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6 results on '"Christophe Leplat"'

1. Genome-wide transcriptome analysis of hydrogen production in the cyanobacterium Synechocystis: Towards the identification of new players

Author

Raphaël Champeimont, Christophe Leplat, Franck Chauvat, Panatda Saenkham, Aude Jean-Christophe, Corinne Cassier-Chauvat, Service de Bioénergétique, Biologie Stucturale, et Mécanismes ( SB2SM ), Centre National de la Recherche Scientifique ( CNRS ) -Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Sud - Paris 11 ( UP11 ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Sud - Paris 11 ( UP11 ), Laboratoire de Biologie Intégrative ( LBI ), Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ), Service de Bioénergétique, Biologie Stucturale, et Mécanismes (SB2SM), Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Biologie Intégrative (LBI), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institut de Biologie Intégrative de la Cellule (I2BC), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Operon, Energy Engineering and Power Technology, Repressor, [ SDV.BBM.BM ] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Computational biology, Cyanobacteria, Plasmid, Genome, Transcriptome, 03 medical and health sciences, Gene, 030304 developmental biology, 0303 health sciences, biology, 030306 microbiology, Renewable Energy, Sustainability and the Environment, Synechocystis, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Condensed Matter Physics, biology.organism_classification, Bioproduction, Metabolism, Fuel Technology, DNA microarray, Hydrogen
Abstract

International audience; We report the development of new tools and methods for facile integration and meaningful representation of high throughput data generated by genome-wide analyses of the model cyanobacterium Synechocystis PCC6803, for future genetic engineering aiming at increasing its level of hydrogen photoproduction. These robust tools comprise new oligonucleotide DNA microarrays to monitor the transcriptomic responses of all 3725 genes of Synechocystis, and the SVGMapping method and custom-made templates to represent the metabolic reprogramming for improved hydrogen production. We show, for the first time, that the AbrB2 repressor of the hydrogenase-encoding operon, also regulates metal transport and protection against oxidative stress, as well as numerous plasmid genes, which have been overlooked so far. This report will stimulate the construction and global analysis of hydrogen production mutants with the prospect of developing powerful cell factories for the sustainable production of hydrogen, as well as investigations of the probable role of plasmids in this process.

Published
2013

2. Recovery of ionizing-radiation damage after high doses of gamma ray in the hyperthermophilic archaeon Thermococcus gammatolerans

Author

Angels Tapias, Fabrice Confalonieri, Christophe Leplat, Institut de génétique et microbiologie [Orsay] (IGM), and Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Time Factors, DNA Repair, Radiation Tolerance, Microbiology, Chromosomes, Ionizing radiation, 03 medical and health sciences, chemistry.chemical_compound, Radiation, Ionizing, Radioresistance, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, 030304 developmental biology, 0303 health sciences, biology, 030306 microbiology, Cell growth, Thermococcus gammatolerans, Dose-Response Relationship, Radiation, General Medicine, biology.organism_classification, Archaea, DNA Repair Kinetics, Cell biology, Thermococcus, Kinetics, DNA, Archaeal, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, chemistry, Gamma Rays, Molecular Medicine, DNA, Bacteria, DNA Damage
Abstract

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.

Published
2009

3. High-throughput transformation method for Yarrowia lipolytica mutant library screening

Author

Christophe Leplat, Tristan Rossignol, Jean-Marc Nicaud, MICrobiologie de l'ALImentation au Service de la Santé (MICALIS), Institut National de la Recherche Agronomique (INRA)-AgroParisTech, and Investments for Future by French Government [ANR-001]

Subjects
Genetics, Microbial, Transformation method, Extracellular protease, Overexpression, gene overexpression, [SDV]Life Sciences [q-bio], Mutant, Yarrowia, Locus (genetics), Applied Microbiology and Biotechnology, Microbiology, Genome, Gateway (R), 03 medical and health sciences, Transformation, Genetic, Plasmid, Genetic Testing, méthode de dépistage, Molecular Biology, surexpression génique, Gene Library, 030304 developmental biology, 0303 health sciences, Mutant screening, biology, 030306 microbiology, Gene Transfer Techniques, Proteolytic enzymes, General Medicine, biology.organism_classification, Molecular biology, Yeast, High-Throughput Screening Assays, Biochemistry, yarrowia lipolytica, Plasmids
Abstract

As a microorganism of major biotechnological importance, the oleaginous yeast Yarrowia lipolytica is subjected to intensive genetic engineering and functional genomic analysis. Future advancements in this area, however, require a system that will generate a large collection of mutants for high-throughput screening. Here, we report a rapid and efficient method for high-throughput transformation of Y. lipolytica in 96-well plates. We developed plasmids and strains for the large-scale screening of overexpression mutant strains, using Gateway (R) vectors that were adapted for specific locus integration in Y. lipolytica. As an example, a collection of mutants that overexpressed the alkaline extracellular protease (AEP) was obtained in a single transformation experiment. The platform strain that we developed to receive the overexpression cassette was designed to constitutively express a fluorescent protein as a convenient growth reporter for screening in non-translucid media. An example of growth comparison in skim milk-based medium between AEP overexpression and deletion mutants is provided.

Published
2015

4. The activity of the Synechocystis PCC6803 AbrB2 regulator of hydrogen production can be post-translationally controlled through glutathionylation

Author

Paulette Decottignies, Marcia Ortega-Ramos, Panatda Saenkham, Stéphane D. Lemaire, Hervé Bottin, Geneviève Guedeney, Jérémy Dutheil, Christophe Leplat, Jean-Christophe Aude, Corinne Cassier-Chauvat, Samer Sakr, Violaine Chapuis, Franck Chauvat, Biologie et Biotechnologie des Cyanobactéries (B2CYA), Département Microbiologie (Dpt Microbio), Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Service de Bioénergétique, Biologie Stucturale, et Mécanismes (SB2SM), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Génétique moléculaire et destin cellulaire (FRE 3377), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Institut de biochimie et biophysique moléculaire et cellulaire (IBBMC), Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), Biologie moléculaire et cellulaire des eucaryotes, Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS), Biologie et Biotechnologie des Cyanobactéries ( B2CYA ), Département Microbiologie ( Dpt Microbio ), Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Sud - Paris 11 ( UP11 ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Sud - Paris 11 ( UP11 ) -Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Sud - Paris 11 ( UP11 ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Sud - Paris 11 ( UP11 ), Service de Bioénergétique, Biologie Stucturale, et Mécanismes ( SB2SM ), Centre National de la Recherche Scientifique ( CNRS ) -Institut de Biologie Intégrative de la Cellule ( I2BC ), Génétique moléculaire et destin cellulaire ( FRE 3377 ), Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Centre National de la Recherche Scientifique ( CNRS ), Institut de biochimie et biophysique moléculaire et cellulaire ( IBBMC ), Université Paris-Sud - Paris 11 ( UP11 ) -Centre National de la Recherche Scientifique ( CNRS ), Université Pierre et Marie Curie - Paris 6 ( UPMC ) -Centre National de la Recherche Scientifique ( CNRS ), Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), and Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Sud - Paris 11 (UP11)

Subjects
Hydrogenase, Operon, [SDV]Life Sciences [q-bio], Regulator, Energy Engineering and Power Technology, Repressor, Cyanobacteria, Conserved sequence, 03 medical and health sciences, Cysteine oxidation, Psychological repression, 030304 developmental biology, 0303 health sciences, biology, [ SDV ] Life Sciences [q-bio], 030306 microbiology, Renewable Energy, Sustainability and the Environment, Chemistry, Synechocystis, Condensed Matter Physics, biology.organism_classification, Bioproduction, Fuel Technology, Biochemistry, Oxidative stress, Cysteine, Hydrogen, Regulation
Abstract

International audience; We show that the Synechocystis AbrB2 repressor of hydrogen production, down regulates the defence against oxidative stress. The single widely conserved cysteine of AbrB2 is also shown to play a crucial role in AbrB2 oligomerisation, and in AbrB2-mediated repression of the hydrogenase encoding operon (hoxEFUYH) and a wealth of other genes. Very interestingly, our results indicate that this cysteine is the target of glutathionylation, which affects the binding of AbrB2 on the hox operon-promoter DNA, as well as the stability of AbrB2 at the non-standard temperature of 39 °C. Similarly, we show that the cysteine of the other hoxEFUYH regulator AbrB1 can also be glutathionylated in vitro. These novel findings will certainly stimulate the in depth analysis of the influence of glutathionylation on the production of hydrogen, a field totally overlooked so far. They also emphasize on the evolutionary conservation of glutathionylation, a process mostly described in eukaryotes, so far.

Published
2013

5. The AbrB2 autorepressor, expressed from an atypical promoter, represses the hydrogenase operon to regulate hydrogen production in Synechocystis strain PCC6803

Author

Corinne Cassier-Chauvat, Samer Sakr, Marcia Ortega-Ramos, Hervé Bottin, Franck Chauvat, Christophe Leplat, Jérémy Dutheil, Panatda Saenkham, Laurent Cournac, Biologie et Biotechnologie des Cyanobactéries ( B2CYA ), Département Microbiologie ( Dpt Microbio ), Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ), Biologie végétale et microbiologie environnementale - UMR7265 ( BVME ), Centre National de la Recherche Scientifique ( CNRS ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Aix Marseille Université ( AMU ), Biologie et Biotechnologie des Cyanobactéries (B2CYA), Département Microbiologie (Dpt Microbio), Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Biologie végétale et microbiologie environnementale - UMR7265 (BVME), Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) (BIAM), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)

Subjects
DNA, Bacterial, DNA, Complementary, Hydrogenase, Operon, [SDV]Life Sciences [q-bio], Molecular Sequence Data, Biology, Microbiology, trp operon, Genetic Determinism, 03 medical and health sciences, gal operon, Promoter Regions, Genetic, Hox gene, Molecular Biology, 030304 developmental biology, Genetics, 0303 health sciences, Base Sequence, [ SDV ] Life Sciences [q-bio], 030306 microbiology, Synechocystis, Promoter, Articles, Gene Expression Regulation, Bacterial, biology.organism_classification, Mutation, L-arabinose operon, Hydrogen
Abstract

We have thoroughly investigated the abrB2 gene (sll0822) encoding an AbrB-like regulator in the wild-type strain of the model cyanobacterium Synechocystis strain PCC6803. We report that abrB2 is expressed from an active but atypical promoter that possesses an extended −10 element (TGTAATAT) that compensates for the absence of a −35 box. Strengthening the biological significance of these data, we found that the occurrence of an extended −10 promoter box and the absence of a −35 element are two well-conserved features in abrB2 genes from other cyanobacteria. We also show that AbrB2 is an autorepressor that is dispensable to cell growth under standard laboratory conditions. Furthermore, we demonstrate that AbrB2 also represses the hox operon, which encodes the Ni-Fe hydrogenase of biotechnological interest, and that the hox operon is weakly expressed even though it possesses the two sequences resembling canonical −10 and −35 promoter boxes. In both the AbrB2-repressed promoters of the abrB2 gene and the hox operon, we found a repeated DNA motif [TT-(N 5 )-AAC], which could be involved in AbrB2 repression. Supporting this hypothesis, we found that a TT-to-GG mutation of one of these elements increased the activity of the abrB2 promoter. We think that our abrB2 -deleted mutant with increased expression of the hox operon and hydrogenase activity, together with the reporter plasmids we constructed to analyze the abrB2 gene and the hox operon, will serve as useful tools to decipher the function and the regulation of hydrogen production in Synechocystis .

Published
2012

6. Genome analysis and genome-wide proteomics of Thermococcus gammatolerans, the most radioresistant organism known amongst the Archaea

Author

Christophe Leplat, Murielle Dutertre, Véronique Anthouard, Fabrice Confalonieri, Patrick Wincker, Yvan Zivanovic, Patrick Forterre, Philippe P. Guerin, Jean Armengaud, Arnaud Lagorce, Institut de génétique et microbiologie [Orsay] (IGM), Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Biochimie des Systèmes Perturbés (LBSP), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institut de Génomique d'Evry (IG), Institut de Biologie François JACOB (JACOB), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay, Université Paris-Saclay-Institut de Biologie François JACOB (JACOB), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)

Subjects
Proteomics, DNA Repair, Proteome, Archaeal Proteins, Codon, Initiator, Biology, Radiation Tolerance, Genome, Chromosomes, Mass Spectrometry, 03 medical and health sciences, Hydrogenase, Genome, Archaeal, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, 030304 developmental biology, Genetics, Whole genome sequencing, 0303 health sciences, Microbial Viability, 030306 microbiology, Research, Cell Membrane, Reproducibility of Results, Thermococcus gammatolerans, Biological Transport, Sequence Analysis, DNA, biology.organism_classification, Thermococcales, Thermococcus, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Gamma Rays, Protein Biosynthesis, DNA Transposable Elements, Protein Processing, Post-Translational, Archaea
Abstract

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.

Published
2009

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