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Accueil > Départements > Microbiologie > Jacques OBERTO : Biologie Cellulaire des Archées

Publications de l’équipe


  • C. Badel, G. Erauso, A. Gomez, R. Catchpole, M. Gonnet, J. Oberto, P. Forterre, et V. Da Cunha, « The global distribution and evolutionary history of the pT26-2 archaeal plasmid family », Environmental Microbiology, sept. 2019.
    Résumé : Although plasmids play an important role in biological evolution, the number of plasmid families well characterized in terms of geographical distribution and evolution remains limited, especially in Archaea. Here, we describe the first systematic study of an archaeal plasmid family, the pT26-2 plasmid family. The in-depth analysis of the distribution, biogeography and host-plasmid co-evolution patterns of 26 integrated and 3 extrachromosomal plasmids of this plasmid family shows that they are widespread in Thermococcales and Methanococcales isolated from around the globe but are restricted to these two orders. All members of the family share 7 core genes but employ different integration and replication strategies. Phylogenetic analysis of the core genes and CRISPR spacer distribution suggest that plasmids of the pT26-2 family evolved with their hosts independently in Thermococcales and Methanococcales, despite these hosts exhibiting similar geographic distribution. Remarkably, core genes are conserved even in integrated plasmids that have lost replication genes and/or replication origins suggesting that they may be beneficial for their hosts. We hypothesise that the core proteins encode for a novel type of DNA/protein transfer mechanism, explaining the widespread oceanic distribution of the pT26-2 plasmid family. This article is protected by copyright. All rights reserved.
    Mots-clés : ARCHEE, MICROBIO.

  • P. Béguin, Y. Chekli, G. Sezonov, P. Forterre, et M. Krupovic, « Sequence motifs recognized by the casposon integrase of Aciduliprofundum boonei », Nucleic Acids Research, mai 2019.
    Résumé : Casposons are a group of bacterial and archaeal DNA transposons encoding a specific integrase, termed casposase, which is homologous to the Cas1 enzyme responsible for the integration of new spacers into CRISPR loci. Here, we characterized the sequence motifs recognized by the casposase from a thermophilic archaeon Aciduliprofundum boonei. We identified a stretch of residues, located in the leader region upstream of the actual integration site, whose deletion or mutagenesis impaired the concerted integration reaction. However, deletions of two-thirds of the target site were fully functional. Various single-stranded 6-FAM-labelled oligonucleotides derived from casposon terminal inverted repeats were as efficiently incorporated as duplexes into the target site. This result suggests that, as in the case of spacer insertion by the CRISPR Cas1-Cas2 integrase, casposon integration involves splaying of the casposon termini, with single-stranded ends being the actual substrates. The sequence critical for incorporation was limited to the five terminal residues derived from the 3' end of the casposon. Furthermore, we characterize the casposase from Nitrosopumilus koreensis, a marine member of the phylum Thaumarchaeota, and show that it shares similar properties with the A. boonei enzyme, despite belonging to a different family. These findings further reinforce the mechanistic similarities and evolutionary connection between the casposons and the adaptation module of the CRISPR-Cas systems.
    Mots-clés : ARCHEE, MICROBIO.

  • R. Catchpole et P. Forterre, « The evolution of Reverse Gyrase suggests a non-hyperthermophilic Last Universal Common Ancestor », Molecular Biology and Evolution, sept. 2019.
    Résumé : Reverse gyrase (RG) is the only protein found ubiquitously in hyperthermophilic organisms, but absent from mesophiles. As such, its simple presence or absence allows us to deduce information about the optimal growth temperature of long-extinct organisms, even as far as the last universal common ancestor of extant life (LUCA). The growth environment and gene content of the LUCA has long been a source of debate in which RG often features. In an attempt to settle this debate, we carried out an exhaustive search for RG proteins, generating the largest RG dataset to date. Comprising 376 sequences, our dataset allows for phylogenetic reconstructions of RG with unprecedented size and detail. These RG phylogenies are strikingly different from those of universal proteins inferred to be present in the LUCA, even when using the same set of species. Unlike such proteins, RG does not form monophyletic archaeal and bacterial clades, suggesting RG emergence after the formation of these domains, and/or significant horizontal gene transfer. Additionally, the branch lengths separating archaeal and bacterial groups are very short, inconsistent with the tempo of evolution from the time of the LUCA. Despite this, phylogenies limited to archaeal RG resolve most archaeal phyla, suggesting predominantly vertical evolution since the time of the last archaeal ancestor. In contrast, bacterial RG indicates emergence after the last bacterial ancestor followed by significant horizontal transfer. Taken together, these results suggest a non-hyperthermophilic LUCA and bacterial ancestor, with hyperthermophily emerging early in the evolution of the archaeal and bacterial domains.
    Mots-clés : ARCHEE, MICROBIO.

  • A. Gorlas, R. Catchpole, E. Marguet, et P. Forterre, « Increase of positive supercoiling in a hyperthermophilic archaeon after UV irradiation », Extremophiles, vol. 23, nᵒ 1, p. 141-149, janv. 2019.
    Résumé : Diverse DNA repair mechanisms are essential to all living organisms. Some of the most widespread repair systems allow recovery of genome integrity in the face of UV radiation. Here, we show that the hyperthermophilic archaeon Thermococcus nautili possesses a remarkable ability to recovery from extreme chromosomal damage. Immediately following UV irradiation, chromosomal DNA of T. nautili is fragmented beyond recognition. However, the extensive UV-induced double-stranded breaks (DSB) are repaired over the course of several hours, allowing restoration of growth. DSBs also disrupted plasmid DNA in this species. Similar to the chromosome, plasmid integrity was restored during an outgrowth period. Intriguingly, the topology of recovered pTN1 plasmids differed from control strain by being more positively supercoiled. As reverse gyrase (RG) is the only enzyme capable of inducing positive supercoiling, our results suggest the activation of RG activity by UV-induced stress. We suggest simple UV stress could be used to study archaeal DNA repair and responses to DSB.
    Mots-clés : ARCHEE, DBG, Double-strand breaks, MICROBIO, Plasmid, RBA, Topology, UV irradiation.

  • M. Krupovic, K. S. Makarova, Y. I. Wolf, S. Medvedeva, D. Prangishvili, P. Forterre, et E. V. Koonin, « Integrated mobile genetic elements in Thaumarchaeota », Environmental Microbiology, vol. 21, nᵒ 6, p. 2056-2078, juin 2019.
    Résumé : To explore the diversity of mobile genetic elements (MGE) associated with archaea of the phylum Thaumarchaeota, we exploited the property of most MGE to integrate into the genomes of their hosts. Integrated MGE (iMGE) were identified in 20 thaumarchaeal genomes amounting to 2 Mbp of mobile thaumarchaeal DNA. These iMGE group into five major classes: (i) proviruses, (ii) casposons, (iii) insertion sequence-like transposons, (iv) integrative-conjugative elements and (v) cryptic integrated elements. The majority of the iMGE belong to the latter category and might represent novel families of viruses or plasmids. The identified proviruses are related to tailed viruses of the order Caudovirales and to tailless icosahedral viruses with the double jelly-roll capsid proteins. The thaumarchaeal iMGE are all connected within a gene sharing network, highlighting pervasive gene exchange between MGE occupying the same ecological niche. The thaumarchaeal mobilome carries multiple auxiliary metabolic genes, including multicopper oxidases and ammonia monooxygenase subunit C (AmoC), and stress response genes, such as those for universal stress response proteins (UspA). Thus, iMGE might make important contributions to the fitness and adaptation of their hosts. We identified several iMGE carrying type I-B CRISPR-Cas systems and spacers matching other thaumarchaeal iMGE, suggesting antagonistic interactions between coexisting MGE and symbiotic relationships with the ir archaeal hosts.
    Mots-clés : ammonia-oxidizing archaeon, ARCHEE, crispr-cas systems, diversity, evolution, genome sequence, insights, MICROBIO, photosystem-i, protein, tailed viruses, transfer agents.

  • J. R. Osman, C. Regeard, C. Badel, G. Fernandes, et M. S. DuBow, « Variation of bacterial biodiversity from saline soils and estuary sediments present near the Mediterranean Sea coast of Camargue (France) », Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology, vol. 112, nᵒ 3, p. 351-365, mars 2019.
    Résumé : Salinity is an important environmental factor influencing microbial community composition. To better understand this influence, we determined the bacterial communities present in 17 different sites of brackish sediment (underwater) and soil (surface) samples from the Camargue region (Rhone river delta) in southern France during the fall of 2013 and 2014 using pyrosequencing of the V3-V4 regions of the 16S rRNA genes amplified by PCR. This region is known for abundant flora and fauna and, though saline, 30% of rice consumed in France is grown here. We found that bacterial abundance in 1g of soil or sediment, calculated by qPCR, was higher in sediments than in surface soil samples. Members belonging to the Proteobacteria, Bacteroidetes, Chloroflexi and Firmicutes phyla dominated the bacterial communities of sediment samples, while members belonging to the Proteobacteria, Bacteroidetes, Gemmatimonadetes, Actinobacteria, Firmicutes and Acidobacteria phyla dominated the bacterial communities of the soil samples. The most abundant bacterial genera present in the saline sediments and soils from the Camargue belonged mostly to halophilic and sulphate reducing bacteria, suggesting that the Camargue may be a valuable system to investigate saline, yet agriculturally productive, sediment and soil microbial ecosystem.
    Mots-clés : ARCHEE, Bacterial biodiversity, community structure, de-giraud, diversity, gen. nov., Halophilic bacteria, hypersaline microbial mat, LGBMB, MICROBIO, molecular analysis, Pyrosequencing, rhone river delta, rice fields, rna, Salinity, temporal variation.


  • R. Catchpole, A. Gorlas, J. Oberto, et P. Forterre, « A series of new E. coli-Thermococcus shuttle vectors compatible with previously existing vectors », Extremophiles: Life Under Extreme Conditions, vol. 22, nᵒ 4, p. 591-598, juill. 2018.
    Résumé : Hyperthermophilic microorganisms are an important asset in the toolkits of biotechnologists, biochemists and evolutionary biologists. The anaerobic archaeon, Thermococcus kodakarensis, has become one of the most useful hyperthermophilic model species, not least due to its natural competence and genetic tractability. Despite this, the range of genetic tools available for T. kodakarensis remains limited. Using sequencing and phylogenetic analyses, we determined that the rolling-circle replication origin of the cryptic mini-plasmid pTP2 from T. prieurii is suitable for plasmid replication in T. kodakarensis. Based on this replication origin, we present a novel series of replicative E. coli-T. kodakarensis shuttle vectors. These shuttle vectors have been constructed with three different selectable markers, allowing selection in a range of T. kodakarensis backgrounds. Moreover, these pTP2-derived plasmids are compatible with the single-existing E. coli-T. kodakarensis shuttle vector, pLC70. We show that both pTP2-derived and pLC70-derived plasmids replicate faithfully while cohabitating in T. kodakarensis cells. These plasmids open the door for new areas of research in plasmid segregation, DNA replication and gene expression.
    Mots-clés : Archaea, ARCHEE, Cloning, DBG, Gene cloning and expression, Genetics of extremophiles, Hyperthermophiles, MICROBIO, Molecular biology, Molecular biology of archaea, RBA.

  • V. Da Cunha, M. Gaia, A. Nasir, et P. Forterre, « Asgard archaea do not close the debate about the universal tree of life topology », PLOS Genetics, vol. 14, nᵒ 3, p. e1007215, mars 2018.

  • R. Fujikane, I. Behm-Ansmant, A. - S. Tillault, C. Loegler, V. Igel-Bourguignon, E. Marguet, P. Forterre, C. Branlant, Y. Motorin, et B. Charpentier, « Contribution of protein Gar1 to the RNA-guided and RNA-independent rRNA:Ψ-synthase activities of the archaeal Cbf5 protein », Scientific Reports, vol. 8, nᵒ 1, p. 13815, sept. 2018.
    Résumé : Archaeal RNA:pseudouridine-synthase (PUS) Cbf5 in complex with proteins L7Ae, Nop10 and Gar1, and guide box H/ACA sRNAs forms ribonucleoprotein (RNP) catalysts that insure the conversion of uridines into pseudouridines (Ψs) in ribosomal RNAs (rRNAs). Nonetheless, in the absence of guide RNA, Cbf5 catalyzes the in vitro formation of Ψ2603 in Pyrococcus abyssi 23S rRNA and of Ψ55 in tRNAs. Using gene-disrupted strains of the hyperthermophilic archaeon Thermococcus kodakarensis, we studied the in vivo contribution of proteins Nop10 and Gar1 to the dual RNA guide-dependent and RNA-independent activities of Cbf5 on 23S rRNA. The single-null mutants of the cbf5, nop10, and gar1 genes are viable, but display a thermosensitive slow growth phenotype. We also generated a single-null mutant of the gene encoding Pus10, which has redundant activity with Cbf5 for in vitro formation of Ψ55 in tRNA. Analysis of the presence of Ψs within the rRNA peptidyl transferase center (PTC) of the mutants demonstrated that Cbf5 but not Pus10 is required for rRNA modification. Our data reveal that, in contrast to Nop10, Gar1 is crucial for in vivo and in vitro RNA guide-independent formation of Ψ2607 (Ψ2603 in P. abyssi) by Cbf5. Furthermore, our data indicate that pseudouridylation at orphan position 2589 (2585 in P. abyssi), for which no PUS or guide sRNA has been identified so far, relies on RNA- and Gar1-dependent activity of Cbf5.
    Mots-clés : ARCHEE, conserved pseudouridine, crystal-structure, escherichia-coli, haloferax-volcanii, hyperthermophilic archaeon, in-vivo, MICROBIO, pseudouridine synthase, substrate rna, sulfolobus-solfataricus, thermococcus-kodakaraensis kod1.

  • S. Gill, R. Catchpole, et P. Forterre, « Extracellular membrane vesicles (EVs) in the three domains of life and beyond », FEMS microbiology reviews, nov. 2018.
    Résumé : Cells from all three domains of life, Archaea, Bacteria and Eukarya, produce extracellular vesicles (EVs) which are sometimes associated to filamentous structures known as nanopods or nanotubes. The mechanisms of EV biogenesis in the three domains remain poorly understood, although studies in Bacteria and Eukarya indicate that the regulation of lipid composition plays a major role in initiating membrane curvature. EVs are increasingly recognized as important mediators of intercellular communication via transfer of a wide variety of molecular cargoes. They have been implicated in many aspects of cell physiology such as stress response, inter-cellular competition, lateral gene transfer (via RNA or DNA), pathogenicity, and detoxification. Their role in various human pathologies and aging has aroused much interest in recent years. EVs can be used as decoys against viral attack but virus infected cells also produce EVs that boost viral infection. Here, we review current knowledge on EVs in the three domains of life and their interactions with the viral world.
    Mots-clés : ARCHEE, MICROBIO.

  • A. Gorlas, P. Jacquemot, J. - M. Guigner, S. Gill, P. Forterre, et F. Guyot, « Greigite nanocrystals produced by hyperthermophilic archaea of Thermococcales order », Plos One, vol. 13, nᵒ 8, p. e0201549, août 2018.
    Résumé : Interactions between hyperthermophilic archaea and minerals occur in hydrothermal deep-ea vents, one of the most extreme environments for life on Earth. These interactions occur in the internal pores and at surfaces of active hydrothermal chimneys. In this study, we show that, at 85 degrees C, Thermococcales, the predominant hyperthermophilic microorganisms inhabiting hot parts of hydrothermal deep-sea vents, produce greigite nanocrystals (Fe3S4) on extracellular polymeric substances, and that an amorphous iron phosphate acts as a precursor phase. Greigite, although a minor component of chimneys, is a recognized catalyst for CO2 reduction thus implying that Thermococcales may influence the balance of CO2 in hydrothermal ecosystems. We propose that observation of greigite nanocrystals on extracellular polymeric substances could provide a signature of hyperthermophilic life in hydrothermal deep-sea vents.
    Mots-clés : ARCHEE, biomineralization, DBG, growth, iron sulfides, life, mechanisms, MICROBIO, mineralization, minerals, oxidizing bacteria, RBA, sequence, vesicles.

  • Y. Ishino, M. Krupovic, et P. Forterre, « History of CRISPR-Cas from Encounter with a Mysterious Repeated Sequence to Genome Editing Technology », Journal of Bacteriology, vol. 200, nᵒ 7, avr. 2018.
    Résumé : Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems are well-known acquired immunity systems that are widespread in archaea and bacteria. The RNA-guided nucleases from CRISPR-Cas systems are currently regarded as the most reliable tools for genome editing and engineering. The first hint of their existence came in 1987, when an unusual repetitive DNA sequence, which subsequently was defined as a CRISPR, was discovered in theEscherichia coligenome during an analysis of genes involved in phosphate metabolism. Similar sequence patterns were then reported in a range of other bacteria as well as in halophilic archaea, suggesting an important role for such evolutionarily conserved clusters of repeated sequences. A critical step toward functional characterization of the CRISPR-Cas systems was the recognition of a link between CRISPRs and the associated Cas proteins, which were initially hypothesized to be involved in DNA repair in hyperthermophilic archaea. Comparative genomics, structural biology, and advanced biochemistry could then work hand in hand, not only culminating in the explosion of genome editing tools based on CRISPR-Cas9 and other class II CRISPR-Cas systems but also providing insights into the origin and evolution of this system from mobile genetic elements denoted casposons. To celebrate the 30th anniversary of the discovery of CRISPR, this minireview briefly discusses the fascinating history of CRISPR-Cas systems, from the original observation of an enigmatic sequence inE. colito genome editing in humans.
    Mots-clés : archaea, ARCHEE, casposon, genome editing, MICROBIO, repeated sequence.

  • L. Meyer, G. Coste, S. Sommer, J. Oberto, F. Confalonieri, P. Servant, et C. Pasternak, « DdrI, a CRP family member, acts as a major regulator for adaptation of Deinococcus radiodurans to various stresses », Journal of Bacteriology, avr. 2018.
    Résumé : The DNA damage response gene ddrI encodes a transcription regulator belonging to the CRP (cAMP Receptor Protein) family. Cells devoid of the DdrI protein exhibit a pleiotropic phenotype, including growth defects, sensitivity to DNA damaging agents and to oxidative stress. Here, we show that the absence of DdrI protein also confers sensitivity to heat shock treatment and several genes involved in heat shock response were shown to be up-regulated in a DdrI dependent manner. Interestingly, expression of the E. coli CRP protein partially compensates the absence of the DdrI protein. Microscopic observations of ΔddrI mutant cells revealed an increased proportion of two-tetrads and anucleated cells in the population as compared to the wild-type strain, indicating that DdrI is crucial for completion of cell division and/or chromosome segregation. We show that DdrI is also involved in the megaplasmid MP1 stability and in efficient plasmid transformation by facilitating the maintenance of the incoming plasmid in the cell. The in silico prediction of putative DdrI binding sites in the D. radiodurans genome suggests that hundreds of genes, belonging to several functional groups, may be regulated by DdrI. In addition, DdrI protein absolutely requires cAMP for in vitro binding to specific target sequences, and acts as a dimer. All these data underline the major role of DdrI for D. radiodurans physiology under normal and stress conditions by regulating, both directly and indirectly, a cohort of genes involved in various cellular processes including central metabolism and specific responses to diverse harmful environments.IMPORTANCEDeinococcus radiodurans has been extensively studied to elucidate the molecular mechanisms responsible for its exceptional ability to withstand lethal effects of various DNA-damaging agents. A complex network, including efficient DNA repair, protein protection against oxidation, as well as diverse metabolic pathways, plays a crucial role for its radioresistance. The regulatory networks orchestrating these various pathways are still missing. Our data provide new insights in the crucial contribution of the transcription factor DdrI for the D. radiodurans ability to withstand harmful conditions, including UV radiation, mitomycin C treatment, heat shock and oxidative stress. Finally, we highlight that DdrI is also required for accurate cell division, for maintenance of plasmid replicons, and for central metabolism processes responsible for the overall cell physiology.
    Mots-clés : ARCHEE, DBG, MICROBIO, RBA.

  • A. Pichard-Kostuch, W. Zhang, D. Liger, M. - C. Daugeron, J. Létoquart, I. Li de la Sierra-Gallay, P. Forterre, B. Collinet, H. van Tilbeurgh, et T. Basta, « Structure-function analysis of Sua5 protein reveals novel functional motifs required for the biosynthesis of the universal t6A tRNA modification », RNA (New York, N.Y.), vol. 24, nᵒ 7, p. 926-938, juill. 2018.
    Résumé : N6-threonyl-carbamoyl adenosine (t6A) is a universal tRNA modification found at position 37, next to the anticodon, in almost all tRNAs decoding ANN codons (where N = A, U, G, or C). t6A stabilizes the codon-anticodon interaction and hence promotes translation fidelity. The first step of the biosynthesis of t6A, the production of threonyl-carbamoyl adenylate (TC-AMP), is catalyzed by the Sua5/TsaC family of enzymes. While TsaC is a single domain protein, Sua5 enzymes are composed of the TsaC-like domain, a linker and an extra domain called SUA5 of unknown function. In the present study, we report structure-function analysis of Pyrococcus abyssi Sua5 (Pa-Sua5). Crystallographic data revealed binding sites for bicarbonate substrate and pyrophosphate product. The linker of Pa-Sua5 forms a loop structure that folds into the active site gorge and closes it. Using structure-guided mutational analysis, we established that the conserved sequence motifs in the linker and the domain-domain interface are essential for the function of Pa-Sua5. We propose that the linker participates actively in the biosynthesis of TC-AMP by binding to ATP/PPi and by stabilizing the N-carboxy-l-threonine intermediate. Hence, TsaC orthologs which lack such a linker and SUA5 domain use a different mechanism for TC-AMP synthesis.
    Mots-clés : anticodon, ARCHEE, B3S, codon, conservation, crystal-structure, escherichia-coli, FAAM, keops-complex, MICROBIO, modified nucleosides, Sua5, t(6)A(37), t6A37, threonylcarbamoyl adenosine, threonylcarbamoyladenosine t(6)a, translation, tRNA modification, TsaC, yeaz-yjee complex.


  • D. A. Braun, J. Rao, G. Mollet, D. Schapiro, M. - C. Daugeron, W. Tan, O. Gribouval, O. Boyer, P. Revy, T. Jobst-Schwan, J. M. Schmidt, J. A. Lawson, D. Schanze, S. Ashraf, J. F. P. Ullmann, C. A. Hoogstraten, N. Boddaert, B. Collinet, G. Martin, D. Liger, S. Lovric, M. Furlano, I. C. Guerrera, O. Sanchez-Ferras, J. F. Hu, A. - C. Boschat, S. Sanquer, B. Menten, S. Vergult, N. De Rocker, M. Airik, T. Hermle, S. Shril, E. Widmeier, H. Y. Gee, W. - I. Choi, C. E. Sadowski, W. L. Pabst, J. K. Warejko, A. Daga, T. Basta, V. Matejas, K. Scharmann, S. D. Kienast, B. Behnam, B. Beeson, A. Begtrup, M. Bruce, G. - S. Ch'ng, S. - P. Lin, J. - H. Chang, C. - H. Chen, M. T. Cho, P. M. Gaffney, P. E. Gipson, C. - H. Hsu, J. A. Kari, Y. - Y. Ke, C. Kiraly-Borri, W. - M. Lai, E. Lemyre, R. O. Littlejohn, A. Masri, M. Moghtaderi, K. Nakamura, F. Ozaltin, M. Praet, C. Prasad, A. Prytula, E. R. Roeder, P. Rump, R. E. Schnur, T. Shiihara, M. D. Sinha, N. A. Soliman, K. Soulami, D. A. Sweetser, W. - H. Tsai, J. - D. Tsai, R. Topaloglu, U. Vester, D. H. Viskochil, N. Vatanavicharn, J. L. Waxler, K. J. Wierenga, M. T. F. Wolf, S. - N. Wong, S. A. Leidel, G. Truglio, P. C. Dedon, A. Poduri, S. Mane, R. P. Lifton, M. Bouchard, P. Kannu, D. Chitayat, D. Magen, B. Callewaert, H. van Tilbeurgh, M. Zenker, C. Antignac, et F. Hildebrandt, « Mutations in KEOPS-complex genes cause nephrotic syndrome with primary microcephaly », Nature Genetics, août 2017.
    Résumé : Galloway-Mowat syndrome (GAMOS) is an autosomal-recessive disease characterized by the combination of early-onset nephrotic syndrome (SRNS) and microcephaly with brain anomalies. Here we identified recessive mutations in OSGEP, TP53RK, TPRKB, and LAGE3, genes encoding the four subunits of the KEOPS complex, in 37 individuals from 32 families with GAMOS. CRISPR-Cas9 knockout in zebrafish and mice recapitulated the human phenotype of primary microcephaly and resulted in early lethality. Knockdown of OSGEP, TP53RK, or TPRKB inhibited cell proliferation, which human mutations did not rescue. Furthermore, knockdown of these genes impaired protein translation, caused endoplasmic reticulum stress, activated DNA-damage-response signaling, and ultimately induced apoptosis. Knockdown of OSGEP or TP53RK induced defects in the actin cytoskeleton and decreased the migration rate of human podocytes, an established intermediate phenotype of SRNS. We thus identified four new monogenic causes of GAMOS, describe a link between KEOPS function and human disease, and delineate potential pathogenic mechanisms.
    Mots-clés : ARCHEE, B3S, FAAM, MICROBIO.

  • M. Cossu, C. Badel, R. Catchpole, D. Gadelle, E. Marguet, V. Barbe, P. Forterre, et J. Oberto, « Flipping chromosomes in deep-sea archaea », PLoS genetics, vol. 13, nᵒ 6, p. e1006847, juin 2017.
    Résumé : One of the major mechanisms driving the evolution of all organisms is genomic rearrangement. In hyperthermophilic Archaea of the order Thermococcales, large chromosomal inversions occur so frequently that even closely related genomes are difficult to align. Clearly not resulting from the native homologous recombination machinery, the causative agent of these inversions has remained elusive. We present a model in which genomic inversions are catalyzed by the integrase enzyme encoded by a family of mobile genetic elements. We characterized the integrase from Thermococcus nautili plasmid pTN3 and showed that besides canonical site-specific reactions, it catalyzes low sequence specificity recombination reactions with the same outcome as homologous recombination events on DNA segments as short as 104bp both in vitro and in vivo, in contrast to other known tyrosine recombinases. Through serial culturing, we showed that the integrase-mediated divergence of T. nautili strains occurs at an astonishing rate, with at least four large-scale genomic inversions appearing within 60 generations. Our results and the ubiquitous distribution of pTN3-like integrated elements suggest that a major mechanism of evolution of an entire order of Archaea results from the activity of a selfish mobile genetic element.
    Mots-clés : ARCHEE, MICROBIO.

  • V. Da Cunha, M. Gaia, D. Gadelle, A. Nasir, et P. Forterre, « Lokiarchaea are close relatives of Euryarchaeota, not bridging the gap between prokaryotes and eukaryotes », PLoS genetics, vol. 13, nᵒ 6, p. e1006810, juin 2017.
    Résumé : 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.
    Mots-clés : Archaeal Proteins, ARCHEE, Eukaryota, Euryarchaeota, Evolution, Molecular, MICROBIO, Phylogeny, Prokaryotic Cells.

  • N. Dautin, C. de Sousa-d'Auria, F. Constantinesco-Becker, C. Labarre, J. Oberto, I. L. de la Sierra-Gallay, C. Dietrich, H. Issa, C. Houssin, et N. Bayan, « Mycoloyltransferases: A large and major family of enzymes shaping the cell envelope of Corynebacteriales », Biochimica et Biophysica Acta (BBA) - General Subjects, vol. 1861, nᵒ 1 Pt B, p. 3581-3592, janv. 2017.
    Résumé : Mycobacterium and Corynebacterium are important genera of the Corynebacteriales order, the members of which are characterized by an atypical diderm cell envelope. Indeed the cytoplasmic membrane of these bacteria is surrounded by a thick mycolic acid-arabinogalactan-peptidoglycan (mAGP) covalent polymer. The mycolic acid-containing part of this complex associates with other lipids (mainly trehalose monomycolate (TMM) and trehalose dimycolate (TDM)) to form an outer membrane. The metabolism of mycolates in the cell envelope is governed by esterases called mycoloyltransferases that catalyze the transfer of mycoloyl chains from TMM to another TMM molecule or to other acceptors such as the terminal arabinoses of arabinogalactan or specific polypeptides. In this review we present an overview of this family of Corynebacteriales enzymes, starting with their expression, localization, structure and activity to finally discuss their putative functions in the cell. In addition, we show that Corynebacteriales possess multiple mycoloyltransferases encoding genes in their genome. The reason for this multiplicity is not known, as their function in mycolates biogenesis appear to be only partially redundant. It is thus possible that, in some species living in specific environments, some mycoloyltransferases have evolved to gain some new functions. In any case, the few characterized mycoloyltransferases are very important for the bacterial physiology and are also involved in adaptation in the host where they constitute major secreted antigens. Although not discussed in this review, all these functions make them interesting targets for the discovery of new antibiotics and promising vaccines candidates. This article is part of a Special Issue entitled "Science for Life" Guest Editor: Dr. Austen Angell, Dr. Salvatore Magazù and Dr. Federica Migliardo.
    Mots-clés : Antigen 85, ARCHEE, CORYNE, Esterase, Fibronectin-binding protein, MICROBIO, Mycobacterium, Mycolyltransferases, Mycomembrane.

  • P. Forterre, « Viruses in the 21st Century: From the Curiosity-Driven Discovery of Giant Viruses to New Concepts and Definition of Life », Clinical Infectious Diseases: An Official Publication of the Infectious Diseases Society of America, vol. 65, nᵒ suppl_1, p. S74-S79, août 2017.
    Résumé : The curiosity-driven discovery of giant DNA viruses infecting amoebas has triggered an intense debate about the origin, nature, and definition of viruses. This discovery was delayed by the current paradigm confusing viruses with small virions. Several new definitions and concepts have been proposed either to reconcile the unique features of giant viruses with previous paradigms or to propose a completely new vision of the living world. I briefly review here how several other lines of research in virology converged during the last 2 decades with the discovery of giant viruses to change our traditional perception of the viral world. This story emphasizes the power of multidisciplinary curiosity-driven research, from the hospital to the field and the laboratory. Notably, some philosophers have now also joined biologists in their quest to make sense of the abundance and diversity of viruses and related capsidless mobile elements in the biosphere.
    Mots-clés : ARCHEE, giant viruses, Life definition, MICROBIO, phage, Virion, Virocell.

  • P. Forterre, V. Da Cunha, et R. Catchpole, « Plasmid vesicles mimicking virions », Nature Microbiology, vol. 2, nᵒ 10, p. 1340-1341, oct. 2017.

  • D. Kazlauskas, G. Sezonov, N. Charpin, Č. Venclovas, P. Forterre, et M. Krupovic, « Novel families of archaeo-eukaryotic primases in mobile genetic elements of Bacteria and Archaea », Journal of Molecular Biology, nov. 2017.
    Résumé : Cellular organisms in different domains of life employ structurally unrelated, non-homologous DNA primases for synthesis of a primer for DNA replication. Archaea and eukaryotes encode enzymes of the archaeo-eukaryotic primase (AEP) superfamily, whereas bacteria uniformly use primases of the DnaG family. However, AEP genes are widespread in bacterial genomes raising questions regarding their provenance and function. Here, using an archaeal primase-polymerase PolpTN2 encoded by pTN2 plasmid as a seed for sequence similarity searches, we recovered over 800 AEP homologs from bacteria belonging to twelve highly diverse phyla. These sequences formed a supergroup, PrimPol-PV1, and could be classified into five novel AEP families which are characterized by a conserved motif containing an arginine residue likely to be involved in nucleotide binding. Functional assays confirm the essentiality of this motif for catalytic activity of the PolpTN2 primase-polymerase. Further analyses showed that bacterial AEP display a range of domain organizations and uncovered several candidates for novel families of helicases. Furthermore, sequence and structure comparisons suggest that PriCT-1 and PriCT-2 domains frequently fused to the AEP domains are related to each other as well as to the non-catalytic, large subunit of archaeal and eukaryotic primases, and to the recently discovered PriX subunit of archaeal primases. Finally, genomic neighborhood analysis indicates that the identified AEP encoded in bacterial genomes are nearly-exclusively associated with highly diverse integrated mobile genetic elements, including integrative conjugative plasmids and prophages.
    Mots-clés : ARCHEE, DNA replication, evolution, helicases, MICROBIO, structural modeling, Thermococcus plasmids.

  • D. Prangishvili, D. H. Bamford, P. Forterre, J. Iranzo, E. V. Koonin, et M. Krupovic, « The enigmatic archaeal virosphere », Nature Reviews. Microbiology, vol. 15, nᵒ 12, p. 724-739, nov. 2017.
    Résumé : One of the most prominent features of archaea is the extraordinary diversity of their DNA viruses. Many archaeal viruses differ substantially in morphology from bacterial and eukaryotic viruses and represent unique virus families. The distinct nature of archaeal viruses also extends to the gene composition and architectures of their genomes and the properties of the proteins that they encode. Environmental research has revealed prominent roles of archaeal viruses in influencing microbial communities in ocean ecosystems, and recent metagenomic studies have uncovered new groups of archaeal viruses that infect extremophiles and mesophiles in diverse habitats. In this Review, we summarize recent advances in our understanding of the genomic and morphological diversity of archaeal viruses and the molecular biology of their life cycles and virus-host interactions, including interactions with archaeal CRISPR-Cas systems. We also examine the potential origins and evolution of archaeal viruses and discuss their place in the global virosphere.
    Mots-clés : ARCHEE, MICROBIO.

  • M. Redrejo-Rodríguez, C. D. Ordóñez, M. Berjón-Otero, J. Moreno-González, C. Aparicio-Maldonado, P. Forterre, M. Salas, et M. Krupovic, « Primer-Independent DNA Synthesis by a Family B DNA Polymerase from Self-Replicating Mobile Genetic Elements », Cell Reports, vol. 21, nᵒ 6, p. 1574-1587, nov. 2017.
    Résumé : Family B DNA polymerases (PolBs) play a central role during replication of viral and cellular chromosomes. Here, we report the discovery of a third major group of PolBs, which we denote primer-independent PolB (piPolB), that might be a link between the previously known protein-primed and RNA/DNA-primed PolBs. PiPolBs are encoded by highly diverse mobile genetic elements, pipolins, integrated in the genomes of diverse bacteria and also present as circular plasmids in mitochondria. Biochemical characterization showed that piPolB displays efficient DNA polymerization activity that can use undamaged and damaged templates and is endowed with proofreading and strand displacement capacities. Remarkably, the protein is also capable of template-dependent de novo DNA synthesis, i.e., DNA-priming activity, thereby breaking the long-standing dogma that replicative DNA polymerases require a pre-existing primer for DNA synthesis. We suggest that piPolBs are involved in self-replication of pipolins and may also contribute to bacterial DNA damage tolerance.
    Mots-clés : ARCHEE, de novo DNA synthesis, DNA Damage, DNA replication, family B DNA polymerase, MICROBIO, primer-independent DNA synthesis, self-replicating mobile element, translesion synthesis.


  • P. Béguin, N. Charpin, E. V. Koonin, P. Forterre, et M. Krupovic, « Casposon integration shows strong target site preference and recapitulates protospacer integration by CRISPR-Cas systems », Nucleic Acids Research, vol. 44, nᵒ 21, p. 10367-10376, déc. 2016.
    Résumé : Casposons are a recently discovered group of large DNA transposons present in diverse bacterial and archaeal genomes. For integration into the host chromosome, casposons employ an endonuclease that is homologous to the Cas1 protein involved in protospacer integration by the CRISPR-Cas adaptive immune system. Here we describe the site-preference of integration by the Cas1 integrase (casposase) encoded by the casposon of the archaeon Aciduliprofundum boonei Oligonucleotide duplexes derived from the terminal inverted repeats (TIR) of the A. boonei casposon as well as mini-casposons flanked by the TIR inserted preferentially at a site reconstituting the original A. boonei target site. As in the A. boonei genome, the insertion was accompanied by a 15-bp direct target site duplication (TSD). The minimal functional target consisted of the 15-bp TSD segment and the adjacent 18-bp sequence which comprises the 3' end of the tRNA-Pro gene corresponding to the TΨC loop. The functional casposase target site bears clear resemblance to the leader sequence-repeat junction which is the target for protospacer integration catalyzed by the Cas1-Cas2 adaptation module of CRISPR-Cas. These findings reinforce the mechanistic similarities and evolutionary connection between the casposons and the adaptation module of the prokaryotic adaptive immunity systems.
    Mots-clés : ARCHEE, MICROBIO.

  • N. Dahmane, D. Gadelle, S. Delmas, A. Criscuolo, S. Eberhard, N. Desnoues, S. Collin, H. Zhang, Y. Pommier, P. Forterre, et G. Sezonov, « topIb, a phylogenetic hallmark gene of Thaumarchaeota encodes a functional eukaryote-like topoisomerase IB », Nucleic Acids Research, vol. 44, nᵒ 6, p. 2795-2805, avr. 2016.
    Résumé : Type IB DNA topoisomerases can eliminate torsional stresses produced during replication and transcription. These enzymes are found in all eukaryotes and a short version is present in some bacteria and viruses. Among prokaryotes, the long eukaryotic version is only observed in archaea of the phylum Thaumarchaeota. However, the activities and the roles of these topoisomerases have remained an open question. Here, we demonstrate that all available thaumarchaeal genomes contain a topoisomerase IB gene that defines a monophyletic group closely related to the eukaryotic enzymes. We show that the topIB gene is expressed in the model thaumarchaeon Nitrososphaera viennensis and we purified the recombinant enzyme from the uncultivated thaumarchaeon Candidatus Caldiarchaeum subterraneum. This enzyme is active in vitro at high temperature, making it the first thermophilic topoisomerase IB characterized so far. We have compared this archaeal type IB enzyme to its human mitochondrial and nuclear counterparts. The archaeal enzyme relaxes both negatively and positively supercoiled DNA like the eukaryotic enzymes. However, its pattern of DNA cleavage specificity is different and it is resistant to camptothecins (CPTs) and non-CPT Top1 inhibitors, LMP744 and lamellarin D. This newly described thermostable topoisomerases IB should be a promising new model for evolutionary, mechanistic and structural studies.
    Mots-clés : Amino Acid Sequence, Archaea, Archaeal Proteins, ARCHEE, Camptothecin, Cloning, Molecular, Coumarins, DNA Topoisomerases, Type I, DNA, Superhelical, Escherichia coli, gene expression, Heterocyclic Compounds, 4 or More Rings, Hot Temperature, Humans, Isoquinolines, MICROBIO, Mitochondrial Proteins, Models, Molecular, Molecular Sequence Data, Phylogeny, Protein Stability, Protein Structure, Secondary, Protein Structure, Tertiary, Recombinant Proteins, Sequence Alignment, Topoisomerase I Inhibitors.

  • P. Forterre, « To be or not to be alive: How recent discoveries challenge the traditional definitions of viruses and life », Studies in History and Philosophy of Biological and Biomedical Sciences, vol. 59, p. 100-108, oct. 2016.
    Résumé : Three major discoveries have recently profoundly modified our perception of the viral world: molecular ecologists have shown that viral particles are more abundant than cells in natural environments; structural biologists have shown that some viruses from the three domains of life, Bacteria, Eukarya and Archaea, are evolutionarily related, and microbiologists have discovered giant viruses that rival with cells in terms of size and gene content. I discuss here the scientific and philosophical impact of these discoveries on the debates over the definition, nature (living or not), and origin of viruses. I suggest that viruses have often been considered non-living, because they are traditionally assimilated to their virions. However, the term virus describes a biological process and should integrate all aspects of the viral reproduction cycle. It is especially important to focus on the intracellular part of this cycle, the virocell, when viral information is actively expressed and reproduced, allowing the emergence of new viral genes. The virocell concept theoretically removes roadblocks that prevent defining viruses as living organisms. However, defining a "living organism" remains challenging, as indicated by the case of organelles that evolved from intracellular bacteria. To bypass this problem, I suggest considering that all biological entities that actively participate in the process of life are living.
    Mots-clés : ARCHEE, Bacteriophage, Life definition, MICROBIO, Organism, Virocell, Virus.

  • P. Forterre et M. Gaïa, « Giant viruses and the origin of modern eukaryotes », Current Opinion in Microbiology, vol. 31, p. 44-49, juin 2016.
    Résumé : Several authors have suggested that viruses from the NucleoCytoplasmic Large DNA Viruses group (NCLDV) have played an important role in the origin of modern eukaryotes. Notably, the viral eukaryogenesis theory posits that the nucleus originated from an ancient NCLDV-related virus. Focusing on the viral factory instead of the virion adds credit to this hypothesis, but also suggests alternative scenarios. Beside a role in the emergence of the nucleus, ancient NCLDV may have provided new genes and/or chromosomes to the proto-eukaryotic lineage. Phylogenetic analyses suggest that NCLDV informational proteins, related to those of Archaea and Eukarya, were either recruited by ancient NCLDV from proto-eukaryotes and/or transferred to proto-eukaryotes, in agreement with the antiquity of NCLDV and their possible role in eukaryogenesis.
    Mots-clés : ARCHEE, MICROBIO.

  • M. Krupovic, S. Shmakov, K. S. Makarova, P. Forterre, et E. V. Koonin, « Recent Mobility of Casposons, Self-Synthesizing Transposons at the Origin of the CRISPR-Cas Immunity », Genome Biology and Evolution, vol. 8, nᵒ 2, p. 375-386, janv. 2016.
    Résumé : Casposons are a superfamily of putative self-synthesizing transposable elements that are predicted to employ a homolog of Cas1 protein as a recombinase and could have contributed to the origin of the CRISPR-Cas adaptive immunity systems in archaea and bacteria. Casposons remain uncharacterized experimentally, except for the recent demonstration of the integrase activity of the Cas1 homolog, and given their relative rarity in archaea and bacteria, original comparative genomic analysis has not provided direct indications of their mobility. Here, we report evidence of casposon mobility obtained by comparison of the genomes of 62 strains of the archaeon Methanosarcina mazei. In these genomes, casposons are variably inserted in three distinct sites indicative of multiple, recent gains, and losses. Some casposons are inserted into other mobile genetic elements that might provide vehicles for horizontal transfer of the casposons. Additionally, many M. mazei genomes contain previously undetected solo terminal inverted repeats that apparently are derived from casposons and could resemble intermediates in CRISPR evolution. We further demonstrate the sequence specificity of casposon insertion and note clear parallels with the adaptation mechanism of CRISPR-Cas. Finally, besides identifying additional representatives in each of the three originally defined families, we describe a new, fourth, family of casposons.
    Mots-clés : Archaeal Proteins, ARCHEE, Base Sequence, casposons, Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR-Cas, DNA Transposable Elements, Endodeoxyribonucleases, Gene Transfer, Horizontal, Genome, Archaeal, Methanosarcina barkeri, MICROBIO, mobile genetic elements, Molecular Sequence Data, Phylogeny, self-synthesizing transposons, transposition.

  • E. R. J. Quemin, P. Chlanda, M. Sachse, P. Forterre, D. Prangishvili, et M. Krupovic, « Eukaryotic-Like Virus Budding in Archaea », mBio, vol. 7, nᵒ 5, sept. 2016.
    Résumé : Similar to many eukaryotic viruses (and unlike bacteriophages), viruses infecting archaea are often encased in lipid-containing envelopes. However, the mechanisms of their morphogenesis and egress remain unexplored. Here, we used dual-axis electron tomography (ET) to characterize the morphogenesis of Sulfolobus spindle-shaped virus 1 (SSV1), the prototype of the family Fuselloviridae and representative of the most abundant archaea-specific group of viruses. Our results show that SSV1 assembly and egress are concomitant and occur at the cellular cytoplasmic membrane via a process highly reminiscent of the budding of enveloped viruses that infect eukaryotes. The viral nucleoprotein complexes are extruded in the form of previously unknown rod-shaped intermediate structures which have an envelope continuous with the host membrane. Further maturation into characteristic spindle-shaped virions takes place while virions remain attached to the cell surface. Our data also revealed the formation of constricted ring-like structures which resemble the budding necks observed prior to the ESCRT machinery-mediated membrane scission during egress of various enveloped viruses of eukaryotes. Collectively, we provide evidence that archaeal spindle-shaped viruses contain a lipid envelope acquired upon budding of the viral nucleoprotein complex through the host cytoplasmic membrane. The proposed model bears a clear resemblance to the egress strategy employed by enveloped eukaryotic viruses and raises important questions as to how the archaeal single-layered membrane composed of tetraether lipids can undergo scission. IMPORTANCE: The replication of enveloped viruses has been extensively studied in eukaryotes but has remained unexplored for enveloped viruses infecting Archaea Here, we provide a sequential view on the assembly and egress of SSV1, a prototypic archaeal virus. The observed process is highly similar to the budding of eukaryotic enveloped viruses, including human immunodeficiency virus, influenza virus, and Ebola virus. The present study is the first to characterize such a phenomenon in archeal cells, showing that membrane budding is not an exclusive feature of eukaryotic viruses. Our results provide significant insights into the biogenesis and architecture of unique, spindle-shaped virions that infect archaea. Furthermore, our findings open doors for future inquiries into (i) the evolution of the virus budding process, (ii) mechanistic details of virus-mediated membrane scission in Archaea, and (iii) elucidation of virus- and host-encoded molecular players responsible for archaeal membrane and surface remodeling.
    Mots-clés : ARCHEE, MICROBIO.

  • G. Richarme, E. Marguet, P. Forterre, S. Ishino, et Y. Ishino, « DJ-1 family Maillard deglycases prevent acrylamide formation », Biochemical and Biophysical Research Communications, vol. 478, nᵒ 3, p. 1111-1116, sept. 2016.
    Résumé : The presence of acrylamide in food is a worldwide concern because it is carcinogenic, reprotoxic and neurotoxic. Acrylamide is generated in the Maillard reaction via condensation of reducing sugars and glyoxals arising from their decomposition, with asparagine, the amino acid forming the backbone of the acrylamide molecule. We reported recently the discovery of the Maillard deglycases (DJ-1/Park7 and its prokaryotic homologs) which degrade Maillard adducts formed between glyoxals and lysine or arginine amino groups, and prevent glycation damage in proteins. Here, we show that these deglycases prevent acrylamide formation, likely by degrading asparagine/glyoxal Maillard adducts. We also report the discovery of a deglycase from the hyperthermophilic archaea Pyrococcus furiosus, which prevents acrylamide formation at 100 °C. Thus, Maillard deglycases constitute a unique enzymatic method to prevent acrylamide formation in food without depleting the components (asparagine and sugars) responsible for its formation.
    Mots-clés : Acrylamide, Advanced glycation endproducts, AGEs, ARCHEE, Asparagine, cancer, Food polluant, Fructose, Glucose, Glycation, Glyoxal, Maillard reaction, Methylglyoxal, MICROBIO.

  • S. Roux, F. Enault, V. Ravet, J. Colombet, Y. Bettarel, J. - C. Auguet, T. Bouvier, S. Lucas-Staat, A. Vellet, D. Prangishvili, P. Forterre, D. Debroas, et T. Sime-Ngando, « Analysis of metagenomic data reveals common features of halophilic viral communities across continents », Environmental Microbiology, vol. 18, nᵒ 3, p. 889-903, mars 2016.
    Résumé : Microbial communities from hypersaline ponds, dominated by halophilic archaea, are considered specific of such extreme conditions. The associated viral communities have accordingly been shown to display specific features, such as similar morphologies among different sites. However, little is known about the genetic diversity of these halophilic viral communities across the Earth. Here, we studied viral communities in hypersaline ponds sampled on the coast of Senegal (8-36% of salinity) using metagenomics approach, and compared them with hypersaline viromes from Australia and Spain. The specificity of hyperhalophilic viruses could first be demonstrated at a community scale, salinity being a strong discriminating factor between communities. For the major viral group detected in all samples (Caudovirales), only a limited number of halophilic Caudovirales clades were highlighted. These clades gather viruses from different continents and display consistent genetic composition, indicating that they represent related lineages with a worldwide distribution. Non-tailed hyperhalophilic viruses display a greater rate of gene transfer and recombination, with uncharacterized genes conserved across different kind of viruses and plasmids. Thus, hypersaline viral communities around the world appear to form a genetically consistent community that are likely to harbour new genes coding for enzymes specifically adapted to these environments.
    Mots-clés : ARCHEE, Australia, Caudovirales, Chromosome Mapping, Genetic Variation, Genome, Viral, metagenomics, MICROBIO, Ponds, Salinity, Senegal, Spain.


  • P. Béguin, S. Gill, N. Charpin, et P. Forterre, « Synergistic template-free synthesis of dsDNA by Thermococcus nautili primase PolpTN2, DNA polymerase PolB, and pTN2 helicase », Extremophiles: Life Under Extreme Conditions, vol. 19, nᵒ 1, p. 69-76, janv. 2015.
    Résumé : A combination of three enzymes from the hyperthermophilic archaeon Thermococcus nautili, DNA primase PolpTN2, DNA polymerase PolB, and pTN2 DNA helicase, was found to synthesize up to 300-400 ng/µl dsDNA from deoxynucleotide triphosphates in less than 30 min in the absence of added template DNA and oligonucleotide primer. The reaction did not occur below 64 °C. No synthesis was observed if PolpTN2 or PolB were left out; helicase was not essential but accelerated the reaction. The DNA synthesized consisted of highly reiterated palindromic sequences reaching up to more that 10 kb. Sequence analysis of three independent reaction products synthesized at different temperatures showed that the palindromes shared a common pentanucleotide core, suggesting that random nucleic acid fragments were not responsible for priming the reaction. When enzymes were added sequentially, preincubation with primase plus helicase followed by PolB led to a shorter delay before the onset of the reaction as compared to preincubation with PolB plus helicase followed by primase. This suggests that the primase generates seeds that are subsequently amplified and elongated in synergy with PolB by a mechanism involving hairpin formation and slippage synthesis.
    Mots-clés : ARCHEE, Base Sequence, Cloning, Molecular, DNA, DNA Helicases, DNA Polymerase II, DNA Primase, DNA Primers, Electrophoresis, Agar Gel, MICROBIO, Micrococcal Nuclease, Molecular Sequence Data, Nucleic Acids, Temperature, Thermococcus.

  • D. Bréchemier-Baey, L. Domínguez-Ramírez, J. Oberto, et J. Plumbridge, « Operator recognition by the ROK transcription factor family members, NagC and Mlc », Nucleic Acids Research, vol. 43, nᵒ 1, p. 361-372, janv. 2015.
    Résumé : NagC and Mlc, paralogous members of the ROK family of proteins with almost identical helix-turn-helix DNA binding motifs, specifically regulate genes for transport and utilization of N-acetylglucosamine and glucose. We previously showed that two amino acids in a linker region outside the canonical helix-turn-helix motif are responsible for Mlc site specificity. In this work we identify four amino acids in the linker, which are required for recognition of NagC targets. These amino acids allow Mlc and NagC to distinguish between a C/G and an A/T bp at positions ±11 of the operators. One linker position, glycine in NagC and arginine in Mlc, corresponds to the major specificity determinant for the two proteins. In certain contexts it is possible to switch repression from Mlc-style to NagC-style, by interchanging this glycine and arginine. Secondary determinants are supplied by other linker positions or the helix-turn-helix motif. A wide genomic survey of unique ROK proteins shows that glycine- and arginine-rich sequences are present in the linkers of nearly all ROK family repressors. Conserved short sequence motifs, within the branches of the ROK evolutionary tree, suggest that these sequences could also be involved in operator recognition in other ROK family members.
    Mots-clés : Amino Acid Motifs, ARCHEE, Binding Sites, Escherichia coli Proteins, Gene Expression Regulation, Bacterial, MICROBIO, Mutation, Operator Regions, Genetic, Protein Binding, Repressor Proteins.

  • M. Cossu, V. Da Cunha, C. Toffano-Nioche, P. Forterre, et J. Oberto, « Comparative genomics reveals conserved positioning of essential genomic clusters in highly rearranged Thermococcales chromosomes », Biochimie, vol. 118, p. 313-321, nov. 2015.
    Résumé : The genomes of the 21 completely sequenced Thermococcales display a characteristic high level of rearrangements. As a result, the prediction of their origin and termination of replication on the sole basis of chromosomal DNA composition or skew is inoperative. Using a different approach based on biologically relevant sequences, we were able to determine oriC position in all 21 genomes. The position of dif, the site where chromosome dimers are resolved before DNA segregation could be predicted in 19 genomes. Computation of the core genome uncovered a number of essential gene clusters with a remarkably stable chromosomal position across species, in sharp contrast with the scrambled nature of their genomes. The active chromosomal reorganization of numerous genes acquired by horizontal transfer, mainly from mobile elements, could explain this phenomenon.
    Mots-clés : Archaea, ARCHEE, Base Sequence, Bioinformatics, Chromosomal landmarks, Chromosomes, Comparative Genomic Hybridization, DBG, Evolution, Molecular, Gene Rearrangement, Genes, Archaeal, Genome, Genome evolution, MICROBIO, Mobile elements, Molecular Sequence Data, SSFA, Thermococcales.

  • P. Forterre, « The universal tree of life: an update », Frontiers in Microbiology, vol. 6, p. 717, 2015.
    Résumé : Biologists used to draw schematic "universal" trees of life as metaphors illustrating the history of life. It is indeed a priori possible to construct an organismal tree connecting the three major domains of ribosome encoding organisms: Archaea, Bacteria and Eukarya, since they originated by cell division from LUCA. Several universal trees based on ribosomal RNA sequence comparisons proposed at the end of the last century are still widely used, although some of their main features have been challenged by subsequent analyses. Several authors have proposed to replace the traditional universal tree with a ring of life, whereas others have proposed more recently to include viruses as new domains. These proposals are misleading, suggesting that endosymbiosis can modify the shape of a tree or that viruses originated from the last universal common ancestor (LUCA). I propose here an updated version of Woese's universal tree that includes several rootings for each domain and internal branching within domains that are supported by recent phylogenomic analyses of domain specific proteins. The tree is rooted between Bacteria and Arkarya, a new name proposed for the clade grouping Archaea and Eukarya. A consensus version, in which each of the three domains is unrooted, and a version in which eukaryotes emerged within archaea are also presented. This last scenario assumes the transformation of a modern domain into another, a controversial evolutionary pathway. Viruses are not indicated in these trees but are intrinsically present because they infect the tree from its roots to its leaves. Finally, I present a detailed tree of the domain Archaea, proposing the sub-phylum neo-Euryarchaeota for the monophyletic group of euryarchaeota containing DNA gyrase. These trees, that will be easily updated as new data become available, could be useful to discuss controversial scenarios regarding early life evolution.
    Mots-clés : Archaea, ARCHEE, bacteria, eukarya, evolution, LUCA, MICROBIO, universal tree.

  • S. Gélis-Jeanvoine, S. Lory, J. Oberto, et N. Buddelmeijer, « Residues located on membrane-embedded flexible loops are essential for the second step of the apolipoprotein N-acyltransferase reaction », Molecular Microbiology, vol. 95, nᵒ 4, p. 692-705, févr. 2015.
    Résumé : Apolipoprotein N-acyltransferase (Lnt) is an essential membrane-bound enzyme that catalyzes the third and last step in the post-translational modification of bacterial lipoproteins. In order to identify essential residues implicated in substrate recognition and/or binding we screened for non-functional variants of Lnt obtained by error-prone polymerase chain reaction in a complementation assay using a lnt depletion strain. Mutations included amino acid substitutions in the active site and of residues located on flexible loops in the catalytic periplasmic domain. All, but one mutation, led to the formation of the thioester acyl-enzyme intermediate and to the accumulation of apo-Lpp, suggesting that these residues are involved in the second step of the reaction. A large cytoplasmic loop contains a highly conserved region and two hydrophobic segments. Accessibility analysis to alkylating reagents of substituted cysteine residues introduced in this region demonstrated that the hydrophobic segments do not completely span the membrane. Two residues in the highly conserved cytoplasmic region were shown to be essential for Lnt function. Together, our data suggest that amino acids located on flexible cytoplasmic and periplasmic loops, predicted to be membrane embedded, are required for efficient N-acylation of lipoproteins.
    Mots-clés : Acylation, Acyltransferases, Amino Acid Substitution, Apolipoproteins, ARCHEE, Catalytic Domain, Cysteine, Escherichia coli, Escherichia coli Proteins, Genetic Complementation Test, Lipoproteins, MICROBIO, Models, Molecular, Mutation, Phospholipids, Polymerase Chain Reaction, Protein Processing, Post-Translational.

  • M. Krupovic et P. Forterre, « Single-stranded DNA viruses employ a variety of mechanisms for integration into host genomes », Annals of the New York Academy of Sciences, vol. 1341, p. 41-53, avr. 2015.
    Résumé : Single-stranded DNA (ssDNA) viruses are widespread in the environment and include economically, medically, and ecologically important pathogens. Recently, it has been discovered that ssDNA virus genomes are also prevalent in the chromosomes of their bacterial, archaeal, and eukaryotic hosts. Sequences originating from viruses of the families Parvoviridae, Circoviridae, and Geminiviridae are particularly widespread in the genomes of eukaryotes, where they are often fossilized as endogenous viral elements. ssDNA viruses have evolved diverse mechanisms to invade cellular genomes, and these principally vary between viruses infecting bacteria/archaea and eukaryotes. Filamentous bacteriophages (Inoviridae) use at least three major mechanisms of integration. Some of these phages encode integrases of serine or tyrosine recombinase superfamilies, while others utilize DDE transposases of the IS3, IS30, or IS110/IS492 families, whereas some inoviruses, and possibly certain members of the Microviridae, hijack the host XerCD recombination machinery. By contrast, eukaryotic viruses for integration rely on the endonuclease activity of their rolling-circle replication-initiation proteins, mimicking the mechanisms used by some bacterial transposons. Certain bacterial and eukaryotic ssDNA viruses have embraced a transposon-like means of propagation, with occasionally dramatic effects on host genome evolution. Here, we review the diversity of experimentally verified and hypothetical mechanisms of genome integration employed by ssDNA viruses, and consider the evolutionary implications of these processes, particularly in the emergence of novel virus groups.
    Mots-clés : Archaea, ARCHEE, bacteria, DNA Viruses, DNA, Single-Stranded, Eukaryotic Cells, Evolution, Molecular, Genome, Viral, Host-Pathogen Interactions, Integrases, MICROBIO, Models, Genetic, ssDNA viruses, transposition, transposons, virus evolution, Virus Integration.

  • J. Lossouarn, S. Dupont, A. Gorlas, C. Mercier, N. Bienvenu, E. Marguet, P. Forterre, et C. Geslin, « An abyssal mobilome: viruses, plasmids and vesicles from deep-sea hydrothermal vents », Research in Microbiology, vol. 166, nᵒ 10, p. 742-752, déc. 2015.
    Résumé : Mobile genetic elements (MGEs) such as viruses, plasmids, vesicles, gene transfer agents (GTAs), transposons and transpovirions, which collectively represent the mobilome, interact with cellular organisms from all three domains of life, including those thriving in the most extreme environments. While efforts have been made to better understand deep-sea vent microbial ecology, our knowledge of the mobilome associated with prokaryotes inhabiting deep-sea hydrothermal vents remains limited. Here we focus on the abyssal mobilome by reviewing accumulating data on viruses, plasmids and vesicles associated with thermophilic and hyperthermophilic Bacteria and Archaea present in deep-sea hydrothermal vents.
    Mots-clés : (Hyper-)thermophiles, Archaea, ARCHEE, bacteria, Deep-sea hydrothermal vent, Hydrothermal Vents, Interspersed Repetitive Sequences, MICROBIO, Mobilome, Phylogeny, Plasmids, Seawater, Viruses.

  • L. Mora, K. Moncoq, P. England, J. Oberto, et M. de Zamaroczy, « The Stable Interaction Between Signal Peptidase LepB of Escherichia coli and Nuclease Bacteriocins Promotes Toxin Entry into the Cytoplasm », The Journal of Biological Chemistry, vol. 290, nᵒ 52, p. 30783-30796, déc. 2015.
    Résumé : LepB is a key membrane component of the cellular secretion machinery, which releases secreted proteins into the periplasm by cleaving the inner membrane-bound leader. We showed that LepB is also an essential component of the machinery hijacked by the tRNase colicin D for its import. Here we demonstrate that this non-catalytic activity of LepB is to promote the association of the central domain of colicin D with the inner membrane before the FtsH-dependent proteolytic processing and translocation of the toxic tRNase domain into the cytoplasm. The novel structural role of LepB results in a stable interaction with colicin D, with a stoichiometry of 1:1 and a nanomolar Kd determined in vitro. LepB provides a chaperone-like function for the penetration of several nuclease-type bacteriocins into target cells. The colicin-LepB interaction is shown to require only a short peptide sequence within the central domain of these bacteriocins and to involve residues present in the short C-terminal Box E of LepB. Genomic screening identified the conserved LepB binding motif in colicin-like ORFs from 13 additional bacterial species. These findings establish a new paradigm for the functional adaptability of an essential inner-membrane enzyme.
    Mots-clés : Amino Acid Motifs, Amino Acid Sequence, ARCHEE, Bacterial Toxins, bacteriocin, Bacteriocins, Biological Transport, colicin, Cytoplasm, Deoxyribonucleases, endonuclease, Escherichia coli, Escherichia coli Proteins, LepB, membrane, Membrane Proteins, MICROBIO, Microbiology, Molecular Sequence Data, protease, Protein Binding, protein import, Protein Structure, Tertiary, Ribonucleases, Sequence Alignment, Serine Endopeptidases, toxicity.

  • D. Prangishvili, S. Gribaldo, B. Franzetti, et P. Forterre, « Special section on Molecular biology of Archaea », Biochimie, vol. 118, p. 253, nov. 2015.
    Mots-clés : Archaea, ARCHEE, MICROBIO, Phylogeny.

  • E. R. J. Quemin, M. K. Pietilä, H. M. Oksanen, P. Forterre, W. I. C. Rijpstra, S. Schouten, D. H. Bamford, D. Prangishvili, et M. Krupovic, « Sulfolobus Spindle-Shaped Virus 1 Contains Glycosylated Capsid Proteins, a Cellular Chromatin Protein, and Host-Derived Lipids », Journal of Virology, vol. 89, nᵒ 22, p. 11681-11691, nov. 2015.
    Résumé : Geothermal and hypersaline environments are rich in virus-like particles, among which spindle-shaped morphotypes dominate. Currently, viruses with spindle- or lemon-shaped virions are exclusive to Archaea and belong to two distinct viral families. The larger of the two families, the Fuselloviridae, comprises tail-less, spindle-shaped viruses, which infect hosts from phylogenetically distant archaeal lineages. Sulfolobus spindle-shaped virus 1 (SSV1) is the best known member of the family and was one of the first hyperthermophilic archaeal viruses to be isolated. SSV1 is an attractive model for understanding virus-host interactions in Archaea; however, the constituents and architecture of SSV1 particles remain only partially characterized. Here, we have conducted an extensive biochemical characterization of highly purified SSV1 virions and identified four virus-encoded structural proteins, VP1 to VP4, as well as one DNA-binding protein of cellular origin. The virion proteins VP1, VP3, and VP4 undergo posttranslational modification by glycosylation, seemingly at multiple sites. VP1 is also proteolytically processed. In addition to the viral DNA-binding protein VP2, we show that viral particles contain the Sulfolobus solfataricus chromatin protein Sso7d. Finally, we provide evidence indicating that SSV1 virions contain glycerol dibiphytanyl glycerol tetraether (GDGT) lipids, resolving a long-standing debate on the presence of lipids within SSV1 virions. A comparison of the contents of lipids isolated from the virus and its host cell suggests that GDGTs are acquired by the virus in a selective manner from the host cytoplasmic membrane, likely during progeny egress. IMPORTANCE: Although spindle-shaped viruses represent one of the most prominent viral groups in Archaea, structural data on their virion constituents and architecture still are scarce. The comprehensive biochemical characterization of the hyperthermophilic virus SSV1 presented here brings novel and significant insights into the organization and architecture of spindle-shaped virions. The obtained data permit the comparison between spindle-shaped viruses residing in widely different ecological niches, improving our understanding of the adaptation of viruses with unusual morphotypes to extreme environmental conditions.
    Mots-clés : Amino Acid Sequence, Archaeal Proteins, ARCHEE, Capsid Proteins, DNA-Binding Proteins, Fuselloviridae, Genome, Viral, Glycosylation, Haloarcula, Host-Pathogen Interactions, Hydrophobic and Hydrophilic Interactions, Membrane Lipids, MICROBIO, Molecular Sequence Data, Sulfolobus solfataricus, Viral Proteins, Virus Assembly.

  • N. Soler, M. Krupovic, E. Marguet, et P. Forterre, « Membrane vesicles in natural environments: a major challenge in viral ecology », The ISME journal, vol. 9, nᵒ 4, p. 793-796, mars 2015.
    Mots-clés : ARCHEE, Carbon, Cell Membrane, DNA, Bacterial, MICROBIO, Prochlorococcus, Seawater.
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