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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.

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.

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.

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.

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.

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.

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.

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.