Hervé Moreau

DNA libraries for sequencing the genome of Ostreococcus tauri (Chlorophyta, prasinophyceae): The smallest free-living eukaryotic cell

Ostreococcus tauri is a marine photosynthetic picoeukaryote presenting a minimal cellular organization with one nucleus, one chloroplast, and one mitochondrion. It has the smallest genome described among free‐living eukaryotic cells, and we showed by pulsed‐field gel electrophoresis (PFGE) that it is divided between 15 bands ranging from 1.2 to 0.15 Mb, giving a total size of 9.7 Mb. A Bacterial Artificial Chromosome (BAC) library was prepared from genomic DNA extracted from a culture of O. tauri. A total of 2457 clones was obtained with an average insert size of around 70 kb, representing an 18‐fold coverage of the genome. The library was spotted on high density filters, and several probes of coding sequences were hybridized to both the high density BAC library filters and directly to the dried PFGE gels of the O. tauri genomic DNA. These hybridizations allowed a preliminary organization of the library and the localization of several markers on the chromosomes. Randomly selected fragments were also sequenced, representing 12% of the O. tauri genome. Many sequences showed significant similarities in data banks, mainly with plant and algae sequences. About 1000 coding sequences could be identified. These data confirmed the position of O. tauri in the green lineage and the hypothesis of a very compact organization of its genome.

The first green lineage cdc25 dual-specificity phosphatase

The Cdc25 protein phosphatase is a key enzyme involved in the regulation of the G2/M transition in metazoans and yeast. However, no Cdc25 ortholog has so far been identified in plants, although functional studies have shown that an activating dephosphorylation of the CDK-cyclin complex regulates the G2/M transition. In this paper, the first green lineage Cdc25 ortholog is described in the unicellular alga Ostreococcus tauri. It encodes a protein which is able to rescue the yeast S. pombe cdc25-22 conditional mutant. Furthermore, microinjection of GST-tagged O. tauri Cdc25 specifically activates prophase-arrested starfish oocytes. In vitro histone H1 kinase assays and anti-phosphotyrosine Western Blotting confirmed the in vivo activating dephosphorylation of starfish CDK1-cyclinB by recombinant O. tauri Cdc25. We propose that there has been co-evolution of the regulatory proteins involved in the control of M-phase entry in the metazoan, yeast and green lineages. Link to supplemental material: http://www.landesbioscience.com/journals/cc/khadarooCC3-4-sup.pdf

Genomic insights into photosynthesis in eukaryotic phytoplankton.

The evolution of photosynthesis completely altered the biogeochemistry of our planet and permitted the evolution of more complex multicellular organisms. Curiously, terrestrial photosynthesis is carried out largely by green algae and their descendents the higher plants, whereas in the ocean the most abundant photosynthetic eukaryotes are microscopic and have red algal affiliations. Although primary productivity is approximately equal between the land and the ocean, the marine microbes represent less than 1% of the photosynthetic biomass found on land. This review focuses on this highly successful and diverse group of organisms collectively known as phytoplankton and reviews how insights from whole genome analyses have improved our understanding of the novel innovations employed by them to maximize photosynthetic efficiency in variable light environments.

Clues about the Genetic Basis of Adaptation Emerge from Comparing the Proteomes of Two Ostreococcus Ecotypes (Chlorophyta, Prasinophyceae)

We compared the proteomes of two picoplanktonic Ostreococcus unicellular green algal ecotypes to analyze the genetic basis of their adaptation with their ecological niches. We first investigated the function of the species-specific genes using Gene Ontology databases and similarity searches. Although most species-specific genes had no known function, we identified several species-specific functions involved in various cellular processes, which could be critical for environmental adaptations. Additionally, we investigated the rate of evolution of orthologous genes and its distribution across chromosomes. We show that faster evolving genes encode significantly more membrane or excreted proteins, consistent with the notion that selection acts on cell surface modifications that is driven by selection for resistance to viruses and grazers, keystone actors of phytoplankton evolution. The relationship between GC content and chromosome length also suggests that both strains have experienced recombination since their divergence and that lack of recombination on the two outlier chromosomes could explain part of their peculiar genomic features, including higher rates of evolution.

Diversity of Viruses Infecting the Green Microalga Ostreococcus lucimarinus

ABSTRACT The functional diversity of eukaryotic viruses infecting a single host strain from seawater samples originating from distant marine locations is unknown. To estimate this diversity, we used lysis plaque assays to detect viruses that infect the widespread species Ostreococcus lucimarinus, which is found in coastal and mesotrophic systems, and O. tauri, which was isolated from coastal and lagoon sites from the northwest Mediterranean Sea. Detection of viral lytic activities against O. tauri was not observed using seawater from most sites, except those close to the area where the host strain was isolated. In contrast, the more cosmopolitan O. lucimarinus species recovered viruses from locations in the Atlantic and Pacific Oceans and the Mediterranean Sea. Six new O. lucimarinus viruses (OlVs) then were characterized and their genomes sequenced. Two subgroups of OlVs were distinguished based on their genetic distances and on the inversion of a central 32-kb-long DNA fragment, but overall their genomes displayed a high level of synteny. The two groups did not correspond to proximity of isolation sites, and the phylogenetic distance between these subgroups was higher than the distances observed among viruses infecting O. tauri. Our study demonstrates that viruses originating from very distant sites are able to infect the same algal host strain and can be more diverse than those infecting different species of the same genus. Finally, distinctive features and evolutionary distances between these different viral subgroups does not appear to be linked to biogeography of the viral isolates. IMPORTANCE Marine eukaryotic phytoplankton virus diversity has yet to be addressed, and more specifically, it is unclear whether diversity is connected to geographical distance and whether differential infection and lysis patterns exist among such viruses that infect the same host strain. Here, we assessed the genetic distance of geographically segregated viruses that infect the ubiquitous green microalga Ostreococcus. This study provides the first glimpse into the diversity of predicted gene functions in Ostreococcus viruses originating from distant sites and provides new insights into potential host distributions and restrictions in the world oceans.

Role of nuclear WW domains and proline-rich proteins in dinoflagellate transcription.

Dinoflagellates are unique among eukaryotes in their lack of histones and nucleosomes, and permanently condensed chromosomes. These unusual features raise questions as how chromatin condensation and gene expression are achieved. In this study, we investigated nuclear proteins potentially implicated in the regulation of the transcription. Dinap1 is a dinoflagellate nuclear protein that has a WW domain and is synthesized mainly in G1 and S phases of the cell cycle. In this study, we found that Dip1, a proline-rich potential ligand of Dinap1, and DapC, a Dip1 potential ligand, were both present in the nucleus of Crypthecodinium cohnii during the G1 phase. Dip1 contained a PPXY motif, and its domain organization was similar to that of the splicing factor FBP21 in that it possessed one zinc finger and two WW domains. Although DapC has no known homolog, 22 repeats of a PPXPXGX heptapeptide were identified at the N-terminus, and this structure is similar to that of the C-terminal part of the mouse splicing factor SAP62. Dinap1 was co-precipitated with Dip1 and DapC in vitro and in vivo, but despite their nuclear location, these three proteins did not bind directly to DNA. Dinap1 activated up to 40% of the basal transcription activity of C. cohnii in an in vitro assay, whereas DapC inhibited it by 40% and Dip1 had no effect. These dinoflagellate proteins appear to be the subunits of a nuclear complex that may be involved in regulating transcription.

A Viral Immunity Chromosome in the Marine Picoeukaryote, Ostreococcus tauri

Micro-algae of the genus Ostreococcus and related species of the order Mamiellales are globally distributed in the photic zone of worlds oceans where they contribute to fixation of atmospheric carbon and production of oxygen, besides providing a primary source of nutrition in the food web. Their tiny size, simple cells, ease of culture, compact genomes and susceptibility to the most abundant large DNA viruses in the sea render them attractive as models for integrative marine biology. In culture, spontaneous resistance to viruses occurs frequently. Here, we show that virus-producing resistant cell lines arise in many independent cell lines during lytic infections, but over two years, more and more of these lines stop producing viruses. We observed sweeping over-expression of all genes in more than half of chromosome 19 in resistant lines, and karyotypic analyses showed physical rearrangements of this chromosome. Chromosome 19 has an unusual genetic structure whose equivalent is found in all of the sequenced genomes in this ecologically important group of green algae.

Host-derived viral transporter protein for nitrogen uptake in infected marine phytoplankton

Significance Viruses often carry genes acquired from their host. In the present work, we show that a virus of a marine alga carries a gene encoding a transporter protein that mediates nutrient uptake. We confirm that the viral transporter protein is expressed during infection and show that the protein functions to take up sources of nitrogen. This is important because acquisition of nutrients often determines the ecological success of phytoplankton populations. This work demonstrates how a virus can amend host–viral dynamics by modulating acquisition of nutrients from the environment. Phytoplankton community structure is shaped by both bottom–up factors, such as nutrient availability, and top–down processes, such as predation. Here we show that marine viruses can blur these distinctions, being able to amend how host cells acquire nutrients from their environment while also predating and lysing their algal hosts. Viral genomes often encode genes derived from their host. These genes may allow the virus to manipulate host metabolism to improve viral fitness. We identify in the genome of a phytoplankton virus, which infects the small green alga Ostreococcus tauri, a host-derived ammonium transporter. This gene is transcribed during infection and when expressed in yeast mutants the viral protein is located to the plasma membrane and rescues growth when cultured with ammonium as the sole nitrogen source. We also show that viral infection alters the nature of nitrogen compound uptake of host cells, by both increasing substrate affinity and allowing the host to access diverse nitrogen sources. This is important because the availability of nitrogen often limits phytoplankton growth. Collectively, these data show that a virus can acquire genes encoding nutrient transporters from a host genome and that expression of the viral gene can alter the nutrient uptake behavior of host cells. These results have implications for understanding how viruses manipulate the physiology and ecology of phytoplankton, influence marine nutrient cycles, and act as vectors for horizontal gene transfer.

Characterization of p80, a Novel Nuclear and Cytoplasmic Protein in Dinoflagellates

The presence of myosin in dinoflagellates was tested using an anti-Acanthamoeba castellanii myosin II polyclonal antibody on the heterotrophic dinoflagellate Crypthecodinium cohnii Seligo. Western blots revealed the presence of a unique band of 80 kDa in total protein extracts and after immunoprecipitation. Expression of this 80 kDa protein appeared constant during the different phases of the cell cycle. In protein extracts from various other dinoflagellates, this 80 kDa protein was detected only in the autotrophic species Prorocentrum micans Ehr. Screening of a C. cohnii cDNA expression library with this antibody revealed a cDNA coding for an amino acid sequence without homology in the databases. However, particular regions were detected: - a polyglutamine repeat domain in the N-terminal part of the protein, - four peptide sequences associated with GTP-binding sites, - a sequence with slight homology to the rod tail of Caenorhabditis elegans myosin II, -a sequence with homology to a human kinesin motor domain. Immunocytolocalization performed on C. cohnii thin sections with a polyclonal antibody raised against the recombinant protein showed p80 to be present both within the nucleus and in the cytoplasm. Labelling was widespread in the nucleoplasm and more concentrated at the periphery of the permanently condensed chromosomes. In the cytoplasm, labelling appeared in a punctate region close to the nucleus and in the flagellum. Potential functions of this novel protein are discussed.

Genomics of Marine Algae

The algae are an extremely diverse group of organisms from several different perspectives; including their phylogeny, their basic biology and biochemistry, the range of complexity they exhibit and their adaptation to a large number of different habitats. As a result, algal research touches on a broad spectrum of questions ranging from the importance of algae as key species in marine ecosystems to algae as a source of novel biomolecules and bioprocesses. Two key developments have accelerated research in this field over the past few years. The first is the application of high-throughput sequencing approaches both to whole genome sequencing and to metagenomic exploration of marine environments. The second is the emergence of model organisms in several of the less well studied algal lineages. These model organisms are important because they permit efficient application of genomic approaches to these groups. This chapter will describe how genomic research is providing new insights into many aspects of algal biology and will attempt to outline where this research is likely to lead in the future.