking mathematical models for pattern formation with information derived from molecular genetic, biochemical, and physiological studies. A major goal of this research is to exploit diatom proficiency in biogenic silica formation to develop strategies for bio-inspired nanofabrication of silicon based materials. Development of high-throughput methods for the functional analysis of diatom genes is a key step toward this goal. In this article we review the different techniques available to investigate gene and protein function in diatoms. Furthermore, to make diatom research as effective as possible the research community must address the question of which diatom species should be developed as a model. Choice of a diatom model organism should be made on the basis of several criteria, such as the ease of genetic manipulation, ecological relevance, or biomineralization capability. Phaeodactylum tricornutum is one of the principal three species that are candidates for such a model. For this species we have accomplished the first large-scale analysis of 12000 expressed sequence tags (ESTs) and have organized it in a queryable database, Phaeodactylum tricornutum database (PtDB). A summary of the functional analysis of this EST collection is presented, and genes of particular interest are highlighted.

Digital expression profiling of novel diatom transcripts provides insight into their biological functions.

BACKGROUND: Diatoms represent the predominant group of eukaryotic phytoplankton in the oceans and are responsible for around 20% of global photosynthesis. Two whole genome sequences are now available. Notwithstanding, our knowledge of diatom biology remains limited because only around half of their genes can be ascribed a function based onhomology-based methods. High throughput tools are needed, therefore, to associate functions with diatom-specific genes. RESULTS: We have performed a systematic analysis of 130,000 ESTs derived from Phaeodactylum tricornutum cells grown in 16 different conditions. These include different sources of nitrogen, different concentrations of carbon dioxide, silicate and iron, and abiotic stresses such as low temperature and low salinity. Based on unbiased statistical methods, we have catalogued transcripts with similar expression profiles and identified transcripts differentially expressed in response to specific treatments. Functional annotation of these transcripts provides insights into expression patterns of genes involved in various metabolic and regulatory pathways and into the roles of novel genes with unknown functions. Specific growth conditions could be associated with enhanced gene diversity, known gene product functions, and over-representation of novel transcripts. Comparative analysis of data from the other sequenced diatom, Thalassiosira pseudonana, helped identify several unique diatom genes that are specifically regulated under particular conditions, thus facilitating studies of gene function, genome annotation and the molecular basis of species diversity. CONCLUSIONS: The digital gene expression database represents a new resource for identifying candidate diatom-specific genes involved in processes of major ecological relevance.

New tools for labeling silica in living diatoms.

Silicon biomineralization is a widespread mechanism found in several kingdoms that concerns both unicellular and multicellular organisms. As a result of genomic and molecular tools, diatoms have emerged as a good model for biomineralization studies and have provided most of the current knowledge on this process. However, the number of techniques available to study its dynamics at the cellular level is still rather limited. Here, new probes were developed specifically to label the pre-existing or the newly synthesized silica frustule of several diatoms species. It is shown that the LysoTracker Yellow HCK-123, which can be used to visualize silica frustules with common filter sets, presents an enhanced signal-to-noise ratio and allows details of the frustules to be imaged without of the use of ionophores. It is also demonstrated that methoxysilane derivatives can be coupled to fluorescein-5-isothiocyanate (FITC) to preferentially label the silica components of living cells. The coupling of labeling procedures might help to address the challenging question of the process of frustule exocytosis.

Biomimetic growth of silica tubes in confined media.

Polymer membranes were used as biomimetic environments to study the effect of confinement on silica formation. Within membrane pores, silica tubes were formed, consisting of a dense silica shell incorporating nanoparticle aggregates. The shell structure does not depend on the membrane pore size, suggesting that its formation proceeds via interfacial interactions with the pore surface. In contrast, the size of primary nanoparticles within core aggregates is influenced by pore dimensions, indicating an effect of confinement on the diffusion-limited growth of silica. A parallel can be drawn with reported roles of confinement in biomineralization processes, providing a basis for future developments in biosilicification mimetic approaches and biofunctional nanomaterials design.

Unravelling the mechanism of RNA-polymerase forward motion by using mechanical force.

Polymerases form a class of enzymes that act as molecular motors as they move along their nucleic acid substrate during catalysis, incorporating nucleotide triphosphates at the end of the growing chain and consuming chemical energy. A debated issue is how the enzyme converts chemical energy into motion [J. Gelles and R. Landick, Cell 93, 13 (1998)]. In a single molecule assay, we studied how an opposing mechanical force affects the translocation rate of T7 RNA polymerase. Our measurements show that force acts as a competitive inhibitor of nucleotide binding. This result is interpreted in the context of possible models, and with respect to published crystal structures of T7 RNA polymerase. The transcribing complex appears to utilize only a small fraction of the energy of hydrolysis to perform mechanical work, with the remainder being converted to heat.

A mutation in T7 RNA polymerase that facilitates promoter clearance.

Like multisubunit RNA polymerases (RNAPs), T7 RNAP frequently releases its transcript over the initial 8-12 transcribed nucleotides, when it still contacts the promoter. This abortive cycling, which is most prominent with initial sequences that deviate from those of T7 late genes, eventually compromises productive transcription. Starting from an in vivo situation where transcription of a target gene by T7 RNAP is virtually abolished because of extensive abortive cycling, we have selected a mutation in RNAP that restores target gene expression. In vitro, this mutation (P266L) weakens promoter binding but markedly reduces abortive cycling over a variety of initial sequences by stabilizing the transcription complex at nucleotides 5-8. Other substitutions of P266 have similar effects. X-ray data show that during the transition from initial to elongation complex, the N-terminal region undergoes a major structural switch of which P266 constitutes one of the hinges. How the mutation might facilitate this switch is tentatively discussed. On the practical side, the mutation can significantly improve in vitro transcription, particularly from templates carrying unfavorable initial sequences.