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Accueil > Départements > Biologie des Génomes > Fabrice CONFALONIERI : Radiorésistance des bactéries et des archées

Publications de l’équipe


  • 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, BDG, MICROBIO, RBA.


  • C. Bouthier de la Tour, M. Mathieu, L. Meyer, P. Dupaigne, F. Passot, P. Servant, S. Sommer, E. Le Cam, et F. Confalonieri, « In vivo and in vitro characterization of DdrC, a DNA damage response protein in Deinococcus radiodurans bacterium », PloS One, vol. 12, nᵒ 5, p. e0177751, 2017.
    Résumé : The bacterium Deinococcus radiodurans possesses a set of Deinococcus-specific genes highly induced after DNA damage. Among them, ddrC (dr0003) was recently re-annotated, found to be in the inverse orientation and called A2G07_00380. Here, we report the first in vivo and in vitro characterization of the corrected DdrC protein to better understand its function in irradiated cells. In vivo, the ΔddrC null mutant is sensitive to high doses of UV radiation and the ddrC deletion significantly increases UV-sensitivity of ΔuvrA or ΔuvsE mutant strains. We show that the expression of the DdrC protein is induced after γ-irradiation and is under the control of the regulators, DdrO and IrrE. DdrC is rapidly recruited into the nucleoid of the irradiated cells. In vitro, we show that DdrC is able to bind single- and double-stranded DNA with a preference for the single-stranded DNA but without sequence or shape specificity and protects DNA from various nuclease attacks. DdrC also condenses DNA and promotes circularization of linear DNA. Finally, we show that the purified protein exhibits a DNA strand annealing activity. Altogether, our results suggest that DdrC is a new DNA binding protein with pleiotropic activities. It might maintain the damaged DNA fragments end to end, thus limiting their dispersion and extensive degradation after exposure to ionizing radiation. DdrC might also be an accessory protein that participates in a single strand annealing pathway whose importance in DNA repair becomes apparent when DNA is heavily damaged.
    Mots-clés : DBG, RBA.

  • S. Li, E. Porcel, H. Remita, S. Marco, M. Réfrégiers, M. Dutertre, F. Confalonieri, et S. Lacombe, « Platinum nanoparticles: an exquisite tool to overcome radioresistance », Cancer Nanotechnology, vol. 8, nᵒ 1, p. 4, 2017.
    Résumé : BACKGROUD: Small metallic nanoparticles are proposed as potential nanodrugs to optimize the performances of radiotherapy. This strategy, based on the enrichment of tumours with nanoparticles to amplify radiation effects in the tumour, aims at increasing the cytopathic effect in tumours while healthy tissue is preserved, an important challenge in radiotherapy. Another major cause of radiotherapy failure is the radioresistance of certain cancers. Surprisingly, the use of nanoparticles to overcome radioresistance has not, to the best of our knowledge, been extensively investigated. The mechanisms of radioresistance have been extensively studied using Deinococcus radiodurans, the most radioresistant organism ever reported, as a model. METHODS: In this work, we investigated the impact of ultra-small platinum nanoparticles (1.7 nm) on this organism, including uptake, toxicity, and effects on radiation responses. RESULTS: We showed that the nanoparticles penetrate D. radiodurans cells, despite the 150 nm cell wall thickness with a minimal inhibition concentration on the order of 4.8 mg L(-1). We also found that the nanoparticles amplify gamma ray radiation effects by >40%. CONCLUSIONS: Finally, this study demonstrates the capacity of metallic nanoparticles to amplify radiation in radioresistant organisms, thus opening the perspective to use nanoparticles not only to improve tumour targeting but also to overcome radioresistance.
    Mots-clés : DBG, Deinococcus radiodurans, Metallic nanoparticles, Radio-enhancement, Radioresistance, Radiosensitization, RBA.

  • R. I. Ponce-Toledo, P. Deschamps, P. López-García, Y. Zivanovic, K. Benzerara, et D. Moreira, « An Early-Branching Freshwater Cyanobacterium at the Origin of Plastids », Current biology: CB, vol. 27, nᵒ 3, p. 386-391, févr. 2017.
    Résumé : Photosynthesis evolved in eukaryotes by the endosymbiosis of a cyanobacterium, the future plastid, within a heterotrophic host. This primary endosymbiosis occurred in the ancestor of Archaeplastida, a eukaryotic supergroup that includes glaucophytes, red algae, green algae, and land plants [1-4]. However, although the endosymbiotic origin of plastids from a single cyanobacterial ancestor is firmly established, the nature of that ancestor remains controversial: plastids have been proposed to derive from either early- or late-branching cyanobacterial lineages [5-11]. To solve this issue, we carried out phylogenomic and supernetwork analyses of the most comprehensive dataset analyzed so far including plastid-encoded proteins and nucleus-encoded proteins of plastid origin resulting from endosymbiotic gene transfer (EGT) of primary photosynthetic eukaryotes, as well as wide-ranging genome data from cyanobacteria, including novel lineages. Our analyses strongly support that plastids evolved from deep-branching cyanobacteria and that the present-day closest cultured relative of primary plastids is Gloeomargarita lithophora. This species belongs to a recently discovered cyanobacterial lineage widespread in freshwater microbialites and microbial mats [12, 13]. The ecological distribution of this lineage sheds new light on the environmental conditions where the emergence of photosynthetic eukaryotes occurred, most likely in a terrestrial-freshwater setting. The fact that glaucophytes, the first archaeplastid lineage to diverge, are exclusively found in freshwater ecosystems reinforces this hypothesis. Therefore, not only did plastids emerge early within cyanobacteria, but the first photosynthetic eukaryotes most likely evolved in terrestrial-freshwater settings, not in oceans as commonly thought.
    Mots-clés : chloroplasts, Cyanobacteria, DBG, evolution, molecular phylogeny, phylogenomics, Plastids, RBA.

  • R. Trias, B. Ménez, P. le Campion, Y. Zivanovic, L. Lecourt, A. Lecoeuvre, P. Schmitt-Kopplin, J. Uhl, S. R. Gislason, H. A. Alfreðsson, K. G. Mesfin, S. Ó. Snæbjörnsdóttir, E. S. Aradóttir, I. Gunnarsson, J. M. Matter, M. Stute, E. H. Oelkers, et E. Gérard, « High reactivity of deep biota under anthropogenic CO2 injection into basalt », Nature Communications, vol. 8, nᵒ 1, p. 1063, oct. 2017.
    Résumé : Basalts are recognized as one of the major habitats on Earth, harboring diverse and active microbial populations. Inconsistently, this living component is rarely considered in engineering operations carried out in these environments. This includes carbon capture and storage (CCS) technologies that seek to offset anthropogenic CO2 emissions into the atmosphere by burying this greenhouse gas in the subsurface. Here, we show that deep ecosystems respond quickly to field operations associated with CO2 injections based on a microbiological survey of a basaltic CCS site. Acidic CO2-charged groundwater results in a marked decrease (by ~ 2.5-4) in microbial richness despite observable blooms of lithoautotrophic iron-oxidizing Betaproteobacteria and degraders of aromatic compounds, which hence impact the aquifer redox state and the carbon fate. Host-basalt dissolution releases nutrients and energy sources, which sustain the growth of autotrophic and heterotrophic species whose activities may have consequences on mineral storage.
    Mots-clés : DBG, RBA.


  • E. Barbier, A. Lagorce, A. Hachemi, M. Dutertre, A. Gorlas, L. Morand, C. Saint-Pierre, J. - L. Ravanat, T. Douki, J. Armengaud, D. Gasparutto, F. Confalonieri, et J. Breton, « Oxidative DNA Damage and Repair in the Radioresistant Archaeon Thermococcus gammatolerans », Chemical Research in Toxicology, vol. 29, nᵒ 11, p. 1796-1809, nov. 2016.
    Résumé : The hyperthermophilic archaeon Thermococcus gammatolerans can resist huge doses of γ-irradiation, up to 5.0 kGy, without loss of viability. The potential to withstand such harsh conditions is probably due to complementary passive and active mechanisms, including repair of damaged chromosomes. In this work, we documented the formation and repair of oxidative DNA lesions in T. gammatolerans. The basal level of the oxidized nucleoside, 8-oxo-2'-deoxyguanosine (8-oxo-dGuo), was established at 9.2 (± 0.9) 8-oxo-dGuo per 10(6) nucleosides, a higher level than those usually measured in eukaryotic cells or bacteria. A significant increase in oxidative damage, i.e., up to 24.2 (± 8.0) 8-oxo-dGuo/10(6) nucleosides, was measured for T. gammatolerans exposed to a 5.0 kGy dose of γ-rays. Surprisingly, the yield of radiation-induced modifications was lower than those previously observed for human cells exposed to doses corresponding to a few grays. One hour after irradiation, 8-oxo-dGuo levels were significantly reduced, indicating an efficient repair. Two putative base excision repair (BER) enzymes, TGAM_1277 and TGAM_1653, were demonstrated both by proteomics and transcriptomics to be present in the cells without exposure to ionizing radiation. Their transcripts were moderately upregulated after gamma irradiation. After heterologous production and purification of these enzymes, biochemical assays based on electrophoresis and MALDI-TOF (matrix-assisted laser desorption ionization-time of flight) mass spectrometry indicated that both have a β-elimination cleavage activity. TGAM_1653 repairs 8-oxo-dGuo, whereas TGAM_1277 is also able to remove lesions affecting pyrimidines (1-[2-deoxy-β-d-erythro-pentofuranosyl]-5-hydroxyhydantoin (5-OH-dHyd) and 1-[2-deoxy-β-d-erythro-pentofuranosyl]-5-hydroxy-5-methylhydantoin (5-OH-5-Me-dHyd)). This work showed that in normal growth conditions or in the presence of a strong oxidative stress, T. gammatolerans has the potential to rapidly reduce the extent of DNA oxidation, with at least these two BER enzymes as bodyguards with distinct substrate ranges.
    Mots-clés : DBG, RBA.

  • A. Devigne, P. Guérin, J. Lisboa, S. Quevillon-Cheruel, J. Armengaud, S. Sommer, C. Bouthier de la Tour, et P. Servant, « PprA Protein Is Involved in Chromosome Segregation via Its Physical and Functional Interaction with DNA Gyrase in Irradiated Deinococcus radiodurans Bacteria », mSphere, vol. 1, nᵒ 1, févr. 2016.
    Résumé : PprA, a radiation-induced Deinococcus-specific protein, was previously shown to be required for cell survival and accurate chromosome segregation after exposure to ionizing radiation. Here, we used an in vivo approach to determine, by shotgun proteomics, putative PprA partners coimmunoprecipitating with PprA when cells were exposed to gamma rays. Among them, we found the two subunits of DNA gyrase and, thus, chose to focus our work on characterizing the activities of the deinococcal DNA gyrase in the presence or absence of PprA. Loss of PprA rendered cells hypersensitive to novobiocin, an inhibitor of the B subunit of DNA gyrase. We showed that treatment of bacteria with novobiocin resulted in induction of the radiation desiccation response (RDR) regulon and in defects in chromosome segregation that were aggravated by the absence of PprA. In vitro, the deinococcal DNA gyrase, like other bacterial DNA gyrases, possesses DNA negative supercoiling and decatenation activities. These two activities are inhibited in vitro by novobiocin and nalidixic acid, whereas PprA specifically stimulates the decatenation activity of DNA gyrase. Together, these results suggest that PprA plays a major role in chromosome decatenation via its interaction with the deinococcal DNA gyrase when D. radiodurans cells are recovering from exposure to ionizing radiation. IMPORTANCE D. radiodurans is one of the most radiation-resistant organisms known. This bacterium is able to cope with high levels of DNA lesions generated by exposure to extreme doses of ionizing radiation and to reconstruct a functional genome from hundreds of radiation-induced chromosomal fragments. Here, we identified partners of PprA, a radiation-induced Deinococcus-specific protein, previously shown to be required for radioresistance. Our study leads to three main findings: (i) PprA interacts with DNA gyrase after irradiation, (ii) treatment of cells with novobiocin results in defects in chromosome segregation that are aggravated by the absence of PprA, and (iii) PprA stimulates the decatenation activity of DNA gyrase. Our results extend the knowledge of how D. radiodurans cells survive exposure to extreme doses of gamma irradiation and point out the link between DNA repair, chromosome segregation, and DNA gyrase activities in the radioresistant D. radiodurans bacterium.
    Mots-clés : B3S, DBG, Deinococcus radiodurans, DNA decatenation, DNA gyrase, FAAM, PprA, RBA.

  • G. Hoff, C. Bertrand, L. Zhang, E. Piotrowski, L. Chipot, C. Bontemps, F. Confalonieri, S. McGovern, F. Lecointe, A. Thibessard, et P. Leblond, « Multiple and Variable NHEJ-Like Genes Are Involved in Resistance to DNA Damage in Streptomyces ambofaciens », Frontiers in Microbiology, vol. 7, p. 1901, 2016.
    Résumé : Non-homologous end-joining (NHEJ) is a double strand break (DSB) repair pathway which does not require any homologous template and can ligate two DNA ends together. The basic bacterial NHEJ machinery involves two partners: the Ku protein, a DNA end binding protein for DSB recognition and the multifunctional LigD protein composed a ligase, a nuclease and a polymerase domain, for end processing and ligation of the broken ends. In silico analyses performed in the 38 sequenced genomes of Streptomyces species revealed the existence of a large panel of NHEJ-like genes. Indeed, ku genes or ligD domain homologues are scattered throughout the genome in multiple copies and can be distinguished in two categories: the "core" NHEJ gene set constituted of conserved loci and the "variable" NHEJ gene set constituted of NHEJ-like genes present in only a part of the species. In Streptomyces ambofaciens ATCC23877, not only the deletion of "core" genes but also that of "variable" genes led to an increased sensitivity to DNA damage induced by electron beam irradiation. Multiple mutants of ku, ligase or polymerase encoding genes showed an aggravated phenotype compared to single mutants. Biochemical assays revealed the ability of Ku-like proteins to protect and to stimulate ligation of DNA ends. RT-qPCR and GFP fusion experiments suggested that ku-like genes show a growth phase dependent expression profile consistent with their involvement in DNA repair during spores formation and/or germination.
    Mots-clés : DBG, DNA Damage, DNA Repair, double strand breaks (DSBs), Ku protein, ligases, non-homologous end joining, RBA, Streptomyces.

  • D. Moreira, R. Tavera, K. Benzerara, F. Skouri-Panet, E. Couradeau, E. Gérard, C. Loussert Fonta, E. Novelo, Y. Zivanovic, et P. López-García, « Description of Gloeomargarita lithophora gen. nov., sp. nov., a thylakoid-bearing basal-branching cyanobacterium with intracellular carbonates, and proposal for Gloeomargaritales ord. nov », International Journal of Systematic and Evolutionary Microbiology, nov. 2016.
    Résumé : A unicellular cyanobacterium, strain Alchichica-D10, was isolated from microbialites of the alkaline Lake Alchichica, Mexico. The cells were short rods (3.9 ± 0.6 μm in length and 1.1 ± 0.1 μm in width) forming biofilms of intense emerald green color. They exhibited red autofluorescence under UV light excitation. UV-visible absorption spectra revealed that they contain chlorophyll a and phycocyanin, and electron microscopy showed the presence of thylakoids. The strain grew within a temperature range of 15-30 °C. Genomic DNA G+C content was 52.2 mol%. The most remarkable feature of this species was its granular cytoplasm, due to the presence of numerous intracellular spherical granules (16-26 per cell) with an average diameter of 270 nm. These granules, easily visible under scanning electron microscopy, were composed of amorphous carbonate containing Ca, Mg, Ba, and Sr. A multi-gene phylogeny based on the analysis of 59 conserved protein markers supported robustly that this strain occupies a deep position in the cyanobacterial tree. Based on its phenotypic characters and phylogenetic position, strain Alchichica-D10 is considered to represent a new genus and novel species of cyanobacteria for which the name Gloeomargarita lithophora gen. nov., sp. nov. is proposed. The type strain is Alchichica-D10 (Culture Collection of Algae and Protozoa CCAP strain 1437/1; Collections de Cyanobactéries et Microalgues Vivantes of the Museum National d'Histoire Naturelle in Paris strain PMC 919.15). Furthermore, a new family, Gloeomargaritaceae, and a new order, Gloeoemargaritales, are proposed to accommodate this species under the International Code of Nomenclature for algae, fungi, and plants.
    Mots-clés : DBG, RBA.

  • A. Saghaï, Y. Zivanovic, D. Moreira, K. Benzerara, P. Bertolino, M. Ragon, R. Tavera, A. I. López-Archilla, et P. López-García, « Comparative metagenomics unveils functions and genome features of microbialite-associated communities along a depth gradient », Environmental Microbiology, vol. 18, nᵒ 12, p. 4990-5004, déc. 2016.
    Résumé : Modern microbialites are often used as analogs of Precambrian stromatolites; therefore, studying the metabolic interplay within their associated microbial communities can help formulating hypotheses on their formation and long-term preservation within the fossil record. We performed a comparative metagenomic analysis of microbialite samples collected at two sites and along a depth gradient in Lake Alchichica (Mexico). The community structure inferred from single-copy gene family identification and long-contig (>10 kb) assignation, consistently with previous rRNA gene surveys, showed a wide prokaryotic diversity dominated by Alphaproteobacteria, Gammaproteobacteria, Cyanobacteria, and Bacteroidetes, while eukaryotes were largely dominated by green algae or diatoms. Functional analyses based on RefSeq, COG and SEED assignations revealed the importance of housekeeping functions, with an overrepresentation of genes involved in carbohydrate metabolism, as compared with other metabolic capacities. The search for genes diagnostic of specific metabolic functions revealed the important involvement of Alphaproteobacteria in anoxygenic photosynthesis and sulfide oxidation, and Cyanobacteria in oxygenic photosynthesis and nitrogen fixation. Surprisingly, sulfate reduction appeared negligible. Comparative analyses suggested functional similarities among various microbial mat and microbialite metagenomes as compared with soil or oceans, but showed differences in microbial processes among microbialite types linked to local environmental conditions.
    Mots-clés : DBG, RBA.


  • C. Bouthier de la Tour, L. Blanchard, R. Dulermo, M. Ludanyi, A. Devigne, J. Armengaud, S. Sommer, et A. de Groot, « The abundant and essential HU proteins in Deinococcus deserti and Deinococcus radiodurans are translated from leaderless mRNA », Microbiology (Reading, England), vol. 161, nᵒ 12, p. 2410-2422, déc. 2015.
    Résumé : HU proteins have an important architectural role in nucleoid organization in bacteria. Compared with HU of many bacteria, HU proteins from Deinococcus species possess an N-terminal lysine-rich extension similar to the eukaryotic histone H1 C-terminal domain involved in DNA compaction. The single HU gene in Deinococcus radiodurans, encoding DrHU, is required for nucleoid compaction and cell viability. Deinococcus deserti contains three expressed HU genes, encoding DdHU1, DdHU2 and DdHU3. Here, we show that either DdHU1 or DdHU2 is essential in D. deserti. DdHU1 and DdHU2, but not DdHU3, can substitute for DrHU in D. radiodurans, indicating that DdHU3 may have a non-essential function different from DdHU1, DdHU2 and DrHU. Interestingly, the highly abundant DrHU and DdHU1 proteins, and also the less expressed DdHU2, are translated in Deinococcus from leaderless mRNAs, which lack a 5'-untranslated region and, hence, the Shine-Dalgarno sequence. Unexpectedly, cloning the DrHU or DdHU1 gene under control of a strong promoter in an expression plasmid, which results in leadered transcripts, strongly reduced the DrHU and DdHU1 protein level in D. radiodurans compared with that obtained from the natural leaderless gene. We also show that the start codon position for DrHU and DdHU1 should be reannotated, resulting in proteins that are 15 and 4 aa residues shorter than initially reported. The expression level and start codon correction were crucial for functional characterization of HU in Deinococcus.
    Mots-clés : 5' Untranslated Regions, Amino Acid Sequence, Bacterial Proteins, Codon, Initiator, DBG, Deinococcus, DNA-Binding Proteins, Gene Expression Regulation, Bacterial, Molecular Sequence Data, Protein Biosynthesis, RBA, RNA, Messenger, Sequence Alignment.

  • A. Devigne, S. Ithurbide, C. Bouthier de la Tour, F. Passot, M. Mathieu, S. Sommer, et P. Servant, « DdrO is an essential protein that regulates the radiation desiccation response and the apoptotic-like cell death in the radioresistant Deinococcus radiodurans bacterium », Molecular Microbiology, vol. 96, nᵒ 5, p. 1069-1084, juin 2015.
    Résumé : Deinococcus radiodurans is known for its extreme radioresistance. Comparative genomics identified a radiation-desiccation response (RDR) regulon comprising genes that are highly induced after DNA damage and containing a conserved motif (RDRM) upstream of their coding region. We demonstrated that the RDRM sequence is involved in cis-regulation of the RDR gene ddrB in vivo. Using a transposon mutagenesis approach, we showed that, in addition to ddrO encoding a predicted RDR repressor and irrE encoding a positive regulator recently shown to cleave DdrO in Deinococcus deserti, two genes encoding α-keto-glutarate dehydrogenase subunits are involved in ddrB regulation. In wild-type cells, the DdrO cell concentration decreased transiently in an IrrE-dependent manner at early times after irradiation. Using a conditional gene inactivation system, we showed that DdrO depletion enhanced expression of three RDR proteins, consistent with the hypothesis that DdrO acts as a repressor of the RDR regulon. DdrO-depleted cells loose viability and showed morphological changes evocative of an apoptotic-like response, including membrane blebbing, defects in cell division and DNA fragmentation. We propose that DNA repair and apoptotic-like death might be two responses mediated by the same regulators, IrrE and DdrO, but differently activated depending on the persistence of IrrE-dependent DdrO cleavage.
    Mots-clés : Amino Acid Sequence, Bacterial Proteins, DBG, Dehydration, Deinococcus, DNA Damage, DNA Repair, Gene Expression Regulation, Bacterial, Genomics, Ketoglutarate Dehydrogenase Complex, Mutagenesis, Open Reading Frames, Promoter Regions, Genetic, Protein Structure, Tertiary, RBA, Regulon.

  • R. Dulermo, T. Onodera, G. Coste, F. Passot, M. Dutertre, M. Porteron, F. Confalonieri, S. Sommer, et C. Pasternak, « Identification of new genes contributing to the extreme radioresistance of Deinococcus radiodurans using a Tn5-based transposon mutant library », PloS One, vol. 10, nᵒ 4, p. e0124358, 2015.
    Résumé : Here, we have developed an extremely efficient in vivo Tn5-based mutagenesis procedure to construct a Deinococcus radiodurans insertion mutant library subsequently screened for sensitivity to genotoxic agents such as γ and UV radiations or mitomycin C. The genes inactivated in radiosensitive mutants belong to various functional categories, including DNA repair functions, stress responses, signal transduction, membrane transport, several metabolic pathways, and genes of unknown function. Interestingly, preliminary characterization of previously undescribed radiosensitive mutants suggests the contribution of cyclic di-AMP signaling in the recovery of D. radiodurans cells from genotoxic stresses, probably by modulating several pathways involved in the overall cell response. Our analyses also point out a new transcriptional regulator belonging to the GntR family, encoded by DR0265, and a predicted RNase belonging to the newly described Y family, both contributing to the extreme radioresistance of D. radiodurans. Altogether, this work has revealed new cell responses involved either directly or indirectly in repair of various cell damage and confirmed that D. radiodurans extreme radiation resistance is determined by a multiplicity of pathways acting as a complex network.
    Mots-clés : Bacterial Proteins, DBG, Deinococcus, Dinucleoside Phosphates, DNA Damage, DNA Repair, DNA Transposable Elements, DNA, Bacterial, Gamma Rays, Gene Deletion, Gene Expression Regulation, Bacterial, Gene Library, Gene Regulatory Networks, Genes, Bacterial, Genetic Complementation Test, Hydrogen Peroxide, Mitomycin, Mutagenesis, Insertional, Mutation, Open Reading Frames, oxidative stress, Radiation Tolerance, RBA, Transcription Factors, Transposases, Ultraviolet Rays.

  • S. Ithurbide, E. Bentchikou, G. Coste, B. Bost, P. Servant, et S. Sommer, « Single Strand Annealing Plays a Major Role in RecA-Independent Recombination between Repeated Sequences in the Radioresistant Deinococcus radiodurans Bacterium », PLoS genetics, vol. 11, nᵒ 10, p. e1005636, oct. 2015.
    Résumé : The bacterium Deinococcus radiodurans is one of the most radioresistant organisms known. It is able to reconstruct a functional genome from hundreds of radiation-induced chromosomal fragments. Our work aims to highlight the genes involved in recombination between 438 bp direct repeats separated by intervening sequences of various lengths ranging from 1,479 bp to 10,500 bp to restore a functional tetA gene in the presence or absence of radiation-induced DNA double strand breaks. The frequency of spontaneous deletion events between the chromosomal direct repeats were the same in recA+ and in ΔrecA, ΔrecF, and ΔrecO bacteria, whereas recombination between chromosomal and plasmid DNA was shown to be strictly dependent on the RecA and RecF proteins. The presence of mutations in one of the repeated sequence reduced, in a MutS-dependent manner, the frequency of the deletion events. The distance between the repeats did not influence the frequencies of deletion events in recA+ as well in ΔrecA bacteria. The absence of the UvrD protein stimulated the recombination between the direct repeats whereas the absence of the DdrB protein, previously shown to be involved in DNA double strand break repair through a single strand annealing (SSA) pathway, strongly reduces the frequency of RecA- (and RecO-) independent deletions events. The absence of the DdrB protein also increased the lethal sectoring of cells devoid of RecA or RecO protein. γ-irradiation of recA+ cells increased about 10-fold the frequencies of the deletion events, but at a lesser extend in cells devoid of the DdrB protein. Altogether, our results suggest a major role of single strand annealing in DNA repeat deletion events in bacteria devoid of the RecA protein, and also in recA+ bacteria exposed to ionizing radiation.
    Mots-clés : BIM, DBG, Deinococcus, DNA Breaks, Double-Stranded, DNA Damage, DNA Repair, Gamma Rays, Genome, Mutation, Radiation Tolerance, RBA, Rec A Recombinases, Recombination, Genetic.

  • P. López-García, Y. Zivanovic, P. Deschamps, et D. Moreira, « Bacterial gene import and mesophilic adaptation in archaea », Nature Reviews. Microbiology, vol. 13, nᵒ 7, p. 447-456, 2015.
    Résumé : It is widely believed that the archaeal ancestor was hyperthermophilic, but during archaeal evolution, several lineages - including haloarchaea and their sister methanogens, the Thaumarchaeota, and the uncultured Marine Group II and Marine Group III Euryarchaeota (MGII/III) - independently adapted to lower temperatures. Recent phylogenomic studies suggest that the ancestors of these lineages were recipients of massive horizontal gene transfer from bacteria. Many of the acquired genes, which are often involved in metabolism and cell envelope biogenesis, were convergently acquired by distant mesophilic archaea. In this Opinion article, we explore the intriguing hypothesis that the import of these bacterial genes was crucial for the adaptation of archaea to mesophilic lifestyles.
    Mots-clés : Adaptation, Biological, Archaea, DBG, Evolution, Molecular, Phylogeny, RBA, Temperature.

  • F. M. Passot, H. H. Nguyen, C. Dard-Dascot, C. Thermes, P. Servant, O. Espéli, et S. Sommer, « Nucleoid organization in the radioresistant bacterium Deinococcus radiodurans », Molecular Microbiology, vol. 97, nᵒ 4, p. 759-774, août 2015.
    Résumé : Processes favoring the exceptional resistance to genotoxic stress of Deinococcus radiodurans are not yet completely characterized. It was postulated that its nucleoid and chromosome(s) organization could participate in the DNA double strand break repair process. Here, we investigated the organization of chromosome 1 by localization of three chromosomal loci including oriC, Ter and a locus located in its left arm. For this purpose, we used a ParB-parS system to visualize the position of the loci before and after exposure to γ-rays. By comparing the number of fluorescent foci with the number of copies of the studied loci present in the cells measured by quantitative polymerase chain reaction (qPCR), we demonstrated that the 4-10 copies of chromosome 1 per cell are dispersed within the nucleoid before irradiation, indicating that the chromosome copies are not prealigned. Chromosome segregation is progressive but not co-ordinated, allowing each locus to be paired with its sister during part of the cell cycle. After irradiation, the nucleoid organization is modified, involving a transient alignment of the loci in the late stage of DNA repair and a delay of segregation of the Ter locus. We discuss how these events can influence DNA double strand break repair.
    Mots-clés : Bacterial Proteins, Chromatin Assembly and Disassembly, Chromosome Mapping, Chromosomes, Bacterial, DBG, Deinococcus, DNA Breaks, Double-Stranded, DNA Damage, DNA Repair, DNA, Bacterial, DNA-Binding Proteins, NGS, PF, Radiation Tolerance, RBA.

  • A. Quaiser, A. Dufresne, F. Ballaud, S. Roux, Y. Zivanovic, J. Colombet, T. Sime-Ngando, et A. - J. Francez, « Diversity and comparative genomics of Microviridae in Sphagnum- dominated peatlands », Frontiers in Microbiology, vol. 6, p. 375, 2015.
    Résumé : Microviridae, a family of bacteria-infecting ssDNA viruses, is one of the still poorly characterized bacteriophage groups, even though it includes phage PhiX174, one of the main models in virology for genomic and capsid structure studies. Recent studies suggest that they are diverse and well represented in marine and freshwater virioplankton as well as in human microbiomes. However, their diversity, abundance, and ecological role are completely unknown in soil ecosystems. Here we present the comparative analysis of 17 completely assembled Microviridae genomes from 12 viromes of a Sphagnum-dominated peatland. Phylogenetic analysis of the conserved major capsid protein sequences revealed the affiliation to Gokushovirinae and Pichovirinae as well as to two newly defined subfamilies, the Aravirinae and Stokavirinae. Additionally, two new distinct prophages were identified in the genomes of Parabacteroides merdae and Parabacteroides distasonis representing a potential new subfamily of Microviridae. The differentiation of the subfamilies was confirmed by gene order and similarity analysis. Relative abundance analysis using the affiliation of the major capsid protein (VP1) revealed that Gokushovirinae, followed by Aravirinae, are the most abundant Microviridae in 11 out of 12 peat viromes. Sequences matching the Gokushovirinae and Aravirinae VP1 matching sequences, respectively, accounted for up to 4.19 and 0.65% of the total number of sequences in the corresponding virome, respectively. In this study we provide new genome information of Microviridae and pave the way toward quantitative estimations of Microviridae subfamilies.
    Mots-clés : Aravirinae, DBG, Gokushovirinae, Microviridae, RBA, ssDNA phage, Stokavirinae, viral metagenomics, virus diversity, virus ecology.

  • A. Saghaï, Y. Zivanovic, N. Zeyen, D. Moreira, K. Benzerara, P. Deschamps, P. Bertolino, M. Ragon, R. Tavera, A. I. López-Archilla, et P. López-García, « Metagenome-based diversity analyses suggest a significant contribution of non-cyanobacterial lineages to carbonate precipitation in modern microbialites », Frontiers in Microbiology, vol. 6, p. 797, 2015.
    Résumé : Cyanobacteria are thought to play a key role in carbonate formation due to their metabolic activity, but other organisms carrying out oxygenic photosynthesis (photosynthetic eukaryotes) or other metabolisms (e.g., anoxygenic photosynthesis, sulfate reduction), may also contribute to carbonate formation. To obtain more quantitative information than that provided by more classical PCR-dependent methods, we studied the microbial diversity of microbialites from the Alchichica crater lake (Mexico) by mining for 16S/18S rRNA genes in metagenomes obtained by direct sequencing of environmental DNA. We studied samples collected at the Western (AL-W) and Northern (AL-N) shores of the lake and, at the latter site, along a depth gradient (1, 5, 10, and 15 m depth). The associated microbial communities were mainly composed of bacteria, most of which seemed heterotrophic, whereas archaea were negligible. Eukaryotes composed a relatively minor fraction dominated by photosynthetic lineages, diatoms in AL-W, influenced by Si-rich seepage waters, and green algae in AL-N samples. Members of the Gammaproteobacteria and Alphaproteobacteria classes of Proteobacteria, Cyanobacteria, and Bacteroidetes were the most abundant bacterial taxa, followed by Planctomycetes, Deltaproteobacteria (Proteobacteria), Verrucomicrobia, Actinobacteria, Firmicutes, and Chloroflexi. Community composition varied among sites and with depth. Although cyanobacteria were the most important bacterial group contributing to the carbonate precipitation potential, photosynthetic eukaryotes, anoxygenic photosynthesizers and sulfate reducers were also very abundant. Cyanobacteria affiliated to Pleurocapsales largely increased with depth. Scanning electron microscopy (SEM) observations showed considerable areas of aragonite-encrusted Pleurocapsa-like cyanobacteria at microscale. Multivariate statistical analyses showed a strong positive correlation of Pleurocapsales and Chroococcales with aragonite formation at macroscale, and suggest a potential causal link. Despite the previous identification of intracellularly calcifying cyanobacteria in Alchichica microbialites, most carbonate precipitation seems extracellular in this system.
    Mots-clés : anoxygenic photosynthesis, biomineralization, Cyanobacteria, DBG, diatoms, green algae, metagenomics, RBA, stromatolite, sulfate-reduction.

  • Y. - S. Yang, B. Fernandez, A. Lagorce, V. Aloin, K. M. De Guillen, J. - B. Boyer, A. Dedieu, F. Confalonieri, J. Armengaud, et C. Roumestand, « Prioritizing targets for structural biology through the lens of proteomics: the archaeal protein TGAM_1934 from Thermococcus gammatolerans », Proteomics, vol. 15, nᵒ 1, p. 114-123, janv. 2015.
    Résumé : ORFans are hypothetical proteins lacking any significant sequence similarity with other proteins. Here, we highlighted by quantitative proteomics the TGAM_1934 ORFan from the hyperradioresistant Thermococcus gammatolerans archaeon as one of the most abundant hypothetical proteins. This protein has been selected as a priority target for structure determination on the basis of its abundance in three cellular conditions. Its solution structure has been determined using multidimensional heteronuclear NMR spectroscopy. TGAM_1934 displays an original fold, although sharing some similarities with the 3D structure of the bacterial ortholog of frataxin, CyaY, a protein conserved in bacteria and eukaryotes and involved in iron-sulfur cluster biogenesis. These results highlight the potential of structural proteomics in prioritizing ORFan targets for structure determination based on quantitative proteomics data. The proteomic data and structure coordinates have been deposited to the ProteomeXchange with identifier PXD000402 ( and Protein Data Bank under the accession number 2mcf, respectively.
    Mots-clés : Amino Acid Sequence, Archaeal Proteins, DBG, Genome annotation, High-throughput proteomics, Iron-Binding Proteins, Microbiology, Models, Molecular, Molecular Sequence Data, NMR structure, Nuclear Magnetic Resonance, Biomolecular, ORFan, Protein Conformation, Protein evolution, proteomics, RBA, Thermococcus.
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Publications avant 2015

- de Groot A, Dulermo R, Ortet P, Blanchard L, Guérin P, Fernandez B, Vacherie B, Dossat C, Jolivet E, Siguier P, Chandler M, Barakat M, Dedieu A, Barbe V, Heulin T, Sommer S, Achouak W, Armengaud J (2009) Alliance of Proteomics and Genomics to Unravel the Specificities of Sahara Bacterium Deinococcus deserti. PLoS Genetics 5 : e1000434

- Zivanovic Y, Armengaud J, Lagorce A, Leplat C¥, Guérin P, Dutertre M, Anthouard V, Forterre P, Wincker P, Confalonieri F (2009) Genome analysis and genome-wide proteomics of Thermococcus gammatolerans, the most radioresistant organism known amongst the Archaea. Genome Biol. 10:R70

- Pasternak C, Ton Hoang B, Coste G, Bailone A, Chandler M*, Sommer S* (2010) Irradiation-induced Deinococcus radiodurans genome fragmentation triggers transposition of a single resident insertion sequence. PloS Genetics 6 : e1000799

- Bentchikou E¥, Servant P, Coste G, Sommer S (2010) A major role of the RecFOR pathway in DNA double-strand-break repair through ESDSA in Deinococcus radiodurans. PloS Genetics 6 : e1000774

- Ton-Hoang B, Pasternak C, Siguier P, Guynet C, Burgess-Hickman A, Dyda F, Sommer S, Chandler M (2010) Single-Stranded DNA Transposition Is Coupled to Host Replication. Cell 142 : 398-408

- Hickman AB, James JA, Barabas O, Pasternak C, Ton-Hoang B, Chandler M, Sommer S, Dyda F (2010) DNA recognition and the precleavage state during single-stranded DNA transposition in D. radiodurans. EMBO Journal 29 : 3840-3852

- Bouthier de la Tour C, Boisnard S, Norais C, Toueille M, Bentchikou E¥, Vannier F, Cox MM, Sommer S, Servant P (2011) The deinococcal DdrB protein is involved in an early step of DNA double strand break repair and in plasmid transformation through its single-strand annealing activity. DNA Repair 10:1223-1231

- Confalonieri F, Sommer S (2011) Bacterial and archaeal resistance to ionizing radiation. J. Phys. : Conf. Ser. 261 doi:10.1088/1742-6596/261/1/012005

- Quaiser A, Zivanovic Y, Moreira D, López-García P. (2011) Comparative metagenomics of bathypelagic plankton and bottom sediment from the Sea of Marmara. ISME J. 5(2):285-304

- Toueille M, Mirabella B, Guérin PP, Bouthier de la Tour C, Boisnard S, Nguyen HH¥, Blanchard L, Servant P, de Groot A*, Sommer S*, Armengaud J* (2012) A comparative proteomic approach to better define Deinococcus nucleoid specificities. Journal of proteomics 75 : 2588-2600

- Lagorce A, Fourçans A, Dutertre M, Bouyssiere B, Zivanovic Y, Confalonieri F. (2012). Genome-wide transcriptional response of the archaeon Thermococcus gammatolerans to cadmium. PloS one 7:e41935

- Pasternak C, Dulermo R, Ton-Hoang B, Debuchy R, Siguier P, Coste G, Chandler M*, Sommer S* ISDra2 transposition in Deinococcus radiodurans is down-regulated by TnpB. Molecular Microbiology 88 : 443-55

- Devigne A¥*, Mersaoui S*, Bouthier-de-la-Tour C, Sommer S, Servant P (2013) The PprA protein is required for accurate cell division of gamma-irradiated Deinococcus radiodurans bacteria. DNA repair 12 : 265-272

- Bouthier de la Tour C, Passot FM, Toueille M, Mirabella B, Guérin P, Blanchard L, Servant P, de Groot A, Sommer S, Armengaud J (2013) Comparative proteomics reveals key proteins recruited at the nucleoid of Deinococcus after irradiation-induced DNA damage. Proteomics 13:3457-3469

- Zivanovic Y, Confalonieri F, Ponchon L, Lurz R, Chami M, Flayhan A, Renouard M, Huet A, Decottignies P, Davidson AR, Breyton C, Boulanger P (2014). Insights into bacteriophage T5 structure from analysis of its morphogenesis genes and protein components. J Virol. 88(2):1162-74.

- Deschamps P, Zivanovic Y, Moreira D, Rodriguez-Valera F, López-García P.(2014) Pangenome evidence for extensive interdomain horizontal transfer affecting lineage core and shell genes in uncultured planktonic thaumarchaeota and euryarchaeota. Genome Biol Evol. 12 ;6(7):1549-63.

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