Search

Large dsDNA viruses are involved in the population control of many globally distributed species of eukaryotic phytoplankton and have a prominent role in bloom termination. The genus Phaeocystis ( Haptophyta , Prymnesiophyceae ) includes several high-biomass-forming phytoplankton species, such as Phaeocystis globosa , the blooms of which occur mostly in the coastal zone of the North Atlantic and the North Sea. Here, we report the 459,984-bp-long genome sequence of P. globosa virus strain PgV-16T, encoding 434 proteins and eight tRNAs and, thus, the largest fully sequenced genome to date among viruses infecting algae. Surprisingly, PgV-16T exhibits no phylogenetic affinity with other viruses infecting microalgae (e.g., phycodnaviruses), including those infecting Emiliania huxleyi , another ubiquitous bloom-forming haptophyte. Rather, PgV-16T belongs to an emerging clade (the Megaviridae) clustering the viruses endowed with the largest known genomes, including Megavirus, Mimivirus (both infecting acanthamoeba), and a virus infecting the marine microflagellate grazer Cafeteria roenbergensis . Seventy-five percent of the best matches of PgV-16T–predicted proteins correspond to two viruses [Organic Lake phycodnavirus (OLPV)1 and OLPV2] from a hypersaline lake in Antarctica (Organic Lake), the hosts of which are unknown. As for OLPVs and other Megaviridae, the PgV-16T sequence data revealed the presence of a virophage-like genome. However, no virophage particle was detected in infected P. globosa cultures. The presence of many genes found only in Megaviridae in its genome and the presence of an associated virophage strongly suggest that PgV-16T shares a common ancestry with the largest known dsDNA viruses, the host range of which already encompasses the earliest diverging branches of domain Eukarya.

The recent discovery of giant viruses exhibiting double-stranded DNA genomes larger than a million base pairs, encoding more than a thousand proteins and packed in near micron-sized icosahedral particles, opened a new and unexpected chapter in virology. As of today, these giant viruses and their closest relatives of lesser dimensions infect unicellular eukaryotes found in aquatic environments, but belonging to a wide diversity of early branching phyla. This broad phylogenetic distribution of hosts is consistent with the hypothesis that giant viruses originated prior to the radiation of the eukaryotic domain and/or might have been involved in the partition of nuclear versus cytoplasmic functions in ancestral cells. The distinctive features of the known giant viruses, in particular the recurrent presence of components of the translation apparatus in their proteome, raise a number of fundamental questions about their origin, their mode of evolution, and the relationship they may entertain with other dsDNA viruses, the genome size of which exhibits the widest distribution among all biological entities, from less than 5 kb to more than 1.25 Mb (a ratio of 1:250). At a more conceptual level, the convergence between the discovery of increasingly reduced parasitic cellular organisms and that of giant viruses exhibiting a widening array of cellular-like functions may ultimately abolish the historical discontinuity between the viral and the cellular world.

The recent discovery of giant viruses exhibiting double-stranded DNA genomes larger than a million base pairs, encoding more than a thousand proteins and packed in near micron-sized icosahedral particles, opened a new and unexpected chapter in virology. As of today, these giant viruses and their closest relatives of lesser dimensions infect unicellular eukaryotes found in aquatic environments, but belonging to a wide diversity of early branching phyla. This broad phylogenetic distribution of hosts is consistent with the hypothesis that giant viruses originated prior to the radiation of the eukaryotic domain and/or might have been involved in the partition of nuclear versus cytoplasmic functions in ancestral cells. The distinctive features of the known giant viruses, in particular the recurrent presence of components of the translation apparatus in their proteome, raise a number of fundamental questions about their origin, their mode of evolution, and the relationship they may entertain with other dsDNA viruses, the genome size of which exhibits the widest distribution among all biological entities, from less than 5 kb to more than 1.25 Mb (a ratio of 1:250). At a more conceptual level, the convergence between the discovery of increasingly reduced parasitic cellular organisms and that of giant viruses exhibiting a widening array of cellular-like functions may ultimately abolish the historical discontinuity between the viral and the cellular world.

Zeus' Revenge Sediment-dwelling amoebae appear to have an unhappy affinity for huge viruses. Giant icosahedral Mimiviruses with genomes of the order of 1 megabase (Mb) were first identified in Acanthamoeba. Digging into antipodean sediments has once again been fruitful where Philippe et al. (p. 281 ; see the cover) discovered some enormous viruses in Acanthamoeba, visible by light microscopy and having genomes up to 2.5 Mb. The Pandoraviruses are phagocytosed by target cells and, after fusing with the phagosome membrane, their contents are released into the cytoplasm where they wreak terrible havoc on its nucleus. These viruses are encased into a tegument-like envelope and lack genes for capsid proteins, and there are no genes for protein translation, adenosine triphosphate generation, or binary fission—confirming their classification as viruses.

X chromosome inactivation in both mouse and human requires the presence of a cis acting locus, the X inactivation centre. This locus is thought to be involved in the initiation and spreading of the inactivation signal in early development. In order to increase our understanding of the mouse X inactivation centre, a 94 kb region immediately distal to the Xist gene has been sequenced and analysed for the presence of transcription units and/or potential cis acting regulatory elements. We have identified a novel gene, Tsx, lying 40 kb 3' from Xist. Tsx is expressed specifically in the testis and shows no convincing homology to proteins currently in the databases. A rat homologue, also X linked, has been isolated. The mouse and rat Tsx sequences are highly divergent, suggesting that part of the X inactivation centre, including both Xist and Tsx are subject to relatively weak evolutionary constraints.

International audience; Phydbac is a web interactive resource based on phylogenomic profiling, designed to help microbiologists to annotate bacterial proteins. Phylogenomic annotation is based on the assumption that functionally linked protein-coding genes must evolve in a coordinated manner. The detection of subsets of co-evolving genes within a given genome involves the computation of protein sequence conservation profiles across a spectrum of microbial species, followed by the identification of significant pairwise correlations between them. Many ongoing studies are devoted to the problem of computing the most biologically significant phylogenomic profiles and how best identifying clusters of 'functionally interacting' genes. Here we introduce a web tool, Phydbac, allowing the dynamic construction of phylogenomic profiles of protein sequences of interest and their interactive display. In addition, Phydbac can identify Escherichia coli proteins exhibiting the evolution pattern most similar to arbitrary query protein sequences, hence providing functional hints for open reading frames (ORFs) of hypothetical or unknown function. The phylogenomic profiles of all E.coli K-12 protein-coding genes are pre-computed, allowing queries about E.coli genes to be answered instantaneously. The profiles and phylogenomic neighborhoods are computed using an original method shown to perform better than previous ones. An extension of Phydbac, including precomputed profiles for all available bacterial genomes (including major pathogens) will soon be available. Phydbac can be accessed at: http://igs-server.cnrs-mrs.fr/phydbac/.

We recently described Megavirus chilensis, a giant virus isolated off the coast of Chile, also replicating in fresh water acanthamoeba. Its 1,259,197-bp genome encodes 1,120 proteins and is the largest known viral genome. Megavirus and its closest relative Mimivirus only share 594 orthologous genes, themselves sharing only 50% of identical residues in average. Despite this divergence, comparable to the maximal divergence exhibited by bacteria within the same division (e.g., gamma proteobacteria), Megavirus retained all of the genomic features unique to Mimivirus, in particular its genes encoding key-elements of the translation apparatus, a trademark of cellular organisms. Besides homologs to the four aminoacyl-tRNA synthetases (aaRS) encoded by Mimivirus, Megavirus added three additional ones, raising the total of known virus-encoded aaRS to seven: IleRS, TrpRS, AsnRS, ArgRS, CysRS, MetRS, TyrRS. This finding strongly suggests that large DNA viruses derived from an ancestral cellular genome by reductive evolution. The nature of this cellular ancestor remains hotly debated.

In this chapter we quickly recapitulate the short history (since 2003) of the giant viruses, the discovery and the progressive characterization of which are deeply shaking the foundation of virology. In the mind of most biologists today, a “virus” remains the most reduced and optimized vehicle to propagate a nucleic acid molecule at the expense of a cellular host, an ultimate parasite at the frontier of (or beyond) the living world. With genome sizes and gene contents larger than many bacteria, as well as particle sizes of the order of half a micron, Mimivirus and Megavirus, collectively referred to as “Megaviridae”, have now clearly made the point that being small and simple should no longer be considered fundamental properties of viruses, nor a testimony to their evolutionary origin. Given what we already know, and what we can reasonably expect from future discoveries, this chapter is exploring which feature, if any, might still provide an absolute criterion to discriminate the most complex viruses from the most reduced parasitic cellular microorganisms.

Mimivirus is one the largest DNA virus identified so far, infecting several Acanthamoeba species. Analysis of its genome revealed the presence of a nine-gene cluster containing genes potentially involved in glycan formation. All of these genes are co-expressed at late stages of infection, suggesting their role in the formation of the long fibers covering the viral surface. Among them, we identified the L136 gene as a pyridoxal phosphate-dependent sugar aminotransferase. This enzyme was shown to catalyze the formation of UDP-4-amino-4,6-dideoxy-D-glucose (UDP-viosamine) from UDP-4-keto-6-deoxy-D-glucose, a key compound involved also in the biosynthesis of L-rhamnose. This finding further supports the hypothesis that Mimivirus encodes a glycosylation system that is completely independent of the amoebal host. Viosamine, together with rhamnose, (N-acetyl)glucosamine, and glucose, was found as a major component of the viral glycans. Most of the sugars were associated with the fibers, confirming a capsular-like nature of the viral surface. Phylogenetic analysis clearly indicated that L136 was not a recent acquisition from bacteria through horizontal gene transfer, but it was acquired very early during evolution. Implications for the origin of the glycosylation machinery in giant DNA virus are also discussed.

The random (shotgun) DNA sequencing strategy is used for most large-scale sequencing projects, including the identification of human disease genes after positional cloning. The principle of the method--sequence assembly from overlap--requires the candidate gene region to be partitioned into 15- to 20-kb pieces (usually λ inserts), themselves randomly subcloned into M13 prior to sequencing with a 6- to 8-fold redundancy. Most often, a time-consuming directed strategy must be invoked to close the remaining gaps. Ultimately, computer-based methods are invoked to locate putative coding exons within the finished genomic sequence. Given the small average size of vertebrate exons, I show here that they can be detected from the computer analysis of the individual runs, much before completion of contiguity. However, the successful assessment of coding potential from the raw data depends on a combination of new sequence masking techniques. When the identification of coding exons is the primary goal, the usual random sequencing strategy can thus be greatly optimized. The streamlined approach requires only a 2- to 2.5-fold sequencing redundancy, can dispense with the subcloning in λ and the closure of gaps, and can be fully automated. The feasibility of this strategy is demonstrated using data from the X-linked Kallmann syndrome gene region.

Single-particle experiments using X-ray Free Electron Lasers produce more than 10(5) snapshots per hour, consisting of an admixture of blank shots (no particle intercepted), and exposures of one or more particles. Experimental data sets also often contain unintentional contamination with different species. We present an unsupervised method able to sort experimental snapshots without recourse to templates, specific noise models, or user-directed learning. The results show 90% agreement with manual classification.

Background Since the discovery of the Malta fever agent, Brucella melitensis, in the 19th century, six terrestrial mammal-associated Brucella species were recognized over the next century. More recently the number of novel Brucella species has increased and among them, isolation of species B. pinnipedialis and B. ceti from marine mammals raised many questions about their origin as well as on the evolutionary history of the whole genus. Results We report here on the first complete genome sequence of a Brucella strain isolated from marine mammals, Brucella pinnipedialis strain B2/94. A whole gene-based phylogenetic analysis shows that five main groups of host-associated Brucella species rapidly diverged from a likely free-living ancestor close to the recently isolated B. microti. However, this tree lacks the resolution required to resolve the order of divergence of those groups. Comparative analyses focusing on a) genome segments unshared between B. microti and B. pinnipedialis, b) gene deletion/fusion events and c) positions and numbers of Brucella specific IS711 elements in the available Brucella genomes provided enough information to propose a branching order for those five groups. Conclusions In this study, it appears that the closest relatives of marine mammal Brucella sp. are B. ovis and Brucella sp. NVSL 07-0026 isolated from a baboon, followed by B. melitensis and B. abortus strains, and finally the group consisting of B. suis strains, including B. canis and the group consisting of the single B. neotomae species. We were not able, however, to resolve the order of divergence of the two latter groups.

The start-up of the Linac Coherent Light Source (LCLS), the new femtosecond hard X-ray laser facility in Stanford, California, has brought high expectations of a new era for biological imaging. The intense, ultrashort X-ray pulses allow diffraction imaging of small structures before radiation damage occurs. Two papers in this issue of Nature present proof-of-concept experiments showing the LCLS in action. Chapman et al. tackle structure determination from nanocrystals of macromolecules that cannot be grown in large crystals. They obtain more than three million diffraction patterns from a stream of nanocrystals of the membrane protein photosystem I, and assemble a three-dimensional data set for this protein. Seibert et al. obtain images of a non-crystalline biological sample, mimivirus, by injecting a beam of cooled mimivirus particles into the X-ray beam. The start-up of the new femtosecond hard X-ray laser facility in Stanford, the Linac Coherent Light Source, has brought high expectations for a new era for biological imaging. The intense, ultrashort X-ray pulses allow diffraction imaging of small structures before radiation damage occurs. This new capability is tested for the problem of imaging a non-crystalline biological sample. Images of mimivirus are obtained, the largest known virus with a total diameter of about 0.75 micrometres, by injecting a beam of cooled mimivirus particles into the X-ray beam. The measurements indicate no damage during imaging and prove the concept of this imaging technique. X-ray lasers offer new capabilities in understanding the structure of biological systems, complex materials and matter under extreme conditions1,2,3,4. Very short and extremely bright, coherent X-ray pulses can be used to outrun key damage processes and obtain a single diffraction pattern from a large macromolecule, a virus or a cell before the sample explodes and turns into plasma1. The continuous diffraction pattern of non-crystalline objects permits oversampling and direct phase retrieval2. Here we show that high-quality diffraction data can be obtained with a single X-ray pulse from a non-crystalline biological sample, a single mimivirus particle, which was injected into the pulsed beam of a hard-X-ray free-electron laser, the Linac Coherent Light Source5. Calculations indicate that the energy deposited into the virus by the pulse heated the particle to over 100,000 K after the pulse had left the sample. The reconstructed exit wavefront (image) yielded 32-nm full-period resolution in a single exposure and showed no measurable damage. The reconstruction indicates inhomogeneous arrangement of dense material inside the virion. We expect that significantly higher resolutions will be achieved in such experiments with shorter and brighter photon pulses focused to a smaller area. The resolution in such experiments can be further extended for samples available in multiple identical copies.

MutS proteins are ubiquitous in cellular organisms and have important roles in DNA mismatch repair or recombination. In the virus world, the amoeba-infecting Mimivirus, as well as the recently sequenced Cafeteria roenbergensis virus are known to encode a MutS related to the homologs found in octocorals and e-proteobacteria. To explore the presence of MutS proteins in other viral genomes, we performed a genomic survey of four giant viruses (‘giruses’) (Pyramimonas orientalis virus (PoV), Phaeocystis pouchetii virus (PpV), Chrysochromulina ericina virus (CeV) and Heterocapsa circularisquama DNA virus (HcDNAV)) that infect unicellular marine algae. Our analysis revealed the presence of a close homolog of Mimivirus MutS in all the analyzed giruses. These viral homologs possess a specific domain structure, including a C-terminal HNH-endonuclease domain, defining the new MutS7 subfamily. We confirmed the presence of conserved mismatch recognition residues in all members of the MutS7 subfamily, suggesting their role in DNA mismatch repair rather than DNA recombination. PoV and PpV were found to contain an additional type of MutS, which we propose to call MutS8. The MutS8 proteins in PoV and PpV were found to be closely related to homologs from ‘Candidatus Amoebophilus asiaticus’, an obligate intracellular amoeba-symbiont belonging to the Bacteroidetes. Furthermore, our analysis revealed that MutS7 and MutS8 are abundant in marine microbial metagenomes and that a vast majority of these environmental sequences are likely of girus origin. Giruses thus seem to represent a major source of the underexplored diversity of the MutS family in the microbial world.

Eric Karsenti et al.-- 5 pages, 2 figures, With biology becoming quantitative, systems-level studies can now be performed at spatial scales ranging from molecules to ecosystems. Biological data generated consistently across scales can be integrated with physico-chemical contextual data for a truly holistic approach, with a profound impact on our understanding of life [1]–[5]. Marine ecosystems are crucial in the regulation of Earth's biogeochemical cycles and climate [6],[7]. Yet their organization, evolution, and dynamics remain poorly understood [8],[9]. The Tara Oceans project was launched in September 2009 for a 3-year study of the global ocean ecosystem aboard the ship Tara. A unique sampling programme encompassing optical and genomic methods to describe viruses, bacteria, archaea, protists, and metazoans in their physico-chemical environment has been implemented. Starting as a grassroots initiative of a few scientists, the project has grown into a global consortium of over 100 specialists from diverse disciplines, including oceanography, microbial ecology, genomics, molecular, cellular, and systems biology, taxonomy, bioinformatics, data management, and ecosystem modeling. This multidisciplinary community aims to generate systematic, open access datasets usable for probing the morphological and molecular makeup, diversity, evolution, ecology, and global impacts of plankton on the Earth system, We are keen to thank the commitment of the following people and sponsors who made this singular expedition possible: CNRS, EMBL, Genoscope/CEA, VIB, Stazione Zoologica Anton Dohrn, UNIMIB, ANR (projects POSEIDON, BIOMARKS, PROMETHEUS, and TARA-GIRUS), FWO, BIO5, Biosphere 2, agnès b., the Veolia Environment Foundation, Region Bretagne, World Courier, Illumina, Cap L'Orient, the EDF Foundation EDF Diversiterre, FRB, the Prince Albert II de Monaco Foundation, Etienne Bourgois, the Tara schooner and its captain and crew. Tara Oceans would not exist without the continuous support of the participating 23 institutes (see http://oceans.taraexpeditions.org). Special thanks also goes to Fabrice Not and Noan Le Bescot for creating the figures of this manuscript. This is contribution number 0001 of the Tara Oceans Expedition 2009–2012

Sets of new gene sequences from human, nematode, and yeast were compared with each other and with a set of Escherichia coli genes in order to detect ancient evolutionarily conserved regions (ACRs) in the encoded proteins. Nearly all of the ACRs so identified were found to be homologous to sequences in the protein databases. This suggests that currently known proteins may already include representatives of most ACRs and that new sequences not similar to any database sequence are unlikely to contain ACRs. Preliminary analyses indicate that moderately expressed genes may be more likely to contain ACRs than rarely expressed genes. It is estimated that there are fewer than 900 ACRs in all.

We have conducted a detailed structural analysis of 90 kilobases (kb) of the HLA Class III region from the Bat2 gene at the centromeric end to 23 kb beyond TNF. A single contig of 80 kb was sequenced entirely with a group of four smaller contigs covering 10 kb being only partly sequenced. This region contains four known genes and a novel telomeric potential coding region. The genes are bracketed by long, dense clusters of Alu repeats belonging to all the major families. At least six new families of MER repeats and one pseudogene are intercalated within and between the Alu clusters. The most telomeric 3.8 kb contains three potential exons, one of which bears strong homology to the ankyrin domain of the DNA binding factors NF kappa B and I kappa B.

The complete assembly of the entire human genome sequence by Venter et al. confirms recent estimates that the total number of human protein coding genes might be less than 30,000--a mere one-third increase over the nematode Caenorhabditis elegans. Such a low gene number constitutes a paradigm change, which could drastically modify our understanding of organism complexity and evolution, as well as our current interpretation of transcriptome analyses. It may also have severe consequences on the long-term sustainability of the biomedical industry in the postgenomic era.

Background The right sampling of homologous sequences for phylogenetic or molecular evolution analyses is a crucial step, the quality of which can have a significant impact on the final interpretation of the study. There is no single way for constructing datasets suitable for phylogenetic analysis, because this task intimately depends on the scientific question we want to address, Moreover, database mining softwares such as BLAST which are routinely used for searching homologous sequences are not specifically optimized for this task. Results To fill this gap, we designed BLAST-Explorer, an original and friendly web-based application that combines a BLAST search with a suite of tools that allows interactive, phylogenetic-oriented exploration of the BLAST results and flexible selection of homologous sequences among the BLAST hits. Once the selection of the BLAST hits is done using BLAST-Explorer, the corresponding sequence can be imported locally for external analysis or passed to the phylogenetic tree reconstruction pipelines available on the Phylogeny.fr platform. Conclusions BLAST-Explorer provides a simple, intuitive and interactive graphical representation of the BLAST results and allows selection and retrieving of the BLAST hit sequences based a wide range of criterions. Although BLAST-Explorer primarily aims at helping the construction of sequence datasets for further phylogenetic study, it can also be used as a standard BLAST server with enriched output. BLAST-Explorer is available at http://www.phylogeny.fr

Viruses are the most abundant biological entities in the sea. Infecting marine organisms from oxygen-producing phytoplankton to whales, viruses regulate the populations of many marine organisms and play important roles in global biogeochemical fluxes. Thanks to the recent improvement of sequencing technology, billions of bases of sequences from marine microbial communities are being determined by different groups of researchers.

It is quite ironic that the uncertainty about the number of human genes (28,000–120,000) (Ewing and Green 2000; Liang et al. 2000; Roest Crollius et al. 2000) appears to increase as the determination of the human genome sequence is nearing completion. I shall contend here that this paradox reveals deep epistemological problems, and that “bioinformatics”—a term coined in 1990 to define the use of computers in sequence analysis—is no longer developing in directions relevant to biology. After the pioneers who established the basic concepts of molecular sequence analysis (Fitch and Margoliash 1967; Needleman and Wunsch 1970; Chou and Fasman 1974), most computational biologists of my generation (the second one) embarked on their journey into the emerging discipline with the ambition to turn it into the bona fide theoretical branch of molecular biology. Having a physicist’s background, I suspect that many of us had the vision of establishing bioinformatics in a leadership role over experimental biology, similar to the supremacy that theoretical physics enjoys over experimental physics. Somewhere along the line, it seems that bioinformatics lost this ambition and became sidetracked onto what physicists would call a “phenomenological” pathway. Let us follow the example of particle physics for a little longer. There, theoretical research has two phases (which, in fact, run in parallel). In the first phase (socalled phenomenological), a large number of physical events are recorded in huge raw databases, classified into separate groups based on statistical regularities, and then utilized to identify the most recurrent objects. Optimal database design, fast classification/ clustering algorithms, and data mining software are the main area of development here. The level of knowledge gained from this phase is, for instance, that objects A and B often appear together except when C is around, or when parameter X is lower than a certain threshold; it is mostly statistical in nature. The parallel with the current state of bioinformatics is clear. However, theoretical physics also has a subsequent, totally different phase, aiming at discovering the basic (few) rules (e.g., E = mc) underlying the relationships between the objects, their individual properties, and thus finally explaining the statistical distributions of the events recorded in the databases. Once known, these rules considerably simplify the description of the database content and, more important, have a predictive power: the realm of the theory may encompass objects or events that have not been observed previously. This part of theoretical endeavor is entirely missing in current bioinformatics. As a consequence, we are still not able to agree on the number of human genes despite having the complete sequence of the human genome at hand. Identifying precisely the 5 and 3 boundaries of genes (the transcription unit) in metazoan genomes, as well as the correct sequences of the resulting mRNA (“exon parsing”) has been a major challenge of bioinformatics for years. Yet, the current program performances are still totally insufficient for a reliable automated annotation (Claverie 1997; Ashburner 2000). It is interesting to recapitulate quickly the research in this area to illustrate the essential limitation plaguing modern bioinformatics. Encoding a protein imposes a variety of constraints on nucleotide sequences, which do not apply to noncoding regions of the genome. These constraints induce statistical biases of various kinds, the most discriminant of which was soon recognized to be the distribution of six nucleotide-long “words” or hexamers (Claverie and Bougueleret 1986; Fickett and Tung 1992). Initial gene parsing methods were then simply based on word frequency computation, eventually combined with the detection of splicing consensus motifs. The next generation of software implemented the same basic principles into a simulated neural network architecture (Uberbacher and Mural 1991). Finally, the last generation of software, based on hidden Markov models, added an additional refinement by computing the likelihood of the predicted gene architectures (e.g., favoring human genes with an average of seven coding exons, each 150 nucleotides long) is added (Kulp et al. 1996; Burge and Karlin, 1997)). These ab initio methods are used in conjunction with a search for sequence similarity with previously characterized genes or expressed sequence tags (EST). Sadly, it is often claimed that matching back cDNA to genomic sequences is the best gene identification protocol; hence, admitting that the best way to find genes is to look them up in a previously established catalog! Thus, the two main principles behind state-of-theart gene prediction software are (1) common statistical regularities and (2) plain sequence similarity. From an E-MAIL Jean.Michel.Claverie@igs.cnrs-mrs.fr; FAX +33-4-91-1645-49. Article and publication are at www.genesdev.org/cgi/doi/10.1101/ gad.155500. Commentary

Heterocapsa circularisquama DNA virus (HcDNAV; previously designated as HcV) is a giant virus (girus) with a ~356-kbp double-stranded DNA (dsDNA) genome. HcDNAV lytically infects the bivalve-killing marine dinoflagellate H. circularisquama, and currently represents the sole DNA virus isolated from dinoflagellates, one of the most abundant protists in marine ecosystems. Its morphological features, genome type, and host range previously suggested that HcDNAV might be a member of the family Phycodnaviridae of Nucleo-Cytoplasmic Large DNA Viruses (NCLDVs), though no supporting sequence data was available. NCLDVs currently include two families found in aquatic environments (Phycodnaviridae, Mimiviridae), one mostly infecting terrestrial animals (Poxviridae), another isolated from fish, amphibians and insects (Iridoviridae), and the last one (Asfarviridae) exclusively represented by the animal pathogen African swine fever virus (ASFV), the agent of a fatal hemorrhagic disease in domestic swine. In this study, we determined the complete sequence of the type B DNA polymerase (PolB) gene of HcDNAV. The viral PolB was transcribed at least from 6 h post inoculation (hpi), suggesting its crucial function for viral replication. Most unexpectedly, the HcDNAV PolB sequence was found to be closely related to the PolB sequence of ASFV. In addition, the amino acid sequence of HcDNAV PolB showed a rare amino acid substitution within a motif containing highly conserved motif: YS DTDS was found in HcDNAV PolB instead of YG DTDS in most dsDNA viruses. Together with the previous observation of ASFV-like sequences in the Sorcerer II Global Ocean Sampling metagenomic datasets, our results further reinforce the ideas that the terrestrial ASFV has its evolutionary origin in marine environments.

Mimivirus, a virus infecting amoebae of the acanthamoeba genus, is the prototype member of the Mimiviridae, the latest addition to the family of the nucleocytoplasmic large DNA viruses, already including the Poxviridae, the Iridoviridae, the Asfarviridae, and the Phycodnaviridae. Because of the size of its particle—a fiber-covered icosahedral protein capsid 0.75 μm in diameter—Mimivirus was initially mistaken for a parasitic bacterium. Its 1.2-Mb genome sequence encodes more than 900 proteins, many of them associated with functions never before encountered in a virus, such as four aminoacyl-tRNA synthetases. These findings revived the debate about the origin of DNA viruses and their possible role in the emergence of the eukaryotic nucleus. The recent isolation of a new type of satellite virus, called a virophage, associated with a second strain of Mimivirus, confirmed its unique position within the virus world. Post-genomic studies are now in progress, slowly shedding some light on the physiology of the most complex virus isolated to date.

Background Using a combination of pyrosequencing and conventional Sanger sequencing, the complete genome sequence of the recently described novel Brucella species, Brucella microti, was determined. B. microti is a member of the genus Brucella within the Alphaproteobacteria, which consists of medically important highly pathogenic facultative intracellular bacteria. In contrast to all other Brucella species, B. microti is a fast growing and biochemically very active microorganism with a phenotype more similar to that of Ochrobactrum, a facultative human pathogen. The atypical phenotype of B. microti prompted us to look for genomic differences compared to other Brucella species and to look for similarities with Ochrobactrum. Results The genome is composed of two circular chromosomes of 2,117,050 and 1,220,319 base pairs. Unexpectedly, we found that the genome sequence of B. microti is almost identical to that of Brucella suis 1330 with an overall sequence identity of 99.84% in aligned regions. The most significant structural difference between the two genomes is a bacteriophage-related 11,742 base pairs insert only present in B. microti. However, this insert is unlikely to have any phenotypical consequence. Only four protein coding genes are shared between B. microti and Ochrobactrum anthropi but impaired in other sequenced Brucella. The most noticeable difference between B. microti and other Brucella species was found in the sequence of the 23S ribosomal RNA gene. This unusual variation could have pleiotropic effects and explain the fast growth of B. microti. Conclusion Contrary to expectations from the phenotypic analysis, the genome sequence of B. microti is highly similar to that of known Brucella species, and is remotely related to the one of O. anthropi. How the few differences in gene content between B. microti and B. suis 1330 could result in vastly different phenotypes remains to be elucidated. This unexpected finding will complicate the task of identifying virulence determinants in the Brucella genus. The genome sequence of B. microti will serve as a model for differential expression analysis and complementation studies. Our results also raise some concerns about the importance given to phenotypical traits in the definition of bacterial species.

Mimivirus, a giant DNA virus infecting Acanthamoeba, is revealing an increasing list of unique features such as a 1.2-Mb genome with numerous genes not found in other viruses, a uniquely conserved promoter signal, and a particle of unmatched complexity using two distinct portals for genome delivery and packaging. Herein, we contribute a further Mimivirus distinctive feature discovered by sequencing a panel of viral cDNAs produced for probing the structure of Mimivirus transcripts. All Mimivirus mRNAs are polyadenylated at a site coinciding exactly with unrelated, but strongly palindromic, genomic sequences. The analysis of 454 Life Sciences (Roche) FLX cDNA tags (150,651) confirmed this finding for all Mimivirus genes independent of their transcription timings and expression levels. The absence of a suitable palindromic signal between adjacent genes results in transcripts encompassing multiple ORFs in the same or even in opposite orientations. Surprisingly, Mimivirus tRNAs are expressed as polyadenylated messengers, including an ORF/tRNA composite mRNA. To our knowledge, both the nature and the stringency of the “hairpin rule” defining the location of polyadenylation sites are unique, raising once more the question of Mimivirus's evolutionary origin. The precise molecular mechanisms implementing the hairpin rule into the 3′-end processing of Mimivirus pre-mRNAs remain to be elucidated.

Mimivirus, a giant DNA virus (i.e. "girus") infecting species of the genus Acanthamoeba, was first identified in 2003. With a particle size of 0.7microm in diameter, and a genome size of 1.2Mb encoding more than 900 proteins, it is the most complex virus described to date. Beyond its unusual size, the Mimivirus genome was found to contain the first viral homologues of many genes thought to be the trademark of cellular organisms, such as central components of the translation apparatus. These findings revived the debate on the origin of DNA viruses, and the role they might have played in the emergence of eukaryotes. Published and ongoing studies on Mimivirus continue to lead to unexpected findings concerning a variety of aspects, such as the structure of its particle, unique features of its replication cycle, or the distribution and abundance of Mimivirus relatives in the oceans. Following a summary of these recent findings, we present preliminary results suggesting that octocorals might have come in close contact with an ancestor of Mimivirus, and that modern sponges might be host to a yet unidentified, even larger, member of the Mimiviridae.

An EST analysis of the phytoplankton Emiliania huxleyi reveals genes involved in haploid- and diploid-specific processes and provides insights into environmental adaptation., Background Eukaryotes are classified as either haplontic, diplontic, or haplo-diplontic, depending on which ploidy levels undergo mitotic cell division in the life cycle. Emiliania huxleyi is one of the most abundant phytoplankton species in the ocean, playing an important role in global carbon fluxes, and represents haptophytes, an enigmatic group of unicellular organisms that diverged early in eukaryotic evolution. This species is haplo-diplontic. Little is known about the haploid cells, but they have been hypothesized to allow persistence of the species between the yearly blooms of diploid cells. We sequenced over 38,000 expressed sequence tags from haploid and diploid E. huxleyi normalized cDNA libraries to identify genes involved in important processes specific to each life phase (2N calcification or 1N motility), and to better understand the haploid phase of this prominent haplo-diplontic organism. Results The haploid and diploid transcriptomes showed a dramatic differentiation, with approximately 20% greater transcriptome richness in diploid cells than in haploid cells and only ≤ 50% of transcripts estimated to be common between the two phases. The major functional category of transcripts differentiating haploids included signal transduction and motility genes. Diploid-specific transcripts included Ca2+, H+, and HCO3- pumps. Potential factors differentiating the transcriptomes included haploid-specific Myb transcription factor homologs and an unusual diploid-specific histone H4 homolog. Conclusions This study permitted the identification of genes likely involved in diploid-specific biomineralization, haploid-specific motility, and transcriptional control. Greater transcriptome richness in diploid cells suggests they may be more versatile for exploiting a diversity of rich environments whereas haploid cells are intrinsically more streamlined.

The interaction between synthetic peptides and A2.1 class I MHC molecules has been investigated using an inhibition of Ag presentation assay and unbiased peptide sets derived of either viral or eucaryotic origin. For the various sets, strong binding (defined as significant inhibition at the 30 micrograms/ml level) was detected in 7 to 46% of the peptides tested, with an overall frequency of 26%. A set of self-peptides derived from human beta 2 microglobulin was also included in the study. In this case, strong binding was detected in 3 of 15 peptides (20%), thus formally demonstrating a lack of self-/non-self-discrimination at the level of class I molecules. When the whole A2.1-binding database of 105 peptides thus generated was examined by sequence analysis, a significant correlation was found with a recently proposed A2.1-binding motif, whereas no particular positive or negative association was detected between the capacity to bind A2.1 and three different class II alleles (DR1, DR5, and DR7). Finally, using this approach, several peptides capable of binding both A2.1 and multiple DR alleles have been identified, suggesting possible candidates for development of peptide vaccines eliciting both class I and class II restricted responses.

We have analysed, in the context of the available structural information, the frequency of occurrence of different amino acids in functional regions of both the class I MHC antigens and of the TCR alpha and beta chains. We found that in class I MHC molecules, charged residues are found frequently among those which are presumably dedicated to interactions with the TCR, while the aromatic side chain residues are found more in the interior of the groove. In the TCR, the Asn residue appears with high frequency in all the CDR equivalents. The TCR CDR3s of both alpha and beta chains are particularly rich in Gly, whereas the CDR1 and CDR2 loops exhibit strong biases in favour of charged residues. Accordingly, the interactions between the MHC molecule and the peptide antigen appear to be essentially mediated by hydrophobic interactions and hydrogen bonding, while electrostatic interactions between charged residues might be important in the association of TCR and MHC molecules. The observation that each CDR1 and CDR2 is biased towards a particular set of amino acids, taken together with the nature of the protruding residues on the MHC helices, allows us to propose, in the frame of a molecular model of the MHC-TCR complex, several plausible configurations.

In an effort to understand how a tick-borne pathogen adapts to the body louse, we sequenced and compared the genomes of the recurrent fever agents Borrelia recurrentis and B. duttonii. The 1,242,163–1,574,910-bp fragmented genomes of B. recurrentis and B. duttonii contain a unique 23-kb linear plasmid. This linear plasmid exhibits a large polyT track within the promoter region of an intact variable large protein gene and a telomere resolvase that is unique to Borrelia. The genome content is characterized by several repeat families, including antigenic lipoproteins. B. recurrentis exhibited a 20.4% genome size reduction and appeared to be a strain of B. duttonii, with a decaying genome, possibly due to the accumulation of genomic errors induced by the loss of recA and mutS. Accompanying this were increases in the number of impaired genes and a reduction in coding capacity, including surface-exposed lipoproteins and putative virulence factors. Analysis of the reconstructed ancestral sequence compared to B. duttonii and B. recurrentis was consistent with the accelerated evolution observed in B. recurrentis. Vector specialization of louse-borne pathogens responsible for major epidemics was associated with rapid genome reduction. The correlation between gene loss and increased virulence of B. recurrentis parallels that of Rickettsia prowazekii, with both species being genomic subsets of less-virulent strains., Author Summary Borreliae are vector-borne spirochetes that are responsible for Lyme disease and recurrent fevers. We completed the genome sequences of the tick-borne Borrelia duttonii and the louse-borne B. recurrentis. The former of these is responsible for emerging infections that mimic malaria in Africa and in travellers, and the latter is responsible for severe recurrent fever in poor African populations. Diagnostic tools for these pathogens remain poor with regard to sensitivity and specificity due, in part, to the lack of genomic sequences. In this study, we show that the genomic content of B. recurrentis is a subset of that of B. duttonii, the genes of which are undergoing a decay process. These phenomena are common to all louse-borne pathogens compared to their tick-borne counterparts. In B. recurrentis, this process may be due to the inactivation of genes encoding DNA repair mechanisms, implying the accumulation of errors in the genome. The increased virulence of B. recurrentis could not be traced back to specific virulence factors, illustrating the lack of correlation between the virulence of a pathogen and so-called virulence genes. Knowledge of these genomes will allow for the development of new molecular tools that provide a more-accurate, sensitive, and specific diagnosis of these emerging infections.

Background In search of new antifungal targets of potential interest for pharmaceutical companies, we initiated a comparative genomics study to identify the most promising protein-coding genes in fungal genomes. One criterion was the protein sequence conservation between reference pathogenic genomes. A second criterion was that the corresponding gene in Saccharomyces cerevisiae should be essential. Since thiamine pyrophosphate is an essential product involved in a variety of metabolic pathways, proteins responsible for its production satisfied these two criteria. Results We report the enzymatic characterization and the crystallographic structure of the Candida albicans Thiamine pyrophosphokinase. The protein was co-crystallized with thiamine or thiamine-PNP. Conclusion The presence of an inorganic phosphate in the crystallographic structure opposite the known AMP binding site relative to the thiamine moiety suggests that a second AMP molecule could be accommodated in the C. albicans structure. Together with the crystallographic structures of the enzyme/substrate complexes this suggests the existence of a secondary, less specific, nucleotide binding site in the Candida albicans thiamine pyrophosphokinase which could transiently serve during the release or the binding of ATP. The structures also highlight a conserved Glutamine residue (Q138) which could interact with the ATP α-phosphate and act as gatekeeper. Finally, the TPK/Thiamine-PNP complex is consistent with a one step mechanism of pyrophosphorylation.

Background Acanthamoeba polyphaga mimivirus is the largest known ds-DNA virus and its 1.2 Mb-genome sequence has revealed many unique features. Mimivirus occupies an independent lineage among eukaryotic viruses and its known hosts include only species from the Acanthamoeba genus. The existence of mimivirus relatives was first suggested by the analysis of the Sargasso Sea metagenomic data. Results We now further demonstrate the presence of numerous "mimivirus-like" sequences using a larger marine metagenomic data set. We also show that the DNA polymerase sequences from three algal viruses (CeV01, PpV01, PoV01) infecting different marine algal species (Chrysochromulina ericina, Phaeocystis pouchetii, Pyramimonas orientalis) are very closely related to their homolog in mimivirus. Conclusion Our results suggest that the numerous mimivirus-related sequences identified in marine environments are likely to originate from diverse large DNA viruses infecting phytoplankton. Micro-algae thus constitute a new category of potential hosts in which to look for new species of Mimiviridae.

Aminoacyl-tRNA synthetases are pivotal in determining how the genetic code is translated in amino acids and in providing the substrate for protein synthesis. As such, they fulfill a key role in a process universally conserved in all cellular organisms from their most complex to their most reduced parasitic forms. In contrast, even complex viruses were not found to encode much translation machinery, with the exception of isolated components such as tRNAs. In this context, the discovery of four aminoacyl-tRNA synthetases encoded in the genome of mimivirus together with a full set of translation initiation, elongation, and termination factors appeared to blur what was once a clear frontier between the cellular and viral world. Functional studies of two mimivirus tRNA synthetases confirmed the MetRS specificity for methionine and the TyrRS specificity for tyrosine and conformity with the identity rules for tRNA Tyr for archea/eukarya. The atomic structure of the mimivirus tyrosyl-tRNA synthetase in complex with tyrosinol exhibits the typical fold and active-site organization of archaeal-type TyrRS. However, the viral enzyme presents a unique dimeric conformation and significant differences in its anticodon binding site. The present work suggests that mimivirus aminoacyl-tRNA synthetases function as regular translation enzymes in infected amoebas. Their phylogenetic classification does not suggest that they have been acquired recently by horizontal gene transfer from a cellular host but rather militates in favor of an intricate evolutionary relationship between large DNA viruses and ancestral eukaryotes.

Background DNA viruses have a wide range of genome sizes (5 kb up to 1.2 Mb, compared to 0.16 Mb to 1.5 Mb for obligate parasitic bacteria) that do not correlate with their virulence or the taxonomic distribution of their hosts. The reasons for such large variation are unclear. According to the traditional view of viruses as gifted "gene pickpockets", large viral genome sizes could originate from numerous gene acquisitions from their hosts. We investigated this hypothesis by studying 67 large DNA viruses with genome sizes larger than 150 kb, including the recently characterized giant mimivirus. Given that horizontally transferred DNA often have anomalous nucleotide compositions differing from the rest of the genome, we conducted a detailed analysis of the inter- and intra-genome compositional properties of these viruses. We then interpreted their compositional heterogeneity in terms of possible causes, including strand asymmetry, gene function/expression, and horizontal transfer. Results We first show that the global nucleotide composition and nucleotide word usage of viral genomes are species-specific and distinct from those of their hosts. Next, we identified compositionally anomalous (cA) genes in viral genomes, using a method based on Bayesian inference. The proportion of cA genes is highly variable across viruses and does not exhibit a significant correlation with genome size. The vast majority of the cA genes were of unknown function, lacking homologs in the databases. For genes with known homologs, we found a substantial enrichment of cA genes in specific functional classes for some of the viruses. No significant association was found between cA genes and compositional strand asymmetry. A possible exogenous origin for a small fraction of the cA genes could be confirmed by phylogenetic reconstruction. Conclusion At odds with the traditional dogma, our results argue against frequent genetic transfers to large DNA viruses from their modern hosts. The large genome sizes of these viruses are not simply explained by an increased propensity to acquire foreign genes. This study also confirms that the anomalous nucleotide compositions of the cA genes is sometimes linked to particular biological functions or expression patterns, possibly leading to an overestimation of recent horizontal gene transfers.

Recent studies on chromosome conformation show that chromosomes colocalize in the nucleus, bringing together active genes in transcription factories. This spatial proximity of actively transcribing genes could provide a means for RNA interaction at the transcript level. We have screened public databases for chimeric EST and mRNA sequences with the intent of mapping transcription-induced interchromosomal interactions. We suggest that chimeric transcripts may be the result of close encounters of active genes, either as functional products or “noise” in the transcription process, and that they could be used as probes for chromosome interactions. We have found a total of 5,614 chimeric ESTs and 587 chimeric mRNAs that meet our selection criteria. Due to their higher quality, the mRNA findings are of particular interest and we hope that they may serve as food for thought for specialists in diverse areas of molecular biology.

Acanthamoeba polyphaga mimivirus is the largest known virus in both particle size and genome complexity. Its 1.2-Mb genome encodes 911 proteins, among which only 298 have predicted functions. The composition of purified isolated virions was analyzed by using a combined electrophoresis/mass spectrometry approach allowing the identification of 114 proteins. Besides the expected major structural components, the viral particle packages 12 proteins unambiguously associated with transcriptional machinery, 3 proteins associated with DNA repair, and 2 topoisomerases. Other main functional categories represented in the virion include oxidative pathways and protein modification. More than half of the identified virion-associated proteins correspond to anonymous genes of unknown function, including 45 “ORFans.” As demonstrated by both Western blotting and immunogold staining, some of these “ORFans,” which lack any convincing similarity in the sequence databases, are endowed with antigenic properties. Thus, anonymous and unique genes constituting the majority of the mimivirus gene complement encode bona fide proteins that are likely to participate in well-integrated processes.

The first viral Nucleoside Diphosphate Kinase was recently identified in the giant double-stranded DNA virus Acanthamoeba polyphag a Mimivirus (ApM). Here we report its expression and detailed biochemical characterization. NDK(apm) exhibits unique features such as a shorter Kpn-loop, a structural motif previously reported to be part of the active site and involved in oligomer formation. Enzymatic activity measurements on the recombinant NDK(apm) revealed its preferential affinity for deoxypyrimidine nucleotides. This property might represent an adaptation of NDK(apm) to the production of the limiting TTP deoxynucleotide required for the replication of the large A+T rich (72%) viral genome. The NDK(apm) might also assume a role in dUTP detoxification to compensate for the surprising absence of Mimivirus dUTPase (deoxyuridine triphosphate pyrophosphatase) an important enzyme conserved in most viruses. Although the phylogenetic analysis of NDK sequences sampled through organisms from the three domains of life is only partially informative, it favors an ancestral origin for NDK(apm) over a recent acquisition from a eukaryotic organism by horizontal gene transfer.

The genomic sequencing of Rickettsia conorii revealed a new family of Rickettsia-specific palindromic elements (RPEs) capable of in-frame insertion in preexisting open reading frames (ORFs). Many of these altered ORFs correspond to proteins with well-characterized or essential functions in other microorganisms. Previous experiments indicated that RPE-containing genes are normally transcribed and that no excision of the repeat occurs at the mRNA level. Using mass spectrometry, we now confirmed the retention of the RPE-derived amino acid residues in 4 proteins successfully expressed in Escherichia coli, raising the general question of the consequences of this common insertion event on the fitness of Rickettsia enzymes. The predicted guanylate kinase activity of the R. conorii gmk gene product was measured both on the RPE-containing and RPE-excised recombinant proteins. We show that the 2 proteins are active but exhibit substantial differences in their affinity for adenosine triphosphate, guanosine monophosphate, and catalytic constants. The distribution of the RPE gmk insert among Rickettsia species indicates that the insertion event is ancient and occurred after the divergence of Rickettsia felis and R. conorii but before that of Rickettsia helvetica and R. conorii. We found no evidence that the gmk gene fixed adaptive changes to compensate the RPE peptide insertion. Furthermore, the analysis of the rates of divergence in 23 RPE-containing genes indicates that coding RPE repeats tend to evolve under weak selective constraint, at a rate similar to intergenic noncoding RPE sequences. Altogether, these results suggest that the insertion of RPE-encoded "selfish peptides," although respecting the original fold and activity of the host proteins, might be slightly detrimental to the enzyme efficiency within limits tolerable for slow-growing intracellular parasites such as Rickettsia.

Advances in genomics have stimulated new hypotheses that DNA viruses were the origin of the eukaryotic cell nucleus, and that they should be considered as living things., The origins of viruses are shrouded in mystery, but advances in genomics and the discovery of highly complex giant DNA viruses have stimulated new hypotheses that DNA viruses were involved in the emergence of the eukaryotic cell nucleus, and that they are worthy of being considered as living organisms.

The Rickettsia genus is a group of obligate intracellular α-proteobacteria representing a paradigm of reductive evolution. Here, we investigate the evolutionary processes that shaped the genomes of the genus. The reconstruction of ancestral genomes indicates that their last common ancestor contained more genes, but already possessed most traits associated with cellular parasitism. The differences in gene repertoires across modern Rickettsia are mainly the result of differential gene losses from the ancestor. We demonstrate using computer simulation that the propensity of loss was variable across genes during this process. We also analyzed the ratio of nonsynonymous to synonymous changes (Ka/Ks) calculated as an average over large sets of genes to assay the strength of selection acting on the genomes of Rickettsia, Anaplasmataceae, and free-living γ-proteobacteria. As a general trend, Ka/Ks were found to decrease with increasing divergence between genomes. The high Ka/Ks for closely related genomes are probably due to a lag in the removal of slightly deleterious nonsynonymous mutations by natural selection. Interestingly, we also observed a decrease of the rate of gene loss with increasing divergence, suggesting a similar lag in the removal of slightly deleterious pseudogene alleles. For larger divergence (Ks > 0.2), Ka/Ks converge toward similar values indicating that the levels of selection are roughly equivalent between intracellular α-proteobacteria and their free-living relatives. This contrasts with the view that obligate endocellular microorganisms tend to evolve faster as a consequence of reduced effectiveness of selection, and suggests a major role of enhanced background mutation rates on the fast protein divergence in the obligate intracellular α-proteobacteria., Author Summary Genome downsizing and fast sequence divergence are frequently observed in bacteria living exclusively within the cells of higher eukaryotes. However, the driving forces and contributions of these processes to the genome diversity of the microorganisms remain poorly understood. The genus Rickettsia, a group of small obligate intracellular pathogens of humans, provides a fascinating model to study the genome downsizing process. In this article, we used seven Rickettsia genomes to reconstruct the genome of their ancestor and inferred the origin and fate of the genes found in today's species. We identify the process of gene loss as the main cause of genome diversification within the genus and show that the rate of gene loss, sequence divergence, and genome rearrangements are highly variable across the various Rickettsia lineages. This heterogeneity likely reflects the intricate effects of specialization to distinct arthropod hosts and critical alterations of the gene repertoire, such as the losses of DNA repair genes and the amplification of mobile genes. In contrast, we did not find evidence for the role of reduced population sizes on the long-term acceleration of sequence evolution. Overall, the data presented in this article shed new light on the fundamental evolutionary processes that drive the evolution of obligate intracellular bacteria.

BackgroundAs numerous diseases involve errors in signal transduction, modern therapeutics often target proteins involved in cellular signaling. Interpretation of the activity of signaling pathways during disease development or therapeutic intervention would assist in drug development, design of therapy, and target identification. Microarrays provide a global measure of cellular response, however linking these responses to signaling pathways requires an analytic approach tuned to the underlying biology. An ongoing issue in pattern recognition in microarrays has been how to determine the number of patterns (or clusters) to use for data interpretation, and this is a critical issue as measures of statistical significance in gene ontology or pathways rely on proper separation of genes into groups.ResultsHere we introduce a method relying on gene annotation coupled to decompositional analysis of global gene expression data that allows us to estimate specific activity on strongly coupled signaling pathways and, in some cases, activity of specific signaling proteins. We demonstrate the technique using the Rosetta yeast deletion mutant data set, decompositional analysis by Bayesian Decomposition, and annotation analysis using ClutrFree. We determined from measurements of gene persistence in patterns across multiple potential dimensionalities that 15 basis vectors provides the correct dimensionality for interpreting the data. Using gene ontology and data on gene regulation in the Saccharomyces Genome Database, we identified the transcriptional signatures of several cellular processes in yeast, including cell wall creation, ribosomal disruption, chemical blocking of protein synthesis, and, criticially, individual signatures of the strongly coupled mating and filamentation pathways.ConclusionThis works demonstrates that microarray data can provide downstream indicators of pathway activity either through use of gene ontology or transcription factor databases. This can be used to investigate the specificity and success of targeted therapeutics as well as to elucidate signaling activity in normal and disease processes.

Acinetobacter baumannii is a species of nonfermentative gram-negative bacteria commonly found in water and soil. This organism was susceptible to most antibiotics in the 1970s. It has now become a major cause of hospital-acquired infections worldwide due to its remarkable propensity to rapidly acquire resistance determinants to a wide range of antibacterial agents. Here we use a comparative genomic approach to identify the complete repertoire of resistance genes exhibited by the multidrug-resistant A. baumannii strain AYE, which is epidemic in France, as well as to investigate the mechanisms of their acquisition by comparison with the fully susceptible A. baumannii strain SDF, which is associated with human body lice. The assembly of the whole shotgun genome sequences of the strains AYE and SDF gave an estimated size of 3.9 and 3.2 Mb, respectively. A. baumannii strain AYE exhibits an 86-kb genomic region termed a resistance island—the largest identified to date—in which 45 resistance genes are clustered. At the homologous location, the SDF strain exhibits a 20 kb-genomic island flanked by transposases but devoid of resistance markers. Such a switching genomic structure might be a hotspot that could explain the rapid acquisition of resistance markers under antimicrobial pressure. Sequence similarity and phylogenetic analyses confirm that most of the resistance genes found in the A. baumannii strain AYE have been recently acquired from bacteria of the genera Pseudomonas, Salmonella, or Escherichia. This study also resulted in the discovery of 19 new putative resistance genes. Whole-genome sequencing appears to be a fast and efficient approach to the exhaustive identification of resistance genes in epidemic infectious agents of clinical significance., Synopsis The bacterial species Acinetobacter baumannii is a major cause of hospital-acquired infection throughout the world, and it is an increasing public health concern due to its increasing resistance to antibiotic treatment. Coincidently, a high incidence of multidrug-resistant A. baumannii bloodstream infections was recently reported in US Army service members injured during Afghanistan and Iraq/Kuwait military operations. A. baumannii exhibits a remarkable ability to rapidly develop antibiotic resistance, which led from fully susceptible to multidrug-resistant strains within three decades. The authors used whole-genome sequencing and bioinformatic analyses to identify the complete repertoire of resistance genes exhibited by the multidrug-resistant A. baumannii strain AYE, which is epidemic in France, and to investigate the mechanisms of their acquisition by comparison with the fully susceptible A. baumannii strain SDF, which is associated with human body lice. This study led to the discovery in the AYE genome of an 86-kb region called a resistance “island”—the largest identified to date—that contains a cluster of 45 resistance genes. The homologous location in the susceptible strain, curiously, exhibited a 20-kb genomic island that is devoid of resistance markers. This ability to “switch” its genomic structure probably explains the unmatched speed at which A. baumannii captures resistance markers when under antibacterial pressure, such as is found in hospital intensive care units.

Mimivirus, a parasite of Acanthamoeba polyphaga , is the largest DNA virus known; it encodes dozens of proteins with imputed functions in nucleic acid transactions. Here we produced, purified, and characterized mimivirus DNA topoisomerase IB (TopIB), which we find to be a structural and functional homolog of poxvirus TopIB and the poxvirus-like topoisomerases discovered recently in bacteria. Arginine, histidine, and tyrosine side chains responsible for TopIB transesterification are conserved and essential in mimivirus TopIB. Moreover, mimivirus TopIB is capable of incising duplex DNA at the 5′-CCCTT cleavage site recognized by all poxvirus topoisomerases. Based on the available data, mimivirus TopIB appears functionally more akin to poxvirus TopIB than bacterial TopIB, despite its greater primary structure similarity to the bacterial TopIB group. We speculate that the ancestral bacterial/viral TopIB was disseminated by horizontal gene transfer within amoebae, which are permissive hosts for either intracellular growth or persistence of many present-day bacterial species that have a type IB topoisomerase.

A chromosomally encoded oxacillinase, OXA-69, was characterized fromAcinetobacter baumanniiAYE. β-Lactamase OXA-69 shared 97% amino acid identity with the recently described OXA-51 enzyme ofA. baumanniiand 62 and 56% amino acid identity with the carbapenem-hydrolyzing oxacillinases OXA-24 and OXA-23, respectively. Biochemical characterization of the purified OXA-69 revealed a narrow-spectrum hydrolysis profile but including, at a low level, imipenem and meropenem. By PCR and sequencingblaOXA-69-like genes were identified in allA. baumanniistrains tested (n= 12), suggesting that this oxacillinase is naturally occurring in that species.

We sequenced the genome of Rickettsia felis, a flea-associated obligate intracellular α-proteobacterium causing spotted fever in humans. Besides a circular chromosome of 1,485,148 bp, R. felis exhibits the first putative conjugative plasmid identified among obligate intracellular bacteria. This plasmid is found in a short (39,263 bp) and a long (62,829 bp) form. R. felis contrasts with previously sequenced Rickettsia in terms of many other features, including a number of transposases, several chromosomal toxin–antitoxin genes, many more spoT genes, and a very large number of ankyrin- and tetratricopeptide-motif-containing genes. Host-invasion-related genes for patatin and RickA were found. Several phenotypes predicted from genome analysis were experimentally tested: conjugative pili and mating were observed, as well as β-lactamase activity, actin-polymerization-driven mobility, and hemolytic properties. Our study demonstrates that complete genome sequencing is the fastest approach to reveal phenotypic characters of recently cultured obligate intracellular bacteria., Rickettsia felis is an obligate intracellular bacterium that lives in fleas and causes spotted fever in humans. Its genome sequence provides the first evidence that such bacteria can undergo conjugation.

The Rickettsia genus is a group of obligate intracellular parasitic alpha-proteobacteria that includes human pathogens responsible for the typhus disease and various types of spotted fevers. rOmpA and rOmpB are two members of the "surface cell antigen" (Sca) autotransporter (AT) protein family that may play key roles in the adhesion of the Rickettsia cells to the host tissue. These molecules are likely determinants for the pathogenicity of the Rickettsia and represent good candidates for vaccine development. We identified the 17 members of this family of outer-membrane proteins in nine fully sequenced Rickettsia genomes. The typical architecture of the Sca proteins is composed of an N-terminal signal peptide and a C-terminal AT domain that promote the export of the central passenger domain to the outside of the bacteria. A characteristic of this family is the frequent degradation of the genes, which results in different subsets of the sca genes being expressed among Rickettsia species. Here, we present a detailed analysis of their phylogenetic relationships and evolution. We provide strong evidence that rOmpA and rOmpB as well as three other members of the Sca protein family--Sca1, Sca2, and Sca4--have evolved under positive selection. The exclusive distribution of the predicted positively selected sites within the passenger domains of these proteins argues that these regions are involved in the interaction with the host and may be locked in "arms race" coevolutionary conflicts.

Giant double-stranded DNA viruses (such as record breaking Acanthamoeba polyphaga Mimivirus), with particle sizes of 0.2 to 0.6 micron, genomes of 300 kbp to 1.200 kbp, and commensurate complex gene contents, constitute an evolutionary mystery. They challenge the common vision of viruses, traditionally seen as highly streamlined genomes optimally fitted to the smallest possible -filterable- package. Such giant viruses are now discovered in increasing numbers through the systematic sampling of ocean waters as well as freshwater aquatic environments, where they play a significant role in controlling phyto- and bacterio- plankton populations. The 4th algal virus workshop showed that the study of these ecologically important viruses is now massively entering the genomic era, promising a better understanding of their diversity and, hopefully, some insights on their origin and the evolutionary forces that shaped their genomes., Comment: Submitted to Virology J

The discovery and genome analysis of Acanthamoeba polyphaga Mimivirus, the largest known DNA virus, challenged much of the accepted dogma regarding viruses. Its particle size (>400 nm), genome length (1.2 million bp) and huge gene repertoire (911 protein coding genes) all contribute to blur the established boundaries between viruses and the smallest parasitic cellular organisms. Phylogenetic analyses also suggested that the Mimivirus lineage could have emerged prior to the individualization of cellular organisms from the three established domains, triggering a debate that can only be resolved by generating and analyzing more data. The next step is then to seek some evidence that Mimivirus is not the only representative of its kind and determine where to look for new Mimiviridae. An exhaustive similarity search of all Mimivirus predicted proteins against all publicly available sequences identified many of their closest homologues among the Sargasso Sea environmental sequences. Subsequent phylogenetic analyses suggested that unknown large viruses evolutionarily closer to Mimivirus than to any presently characterized species exist in abundance in the Sargasso Sea. Their isolation and genome sequencing could prove invaluable in understanding the origin and diversity of large DNA viruses, and shed some light on the role they eventually played in the emergence of eukaryotes., see also http://www.giantvirus.org