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Accueil > Publications

Publications Département Biologie des Génomes

2019


  • A. Aliouat, I. Hatin, P. Bertin, P. François, V. Stierlé, O. Namy, S. Salhi, et O. Jean-Jean, « Divergent effects of translation termination factor eRF3A and nonsense-mediated mRNA decay factor UPF1 on the expression of uORF carrying mRNAs and ribosome protein genes », RNA biology, p. 1-13, oct. 2019.
    Résumé : In addition to its role in translation termination, eRF3A has been implicated in the nonsense-mediated mRNA decay (NMD) pathway through its interaction with UPF1. NMD is a RNA quality control mechanism, which detects and degrades aberrant mRNAs as well as some normal transcripts including those that harbour upstream open reading frames in their 5' leader sequence. In this study, we used RNA-sequencing and ribosome profiling to perform a genome wide analysis of the effect of either eRF3A or UPF1 depletion in human cells. Our bioinformatics analyses allow to delineate the features of the transcripts controlled by eRF3A and UPF1 and to compare the effect of each of these factors on gene expression. We find that eRF3A and UPF1 have very different impacts on the human transcriptome, less than 250 transcripts being targeted by both factors. We show that eRF3A depletion globally derepresses the expression of mRNAs containing translated uORFs while UPF1 knockdown derepresses only the mRNAs harbouring uORFs with an AUG codon in an optimal context for translation initiation. Finally, we also find that eRF3A and UPF1 have opposite effects on ribosome protein gene expression. Together, our results provide important elements for understanding the impact of translation termination and NMD on the human transcriptome and reveal novel determinants of ribosome biogenesis regulation.
    Mots-clés : DBG, eRF3, GSPT1, GST, nonsense-mediated mRNA decay, ribosome protein genes, translation termination, uORF, UPF1.

  • I. Altinoglu, C. J. Merrifield, et Y. Yamaichi, « Single molecule super-resolution imaging of bacterial cell pole proteins with high-throughput quantitative analysis pipeline », Scientific Reports, vol. 9, nᵒ 1, p. 6680, avr. 2019.
    Résumé : Bacteria show sophisticated control of their cellular organization, and many bacteria deploy different polar landmark proteins to organize the cell pole. Super-resolution microscopy, such as Photo-Activated Localization Microscopy (PALM), provides the nanoscale localization of molecules and is crucial for better understanding of organization and dynamics in single-molecule. However, analytical tools are not fully available yet, in particular for bacterial cell biology. For example, quantitative and statistical analyses of subcellular localization with multiple cells from multiple fields of view are lacking. Furthermore, brightfield images are not sufficient to get accurate contours of small and low contrast bacterial cells, compared to subpixel presentation of target molecules. Here we describe a novel analytic tool for PALM which integrates precisely drawn cell outlines, of either inner membrane or periplasm, labelled by PALM-compatible fluorescent protein fusions, with molecule data for >10,000 molecules from >100 cells by fitting each cell into an oval arc. In the vibrioid bacterium Vibrio cholerae, the polar anchor HubP constitutes a big polar complex which includes multiple proteins involved in chemotaxis and the flagellum. With this pipeline, HubP is shown to be slightly skewed towards the inner curvature side of the cell, while its interaction partners showed rather loose polar localization.
    Mots-clés : BIOCELL, DBG, EQYY.

  • M. Bakail, A. Gaubert, J. Andreani, G. Moal, G. Pinna, E. Boyarchuk, M. - C. Gaillard, R. Courbeyrette, C. Mann, J. - Y. Thuret, B. Guichard, B. Murciano, N. Richet, A. Poitou, C. Frederic, M. - H. Le Du, M. Agez, C. Roelants, Z. A. Gurard-Levin, G. Almouzni, N. Cherradi, R. Guerois, et F. Ochsenbein, « Design on a Rational Basis of High-Affinity Peptides Inhibiting the Histone Chaperone ASF1 », Cell Chemical Biology, sept. 2019.
    Résumé : Anti-silencing function 1 (ASF1) is a conserved H3-H4 histone chaperone involved in histone dynamics during replication, transcription, and DNA repair. Overexpressed in proliferating tissues including many tumors, ASF1 has emerged as a promising therapeutic target. Here, we combine structural, computational, and biochemical approaches to design peptides that inhibit the ASF1-histone interaction. Starting from the structure of the human ASF1-histone complex, we developed a rational design strategy combining epitope tethering and optimization of interface contacts to identify a potent peptide inhibitor with a dissociation constant of 3 nM. When introduced into cultured cells, the inhibitors impair cell proliferation, perturb cell-cycle progression, and reduce cell migration and invasion in a manner commensurate with their affinity for ASF1. Finally, we find that direct injection of the most potent ASF1 peptide inhibitor in mouse allografts reduces tumor growth. Our results open new avenues to use ASF1 inhibitors as promising leads for cancer therapy.
    Mots-clés : AMIG, B3S, Cancer, Cell Penetrating Peptide, Chromatin, DBG, Drug Design, Epigenetics, INTGEN, PARI, Peptide Inhibitor, PF, Protein Binding, Protein-Protein Interaction, Rosetta Design, SEN, X-Ray Crystallography.

  • A. Briquet, R. Vong, J. - B. Roseau, E. Javelle, N. Cazes, F. Rivière, M. Aletti, M. - P. Otto, C. Ficko, S. Duron, M. Fabre, C. Pourcel, F. Simon, et C. Soler, « Clinical features of Mycobacterium canettii infection: a retrospective study of 20 cases among French soldiers and relatives », Clinical Infectious Diseases: An Official Publication of the Infectious Diseases Society of America, févr. 2019.
    Résumé : Background: Mycobacterium canettii forms part of the Mycobacterium tuberculosis complex. M. canettii infections are mainly described in the Horn of Africa. The permanent presence of French soldiers in Djibouti raises the question of the risk of being infected with M. canettii. Our study aims to describe M. canettii infections among French military or their families between 1998 and 2015. Methods: This retrospective study relied on 3 sources of data: the reference centre for mycobacteria in the Biology Department at Percy military hospital in Paris, the French Military Center for Epidemiology and Public Health, and the scientific literature. After an exhaustive census of the strains, we studied the epidemiological data on 20 cases among French soldiers and their families. Results: 20 cases of M. canettii infections are reported, including 5 unpublished cases. Adenitis predominates (n = 15), especially in the cervico facial area and among children; one case was observed one month after dental care in Djibouti. The pulmonary forms were less frequent (n = 6) and 3 atypical forms are described. All patients had stayed in Djibouti. Conclusions: Cases of M. canettii infection among the French military consisted mainly of adenitis; disseminated forms were possible with immunodeficiency. Their evolution under specific treatments were comparable to tuberculosis. The presumed origin of the infection seemed to be environmental, possibly a water reservoir, and not due to human-to-human contagion.
    Mots-clés : DBG, LGBMB, MICROBIO, SSFA.

  • E. M. S. Brito, V. M. Romero-Núñez, C. A. Caretta, P. Bertin, J. C. Valerdi-Negreros, R. Guyoneaud, et M. Goñi-Urriza, « The bacterial diversity on steam vents from Paricutín and Sapichu volcanoes », Extremophiles: Life Under Extreme Conditions, févr. 2019.
    Résumé : Vapor steam vents are prevailing structures on geothermal sites in which local geochemical conditions allow the development of extremophilic microorganisms. We describe the structure of the prokaryotic community able to grow on the walls and rocks of such microecosystems in two terrestrial Mexican volcanoes: Paricutín (PI and PII samples) and its satellite Sapichu (S sample). The investigated samples showed similar diversity indices, with few dominant OTUs (abundance > 1%): 21, 16 and 23, respectively for PI, PII and S. However, each steam vent showed a particular community profile: PI was dominated by photosynthetic bacteria (Cyanobacteria and Chloroflexia class), PII by Actinobacteria and Proteobacteria, and S by Ktedonobacteria class, Acidobacteria and Cyanobacteria phyla. Concerning the predicted metabolic potential, we found a dominance of cellular pathways, especially the ones for energy generation with metabolisms for sulfur respiration, nitrogen fixation, methanogenesis, carbon fixation, photosynthesis, and metals, among others. We suggest a different maturity stage for the three studied fumaroles, from the youngest (PI) to the oldest (S and PII), also influenced by the temperature and other geochemical parameters. Furthermore, four anaerobic strains were isolated, belonging to Clostridia class (Clostridium sphenoides, C. swellfunanium and Anaerocolumna cellulosilytica) and to Bacilli class (Paenibacillus azoreducens).
    Mots-clés : Anaerobic bacteria, DBG, Extreme environment, GST, Microbial biodiversity, Predictive metagenomics profiling, Volcanic fumaroles.

  • C. Carvalho, V. L'Hôte, R. Courbeyrette, G. Kratassiouk, G. Pinna, J. - C. Cintrat, C. Denby-Wilkes, C. Derbois, R. Olaso, J. - F. Deleuze, C. Mann, et J. - Y. Thuret, « Glucocorticoids delay RAF-induced senescence promoted by EGR1 », Journal of Cell Science, vol. 132, nᵒ 16, p. UNSP jcs230748, août 2019.
    Résumé : Expression of hyperactive RAF kinases, such as the oncogenic B-RAF-V600E mutant, in normal human cells triggers a proliferative arrest that blocks tumor formation. We discovered that glucocorticoids delayed the entry into senescence induced by B-RAF-V600E in human fibroblasts, and allowed senescence bypass when the cells were regularly passaged, but that they did not allow proliferation of cells that were already senescent. Transcriptome and siRNA analyses revealed that the EGR1 gene is one target of glucocorticoid action. Transcription of the EGR1 gene is activated by the RAF-MEK-ERK MAPK pathway and acts as a sensor of hyper-mitogenic pathway activity. The EGR1 transcription factor regulates the expression of p15 and p21 (encoded by CDKN2B and CDKN1A, respectively) that are redundantly required for the proliferative arrest of BJ fibroblasts upon expression of B-RAF-V600E. Our results highlight the need to evaluate the action of glucocorticoid on cancer progression in melanoma, thyroid and colon carcinoma in which B-RAF-V600E is a frequent oncogene, and cancers in which evasion from senescence has been shown.
    Mots-clés : B-RAF-V600E, CDKN1A, CDKN2B, DBG, EGR1, Glucocorticoid, GTR, Oncogene-induced senescence, PARI, PF, SEN.

  • D. Ciardo, A. Goldar, et K. Marheineke, « On the Interplay of the DNA Replication Program and the Intra-S Phase Checkpoint Pathway », Genes, vol. 10, nᵒ 2, p. 94, janv. 2019.
    Résumé : DNA replication in eukaryotes is achieved by the activation of multiple replication origins which needs to be precisely coordinated in space and time. This spatio-temporal replication program is regulated by many factors to maintain genome stability, which is frequently threatened through stresses of exogenous or endogenous origin. Intra-S phase checkpoints monitor the integrity of DNA synthesis and are activated when replication forks are stalled. Their activation leads to the stabilization of forks, to the delay of the replication program by the inhibition of late firing origins, and the delay of G2/M phase entry. In some cell cycles during early development these mechanisms are less efficient in order to allow rapid cell divisions. In this article, we will review our current knowledge of how the intra-S phase checkpoint regulates the replication program in budding yeast and metazoan models, including early embryos with rapid S phases. We sum up current models on how the checkpoint can inhibit origin firing in some genomic regions, but allow dormant origin activation in other regions. Finally, we discuss how numerical and theoretical models can be used to connect the multiple different actors into a global process and to extract general rules.
    Mots-clés : ataxia-telangiectasia, ATR, Chk1, DBG, DNA replication, DYNREP, GTR, MBT.

  • I. Corcoles-Saez, J. - L. Ferat, M. Costanzo, C. M. Boone, et R. S. Cha, « Functional link between mitochondria and Rnr3, the minor catalytic subunit of yeast ribonucleotide reductase », Microbial Cell (Graz, Austria), vol. 6, nᵒ 6, p. 286-294, mai 2019.
    Résumé : Ribonucleotide reductase (RNR) is an essential holoenzyme required for de novo synthesis of dNTPs. The Saccharomyces cerevisiae genome encodes for two catalytic subunits, Rnr1 and Rnr3. While Rnr1 is required for DNA replication and DNA damage repair, the function(s) of Rnr3 is unknown. Here, we show that carbon source, an essential nutrient, impacts Rnr1 and Rnr3 abundance: Non-fermentable carbon sources or limiting concentrations of glucose down regulate Rnr1 and induce Rnr3 expression. Oppositely, abundant glucose induces Rnr1 expression and down regulates Rnr3. The carbon source dependent regulation of Rnr3 is mediated by Mec1, the budding yeast ATM/ATR checkpoint response kinase. Unexpectedly, this regulation is independent of all currently known components of the Mec1 DNA damage response network, including Rad53, Dun1, and Tel1, implicating a novel Mec1 signalling axis. rnr3Δ leads to growth defects under respiratory conditions and rescues temperature sensitivity conferred by the absence of Tom6, a component of the mitochondrial TOM (translocase of outer membrane) complex responsible for mitochondrial protein import. Together, these results unveil involvement of Rnr3 in mitochondrial functions and Mec1 in mediating the carbon source dependent regulation of Rnr3.
    Mots-clés : atr, autophagy, carbon source, cell-cycle, checkpoint, DBG, dna-damage response, dNTP, EMC2, gene, kinase, Mec1, Rnr1, Rnr3.

  • A. de Groot, M. I. Siponen, R. Magerand, N. Eugénie, R. Martin-Arevalillo, J. Doloy, D. Lemaire, G. Brandelet, F. Parcy, R. Dumas, P. Roche, P. Servant, F. Confalonieri, P. Arnoux, D. Pignol, et L. Blanchard, « Crystal structure of the transcriptional repressor DdrO: insight into the metalloprotease/repressor-controlled radiation response in Deinococcus », Nucleic Acids Research, oct. 2019.
    Résumé : Exposure to harmful conditions such as radiation and desiccation induce oxidative stress and DNA damage. In radiation-resistant Deinococcus bacteria, the radiation/desiccation response is controlled by two proteins: the XRE family transcriptional repressor DdrO and the COG2856 metalloprotease IrrE. The latter cleaves and inactivates DdrO. Here, we report the biochemical characterization and crystal structure of DdrO, which is the first structure of a XRE protein targeted by a COG2856 protein. DdrO is composed of two domains that fold independently and are separated by a flexible linker. The N-terminal domain corresponds to the DNA-binding domain. The C-terminal domain, containing three alpha helices arranged in a novel fold, is required for DdrO dimerization. Cleavage by IrrE occurs in the loop between the last two helices of DdrO and abolishes dimerization and DNA binding. The cleavage site is hidden in the DdrO dimer structure, indicating that IrrE cleaves DdrO monomers or that the interaction with IrrE induces a structural change rendering accessible the cleavage site. Predicted COG2856/XRE regulatory protein pairs are found in many bacteria, and available data suggest two different molecular mechanisms for stress-induced gene expression: COG2856 protein-mediated cleavage or inhibition of oligomerization without cleavage of the XRE repressor.
    Mots-clés : DBG, RBA.


  • A. Demené, L. Legrand, J. Gouzy, R. Debuchy, G. Saint-Jean, O. Fabreguettes, et C. Dutech, « Whole-genome sequencing reveals recent and frequent genetic recombination between clonal lineages of Cryphonectria parasitica in western Europe », Fungal Genetics and Biology, vol. 130, p. 122-133, sept. 2019.
    Résumé : Changes in the mode of reproduction are frequently observed in invasive fungal populations. The ascomycete Cryphonectria parasitica, which causes Chestnut Blight, was introduced to Europe from North America and Asia in the 20th century. Previous genotyping studies based on ten microsatellite markers have identified several clonal lineages which have spread throughout western Europe, suggesting that asexuality was the main reproductive mode of this species during colonization, although occasional sexual reproduction is not excluded. Based on the whole-genome sequences alignment of 46 C. parasitica isolates from France, North America and Asia, genealogy and population structure analyses mostly confirmed these lineages as clonal. However, one of these clonal lineages showed a signal of strong recombination, suggesting different strategies of reproduction in western Europe. Signatures of several recent recombination events within all the French clonal lineages studied here were also identified, indicating that gene flow is regular between these lineages. In addition, haplotype identification of seven French clonal lineages revealed that emergences of new clonal lineages during colonization were the result of hybridization between the main expanding clonal lineages and minor haplotypes non-sequenced in the present study. This whole-genome sequencing study underlines the importance of recombination events in the invasive success of these clonal populations, and suggests that sexual reproduction may be more frequent within and between the western European clonal lineages of C. parasitica than previously assumed using few genetic markers.
    Mots-clés : Bayesian inferences, Clonal evolution, DBG, Intra-haploid mating, MRP, Recombination rates, Whole genome sequencing.

  • T. Denecker, W. Durand, J. Maupetit, C. Hébert, J. - M. Camadro, P. Poulain, et G. Lelandais, « Pixel: a content management platform for quantitative omics data », PeerJ, vol. 7, p. e6623, 2019.
    Résumé : Background: In biology, high-throughput experimental technologies, also referred as "omics" technologies, are increasingly used in research laboratories. Several thousands of gene expression measurements can be obtained in a single experiment. Researchers are routinely facing the challenge to annotate, store, explore and mine all the biological information they have at their disposal. We present here the Pixel web application (Pixel Web App), an original content management platform to help people involved in a multi-omics biological project. Methods: The Pixel Web App is built with open source technologies and hosted on the collaborative development platform GitHub (https://github.com/Candihub/pixel). It is written in Python using the Django framework and stores all the data in a PostgreSQL database. It is developed in the open and licensed under the BSD 3-clause license. The Pixel Web App is also heavily tested with both unit and functional tests, a strong code coverage and continuous integration provided by CircleCI. To ease the development and the deployment of the Pixel Web App, Docker and Docker Compose are used to bundle the application as well as its dependencies. Results: The Pixel Web App offers researchers an intuitive way to annotate, store, explore and mine their multi-omics results. It can be installed on a personal computer or on a server to fit the needs of many users. In addition, anyone can enhance the application to better suit their needs, either by contributing directly on GitHub (encouraged) or by extending Pixel on their own. The Pixel Web App does not provide any computational programs to analyze the data. Still, it helps to rapidly explore and mine existing results and holds a strategic position in the management of research data.
    Mots-clés : BIM, candida-glabrata, Data cycle analyses, DBG, Omics, Open source, Pixel Web App.

  • Y. Deng, X. Luo, M. Xie, P. Bouloc, C. Chen, et A. Jacq, « The ilvGMEDA Operon Is Regulated by Transcription Attenuation in Vibrio alginolyticus ZJ-T », Applied and Environmental Microbiology, vol. 85, nᵒ 19, oct. 2019.
    Résumé : Bacteria synthesize amino acids according to their availability in the environment or, in the case of pathogens, within the host. We explored the regulation of the biosynthesis of branched-chain amino acids (BCAAs) (l-leucine, l-valine, and l-isoleucine) in Vibrio alginolyticus, a marine fish and shellfish pathogen and an emerging opportunistic human pathogen. In this species, the ilvGMEDA operon encodes the main pathway for biosynthesis of BCAAs. Its upstream regulatory region shows no sequence similarity to the corresponding region in Escherichia coli or other Enterobacteriaceae, and yet we show that this operon is regulated by transcription attenuation. The translation of a BCAA-rich peptide encoded upstream of the structural genes provides an adaptive response similar to the E. coli canonical model. This study of a nonmodel Gram-negative organism highlights the mechanistic conservation of transcription attenuation despite the absence of primary sequence conservation.IMPORTANCE This study analyzes the regulation of the biosynthesis of branched-chain amino acids (leucine, valine, and isoleucine) in Vibrio alginolyticus, a marine bacterium that is pathogenic to fish and humans. The results highlight the conservation of the main regulatory mechanism with that of the enterobacterium Escherichia coli, suggesting that such a mechanism appeared early during the evolution of Gram-negative bacteria, allowing adaptation to a wide range of environments.
    Mots-clés : acetolactate synthase (AHAS), branched-chain amino acids, DBG, ilvGMEDA operon, leader attenuator, SRRB, transcription attenuation, Vibrio alginolyticus.

  • A. Devigne, L. Meyer, C. B. de la Tour, N. Eugenie, S. Sommer, et P. Servant, « The absence of the RecN protein suppresses the cellular defects of Deinococcus radiodurans irradiated cells devoid of the PprA protein by Cheek tot limiting recombinational repair of DNA lesions », Dna Repair, vol. 73, p. 144-154, janv. 2019.
    Résumé : The Deinococcus radiodurans bacterium is one of the most radioresistant organisms known. It can repair hundreds of radiation-induced DNA double-strand breaks without loss of viability and reconstitute an intact genome through RecA-dependent and RecA-independent DNA repair pathways. Among the Deinococcus specific proteins required for radioresistance, the PprA protein was shown to play a major role for accurate chromosome segregation and cell division after completion of DNA repair. Here, we analyzed the cellular role of the deinococcal RecN protein belonging to the SMC family and, surprisingly, observed that the absence of the RecN protein suppressed the sensitivity of cells devoid of the PprA protein to gamma- and UV-irradiation and to treatment with MMC or DNA gyrase inhibitors. This suppression was not observed when Delta pprA cells were devoid of SMC or SbcC, two other proteins belonging to the SMC family. The absence of RecN also alleviated the DNA segregation defects displayed by Delta pprA cells recovering from y-irradiation. When exposed to 5 kGy gamma-irradiation, Delta pprA, Delta recN and Delta pprA Delta recN cells repaired their DNA with a delay of about one hour, as compared to the wild type cells. After irradiation, the absence of RecN reduced recombination between chromosomal and plasmid DNA, indicating that the deinococcal RecN protein is important for recombinational repair of DNA lesions. The transformation efficiency of genomic DNA was also reduced in the absence of the RecN protein. Here, we propose a model in which RecN, via its cohesin activity, might favor recombinational repair of DNA double strand breaks. This might increase, in irradiated cells, DNA constraints with PprA protein being required to resolve them via its ability to recruit DNA gyrase and to stimulate its decatenation activity.
    Mots-clés : chromosomes, complex, DBG, Deinococcus radiodurans, DNA segregation, DNA segregation, double-strand breaks, dynamics, Homologous recombination, identification, PprA, RBA, RecN, recruitment, replication, segregation, smc protein, structural maintenance.

  • E. Dubois, A. De Muyt, J. L. Soyer, K. Budin, M. Legras, T. Piolot, R. Debuchy, N. Kleckner, D. Zickler, et E. Espagne, « Building bridges to move recombination complexes », Proceedings of the National Academy of Sciences of the United States of America, mai 2019.
    Résumé : A central feature of meiosis is pairing of homologous chromosomes, which occurs in two stages: coalignment of axes followed by installation of the synaptonemal complex (SC). Concomitantly, recombination complexes reposition from on-axis association to the SC central region. We show here that, in the fungus Sordaria macrospora, this critical transition is mediated by robust interaxis bridges that contain an axis component (Spo76/Pds5), DNA, plus colocalizing Mer3/Msh4 recombination proteins and the Zip2-Zip4 mediator complex. Mer3-Msh4-Zip2-Zip4 colocalizing foci are first released from their tight axis association, dependent on the SC transverse-filament protein Sme4/Zip1, before moving to bridges and thus to a between-axis position. Ensuing shortening of bridges and accompanying juxtaposition of axes to 100 nm enables installation of SC central elements at sites of between-axis Mer3-Msh4-Zip2-Zip4 complexes. We show also that the Zip2-Zip4 complex has an intrinsic affinity for chromosome axes at early leptotene, where it localizes independently of recombination, but is dependent on Mer3. Then, later, Zip2-Zip4 has an intrinsic affinity for the SC central element, where it ultimately localizes to sites of crossover complexes at the end of pachytene. These and other findings suggest that the fundamental role of Zip2-Zip4 is to mediate the recombination/structure interface at all post-double-strand break stages. We propose that Zip2-Zip4 directly mediates a molecular handoff of Mer3-Msh4 complexes, from association with axis components to association with SC central components, at the bridge stage, and then directly mediates central region installation during SC nucleation.
    Mots-clés : chromosome structure, DBG, interaxis bridges, meiotic recombination, MRP, synaptonemal complex, Zip2-Zip4.

  • E. Durand, I. Gagnon-Arsenault, J. Hallin, I. Hatin, A. K. Dube, L. Nielly-Thibault, O. Namy, et C. R. Landry, « Turnover of ribosome-associated transcripts from de novo ORFs produces gene-like characteristics available for de novo gene emergence in wild yeast populations », Genome Research, vol. 29, nᵒ 6, p. 932-943, juin 2019.
    Résumé : Little is known about the rate of emergence of de novo genes, what their initial properties are, and how they spread in populations. We examined wild yeast populations (Saccharomyces paradoxus) to characterize the diversity and turnover of intergenic ORFs over short evolutionary timescales. We find that hundreds of intergenic ORFs show translation signatures similar to canonical genes, and we experimentally confirmed the translation of many of these ORFs in laboratory conditions using a reporter assay. Compared with canonical genes, intergenic ORFs have lower translation efficiency, which could imply a lack of optimization for translation or a mechanism to reduce their production cost. Translated intergenic ORFs also tend to have sequence properties that are generally close to those of random intergenic sequences. However, some of the very recent translated intergenic ORFs, which appeared <110 kya, already show gene-like characteristics, suggesting that the raw material for functional innovations could appear over short evolutionary timescales.
    Mots-clés : DBG, drosophila-yakuba, evolution, genome, GST, in-vivo, map, origin, sequences, translation.

  • C. Essoh, J. - P. Vernadet, G. Vergnaud, A. Coulibaly, A. Kakou-N'Douba, A. S. - P. N'Guetta, G. Resch, et C. Pourcel, « Complete Genome Sequences of Five Acinetobacter baumannii Phages from Abidjan, Côte d'Ivoire », Microbiology Resource Announcements, vol. 8, nᵒ 1, janv. 2019.
    Résumé : Five bacteriophages of Acinetobacter baumannii were isolated from sewage water in Abidjan, Côte d'Ivoire. Phages Aci01-1, Aci02-2, and Aci05 belong to an unclassified genus of the Myoviridae family, with double-stranded DNA (dsDNA) genomes, whereas Aci07 and Aci08 belong to the Fri1virus genus of the Podoviridae family of phages.
    Mots-clés : DBG, LGBMB, MICROBIO, SSFA.

  • A. Ferrandi, F. Gastoni, M. Pitaro, S. Tagliaferri, C. B. de la Tour, R. Alduina, S. Sommer, M. Fasano, P. Barbieri, M. Mancini, et I. M. Bonapace, « Deinococcus radiodurans' SRA-HNH domain containing protein Shp (Dr1533) is involved in faithful genome inheritance maintenance following DNA damage », Biochimica Et Biophysica Acta-General Subjects, vol. 1863, nᵒ 1, p. 118-129, janv. 2019.
    Résumé : Background: Deinococcus radiodurans R1 (DR) survives conditions of extreme desiccation, irradiation and exposure to genotoxic chemicals, due to efficient DNA breaks repair, also through Mn2+ protection of DNA repair enzymes. Methods: Possible annotated domains of the DR1533 locus protein (Shp) were searched by bioinformatic analysis. The gene was cloned and expressed as fusion protein. Band-shift assays of Shp or the SRA and HNH domains were performed on oligonucleotides, genomic DNA from E. coif and DR. slip knock-out mutant was generated by homologous recombination with a kanamycin resistance cassette. Results: DR1533 contains an N-terminal SRA domain and a C-terminal HNH motif (SRA-HNH Protein, Shp). Through its SRA domain, Shp binds double-strand oligonucleotides containing 5mC and 5hmC, but also unmethylated and mismatched cytosines in presence of Mn2+. Shp also binds to Escherichia coli dcm(+) genomic DNA, and to cytosine unmethylated DR and E. coli dcm(-) genomic DNAs, but only in presence of Mn2+. Under these binding conditions, Shp displays DNAse activity through its HNH domain. Shp KO enhanced > 100 fold the number of spontaneous mutants, whilst the treatment with DNA double strand break inducing agents enhanced up to 3-log the number of survivors. Conclusions: The SRA-HNH containing protein Shp binds to and cuts 5mC DNA, and unmethylated DNA in a Mn2+ dependent manner, and might be involved in faithful genome inheritance maintenance following DNA damage. General significance: Our results provide evidence for a potential role of DR Shp protein for genome integrity maintenance, following DNA double strand breaks induced by genotoxic agents.
    Mots-clés : cytosine, DBG, DNA cytosine-methylation, DNA damage, DR1533 locus, features, Genotoxic agents, manganese(ii), Mn2+, oxidation, perspective, RBA, recognition, repair, resistance, SRA domain, uhrf1.

  • K. Floc'h, F. Lacroix, P. Servant, Y. - S. Wong, J. - P. Kleman, D. Bourgeois, et J. Timmins, « Cell morphology and nucleoid dynamics in dividing Deinococcus radiodurans », Nature Communications, vol. 10, nᵒ 1, p. 3815, août 2019.
    Résumé : Our knowledge of bacterial nucleoids originates mostly from studies of rod- or crescent-shaped bacteria. Here we reveal that Deinococcus radiodurans, a relatively large spherical bacterium with a multipartite genome, constitutes a valuable system for the study of the nucleoid in cocci. Using advanced microscopy, we show that D. radiodurans undergoes coordinated morphological changes at both the cellular and nucleoid level as it progresses through its cell cycle. The nucleoid is highly condensed, but also surprisingly dynamic, adopting multiple configurations and presenting an unusual arrangement in which oriC loci are radially distributed around clustered ter sites maintained at the cell centre. Single-particle tracking and fluorescence recovery after photobleaching studies of the histone-like HU protein suggest that its loose binding to DNA may contribute to this remarkable plasticity. These findings demonstrate that nucleoid organization is complex and tightly coupled to cell cycle progression in this organism.
    Mots-clés : DBG, RBA.

  • A. Frapporti, C. Miró Pina, O. Arnaiz, D. Holoch, T. Kawaguchi, A. Humbert, E. Eleftheriou, B. Lombard, D. Loew, L. Sperling, K. Guitot, R. Margueron, et S. Duharcourt, « The Polycomb protein Ezl1 mediates H3K9 and H3K27 methylation to repress transposable elements in Paramecium », Nature Communications, vol. 10, nᵒ 1, p. 2710, 2019.
    Résumé : In animals and plants, the H3K9me3 and H3K27me3 chromatin silencing marks are deposited by different protein machineries. H3K9me3 is catalyzed by the SET-domain SU(VAR)3-9 enzymes, while H3K27me3 is catalyzed by the SET-domain Enhancer-of-zeste enzymes, which are the catalytic subunits of Polycomb Repressive Complex 2 (PRC2). Here, we show that the Enhancer-of-zeste-like protein Ezl1 from the unicellular eukaryote Paramecium tetraurelia, which exhibits significant sequence and structural similarities with human EZH2, catalyzes methylation of histone H3 in vitro and in vivo with an apparent specificity toward K9 and K27. We find that H3K9me3 and H3K27me3 co-occur at multiple families of transposable elements in an Ezl1-dependent manner. We demonstrate that loss of these histone marks results in global transcriptional hyperactivation of transposable elements with modest effects on protein-coding gene expression. Our study suggests that although often considered functionally distinct, H3K9me3 and H3K27me3 may share a common evolutionary history as well as a common ancestral role in silencing transposable elements.
    Mots-clés : chromatin, complex, DBG, DNA Methylation, DNA Transposable Elements, Gene Silencing, genes, heterochromatin formation, histone methyltransferase activity, Histones, mechanisms, MICMAC, Paramecium tetraurelia, pluripotent, Polycomb Repressive Complex 2, Protein Processing, Post-Translational, specificity, states, structural basis, Transcriptional Activation.
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  • E. Galli, J. - L. Ferat, J. - M. Desfontaines, M. - E. Val, O. Skovgaard, F. - X. Barre, et C. Possoz, « Replication termination without a replication fork trap », Scientific Reports, vol. 9, nᵒ 1, p. 8315, juin 2019.
    Résumé : Bacterial chromosomes harbour a unique origin of bidirectional replication, oriC. They are almost always circular, with replication terminating in a region diametrically opposite to oriC, the terminus. The oriC-terminus organisation is reflected by the orientation of the genes and by the disposition of DNA-binding protein motifs implicated in the coordination of chromosome replication and segregation with cell division. Correspondingly, the E. coli and B. subtilis model bacteria possess a replication fork trap system, Tus/ter and RTP/ter, respectively, which enforces replication termination in the terminus region. Here, we show that tus and rtp are restricted to four clades of bacteria, suggesting that tus was recently domesticated from a plasmid gene. We further demonstrate that there is no replication fork system in Vibrio cholerae, a bacterium closely related to E. coli. Marker frequency analysis showed that replication forks originating from ectopic origins were not blocked in the terminus region of either of the two V. cholerae chromosomes, but progressed normally until they encountered an opposite fork. As expected, termination synchrony of the two chromosomes is disrupted by these ectopic origins. Finally, we show that premature completion of the primary chromosome replication did not modify the choreography of segregation of its terminus region.
    Mots-clés : binding, cell-division, DBG, dna-replication, EMC2, escherichia-coli-chromosome, ftsk, protein, segregation, terminus region, tus, vibrio-cholerae.

  • A. Georges, D. Gopaul, C. Denby Wilkes, N. Giordanengo Aiach, E. Novikova, M. - B. Barrault, O. Alibert, et J. Soutourina, « Functional interplay between Mediator and RNA polymerase II in Rad2/XPG loading to the chromatin », Nucleic Acids Research, juill. 2019.
    Résumé : Transcription and maintenance of genome integrity are fundamental cellular functions. Deregulation of transcription and defects in DNA repair lead to serious pathologies. The Mediator complex links RNA polymerase (Pol) II transcription and nucleotide excision repair via Rad2/XPG endonuclease. However, the functional interplay between Rad2/XPG, Mediator and Pol II remains to be determined. In this study, we investigated their functional dynamics using genomic and genetic approaches. In a mutant affected in Pol II phosphorylation leading to Mediator stabilization on core promoters, Rad2 genome-wide occupancy shifts towards core promoters following that of Mediator, but decreases on transcribed regions together with Pol II. Specific Mediator mutations increase UV sensitivity, reduce Rad2 recruitment to transcribed regions, lead to uncoupling of Rad2, Mediator and Pol II and to colethality with deletion of Rpb9 Pol II subunit involved in transcription-coupled repair. We provide new insights into the functional interplay between Rad2, Mediator and Pol II and propose that dynamic interactions with Mediator and Pol II are involved in Rad2 loading to the chromatin. Our work contributes to the understanding of the complex link between transcription and DNA repair machineries, dysfunction of which leads to severe diseases.
    Mots-clés : DBG, GTR.

  • E. Godat, J. - C. Preiser, J. - C. Aude, et P. Kalfon, « Dynamic properties of glucose complexity during the course of critical illness: a pilot study », Journal of Clinical Monitoring and Computing, mars 2019.
    Résumé : Methods to control the blood glucose (BG) levels of patients in intensive care units (ICU) improve the outcomes. The development of continuous BG levels monitoring devices has also permitted to optimize these processes. Recently it was shown that a complexity loss of the BG signal is linked to poor clinical outcomes. Thus, it becomes essential to decipher this relation to design efficient BG level control methods. In previous studies the BG signal complexity was calculated as a single index for the whole ICU stay. Although, these approaches did not grasp the potential variability of the BG signal complexity. Therefore, we setup this pilot study using a continuous monitoring of central venous BG levels in ten critically ill patients (EIRUS platform, Maquet Critical CARE AB, Solna, Sweden). Data were processed and the complexity was assessed by the detrended fluctuation analysis and multiscale entropy (MSE) methods. Finally, recordings were split into 24 h overlapping intervals and a MSE analysis was applied to each of them. The MSE analysis on time intervals revealed an entropy variation and allowed periodic BG signal complexity assessments. To highlight differences of MSE between each time interval we calculated the MSE complexity index defined as the area under the curve. This new approach could pave the way to future studies exploring new strategies aimed at restoring blood glucose complexity during the ICU stay.
    Mots-clés : BIM, Continuous glucose monitoring, Critically ill patients, DBG, Glucose control, Multiscale entropy, Signal complexity.

  • J. Godau, L. P. Ferretti, A. Trenner, E. Dubois, C. von Aesch, A. Marmignon, L. Simon, A. Kapusta, R. Guérois, M. Bétermier, et A. A. Sartori, « Identification of a miniature Sae2/Ctp1/CtIP ortholog from Paramecium tetraurelia required for sexual reproduction and DNA double-strand break repair », DNA repair, vol. 77, p. 96-108, mars 2019.
    Résumé : DNA double-strand breaks (DSBs) induced by genotoxic agents can cause cell death or contribute to chromosomal instability, a major driving force of cancer. By contrast, Spo11-dependent DSBs formed during meiosis are aimed at generating genetic diversity. In eukaryotes, CtIP and the Mre11 nuclease complex are essential for accurate processing and repair of both unscheduled and programmed DSBs by homologous recombination (HR). Here, we applied bioinformatics and genetic analysis to identify Paramecium tetraurelia CtIP (PtCtIP), the smallest known Sae2/Ctp1/CtIP ortholog, as a key factor for the completion of meiosis and the recovery of viable sexual progeny. Using in vitro assays, we find that purified recombinant PtCtIP preferentially binds to double-stranded DNA substrates but does not contain intrinsic nuclease activity. Moreover, mutation of the evolutionarily conserved C-terminal 'RHR' motif abrogates DNA binding of PtCtIP but not its ability to functionally interact with Mre11. Translating our findings into mammalian cells, we provide evidence that disruption of the 'RHR' motif abrogates accumulation of human CtIP at sites of DSBs. Consequently, cells expressing the DNA binding mutant CtIPR837A/R839A are defective in DSB resection and HR. Collectively, our work highlights minimal structural requirements for CtIP protein family members to facilitate the processing of DSBs, thereby maintaining genome stability as well as enabling sexual reproduction.
    Mots-clés : AMIG, B3S, CtIP, ctp1, damage response, DBG, DNA double-strand breaks, DNA end resection, end-resection, endonuclease, gene, Homologous recombination, human ctip, Meiosis, MICMAC, mre11 complex, Paramecium tetraurelia, protein, rad32(mre11) nuclease, sae2.


  • 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. V. C. Greenberg, A. Teissandier, M. Walter, D. Noordermeer, et D. Bourc'his, « Dynamic enhancer partitioning instructs activation of a growth-related gene during exit from naïve pluripotency », eLife, vol. 8, avr. 2019.
    Résumé : During early mammalian development, the chromatin landscape undergoes profound transitions. The Zdbf2 gene-involved in growth control-provides a valuable model to study this window: upon exit from naïve pluripotency and prior to tissue differentiation, it undergoes a switch from a distal to a proximal promoter usage, accompanied by a switch from polycomb to DNA methylation occupancy. Using an embryonic stem cell (ESC) system to mimic this period, we show here that four enhancers contribute to the Zdbf2 promoter switch, concomitantly with dynamic changes in chromatin architecture. In ESCs, the locus is partitioned to facilitate enhancer contacts with the distal Zdbf2 promoter. Relieving the partition enhances proximal Zdbf2 promoter activity, as observed during differentiation or with genetic mutants. Importantly, we show that 3D regulation occurs upstream of the polycomb and DNA methylation pathways. Our study reveals the importance of multi-layered regulatory frameworks to ensure proper spatio-temporal activation of developmentally important genes.
    Mots-clés : CHRODY, chromosomes, DBG, differentiation, DNA methylation, enhancers, epigenetics, gene expression, genetics, genomics, mouse, polycomb, stem cells.

  • P. Grognet, H. Timpano, F. Carlier, J. Aït-Benkhali, V. Berteaux-Lecellier, R. Debuchy, F. Bidard, et F. Malagnac, « A RID-like putative cytosine methyltransferase homologue controls sexual development in the fungus Podospora anserina », PLoS genetics, vol. 15, nᵒ 8, p. e1008086, août 2019.
    Résumé : DNA methyltransferases are ubiquitous enzymes conserved in bacteria, plants and opisthokonta. These enzymes, which methylate cytosines, are involved in numerous biological processes, notably development. In mammals and higher plants, methylation patterns established and maintained by the cytosine DNA methyltransferases (DMTs) are essential to zygotic development. In fungi, some members of an extensively conserved fungal-specific DNA methyltransferase class are both mediators of the Repeat Induced Point mutation (RIP) genome defense system and key players of sexual reproduction. Yet, no DNA methyltransferase activity of these purified RID (RIP deficient) proteins could be detected in vitro. These observations led us to explore how RID-like DNA methyltransferase encoding genes would play a role during sexual development of fungi showing very little genomic DNA methylation, if any. To do so, we used the model ascomycete fungus Podospora anserina. We identified the PaRid gene, encoding a RID-like DNA methyltransferase and constructed knocked-out ΔPaRid defective mutants. Crosses involving P. anserina ΔPaRid mutants are sterile. Our results show that, although gametes are readily formed and fertilization occurs in a ΔPaRid background, sexual development is blocked just before the individualization of the dikaryotic cells leading to meiocytes. Complementation of ΔPaRid mutants with ectopic alleles of PaRid, including GFP-tagged, point-mutated and chimeric alleles, demonstrated that the catalytic motif of the putative PaRid methyltransferase is essential to ensure proper sexual development and that the expression of PaRid is spatially and temporally restricted. A transcriptomic analysis performed on mutant crosses revealed an overlap of the PaRid-controlled genetic network with the well-known mating-types gene developmental pathway common to an important group of fungi, the Pezizomycotina.
    Mots-clés : DBG, EDC, MRP.
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  • M. A. Hanson, A. Dostálová, C. Ceroni, M. Poidevin, S. Kondo, et B. Lemaitre, « Synergy and remarkable specificity of antimicrobial peptides in vivo using a systematic knockout approach », eLife, vol. 8, févr. 2019.
    Résumé : Antimicrobial peptides (AMPs) are host-encoded antibiotics that combat invading microorganisms. These short, cationic peptides have been implicated in many biological processes, primarily involving innate immunity. In vitro studies have shown AMPs kill bacteria and fungi at physiological concentrations, but little validation has been done in vivo. We utilized CRISPR gene editing to delete all known immune-inducible AMPs of Drosophila, namely: 4 Attacins, 4 Cecropins, 2 Diptericins, Drosocin, Drosomycin, Metchnikowin and Defensin. Using individual and multiple knockouts, including flies lacking all 14 AMP genes, we characterize the in vivo function of individual and groups of AMPs against diverse bacterial and fungal pathogens. We found that Drosophila AMPs act primarily against Gram-negative bacteria and fungi, contributing either additively or synergistically. We also describe remarkable specificity wherein certain AMPs contribute the bulk of microbicidal activity against specific pathogens, providing functional demonstrations of highly specific AMP-pathogen interactions in an in vivo setting.
    Mots-clés : AMP, D. melanogaster, DBG, Diptericin, Drosocin, EQYY, Imd, immunology, inflammation, PF, systemic immunity, Toll.

  • D. Knierim, Q. Barrière, I. Grigoras, S. Winter, H. - J. Vetten, M. Schwinghamer, J. Thomas, P. Chu, B. Gronenborn, et T. Timchenko, « Subterranean Clover Stunt Virus Revisited: Detection of Two Missing Genome Components », Viruses, vol. 11, nᵒ 2, p. 138, févr. 2019.
    Résumé : Subterranean clover stunt virus (SCSV) is a type species of the genus Nanovirus in the family Nanoviridae. It was the first single-stranded DNA plant virus with a multipartite genome, of which genomic DNA sequences had been determined. All nanoviruses have eight genome components except SCSV, for which homologs of two genome components present in all other nanovirus genomes, DNA-U2 and DNA-U4, were lacking. We analysed archived and more recent samples from SCSV-infected legume plants to verify its genome composition and found the missing genome components. These results indicated that SCSV also has eight genome components and is a typical member of the genus Nanovirus.
    Mots-clés : circular ssdna components, DBG, dna, dwarf virus, high-throughput sequencing, high-throughput sequencing, identification, MICROBIO, nanovirus, nanovirus-alphasatellite complex, necrotic yellows virus, PBI, pea, PROMTI, proteins, recombination, replication, rolling circle replication, virus evolution.

  • L. Latino, C. Midoux, G. Vergnaud, et C. Pourcel, « Investigation of Pseudomonas aeruginosa strain PcyII-10 variants resisting infection by N4-like phage Ab09 in search for genes involved in phage adsorption », PloS One, vol. 14, nᵒ 4, p. e0215456, 2019.
    Résumé : Bacteria and their bacteriophages coexist and coevolve for the benefit of both in a mutualistic association. Multiple mechanisms are used by bacteria to resist phages in a trade-off between survival and maintenance of fitness. In vitro studies allow inquiring into the fate of virus and host in different conditions aimed at mimicking natural environment. We analyse here the mutations emerging in a clinical Pseudomonas aeruginosa strain in response to infection by Ab09, a N4-like lytic podovirus and describe a variety of chromosomal deletions and mutations conferring resistance. Some deletions result from illegitimate recombination taking place during long-term maintenance of the phage genome. Phage variants with mutations in a tail fiber gene are selected during pseudolysogeny with the capacity to infect resistant cells and produce large plaques. These results highlight the complex host/phage association and suggest that phage Ab09 promotes bacterial chromosome rearrangements. Finally this study points to the possible role of two bacterial genes in Ab09 phage adhesion to the cell, rpsB encoding protein S2 of the 30S ribosomal subunit and ORF1587 encoding a Wzy-like membrane protein involved in LPS biosynthesis.
    Mots-clés : bacteriophages, chromosomal deletion, comparative genomics, DBG, escherichia-coli, fine-structure, mechanisms, o-antigen, pseudolysogeny, replication, resistance, SSFA.

  • L. Latino, D. Patin, D. Cherier, T. Touze, C. Pourcel, H. Barreteau, et D. Mengin-Lecreulx, « Impact of FiuA Outer Membrane Receptor Polymorphism on the Resistance of Pseudomonas aeruginosa toward Peptidoglycan Lipid II-Targeting PaeM Pyocins », Journal of Bacteriology, vol. 201, nᵒ 13, p. e00164, juill. 2019.
    Résumé : Certain Pseudomonas aeruginosa strains produce a homolog of colicin M, namely, PaeM, that specifically inhibits peptidoglycan biosynthesis of susceptible P. aeruginosa strains by hydrolyzing the lipid II intermediate precursor. Two variants of this pyocin were identified whose sequences mainly differed in the N-terminal protein moiety, i.e., the region involved in the binding to the FiuA outer membrane receptor and translocation into the periplasm. The antibacterial activity of these two variants, PaeM1 and PaeM2, was tested against various P. aeruginosa strains comprising reference strains PAO1 and PA14, PaeM-producing strains, and 60 clinical isolates. Seven of these strains, including PAO1, were susceptible to only one variant (2 to PaeM1 and 5 to PaeM2), and 11 were affected by both. The remaining strains, including PA14 and four PaeM1 producers, were resistant to both variants. The differences in the antibacterial spectra of the two PaeM homologs prompted us to investigate the molecular determinants allowing their internalization into P. aeruginosa cells, taking the PAO1 strain that is susceptible to PaeM2 but resistant to PaeM1 as the indicator strain. Heterologous expression of fiuA gene orthologs from different strains into PAO1, site-directed mutagenesis experiments, and construction of PaeM chimeric proteins provided evidence that the cell susceptibility and discrimination differences between the PaeM variants resulted from a polymorphism of both the pyocin and the outer membrane receptor FiuA. Moreover, we found that a third component, TonB1, a protein involved in iron transport in P. aeruginosa, working together with FiuA and the ExbB/ExbD complex, was directly implicated in this discrimination. IMPORTANCE Bacterial antibiotic resistance constitutes a threat to human health, imposing the need for identification of new targets and development of new strategies to fight multiresistant pathogens. Bacteriocins and other weapons that bacteria have themselves developed to kill competitors are therefore of great interest and a valuable source of inspiration for us. Attention was paid here to two variants of a colicin M homolog (PaeM) produced by certain strains of P. aeruginosa that inhibit the growth of their congeners by blocking cell wall peptidoglycan synthesis. Molecular determinants allowing recognition of these pyocins by the outer membrane receptor FiuA were identified, and a receptor polymorphism affecting the susceptibility of P. aeruginosa clinical strains was highlighted, providing new insights into the potential use of these pyocins as an alternative to antibiotics.
    Mots-clés : acquisition, bacteriocins, cell wall, colicin M, colicin-m, crystal-structure, DBG, emergence, ENVBAC, escherichia-coli, FiuA, lipid II, locus variable-number, mechanism, MICROBIO, PaeM pyocins, peptidoglycan, protein, Pseudomonas aeruginosa, receptor structure-function, sequence, SSFA, TonB, tonb gene.

  • B. Le Cam, D. Sargent, J. Gouzy, J. Amselem, M. - N. Bellanger, O. Bouchez, S. Brown, V. Caffier, M. De Gracia Coquerel, R. Debuchy, L. Duvaux, T. Payen, M. Sannier, J. Shiller, J. Collemare, et C. Lemaire, « Population Genome Sequencing of the Scab Fungal Species Venturia inaequalis, Venturiapirina, Venturia aucupariae and Venturia asperata », G3-Genes Genomes Genetics, vol. 9, nᵒ 8, p. 2405-2414, août 2019.
    Résumé : The Venturia genus comprises fungal species that are pathogens on Rosaceae host plants, including V. inaequalis and V. asperata on apple, V. aucupariae on sorbus and V. pirina on pear. Although the genetic structure of V. inaequalis populations has been investigated in detail, genomic features underlying these subdivisions remain poorly understood. Here, we report whole genome sequencing of 87 Venturia strains that represent each species and each population within V. inaequalis We present a PacBio genome assembly for the V. inaequalis EU-B04 reference isolate. The size of selected genomes was determined by flow cytometry, and varied from 45 to 93 Mb. Genome assemblies of V. inaequalis and V. aucupariae contain a high content of transposable elements (TEs), most of which belong to the Gypsy or Copia LTR superfamilies and have been inactivated by Repeat-Induced Point mutations. The reference assembly of V. inaequalis presents a mosaic structure of GC-equilibrated regions that mainly contain predicted genes and AT-rich regions, mainly composed of TEs. Six pairs of strains were identified as clones. Single-Nucleotide Polymorphism (SNP) analysis between these clones revealed a high number of SNPs that are mostly located in AT-rich regions due to misalignments and allowed determining a false discovery rate. The availability of these genome sequences is expected to stimulate genetics and population genomics research of Venturia pathogens. Especially, it will help understanding the evolutionary history of Venturia species that are pathogenic on different hosts, a history that has probably been substantially influenced by TEs.
    Mots-clés : apple, apple scab, DBG, effectors, formae specialis, Fusicladium, MRP, pear, transposable elements, Venturia.

  • M. B. Ledwaba, C. Gomo, K. E. Lekota, P. Le Flèche, A. Hassim, G. Vergnaud, et H. van Heerden, « Molecular characterization of Brucella species from Zimbabwe », PLoS neglected tropical diseases, vol. 13, nᵒ 5, p. e0007311, mai 2019.
    Résumé : Brucella abortus and B. melitensis have been reported in several studies in animals in Zimbabwe but the extent of the disease remains poorly known. Thus, characterizing the circulating strains is a critical first step in understanding brucellosis in the country. In this study we used an array of molecular assays including AMOS-PCR, Bruce-ladder, multiple locus variable number tandem repeats analysis (MLVA) and single nucleotide polymorphisms from whole genome sequencing (WGS-SNP) to characterize Brucella isolates to the species, biovar, and individual strain level. Sixteen Brucella strains isolated in Zimbabwe at the Central Veterinary laboratory from various hosts were characterized using all or some of these assays. The strains were identified as B. ovis, B. abortus, B. canis and B. suis, with B. canis being the first report of this species in Zimbabwe. Zimbabwean strains identified as B. suis and B. abortus were further characterized with whole genome sequencing and were closely related to reference strains 1330 and 86/8/59, respectively. We demonstrate the range of different tests that can be performed from simple assays that can be run in laboratories lacking sophisticated instrumentation to whole genome analyses that currently require substantial expertise and infrastructure often not available in the developing world.
    Mots-clés : abortus, DBG, differentiation, genome sequence, identification, ladder, melitensis, multiplex pcr assay, reveals, SSFA, strains, suis.

  • G. Lelandais, T. Denecker, C. Garcia, N. Danila, T. Léger, et J. - M. Camadro, « Label-free quantitative proteomics in Candida yeast species: technical and biological replicates to assess data reproducibility », BMC research notes, vol. 12, nᵒ 1, p. 470, août 2019.
    Résumé : OBJECTIVE: Label-free quantitative proteomics has emerged as a powerful strategy to obtain high quality quantitative measures of the proteome with only a very small quantity of total protein extract. Because our research projects were requiring the application of bottom-up shotgun mass spectrometry proteomics in the pathogenic yeasts Candida glabrata and Candida albicans, we performed preliminary experiments to (i) obtain a precise list of all the proteins for which measures of abundance could be obtained and (ii) assess the reproducibility of the results arising respectively from biological and technical replicates. DATA DESCRIPTION: Three time-courses were performed in each Candida species, and an alkaline pH stress was induced for two of them. Cells were collected 10 and 60 min after stress induction and proteins were extracted. Samples were analysed two times by mass spectrometry. Our final dataset thus comprises label-free quantitative proteomics results for 24 samples (two species, three time-courses, two time points and two runs of mass spectrometry). Statistical procedures were applied to identify proteins with differential abundances between stressed and unstressed situations. Considering that C. glabrata and C. albicans are human pathogens, which face important pH fluctuations during a human host infection, this dataset has a potential value to other researchers in the field.
    Mots-clés : Alkaline pH, Candida albicans, Candida glabrata, DBG, EDC, Label-free quantitative proteomics, Mass spectrometry.
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  • C. Loiseau, D. Brites, I. Moser, F. Coll, C. Pourcel, S. Robbe-Austerman, V. Escuyer, K. A. Musser, S. J. Peacock, S. Feuerriegel, T. A. Kohl, S. Niemann, S. Gagneux, et C. U. Koser, « Revised Interpretation of the Hain Lifescience GenoType MTBC To Differentiate Mycobacterium canettii and Members of the Mycobacterium tuberculosis Complex », Antimicrobial Agents and Chemotherapy, vol. 63, nᵒ 6, p. e00159-19, juin 2019.
    Résumé : Using 894 phylogenetically diverse genomes of the Mycobacterium tuberculosis complex (MTBC), we simulated in silico the ability of the Hain Lifescience GenoType MTBC assay to differentiate the causative agents of tuberculosis. Here, we propose a revised interpretation of this assay to reflect its strengths (e.g., it can distinguish some strains of Mycobacterium canettii and variants of Mycobacterium bovis that are not intrinsically resistant to pyrazinamide) and limitations (e.g., Mycobacterium orygis cannot be differentiated from Mycobacterium africanum).
    Mots-clés : assay, DBG, genotyping, intrinsic antibiotic resistance, Mycobacterium tuberculosis, orygis, polymorphisms, pyrazinamide, SSFA.


  • L. Marichal, G. Giraudon--Colas, F. Cousin, A. Thill, J. Labarre, Y. Boulard, J. - C. Aude, S. Pin, et J. P. Renault, « Protein–Nanoparticle Interactions: What Are the Protein–Corona Thickness and Organization? », Langmuir, vol. 35, nᵒ 33, p. 10831-10837, août 2019.
    Résumé : Protein adsorption on a surface is generally evaluated in terms of the evolution of the proteins’ structures and functions. However, when the surface is that of a nanoparticle, the protein corona formed around it possesses a particular supramolecular structure that gives a “biological identity” to the new object. Little is known about the actual shape of the protein corona. Here, the protein corona formed by the adsorption of model proteins (myoglobin and hemoglobin) on silica nanoparticles was studied. Small-angle neutron scattering and oxygenation studies were combined to assess both the structural and functional impacts of the adsorption on proteins. Large differences in the oxygenation properties could be found while no significant global shape changes were seen after adsorption. Moreover, the structural study showed that the adsorbed proteins form an organized yet discontinuous monolayer around the nanoparticles.
    Mots-clés : B3S, BIM, DBG, IMAPP, PEPS.
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  • V. P. Masamsetti, R. R. J. Low, K. S. Mak, A. O'Connor, C. D. Riffkin, N. Lamm, L. Crabbe, J. Karlseder, D. C. S. Huang, M. T. Hayashi, et A. J. Cesare, « Replication stress induces mitotic death through parallel pathways regulated by WAPL and telomere deprotection », Nature Communications, vol. 10, nᵒ 1, p. 4224, sept. 2019.
    Résumé : Mitotic catastrophe is a broad descriptor encompassing unclear mechanisms of cell death. Here we investigate replication stress-driven mitotic catastrophe in human cells and identify that replication stress principally induces mitotic death signalled through two independent pathways. In p53-compromised cells we find that lethal replication stress confers WAPL-dependent centromere cohesion defects that maintain spindle assembly checkpoint-dependent mitotic arrest in the same cell cycle. Mitotic arrest then drives cohesion fatigue and triggers mitotic death through a primary pathway of BAX/BAK-dependent apoptosis. Simultaneously, a secondary mitotic death pathway is engaged through non-canonical telomere deprotection, regulated by TRF2, Aurora B and ATM. Additionally, we find that suppressing mitotic death in replication stressed cells results in distinct cellular outcomes depending upon how cell death is averted. These data demonstrate how replication stress-induced mitotic catastrophe signals cell death with implications for cancer treatment and cancer genome evolution.
    Mots-clés : DBG, TENOR.
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  • M. - C. Maurel, F. Leclerc, J. Vergne, et G. Zaccai, « RNA Back and Forth: Looking through Ribozyme and Viroid Motifs », Viruses, vol. 11, nᵒ 3, p. 283, mars 2019.
    Résumé : Current cellular facts allow us to follow the link from chemical to biochemical metabolites, from the ancient to the modern world. In this context, the "RNA world" hypothesis proposes that early in the evolution of life, the ribozyme was responsible for the storage and transfer of genetic information and for the catalysis of biochemical reactions. Accordingly, the hammerhead ribozyme (HHR) and the hairpin ribozyme belong to a family of endonucleolytic RNAs performing self-cleavage that might occur during replication. Furthermore, regarding the widespread occurrence of HHRs in several genomes of modern organisms (from mammals to small parasites and elsewhere), these small ribozymes have been regarded as living fossils of a primitive RNA world. They fold into 3D structures that generally require long-range intramolecular interactions to adopt the catalytically active conformation under specific physicochemical conditions. By studying viroids as plausible remains of ancient RNA, we recently demonstrated that they replicate in non-specific hosts, emphasizing their adaptability to different environments, which enhanced their survival probability over the ages. All these results exemplify ubiquitous features of life. Those are the structural and functional versatility of small RNAs, ribozymes, and viroids, as well as their diversity and adaptability to various extreme conditions. All these traits must have originated in early life to generate novel RNA populations.
    Mots-clés : catalytic-activity, circular rnas, crystal-structure, DBG, evolution, hammerhead ribozymes, in-vitro, origins of life, replication, ribozyme, RNA world, rolling-circles, self-cleavage, SSFA, viroid, virus.

  • A. Mercier, C. Clairet, R. Debuchy, D. Morais, P. Silar, et S. Brun, « The mitochondrial translocase of the inner membrane PaTim54 is involved in defense response and longevity in Podospora anserina », Fungal genetics and biology: FG & B, vol. 132, p. 103257, juill. 2019.
    Résumé : Fungi are very successful microorganisms capable of colonizing virtually any ecological niche where they must constantly cope with competitors including fungi, bacteria and nematodes. We have shown previously that the ascomycete Podopora anserina exhibits Hyphal Interference (HI), an antagonistic response triggered by direct contact of competing fungal hyphae. When challenged with Penicillium chrysogenum, P. anserina produces hydrogen peroxide at the confrontation and kills the hyphae of P. chrysogenum. Here, we report the characterization of the PDC2218 mutant affected in HI. When challenged with P. chrysogenum, the PDC2218 mutant produces a massive oxidative burst at the confrontation. However, this increased production of hydrogen peroxide is not correlated to increased cell death in P. chrysogenum. Hence, the oxidative burst and cell death in the challenger are uncoupled in PDC2218. The gene affected in PDC2218 is PaTim54, encoding the homologue of the budding yeast mitochondrial inner membrane import machinery component Tim54p. We show that PaTim54 is essential in P. anserina and that the phenotypes displayed by the PDC2218 mutant, renamed PaTim542218, are the consequence of a drastic reduction in the expression of PaTim54. Among these pleiotropic phenotypes, PDC2218-PaTim542218- displays increased lifespan, a phenotype in line with the observed mitochondrial defects in the mutant.
    Mots-clés : Aging, DBG, Fungi, Hyphal Interference, Mitochondria, MRP, Podospora anserina, Tim54.

  • M. Meyer, H. Walbott, V. Olieric, J. Kondo, M. Costa, et B. Masquida, « Conformational adaptation of UNCG loops upon crowding », RNA (New York, N.Y.), août 2019.
    Résumé : If the A-form helix is the major structural motif found in RNA, the loops that cap them constitute the second most important family of motifs. Among those, two are over-represented, the GNRA and the UNCG tetraloops. Although one might think that these consensus sequences imply distinct and specific architectures, such is not the case. Recent surveys of RNA structures deposited in the PDB show that GNRA and UNCG tetraloops can adopt tertiary folds that are very different from their canonical conformations, characterized by the presence of a U-turn of a Z-turn, respectively. In this study, crystallographic data derived from both a Lariat-Capping (LC) ribozyme and a group II intron ribozyme reveal that a given UUCG tetraloop can adopt a distinct fold depending on its direct structural environment. Specifically, when the crystal packing applies relaxed constraints on the loop, the canonical Z-turn conformation is observed. In contrast, a highly-packed environment induces "squashing" of the tetraloop by distorting its sugar-phosphate backbone in a specific way that expels the first and fourth nucleobases out of the loop, and falls in van der Waals distance of the last base pair of the helix, taking the place of the pair formed between the first and fourth residues in Z-turn loops. Importantly, the biological relevance of our observations is supported by the presence of similarly deformed loops in the highly-packed environment of the ribosome and in a complex between a dsRNA and a yeast RNase III. The finding that Z-turn loops can change conformation under higher molecular packing suggests that, in addition to their early demonstrated role in stabilizing RNA folding, they may also contribute to the three-dimensional structure of RNA by mediating tertiary interactions with distal residues.
    Mots-clés : DBG, group II intron, Lariat-Capping ribozyme, RIBOZYMO, UNCG tetraloop, Z-turn loop.

  • C. Midonet, S. Miele, E. Paly, R. Guerois, et F. - X. Barre, « The TLCΦ satellite phage harbors a Xer recombination activation factor », Proceedings of the National Academy of Sciences of the United States of America, août 2019.
    Résumé : The circular chromosomes of bacteria can be concatenated into dimers by homologous recombination. Dimers are solved by the addition of a cross-over at a specific chromosomal site, dif, by 2 related tyrosine recombinases, XerC and XerD. Each enzyme catalyzes the exchange of a specific pair of strands. Some plasmids exploit the Xer machinery for concatemer resolution. Other mobile elements exploit it to integrate into the genome of their host. Chromosome dimer resolution is initiated by XerD. The reaction is under the control of a cell-division protein, FtsK, which activates XerD by a direct contact. Most mobile elements exploit FtsK-independent Xer recombination reactions initiated by XerC. The only notable exception is the toxin-linked cryptic satellite phage of Vibrio cholerae, TLCΦ, which integrates into and excises from the dif site of the primary chromosome of its host by a reaction initiated by XerD. However, the reaction remains independent of FtsK. Here, we show that TLCΦ carries a Xer recombination activation factor, XafT. We demonstrate in vitro that XafT activates XerD catalysis. Correspondingly, we found that XafT specifically interacts with XerD. We further show that integrative mobile elements exploiting Xer (IMEXs) encoding a XafT-like protein are widespread in gamma- and beta-proteobacteria, including human, animal, and plant pathogens.
    Mots-clés : AMIG, B3S, cholera, DBG, EMC2, IMEX, integrative mobile element, lysogenic conversion, site-specific recombination.
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  • C. Midonet, S. Miele, E. Paly, R. Guerois, et F. - X. Barre, « Insights into TLCΦ lysogeny: A twist in the mechanism of IMEX integration », Proceedings of the National Academy of Sciences, vol. 116, nᵒ 37, p. 18159-18161, sept. 2019.
    Résumé : Many organisms have established symbiotic relationships with acquired mobile genetic elements (MGEs) integrated in their genomes (1). MGEs spread among genomes within and across microbial species through horizontal gene transfer and, once integrated into host chromosome, are disseminated vertically to the progeny, causing rapid evolution of drug resistance, pathogenicity, and virulence traits (2, 3). The MGEs that integrates into the host bacterial chromosomes (IMGEs) either carry their own DNA integration machineries or exploit machineries already existing in the host organisms for integration (4). The latter elements are of interest, in part, because of their contribution to pathogenesis, antimicrobial resistance, and other medically relevant properties (5, 6). One of the host site-specific recombination systems frequently exploited by IMGEs is the widely distributed bacterial chromosome dimer-resolving Xer recombination system, a system that recombines chromosomes at dif site located near where DNA replication terminates (7). As in all organisms, during DNA replication of bacteria, many DNA damages need to be repaired by homologous recombination reaction. For bacteria having circular chromosomes, this often generates circular dimer chromosome, causing problems when the cell divides. Hence, when a pair of unresolved chromosome dimer junctions get trapped at the closing cell division septum, the pair of dif sites with XerC and XerD recombinases bound across the recombination junction encounter FtsK DNA translocation pump, a component of the closing septum complex, whose job is to clear trapped DNA out of the septum. This encounter triggers initiation of recombination by activating XerD to carry out the first strand exchange, generating a Holliday junction recombination intermediate, which is resolved by XerC-mediated second pair of strand exchange (8). XerC is an efficient resolver of the recombination intermediate but a poor recombination initiator. Without FtsK activation, Xer remains essentially silent, avoiding formation of chromosome dimer out of 2 separable replicated … [↵][1]1Email: bhabatosh{at}thsti.res.in. [1]: #xref-corresp-1-1
    Mots-clés : AMIG, B3S, DBG, EMC2.


  • A. H. Millar, J. L. Heazlewood, C. Giglione, M. J. Holdsworth, A. Bachmair, et W. X. Schulze, « The Scope, Functions, and Dynamics of Posttranslational Protein Modifications », in Annual Review of Plant Biology, vol. 70, Annual Reviews, 2019, p. 119-151.
    Résumé : Assessing posttranslational modification (PTM) patterns within protein molecules and reading their functional implications present grand challenges for plant biology. We combine four perspectives on PTMs and their roles by considering five classes of PTMs as examples of the broader context of PTMs. These include modifications of the N terminus, glycosylation, phosphorylation, oxidation, and N-terminal and protein modifiers linked to protein degradation. We consider the spatial distribution of PTMs, the subcellular distribution of modifying enzymes, and their targets throughout the cell, and we outline the complexity of compartmentation in understanding of PTM function. We also consider PTMs temporally in the context of the lifetime of a protein molecule and the need for different PTMs for assembly, localization, function, and degradation. Finally, we consider the combined action of PTMs on the same proteins, their interactions, and the challenge ahead of integrating PTMs into an understanding of protein function in plants. Expected final online publication date for the Annual Review of Plant Biology Volume 70 is April 29, 2019. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
    Mots-clés : DBG, PROMTI.

  • A. Morillon et D. Gautheret, « Bridging the gap between reference and real transcriptomes », Genome Biology, vol. 20, nᵒ 1, p. 112, juin 2019.
    Résumé : Genetic, transcriptional, and post-transcriptional variations shape the transcriptome of individual cells, rendering establishing an exhaustive set of reference RNAs a complicated matter. Current reference transcriptomes, which are based on carefully curated transcripts, are lagging behind the extensive RNA variation revealed by massively parallel sequencing. Much may be missed by ignoring this unreferenced RNA diversity. There is plentiful evidence for non-reference transcripts with important phenotypic effects. Although reference transcriptomes are inestimable for gene expression analysis, they may turn limiting in important medical applications. We discuss computational strategies for retrieving hidden transcript diversity.
    Mots-clés : DBG, SSFA.

  • I. Nekrasova, V. Nikitashina, S. Bhullar, O. Arnaiz, D. P. Singh, E. Meyer, et A. Potekhin, « Loss of a Fragile Chromosome Region leads to the Screwy Phenotype in Paramecium tetraurelia », Genes, vol. 10, nᵒ 7, juill. 2019.
    Résumé : A conspicuous cell-shape phenotype known as "screwy" was reported to result from mutations at two or three uncharacterized loci in the ciliate Paramecium tetraurelia. Here, we describe a new screwy mutation, Spinning Top, which appeared spontaneously in the cross of an unrelated mutant with reference strain 51. The macronuclear (MAC) genome of the Spinning Top mutant is shown to lack a ~28.5-kb segment containing 18 genes at the end of one chromosome, which appears to result from a collinear deletion in the micronuclear (MIC) genome. We tested several candidate genes from the deleted locus by dsRNA-induced silencing in wild-type cells, and identified a single gene responsible for the phenotype. This gene, named Spade, encodes a 566-aa glutamine-rich protein with a C2HC zinc finger. Its silencing leads to a fast phenotype switch during vegetative growth, but cells recover a wild-type phenotype only 5-6 divisions after silencing is stopped. We analyzed 5 independently-obtained mutant alleles of the Sc1 locus, and concluded that all of them also lack the Spade gene and a number of neighboring genes in the MAC and MIC genomes. Mapping of the MAC deletion breakpoints revealed two different positions among the 5 alleles, both of which differ from the Spinning Top breakpoint. These results suggest that this MIC chromosome region is intrinsically unstable in strain 51.
    Mots-clés : chromosome fragile sites, cortical inheritance, DBG, epimutation, maternal inheritance, MICMAC, micronuclear deletion, Paramecium, trichocysts.
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  • T. B. Nesterova, G. Wei, H. Coker, G. Pintacuda, J. S. Bowness, T. Zhang, M. Almeida, B. Bloechl, B. Moindrot, E. J. Carter, I. Alvarez Rodrigo, Q. Pan, Y. Bi, C. - X. Song, et N. Brockdorff, « Systematic allelic analysis defines the interplay of key pathways in X chromosome inactivation », Nature Communications, vol. 10, nᵒ 1, p. 3129, juill. 2019.
    Résumé : Xist RNA, the master regulator of X chromosome inactivation, acts in cis to induce chromosome-wide silencing. Whilst recent studies have defined candidate silencing factors, their relative contribution to repressing different genes, and their relationship with one another is poorly understood. Here we describe a systematic analysis of Xist-mediated allelic silencing in mouse embryonic stem cell-based models. Using a machine learning approach we identify distance to the Xist locus and prior gene expression levels as key determinants of silencing efficiency. We go on to show that Spen, recruited through the Xist A-repeat, plays a central role, being critical for silencing of all except a subset of weakly expressed genes. Polycomb, recruited through the Xist B/C-repeat, also plays a key role, favouring silencing of genes with pre-existing H3K27me3 chromatin. LBR and the Rbm15/m6A-methyltransferase complex make only minor contributions to gene silencing. Together our results provide a comprehensive model for Xist-mediated chromosome silencing.
    Mots-clés : CHRODY, DBG.
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  • N. Paleiron, C. Soler, M. O. Hassan, D. Andriamanantena, R. Vong, C. Pourcel, et J. - B. Roseau, « First description of Mycobacterium tuberculosis and M. canettii concomitant infection: report of two cases », International Journal of Tuberculosis and Lung Disease, vol. 23, nᵒ 2, p. 232-+, janv. 2019.
    Résumé : <h2>SUMMARY</h2>We report the first two cases of tuberculous coinfection with Mycobacterium tuberculosis and M. canettii. Both patients were young Djiboutian females with pulmonary tuberculosis (TB). One had a miliary pattern with concomitant human immunodeficiency virus infection. Both recovered completely with a standard four-drug anti-tuberculosis treatment regimen. Due to the different natural reservoirs and routes of infection of these two strains, our study supports the common belief that multiple strains of infection in TB are related to superinfection rather than concomitant infection.
    Mots-clés : CHERDIR, coinfection, DBG, Mycobacterium canettii, SSFA, tuberculosis.

  • A. Petitalot, E. Dardillac, E. Jacquet, N. Nhiri, J. Guirouilh-Barbat, P. Julien, I. Bouazzaoui, D. Bonte, J. Feunteun, J. A. Schnell, P. Lafitte, J. - C. Aude, C. Nogues, E. Rouleau, R. Lidereau, B. S. Lopez, S. Zinn-Justin, S. M. Caputo, F. Bonnet, N. Jones, V. Bubien, M. Longy, N. Sevenet, S. Krieger, L. Casters, D. Vaur, N. Uhrhammer, Y. J. Bignon, S. Lizard, A. Dumont, F. Revillion, M. Leone, N. Boutry-Kryza, O. Sinilnikova, A. Remenieras, V. Bourdon, T. Noguchi, H. Sobol, P. - O. Harmand, P. Vilquin, P. Pujol, P. Jonveaux, M. Bronner, J. Sokolowska, C. Delnatte, V. Guibert, C. Garrec, S. Bezieau, F. Soubrier, E. Guillerm, F. Coulet, C. Lefol, V. Caux-Moncoutier, L. Golmard, C. Houdayer, D. Stoppa-Lyonnet, C. Delvincoun, O. Beaudoux, D. Muller, C. Toulas, M. Guillaud-Bataille, et B. Bressac-De Paillerets, « Combining Homologous Recombination and Phosphopeptide-binding Data to Predict the Impact of BRCA1 BRCT Variants on Cancer Risk », Molecular Cancer Research, vol. 17, nᵒ 1, p. 54-69, janv. 2019.
    Résumé : BRCA1 mutations have been identified that increase the risk of developing hereditary breast and ovarian cancers. Genetic screening is now offered to patients with a family history of cancer, to adapt their treatment and the management of their relatives. However, a large number of BRCA1 variants of uncertain significance (VUS) are detected. To better understand the significance of these variants, a high-throughput structural and functional analysis was performed on a large set of BRCA1 VUS. Information on both cellular localization and homology-directed DNA repair (HR) capacity was obtained for 78 BRCT missense variants in the UMD-BRCA1 database and measurement of the structural stability and phosphopeptide-binding capacities was performed for 42 mutated BRCT domains. This extensive and systematic analysis revealed that most characterized causal variants affect BRCT-domain solubility in bacteria and all impair BRCA1 HR activity in cells. Furthermore, binding to a set of 5 different phosphopeptides was tested: all causal variants showed phosphopeptide-binding defects and no neutral variant showed such defects. A classification is presented on the basis of mutated BRCT domain solubility, phosphopeptide-binding properties, and VUS HR capacity. These data suggest that HR-defective variants, which present, in addition, BRCT domains either insoluble in bacteria or defective for phosphopeptide binding, lead to an increased cancer risk. Furthermore, the data suggest that variants with a WT HR activity and whose BRCT domains bind with a WT affinity to the 5 phosphopeptides are neutral. The case of variants with WT HR activity and defective phosphopeptide binding should be further characterized, as this last functional defect might be sufficient per se to lead to tumorigenesis. Implications: The analysis of the current study on BRCA1 structural and functional defects on cancer risk and classification presented may improve clinical interpretation and therapeutic selection.
    Mots-clés : B3S, bach1 phosphopeptide, BIM, classification, DBG, dna-damage, domain, functional-analysis, hereditary breast, INTGEN, missense variants, ovarian, repair, structural basis.

  • M. Platel, H. Narassimprakash, D. Ciardo, O. Haccard, et K. Marheineke, « Genome wide decrease of DNA replication eye density at the midblastula transition of Xenopus laevis », Cell Cycle (Georgetown, Tex.), vol. 18, nᵒ 13, p. 1458-1472, juill. 2019.
    Résumé : During the first rapid divisions of early development in many species, the DNA:cytoplasm ratio increases until the midblastula transition (MBT) when transcription resumes and cell cycles lengthen. S phase is very rapid in early embryos, about 20-30 times faster than in differentiated cells. Using a combination of DNA fiber studies and a Xenopus laevis embryonic in vitro replication system, we show that S phase slows down shortly after the MBT owing to a genome wide decrease of replication eye density. Increasing the dNTP pool did not accelerate S phase or increase replication eye density implying that dNTPs are not rate limiting for DNA replication at the Xenopus MBT. Increasing the ratio of DNA:cytoplasm in egg extracts faithfully recapitulates changes in the spatial replication program in embryos, supporting the hypothesis that titration of soluble limiting factors could explain the observed changes in the DNA replication program at the MBT in Xenopus laevis.
    Mots-clés : DBG, DNA combing, DNA replication, DYNREP, midblastula transition (MBT), replication origins, S-phase, Xenopus laevis.

  • C. Pourcel, M. Touchon, N. Villeriot, J. - P. Vernadet, D. Couvin, C. Toffano-Nioche, et G. Vergnaud, « CRISPRCasdb a successor of CRISPRdb containing CRISPR arrays and cas genes from complete genome sequences, and tools to download and query lists of repeats and spacers », Nucleic Acids Research, oct. 2019.
    Résumé : In Archaea and Bacteria, the arrays called CRISPRs for 'clustered regularly interspaced short palindromic repeats' and the CRISPR associated genes or cas provide adaptive immunity against viruses, plasmids and transposable elements. Short sequences called spacers, corresponding to fragments of invading DNA, are stored in-between repeated sequences. The CRISPR-Cas systems target sequences homologous to spacers leading to their degradation. To facilitate investigations of CRISPRs, we developed 12 years ago a website holding the CRISPRdb. We now propose CRISPRCasdb, a completely new version giving access to both CRISPRs and cas genes. We used CRISPRCasFinder, a program that identifies CRISPR arrays and cas genes and determine the system's type and subtype, to process public whole genome assemblies. Strains are displayed either in an alphabetic list or in taxonomic order. The database is part of the CRISPR-Cas++ website which also offers the possibility to analyse submitted sequences and to download programs. A BLAST search against lists of repeats and spacers extracted from the database is proposed. To date, 16 990 complete prokaryote genomes (16 650 bacteria from 2973 species and 340 archaea from 300 species) are included. CRISPR-Cas systems were found in 36% of Bacteria and 75% of Archaea strains. CRISPRCasdb is freely accessible at https://crisprcas.i2bc.paris-saclay.fr/.
    Mots-clés : DBG, SSFA.

  • T. Rubio, D. Oyanedel, Y. Labreuche, E. Toulza, X. Luo, M. Bruto, C. Chaparro, M. Torres, J. de Lorgeril, P. Haffner, J. Vidal-Dupiol, A. Lagorce, B. Petton, G. Mitta, A. Jacq, F. Le Roux, G. M. Charrière, et D. Destoumieux-Garzón, « Species-specific mechanisms of cytotoxicity toward immune cells determine the successful outcome of Vibrio infections », Proceedings of the National Academy of Sciences of the United States of America, juin 2019.
    Résumé : Vibrio species cause infectious diseases in humans and animals, but they can also live as commensals within their host tissues. How Vibrio subverts the host defenses to mount a successful infection remains poorly understood, and this knowledge is critical for predicting and managing disease. Here, we have investigated the cellular and molecular mechanisms underpinning infection and colonization of 2 virulent Vibrio species in an ecologically relevant host model, oyster, to study interactions with marine Vibrio species. All Vibrio strains were recognized by the immune system, but only nonvirulent strains were controlled. We showed that virulent strains were cytotoxic to hemocytes, oyster immune cells. By analyzing host and bacterial transcriptional responses to infection, together with Vibrio gene knock-outs, we discovered that Vibrio crassostreae and Vibrio tasmaniensis use distinct mechanisms to cause hemocyte lysis. Whereas V. crassostreae cytotoxicity is dependent on a direct contact with hemocytes and requires an ancestral gene encoding a protein of unknown function, r5.7, V. tasmaniensis cytotoxicity is dependent on phagocytosis and requires intracellular secretion of T6SS effectors. We conclude that proliferation of commensal vibrios is controlled by the host immune system, preventing systemic infections in oysters, whereas the successful infection of virulent strains relies on Vibrio species-specific molecular determinants that converge to compromise host immune cell function, allowing evasion of the host immune system.
    Mots-clés : cytolysis, DBG, dual RNA-seq, pathogenesis, SRRB, T6SS, toxin.

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