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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.

Deinococcus radiodurans is a spherical bacterium well-known for its outstanding resistance to DNA-damaging agents. Exposure to such agents leads to drastic changes in the transcriptome of D. radiodurans. In particular, four Deinococcus-specific genes, known as DNA Damage Response genes, are strongly up-regulated and have been shown to contribute to the resistance phenotype of D. radiodurans. One of these, DdrC, is expressed shortly after exposure to γ-radiation and is rapidly recruited to the nucleoid. In vitro, DdrC has been shown to compact circular DNA, circularize linear DNA, anneal complementary DNA strands and protect DNA from nucleases. To shed light on the possible functions of DdrC in D. radiodurans, we determined the crystal structure of the domain-swapped DdrC dimer at a resolution of 2.2 Å and further characterized its DNA binding and compaction properties. Notably, we show that DdrC bears two asymmetric DNA binding sites located on either side of the dimer and can modulate the topology and level of compaction of circular DNA. These findings suggest that DdrC may be a DNA damage-induced nucleoid-associated protein that enhances nucleoid compaction to limit the dispersion of the fragmented genome and facilitate DNA repair after exposure to severe DNA damaging conditions.

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.

Horizontal gene transfer is a major driver of bacterial evolution and adaptation to environmental stresses, occurring notably via transformation of naturally competent organisms. The Deinococcus radiodurans bacterium, characterized by its extreme radioresistance, is also naturally competent. Here, we investigated the role of D. radiodurans players involved in different steps of natural transformation. First, we identified the factors (PilQ, PilD, type IV pilins, PilB, PilT, ComEC-ComEA, and ComF) involved in DNA uptake and DNA translocation across the external and cytoplasmic membranes and showed that the DNA-uptake machinery is similar to that described in the Gram negative bacterium Vibrio cholerae. Then, we studied the involvement of recombination and DNA repair proteins, RecA, RecF, RecO, DprA, and DdrB into the DNA processing steps of D. radiodurans transformation by plasmid and genomic DNA. The transformation frequency of the cells devoid of DprA, a highly conserved protein among competent species, strongly decreased but was not completely abolished whereas it was completely abolished in ΔdprA ΔrecF, ΔdprA ΔrecO, and ΔdprA ΔddrB double mutants. We propose that RecF and RecO, belonging to the recombination mediator complex, and DdrB, a specific deinococcal DNA binding protein, can replace a function played by DprA, or alternatively, act at a different step of recombination with DprA. We also demonstrated that a ΔdprA mutant is as resistant as wild type to various doses of γ-irradiation, suggesting that DprA, and potentially transformation, do not play a major role in D. radiodurans radioresistance. SI, NE, and JL were supported by a Ph.D. fellowship from the French Ministry of Education. This work was supported by the Centre National de la Recherche Scientifique, the University Paris-Sud, the Agence Nationale de la Recherche (ANR Radioresistance-11-BSV3-01701 to SS), and by the French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INSB-05-01.

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.

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.

International audience; The recently discovered hyperthermophilic and radioresistant archaeon Thermococcus gammatolerans is of great interest to compare and contrast the impact of its physiology on radioresistance and its ability to repair damaged chromosomes after exposure to gamma irradiation with radioresistant bacteria. We showed that, in contrast to other organisms, cell survival was not modified by the cellular growth phase under optimal growth conditions but nutrient-limited conditions did affect the T. gammatolerans radioresistance. We determined the first kinetics of damaged DNA recovery in an archaeon after exposure to massive doses of gamma irradiation and compared the efficiency of chromosomal DNA repair according to the cellular growth phase, nutrient availability and culture conditions. Chromosomal DNA repair kinetics showed that stationary phase cells reconstitute disrupted chromosomes more rapidly than exponential phase cells. Our data also revealed that this radioresistant archaeon was proficient to reconstitute shattered chromosomes either slowly or rapidly without any loss of viability. These results suggest that rapid DNA repair is not required for the extreme radioresistance of T. gammatolerans.

International audience; 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 (http://proteomecentral.proteomexchange.org/dataset/PXD000402) and Protein Data Bank under the accession number 2mcf, respectively.

We sequenced an 80 kb DNA region containing the complete sequence of the silkworm Bombyx mori fibroin gene and its flanking, especially the upstream, regions (-62 kb). About 30% of the 62 kb upstream region is composed of repetitive elements including short interspersed elements Bm1, long interspersed elements L1Bm and mariner-like elements Bmmar1 which are widespread over the silkworm genome. This 62 kb region is also enriched of commonly considered matrix association region (MAR) motifs. A total of 25 individual MAR recognition signatures (MRSs) were identified, with 24 at the upstream and one at the downstream region. Combining two newly developed MAR prediction programs (MAR-finder and Chrclass), ten candidate MARs were predicted, with five containing MRS and seven related to the repetitive elements. The wide distribution of nested repetitive elements, candidate MARs, DNase I hypersensitive sites and other potential regulatory factors recognition sites indicates this region is probably a unique huge cis-acting element contributing to the regulation of the spatial and temporal specificity and efficiency of fibroin gene expression.

The recent availability of several archaeal genome sequences has provided a basis for detailed analyses of the frequency, location and phylogeny of archaeal mobile elements. All the known elements fall into two main types, autonomous insertion sequence (IS) elements and the non-autonomous miniature inverted repeat element (MITE)-like elements. Both classes are considered to be mobilized via transposases that are encoded by the IS elements, although mobility has only been demonstrated experimentally for a few elements. The number, and diversity, of the elements differs greatly between the genomes. At one extreme Sulfolobus solfataricus P2 and Halobacterium NRC-1 are very rich in elements while Methanobacterium thermoautotrophicum contains none. The former also show examples of complex clusters of interwoven elements. An analysis of the genomic distribution in S. solfataricus suggests that the putative oriC and terC regions act as barriers for the mobility of both IS and MITE-like elements. Moreover, the very high level of truncated IS elements in the genomes of S. solfataricus, Sulfolobus tokodaii and Thermoplasma volcanium suggests that there may be a cellular mechanism for selectively inactivating IS elements at a point when they become too numerous and disadvantageous for the cell. Phylogenetically, archaeal IS elements are confined to 11 of the 17 known families of bacterial and eukaryal IS elements where some generate distinct subgroups. Finally, DNA viruses, plasmids and DNA fragments can also be inserted into, and excised from, archaeal genomes by means of an integrase-mediated mechanism that has special archaeal characteristics.

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.

Reverse gyrases are atypical topoisomerases present in hyperthermophiles and are able to positively supercoil a circular DNA. Despite a number of studies, the mechanism by which they perform this peculiar activity is still unclear. Sequence data suggested that reverse gyrases are composed of two putative domains, a helicase-like and a topoisomerase I, usually in a single polypeptide. Based on these predictions, we have separately expressed the putative domains and the full-length polypeptide of Sulfolobus acidocaldarius reverse gyrase as recombinant proteins in Escherichia coli. We show the following. (i) The full-length recombinant enzyme sustains ATP-dependent positive supercoiling as efficiently as the wild type reverse gyrase. (ii) The topoisomerase domain exhibits a DNA relaxation activity by itself, although relatively low. (iii) We failed to detect helicase activity for both the N-terminal domain and the full-length reverse gyrase. (iv) Simple mixing of the two domains reconstitutes positive supercoiling activity at 75 degrees C. The cooperation between the domains seems specific, as the topoisomerase domain cannot be replaced by another thermophilic topoisomerase I, and the helicase-like cannot be replaced by a true helicase. (v) The helicase-like domain is not capable of promoting stoichiometric DNA unwinding by itself; like the supercoiling activity, unwinding requires the cooperation of both domains, either separately expressed or in a single polypeptide. However, unwinding occurs in the absence of ATP and DNA cleavage, indicating a structural effect upon binding to DNA. These results suggest that the N-terminal domain does not directly unwind DNA but acts more likely by driving ATP-dependent conformational changes within the whole enzyme, reminiscent of a protein motor.

The original strategy used in the Sulfolobus solfataricus genome project was to sequence non overlapping, or minimally overlapping, cosmid or lambda inserts without constructing a physical map. However, after only about two thirds of the genome sequence was completed, this approach became counter-productive because there was a high sequence bias in the cosmid and lambda libraries. Therefore, a new approach was devised for linking the sequenced regions which may be generally applicable. BAC libraries were constructed and terminal sequences of the clones were determined and used for both end mapping and PCR screening. The PCR approaches included a novel chromosome walking method termed "paired-PCR". 21 gaps were filled by BAC end sequence analyses and 6 gaps were filled by PCR including three large ones by paired-PCR. The complete map revealed that 0.9 Mb remained to be sequenced and 34 BAC clones were selected for walking over small gaps and preparing template libraries for larger ones. It is concluded that an optimal strategy for sequencing microorganism genomes involves construction of a high-resolution physical map by BAC end analyses, PCR screening and paired-PCR chromosome walking after about half the genome sequence has been accumulated.

Summary The gene encoding the DNA topoisomerase I from the thermophilic bacterium Fervidobacterium islandicum has been cloned by using oligonucleotides derived from internal microsequencing of the purified protein ( Bouthier de la Tour et al., 1993). The corresponding nucleotide sequence has been determined, revealing an open reading frame of 2097 nucleotides. The deduced amino acid sequence (699 residues) clearly showed that the gene belongs to E. coli TopA family, and has 53% identity with Thermotoga maritima TopA ( Bouthier de la Tour et al., 1995). Comparison of the F. islandicum sequence to the topoisomerase I sequences of other species revealed several interesting features: (1) a putative N-terminal tail of 33 amino acids, whose function is unknown. (2) a 8 amino acids insertion close to the active site tyrosine, shared by 3 other bacterial TopA sequences. (3) a unique putative zinc finger that is conserved in 7 out of 9 sequences. However, no significant feature linked to thermophily could distinguish the two thermotogales topoisomerases from their mesophilic counterparts. Finally, the analysis of the gene organization in the vicinity of the TopA locus in different bacterial species showed that it was not conserved.

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.

Summary We review our present knowledge on DNA topoisomerase and DNA polymerase evolution, with emphasis on information obtained by studying these enzymes in Archaea. Two archaeal DNA topoisomerase genes have been sequenced: the reverse gyrase from Sulfolobus acidocaldarius turns out to be a completely novel type of enzyme, likely originating from the fusion of a helicase and a type I DNA topoisomerase, whereas a novobiocin sensitive type II DNA topoisomerase from Haloferax is closely related to bacterial DNA gyrases. Beside reverse gyrase, we recently isolated from Sulfolobus shibatae a type II DNA topoisomerase which has no gyrase activity and exhibits the pattern of drug sensitivity specific for its eukaryotic counterpart. Several archaeal DNA polymerase genes have now been sequenced: they all belong to the DNA polymerase B family, together with the three eucaryal DNA replicases and Escherichia coli DNA pol II, a repair enzyme. All DNA polymerases from family B described so far are sensitive to aphidicolin. We present recent data suggesting that archaeal DNA polymerases resistant to aphidicolin also belong to family B. Phylogenetic trees of DNA topoisomerases and DNA polymerases turn out to be all noncongruent with the rRNA tree and exhibit different and unusual topologies. We discuss several hypotheses which can explain this observation and their implications concerning the nature of the last common ancestor to the three domains. We conclude that this universal ancestor was probably an advanced member of the DNA world that already contained several DNA polymerases and DNA topoisomerases.

The nature of the last universal ancestor to all extent cellular organisms and the rooting of the universal tree of life are fundamental questions which can now be adressed by molecular evolutionists. Several scenarios have been proposed during the last years, based on the phylogenies of ribosomal RNA and of duplicated proteins, which suggest that the last universal ancestor was either an RNA progenote or an hyperthermophilic prokaryote. We discuss these hypotheses in the light of new data on the evolution of DNA metabolizing enzymes and of contradictions between different protein phylogenies. We conclude that the last universal ancestor was a member of the DNA world already containing several DNA polymerases and DNA topoisomerases. Furthermore, we criticize current data which suggest that the rooting of the universal tree of life is located in the eubacterial branch and we conclude that both rooting the universal tree and the nature of the last universal ancestor are still open questions.

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

An analysis of the position of Nanoarcheum equitans in the archaeal phylogeny using a large dataset of concatenated ribosomal proteins from 25 archaeal genomes suggests that N. equitans is likely to be the representative of a fast-evolving euryarchaeal lineage., Background Cultivable archaeal species are assigned to two phyla - the Crenarchaeota and the Euryarchaeota - by a number of important genetic differences, and this ancient split is strongly supported by phylogenetic analysis. The recently described hyperthermophile Nanoarchaeum equitans, harboring the smallest cellular genome ever sequenced (480 kb), has been suggested as the representative of a new phylum - the Nanoarchaeota - that would have diverged before the Crenarchaeota/Euryarchaeota split. Confirming the phylogenetic position of N. equitans is thus crucial for deciphering the history of the archaeal domain. Results We tested the placement of N. equitans in the archaeal phylogeny using a large dataset of concatenated ribosomal proteins from 25 archaeal genomes. We indicate that the placement of N. equitans in archaeal phylogenies on the basis of ribosomal protein concatenation may be strongly biased by the coupled effect of its above-average evolutionary rate and lateral gene transfers. Indeed, we show that different subsets of ribosomal proteins harbor a conflicting phylogenetic signal for the placement of N. equitans. A BLASTP-based survey of the phylogenetic pattern of all open reading frames (ORFs) in the genome of N. equitans revealed a surprisingly high fraction of close hits with Euryarchaeota, notably Thermococcales. Strikingly, a specific affinity of N. equitans and Thermococcales was strongly supported by phylogenies based on a subset of ribosomal proteins, and on a number of unrelated molecular markers. Conclusion We suggest that N. equitans may more probably be the representative of a fast-evolving euryarchaeal lineage (possibly related to Thermococcales) than the representative of a novel and early diverging archaeal phylum.

The surface sands of the Sahara Desert are exposed to extremes of ultraviolet light irradiation, desiccation and temperature variation. Nonetheless, the presence of bacteria has recently been demonstrated in this environment by cultivation methods and by 16S rDNA analyses from total DNA isolated from surface sands. To discern the presence of bacteriophages in this harsh environment, we searched for extracellular phages and intracellularly located phages present as prophages or within pseudolysogens. Mild sonication of the sand, in different liquid culture media, incubated with and without Mitomycin-C, was followed by differential centrifugation to enrich for dsDNA phages. The resulting preparations, examined by electron microscopy, revealed the presence of virus-like particles with a diversity of morphotypes representative of all three major double-stranded DNA bacteriophage families (Myoviridae, Siphoviridae and Podoviridae). Moreover, pulsed-field gel electrophoresis of DNA, extracted from the enriched bacteriophage preparations, revealed the presence of distinct bands suggesting the presence of putative dsDNA phage genomes ranging in size from 45 kb to 270 kb. Characterization of the bacteriophages present in the surface sands of the Sahara Desert extends the range of environments from which bacteriophages can be isolated, and provides an important point of departure for the study of phages in extreme terrestrial environments.

The amino acid sequence of the heavy chain of Bombyx mori silk fibroin was derived from the gene sequence. The 5,263-residue (391-kDa) polypeptide chain comprises 12 low-complexity "crystalline" domains made up of Gly-X repeats and covering 94% of the sequence; X is Ala in 65%, Ser in 23%, and Tyr in 9% of the repeats. The remainder includes a nonrepetitive 151-residue header sequence, 11 nearly identical copies of a 43-residue spacer sequence, and a 58-residue C-terminal sequence. The header sequence is homologous to the N-terminal sequence of other fibroins with a completely different crystalline region. In Bombyx mori, each crystalline domain is made up of subdomains of approximately 70 residues, which in most cases begin with repeats of the GAGAGS hexapeptide and terminate with the GAAS tetrapeptide. Within the subdomains, the Gly-X alternance is strict, which strongly supports the classic Pauling-Corey model, in which beta-sheets pack on each other in alternating layers of Gly/Gly and X/X contacts. When fitting the actual sequence to that model, we propose that each subdomain forms a beta-strand and each crystalline domain a two-layered beta-sandwich, and we suggest that the beta-sheets may be parallel, rather than antiparallel, as has been assumed up to now.

The genome of the crenarchaeon Sulfolobus solfataricus P2 contains 2,992,245 bp on a single chromosome and encodes 2,977 proteins and many RNAs. One-third of the encoded proteins have no detectable homologs in other sequenced genomes. Moreover, 40% appear to be archaeal-specific, and only 12% and 2.3% are shared exclusively with bacteria and eukarya, respectively. The genome shows a high level of plasticity with 200 diverse insertion sequence elements, many putative nonautonomous mobile elements, and evidence of integrase-mediated insertion events. There are also long clusters of regularly spaced tandem repeats. Different transfer systems are used for the uptake of inorganic and organic solutes, and a wealth of intracellular and extracellular proteases, sugar, and sulfur metabolizing enzymes are encoded, as well as enzymes of the central metabolic pathways and motility proteins. The major metabolic electron carrier is not NADH as in bacteria and eukarya but probably ferredoxin. The essential components required for DNA replication, DNA repair and recombination, the cell cycle, transcriptional initiation and translation, but not DNA folding, show a strong eukaryal character with many archaeal-specific features. The results illustrate major differences between crenarchaea and euryarchaea, especially for their DNA replication mechanism and cell cycle processes and their translational apparatus.

The sequence of a 281-kbp contig from the crenarchaeote Sulfolobus solfataricus P2 was determined and analysed. Notable features in this region include 29 ribosomal protein genes, 12 tRNA genes (four of which contain archaeal-type introns), operons encoding enzymes of histidine biosynthesis, pyrimidine biosynthesis, and arginine biosynthesis, an ATPase operon, numerous genes for enzymes of lipopolysaccharide biosynthesis, and six insertion sequences. The content and organization of this contig are compared with sequences from crenarchaeotes, euryarchaeotes, bacteria, and eukaryotes. Key words: Archaea, Sulfolobus Genome Project, comparative genomics, bioinformatics.

A 2767 bp fragment containing a gene coding for a topoisomerase I from the extremely thermophilic eubacterium Thermotoga maritima (Tm TopA) has been cloned and sequenced. The protein is composed of 633 amino acids with a calculated molecular mass of 72,695 Da. It shares significant similarity with the topoisomerases I of mesophilic eubacteria. The highest score is obtained with Bacillus subtilis (44% identity); in particular, T. maritima and B. subtilis possess an insertion of 7-8 amino acids in the vicinity of the active site, that is absent in topoisomerases of other organisms. A specific feature of T. maritima topoisomerase I is its low cysteine content compared to its mesophilic homologs. It contains 5 cysteine residues, of which 4 could constitute a zinc finger motif. Finally, analysis of the regions flanking the gene reveals that Tm TopA is surrounded by two other ORFs, suggesting the occurrence of a polycistronic transcriptional unit.

A fast growing family of ATPases has recently been highlighted. It was named the AAA family, for ATPases Associated to a variety of cellular Activities. The key feature of the family is a highly conserved module of 230 amino acids present in one or two copies in each protein. Despite extensive sequence conservation, the members of the family fulfil a large diversity of cellular functions: cell cycle regulation, gene expression in yeast and HIV, vesicle-mediated transport, peroxisome assembly, 26S protease function etc. In addition, several members of this family can be found in the same organism (up to 17 in S. cerevisiae). The contrast between functional diversity and structural conservation of the module, from archaebacteria to mammals, suggests that it plays an essential, but as yet unknown, role at key points of the cellular machinery. Two (non-exclusive) such possibilities are: (1) ATP-dependent proteasome function and (2) ATP-dependent anchorage of proteins. Finally, the basic biochemical activity of the AAA module is still a matter of speculation, and we propose that it acts as an ATP-dependent protein clamp.

Nucleotide sequencing of a region of the hyperthermophilic archaebacterium Sulfolobus acidocaldarius allowed us to identify an open reading frame of 780 amino acids strikingly similar to a family of eukaryotic ATPases, involved in a variety of biological functions. Sequence analysis of the predicted polypeptide revealed 63 to 66% similarity with S. cerevisiae CDC 48p and its related genes in amphibians (p97ATPase) and mammals (Valosin Containing Protein, VCP), all possibly involved in the regulation of the cell cycle. The finding of an archaebacterial equivalent of these proteins with a high degree of similarity suggests that it represents the same gene in these various species. The new archaebacterial ORF, called SAV (S. acidocaldarius VCP-like) exhibited the usual signature of all members of the family, a highly conserved domain of about 200 amino acids, which is duplicated. Thus, apart from the VCP-like proteins, SAV also appeared similar, although less clearly, to other ATPases, members of the family, involved in vesicle-mediated transport (NSF, Sec18p), peroxysome assembly (PAS1p), and gene expression in yeast (SUG1p) and in human immunodeficiency virus (TBP-1). Finally, the discovery of the archaebacterial gene could enlighten not only the evolutionary relationships between the members of this complex ATPase family, but also the cellular function of these proteins, that is presently obscure.

Cultivable archaeal species are assigned to two phyla - the Crenarchaeota and the Euryarchaeota - by a number of important genetic differences, and this ancient split is strongly supported by phylogenetic analysis. The recently described hyperthermophile Nanoarchaeum equitans, harboring the smallest cellular genome ever sequenced (480 kb), has been suggested as the representative of a new phylum - the Nanoarchaeota - that would have diverged before the Crenarchaeota/Euryarchaeota split. Confirming the phylogenetic position of N. equitans is thus crucial for deciphering the history of the archaeal domain. We tested the placement of N. equitans in the archaeal phylogeny using a large dataset of concatenated ribosomal proteins from 25 archaeal genomes. We indicate that the placement of N. equitans in archaeal phylogenies on the basis of ribosomal protein concatenation may be strongly biased by the coupled effect of its above-average evolutionary rate and lateral gene transfers. Indeed, we show that different subsets of ribosomal proteins harbor a conflicting phylogenetic signal for the placement of N. equitans. A BLASTP-based survey of the phylogenetic pattern of all open reading frames (ORFs) in the genome of N. equitans revealed a surprisingly high fraction of close hits with Euryarchaeota, notably Thermococcales. Strikingly, a specific affinity of N. equitans and Thermococcales was strongly supported by phylogenies based on a subset of ribosomal proteins, and on a number of unrelated molecular markers. We suggest that N. equitans may more probably be the representative of a fast-evolving euryarchaeal lineage (possibly related to Thermococcales) than the representative of a novel and early diverging archaeal phylum.

Reverse gyrase is a type I DNA topoisomerase able to positively supercoil DNA and is found in thermophilic archaebacteria and eubacteria. The gene coding for this protein was cloned from Sulfolobus acidocaldarius DSM 639. Analysis of the 1247-amino acid sequence and comparison of it with available sequence data suggest that reverse gyrase is constituted of two distinct domains: (i) a C-terminal domain of approximately 630 amino acids clearly related to eubacterial topoisomerase I (Escherichia coli topA and topB gene products) and to Saccharomyces cerevisiae top3; (ii) an N-terminal domain without any similarity to other known topoisomerases but containing several helicase motifs, including an ATP-binding site. These results are consistent with those from our previous mechanistic studies of reverse gyrase and suggest a model in which positive supercoiling is driven by the concerted action of helicase and topoisomerase in the same polypeptide: this constitutes an example of a composite gene formed by a helicase domain and a topoisomerase domain.

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.