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Family A DNA polymerases (PolAs) form an important and well-studied class of extant polymerases participating in DNA replication and repair. Nonetheless, despite the characterization of multiple subfamilies in independent, dedicated works, their comprehensive classification thus far is missing. We therefore re-examine all presently available PolA sequences, converting their pairwise similarities into positions in Euclidean space, separating them into 19 major clusters. While 11 of them correspond to known subfamilies, eight had not been characterized before. For every group, we compile their general characteristics, examine their phylogenetic relationships and perform conservation analysis in the essential sequence motifs. While most subfamilies are linked to a particular domain of life (including phages), one subfamily appears in Bacteria, Archaea and Eukaryota. We also show that two new bacterial subfamilies contain functional enzymes. We use AlphaFold2 to generate high-confidence prediction models for all clusters lacking an experimentally determined structure. We identify new, conserved features involving structural alterations, ordered insertions and an apparent structural incorporation of a uracil-DNA glycosylase (UDG) domain. Finally, genetic and structural analyses of a subset of T7-like phages indicate a splitting of the 3′–5′ exo and pol domains into two separate genes, observed in PolAs for the first time.

International audience; The discovery of giant viruses, with capsids as large as some bacteria, megabase-range genomes and a variety of traits typically found only in cellular organisms, was one of the most remarkable breakthroughs in biology. Until recently, most of our knowledge of giant viruses came from ~100 species-level isolates for which genome sequences were available. However, these isolates were primarily derived from laboratory-based co-cultivation with few cultured protists and algae and, thus, did not reflect the true diversity of giant viruses. Although virus co-cultures enabled valuable insights into giant virus biology, many questions regarding their origin, evolution and ecological importance remain unanswered. With advances in sequencing technologies and bioinformatics, our understanding of giant viruses has drastically expanded. In this Review, we summarize our understanding of giant virus diversity and biology based on viral isolates as laboratory cultivation has enabled extensive insights into viral morphology and infection strategies. We then explore how cultivation-independent approaches have heightened our understanding of the coding potential and diversity of the Nucleocytoviricota. We discuss how metagenomics has revolutionized our perspective of giant viruses by revealing their distribution across our planet's biomes, where they impact the biology and ecology of a wide range of eukaryotic hosts and ultimately affect global nutrient cycles.

Legionella pneumophila is an intracellular bacterial pathogen that can cause a severe form of pneumonia in humans, a phenotype evolved through interactions with aquatic protozoa in the environment. Here, we show that L. pneumophila uses extracellular vesicles to translocate bacterial small RNAs (sRNAs) into host cells that act on host defence signalling pathways. The bacterial sRNA RsmY binds to the UTR of ddx58 (RIG-I encoding gene) and cRel, while tRNA-Phe binds ddx58 and irak1 collectively reducing expression of RIG-I, IRAK1 and cRel, with subsequent downregulation of IFN-β. Thus, RsmY and tRNA-Phe are bacterial trans-kingdom regulatory RNAs downregulating selected sensor and regulator proteins of the host cell innate immune response. This miRNA-like regulation of the expression of key sensors and regulators of immunity is a feature of L. pneumophila host-pathogen communication and likely represents a general mechanism employed by bacteria that interact with eukaryotic hosts.

International audience; One sentence summary: This review aims to dissect the evolutionary processes that may lead to an enrichment of eukaryotic-like proteins in environmental intracellular bacteria through continuous adaptation to multiple eukaryotic hosts. We reveal that chromatin and ubiquitin modulation functions, as well as adaptable tandem repeat domains, are crucial for their virulence in protozoa and accidentally also in humans.Intracellular pathogens that are able to thrive in different environments, such as Legionella spp. that preferentially live in protozoa in aquatic environments or environmental Chlamydiae that replicate either within protozoa or a range of animals, possess a plethora of cellular biology tools to influence their eukaryotic host. The host manipulation tools that evolved in the interaction with protozoa confer these bacteria the capacity to also infect phylogenetically distinct eukaryotic cells, such as macrophages, and thus they can also be human pathogens. To manipulate the host cell, bacteria use protein secretion systems and molecular effectors. Although these molecular effectors are encoded in bacteria, they are expressed and function in a eukaryotic context often mimicking or inhibiting eukaryotic proteins. Indeed, many of these effectors have eukaryotic-like domains. In this review, we propose that the main pathways that environmental intracellular bacteria need to subvert in order to establish the host eukaryotic cell as a replication niche are chromatin remodelling, ubiquitination signalling and modulation of protein–protein interactions via tandem repeat domains. We then provide mechanistic insight into how these proteins might have evolved. Finally, we highlight that in environmental intracellular bacteria the number of eukaryotic-like domains and proteins is considerably higher than in intracellular bacteria specialized to an isolated niche, such as obligate intracellular human pathogens. As mimics of eukaryotic proteins are critical components of host–pathogen interactions, this distribution of eukaryotic-like domains suggests that the environment has selected them.

The type 2 secretion system (T2SS) is present in some Gram-negative eubacteria and used to secrete proteins across the outer membrane. Here we report that certain representative heteroloboseans, jakobids, malawimonads and hemimastigotes unexpectedly possess homologues of core T2SS components. We show that at least some of them are present in mitochondria, and their behaviour in biochemical assays is consistent with the presence of a mitochondrial T2SS-derived system (miT2SS). We additionally identified 23 protein families co-occurring with miT2SS in eukaryotes. Seven of these proteins could be directly linked to the core miT2SS by functional data and/or sequence features, whereas others may represent different parts of a broader functional pathway, possibly also involving the peroxisome. Its distribution in eukaryotes and phylogenetic evidence together indicate that the miT2SS-centred pathway is an ancestral eukaryotic trait. Our findings thus have direct implications for the functional properties of the early mitochondrion., Bacteria use the type 2 secretion system to secrete enzymes and toxins across the outer membrane to the environment. Here the authors analyse the T2SS pathway in three protist lineages and suggest that the early mitochondrion may have been capable of secreting proteins into the cytosol.

International audience; In the long co-evolution of host-pathogen interaction, bacteria have developed sophisticated strategies to manipulate host cell mechanisms and reprogram host transcription. Targeting chromatin, mainly through post-translational modification (PTM) of histone proteins, is one strategy that has been revealed over the last decade. Indeed, histone modifications play a crucial role in regulating transcription during cell type and stimulus specific responses, making them good targets during infection. Therefore, the study of host-pathogen interactions provides breakthroughs in understanding virulence mechanisms, but also in host cell mechanisms. Although chromatin is regulated by DNA methylation, noncoding RNAs, and post-translational modifications of histones, most studies have concentrated on bacteria-induced histone modifications, which will be the focus of this review. We will discuss the different mechanisms used by bacteria to induce histone PTMs, whether it is through direct targeting of pathogen effector enzymes, or indirectly through modulation of cellular signaling cascade. We will summarize the concepts we learned in cell biology from exploring bacteria-triggered histone modifications, by focusing on the signaling cascades modified by bacteria, bacterial mimics of eukaryotic enzymes, and the novel histone marks imposed upon infection.

International audience; Since the discovery of c-di-GMP almost three decades ago, cyclic dinucleotides (CDNs) have emerged as widely used signaling molecules in most kingdoms of life. The family of second messengers now includes c-di-AMP and distinct versions of mixed cyclic GMP-AMP (cGAMP) compounds. In addition to these nucleotides, a vast number of proteins for the production and turnover of these molecules have been described, as well as effectors that translate the signals into physiological responses. The latter include, but are not limited to, mechanisms for adaptation and survival in prokaryotes, persistence and virulence of bacterial pathogens, and immune responses to viral and bacterial invasion in eukaryotes. In this review, we will focus on recent discoveries and emerging themes that illustrate the ubiquity and versatility of cyclic dinucleotide function at the transcriptional and post-translational levels and, in particular, on insights gained through mechanistic structure-function analyses.

International audience; Self-synthesizing transposons are the largest known transposable elements that encode their own DNA polymerases (DNAP). The Polinton/Maverick family of self-synthesizing transposons is widespread in eukaryotes and abundant in the genomes of some protists. In addition to the DNAP and a retrovirus-like integrase, most of the polintons encode homologs of the major and minor jelly-roll capsid proteins, DNA-packaging ATPase and capsid maturation protease. Therefore, polintons are predicted to alternate between the transposon and viral lifestyles although virion formation remains to be demonstrated. Polintons are related to a group of eukaryotic viruses known as virophages that parasitize on giant viruses of the family Mimiviridae and another recently identified putative family of polinton-like viruses (PLV) predicted to lead a similar, dual life style. Comparative genomic analysis of polintons, virophages, PLV and the other viruses with double-stranded (ds)DNA genomes infecting eukaryotes and prokaryotes suggests that the polintons evolved from bacterial tectiviruses and could have been the ancestors of a broad range of eukaryotic viruses including adenoviruses and members of the proposed order 'Megavirales' as well as linear cytoplasmic plasmids. Recently, a group of predicted self-synthesizing transposons was discovered also in prokaryotes. These elements, denoted casposons, encode a DNAP and a homolog of the CRISPR-associated Cas1 endonuclease that has an integrase activity but no capsid proteins. Thus, unlike polintons, casposons appear to be limited to the transposon life style although they could have evolved from viruses. The casposons are thought to have played a pivotal role in the origin of the prokaryotic adaptive immunity, giving rise to the adaptation module of the CRISPR-Cas systems.

Replicative DNA polymerases are nano-machines essential to life, which have evolved the ability to copy the genome with high fidelity and high processivity. In contrast with cellular transcriptases and ribosome machines, which evolved by accretion of complexity from a conserved catalytic core, no replicative DNA polymerase is universally conserved. Strikingly, four different families of DNA polymerases have evolved to perform DNA replication in the three domains of life. In Bacteria, the genome is replicated by DNA polymerases belonging to the A- and C-families. In Eukarya, genomic DNA is copied mainly by three distinct replicative DNA polymerases, Polα, Polδ, and Polε, which all belong to the B-family. Matters are more complicated in Archaea, which contain an unusual D-family DNA polymerase (PolD) in addition to PolB, a B-family replicative DNA polymerase that is homologous to the eukaryotic ones. PolD is a heterodimeric DNA polymerase present in all Archaea discovered so far, except Crenarchaea. While PolD is an essential replicative DNA polymerase, it is often underrepresented in the literature when the diversity of DNA polymerases is discussed. Recent structural studies have shown that the structures of both polymerase and proofreading active sites of PolD differ from other structurally characterized DNA polymerases, thereby extending the repertoire of folds known to perform DNA replication. This review aims to provide an updated structural classification of all replicative DNAPs and discuss their evolutionary relationships, both regarding the DNA polymerase and proofreading active sites.

International audience; Biodiversity monitoring is the standard for environmental impact assessment of anthropogenic activities. Several recent studies showed that high-throughput amplicon sequencing of environmental DNA (eDNA metabarcoding) could overcome many limitations of the traditional morphotaxonomy-based bioassessment. Recently, we demonstrated that supervised machine learning (SML) can be used to predict accurate biotic indices values from eDNA metabarcoding data, regardless of the taxonomic affiliation of the sequences. However, it is unknown to which extent the accuracy of such models depends on taxonomic resolution of molecular markers or how SML compares with metabarcoding approaches targeting well-established bioindicator species. In this study, we address these issues by training predictive models upon five different ribosomal bacterial and eukaryotic markers and measuring their performance to assess the environmental impact of marine aquaculture on independent data sets. Our results show that all tested markers are yielding accurate predictive models and that they all outperform the assessment relying solely on taxonomically assigned sequences. Remarkably, we did not find any significant difference in the performance of the models built using universal eukaryotic or prokaryotic markers. Using any molecular marker with a taxonomic range broad enough to comprise different potential bioindicator taxa, SML approach can overcome the limits of taxonomy-based eDNA bioassessment.

HAPLESS2 (HAP2) is a broadly conserved, gamete-expressed transmembrane protein that was shown recently to be structurally homologous to viral class II fusion proteins, which initiate fusion with host cells via insertion of fusion loops into the host membrane. However, the functional conformation of the HAP2 fusion loops has remained unknown, as the reported X-ray structure of Chlamydomonas reinhardtii HAP2 lacked this critical region. Here, we report a structure-guided alignment that reveals diversification of the proposed HAP2 fusion loops. Representative crystal structures show that in flowering plants, HAP2 has a single prominent fusion loop projecting an amphipathic helix at its apex, while in trypanosomes, three small nonpolar loops of HAP2 are poised to interact with the target membrane. A detailed structure-function analysis of the Arabidopsis HAP2 amphipathic fusion helix defines key residues that are essential for membrane insertion and for gamete fusion. Our study suggests that HAP2 may have evolved multiple modes of membrane insertion to accommodate the diversity of membrane environments it has encountered during eukaryotic evolution., Author summary The fusion of gamete plasma membranes is the fundamental cellular event that brings two parental cells together to form a new individual, yet we know surprisingly little about this process at the molecular level. HAPLESS 2 (HAP2) is a conserved sperm plasma membrane protein that is essential for gamete fusion in a diverse array of eukaryotes. It was recently shown to share a common ancestor with viral proteins that drive fusion of the viral envelope with host membranes, but its mechanism of action remained elusive, since the reported structure did not resolve the proposed membrane interaction surface. Here, we report two new HAP2 structures revealing that HAP2 has evolved diverse membrane interaction surfaces. In the flowering plants, HAP2 uses an amphipathic helix that presents nonpolar residues to the target membrane; in trypanosomes, the membrane interaction surface comprises three shallow nonpolar loops.

International audience; Viruses and other selfish genetic elements are dominant entities in the biosphere, with respect to both physical abundance and genetic diversity. Various selfish elements parasitize on all cellular life forms. The relative abundances of different classes of viruses are dramatically different between prokaryotes and eukaryotes. In prokaryotes, the great majority of viruses possess double-stranded (ds) DNA genomes, with a substantial minority of single-stranded (ss) DNA viruses and only limited presence of RNA viruses. In contrast, in eukaryotes, RNA viruses account for the majority of the virome diversity although ssDNA and dsDNA viruses are common as well. Phylogenomic analysis yields tangible clues for the origins of major classes of eukaryotic viruses and in particular their likely roots in prokaryotes. Specifically, the ancestral genome of positive-strand RNA viruses of eukaryotes might have been assembled de novo from genes derived from prokaryotic retroelements and bacteria although a primordial origin of this class of viruses cannot be ruled out. Different groups of double-stranded RNA viruses derive either from dsRNA bacteriophages or from positive-strand RNA viruses. The eukaryotic ssDNA viruses apparently evolved via a fusion of genes from prokaryotic rolling circle-replicating plasmids and positive-strand RNA viruses. Different families of eukaryotic dsDNA viruses appear to have originated from specific groups of bacteriophages on at least two independent occasions. Polintons, the largest known eukaryotic transposons, predicted to also form virus particles, most likely, were the evolutionary intermediates between bacterial tectiviruses and several groups of eukaryotic dsDNA viruses including the proposed order "Megavirales" that unites diverse families of large and giant viruses. Strikingly, evolution of all classes of eukaryotic viruses appears to have involved fusion between structural and replicative gene modules derived from different sources along with additional acquisitions of diverse genes.

International audience; What differentiates Mycobacterium abscessus from other saprophytic mycobacteria is the ability to resist phagocytosis by human macrophages and the ability to multiply inside such cells. These virulence traits render M. abscessus pathogenic, especially in vulnerable hosts with underlying structural lung disease, such as cystic fibrosis, bronchiectasis or tuberculosis. How patients become infected with M. abscessus remains unclear. Unlike many mycobacteria, M. abscessus is not found in the environment but might reside inside amoebae, environmental phagocytes that represent a potential reservoir for M. abscessus. Indeed, M. abscessus is resistant to amoebal phagocytosis and the intra-amoeba life seems to increase M. abscessus virulence in an experimental model of infection. However, little is known about M. abscessus virulence in itself. To decipher the genes conferring an advantage to M. abscessus intracellular life, a screening of a M. abscessus transposon mutant library was developed. In parallel, a method of RNA extraction from intracellular Mycobacteria after co-culture with amoebae was developed. This method was validated and allowed the sequencing of whole M. abscessus transcriptomes inside the cells; providing, for the first time, a global view on M. abscessus adaptation to intracellular life. Both approaches give us an insight into M. abscessus virulence factors that enable M. abscessus to colonize the airways in humans.

International audience; Virophages are satellite DNA viruses that depend for their replication on giant viruses of the family Mimiviridae. An evolutionary relationship exists between the virophages and Polintons, large self-synthesizing transposons that are wide spread in the genomes of diverse eukaryotes. Most of the Polintons encode homologs of major and minor icosahedral virus capsid proteins and accordingly are predicted to form virions. Additionally, metagenome analysis has led to the discovery of an expansive family of Polinton-like viruses (PLV) that are more distantly related to bona fide Polintons and virophages. Another group of giant virus parasites includes small, linear, double-stranded DNA elements called transpovirons. Recent in-depth comparative genomic analysis has yielded evidence of the origin of the PLV and the transpovirons from Polintons. Integration of virophage genomes into genomes of both giant viruses and protists has been demonstrated. Furthermore, in an experimental coinfection system that consisted of a protist host, a giant virus and an associated virophage, the virophage integrated into the host genome and, after activation of its expression by a superinfecting giant virus, served as an agent of adaptive immunity. There is a striking analogy between this mechanism and the CRISPR-Cas system of prokaryotic adaptive immunity. Taken together, these findings show that Polintons, PLV, virophages and transpovirons form a dynamic network of integrating mobile genetic elements that contribute to the cellular antivirus defense and host-virus coevolution.

The eocyte hypothesis, in which Eukarya emerged from within Archaea, has been boosted by the description of a new candidate archaeal phylum, “Lokiarchaeota”, from metagenomic data. Eukarya branch within Lokiarchaeota in a tree reconstructed from the concatenation of 36 universal proteins. However, individual phylogenies revealed that lokiarchaeal proteins sequences have different evolutionary histories. The individual markers phylogenies revealed at least two subsets of proteins, either supporting the Woese or the Eocyte tree of life. Strikingly, removal of a single protein, the elongation factor EF2, is sufficient to break the Eukaryotes-Lokiarchaea affiliation. Our analysis suggests that the three lokiarchaeal EF2 proteins have a chimeric organization that could be due to contamination and/or homologous recombination with patches of eukaryotic sequences. A robust phylogenetic analysis of RNA polymerases with a new dataset indicates that Lokiarchaeota and related phyla of the Asgard superphylum are sister group to Euryarchaeota, not to Eukarya, and supports the monophyly of Archaea with their rooting in the branch leading to Thaumarchaeota., Author summary Two scenarios have been proposed to describe the history of cellular life on our planet. For some authors, two lineages emerged from the last universal cellular ancestor, one leading to Bacteria, the other one leading to a common ancestor of Archaea and Eukarya (Woese’s hypothesis), while others suggest that Eukaryotes emerged from within an archaeal subgroup (eocyte hypothesis). This latter hypothesis has been boosted by the reconstruction of new archaeal genomes from environmental DNA. These analyses have suggested that eukaryotes originated from complex archaea, called Lokiarchaeota, the first described members of the recently proposed Asgard superphylum. Considering the importance of this question, we performed new analyses of the universal proteins from Lokiarchaea and realized that their affiliation to Eukaryotes was most probably due to different biases, including chimeric sequences and unequal rate of protein evolution. From our results, we suggest here that Lokiarchaea and close relatives are sister group to Euryarchaeota, not to Eukarya. Notably, we also show that the choices of the universal markers to include in one’s analysis will critically impact the scenario supported and that some markers as the RNA polymerase support the traditional Woese’s tree.

International audience; Viruses are the most abundant biological entities on earth and show remarkable diversity of genome sequences, replication and expression strategies, and virion structures. Evolutionary genomics of viruses revealed many unexpected connections but the general scenario(s) for the evolution of the virosphere remains a matter of intense debate among proponents of the cellular regression, escaped genes, and primordial virus world hypotheses. A comprehensive sequence and structure analysis of major virion proteins indicates that they evolved on about 20 independent occasions, and in some of these cases likely ancestors are identifiable among the proteins of cellular organisms. Virus genomes typically consist of distinct structural and replication modules that recombine frequently and can have different evolutionary trajectories. The present analysis suggests that, although the replication modules of at least some classes of viruses might descend from primordial selfish genetic elements, bona fide viruses evolved on multiple, independent occasions throughout the course of evolution by the recruitment of diverse host proteins that became major virion components.

Unlike superoxide dismutases (SODs), superoxide reductases (SORs) eliminate superoxide anion (O(2)(*-)) not through its dismutation, but via reduction to hydrogen peroxide (H(2)O(2)) in the presence of an electron donor. The microaerobic protist Giardia intestinalis, responsible for a common intestinal disease in humans, though lacking SOD and other canonical reactive oxygen species-detoxifying systems, is among the very few eukaryotes encoding a SOR yet identified. In this study, the recombinant SOR from Giardia (SOR(Gi)) was purified and characterized by pulse radiolysis and stopped-flow spectrophotometry. The protein, isolated in the reduced state, after oxidation by superoxide or hexachloroiridate(IV), yields a resting species (T(final)) with Fe(3+) ligated to glutamate or hydroxide depending on pH (apparent pK(a)=8.7). Although showing negligible SOD activity, reduced SOR(Gi) reacts with O(2)(*-) with a pH-independent second-order rate constant k(1)=1.0×10(9) M(-1) s(-1) and yields the ferric-(hydro)peroxo intermediate T(1); this in turn rapidly decays to the T(final) state with pH-dependent rates, without populating other detectable intermediates. Immunoblotting assays show that SOR(Gi) is expressed in the disease-causing trophozoite of Giardia. We propose that the superoxide-scavenging activity of SOR in Giardia may promote the survival of this air-sensitive parasite in the fairly aerobic proximal human small intestine during infection.

The origin of eukaryotes and their evolutionary relationship with the Archaea is a major biological question and the subject of intense debate. In the context of the classical view of the universal tree of life, the Archaea and the Eukarya have a common ancestor, the nature of which remains undetermined. Alternative views propose instead that the Eukarya evolved directly from a bona fide archaeal lineage. Several recent large-scale phylogenomic studies using an array of approaches are divided in supporting either one or the other scenario, despite analysing largely overlapping data sets of universal genes. We examine the reasons for such a lack of consensus and consider how alternative approaches may enable progress in answering this fascinating and as-yet-unresolved question.

International audience; In photosynthesis, electron transfer along the photosynthetic chain results in a vectorial transfer of protons from the stroma to the lumenal space of the thylakoids. This promotes the generation of an electrochemical proton gradient (Δμ(H)(+)), which comprises a gradient of electric potential (ΔΨ) and of proton concentration (ΔpH). The Δμ(H)(+) has a central role in the photosynthetic process, providing the energy source for ATP synthesis. It is also involved in many regulatory mechanisms. The ΔpH modulates the rate of electron transfer and triggers deexcitation of excess energy within the light harvesting complexes. The ΔΨ is required for metabolite and protein transport across the membranes. Its presence also induces a shift in the absorption spectra of some photosynthetic pigments, resulting in the so-called ElectroChromic Shift (ECS). In this review, we discuss the characteristic features of the ECS, and illustrate possible applications for the study of photosynthetic processes in vivo.

Aims: We performed a preliminary assessment of the eukaryotic 18S rDNA diversity present in finished drinking water samples from three different surface water treatment plants supplying water to the city of Paris (France). Methods and Results: A molecular analysis was performed on a sample from each site based on sequencing of PCR amplified and cloned 18S ribosomal RNA genes. Overall, the 18S rDNA sequences combined from all samples could be affiliated to the Amoebozoa (20·8% of the phylotypes), Ciliophora (25%), Metazoa (33·3%), Fungi (8·3%), Cercozoa (4·2%) and unclassified eukaryotes (8·3%) groups. Conclusions: The 18S rDNA sequences affiliated to the Amoebozoa, Ciliophora and Metazoa lineages were found to be the most abundant phylotypes observed in the drinking water samples. Phylotypes found to be present in two, or all three, samples (41·7% of the total) may represent groups with members adapted to drinking water treatment plant (DWTP) ecosystem conditions. Significance and Impact of the Study: This study shows that finished drinking water can contain 18S rDNA sequences representing a variety of eukaryotic taxa. Further research is needed to better characterize the eukaryotic biodiversity of DWTPs and the effects of the finished drinking water diversity on the downstream water distribution network.

Virus genomes are prone to extensive gene loss, gain, and exchange and share no universal genes. Therefore, in a broad-scale study of virus evolution, gene and genome network analyses can complement traditional phylogenetics. We performed an exhaustive comparative analysis of the genomes of double-stranded DNA (dsDNA) viruses by using the bipartite network approach and found a robust hierarchical modularity in the dsDNA virosphere. Bipartite networks consist of two classes of nodes, with nodes in one class, in this case genomes, being connected via nodes of the second class, in this case genes. Such a network can be partitioned into modules that combine nodes from both classes. The bipartite network of dsDNA viruses includes 19 modules that form 5 major and 3 minor supermodules. Of these modules, 11 include tailed bacteriophages, reflecting the diversity of this largest group of viruses. The module analysis quantitatively validates and refines previously proposed nontrivial evolutionary relationships. An expansive supermodule combines the large and giant viruses of the putative order “Megavirales” with diverse moderate-sized viruses and related mobile elements. All viruses in this supermodule share a distinct morphogenetic tool kit with a double jelly roll major capsid protein. Herpesviruses and tailed bacteriophages comprise another supermodule, held together by a distinct set of morphogenetic proteins centered on the HK97-like major capsid protein. Together, these two supermodules cover the great majority of currently known dsDNA viruses. We formally identify a set of 14 viral hallmark genes that comprise the hubs of the network and account for most of the intermodule connections., IMPORTANCE Viruses and related mobile genetic elements are the dominant biological entities on earth, but their evolution is not sufficiently understood and their classification is not adequately developed. The key reason is the characteristic high rate of virus evolution that involves not only sequence change but also extensive gene loss, gain, and exchange. Therefore, in the study of virus evolution on a large scale, traditional phylogenetic approaches have limited applicability and have to be complemented by gene and genome network analyses. We applied state-of-the art methods of such analysis to reveal robust hierarchical modularity in the genomes of double-stranded DNA viruses. Some of the identified modules combine highly diverse viruses infecting bacteria, archaea, and eukaryotes, in support of previous hypotheses on direct evolutionary relationships between viruses from the three domains of cellular life. We formally identify a set of 14 viral hallmark genes that hold together the genomic network.

Electron crystallography of the chloroplastic b6f complex allowed the calculation of projection maps of crystals negatively stained or embedded in glucose. This gives insights into the overall structure of the extra- and transmembrane domains of the complex. A comparison with the structure of the bc1 complex, the mitochondrial homologue of the b6f complex, suggests that the transmembrane domains of the two complexes are very similar, confirming the structural homology deduced from sequence analysis. On the other hand, the extramembrane organisation of the c-type cytochrome and of the Rieske protein seems quite different. Nevertheless, the same type of movement of the Rieske protein is observed in the b6f as in the bc1 complex upon the binding of the quinol analogue stigmatellin. Crystallographic data also suggest movements in the transmembrane domains of the b6f complex, which would be specific of the b6f complex. fl 2000 Elsevier Science B.V. All rights reserved.

Opinion; International audience; It has been claimed that giant DNA viruses represent a separate, fourth domain of life in addition to the domains of Bacteria, Archaea, and Eukarya. Such classification disregards fundamental differences between the two types of living entities - viruses and cells - and results in confusion and controversies in evolutionary scenarios. We highlight these problems and emphasize the importance of restricting the term 'domain' to the descendants of the last universal cellular ancestor (LUCA), based on the shared ribosome structure. We suggest tracing phylogeny of viruses along evolutionary lineages primarily defined by virion architectures and the structures of the major capsid proteins.

International audience; The replicon model has initiated a major research line in molecular biology: the study of DNA replication mechanisms. Until now, the majority of studies have focused on a limited set of model organisms, mainly from Bacteria or Opisthokont eukaryotes (human, yeasts) and a few viral systems. However, molecular evolutionists have shown that the living world is more complex and diverse than believed when the operon model was proposed. Comparison of DNA replication proteins in the three domains, Archaea, Bacteria, and Eukarya, have surprisingly revealed the existence of two distinct sets of non-homologous cellular DNA replication proteins, one in Bacteria and the other in Archaea and Eukarya, suggesting that the last universal common ancestor possibly still had an RNA genome. A major puzzle is the presence in eukaryotes of the unfaithful DNA polymerase alpha (Pol α) to prime Okazaki fragments. Interestingly, Pol α is specifically involved in telomere biosynthesis, and its absence in Archaea correlates with the absence of telomeres. The recent discovery of telomere-like GC quartets in eukaryotic replication origins suggests a link between Pol α and the overall organization of the eukaryotic chromosome. As previously proposed by Takemura, Pol α might have originated from a mobile element of viral origin that played a critical role in the emergence of the complex eukaryotic genomes. Notably, most large DNA viruses encode DNA replication proteins very divergent from their cellular counterparts. The diversity of viral replication machineries compared to cellular ones suggests that DNA and DNA replication mechanisms first originated and diversified in the ancient virosphere, possibly explaining why they are so many different types of replication machinerie.

International audience; While the basal transcription machinery in archaea is eukaryal-like, transcription factors in archaea and their viruses are usually related to bacterial transcription factors. Nevertheless, some of these organisms show predicted classical zinc fingers motifs of the C2H2 type, which are almost exclusively found in proteins of eukaryotes and most often associated with transcription regulators. In this work, we focused on the protein AFV1p06 from the hyperthermophilic archaeal virus AFV1. The sequence of the protein consists of the classical eukaryotic C2H2 motif with the fourth histidine coordinating zinc missing, as well as of N- and C-terminal extensions. We showed that the protein AFV1p06 binds zinc and solved its solution structure by NMR. AFV1p06 displays a zinc finger fold with a novel structure extension and disordered N- and C-termini. Structure calculations show that a glutamic acid residue that coordinates zinc replaces the fourth histidine of the C2H2 motif. Electromobility gel shift assays indicate that the protein binds to DNA with different affinities depending on the DNA sequence. AFV1p06 is the first experimentally characterised archaeal zinc finger protein with a DNA binding activity. The AFV1p06 protein family has homologues in diverse viruses of hyperthermophilic archaea. A phylogenetic analysis points out a common origin of archaeal and eukaryotic C2H2 zinc fingers.

International audience; Viruses are ubiquitous and can infect any of the three existing cellular lineages (Archaea, Bacteria and Eukarya). Despite the persisting negative public perception of these entities, scientists learnt how to domesticate some of them. The study of molecular mechanisms essential to the completion of viral cycles has greatly contributed to deciphering fundamental processes in biology. Nowadays, viruses have entered the biotechnological era and numerous applications have already been developed. Viral-derived tools are used to manipulate genetic information, detect, diagnose, control and cure infectious diseases, or even design new structural assemblies. With the recent advances in the field of metagenomics, an overwhelming amount of information on novel viruses has become available. As current tools have been historically developed from a limited number of viruses, the potential of discoveries from new archaeal, bacterial and eukaryotic viruses may be limited only by our understanding of the multiple facets of viral cycles.

International audience; Due to the environmentally harmful impact of tributyltin self-polishing paints, there is a critical need of more ecological alternatives. The aim of the present work is to study the joint-action of three molecules chosen in order to combine the two modes of prevention: chemical and physical repelling of biofouling. This "hybrid" system is principally dedicated to disturb durable settlement of microfouling. Each component was chosen according to its specific properties: chlorhexidine is a bisdiguanide antiseptic with antibacterial activity, zinc peroxide is an inorganic precursor of high instable entities which react with seawater to create hydrogen peroxide, Tween 85 is a non ionic surfactant disturbing interactions between colonizing organisms and surface. Obtained results highlighted the interest on mixing such molecules to get additive action on antifouling efficiency.

Legionella species are frequently detected in hot water systems, attached to the surface as a biofilm. In this work, the dynamics of Legionella spp. and diverse bacteria and eukarya associated together in the biofilm, coming from a pilot scale 1 system simulating a real hot water system, were investigated throughout 6 months after two successive heat shock treatments followed by three successive chemical treatments. Community structure was assessed by a fingerprint technique, single-strand conformation polymorphism (SSCP). In addition, the diversity and dynamics of Legionella and eukarya were investigated by small-subunit (SSU) ribosomal cloning and sequencing. Our results showed that pathogenic Legionella species remained after the heat shock and chemical treatments ( Legionella pneumophila and Legionella anisa , respectively). The biofilm was not removed, and the bacterial community structure was transitorily affected by the treatments. Moreover, several amoebae had been detected in the biofilm before treatments ( Thecamoebae sp., Vannella sp., and Hartmanella vermiformis ) and after the first heat shock treatment, but only H. vermiformis remained. However, another protozoan affiliated with Alveolata, which is known as a host cell for Legionella , dominated the eukaryal species after the second heat shock and chemical treatment tests. Therefore, effective Legionella disinfection may be dependent on the elimination of these important microbial components. We suggest that eradicating Legionella in hot water networks requires better study of bacterial and eukaryal species associated with Legionella in biofilms.

International audience; 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.

International audience; Understanding early evolution is a major challenge for the post-genomic era. A promising way to tackle this issue is to analyze the evolutionary history of key cellular systems through phylogenomic approaches. The current availability of genomic data from representatives of diverse lineages (especially eukaryotes), together with the ever growing number of proteomic characterizations now provides ample material to apply this type of analyses to trace back the origin and evolution of the three domains of life. Here, we have reconstructed the composition of the ancestral mitochondrial ribosome in the Last Eukaryotic Common Ancestor (LECA) and investigated its subsequent evolution in six major eukaryotic supergroups. We infer that LECA possessed a mitochondrial ribosome that was already much larger than its bacterial ancestor, with 19 additional specific proteins, indicating that a certain amount of time occurred between initial endosymbiosis at the origin of the mitochondrion and the diversification of present-day eukaryotic supergroups. Subsequently, mitochondrial ribosomes appear to have undergone a very dynamic evolutionary history in the different eukaryotic lineages, involving the loss of different sets of ribosomal protein-coding genes, their transfer to the host genome, as well as the acquisition of many novel components. This chaotic history for a such fundamental cellular machinery is puzzling, especially when compared to cytosolic, bacterial or chloroplastic ribosomes, which are much more stable. Intriguingly, archaeal ribosomes also show a very dynamic nature, with multiple independent losses among lineages.

oceanographic cruise MARNAUT; International audience; To extend comparative metagenomic analyses of the deep-sea, we produced metagenomic data by direct 454 pyrosequencing from bathypelagic plankton (1000m depth) and bottom sediment of the Sea of Marmara, the gateway between the Eastern Mediterranean and the Black Seas. Data from small subunit ribosomal RNA (SSU rRNA) gene libraries and direct pyrosequencing of the same samples indicated that Gamma- and Alpha-proteobacteria, followed by Bacteroidetes, dominated the bacterial fraction in Marmara deep-sea plankton, whereas Planctomycetes, Delta- and Gammaproteobacteria were the most abundant groups in high bacterial-diversity sediment. Group I Crenarchaeota/Thaumarchaeota dominated the archaeal plankton fraction, although group II and III Euryarchaeota were also present. Eukaryotes were highly diverse in SSU rRNA gene libraries, with group I (Duboscquellida) and II (Syndiniales) alveolates and Radiozoa dominating plankton, and Opisthokonta and Alveolates, sediment. However, eukaryotic sequences were scarce in pyrosequence data. Archaeal amo genes were abundant in plankton, suggesting that Marmara planktonic Thaumarchaeota are ammonia oxidizers. Genes involved in sulfate reduction, carbon monoxide oxidation, anammox and sulfatases were over-represented in sediment. Genome recruitment analyses showed that Alteromonas macleodii ‘surface ecotype', Pelagibacter ubique and Nitrosopumilus maritimus were highly represented in 1000 m-deep plankton. A comparative analysis of Marmara metagenomes with ALOHA deep-sea and surface plankton, whale carcasses, Peru subsurface sediment and soil metagenomes clustered deep-sea Marmara plankton with deep-ALOHA plankton and whale carcasses, likely because of the suboxic conditions in the deep Marmara water column. The Marmara sediment clustered with the soil metagenome, highlighting the common ecological role of both types of microbial communities in the degradation of organic matter and the completion of biogeochemical cycles.

Background The production of stable and soluble proteins is one of the most important steps prior to structural and functional studies of biological importance. We investigated the parallel production in a medium throughput strategy of genes coding for proteins from various marine organisms, using protocols that involved recombinatorial cloning, protein expression screening and batch purification. This strategy was applied in order to respond to the need for post-genomic validation of the recent success of a large number of marine genomic projects. Indeed, the upcoming challenge is to go beyond the bioinformatic data, since the bias introduced through the genomes of the so called model organisms leads to numerous proteins of unknown function in the still unexplored world of the oceanic organisms. Results We present here the results of expression tests for 192 targets using a 96-well plate format. Genes were PCR amplified and cloned in parallel into expression vectors pFO4 and pGEX-4T-1, in order to express proteins N-terminally fused to a six-histidine-tag and to a GST-tag, respectively. Small-scale expression and purification permitted isolation of 84 soluble proteins and 34 insoluble proteins, which could also be used in refolding assays. Selected examples of proteins expressed and purified to a larger scale are presented. Conclusions The objective of this program was to get around the bottlenecks of soluble, active protein expression and crystallization for post-genomic validation of a number of proteins that come from various marine organisms. Multiplying the constructions, vectors and targets treated in parallel is important for the success of a medium throughput strategy and considerably increases the chances to get rapid access to pure and soluble protein samples, needed for the subsequent biochemical characterizations. Our set up of a medium throughput strategy applied to genes from marine organisms had a mean success rate of 44% soluble protein expression from marine bacteria, archaea as well as eukaryotic organisms. This success rate compares favorably with other protein screening projects, particularly for eukaryotic proteins. Several purified targets have already formed the base for experiments aimed at post-genomic validation.

International audience; Eukaryotic pathogens (e.g. Plasmodium, Leishmania, Trypanosomes, etc.) are a major source of morbidity and mortality worldwide. In Africa, one of the most impacted continents, they cause millions of deaths and constitute an immense economic burden. While the genome sequence of several of these organisms is now available, the biological functions of more than half of their proteins are still unknown. This is a serious issue for bringing to the foreground the expected new therapeutic targets. In this context, the identification of protein domains is a key step to improve the functional annotation of the proteins. However, several domains are missed in eukaryotic pathogens because of the high phylogenetic distance of these organisms from the classical eukaryote models. We recently proposed a method, co-occurrence domain detection (CODD), that improves the sensitivity of Pfam domain detection by exploiting the tendency of domains to appear preferentially with a few other favorite domains in a protein. In this paper, we present EuPathDomains (http://www.atgc-montpellier.fr/EuPathDomains/), an extended database of protein domains belonging to ten major eukaryotic human pathogens. EuPathDomains gathers known and new domains detected by CODD, along with the associated confidence measurements and the GO annotations that can be deduced from the new domains. This database significantly extends the Pfam domain coverage of all selected genomes, by proposing new occurrences of domains as well as new domain families that have never been reported before. For example, with a false discovery rate lower than 20%, EuPathDomains increases the number of detected domains by 13% in Toxoplasma gondii genome and up to 28% in Cryptospordium parvum, and the total number of domain families by 10% in Plasmodium falciparum and up to 16% in C. parvum genome. The database can be queried by protein names, domain identifiers, Pfam or Interpro identifiers, or organisms, and should become a valuable resource to decipher the protein functions of eukaryotic pathogens.

International audience; Sulfatases form a major group of enzymes present in prokaryotes and eukaryotes. This class of hydrolases is unique in requiring essential post-translational modification of a critical active-site cysteinyl or seryl residue to C(alpha)-formylglycine (FGly). Herein, we report mechanistic investigations of a unique class of radical-S-adenosyl-L-methionine (AdoMet) enzymes, namely anaerobic sulfatase-maturating enzymes (anSMEs), which catalyze the oxidation of Cys-type and Ser-type sulfatases and possess three [4Fe-4S](2+,+) clusters. We were able to develop a reliable quantitative enzymatic assay that allowed the direct measurement of FGly production and AdoMet cleavage. The results demonstrate stoichiometric coupling of AdoMet cleavage and FGly formation using peptide substrates with cysteinyl or seryl active-site residues. Analytical and EPR studies of the reconstituted wild-type enzyme and cysteinyl cluster mutants indicate the presence of three almost isopotential [4Fe-4S](2+,+) clusters, each of which is required for the generation of FGly in vitro. More surprisingly, our data indicate that the two additional [4Fe-4S](2+,+) clusters are required to obtain efficient reductive cleavage of AdoMet, suggesting their involvement in the reduction of the radical AdoMet [4Fe-4S](2+,+) center. These results, in addition to the recent demonstration of direct abstraction by anSMEs of the C(beta) H-atom from the sulfatase active-site cysteinyl or seryl residue using a 5'-deoxyadenosyl radical, provide new insights into the mechanism of this new class of radical-AdoMet enzymes.

The data presented in this paper suggest that pheromone-induced receptor phosphorylation and internalization, but not actin-dependent directed secretion, are required to establish receptor polarity., In the best understood models of eukaryotic directional sensing, chemotactic cells maintain a uniform distribution of surface receptors even when responding to chemical gradients. The yeast pheromone receptor is also uniformly distributed on the plasma membrane of vegetative cells, but pheromone induces its polarization into “crescents” that cap the future mating projection. Here, we find that in pheromone-treated cells, receptor crescents are visible before detectable polarization of actin cables and that the receptor can polarize in the absence of actin-dependent directed secretion. Receptor internalization, in contrast, seems to be essential for the generation of receptor polarity, and mutations that deregulate this process confer dramatic defects in directional sensing. We also show that pheromone induces the internalization and subsequent polarization of the mating-specific Gα and Gβ proteins and that the changes in G protein localization depend on receptor internalization and receptor–Gα coupling. Our data suggest that the polarization of the receptor and its G protein precedes actin polarization and is important for gradient sensing. We propose that the establishment of receptor/G protein polarity depends on a novel mechanism involving differential internalization and that this serves to amplify the shallow gradient of activated receptor across the cell.

International audience; A transcription reaction relies on the specific recognition of cis-regulatory regions containing short DNA motifs. Such sequences are bound by transcription factors (TFs) involved in the recruitment, direct or not, of the transcriptional machinery. A eukaryotic genome can contain tens of genes encoding TFs that recognize very similar consensus DNA target sequences. In this review, we explore in a simple way how TFs coexpressed in the same cells and recognizing generic consensus sites with generic DNA-binding domains can achieve a specific modulation of target gene expression. We dissect the strategy followed by eukaryotes, which involves the formation of complex nucleoprotein structures involving many TFs and their cognate binding sites. This multiplicity of actors increases the effective length of the target DNA recognized by the TFs and might help paralogous TFs establish specific interactions. From this perspective, eukaryotic gene regulation implies the cooperation of several TFs, which is also the basis of information integration. Such cooperative TFs are likely to form a combinatorial partner code whose ultimate molecular hallmark is the assembly of enhanceosome-like structures ensuring the formation of an activation surface that is complementary to other coactivators and to the transcriptional machinery itself.

International audience; Eukaryotic/archaeal translation initiation factor 2 (e/aIF2) is a heterotrimeric GTPase that plays a key role in selection of the correct start codon on messenger RNA. This review integrates structural and functional data to discuss the involvement of the three subunits in initiator tRNA binding. A possible role of the peripheral subunits in modulating the guanine nucleotide cycle on the core subunit is also addressed.

International audience; The strategies organisms use to decode synonymous codons in cytosolic protein synthesis are not uniform. The complete isoacceptor tRNA repertoire and the type of modified nucleoside found at the wobble position 34 of their anticodons were analyzed in all kingdoms of life. This led to the identification of four main decoding strategies that are diversely used in Bacteria, Archaea and Eukarya. Many of the modern tRNA modification enzymes acting at position 34 of tRNAs are present only in specific domains and obviously have arisen late during evolution. In an evolutionary fine-tuning process, these enzymes must have played an essential role in the progressive introduction of new amino acids, and in the refinement and standardization of the canonical nuclear genetic code observed in all extant organisms (functional convergent evolutionary hypothesis).

Background Phylogenetic profiling encompasses an important set of methodologies for in silico high throughput inference of functional relationships between genes. The simplest profiles represent the distribution of gene presence-absence in a set of species as a sequence of 0's and 1's, and it is assumed that functionally related genes will have more similar profiles. The methodology has been successfully used in numerous studies of prokaryotic genomes, although its application in eukaryotes appears problematic, with reported low accuracy due to the complex genomic organization within this domain of life. Recently some groups have proposed an alternative approach based on the correlation of homologous gene group sizes, taking into account all potentially informative genetic events leading to a change in group size, regardless of whether they result in a de novo group gain or total gene group loss. Results We have compared the performance of classical presence-absence and group size based approaches using a large, diverse set of eukaryotic species. In contrast to most previous comparisons in Eukarya, we take into account the species phylogeny. We also compare the approaches using two different group categories, based on orthology and on domain-sharing. Our results confirm a limited overall performance of phylogenetic profiling in eukaryotes. Although group size based approaches initially showed an increase in performance for the domain-sharing based groups, this seems to be an overestimation due to a simplistic negative control dataset and the choice of null hypothesis rejection criteria. Conclusion Presence-absence profiling represents a more accurate classifier of related versus non-related profile pairs, when the profiles under consideration have enough information content. Group size based approaches provide a complementary means of detecting domain or family level co-evolution between groups that may be elusive to presence-absence profiling. Moreover positive correlation between co-evolution scores and functional links imply that these methods could be used to estimate functional distances between gene groups and to cluster them based on their functional relatedness. This study should have important implications for the future development and application of phylogenetic profiling methods, not only in eukaryotic, but also in prokaryotic datasets.

Background Recent advances in sequencing strategies make possible unprecedented depth and scale of sampling for molecular detection of microbial diversity. Two major paradigm-shifting discoveries include the detection of bacterial diversity that is one to two orders of magnitude greater than previous estimates, and the discovery of an exciting 'rare biosphere' of molecular signatures ('species') of poorly understood ecological significance. We applied a high-throughput parallel tag sequencing (454 sequencing) protocol adopted for eukaryotes to investigate protistan community complexity in two contrasting anoxic marine ecosystems (Framvaren Fjord, Norway; Cariaco deep-sea basin, Venezuela). Both sampling sites have previously been scrutinized for protistan diversity by traditional clone library construction and Sanger sequencing. By comparing these clone library data with 454 amplicon library data, we assess the efficiency of high-throughput tag sequencing strategies. We here present a novel, highly conservative bioinformatic analysis pipeline for the processing of large tag sequence data sets. Results The analyses of ca. 250,000 sequence reads revealed that the number of detected Operational Taxonomic Units (OTUs) far exceeded previous richness estimates from the same sites based on clone libraries and Sanger sequencing. More than 90% of this diversity was represented by OTUs with less than 10 sequence tags. We detected a substantial number of taxonomic groups like Apusozoa, Chrysomerophytes, Centroheliozoa, Eustigmatophytes, hyphochytriomycetes, Ichthyosporea, Oikomonads, Phaeothamniophytes, and rhodophytes which remained undetected by previous clone library-based diversity surveys of the sampling sites. The most important innovations in our newly developed bioinformatics pipeline employ (i) BLASTN with query parameters adjusted for highly variable domains and a complete database of public ribosomal RNA (rRNA) gene sequences for taxonomic assignments of tags; (ii) a clustering of tags at k differences (Levenshtein distance) with a newly developed algorithm enabling very fast OTU clustering for large tag sequence data sets; and (iii) a novel parsing procedure to combine the data from individual analyses. Conclusion Our data highlight the magnitude of the under-sampled 'protistan gap' in the eukaryotic tree of life. This study illustrates that our current understanding of the ecological complexity of protist communities, and of the global species richness and genome diversity of protists, is severely limited. Even though 454 pyrosequencing is not a panacea, it allows for more comprehensive insights into the diversity of protistan communities, and combined with appropriate statistical tools, enables improved ecological interpretations of the data and projections of global diversity.

International audience; Contrary to a widespread opinion, horizontal gene transfer (HGT) between distantly related microorganisms (such as Bacteria and Archaea) has not been demonstrated to occur on a large scale. Except for transfer of mobile elements between closely related organisms, most alleged HGT events reflect phylogenetic discrepancies that can be explained by a variety of artefacts or by the differential loss of paralogous gene copies either originally present in the Last Universal Common Ancestor (LUCA) to the three Domains (a sophisticated, genetically redundant and promiscuous community of protoeukaryotes), or created by duplications having occurred at later times. Besides, (i) there is no experimental evidence for the facile acquisition of foreign DNA between distant taxa and (ii) important biological constraints operate on the phenotypic success of genetic exchange at several levels, including protein-protein interactions involved in metabolic channelling; stable integration and expression of foreign DNA is, therefore, expected to require strong selection. Explaining phylogenetic discrepancies by artefacts or loss of paralogs does not eliminate difficulties in retracing species genealogy but maintains the picture of a universal tree of life, HGT between distant organisms being reduced to a trickle. We illustrate our thesis by the phylogenetic analysis of carbamoyltransferases, a family of paralogous proteins. Among higher eukaryotes HGT appears of limited scope except in asexual organisms. We suggest that meiotic sexuality (a hallmark of eukaryotes) emerged in the genetically redundant and protoeukaryotic LUCA as a molecular identity check providing a defence mechanism against the deleterious effects of HGT.

International audience; It is generally agreed that autotrophic organisms and especially phytoplanktonic species can be harmed by copper through its effect on photosystem. However, the impact of copper on other components of the pelagic food web, such as the microbial loop (autotrophic and heterotrophic picoplankton, pigmented and non-pigmented flagellates and ciliates) has received little attention. Indoor experiments were conducted to evaluate the direct and indirect effects of copper, supplied in the range of concentrations used to control cyanobacteria growth in ponds, on non-targeted organisms of natural microbial loop communities sampled in spring and summer. Two copper concentrations were tested (80microgL(-1) and 160microgL(-1) final concentrations), set, respectively, below and above the ligand binding capacity of the water samples. Both caused a significant decrease in the biomass and diversity of pigmented organisms (picophytoplankton and pigmented flagellates). Conversely, the heterotrophic bacterioplankton and the heterotrophic flagellates did not seem to be directly affected by either copper treatment in terms of biomass or diversity, according to the descriptor chosen. The ciliate biomass was significantly reduced with increasing copper concentrations, but differences in sensitivity appeared between spring and summer communities. Potential mixotrophic and nanoplanktorivorous ciliates appeared to be more sensitive to copper treatments than bacterivorous ciliates, suggesting a stronger direct and (or) indirect effect of copper on the former. Copper sulphate treatments had a significant restructuring effect on the microbial loop communities, resulting in a dominance of heterotrophic bacterioplankton among microbial microorganisms 27 days after the beginning of the treatment. The spring microbial communities exhibited a greater sensitivity than the summer communities with respect to their initial compositions.

The structure and dynamics of small eukaryotes (cells with a diameter less than 5 μm) were studied over two consecutive years in an oligomesotrophic lake (Lake Pavin in France). Water samples were collected at 5 and 30 m below the surface; when the lake was stratified, these depths corresponded to the epilimnion and hypolimnion. Changes in small-eukaryote structure were analyzed using terminal restriction fragment length polymorphism (T-RFLP) and cloning and sequencing of the 18S rRNA genes. Terminal restriction fragments from clones were used to reveal the dominant taxa in T-RFLP profiles of the environmental samples. Spumella -like cells (Chrysophyceae) did not dominate the small eukaryote community identified by molecular techniques in lacustrine ecosystems. Small eukaryotes appeared to be dominated by heterotrophic cells, particularly Cercozoa, which represented nearly half of the identified phylotypes, followed by the Fungi-LKM11 group (25%), choanoflagellates (10.3%) and Chrysophyceae (8.9%). Bicosoecida, Cryptophyta, and ciliates represented less than 9% of the community studied. No seasonal reproducibility in temporal evolution of the small-eukaryote community was observed from 1 year to the next. The T-RFLP patterns were related to bottom-up (resources) and top-down (grazing) variables using canonical correspondence analysis. The results showed a strong top-down regulation of small eukaryotes by zooplankton, more exactly, by cladocerans at 5 m and copepods at 30 m. Among bottom-up factors, temperature had a significant effect at both depths. The concentrations of nitrogenous nutrients and total phosphorus also had an effect on small-eukaryote dynamics at 5 m, whereas bacterial abundance and dissolved oxygen played a more important structuring role in the deeper zone.

International audience; Antibodies are characteristically induced in many parasitic infection processes. The class and subclass of the antibody response is instrumental because each isotype has a distinct biological function. It is thus crucially important for an infected individual to mount the most appropriate secondary antibody response--that is the response that has the best chance of clearing the infection and/or controlling disease. This represents a fundamental of vaccine strategies. Immuno-epidemiological surveys and in vitro models of antibody production have helped to understand some of the goals which should be achieved when designing antiparasitic vaccines.

International audience; Comparative electron microscopic studies of Collodictyon triciliatum and Diphylleia rotans (=Aulacomonas submarina) showed that they share a distinctive flagellar transitional zone and a very similar flagellar apparatus. In both species, the basic couple of basal bodies and flagella #1 and #2 are connected to the dorsal and ventral roots, respectively. Collodictyon triciliatum has two additional basal bodies and flagella, #3 and #4, situated on each side of the basic couple, each of which also bears a dorsal root. The horseshoe-shaped arrangement of dictyosomes, mitochondria with tubular cristae and the deep ventral groove are very similar to those of Diphylleia rotans. These two genera have very specific features and are placed in a new family, Collodictyonidae, distinct from other eukaryotic groups. Electron microscopic observation of mitotic telophase in Diphylleia rotans revealed two chromosomal masses, surrounded by the nuclear envelope, within the dividing parental nucleus, as in the telophase stage of the heliozoan Actinophrys and the helioflagellate Dimorpha. Spindle microtubules arise from several MTOCs outside the nucleus, and several microtubules penetrate within the dividing nucleus, via pores at the poles. This semi-open type of orthomitosis is reminiscent of that of actinophryids. The SSU rDNA sequence of Diphylleia rotans was compared with that of all the eukaryotic groups that have a slow-evolving rDNA. Diphylleia did not strongly assemble with any group and emerged in a very poorly resolved part of the eukaryotic phylogenetic tree.

H/ACA small nucleolar and Cajal body ribonucleoproteins (RNPs) function in site-specific pseudouridylation of eukaryotic rRNA and snRNA, rRNA processing, and vertebrate telomerase biogenesis. Nhp2, one of four essential protein components of eukaryotic H/ACA RNPs, forms a core trimer with the pseudouridylase Cbf5 and Nop10 that specifically binds to H/ACA RNAs. Crystal structures of archaeal H/ACA RNPs have revealed how the protein components interact with each other and with the H/ACA RNA. However, in place of Nhp2p, archaeal H/ACA RNPs contain L7Ae, which binds specifically to an RNA K-loop motif absent in eukaryotic H/ACA RNPs, while Nhp2 binds a broader range of RNA structures. We report solution NMR studies of S. cerevisiae Nhp2 (Nhp2p), which reveal that Nhp2p exhibits two major conformations in solution due to cis/trans isomerization of the evolutionarily conserved Pro83. The equivalent proline is in the cis conformation in all reported structures of L7Ae and other homologous proteins. Nhp2p has the expected α-β-α fold, but the solution structures of the major conformation of Nhp2p with trans Pro83 and of Nhp2p-S82W with cis Pro83 reveal that Pro83 cis/trans isomerization affects the positions of numerous residues at the Nop10- and RNA-binding interface. An S82W substitution, which stabilizes the cis conformation, also stabilizes the association of Nhp2p with H/ACA snoRNPs in vivo. We propose that Pro83 plays a key role in the assembly of the eukaryotic H/ACA RNP, with the cis conformation locking in a stable Cbf5-Nop10-Nhp2 ternary complex and positioning the protein backbone to interact with the H/ACA RNA.

International audience; The cytopharyngeal apparatus in the Nassulinid ciliates Nassula and Furgasonia is a highly specialized microtubular/filamentous organelle designed for ingestion of organisms such as filamentous bacteria. From studies on living cells, it was previously shown that this organelle, also called "feeding basket," guides the filamentous bacteria and manipulates them to some extent during the early steps of ingestion. This results in a complex sequence of movements where the basket is successively dilated and constricted in its upper part. Whereas some of these movements (dilation) seem to be intrinsic to the microtubular components of the basket, others (constriction) are believed to be mediated by contractile filamentous structures [Tucker, 1968: J. Cell Sci. 3:493-514]. In this study, we have used antibodies raised against ciliate centrins to demonstrate these proteins by Western blot and immunocytochemical methods in Nassula and Furgasonia. In both ciliates, a 20-kDa centrin immunoanalog was localized in the upper (contractile) part of the cytopharyngeal apparatus. Immunoelectron microscopy revealed that cytopharyngeal centrin is engaged in filamentous material, forming a sphincter-like structure possibly involved in the movements of contraction. Interestingly, physical links were noted between filaments labeled for centrin and cytopharyngeal microtubules. The mechanistic implications of these findings are discussed.

Human genes contain a 10 bp repeat of RR dinucleotides focused around the first nucleosome position suggesting a role in transcriptional control., Background The position of a nucleosome, both translational along the DNA molecule and rotational between the histone core and the DNA, is controlled by many factors, including the regular occurrence of specific dinucleotides with a period of approximately 10 bp, important for the rotational setting of the DNA around the histone octamer. Results We show that such a 10 bp periodic signal of purine-purine dinucleotides occurs in phase with the transcription start site (TSS) of human genes and is centered on the position of the first (+1) nucleosome downstream of the TSS. These data support a direct link between transcription and the rotational setting of the nucleosome. The periodic signal is most prevalent in genes that contain CpG islands that are expressed at low levels in a tissue-specific manner and are involved in the control of transcription. Conclusions These results, together with several lines of evidence from the recent literature, support a new model whereby the +1 nucleosome could be more efficiently disassembled from gene promoters by H3K56 acetylation marks if the periodic signal specifies an optimal rotational setting.