The Complete Genome Sequence of Haloferax volcanii DS2, a Model Archaeon

a key model organism, not only for the study of halophilicity, but also for archaeal biology in general. DS2, the type strain of this species. The genome contains a main 2.848 Mb chromosome, three smaller chromosomes pHV1, 3, 4 (85, 438, 636 kb, respectively) and the pHV2 plasmid (6.4 kb).

Etude de la réplication de l'ADN chez l'archaea halophile Haloferax volcanii.

J'ai étudié, durant mon travail de thèse, la réplication de l'ADN chez l'archaea halophile Haloferax volcanii. Le premier objectif de ma thèse était de mettre en place l'utilisation des outils génétiques disponibles chez H. volcanii, dont la méthode de délétion de gène par intégration/excision de plasmide (pop-in/pop-out), pour un usage routinier au laboratoire. L'annotation partielle des gènes impliqués dans les mécanismes de réplication et réparation de l'ADN a permis d'identifier 16 protéines Initiator Cdc6/Orc1 putatives. L'utilisation des outils génétiques, combinée à une analyse des biais de représentation des nucléotides, nous ont permis d'identifier cinq origines de réplication. Le chromosome majeur possède au moins deux origines de réplication tandis qu'une autre est retrouvée sur les deux épisomes pHV1 et pHV4. L'activité in vivo de ces origines a pu être confirmée par cartographie des sites d'initiation de la réplication (RIP mapping) et par gel bidimensionnels d'ADN. L'étude des peptides interagissant avec PCNA nous a permis d'identifier que RnaseH interagit avec PCNA pour former un complexe inactif. Nos analyses génétiques chez H. volcanii ont permis d'illustrer l'implication dans les processus de réparation de l'ADN des protéines Fen1 et de manière surprenante de RNAseH I et RnaseHII. Ces analyses ont montré que Fen1 intervient aussi dans la réplication de l'ADN. J'ai pu confirmer que les fragments d'Okazaki font moins de 200 bases et portent une amorce ARN synthétisée par la primase de type eucaryote PriS/L qui est essentielle chez H. volcanii. En revanche, la primase de type bactérienne putative DnaG peut être inactivée, mais son rôle reste à caractériser. Mes travaux ont démontré qu'avec ses multiples réplicons et des outils génétiques efficaces permettant la caractérisation des gènes, H. volcanii est un modèle novateur et pertinent pour l'étude de la réplication de l'ADN chez les archaea.During my doctoral work, I have studied DNA replication in the halophilic archaeon Haloferax volcanii. The first aim of this study was to establish the use of available genetic tools for H. volcanii, including the pop-in/pop-out gene deletion system, for routine work at the laboratory. A partial annotation of the genes implicated in DNA replication and repair allowed the identification of 16 putative Initiator Cdc6/Orc1. The use of genetics combined with nucleotide skews analyses allowed the identification of five replication origins. The main chromosome carries at least two replication origins whereas another origin is used to replicate both pHV1 and pHV4. The in vivo activity of these origins could be confirmed by replication initiation point mapping and DNA two-dimensional gels. The study of PCNA interacting peptides revealed that archaeal RNAseH interacts with PCNA to form an inactive complex. Genetic analyses with H. volcanii revealed the implication in DNA repair of Fen1 and surprisingly RnaseHI and RnaseHII. These studies also showed that Fen1 is required for DNA replication. I confirmed that H. volcanii Okazaki fragments are less than 200 bases long and carry an RNA primer synthesized by the essential PriS/L eukaryotic-like primase. On the other hand, the putative bacterial-like primase DnaG can be deleted and its role remains to be characterized. My studies have demonstrated that with its multi-replicon structure and efficient genetic tools for gene characterization, H. volcanii is a novel and pertinent model for the study of archaeal DNA replication.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Diversity of CRISPR system in the euryarchaeal pyrococcales

National audienc

Diversity of CRISPR systems in the euryarchaeal Pyrococcales

International audiencePyrococcales are members of the order Thermococcales, a group of hyperthermophilic euryarchaea that are frequently found in deep sea hydrothermal vents. Infectious genetic elements, such as plasmids and viruses, remain a threat even in this remote environment and these microorganisms have developed several ways to fight their genetic invaders. Among these are the recently discovered CRISPR systems. In this review, we have combined and condensed available information on genetic elements infecting the Thermococcales and on the multiple CRISPR systems found in the Pyrococcales to fight them. Their organization and mode of action will be presented with emphasis on the Type III-B system that is the only CRISPR system known to target RNA molecules in a process reminiscent of RNA interference. The intriguing case of Pyrococcus abyssi, which is among the rare strains to present a CRISPR system devoid of the universal cas1 and cas2 genes, is also discussed

Characterization of the DdrD protein from the extremely radioresistant bacterium Deinococcus radiodurans

International audienceHere, 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