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

The Gut Microbiota Regulates Intestinal CD4 T Cells Expressing RORγt and Controls Metabolic Disease

SummaryA high-fat diet (HFD) induces metabolic disease and low-grade metabolic inflammation in response to changes in the intestinal microbiota through as-yet-unknown mechanisms. Here, we show that a HFD-derived ileum microbiota is responsible for a decrease in Th17 cells of the lamina propria in axenic colonized mice. The HFD also changed the expression profiles of intestinal antigen-presenting cells and their ability to generate Th17 cells in vitro. Consistent with these data, the metabolic phenotype was mimicked in RORγt-deficient mice, which lack IL17 and IL22 function, and in the adoptive transfer experiment of T cells from RORγt-deficient mice into Rag1-deficient mice. We conclude that the microbiota of the ileum regulates Th17 cell homeostasis in the small intestine and determines the outcome of metabolic disease

Prevalence and temporal trends of crack injection among injection drug users in eastern central Canada

Background: Little is known about crack injection and its temporal trends in North America. This article describes the extent of crack injection and examines temporal trends among injection drug users (IDUs) recruited from 2003 to 2010 in the SurvUDI network. Methods: IDUs who injected recently (past 6 months) were recruited in harm reduction and health programs in eastern central Canada. Trend analyses were performed using generalized estimating equations. Some IDUs participated multiple times; first interview was retained for the descriptive analyses, while first interview per year was retained for the trend analyses. Results: Of the 4088 IDUs recruited, 15.2% (621) reported crack injection; large variations across sites were noted (range: 0.3%-39.5%). Trend analyses were limited to Ottawa (449 crack injectors) and Montréal (121). For Ottawa, a significant decline was observed, from 48.3% to 36.9%, with a prevalence ratio (PR) of 0.97 per year (95%CI: 0.94-0.99). For Montréal, a significant rise was observed, from 6.0% to 18.4%, with a PR of 1.29 per year (95%CI: 1.19-1.40). Conclusions: Strong variations in crack injection exist throughout the SurvUDI network, and reversed temporal trends have been observed in Ottawa and Montréal. These data will be useful to local harm reduction programs to evaluate the need to distribute items required by crack injectors and to develop prevention messages

Tye7 regulates yeast Ty1 retrotransposon sense and antisense transcription in response to adenylic nucleotides stress

Transposable elements play a fundamental role in genome evolution. It is proposed that their mobility, activated under stress, induces mutations that could confer advantages to the host organism. Transcription of the Ty1 LTR-retrotransposon of Saccharomyces cerevisiae is activated in response to a severe deficiency in adenylic nucleotides. Here, we show that Ty2 and Ty3 are also stimulated under these stress conditions, revealing the simultaneous activation of three active Ty retrotransposon families. We demonstrate that Ty1 activation in response to adenylic nucleotide depletion requires the DNA-binding transcription factor Tye7. Ty1 is transcribed in both sense and antisense directions. We identify three Tye7 potential binding sites in the region of Ty1 DNA sequence where antisense transcription starts. We show that Tye7 binds to Ty1 DNA and regulates Ty1 antisense transcription. Altogether, our data suggest that, in response to adenylic nucleotide reduction, TYE7 is induced and activates Ty1 mRNA transcription, possibly by controlling Ty1 antisense transcription. We also provide the first evidence that Ty1 antisense transcription can be regulated by environmental stress conditions, pointing to a new level of control of Ty1 activity by stress, as Ty1 antisense RNAs play an important role in regulating Ty1 mobility at both the transcriptional and post-transcriptional stages

A Major Role of the RecFOR Pathway in DNA Double-Strand-Break Repair through ESDSA in Deinococcus radiodurans

In Deinococcus radiodurans, the extreme resistance to DNA–shattering treatments such as ionizing radiation or desiccation is correlated with its ability to reconstruct a functional genome from hundreds of chromosomal fragments. The rapid reconstitution of an intact genome is thought to occur through an extended synthesis-dependent strand annealing process (ESDSA) followed by DNA recombination. Here, we investigated the role of key components of the RecF pathway in ESDSA in this organism naturally devoid of RecB and RecC proteins. We demonstrate that inactivation of RecJ exonuclease results in cell lethality, indicating that this protein plays a key role in genome maintenance. Cells devoid of RecF, RecO, or RecR proteins also display greatly impaired growth and an important lethal sectoring as bacteria devoid of RecA protein. Other aspects of the phenotype of recFOR knock-out mutants paralleled that of a ΔrecA mutant: ΔrecFOR mutants are extremely radiosensitive and show a slow assembly of radiation-induced chromosomal fragments, not accompanied by DNA synthesis, and reduced DNA degradation. Cells devoid of RecQ, the major helicase implicated in repair through the RecF pathway in E. coli, are resistant to γ-irradiation and have a wild-type DNA repair capacity as also shown for cells devoid of the RecD helicase; in contrast, ΔuvrD mutants show a markedly decreased radioresistance, an increased latent period in the kinetics of DNA double-strand-break repair, and a slow rate of fragment assembly correlated with a slow rate of DNA synthesis. Combining RecQ or RecD deficiency with UvrD deficiency did not significantly accentuate the phenotype of ΔuvrD mutants. In conclusion, RecFOR proteins are essential for DNA double-strand-break repair through ESDSA whereas RecJ protein is essential for cell viability and UvrD helicase might be involved in the processing of double stranded DNA ends and/or in the DNA synthesis step of ESDSA

Activation de la transcription du rétrotransposon Ty1 de la levure Saccharomyces cerevisiae par la carence sévère en adénine

Les rétrotransposons sont des entités génétiques capables de se déplacer dans les génomes eucaryotes. Activés par des stress environnementaux, ils jouent un rôle majeur dans la structure, l évolution et l expression des génomes. Le rétrotransposon Ty1 de la levure S. cerevisiae est un modèle de l étude de la régulation et de l impact des rétrotransposons. La transcription et la mobilité de Ty1 sont activées dans une souche privée d adénine, déficiente dans la voie de biosynthèse de l AMP, conditions nommées carence sévère en adénine . L objectif de ma thèse a été de comprendre l activation de l expression de Ty1 par cette carence et d étudier l impact de cette activation sur le génome de la levure. Nous avons montré que la région longue terminale de Ty1, où démarre la transcription, est suffisante pour permettre la régulation. Nous avons aussi découvert que la carence modifie la transcription de gènes adjacents au promoteur de Ty1. Nous avons montré une corrélation entre l activation de la transcription de Ty1 et la diminution de l ATP cellulaire. De plus, un mutant qui crée directement un déficit en ATP, induit l activation de la transcription de Ty1 et mime les conditions de carence sévère en adénine. Le déficit en ATP pourrait donc être le signal de l activation de Ty1PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Disruption of hspR , the repressor gene of the dnaK operon in Streptomyces albus G

International audiencehspR is the distal gene of the Streptomyces albus dnaK operon. It encodes a protein similar to GlnR, the repressor of the Bacillus subtilis glutamine synthetase gene. Transcriptional analysis showed that disruption of hspR led to constitutive high-level expression of the dnaK operon, SDS-PAGE analysis revealed over-production and accumulation of the chaperone DnaK at low temperature HSP94, a heat-inducible protein cross-reacting with anti-CipB antibodies, was also shown to be constitutively overexpressed at low temperature in the hspR mutant. Those features were lost when the mutant was complemented in trans by an intact copy of hspR. The hspR mutant was impaired in its growth on solid rich medium: colonies grow slowly at 30 degrees C. However, formation of aerial mycelium and sporulation was not prevented. In liquid culture growth curves of the mutant and the wild type were similar. The kinetics of groEL gene induction were not modified by the hspR null mutation, indicating that HspR was not directly involved in the control of groEL transcription. Thus, in contrast with B. subtilis and other Gram-positive bacteria, transcription of Streptomyces dnaK and groEL operons is not controlled by the same regulator

The RheA repressor is the thermosensor of the HSP18 heat shock response in Streptomyces albus

Microorganisms have mechanisms to sense their environment and rapidly adapt to survive changes in conditions. In Streptomyces albus, various transcriptional repressors mediate the induction of heat shock genes. The RheA repressor regulates the synthesis of HSP18, a small heat shock protein, which plays a role in thermotolerance. The RheA protein was purified to determine how it responds rapidly to temperature. Gel retardation assays and footprinting experiments identified the specific target of RheA as an inverted repeat (TGTCATC 5N GATGACA) located in Phsp18, PrheA which is the common promoter region of the divergon. Gel retardation assays detected RheA-complexes formed with the hsp18-rheA promoters. The complexes did not form at higher temperature. In vitro transcription experiments showed that RheA is an autoregulatory protein and that its activity is inhibited by high temperature. The temperature-induced derepression by RheA is reversible. Dichroism circular spectroscopy revealed a reversible change of RheA conformation in relation with the temperature that could represent a transition between an active and an inactive form. Our experiments demonstrate that RheA acts as a cellular thermometer in hsp18 regulation

Remodeling Yeast Gene Transcription by Activating the Ty1 Long Terminal Repeat Retrotransposon under Severe Adenine Deficiency▿

The Ty1 long terminal repeat (LTR) retrotransposon of Saccharomyces cerevisiae is a powerful model to understand the activation of transposable elements by stress and their impact on genome expression. We previously discovered that Ty1 transcription is activated under conditions of severe adenine starvation. The mechanism of activation is independent of the Bas1 transcriptional activator of the de novo AMP biosynthesis pathway and probably involves chromatin remodeling at the Ty1 promoter. Here, we show that the 5′ LTR has a weak transcriptional activity and is sufficient for the activation by severe adenine starvation. Furthermore, we demonstrate that Ty1 insertions that bring Ty1 promoter sequences into the vicinity of a reporter gene confer adenine starvation regulation on it. We provide evidence that similar coactivation of genes adjacent to Ty1 sequences occurs naturally in the yeast genome, indicating that Ty1 insertions can mediate transcriptional control of yeast gene expression under conditions of severe adenine starvation. Finally, the transcription pattern of genes adjacent to Ty1 insertions suggests that severe adenine starvation facilitates the initiation of transcription at alternative sites, partly located in the 5′ LTR. We propose that Ty1-driven transcription of coding and noncoding sequences could regulate yeast gene expression in response to stress