Variabilité génétique chez la bactérie radiorésistante Deinococcus radiodurans : la recombinaison entre séquences répétées et la transformation naturelle

The bacterium Deinococcus radiodurans is known for its ability to withstand a large number of genotoxic treatments, including exposure to ionizing or ultraviolet radiation, mitomycin C, desiccation, and oxidative stress. It is able, upon exposure to extreme doses of γ-radiation generating hundreds of DNA breaks, to reconstitute an intact genome in only 2 to 3 hours via an ESDSA mechanism, involving massive DNA synthesis during DNA double strand break repair. Together with efficient DNA repair mechanisms, D. radiodurans possesses a survival kit comprising significant compaction of its nucleoid, protection mechanisms against protein oxidation, an original response to DNA damage and specific proteins induced after irradiation. All of these contribute to the maintenance of genomic integrity and cell survival upon exposure to various genotoxic agents. In spite of the idea that D. radiodurans is an organism with outstanding genomic stability, this bacterium has in its genome a large number of repeat sequences and mobile elements and is also naturally competent. All these factors contribute to the genetic variability of species. I was interested in two processes that can play a role in genetic variability in D. radiodurans: recombination between repeated sequences and natural transformation.The introduction, into the genome of D. radiodurans, of 438 bp direct repeated sequences separated by DNA regions ranging from 1,479 bp to 10,500 bp in length allowed me to demonstrate the major role of Single Strand Annealing (SSA) involving the DdrB protein specific for Deinococcaceae, in the "spontaneous" recombination between the repeated sequences in the absence of the RecA recombinase. The absence of DdrB in strains deficient for recombination further increased the loss of viability observed in these strains, suggesting that SSA is required for the management of blocked replication forks, a major source of genetic instability in the absence of external stress when these forks cannot be rescued by pathways involving recombination proteins.I was also interested in the natural transformation and proteins involved in this process in D. radiodurans. I demonstrated that DprA protein involved in DNA single strand protection and loading of RecA on single-stranded DNA internalized during transformation of many species such as Streptococcus pneumoniae, Helicobacter pylori, or Bacillus subtilis, is also involved in this process in D. radiodurans. I also showed that, in addition to playing a major role in transformation by plasmid DNA, DdrB is also involved in transformation by genomic DNA of cells devoid of the DprA protein.La bactérie Deinococcus radiodurans est connue pour sa capacité à résister à un grand nombre de traitements génotoxiques parmi lesquels on peut citer l’exposition aux rayons ionisants, aux ultra-violets, à la mitomycine C, à la dessication et au stress oxydant. Elle est capable lors d’une exposition à des doses extrêmes de rayons γ générant des centaines de cassures de l’ADN de reconstituer un génome intact en seulement 2 à 3 heures via un mécanisme original, l’ESDSA, impliquant une synthèse massive d’ADN pendant la phase de réparation des cassures de l’ADN. En plus de mécanismes efficaces de réparation de l’ADN, elle possède un kit de survie comprenant une compaction importante du nucléoïde, des mécanismes de protection des protéines contre l’oxydation, une réponse originale aux lésions de l’ADN et des protéines spécifiques induites après irradiation. Tous ces facteurs contribuent au maintien de l’intégrité du génome et à la survie de la cellule lors de l’exposition à différents agents génotoxiques. Souvent considéré comme un organisme ayant une stabilité génomique exceptionnelle, cette bactérie possède dans son génome un grand nombre de séquences répétées et des éléments mobiles et est par ailleurs naturellement compétente. Ce sont autant de facteurs pouvant participer à la variabilité génétique de cette espèce. Je me suis donc intéressée lors de ma thèse à deux processus pouvant participer à l’instabilité génétique chez D. radiodurans : la recombinaison entre séquences répétées et la transformation naturelle.L’introduction dans le génome de D. radiodurans de séquences répétées directes de 438 pb séparées par des régions d’ADN d’une longueur allant de 1479 pb à 10 500 pb m’a permis de mettre en évidence le rôle majeur joué par l’appariement simple brin (Single Strand Annealing ou SSA) impliquant la protéine DdrB, spécifique des Deinococcaceae, joue un rôle majeur dans la recombinaison « spontanée » entre les séquences répétées en absence de la recombinase RecA. L’absence de DdrB dans des souches déficientes pour la recombinaison augmente davantage la perte de viabilité observée dans ces souches ce qui suggère que le SSA participe à la prise en charge de fourches de réplication bloquées, source majeure d’instabilité génétique en absence de stress extérieur, si ces fourches ne peuvent être prise en charge par des voies impliquant des protéines de recombinaison. Je me suis également intéressée à la transformation naturelle et aux protéines impliquées dans ce processus chez D. radiodurans. J’ai pu démontrer que la protéine DprA impliquée dans la protection de l’ADN simple brin et le chargement de RecA sur l’ADN simple brin internalisé lors de la transformation de nombreuses espèces comme Streptococcus pneumoniae, Bacillus subtilis ou Helicobacter pylori, est également impliquée dans la transformation chez D. radiodurans. J’ai pu montrer également qu’en plus de jouer un rôle majeur dans la transformation par de l’ADN plasmidique, DdrB est impliquée dans la transformation par de l’ADN génomique si la protéine DprA est absente

Insights into the role of three Endonuclease III enzymes for oxidative stress resistance in the extremely radiation resistant bacterium Deinococcus radiodurans

The extremely radiation and desiccation resistant bacterium Deinococcus radiodurans possesses three genes encoding Endonuclease III-like enzymes (DrEndoIII1, DrEndoIII2, DrEndoIII3). In vitro enzymatic activity measurements revealed that DrEndoIII2 is the main Endonuclease III in this organism, while DrEndoIII1 and 3 possess unusual and, so far, no detectable EndoIII activity, respectively. In order to understand the role of these enzymes at a cellular level, DrEndoIII knockout mutants were constructed and subjected to various oxidative stress related conditions. The results showed that the mutants are as resistant to ionizing and UV-C radiation as well as H2O2 exposure as the wild type. However, upon exposure to oxidative stress induced by methyl viologen, the knockout strains were more resistant than the wild type. The difference in resistance may be attributed to the observed upregulation of the EndoIII homologs gene expression upon addition of methyl viologen. In conclusion, our data suggest that all three EndoIII homologs are crucial for cell survival in stress conditions, since the knockout of one of the genes tend to be compensated for by overexpression of the genes encoding the other two

“Influence of plasmids, selection markers and auxotrophic mutations on Haloferax volcanii cell shape plasticity”

Haloferax volcanii and other Haloarchaea can be pleomorphic, adopting different shapes, which vary with growth stages. Several studies have shown that H. volcanii cell shape is sensitive to various external factors including growth media and physical environment. In addition, several studies have noticed that the presence of a recombinant plasmid in the cells is also a factor impacting H. volcanii cell shape, notably by favoring the development of rods in early stages of growth. Here we investigated the reasons for this phenomenon by first studying the impact of auxotrophic mutations on cell shape in strains that are commonly used as genetic backgrounds for selection during strain engineering (namely: H26, H53, H77, H98, and H729) and secondly, by studying the effect of the presence of different plasmids containing selection markers on the cell shape of these strains. Our study showed that most of these auxotrophic strains have variation in cell shape parameters including length, aspect ratio, area and circularity and that the plasmid presence is impacting these parameters too. Our results indicated that ΔhdrB strains and hdrB selection markers have the most influence on H. volcanii cell shape, in addition to the sole presence of a plasmid. Finally, we discuss limitations in studying cell shape in H. volcanii and make recommendations based on our results for improving reproducibility of such studies

Genetic variability in the radioresistant Deinococcus radiodurans bacterium : recombination between direct repeats and natural transformation

La bactérie Deinococcus radiodurans est connue pour sa capacité à résister à un grand nombre de traitements génotoxiques parmi lesquels on peut citer l’exposition aux rayons ionisants, aux ultra-violets, à la mitomycine C, à la dessication et au stress oxydant. Elle est capable lors d’une exposition à des doses extrêmes de rayons γ générant des centaines de cassures de l’ADN de reconstituer un génome intact en seulement 2 à 3 heures via un mécanisme original, l’ESDSA, impliquant une synthèse massive d’ADN pendant la phase de réparation des cassures de l’ADN. En plus de mécanismes efficaces de réparation de l’ADN, elle possède un kit de survie comprenant une compaction importante du nucléoïde, des mécanismes de protection des protéines contre l’oxydation, une réponse originale aux lésions de l’ADN et des protéines spécifiques induites après irradiation. Tous ces facteurs contribuent au maintien de l’intégrité du génome et à la survie de la cellule lors de l’exposition à différents agents génotoxiques. Souvent considéré comme un organisme ayant une stabilité génomique exceptionnelle, cette bactérie possède dans son génome un grand nombre de séquences répétées et des éléments mobiles et est par ailleurs naturellement compétente. Ce sont autant de facteurs pouvant participer à la variabilité génétique de cette espèce. Je me suis donc intéressée lors de ma thèse à deux processus pouvant participer à l’instabilité génétique chez D. radiodurans : la recombinaison entre séquences répétées et la transformation naturelle.L’introduction dans le génome de D. radiodurans de séquences répétées directes de 438 pb séparées par des régions d’ADN d’une longueur allant de 1479 pb à 10 500 pb m’a permis de mettre en évidence le rôle majeur joué par l’appariement simple brin (Single Strand Annealing ou SSA) impliquant la protéine DdrB, spécifique des Deinococcaceae, joue un rôle majeur dans la recombinaison « spontanée » entre les séquences répétées en absence de la recombinase RecA. L’absence de DdrB dans des souches déficientes pour la recombinaison augmente davantage la perte de viabilité observée dans ces souches ce qui suggère que le SSA participe à la prise en charge de fourches de réplication bloquées, source majeure d’instabilité génétique en absence de stress extérieur, si ces fourches ne peuvent être prise en charge par des voies impliquant des protéines de recombinaison. Je me suis également intéressée à la transformation naturelle et aux protéines impliquées dans ce processus chez D. radiodurans. J’ai pu démontrer que la protéine DprA impliquée dans la protection de l’ADN simple brin et le chargement de RecA sur l’ADN simple brin internalisé lors de la transformation de nombreuses espèces comme Streptococcus pneumoniae, Bacillus subtilis ou Helicobacter pylori, est également impliquée dans la transformation chez D. radiodurans. J’ai pu montrer également qu’en plus de jouer un rôle majeur dans la transformation par de l’ADN plasmidique, DdrB est impliquée dans la transformation par de l’ADN génomique si la protéine DprA est absente.The bacterium Deinococcus radiodurans is known for its ability to withstand a large number of genotoxic treatments, including exposure to ionizing or ultraviolet radiation, mitomycin C, desiccation, and oxidative stress. It is able, upon exposure to extreme doses of γ-radiation generating hundreds of DNA breaks, to reconstitute an intact genome in only 2 to 3 hours via an ESDSA mechanism, involving massive DNA synthesis during DNA double strand break repair. Together with efficient DNA repair mechanisms, D. radiodurans possesses a survival kit comprising significant compaction of its nucleoid, protection mechanisms against protein oxidation, an original response to DNA damage and specific proteins induced after irradiation. All of these contribute to the maintenance of genomic integrity and cell survival upon exposure to various genotoxic agents. In spite of the idea that D. radiodurans is an organism with outstanding genomic stability, this bacterium has in its genome a large number of repeat sequences and mobile elements and is also naturally competent. All these factors contribute to the genetic variability of species. I was interested in two processes that can play a role in genetic variability in D. radiodurans: recombination between repeated sequences and natural transformation.The introduction, into the genome of D. radiodurans, of 438 bp direct repeated sequences separated by DNA regions ranging from 1,479 bp to 10,500 bp in length allowed me to demonstrate the major role of Single Strand Annealing (SSA) involving the DdrB protein specific for Deinococcaceae, in the "spontaneous" recombination between the repeated sequences in the absence of the RecA recombinase. The absence of DdrB in strains deficient for recombination further increased the loss of viability observed in these strains, suggesting that SSA is required for the management of blocked replication forks, a major source of genetic instability in the absence of external stress when these forks cannot be rescued by pathways involving recombination proteins.I was also interested in the natural transformation and proteins involved in this process in D. radiodurans. I demonstrated that DprA protein involved in DNA single strand protection and loading of RecA on single-stranded DNA internalized during transformation of many species such as Streptococcus pneumoniae, Helicobacter pylori, or Bacillus subtilis, is also involved in this process in D. radiodurans. I also showed that, in addition to playing a major role in transformation by plasmid DNA, DdrB is also involved in transformation by genomic DNA of cells devoid of the DprA protein

Impaired growth and stationary-phase lethality of recombination-deficient mutant cells.

<p>GY9613 (WT) (black diamonds), GY13915 (Δ<i>ddrB</i>) (grey squares), GY15125 (Δ<i>recO</i>) (green triangles), GY12968 (Δ<i>recA</i>) (blue squares), GY16626 (Δ<i>ddrB</i> Δ<i>recA</i>) (red squares and interrupted lines), GY16636 (Δ<i>ddrB</i> Δ<i>recO</i>) (orange circles) were grown from independent colonies at 30°C to an OD_650nm = 0.1 (time 0 of the growth curves). <b>A.</b> OD_650nm as a function of time. <b>B.</b> Colony forming units as a function of time. <b>C.</b> Colony forming units as a function of OD_650nm.</p

Recombination between chromosomal and plasmid DNA is RecA- and RecF-dependent.

<p><b>A.</b> Schematic representation of the recombination assay between chromosomal and plasmid DNA. The 5’<i>tetA</i> and the 3’<i>tetA</i> regions of the <i>tetA</i> gene, containing 438 bp repeats, were introduced into the chromosomal dispensable <i>amyE</i> gene and into the p11554 plasmid giving rise to plasmid p15002, respectively. One crossing over between the two 438 bp repeated sequences (black boxes) leads to the reconstitution of a functional <i>tetA</i> gene and the integration of the plasmid into chromosomal DNA. <b>B.</b> Medians of [Tet^R] frequencies in WT (GY15147), Δ<i>recA</i> (GY15158), Δ<i>recF</i> (GY15160), Δ<i>radA</i> (GY15149), and Δ<i>uvrD</i> (GY15156) bacteria, all containing the p15002 plasmid, are calculated from at least 3 independent values and represented by Tukey boxplots. Outliers are represented by open circles. The small arrows attached to the horizontal line representing the upper limit of detectable [Tet^R] frequencies indicate that [Tet^R] frequencies were < 5 10⁻⁷ for Δ<i>recA</i> and Δ<i>recF</i> bacteria.</p

Deletion frequencies between repeated sequences separated by 3,500, 6,500, and 10,500 bp.

<p><b>A.</b> Schematic representation of the constructions used. <b>B., C., D.</b> Bacteria contain 3,500 bp (panel B), 6,500 bp (panel C), and 10,500 bp (panel D) intervening sequences between the <i>tetA</i> repeats. The medians of [Tet^R] frequencies calculated from 10 to 35 independent values in the tested strains are represented by Tukey boxplots. Outliers are represented by open circles. Statistically significant differences in the medians of recombination frequencies of the mutants compared to the WT GY16209, GY16227, and GY16235 in panel B, C, and D, respectively, were calculated using the non-parametric Dunn's multiple comparison test: * P < 0.05; ** P < 0.01; *** P < 0.001; (ns) if P > 0.05. <b>B.</b> WT (GY16209), Δ<i>recA</i> (GY16238), Δ<i>recO</i> (GY16262), Δ<i>recF</i> (GY16264), Δ<i>uvrD</i> (GY16608), Δ<i>ddrB</i> (GY16268), Δ<i>ddrB</i> Δ<i>recA</i> (GY16630), Δ<i>ddrB</i> Δ<i>recO</i> (GY16640) <b>C.</b> WT (GY16227), Δ<i>recA</i> (GY16244), Δ<i>recO</i> (GY16278), Δ<i>recF</i> (GY16276), Δ<i>uvrD</i> (GY16612), Δ<i>ddrB</i> (GY16282), Δ<i>ddrB</i> Δ<i>recA</i> (GY16632), Δ<i>ddrB</i> Δ<i>recO</i> (GY16642) <b>D.</b> WT (GY16235), Δ<i>recA</i> (GY16252), Δ<i>recO</i> (GY16290), Δ<i>recF</i> (GY16292), Δ<i>uvrD</i> (GY16614), Δ<i>ddrB</i> (GY16296), Δ<i>ddrB ΔrecA</i> (GY16634), Δ<i>ddrB ΔrecO</i> (GY16644).</p

γ-irradiation induced recombination between repeated sequences.

<p><b>A.</b> Induction of recombination between repeated sequences separated by 1,479 bp as a function of the radiation dose. [Tet^R] frequencies were measured in at least 5 independent cultures after 20 hours of post irradiation incubation of GY12949 in TGY medium after exposure to different γ-irradiation doses. <b>B.</b> Induction by exposure to γ-irradiation of recombination between repeats separated by intervening sequences of increasing length: 1,479 bp (GY12949), 3,500 bp (GY16209), 6,500 bp (GY16227) and 10,500 bp (GY16235). <b>C.</b> γ-promoted induction of recombination between overlapping sequences separated by 1,479 bp in WT (GY12949), Δ<i>ddrB</i> (GY16016), and Δ<i>uvrD</i> (GY12953) bacteria. <b>D.</b> γ-promoted induction of recombination between overlapping sequences separated by 10,500 bp in WT (GY16235), Δ<i>ddrB</i>(GY16296), Δ<i>uvrD</i>(GY16614). <b>B.</b>, <b>C.</b>, <b>D.</b> Medians of the [Tet^R] frequencies calculated from 5 to 30 independent values are represented by Tukey boxplots. Outliers were represented by open circles. Statistically significant differences in the medians of recombination frequencies between irradiated and the corresponding non-irradiated bacteria were calculated using the non-parametric Mann-Withney test: * P < 0.05; ** P < 0.01; *** P < 0.001; (ns) if P> 0.05. NI: non-irradiated bacteria. IR: irradiated bacteria.</p

Effect of base pair changes in the repeats on their recombination frequencies.

<p><b>A.</b> Schematic representation of the recombination substrates containing 1 or 3 bp changes. The positions of the base pair changes calculated from the initiation codon in the 5’<i>tetA</i> region are indicated. <b>B.</b> Medians of [Tet^R] frequencies calculated from 14 to 43 independent values in WT (GY12949), Δ<i>recA</i> (GY15184), Δ<i>mutS</i> (GY12978), Δ<i>mutS ΔrecA</i> (GY16620) bacteria containing identical repeated sequences, WT (GY12950), Δ<i>recA</i> (GY16624), Δ<i>mutS</i> (GY12980), Δ<i>mutS</i> Δ<i>recA</i> (GY16618) containing one base difference in the repeated sequences, and WT (GY12948), Δ<i>recA</i> (GY16622), Δ<i>mutS</i> (GY12979), Δ<i>mutS</i> Δ<i>recA</i> (GY16616) containing 3 base differences in the repeated sequences, are represented by Tukey boxplots. Outliers are represented by open circles. Statistically significant differences in the medians of recombination frequencies of the mutants and WT containing sequence differences in the repeated sequence compared to GY12949 were calculated using the non-parametric Dunn's multiple comparison test: * P < 0.05; ** P < 0.01; *** P < 0.001; ns if P > 0.05.</p

Bacterial strains and plasmids.

<p>^(a) 5’<i>tetA</i> and the 3’<i>tetA</i> contain the 1–779 and the 342–1190 part of the <i>tetA</i> coding region, respectively. They contain a 342–779 overlapping region. The positions are numbered relatively to the first base of the initiation codon of <i>tetA</i>.</p><p>^(b) The positions of the mutations in the 5’<i>tetA</i> fragment are numbered relatively to the first base of the initiation codon of <i>tetA</i>.</p><p>Bacterial strains and plasmids.</p