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International audience; Abstract 8-Oxoguanine (GO) is a major purine oxidation product in DNA. Because of its highly mutagenic properties, GO absolutely must be eliminated from DNA. To do this, aerobic and anaerobic organisms from the three kingdoms of life have evolved repair mechanisms to prevent its deleterious effect on genetic integrity. The major way to remove GO is the base excision repair pathway, usually initiated by a GO-DNA glycosylase. First identified in bacteria (Fpg) and eukaryotes (OGG1), GO-DNA glycosylases were more recently identified in archaea (OGG2 and AGOG). AGOG is the less documented enzyme and its mode of damage recognition and removing remains to be clarified at the molecular and atomic levels. This study presents a complete structural characterisation of apo AGOGs from Pyrococcus abyssi (Pab) and Thermococcus gammatolerans (Tga) and the first structure of Pab-AGOG bound to lesion-containing single- or double-stranded DNA. By combining X-ray structure analysis, site directed mutagenesis and biochemistry experiments, we identified key amino acid residues of AGOGs responsible for the specific recognition of the lesion and the base opposite the lesion and for catalysis. Moreover, a unique binding mode of GO, involving double base flipping, never observed for any other DNA glycosylases, is revealed. In addition to unravelling the properties of AGOGs, our study, through comparative biochemical and structural analysis, offers new insights into the evolutionary plasticity of DNA glycosylases across all three kingdoms of life.

Numerous genes are overexpressed in the radioresistant bacterium Deinococcus radiodurans after exposure to radiation or prolonged desiccation. The DdrO and IrrE proteins play a major role in regulating the expression of approximately predicted twenty of these genes. The transcriptional repressor DdrO blocks the expression of these genes under normal growth conditions. After exposure to genotoxic agents, the IrrE metalloprotease cleaves DdrO and relieves gene repression. Bioinformatic analyzes showed that this mechanism seems to be conserved in several species of Deinococcus, but many questions remain as such the number of genes regulated by DdrO. Here, by RNA-seq and CHiP-seq assays performed at a genome-wide scale coupled with bioinformatic analyses, we show that, the DdrO regulon in D. radiodurans includes many other genes than those previously described. These results thus pave the way to better understand the radioresistance mechanisms encoded by this bacterium.Author SummaryThe main response pathway to genotoxic conditions in the radioresistant bacterium Deinococcus radiodurans is regulated by the constitutively expressed metalloprotease IrrE that cleaves the transcriptional repressor DdrO, leading to the expression of the genes repressed by DdrO. One of the major goals to better understand how pathways involved in radioresistance are coordinated into this fascinating bacterium is to highlight genes regulated by DdrO. In this study, we mapped in vivo the DdrO regulon in D. radiodurans by using two genome-scale approaches, ChIP-seq and RNA-seq analyses, coupled with bioinformatic analyses. As homologs of these two proteins are also found in many other bacteria, these results also pave the way to compare the stress-induced responses mediated by this couple of proteins in diverse bacteria.

Here, we report the in vitro and in vivo characterization of the DdrD protein from the extraordinary stress-resistant bacterium, D. radiodurans. DdrD is one of the most highly induced proteins following cellular irradiation or desiccation. We confirm that DdrD belongs to the Radiation Desiccation Response (RDR) regulon protein family whose expression is regulated by the IrrE/DdrO proteins after DNA damage. We show that DdrD is a DNA binding protein that binds to single-stranded DNA In vitro, but not to duplex DNA unless it has a 5′ single-stranded extension. In vivo, we observed no significant effect of the absence of DdrD on the survival of D. radiodurans cells after exposure to γ-rays or UV irradiation in different genetic contexts. However, genome reassembly is affected in a ∆ddrD mutant when cells recover from irradiation in the absence of nutrients. Thus, DdrD likely contributes to genome reconstitution after irradiation, but only under starvation conditions. Lastly, we show that the absence of the DdrD protein partially restores the frequency of plasmid transformation of a ∆ddrB mutant, suggesting that DdrD could also be involved in biological processes other than the response to DNA damage.

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.

The D NA d amage r esponse ddrI gene encodes a transcription regulator belonging to the cAMP receptor protein (CRP) family. Cells devoid of the DdrI protein exhibit a pleiotropic phenotype, including growth defects and sensitivity to DNA-damaging agents and to oxidative stress. Here, we show that the absence of the DdrI protein also confers sensitivity to heat shock treatment, and several genes involved in heat shock response were shown to be upregulated in a DdrI-dependent manner. Interestingly, expression of the Escherichia coli CRP partially compensates for the absence of the DdrI protein. Microscopic observations of Δ ddrI mutant cells revealed an increased proportion of two-tetrad and anucleated cells in the population compared to the wild-type strain, indicating that DdrI is crucial for the 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, the DdrI protein absolutely requires cAMP for in vitro binding to specific target sequences, and it acts as a dimer. All these data underline the major role of DdrI in 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. IMPORTANCE Deinococcus 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, and 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 into 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.

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. Electronic supplementary material The online version of this article (doi:10.1186/s12645-017-0028-y) contains supplementary material, which is available to authorized users.

International audience; 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.

International audience; 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 106 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/106 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.

Bacteriophage T5 represents a large family of lytic Siphoviridae infecting Gram-negative bacteria. The low-resolution structure of T5 showed the T=13 geometry of the capsid and the unusual trimeric organization of the tail tube, and the assembly pathway of the capsid was established. Although major structural proteins of T5 have been identified in these studies, most of the genes encoding the morphogenesis proteins remained to be identified. Here, we combine a proteomic analysis of T5 particles with a bioinformatic study and electron microscopic immunolocalization to assign function to the genes encoding the structural proteins, the packaging proteins, and other nonstructural components required for T5 assembly. A head maturation protease that likely accounts for the cleavage of the different capsid proteins is identified. Two other proteins involved in capsid maturation add originality to the T5 capsid assembly mechanism: the single head-to-tail joining protein, which closes the T5 capsid after DNA packaging, and the nicking endonuclease responsible for the single-strand interruptions in the T5 genome. We localize most of the tail proteins that were hitherto uncharacterized and provide a detailed description of the tail tip composition. Our findings highlight novel variations of viral assembly strategies and of virion particle architecture. They further recommend T5 for exploring phage structure and assembly and for deciphering conformational rearrangements that accompany DNA transfer from the capsid to the host cytoplasm.

International audience; Thermococcus gammatolerans, the most radioresistant archaeon known to date, is an anaerobic and hyperthermophilic sulfur-reducing organism living in deep-sea hydrothermal vents. Knowledge of mechanisms underlying archaeal metal tolerance in such metal-rich ecosystem is still poorly documented. We showed that T. gammatolerans exhibits high resistance to cadmium (Cd), cobalt (Co) and zinc (Zn), a weaker tolerance to nickel (Ni), copper (Cu) and arsenate (AsO(4)) and that cells exposed to 1 mM Cd exhibit a cellular Cd concentration of 67 µM. A time-dependent transcriptomic analysis using microarrays was performed at a non-toxic (100 µM) and a toxic (1 mM) Cd dose. The reliability of microarray data was strengthened by real time RT-PCR validations. Altogether, 114 Cd responsive genes were revealed and a substantial subset of genes is related to metal homeostasis, drug detoxification, re-oxidization of cofactors and ATP production. This first genome-wide expression profiling study of archaeal cells challenged with Cd showed that T. gammatolerans withstands induced stress through pathways observed in both prokaryotes and eukaryotes but also through new and original strategies. T. gammatolerans cells challenged with 1 mM Cd basically promote: 1) the induction of several transporter/permease encoding genes, probably to detoxify the cell; 2) the upregulation of Fe transporters encoding genes to likely compensate Cd damages in iron-containing proteins; 3) the induction of membrane-bound hydrogenase (Mbh) and membrane-bound hydrogenlyase (Mhy2) subunits encoding genes involved in recycling reduced cofactors and/or in proton translocation for energy production. By contrast to other organisms, redox homeostasis genes appear constitutively expressed and only a few genes encoding DNA repair proteins are regulated. We compared the expression of 27 Cd responsive genes in other stress conditions (Zn, Ni, heat shock, γ-rays), and showed that the Cd transcriptional pattern is comparable to other metal stress transcriptional responses (Cd, Zn, Ni) but not to a general stress response.

The genome sequence of Thermococcus gammatolerans, a radioresistant archaeon, is described; a proteomic analysis reveals that radioresistance may be due to unknown DNA repair enzymes., Background Thermococcus gammatolerans was isolated from samples collected from hydrothermal chimneys. It is one of the most radioresistant organisms known amongst the Archaea. We report the determination and annotation of its complete genome sequence, its comparison with other Thermococcales genomes, and a proteomic analysis. Results T. gammatolerans has a circular chromosome of 2.045 Mbp without any extra-chromosomal elements, coding for 2,157 proteins. A thorough comparative genomics analysis revealed important but unsuspected genome plasticity differences between sequenced Thermococcus and Pyrococcus species that could not be attributed to the presence of specific mobile elements. Two virus-related regions, tgv1 and tgv2, are the only mobile elements identified in this genome. A proteogenome analysis was performed by a shotgun liquid chromatography-tandem mass spectrometry approach, allowing the identification of 10,931 unique peptides corresponding to 951 proteins. This information concurrently validates the accuracy of the genome annotation. Semi-quantification of proteins by spectral count was done on exponential- and stationary-phase cells. Insights into general catabolism, hydrogenase complexes, detoxification systems, and the DNA repair toolbox of this archaeon are revealed through this genome and proteome analysis. Conclusions This work is the first archaeal proteome investigation done at the stage of primary genome annotation. This archaeon is shown to use a large variety of metabolic pathways even under a rich medium growth condition. This proteogenomic study also indicates that the high radiotolerance of T. gammatolerans is probably due to proteins that remain to be characterized rather than a larger arsenal of known DNA repair enzymes.

Summary 1. We investigated a sediment sample of the Seine River downstream of Paris for morphotype and genome diversity of bacteriophages using electron microscopy, pulsed-field gel electrophoresis and a metagenomic analysis. 2. Short-term incubations of the sediment in bacterial culture media, with or without addition of mitomycin C, were used to study the influence of pseudolysogens and induced lysis of lysogenic cells. 3. Electron microscopy of phages from the sediment revealed five morphotypes, whereas 13 morphotypes were observed after sediment incubation both with and without mitomycin C added. Pulsed-field gel electrophoresis yielded seven size-classes of double-stranded DNA genomes. 4. Representatives of the three common families of tailed bacteriophages (Myoviridae, Siphoviridae or Podoviridae) were observed. Capsid sizes ranged from 85 to 200 nm in diameter and tail sizes from

Les différentes recherches que je mène depuis ma thèse portent sur la caractérisation de mécanismes mis en jeu dans différents aspects du métabolisme des acides nucléiques chez les microbes. L'étude des protéines isolées impliquées dans ces processus, bien qu'essentielle, s'est avérée rapidement limitée pour comprendre les mécanismes dans leur ensemble et expliquer des processus, même simples. Suite à mon recrutement à l'INRA, je me suis donc focalisé sur l'identification des machines moléculaires, i.e. des complexes protéiques ou nucléoprotéiques, assurant ces mécanismes, et sur la caractérisation des interactions assurant leur cohésion et leur dynamique.J’ai tout d’abord étudié la recombinaison homologue et le redémarrage de la réplication chez Bacillus subtilis via l'étude du partenariat de la protéine de fixation à l'ADN simple brin, SSB, ainsi que la voie de réparation par recombinaison non homologue (NHEJ), via l'étude de la protéine Ku et de son partenaire LigD. J’ai alors pu établir un modèle de leurs rôles respectifs dans ces mécanismes de réparation.Plus récemment, je me suis intéressé aux machines moléculaires impliquées dans les mécanismes de recombinaison de l’ADN chez les phages (virus de bactérie). En effet, ces entités suscitent un regain d’intérêt ces dernières années en raison de leur potentiel thérapeutique pour lutter contre le phénomène d'antibiorésistance des bactéries pathogènes, apparu rapidement après l'utilisation massive des antibiotiques au cours du XXème siècle, et qui représente désormais un problème de santé publique majeur. Un inconvénient possible à l'emploi de la phagothérapie est lié à l’évolution génétique rapide des phages, due en partie à leur capacité à coder des systèmes de recombinaison de l'ADN particulièrement divers et efficaces. Ces systèmes restent cependant pour la plupart à caractériser. De plus, et contrairement aux organismes cellulaires, ces systèmes ne sont pas ubiquitaires, soulevant la question de leur rôle, à la fois pour le phage lui-même, mais également potentiellement pour son hôte bactérien.Ainsi, ma recherche actuelle vise premièrement à caractériser des mécanismes de recombinaison phagiques originaux, via l'étude des machines impliquées. Deuxièmement, je cherche à comprendre l'impact de ces systèmes de réparation sur le cycle de vie des phages et en quoi ils influencent la physiologie des bactéries à l'état lysogénique.Pour répondre à ces objectifs, deux machines moléculaires sont en cours d'étude. La première implique la recombinase Sak4 et sa SSB, codées par le phage HK620 d'Escherichia coli. Nous avons montré que l'association des deux est requise pour une recombinaison homologue par appariement simple brin efficace. Cette caractéristique est inédite puisque les SSB sont connues pour inhiber cette activité chez les autres recombinases de la même famille (type RAD51), faisant de Sak4 une recombinase particulièrement intéressante à caractériser d'un point de vue évolution et fonction. Nous cherchons aussi à déterminer l'influence de ce couple dans le processus d'induction du cycle lytique du phage. La deuxième machine est composée a minima des protéines Mu-Gam et RecT du phage Gally, potentiellement impliquées respectivement dans des évènements de recombinaison non homologue et homologue. Cette combinatoire au sein d'un même phage et la résultante en terme de mécanisme de recombinaison n'a jamais été étudiée chez les phages et nos premiers résultats indiquent que ces deux protéines fonctionnent ensemble dans un mécanisme en cours de caractérisation. Ce mécanisme n'influe que très peu sur la capacité de Gally à produire des virions. A l'inverse, des données préliminaires nous indique que Mu-Gam et RecT pourraient être impliquées dans la virulence de la souche E. coli LF82, une souche porteuse du prophage Gally et qui est impliquée dans la maladie de Crohn. Nous cherchons maintenant à valider ces résultats et à comprendre le lien entre recombinaison phagique et virulence bactérienne.L'ensemble des résultats générés au cours de ces études permet de mieux comprendre les mécanismes de réparation de l'ADN chez les phages, et une partie de ces connaissances devraient être transposables chez les organismes cellulaires. Ces études impliquent également que des mécanismes de réparation phagiques, à l'origine de leur évolution et du bon déroulement de leur cycle de vie, pourraient être adoptés par leur hôte bactérien pour leur propre bénéfice dans des environnements génotoxiques.