Author: "David Pignol" / Language: english - Searchworks@Jio Institute Digital Library Search Results

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54 results on '"David Pignol"'

1. Reduction of Protein Bound Methionine Sulfoxide by a Periplasmic Dimethyl Sulfoxide Reductase

Author: Lionel Tarrago, Sandrine Grosse, David Lemaire, Laetitia Faure, Mathilde Tribout, Marina I. Siponen, Mila Kojadinovic-Sirinelli, David Pignol, Pascal Arnoux, and Monique Sabaty
Subjects: dimethyl sulfoxide reductase, enzyme kinetics, methionine sulfoxide, oxidative stress, protein oxidation, protein quality control, Therapeutics. Pharmacology, RM1-950
Abstract: In proteins, methionine (Met) can be oxidized into Met sulfoxide (MetO). The ubiquitous methionine sulfoxide reductases (Msr) A and B are thiol-oxidoreductases reducing MetO. Reversible Met oxidation has a wide range of consequences, from protection against oxidative stress to fine-tuned regulation of protein functions. Bacteria distinguish themselves by the production of molybdenum-containing enzymes reducing MetO, such as the periplasmic MsrP which protects proteins during acute oxidative stress. The versatile dimethyl sulfoxide (DMSO) reductases were shown to reduce the free amino acid MetO, but their ability to reduce MetO within proteins was never evaluated. Here, using model oxidized proteins and peptides, enzymatic and mass spectrometry approaches, we showed that the Rhodobacter sphaeroides periplasmic DorA-type DMSO reductase reduces protein bound MetO as efficiently as the free amino acid L-MetO and with catalytic values in the range of those described for the canonical Msrs. The identification of this fourth type of enzyme able to reduce MetO in proteins, conserved across proteobacteria and actinobacteria, suggests that organisms employ enzymatic systems yet undiscovered to regulate protein oxidation states.
Published: 2020
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2. Heterologous expression of membrane proteins: choosing the appropriate host.

Author: Florent Bernaudat, Annie Frelet-Barrand, Nathalie Pochon, Sébastien Dementin, Patrick Hivin, Sylvain Boutigny, Jean-Baptiste Rioux, Daniel Salvi, Daphné Seigneurin-Berny, Pierre Richaud, Jacques Joyard, David Pignol, Monique Sabaty, Thierry Desnos, Eva Pebay-Peyroula, Elisabeth Darrouzet, Thierry Vernet, and Norbert Rolland
Subjects: Medicine, Science
Abstract: BackgroundMembrane proteins are the targets of 50% of drugs, although they only represent 1% of total cellular proteins. The first major bottleneck on the route to their functional and structural characterisation is their overexpression; and simply choosing the right system can involve many months of trial and error. This work is intended as a guide to where to start when faced with heterologous expression of a membrane protein.Methodology/principal findingsThe expression of 20 membrane proteins, both peripheral and integral, in three prokaryotic (E. coli, L. lactis, R. sphaeroides) and three eukaryotic (A. thaliana, N. benthamiana, Sf9 insect cells) hosts was tested. The proteins tested were of various origins (bacteria, plants and mammals), functions (transporters, receptors, enzymes) and topologies (between 0 and 13 transmembrane segments). The Gateway system was used to clone all 20 genes into appropriate vectors for the hosts to be tested. Culture conditions were optimised for each host, and specific strategies were tested, such as the use of Mistic fusions in E. coli. 17 of the 20 proteins were produced at adequate yields for functional and, in some cases, structural studies. We have formulated general recommendations to assist with choosing an appropriate system based on our observations of protein behaviour in the different hosts.Conclusions/significanceMost of the methods presented here can be quite easily implemented in other laboratories. The results highlight certain factors that should be considered when selecting an expression host. The decision aide provided should help both newcomers and old-hands to select the best system for their favourite membrane protein.
Published: 2011
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3. Single-step production of a recyclable nanobiocatalyst for organophosphate pesticides biodegradation using functionalized bacterial magnetosomes.

Author: Nicolas Ginet, Romain Pardoux, Géraldine Adryanczyk, Daniel Garcia, Catherine Brutesco, and David Pignol
Subjects: Medicine, Science
Abstract: Enzymes are versatile catalysts in laboratories and on an industrial scale; improving their immobilization would be beneficial to broadening their applicability and ensuring their (re)use. Lipid-coated nano-magnets produced by magnetotactic bacteria are suitable for a universally applicable single-step method of enzyme immobilization. By genetically functionalizing the membrane surrounding these magnetite particles with a phosphohydrolase, we engineered an easy-to-purify, robust and recyclable biocatalyst to degrade ethyl-paraoxon, a commonly used pesticide. For this, we genetically fused the opd gene from Flavobacterium sp. ATCC 27551 encoding a paraoxonase to mamC, an abundant protein of the magnetosome membrane in Magnetospirillum magneticum AMB-1. The MamC protein acts as an anchor for the paraoxonase to the magnetosome surface, thus producing magnetic nanoparticles displaying phosphohydrolase activity. Magnetosomes functionalized with Opd were easily recovered from genetically modified AMB-1 cells: after cellular disruption with a French press, the magnetic nanoparticles are purified using a commercially available magnetic separation system. The catalytic properties of the immobilized Opd were measured on ethyl-paraoxon hydrolysis: they are comparable with the purified enzyme, with K(m) (and k(cat)) values of 58 µM (and 178 s(-1)) and 43 µM (and 314 s(-1)) for the immobilized and purified enzyme respectively. The Opd, a metalloenzyme requiring a zinc cofactor, is thus properly matured in AMB-1. The recycling of the functionalized magnetosomes was investigated and their catalytic activity proved to be stable over repeated use for pesticide degradation. In this study, we demonstrate the easy production of functionalized magnetic nanoparticles with suitably genetically modified magnetotactic bacteria that are efficient as a reusable nanobiocatalyst for pesticides bioremediation in contaminated effluents.
Published: 2011
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4. A second actin-like MamK protein in Magnetospirillum magneticum AMB-1 encoded outside the genomic magnetosome island.

Author: Jean-Baptiste Rioux, Nadège Philippe, Sandrine Pereira, David Pignol, Long-Fei Wu, and Nicolas Ginet
Subjects: Medicine, Science
Abstract: Magnetotactic bacteria are able to swim navigating along geomagnetic field lines. They synthesize ferromagnetic nanocrystals that are embedded in cytoplasmic membrane invaginations forming magnetosomes. Regularly aligned in the cytoplasm along cytoskeleton filaments, the magnetosome chain effectively forms a compass needle bestowing on bacteria their magnetotactic behaviour. A large genomic island, conserved among magnetotactic bacteria, contains the genes potentially involved in magnetosome formation. One of the genes, mamK has been described as encoding a prokaryotic actin-like protein which when it polymerizes forms in the cytoplasm filamentous structures that provide the scaffold for magnetosome alignment. Here, we have identified a series of genes highly similar to the mam genes in the genome of Magnetospirillum magneticum AMB-1. The newly annotated genes are clustered in a genomic islet distinct and distant from the known magnetosome genomic island and most probably acquired by lateral gene transfer rather than duplication. We focused on a mamK-like gene whose product shares 54.5% identity with the actin-like MamK. Filament bundles of polymerized MamK-like protein were observed in vitro with electron microscopy and in vivo in E. coli cells expressing MamK-like-Venus fusions by fluorescence microscopy. In addition, we demonstrate that mamK-like is transcribed in AMB-1 wild-type and DeltamamK mutant cells and that the actin-like filamentous structures observed in the DeltamamK strain are probably MamK-like polymers. Thus MamK-like is a new member of the prokaryotic actin-like family. This is the first evidence of a functional mam gene encoded outside the magnetosome genomic island.
Published: 2010
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5. Repeated horizontal gene transfers triggered parallel evolution of magnetotaxis in two evolutionary divergent lineages of magnetotactic bacteria

Author: Christopher T. Lefèvre, Caroline L. Monteil, Béatrice Alonso, Denis S. Grouzdev, Zoé Rouy, Stéphane Cruveiller, David Pignol, Nicolas Ginet, Guy Perrière, 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), Centre National de la Recherche Scientifique (CNRS), Genoscope - Centre national de séquençage [Evry] (GENOSCOPE), Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Microbiologie Environnementale et Moléculaire (MEM), 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)), ANR-18-CE31-0003,SIGMAG,SIGNATURE DES MAGNETITES PRODUITES PAR LES BACTERIES MAGNETOTACTIQUES : PERSPECTIVES CHIMIQUES ET ISOTOPIQUES(2018), 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: Gene Transfer, Horizontal, Magnetotactic bacteria, [SDV.BID.SPT]Life Sciences [q-bio]/Biodiversity/Systematics, Phylogenetics and taxonomy, Microbiology, Article, 03 medical and health sciences, Phylogenetics, Gram-Negative Bacteria, Magnetotaxis, Magnetospirillum, Ecology, Evolution, Behavior and Systematics, ComputingMilieux_MISCELLANEOUS, Alphaproteobacteria, 030304 developmental biology, 0303 health sciences, Experimental evolution, Bacteria, biology, 030306 microbiology, Clonal interference, [SDV.BID.EVO]Life Sciences [q-bio]/Biodiversity/Populations and Evolution [q-bio.PE], biology.organism_classification, Evolutionary biology, Parallel evolution, Adaptation
Abstract: Under the same selection pressures, two genetically divergent populations may evolve in parallel toward the same adaptive solutions. Here, we hypothesized that magnetotaxis (i.e., magnetically guided chemotaxis) represents a key adaptation to micro-oxic habitats in aquatic sediments and that its parallel evolution homogenized the phenotypes of two evolutionary divergent clusters of freshwater spirilla. All magnetotactic bacteria affiliated to the Magnetospirillum genus (Alphaproteobacteria class) biomineralize the same magnetic particle chains and share highly similar physiological and ultrastructural features. We looked for the processes that could have contributed at shaping such an evolutionary pattern by reconciling species and gene trees using newly sequenced genomes of Magnetospirillum related bacteria. We showed that repeated horizontal gene transfers and homologous recombination of entire operons contributed to the parallel evolution of magnetotaxis. We propose that such processes could represent a more parsimonious and rapid solution for adaptation compared with independent and repeated de novo mutations, especially in the case of traits as complex as magnetotaxis involving tens of interacting proteins. Besides strengthening the idea about the importance of such a function in micro-oxic habitats, these results reinforce previous observations in experimental evolution suggesting that gene flow could alleviate clonal interference and speed up adaptation under some circumstances.
Published: 2020

6. Transformation Cycle of Magnetosomes in Human Stem Cells: From Degradation to Biosynthesis of Magnetic Nanoparticles Anew

Author: Christine Péchoux, Claire Wilhelm, Sandra Prévéral, Christopher T. Lefèvre, Aurore Van de Walle, David Pignol, Nicolas Menguy, Aida Serrano, Alberto Curcio, Ana Espinosa, Matière et Systèmes Complexes (MSC), Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), CSIC, Inst Ciencia Mat Madrid, Cantoblanco, E-28049 Madrid, Spain, 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), Unité de recherche génomique et physiologie de la lactation (GPL), Institut National de la Recherche Agronomique (INRA), Génétique Animale et Biologie Intégrative (GABI), AgroParisTech-Université Paris-Saclay-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Université Paris-Saclay, Institut de minéralogie, de physique des matériaux et de cosmochimie (IMPMC), Muséum national d'Histoire naturelle (MNHN)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de recherche pour le développement [IRD] : UR206-Centre National de la Recherche Scientifique (CNRS), Microbiologie Environnementale et Moléculaire (MEM), 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)), Comunidad de Madrid 2018-T1/1ND-100S, European Project: 648779,H2020,ERC-2014-CoG,MaTissE(2015), Matière et Systèmes Complexes (MSC (UMR_7057)), Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7), 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), Université Paris-Saclay-AgroParisTech-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), and Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de recherche pour le développement [IRD] : UR206-Muséum national d'Histoire naturelle (MNHN)-Centre National de la Recherche Scientifique (CNRS)
Subjects: magnetite, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, Magnetotactic bacteria, Surface Properties, Magnetism, Magnetosome, General Physics and Astronomy, Nanoparticle, 02 engineering and technology, 010402 general chemistry, 01 natural sciences, magnetosomes, biodegradation, ferrihydrite, chemistry.chemical_compound, Ferrihydrite, Humans, General Materials Science, Particle Size, Magnetite Nanoparticles, Lipid bilayer, Cells, Cultured, Magnetite, Chemistry, General Engineering, Mesenchymal Stem Cells, 021001 nanoscience & nanotechnology, equipment and supplies, 0104 chemical sciences, Biophysics, Magnetic nanoparticles, nanoparticles, biosynthesis, 0210 nano-technology
Abstract: International audience; The nanoparticles produced by magnetotactic bacteria, called magnetosomes, are made of a magnetite core with high levels of crystallinity surrounded by a lipid bilayer. This organized structure has been developed during the course of evolution of these organisms to adapt to their specific habitat and is assumed to resist degradation and to be able to withstand the demanding biological environment. Herein, we investigated magnetosomes' structural fate upon internalization in human stem cells using magnetic and photothermal measurements, electron microscopy, and X-ray absorption spectroscopy. All measurements first converge to the demonstration that intracellular magnetosomes can experience an important biodegradation, with up to 70% of their initial content degraded, which is associated with the progressive storage of the released iron in the ferritin protein. It correlates with an extensive magnetite to ferrihydrite phase transition. The ionic species delivered by this degradation could then be used by the cells to biosynthesize magnetic nanoparticles anew. In this case, cell magnetism first decreased with magnetosomes being dissolved, but then cells remagnetized entirely, evidencing the neo-synthesis of biogenic magnetic nanoparticles. Bacteria-made biogenic magnetosomes can thus be totally remodeled by human stem cells, into human cells-made magnetic nanoparticles.
Published: 2020

7. RGD-functionalized magnetosomes are efficient tumor radioenhancers for X-rays and protons

Author: Hervé Michel, David Pignol, Géraldine Adryanczyk Perrier, Joel Herault, Olivier Humbert, Juliette Thariat, Christopher T. Lefèvre, Jérôme Doyen, Béatrice Cambien, Christopher Hoog, Sandra Prévéral, Thierry Pourcher, Maha Hafsi, Transporteurs et Imagerie, Radiothérapie en Oncologie et Mécanismes biologiques des Altérations du Tissu Osseux (TIRO-MATOs - UMR E4320), UMR E4320 (TIRO-MATOs), Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Côte d'Azur (UCA)-Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Côte d'Azur (UCA)-Service Hospitalier Frédéric Joliot (SHFJ), 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-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, Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) (BIAM), 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), Centre de Lutte contre le Cancer Antoine Lacassagne [Nice] (UNICANCER/CAL), UNICANCER-Université Côte d'Azur (UCA), Institut de Chimie de Nice (ICN), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA), Métropole Nice Côte d'Azur (NCA), Centre Régional de Lutte contre le Cancer François Baclesse [Caen] (UNICANCER/CRLC), UNICANCER-Tumorothèque de Caen Basse-Normandie (TCBN)-Normandie Université (NU), Service Hospitalier Frédéric Joliot (SHFJ), Université Paris-Saclay-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)-UMR E4320 (TIRO-MATOs), Université Nice Sophia Antipolis (1965 - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Côte d'Azur (UCA)-Université Nice Sophia Antipolis (1965 - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Côte d'Azur (UCA), 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), and Normandie Université (NU)-UNICANCER-Tumorothèque de Caen Basse-Normandie (TCBN)
Subjects: Radiation-Sensitizing Agents, media_common.quotation_subject, medicine.medical_treatment, [SDV]Life Sciences [q-bio], Magnetosome, Biomedical Engineering, Mice, Nude, Pharmaceutical Science, Medicine (miscellaneous), Bioengineering, 02 engineering and technology, X-Ray Therapy, Mice, 03 medical and health sciences, Cell Line, Tumor, Proton Therapy, medicine, Animals, Humans, General Materials Science, Magnetospirillum, Internalization, Proton therapy, 030304 developmental biology, media_common, Mice, Inbred BALB C, 0303 health sciences, Chemistry, Cancer, Neoplasms, Experimental, 021001 nanoscience & nanotechnology, medicine.disease, 3. Good health, Radiation therapy, Cancer cell, Biophysics, Molecular Medicine, Magnetic nanoparticles, Nanomedicine, Female, Magnetosomes, 0210 nano-technology, Oligopeptides
Abstract: International audience; Although chemically synthesized ferro/ferrimagnetic nanoparticles have attracted great attention in cancer theranostics, they lack radio-enhancement efficacy due to low targeting and internalization ability. Herein, we investigated the potential of RGD-tagged magnetosomes, bacterial biogenic magnetic nanoparticles naturally coated with a biological membrane and genetically engineered to express an RGD peptide, as tumor radioenhancers for conventional radiotherapy and proton therapy. Although native and RGD-magnetosomes similarly enhanced radiation-induced damage to plasmid DNA, RGD-magnetoprobes were able to boost the efficacy of radiotherapy to a much larger extent than native magnetosomes both on cancer cells and in tumors. Combined to magnetosomes@RGD, proton therapy exceeded the efficacy of X-rays at equivalent doses. Also, increased secondary emissions were measured after irradiation of magnetosomes with protons versus photons. Our results indicate the therapeutic advantage of using functionalized magnetoparticles to sensitize tumors to both X-rays and protons and strengthen the case for developing biogenic magnetoparticles for multimodal nanomedicine in cancer therapy.
Published: 2020

8. Crystal structure of the transcriptional repressor DdrO: insight into the metalloprotease/repressor-controlled radiation response in Deinococcus

Author: Philippe Roche, Fabrice Confalonieri, Pascale Servant, Romaric Magerand, David Lemaire, David Pignol, François Parcy, Pascal Arnoux, Géraldine Brandelet, Raquel Martin-Arevalillo, Laurence Blanchard, Jade Doloy, Nicolas Eugénie, Arjan de Groot, Marina I. Siponen, Renaud Dumas, Microbiologie Environnementale et Moléculaire (MEM), 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), 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), Radiorésistance des bactéries et des archées (RBA), Département Biologie des Génomes (DBG), 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), Régulateurs du développement de la fleur (Flo_RE), Physiologie cellulaire et végétale (LPCV), Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), 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)-Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Interactions Protéine Métal (IPM), Institut Paoli-Calmettes, Fédération nationale des Centres de lutte contre le Cancer (FNCLCC), ANR-19-CE12-0010,NOVOREP,Réparation des dommages ou mort cellulaire chez les bactéries exposées aux stress, 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)-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), Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), ANR-19-CE12-0010,NOVOREP,Réparation des dommages ou mort cellulaire chez les bactéries exposées aux stress(2019), 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), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Saclay, Université Paris-Saclay, Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Centre National de la Recherche Scientifique (CNRS), Centre de Recherche en Cancérologie de Marseille (CRCM), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Paoli-Calmettes, Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Aix Marseille Université (AMU), MARAVAL, Alexandra, and Réparation des dommages ou mort cellulaire chez les bactéries exposées aux stress - - NOVOREP2019 - ANR-19-CE12-0010 - AAPG2019 - VALID
Subjects: Models, Molecular, DNA damage, [SDV]Life Sciences [q-bio], Repressor, [SDV.CAN]Life Sciences [q-bio]/Cancer, Cleavage (embryo), Crystallography, X-Ray, DNA-binding protein, Protein Structure, Secondary, 03 medical and health sciences, Protein structure, Structural Biology, Stress, Physiological, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Genetics, Deinococcus, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Amino Acid Sequence, Peptide sequence, 030304 developmental biology, 0303 health sciences, [SDV.MHEP] Life Sciences [q-bio]/Human health and pathology, biology, 030306 microbiology, Gene Expression Regulation, Bacterial, biology.organism_classification, Protein Structure, Tertiary, [SDV] Life Sciences [q-bio], Repressor Proteins, Biophysics, Metalloproteases, Alpha helix, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology, DNA Damage, Transcription Factors
Abstract: 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.
Published: 2019

9. Ectosymbiotic bacteria at the origin of magnetoreception in a marine protist

Author: David Vallenet, Eric Viollier, Damien Faivre, Nathalie Leonhardt, Stéphanie Fouteau, Géraldine Adryanczyk, Corinne Cruaud, Christopher T. Lefèvre, Nicolas Menguy, Purificación López-García, David Pignol, Richard J. Weld, Karim Benzerara, Valérie Barbe, Caroline L. Monteil, Magali Floriani, Microbiologie Environnementale et Moléculaire (MEM), 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), Génomique métabolique (UMR 8030), Genoscope - Centre national de séquençage [Evry] (GENOSCOPE), Université Paris-Saclay-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-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 d'Évry-Val-d'Essonne (UEVE)-Centre National de la Recherche Scientifique (CNRS), Institut de minéralogie, de physique des matériaux et de cosmochimie (IMPMC), Muséum national d'Histoire naturelle (MNHN)-Institut de recherche pour le développement [IRD] : UR206-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Laboratoire d'écotoxicologie des radionucléides (IRSN/PSE-ENV/SRTE/LECO), Service de recherche sur les transferts et les effets des radionucléides sur les écosystèmes (IRSN/PSE-ENV/SRTE), Institut de Radioprotection et de Sûreté Nucléaire (IRSN)-Institut de Radioprotection et de Sûreté Nucléaire (IRSN), Institut de Physique du Globe de Paris (IPGP), Institut national des sciences de l'Univers (INSU - CNRS)-Université Paris Diderot - Paris 7 (UPD7)-Université de La Réunion (UR)-Institut de Physique du Globe de Paris (IPG Paris)-Centre National de la Recherche Scientifique (CNRS), Plant Environmental Physiology and Stress Signaling (PEPSS), Department of Biomaterials [Potsdam], Max Planck Institute of Colloids and Interfaces, Max-Planck-Gesellschaft-Max-Planck-Gesellschaft, Ecologie Systématique et Evolution (ESE), Université Paris-Sud - Paris 11 (UP11)-AgroParisTech-Centre National de la Recherche Scientifique (CNRS), Lincoln University, New Zealand, Dumont d’Urville Science and Technology Programme (grant no. DDU-LVL1501), New Zealand Ministry for Business, Innovation and Employment (grant no. LVLX1703), ANR-16-TERC-0025,BIOMAGNET,Exploration de la biodiversité des bactéries magnétotactiques(2016), ANR-14-CE35-0018,GroMa,Greigite or magnetite: Environmental and genetic determinants controlling biomineralization in magnetotactic bacteria(2014), 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), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université d'Évry-Val-d'Essonne (UEVE), PSE-ENV/SRTE/LECO, Institut de Radioprotection et de Sûreté Nucléaire (IRSN), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut national des sciences de l'Univers (INSU - CNRS)-IPG PARIS-Université Paris Diderot - Paris 7 (UPD7)-Université de La Réunion (UR)-Centre National de la Recherche Scientifique (CNRS), Signalisation de l'Adaptation des Végétaux à l'Environnement (SAVE), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-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)-Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), and Laboratoire de Radioécologie et d'Ecotoxicologie (DEI/SECRE/LRE)
Subjects: Microbiology (medical), [SDV]Life Sciences [q-bio], Immunology, Magnetosome, Biology, medicine.disease_cause, Deltaproteobacteria, Applied Microbiology and Biotechnology, Microbiology, 03 medical and health sciences, Symbiosis, Genetics, medicine, Euglenozoa, 14. Life underwater, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, Mutualism (biology), 0303 health sciences, 030306 microbiology, Protist, Magnetoreception, Cell Biology, biology.organism_classification, Anoxic waters, Evolutionary biology
Abstract: Mutualistic symbioses are often a source of evolutionary innovation and drivers of biological diversification1. Widely distributed in the microbial world, particularly in anoxic settings2,3, they often rely on metabolic exchanges and syntrophy2,4. Here, we report a mutualistic symbiosis observed in marine anoxic sediments between excavate protists (Symbiontida, Euglenozoa)5 and ectosymbiotic Deltaproteobacteria biomineralizing ferrimagnetic nanoparticles. Light and electron microscopy observations as well as genomic data support a multi-layered mutualism based on collective magnetotactic motility with division of labour and interspecies hydrogen-transfer-based syntrophy6. The guided motility of the consortia along the geomagnetic field is allowed by the magnetic moment of the non-motile ectosymbiotic bacteria combined with the protist motor activity, which is a unique example of eukaryotic magnetoreception7 acquired by symbiosis. The nearly complete deltaproteobacterial genome assembled from a single consortium contains a full magnetosome gene set8, but shows signs of reduction, with the probable loss of flagellar genes. Based on the metabolic gene content, the ectosymbiotic bacteria are anaerobic sulfate-reducing chemolithoautotrophs that likely reduce sulfate with hydrogen produced by hydrogenosome-like organelles6 underlying the plasma membrane of the protist. In addition to being necessary hydrogen sinks, ectosymbionts may provide organics to the protist by diffusion and predation, as shown by magnetosome-containing digestive vacuoles. Phylogenetic analyses of 16S and 18S ribosomal RNA genes from magnetotactic consortia in marine sediments across the Northern and Southern hemispheres indicate a host–ectosymbiont specificity and co-evolution. This suggests a historical acquisition of magnetoreception by a euglenozoan ancestor from Deltaproteobacteria followed by subsequent diversification. It also supports the cosmopolitan nature of this type of symbiosis in marine anoxic sediments. Here, the authors identify a mutualistic symbiosis between a non-motile, magnetic deltaproteobacterium and a protist, resulting in eukaryotic magnetoreception in marine anoxic sediments.
Published: 2019

10. Tuning the redox properties of a [4Fe-4S] center to modulate the activity of Mo-bisPGD periplasmic nitrate reductase

Author: Bruno Guigliarelli, David Pignol, Kamal Zeamari, Guillaume Gerbaud, Florence Chaspoul, Pascal Arnoux, Bénédicte Burlat, Monique Sabaty, Sandrine Grosse, Vincent Fourmond, Frédéric Biaso, Bioénergétique et Ingénierie des Protéines (BIP ), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) (BIAM), 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), Microbiologie Environnementale et Moléculaire (MEM), 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)), Institut méditerranéen de biodiversité et d'écologie marine et continentale (IMBE), Avignon Université (AU)-Aix Marseille Université (AMU)-Institut de recherche pour le développement [IRD] : UMR237-Centre National de la Recherche Scientifique (CNRS), 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), and ANR-16-CE29-0010,MOLYERE,Comprendre et Contrôler la Réactivité du Cofacteur à Molybdène dans les Enzymes et les Protéines Maquettes(2016)
Subjects: Iron-Sulfur Proteins, 0301 basic medicine, Stereochemistry, [SDV]Life Sciences [q-bio], Mutation, Missense, Biophysics, Rhodobacter sphaeroides, 010402 general chemistry, Nitrate reductase, Nitrate Reductase, 01 natural sciences, Biochemistry, Redox, Electron Transport, 03 medical and health sciences, Electron transfer, Catalytic Domain, Redox titration, [CHIM]Chemical Sciences, ComputingMilieux_MISCELLANEOUS, biology, Chemistry, Active site, Cell Biology, Periplasmic space, biology.organism_classification, Electron transport chain, 0104 chemical sciences, [CHIM.THEO]Chemical Sciences/Theoretical and/or physical chemistry, 030104 developmental biology, Amino Acid Substitution, biology.protein, Periplasmic Proteins, Oxidation-Reduction
Abstract: Molybdoenzymes are ubiquitous in living organisms and catalyze, for most of them, oxidation-reduction reactions using a large range of substrates. Periplasmic nitrate reductase (NapAB) from Rhodobacter sphaeroides catalyzes the 2-electron reduction of nitrate into nitrite. Its active site is a Mo bis-(pyranopterin guanine dinucleotide), or Mo-bisPGD, found in most prokaryotic molybdoenzymes. A [4Fe-4S] cluster and two c-type hemes form an intramolecular electron transfer chain that deliver electrons to the active site. Lysine 56 is a highly conserved amino acid which connects, through hydrogen-bonds, the [4Fe-4S] center to one of the pyranopterin ligands of the Mo-cofactor. This residue was proposed to be involved in the intramolecular electron transfer, either defining an electron transfer pathway between the two redox cofactors, and/or modulating their redox properties. In this work, we investigated the role of this lysine by combining site-directed mutagenesis, activity assays, redox titrations, EPR and HYSCORE spectroscopies. Removal of a positively-charged residue at position 56 strongly decreased the redox potential of the [4Fe-4S] cluster at pH 8 by 230 mV to 400 mV in the K56H and K56M mutants, respectively, thus affecting the kinetics of electron transfer from the hemes to the [4Fe-4S] center up to 5 orders of magnitude. This effect was partly reversed at acidic pH in the K56H mutant likely due to protonation of the imidazole ring of the histidine. Overall, our study demonstrates the critical role of a charged residue from the second coordination sphere in tuning the reduction potential of the [4Fe-4S] cluster in RsNapAB and related molybdoenzymes.
Published: 2019

11. Magnetosomes: biogenic iron nanoparticles produced by environmental bacteria

Author: Sandra Prévéral, Anissa Dieudonné, David Pignol, Microbiologie Environnementale et Moléculaire (MEM), Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) (BIAM), 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)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-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)
Subjects: 0303 health sciences, Magnetotactic bacteria, biology, Bacteria, 030306 microbiology, Chemistry, Iron, [SDV]Life Sciences [q-bio], Magnetosome, Nanotechnology, General Medicine, biology.organism_classification, Applied Microbiology and Biotechnology, Cancer treatment, 03 medical and health sciences, Environmental Microbiology, Nanoparticles, Magnetosomes, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, Biotechnology, Biomineralization
Abstract: The scientific community's interest in magnetotactic bacteria has increased substantially in recent decades. These prokaryotes have the particularity of synthesizing nanomagnets, called magnetosomes. The majority of research is based on several scientific questions. Where do magnetotactic bacteria live, what are their characteristics, and why are they magnetic? What are the molecular phenomena of magnetosome biomineralization and what are the physical characteristics of magnetosomes? In addition to scientific curiosity to better understand these stunning organisms, there are biotechnological opportunities to consider. Magnetotactic bacteria, as well as magnetosomes, are used in medical applications, for example cancer treatment, or in environmental ones, for example bioremediation. In this mini-review, we investigated all the aspects mentioned above and summarized the currently available knowledge.
Published: 2019

12. Control by Metals of Staphylopine Dehydrogenase Activity during Metallophore Biosynthesis

Author: Christine Hajjar, Roberto Fanelli, Clémentine Laffont, Catherine Brutesco, Gregorio Cullia, Mathilde Tribout, Didier Nurizzo, Elise Borezée-Durant, Romé Voulhoux, David Pignol, Jérôme Lavergne, Florine Cavelier, Pascal Arnoux, Microbiologie Environnementale et Moléculaire (MEM), 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), Institut des Biomolécules Max Mousseron [Pôle Chimie Balard] (IBMM), Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), European Synchrotron Radiation Facility (ESRF), MICrobiologie de l'ALImentation au Service de la Santé (MICALIS), Institut National de la Recherche Agronomique (INRA)-AgroParisTech, Laboratoire de chimie bactérienne (LCB), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Association Vaincre la Mucoviscidose VLM, financement RFI20160501495, ANR-14-CE09-0007,ANIBAL,Un nouveau metallophore dérivé de la nicotianamine chez des bactéries pathogènes(2014), Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM)-Institut de Chimie du CNRS (INC)-Université de Montpellier (UM)-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), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-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)-Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Direction de Recherche Fondamentale (CEA) (DRF (CEA))
Subjects: Models, Molecular, 0303 health sciences, Staphylococcus aureus, pseudomonas-aeruginosa, 030306 microbiology, [SDV]Life Sciences [q-bio], Imidazoles, Molecular Conformation, crystal-structure, General Chemistry, nutritional immunity, Biochemistry, Catalysis, 03 medical and health sciences, Colloid and Surface Chemistry, iron, Metals, Heavy, transport, escherichia-coli, ions, Oxidoreductases, roles, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology
Abstract: International audience; Enzymatic regulations are central processes for the adaptation to changing environments. In the particular case of metallophore-dependent metal uptake, there is a need to quickly adjust the production of these metallophores to the metal level outside the cell, to avoid metal shortage or overload, as well as waste of metallophores. In Staphylococcus aureus, CntM catalyzes the last biosynthetic step in the production of staphylopine, a broad-spectrum metallophore, through the reductive condensation of a pathway intermediate (xNA) with pyruvate. Here, we describe the chemical synthesis of this intermediate, which was instrumental in the structural and functional characterization of CntM and confirmed its opine synthase properties. The three-dimensional structure of CntM was obtained in an "open" form, in the apo state or as a complex with substrate or product. The xNA substrate appears mainly stabilized by its imidazole ring through a pi-pi interaction with the side chain of Tyr240. Intriguingly, we found that metals exerted various and sometime antagonistic effects on the reaction catalyzed by CntM: zinc and copper are moderate activators at low concentration and then total inhibitors at higher concentration, whereas manganese is only an activator and cobalt and nickel are only inhibitors. We propose a model in which the relative affinity of a metal toward xNA and an inhibitory binding site on the enzyme controls activation, inhibition, or both as a function of metal concentration. This metal-dependent regulation of a metallophore-producing enzyme might also take place in vivo, which could contribute to the adjustment of metallophore production to the internal metal level.
Published: 2019

13. $Rhodobacter\ sphaeroides$ methionine sulfoxide reductase P reduces $R$ ‑ and $S$ ‑diastereomers of methionine sulfoxide from a broad‑spectrum of protein substrates

Author: Sandrine Grosse, Guylaine Miotello, Pascal Arnoux, David Lemaire, Lionel Tarrago, Marina I. Siponen, Jean Armengaud, David Pignol, Béatrice Alonso, Monique Sabaty, 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), Microbiologie Environnementale et Moléculaire (MEM), 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)), Interactions Protéine Métal (IPM), Laboratoire Innovations technologiques pour la Détection et le Diagnostic (LI2D), Service de Pharmacologie et Immunoanalyse (SPI), Médicaments et Technologies pour la Santé (MTS), Université Paris-Saclay-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)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Paris-Saclay-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)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Médicaments et Technologies pour la Santé (MTS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Commissariat a l'Energie Atomique et aux Energies Alternatives (CEA) [ANR 16-CE11-0012], ANR-16-CE11-0012,METOXIC,Enzyme de réparation des protéines oxydées: un nouveau système de contrôle qualité dans le périplasme(2016), 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: 0301 basic medicine, proteomics 15, [SDV]Life Sciences [q-bio], Protein oxidation, Biochemistry, 03 medical and health sciences, chemistry.chemical_compound, Rhodobacter sphaeroides, protein oxidation, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Molecular Biology, periplasm, Methionine, biology, Methionine sulfoxide, microbiology, Enzyme kinetics, molybdoenzyme, Cell Biology, Periplasmic space, biology.organism_classification, oxidative 14 stress, 030104 developmental biology, chemistry, methionine sulfoxide reductase, Methionine sulfoxide reductase, Photosynthetic bacteria, MSRA
Abstract: International audience; Methionine (Met) is prone to oxidation and can be converted to Met sulfoxide (MetO), which exists as $R$- and $S$-diastereomers. MetO can be reduced back to Met by the ubiquitous methionine sulfoxide reductase (Msr) enzymes. Canonical MsrA and MsrB were shown to be absolutely stereospecific for the reduction of S ‑ and R‑ diastereomer, respectively. Recently, a new enzymatic system, MsrQ/MsrP which is conserved in all gram‑negative bacteria, was identified as a key actor in the reduction of oxidized periplasmic proteins. The haem‑binding membrane protein MsrQ transmits reducing power from the electron transport chains to the molybdoenzyme MsrP, which acts as a protein‑MetO reductase. The MsrQ/MsrP function was well established genetically, but the identity and biochemical properties of MsrP substrates remain unknown. In this work, using the purified MsrP enzyme from the photosynthetic bacteria $Rhodobacter\ sphaeroides$ as a model, we show that it can reduce a broad spectrum of protein substrates. The most efficiently reduced MetO are found in clusters of amino acid sequences devoid of threonine and proline on the C‑terminal side. Moreover, $R.\ sphaeroides$ MsrP lacks stereospecificity as it can reduce both R‑ and S‑ diastereomers of MetO, similarly to its $Escherichia\ coli$ homolog, and preferentially acts on unfolded oxidized proteins. Overall, these results provide important insights into the function of a bacterial envelop protecting system, which should help understand how bacteria cope in harmful environments.
Published: 2018

14. Genomic study of a novel magnetotactic Alphaproteobacteria uncovers the multiple ancestry of magnetotaxis

Author: Béatrice Alonso, Christopher T. Lefèvre, Nicolas Menguy, Caroline L. Monteil, Nicolas Ginet, Stéphane Cruveiller, Guy Perrière, David Pignol, Nicolas Waisbord, 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), Microbiologie Environnementale et Moléculaire (MEM), 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)), Bioinformatique, phylogénie et génomique évolutive (BPGE), Département PEGASE [LBBE] (PEGASE), Laboratoire de Biométrie et Biologie Evolutive - UMR 5558 (LBBE), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de Recherche en Informatique et en Automatique (Inria)-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de Recherche en Informatique et en Automatique (Inria)-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS)-Laboratoire de Biométrie et Biologie Evolutive - UMR 5558 (LBBE), Université de Lyon-Université de Lyon-Institut National de Recherche en Informatique et en Automatique (Inria)-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS), Institut de minéralogie, de physique des matériaux et de cosmochimie (IMPMC), Muséum national d'Histoire naturelle (MNHN)-Institut de recherche pour le développement [IRD] : UR206-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Tufts University [Medford], Genoscope - Centre national de séquençage [Evry] (GENOSCOPE), Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Génomique métabolique (UMR 8030), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-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 d'Évry-Val-d'Essonne (UEVE)-Centre National de la Recherche Scientifique (CNRS), ANR-14-CE35-0018,GroMa,Greigite or magnetite: Environmental and genetic determinants controlling biomineralization in magnetotactic bacteria(2014), 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), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université d'Évry-Val-d'Essonne (UEVE)
Subjects: 0301 basic medicine, Gene Transfer, Horizontal, Magnetotactic bacteria, [SDV]Life Sciences [q-bio], 030106 microbiology, Magnetosome, Microbiology, Evolution, Molecular, Magnetics, 03 medical and health sciences, Mediterranean Sea, Magnetotaxis, 14. Life underwater, Magnetospirillum, Ecology, Evolution, Behavior and Systematics, Alphaproteobacteria, Comparative genomics, biology, Phylum, biology.organism_classification, Evolutionary biology, France, Magnetosomes, Proteobacteria, Water Microbiology, Genome, Bacterial
Abstract: Ecological and evolutionary processes involved in magnetotactic bacteria (MTB) adaptation to their environment have been a matter of debate for many years. Ongoing efforts for their characterization are progressively contributing to understand these processes, including the genetic and molecular mechanisms responsible for biomineralization. Despite numerous culture-independent MTB characterizations, essentially within the Proteobacteria phylum, only few species have been isolated in culture because of their complex growth conditions. Here, we report a newly cultivated magnetotactic, microaerophilic and chemoorganoheterotrophic bacterium isolated from the Mediterranean Sea in Marseille, France: Candidatus Terasakiella magnetica strain PR-1 that belongs to an Alphaproteobacteria genus with no magnetotactic relative. By comparing the morphology and the whole genome shotgun sequence of this MTB with those of closer relatives, we brought further evidence that the apparent vertical ancestry of magnetosome genes suggested by previous studies within Alphaproteobacteria hides a more complex evolutionary history involving horizontal gene transfers and/or duplication events before and after the emergence of Magnetospirillum, Magnetovibrio and Magnetospira genera. A genome-scale comparative genomics analysis identified several additional candidate functions and genes that could be specifically associated to MTB lifestyle in this class of bacteria.
Published: 2018

15. Targeted thermal therapy with genetically engineered magnetite magnetosomes@RGD: Photothermia is far more efficient than magnetic hyperthermia

Author: Anouchka Plan Sangnier, Yoann Lalatonne, Sandra Prévéral, Chistopher T. Lefèvre, David Pignol, Alberto Curcio, Claire Wilhelm, Amanda K. A. Silva, CNRS UMR 7057 - Laboratoire Matières et Systèmes Complexes (MSC) (MSC), Centre National de la Recherche Scientifique (CNRS), Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) (BIAM), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-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), Microbiologie Environnementale et Moléculaire (MEM), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Matière et Systèmes Complexes (MSC (UMR_7057)), Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Recherche Vasculaire Translationnelle (LVTS (UMR_S_1148 / U1148)), Université Paris 13 (UP13)-Université Paris Diderot - Paris 7 (UPD7)-Institut National de la Santé et de la Recherche Médicale (INSERM), Matière et Systèmes Complexes (MSC), 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)), 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), and Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7)
Subjects: Male, Hot Temperature, Magnetotactic bacteria, [SDV]Life Sciences [q-bio], Magnetosome, Pharmaceutical Science, Mice, Nude, Uterine Cervical Neoplasms, Antineoplastic Agents, 02 engineering and technology, 010402 general chemistry, 01 natural sciences, HeLa, Mice, Animals, Humans, Magnetite Nanoparticles, ComputingMilieux_MISCELLANEOUS, biology, Chemistry, Prostatic Neoplasms, 021001 nanoscience & nanotechnology, biology.organism_classification, 0104 chemical sciences, 3. Good health, Magnetic hyperthermia, Tumor progression, Heat generation, Cancer cell, PC-3 Cells, Biophysics, Magnetic nanoparticles, Female, Magnetosomes, 0210 nano-technology, Genetic Engineering, Oligopeptides, HeLa Cells
Abstract: Providing appropriate means for heat generation by low intratumoral nanoparticle concentrations is a major challenge for cancer nanotherapy. Here we propose RGD-tagged magnetosomes (magnetosomes@RGD) as a biogenic, genetically engineered, inorganic platform for multivalent thermal cancer treatment. Magnetosomes@RGD are biomagnetite nanoparticles synthesized by genetically modified magnetotactic bacteria thanks to a translational fusion of the RGD peptide with the magnetosomal protein MamC. Magnetosomes@RGD thus combine the high crystallinity of their magnetite core with efficient surface functionalization. The specific affinity of RGD was first quantified by single-cell magnetophoresis with a variety of cell types, including immune, muscle, endothelial, stem and cancer cells. The highest affinity and cellular uptake was observed with PC3 prostatic and HeLa uterine cancer cells. The efficiency of photothermia and magnetic hyperthermia was then compared on PC3 cells. Unexpectedly, photothermia was far more efficient than magnetic hyperthermia, which was almost totally inhibited by the cellular environment. RGD targeting was then assessed in vivo at tumor site, in mice bearing PC3 tumors. As a result, we demonstrate that targeted magnetic nanoparticles could generate heat on a therapeutic level after systemic administration, but only under laser excitation, and successfully inhibit tumor progression.
Published: 2018

16. Accumulation and Dissolution of Magnetite Crystals in a Magnetically Responsive Ciliate

Author: David Pignol, Caroline L. Monteil, Christopher T. Lefèvre, Nicolas Menguy, Sandra Prévéral, Alan Warren, 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), Microbiologie Environnementale et Moléculaire (MEM), 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)), Institut de minéralogie, de physique des matériaux et de cosmochimie (IMPMC), Muséum national d'Histoire naturelle (MNHN)-Institut de recherche pour le développement [IRD] : UR206-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), National History Museum of London, ANR-14-CE35-0018,GroMa,Greigite or magnetite: Environmental and genetic determinants controlling biomineralization in magnetotactic bacteria(2014), ANR-16-TERC-0025,BIOMAGNET,Exploration de la biodiversité des bactéries magnétotactiques(2016), 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: 0301 basic medicine, iron cycling, Food Chain, magnetite, Magnetotactic bacteria, Microorganism, [SDV]Life Sciences [q-bio], Magnetosome, Applied Microbiology and Biotechnology, 03 medical and health sciences, Environmental Microbiology, grazing, 14. Life underwater, Ciliate, Ecology, biology, Bacteria, Chemistry, Aquatic ecosystem, magnetotactic, 15. Life on land, equipment and supplies, biology.organism_classification, biomineralization, Ferrosoferric Oxide, 030104 developmental biology, Solubility, 13. Climate action, Environmental chemistry, Oligohymenophorea, Protozoa, France, Magnetosomes, Food Science, Biotechnology, Biomineralization
Abstract: Magnetotactic bacteria (MTB) represent a group of microorganisms that are widespread in aquatic habitats and thrive at oxic-anoxic interfaces. They are able to scavenge high concentrations of iron thanks to the biomineralization of magnetic crystals in their unique organelles, the so-called magnetosome chains. Although their biodiversity has been intensively studied, their ecology and impact on iron cycling remain largely unexplored. Predation by protozoa was suggested as one of the ecological processes that could be involved in the release of iron back into the ecosystem. Magnetic protozoa were previously observed in aquatic environments, but their diversity and the fate of particulate iron during grazing are poorly documented. In this study, we report the morphological and molecular characterizations of a magnetically responsive MTB-grazing protozoan able to ingest high quantities of MTB. This protozoan is tentatively identified as Uronema marinum , a ciliate known to be a predator of bacteria. Using light and electron microscopy, we investigated in detail the vacuoles in which the lysis of phagocytized prokaryotes occurs. We carried out high-resolution observations of aligned magnetosome chains and ongoing dissolution of crystals. Particulate iron in the ciliate represented approximately 0.01% of its total volume. We show the ubiquity of this interaction in other types of environments and describe different grazing strategies. These data contribute to the mounting evidence that the interactions between MTB and protozoa might play a significant role in iron turnover in microaerophilic habitats. IMPORTANCE Identifying participants of each biogeochemical cycle is a prerequisite to our understanding of ecosystem functioning. Magnetotactic bacteria (MTB) participate in iron cycling by concentrating large amounts of biomineralized iron minerals in their cells, which impacts their chemical environment at, or below, the oxic-anoxic transition zone in aquatic habitats. It was shown that some protozoa inhabiting this niche could become magnetic by the ingestion of magnetic crystals biomineralized by grazed MTB. In this study, we show that magnetic MTB grazers are commonly observed in marine and freshwater sediments and can sometimes accumulate very large amounts of particulate iron. We describe here different phagocytosis strategies, determined using magnetic particles from MTB as tracers after their ingestion by the protozoa. This study paves the way for potential scientific or medical applications using MTB grazers as magnetosome hyperaccumulators.
Published: 2018

17. The Rhodobacter sphaeroides methionine sulfoxide reductase MsrP can reduce R- and S-diastereomers of methionine sulfoxide from a broad-spectrum of protein substrates

Author: David Lemaire, Monique Sabaty, Jean Armengaud, David Pignol, Lionel Tarrago, Pascal Arnoux, Sandrine Grosse, Guylaine Miotello, Marina I. Siponen, and Béatrice Alonso
Subjects: Methionine, biology, Chemistry, Methionine sulfoxide, Sulfoxide, Periplasmic space, medicine.disease_cause, biology.organism_classification, chemistry.chemical_compound, Rhodobacter sphaeroides, Biochemistry, medicine, Methionine sulfoxide reductase, Escherichia coli, MSRA
Abstract: SummaryMethionine (Met) is prone to oxidation and can be converted to Met sulfoxide (MetO), which exists as R- and S-diastereomers. MetO can be reduced back to Met by the ubiquitous methionine sulfoxide reductase (Msr) enzymes. Canonical MsrA and MsrB were shown as absolutely stereospecific for the reduction of S- and R-diastereomer, respectively. Recently, the molybdenum-containing protein MsrP, conserved in all gram-negative bacteria, was shown to be able to reduce MetO of periplasmic proteins without apparent stereospecificity inEscherichia coli.Here, we describe the substrate specificity of theRhodobacter sphaeroidesMsrP. Proteomics analysis coupled to enzymology approaches indicate that it reduces a broad spectrum of periplasmic oxidized proteins. Moreover, using model proteins, we demonstrated that RsMsrP preferentially reduces unfolded oxidized proteins and we confirmed that this enzyme, like itsE. colihomolog, can reduce bothR-andS-diastereomers of MetO with similar efficiency.
Published: 2018
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18. An algal photoenzyme converts fatty acids to hydrocarbons

Author: Bertrand Légeret, Pavel Müller, Yonghua Li-Beisson, Yohann Couté, David Pignol, Fred Beisson, Gilles Peltier, Damien Sorigué, Emmanuelle Billon, Stéphanie Blangy, Didier Nurizzo, Pascal Arnoux, Pierre Richaud, Solène L. Y. Moulin, Klaus Brettel, Stéphan Cuiné, Sabine Brugière, 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), Synthèse et étude de systèmes à intêret biologique (SEESIB), Université Blaise Pascal - Clermont-Ferrand 2 (UBP)-Centre National de la Recherche Scientifique (CNRS), Troubles cognitifs dégénératifs et vasculaires (DN2M), Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-INSERM, Bioénergie et Microalgues (EBM), 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)), Etude de la dynamique des protéomes (EDyP), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), 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)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA), European Synchrotron Radiation Facility (ESRF), Institut de Biologie Intégrative de la Cellule (I2BC), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Saclay, Microbiologie Environnementale et Moléculaire (MEM), 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), Etude de la dynamique des protéomes (EDyP ), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), ANR-13-JSV5-0005,MUsCA,Ingénierie métabolique d'une microalgue verte en vue de la production d'alcanes à chaine moyenne(2013), ANR-11-IDEX-0001,Amidex,INITIATIVE D'EXCELLENCE AIX MARSEILLE UNIVERSITE(2011), ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), ANR-10-INBS-0008,ProFI,Infrastructure Française de Protéomique(2010), 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 ), Laboratoire de Biologie à Grande Échelle ( BGE - UMR S1038 ), Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Grenoble Alpes [Saint Martin d'Hères]-Institut National de la Santé et de la Recherche Médicale ( INSERM ), European Synchrotron Radiation Facility ( ESRF ), 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 ), 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 ), and Environnement, Bioénergie, Microalgues et Plantes (EBMP)
Subjects: 0301 basic medicine, Photosynthetic reaction centre, Light, Carboxy-Lyases, Decarboxylation, [SDV]Life Sciences [q-bio], Chlorella, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Alkenes, 010402 general chemistry, 01 natural sciences, Cofactor, 03 medical and health sciences, chemistry.chemical_compound, Oxidoreductase, Alkanes, Phylogeny, ComputingMilieux_MISCELLANEOUS, Plant Proteins, chemistry.chemical_classification, Flavin adenine dinucleotide, Multidisciplinary, biology, [ SDV.BC ] Life Sciences [q-bio]/Cellular Biology, Fatty Acids, Fatty acid, Lipid metabolism, Lipid Metabolism, Photochemical Processes, 0104 chemical sciences, 030104 developmental biology, Enzyme, chemistry, Biochemistry, Biocatalysis, Flavin-Adenine Dinucleotide, biology.protein, Oxidoreductases
Abstract: Algal enzyme driven by blue light Microalgae make hydrocarbons. In searching for the enzyme responsible, Sorigué et al. found a glucose-methanolcholine oxidoreductase (see the Perspective by Scrutton). Expression of the enzyme in Escherichia coli showed that hydrocarbon production requires visible light. In fact, the enzyme requires a constant input of blue photons to carry out its catalytic reaction. A long hydrophobic tunnel in the enzyme stabilizes the fatty acid substrates in proximity to the flavin adenine dinucleotide cofactor. Science , this issue p. 903 ; see also p. 872
Published: 2017

19. Pseudopaline, a staphylopine-like metallophore involved in zinc and nickel uptake in Pseudomonas aeruginosa

Author: Ryszard Lobinski, Ahmed Liratni, Ghassan Ghssein, Nicolas Gomez, Pierre Richaud, Shuanglong Wang, Elise Borezée-Durant, Geneviève Ball, Catherine Brutesco, Christine Hajjar, Romé Voulhoux, Sophie Bleves, David Pignol, Laurent Ouerdane, Pascal Arnoux, and Sébastien Lhospice
Subjects: 0303 health sciences, 030306 microbiology, Operon, Repressor, chemistry.chemical_element, Zinc, Biology, 03 medical and health sciences, chemistry.chemical_compound, Alpha ketoglutarate, chemistry, Biosynthesis, Biochemistry, Moiety, Nicotianamine, Histidine, 030304 developmental biology
Abstract: Metal uptake is vital for all living organisms. In metal scarce conditions, a common bacterial strategy consists in the biosynthesis of metallophores, their export in the extracellular medium and the recovery of a metal-metallophore complex through dedicated membrane transporters. Staphylopine is a recently described metallophore distantly related to plant nicotianamine that contributes to the broad-spectrum metal uptake capabilities of Staphylococcus aureus. Here, we characterize a four genes operon (PA4837–PA4834) in Pseudomonas aeruginosa involved in the biosynthesis and trafficking of a staphylopine-like metallophore named pseudopaline. Pseudopaline differs from staphylopine with regard to the stereochemistry of its histidine moiety associated to an alpha ketoglutarate moiety instead of pyruvate. In vivo, the pseudopaline operon is regulated by zinc through the Zur repressor. The metal-uptake property of the pseudopaline system appears different from that of staphylopine with a predominant effect on nickel uptake, and on zinc uptake in metal scarce conditions mimicking a chelating environment, thus reconciling the regulation of the cnt operon by zinc with its function as a zinc importer under metal scarce conditions.AUTHOR SUMMARYZinc is an essential micronutrients for bacteria, particularly important at the host-pathogen interface where the host tends to sequester metals in a so called nutritional immunity framework, and the pathogenic bacterium increases its metal uptake efforts in order to keep up with its metal requirements. Here we reveal a novel metallophore, named pseudopaline, which is synthesized and exported by Pseudomonas aeruginosa and serves for the uptake of nickel in metal poor media, and for the uptake of zinc in metal scarce conditions that mimic the chelating environment that presumably prevails within a host.
Published: 2017
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20. Desulfamplus magnetovallimortis gen. nov., sp nov., a magnetotactic bacterium from a brackish desert spring able to biomineralize greigite and magnetite, that represents a novel lineage in the Desulfobacteraceae

Author: Caroline L. Monteil, Christopher T. Lefèvre, David Pignol, Nicolas Menguy, Nicolas Ginet, Elodie C. T. Descamps, Dennis A. Bazylinski, 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), Microbiologie Environnementale et Moléculaire (MEM), 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)), Institut de minéralogie, de physique des matériaux et de cosmochimie (IMPMC), Muséum national d'Histoire naturelle (MNHN)-Institut de recherche pour le développement [IRD] : UR206-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de chimie bactérienne (LCB), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), University of Nevada [Reno], U.S. National Science Foundation grant EAR-1423939, ANR-14-CE35-0018,GroMa,Greigite or magnetite: Environmental and genetic determinants controlling biomineralization in magnetotactic bacteria(2014), ANR-12-NANO-0013,MEFISTO,Nano-aimants biologiques fonctionnalisés pour la théranostique(2012), 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: 0301 basic medicine, Greigite, DNA, Bacterial, Deltaproteobacteria, Magnetotactic bacteria, Iron, Magnetosome, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Sulfides, Applied Microbiology and Biotechnology, Microbiology, California, 03 medical and health sciences, Fumarates, Botany, Magnetotaxis, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Ecology, Evolution, Behavior and Systematics, Base Composition, [SDV.GEN]Life Sciences [q-bio]/Genetics, biology, Strain (chemistry), Obligate anaerobe, Sequence Analysis, DNA, biology.organism_classification, Ferrosoferric Oxide, Bacterial Typing Techniques, 030104 developmental biology, [SDE]Environmental Sciences, Desulfobacteraceae, Magnetosomes, Genome, Bacterial
Abstract: International audience; A magnetotactic bacterium, designated strain BW-1(T), was isolated from a brackish spring in Death Valley National Park (California, USA) and cultivated in axenic culture. The Gram-negative cells of strain BW-1(T) are relatively large and rod-shaped and possess a single polar flagellum (monotrichous). This strain is the first magnetotactic bacterium isolated in axenic culture capable of producing greigite and/or magnetite nanocrystals aligned in one or more chains per cell. Strain BW-1(T) is an obligate anaerobe that grows chemoorganoheterotrophically while reducing sulfate as a terminal electron acceptor. Optimal growth occurred at pH 7.0 and 28 degrees C with fumarate as electron donor and carbon source. Based on its genome sequence, the G + C content is 40.72 mol %. Phylogenomic and phylogenetic analyses indicate that strain BW-1(T) belongs to the Desulfobacteraceae family within the Deltaproteobacteria class. Based on average amino acid identity, strain BW-1(T) can be considered as a novel species of a new genus, for which the name Desulfamplus magnetovallimortis is proposed. The type strain of D. magnetovallimortis is BW-1(T) (JCM 18010(T)-DSM 103535(T)). (C) 2017 Elsevier GmbH. All rights reserved.
Published: 2017

21. Genetically tailored magnetosomes used as MRI probe for molecular imaging of brain tumor

Author: Sandra Prévéral, Erwan Selingue, Sébastien Mériaux, Marianne Boucher, David Pignol, Christopher T. Lefèvre, Michel Pean, Géraldine Adryanczyk-Perrier, Françoise Geffroy, Nicolas Ginet, L. Bellanger, HYBIS, Laboratoire d'étude des transferts en hydrologie et environnement (LTHE), Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique de Grenoble (INPG)-Institut national des sciences de l'Univers (INSU - CNRS)-Université Joseph Fourier - Grenoble 1 (UJF)-Observatoire des Sciences de l'Univers de Grenoble (OSUG), Université Savoie Mont Blanc (USMB [Université de Savoie] [Université de Chambéry])-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP)-Institut national de recherche en sciences et technologies pour l'environnement et l'agriculture (IRSTEA)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut national des sciences de l'Univers (INSU - CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Université Savoie Mont Blanc (USMB [Université de Savoie] [Université de Chambéry])-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP)-Institut national de recherche en sciences et technologies pour l'environnement et l'agriculture (IRSTEA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique de Grenoble (INPG)-Institut national des sciences de l'Univers (INSU - CNRS)-Université Joseph Fourier - Grenoble 1 (UJF)-Observatoire des Sciences de l'Univers de Grenoble (OSUG), Université Savoie Mont Blanc (USMB [Université de Savoie] [Université de Chambéry])-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP)-Institut national de recherche en sciences et technologies pour l'environnement et l'agriculture (IRSTEA)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut national des sciences de l'Univers (INSU - CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Université Savoie Mont Blanc (USMB [Université de Savoie] [Université de Chambéry])-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP)-Institut national de recherche en sciences et technologies pour l'environnement et l'agriculture (IRSTEA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Department of Applied Geophysics, Technische Universität Berlin (TUB)-Technische Universität Berlin (TUB), Unité d'imagerie par résonance magnétique à très haut champ et de spectroscopie (UNIRS), Service NEUROSPIN (NEUROSPIN), 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-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, Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) (BIAM), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-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), Microbiologie Environnementale et Moléculaire (MEM), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Service de Pharmacologie et Immunoanalyse (SPI), Médicaments et Technologies pour la Santé (MTS), Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Laboratoire de chimie bactérienne (LCB), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Observatoire des Sciences de l'Univers de Grenoble (OSUG ), Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-Institut national des sciences de l'Univers (INSU - CNRS)-Institut national de recherche en sciences et technologies pour l'environnement et l'agriculture (IRSTEA)-Université Savoie Mont Blanc (USMB [Université de Savoie] [Université de Chambéry])-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), 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), 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)), Service de Pharmacologie et d'Immunoanalyse (SPI), Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay, 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)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Paris-Saclay-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)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)
Subjects: 0301 basic medicine, Materials science, media_common.quotation_subject, Magnetosome, Biophysics, Brain tumor, Contrast Media, Molecular Probe Techniques, Bioengineering, Single step, Nanoconjugates, 02 engineering and technology, Article, Biomaterials, Mice, 03 medical and health sciences, Nuclear magnetic resonance, Cell Line, Tumor, Biomarkers, Tumor, medicine, Animals, Tissue Distribution, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Internalization, media_common, medicine.diagnostic_test, Brain Neoplasms, RGD peptide, Magnetic resonance imaging, 021001 nanoscience & nanotechnology, medicine.disease, Magnetic Resonance Imaging, Molecular Imaging, Genetic Enhancement, 030104 developmental biology, Mechanics of Materials, Molecular Probes, Ceramics and Composites, Magnetosomes, Lipid vesicle, Molecular imaging, 0210 nano-technology, Oligopeptides
Abstract: International audience; We investigate here the potential of single step production of genetically engineeredmagnetosomes, bacterial biogenic iron-oxide nanoparticles embedded in a lipid vesicle, as a new tailorable magnetic resonance molecular imaging probe. We demonstrate in vitro the specific binding and the significant internalization into U87 cells of magnetosomes decorated with RGD peptide. After injection at the tail vein of glioblastoma-bearing mice, we evidence in the first 2 h the rapid accumulation of both unlabeled and functionalized magnetosomes inside the tumor by Enhanced Permeability and Retention effects. 24 h after the injection, a specific enhancement of the tumor contrast is observed on MR images only for RGD-labeled magnetosomes. Post mortem acquisition of histological data confirms MRI results with more magnetosomes found into the tumor treated with functionalized magnetosomes. This work establishes the first proof-of-concept of a successful bio-integrated production of molecular imaging probe for MRI.
Published: 2017

22. Toward inline multiplex biodetection of metals, bacteria, and toxins in water networks: the COMBITOX project

Author: Pierre Cholat, Agnès Rodrigue, Mireille Ansaldi, David Pignol, Ingrid Bazin, Laboratoire de chimie bactérienne (LCB), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Institut de Microbiologie de la Méditerranée (IMM), Laboratoire de Génie de l'Environnement Industriel (LGEI), IMT - MINES ALES (IMT - MINES ALES), Institut Mines-Télécom [Paris] (IMT)-Institut Mines-Télécom [Paris] (IMT), Trafic et signalisation membranaires chez les bactéries (MTSB), Microbiologie, adaptation et pathogénie (MAP), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-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), Microbiologie Environnementale et Moléculaire (MEM), ANR-11-ECOT-0009,COMBITOX,COnception d'un instrument pour la Mesure BIologique multiparamètrique en continu de TOXiques(2011), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université de Lyon-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon, 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: Engineering, Health, Toxicology and Mutagenesis, 02 engineering and technology, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, 010501 environmental sciences, Bioinformatics, 01 natural sciences, Human health, Time response, Environmental Chemistry, Multiplex, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, ComputingMilieux_MISCELLANEOUS, 0105 earth and related environmental sciences, Light detector, [SDV.GEN]Life Sciences [q-bio]/Genetics, Physical conditioning, business.industry, General Medicine, 021001 nanoscience & nanotechnology, Pollution, 6. Clean water, 13. Climate action, [SDE]Environmental Sciences, Biochemical engineering, 0210 nano-technology, business, Biosensor, Conceptual level, Field conditions
Abstract: This special issue of Environmental Science and Pollution Research highlights selected papers whose results have been obtained in the course of the COMBITOX project. COMBITIOX is an interdisciplinary research project funded by the French National Research Agency (ANR) aiming at conceiving an inline multiparametric device for the surveillance of water networks using biosensors. This device is not intended to fully replace chemical methods, but when compared to analytical chromatographic methodologies, biological sensors can offer rapid and on-site monitoring of even trace levels of targeted compounds (Sun et al. 2015) and can quickly raise the alarm in the event of an accidental or intentional pollution. Numerous developments have been published to improve the sensitivity, specificity, and time response of various biosensors in laboratory conditions (Xiong et al. 2012) (der Meer et al. 2010), but their actual transfer into technological devices for the surveillance of water networks remains at a conceptual level. Thus, the challenge here is to go a step beyond and validate biosensors under real-life field conditions by incorporating them in a single inline detector. During the course of COMBITOX, we could define the interface between the biosensors and a common light detector as well as the physical conditioning of the bioreagents and usage protocol. Our resulting prototype allow the detection of bioavailable toxic compounds as well as microorganisms, impacting human health through the drinking water network or interfering with the biological process of modern wastewater treatment plants. We also plan to propose this system to meet the emerging threats such as bioterrorism. COMBITOX focuses on three families of Bobjects^ to detect: metals (cadmium, mercury, arsenic, nickel, etc.), environmental and/or food toxins, and pathogenic microorganisms. Whole-cell biosensors based on reporter gene under the control of an inducible promoter are used to detect various metals (Hynninen and Virta 2010), the antibody/antigen interaction for toxins (Makaraviciute and Ramanaviciene 2013), and the specific infection of bacteria by bacteriophages for pathogenic microorganisms (Smartt et al. 2012) (Vinay et al. 2015). In all cases, the signal measured is photochemical (fluorescence, bioluminescence, or chemo-luminescence): such a method to transduce the biological recognition is very sensitive and a single photodetector can be used for all biosensors included in the device. The challenge here rather lies in the design and the optimization of the different biological compounds for their use in the field while maintaining a high sensibility and robustness. As a consequence, the different articles presented in this special issue focus on original strategies for the optimization and the adaptation of the three types of biosensors for their use in a semi-autonomous inline water analyzer. In the case of whole-cell biosensors, improvement of the dose-responses Responsible editor: Philippe Garrigues
Published: 2017

23. Semi-autonomous inline water analyzer: design of a common light detector for bacterial, phage, and immunological biosensors

Author: Nicolas Ginet, Mireille Ansaldi, Agnès Rodrigue, Sandra Prévéral, Damien Meunier, Bertrand Miclot, Camille Escoffier, Jean-Raphaël Fantino, Nathalie Franche, Philippe Larosa, Ingrid Bazin, Elodie C. T. Descamps, David Pignol, Pierre Cholat, Catherine Brutesco, Daniel Garcia, 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), Microbiologie Environnementale et Moléculaire (MEM), 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)), Laboratoire de chimie bactérienne (LCB), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Institut de Microbiologie de la Méditerranée (IMM), Laboratoire de Génie de l'Environnement Industriel (LGEI), IMT - MINES ALES (IMT - MINES ALES), Institut Mines-Télécom [Paris] (IMT)-Institut Mines-Télécom [Paris] (IMT), Trafic et signalisation membranaires chez les bactéries (MTSB), Microbiologie, adaptation et pathogénie (MAP), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-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), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université de Lyon-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées de Lyon (INSA Lyon), and Université de Lyon
Subjects: 0301 basic medicine, Spectrum analyzer, Engineering, Light, Health, Toxicology and Mutagenesis, Environmental pollution, Nanotechnology, Biosensing Techniques, Technology readiness level, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, 010501 environmental sciences, 01 natural sciences, 03 medical and health sciences, Water Quality, Environmental Chemistry, Bacteriophages, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Reaction chamber, 0105 earth and related environmental sciences, Light detector, [SDV.GEN]Life Sciences [q-bio]/Genetics, Bacteria, business.industry, Water, General Medicine, Pollution, 030104 developmental biology, [SDE]Environmental Sciences, Water Microbiology, business, Biosensor, Food Analysis, Computer hardware, Environmental Monitoring
Abstract: International audience; The use of biosensors as sensitive and rapid alert systems is a promising perspective to monitor accidental or intentional environmental pollution, but their implementation in the field is limited by the lack of adapted inline water monitoring devices. We describe here the design and initial qualification of an analyzer prototype able to accommodate three types of biosensors based on entirely different methodologies (immunological, whole-cell, and bacteriophage biosensors), but whose responses rely on the emission of light. We developed a custom light detector and a reaction chamber compatible with the specificities of the three systems and resulting in statutory detection limits. The water analyzer prototype resulting from the COMBITOX project can be situated at level 4 on the Technology Readiness Level (TRL) scale and this technical advance paves the way to the use of biosensors on-site.
Published: 2017

24. Bacterial host and reporter gene optimization for genetically encoded whole cell biosensors

Author: Nicolas Ginet, Elodie C. T. Descamps, Géraldine Adryanczyk-Perrier, Louis Dumas, Agnès Rodrigue, David Pignol, Arjan de Groot, Julien Cayron, Elsa Prudent, Sandra Prévéral, Camille Escoffier, Manon Ricquebourg, Catherine Brutesco, Daniel Garcia, Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) (BIAM), 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), Microbiologie Environnementale et Moléculaire (MEM), 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)), Microbiologie, adaptation et pathogénie (MAP), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), Bioénergie et Microalgues (EBM), Trafic et signalisation membranaires chez les bactéries (MTSB), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), ANR-11-ECOT-0009,COMBITOX,COnception d'un instrument pour la Mesure BIologique multiparamètrique en continu de TOXiques(2011), 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 ), Ecologie microbienne ( EM ), Centre National de la Recherche Scientifique ( CNRS ) -Ecole Nationale Vétérinaire de Lyon ( ENVL ) -Université Claude Bernard Lyon 1 ( UCBL ), Université de Lyon-Université de Lyon-Institut National de la Recherche Agronomique ( INRA ) -VetAgro Sup ( VAS ), Centre Sciences des Processus Industriels et Naturels ( SPIN-ENSMSE ), École des Mines de Saint-Étienne ( Mines Saint-Étienne MSE ), Institut Mines-Télécom [Paris]-Institut Mines-Télécom [Paris], Département GéoSciences et Environnement ( GSE-ENSMSE ), Institut Mines-Télécom [Paris]-Institut Mines-Télécom [Paris]-SPIN, Environnement Ville Société ( EVS ), École normale supérieure - Lyon ( ENS Lyon ) -Université Lumière - Lyon 2 ( UL2 ) -Université Jean Moulin - Lyon III-Institut National des Sciences Appliquées de Lyon ( INSA Lyon ), Université de Lyon-Institut National des Sciences Appliquées ( INSA ) -Université de Lyon-Institut National des Sciences Appliquées ( INSA ) -Université Jean Monnet [Saint-Étienne] ( UJM ) -École Nationale des Travaux Publics de l'État ( ENTPE ) -Ecole Nationale Supérieure des Mines de Saint-Etienne-École nationale supérieure d'architecture de Lyon ( ENSAL ) -Centre National de la Recherche Scientifique ( CNRS ), Microbiologie, adaptation et pathogénie ( MAP ), Université Claude Bernard Lyon 1 ( UCBL ), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon ( INSA Lyon ), Université de Lyon-Institut National des Sciences Appliquées ( INSA ) -Institut National des Sciences Appliquées ( INSA ) -Centre National de la Recherche Scientifique ( CNRS ), Laboratoire de chimie bactérienne ( LCB ), Aix Marseille Université ( AMU ) -Centre National de la Recherche Scientifique ( CNRS ), 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), Environnement, Bioénergie, Microalgues et Plantes (EBMP), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon, and Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées de Lyon (INSA Lyon)
Subjects: 0301 basic medicine, Arsenites, Health, Toxicology and Mutagenesis, Biosensing Techniques, macromolecular substances, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, 010501 environmental sciences, medicine.disease_cause, 01 natural sciences, Gene Expression Regulation, Enzymologic, Microbiology, [ SDE ] Environmental Sciences, 03 medical and health sciences, chemistry.chemical_compound, Genes, Reporter, Metals, Heavy, Escherichia coli, medicine, Environmental Chemistry, Bioluminescence, Deinococcus, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Promoter Regions, Genetic, [ SDV.BBM ] Life Sciences [q-bio]/Biochemistry, Molecular Biology, 0105 earth and related environmental sciences, Arsenite, Reporter gene, [SDV.GEN]Life Sciences [q-bio]/Genetics, biology, [ SDV.BC ] Life Sciences [q-bio]/Cellular Biology, technology, industry, and agriculture, Water, Gene Expression Regulation, Bacterial, General Medicine, biology.organism_classification, Deinococcus deserti, Pollution, Luciferases, Bacterial, 030104 developmental biology, chemistry, Biochemistry, [SDE]Environmental Sciences, [ SDV.GEN ] Life Sciences [q-bio]/Genetics, Biosensor, Water Pollutants, Chemical, Bacteria, Environmental Monitoring
Abstract: International audience; Whole-cell biosensors based on reporter genes allow detection of toxic metals in water with high selectivity and sensitivity under laboratory conditions; nevertheless, their transfer to a commercial inline water analyzer requires specific adaptation and optimization to field conditions as well as economical considerations. We focused here on both the influence of the bacterial host and the choice of the reporter gene by following the responses of global toxicity biosensors based on constitutive bacterial promoters as well as arsenite biosensors based on the arsenite-inducible P-ars promoter. We observed important variations of the bioluminescence emission levels in five different Escherichia coli strains harboring two different lux-based biosensors, suggesting that the best host strain has to be empirically selected for each new biosensor under construction. We also investigated the bioluminescence reporter gene system transferred into Deinococcus deserti, an environmental, desiccation- and radiation-tolerant bacterium that would reduce the manufacturing costs of bacterial biosensors for commercial water analyzers and open the field of biodetection in radioactive environments. We thus successfully obtained a cell survival biosensor and a metal biosensor able to detect a concentration as low as 100 nM of arsenite in D. deserti. We demonstrated that the arsenite biosensor resisted desiccation and remained functional after 7 days stored in air-dried D. deserti cells. We also report here the use of a new near-infrared (NIR) fluorescent reporter candidate, a bacteriophytochrome from the magnetotactic bacterium Magnetospirillum magneticum AMB-1, which showed a NIR fluorescent signal that remained optimal despite increasing sample turbidity, while in similar conditions, a drastic loss of the lux-based biosensors signal was observed.
Published: 2017

25. Biosynthesis of a broad-spectrum nicotianamine-like metallophore in Staphylococcus aureus

Author: Ryszard Lobinski, Elise Borezée-Durant, Felipe Cava, David Lemaire, Ghassan Ghssein, Christine Hajjar, Catherine Brutesco, Amélie Izaute, David Pignol, Romé Voulhoux, Pierre Richaud, Pascal Arnoux, Akbar Espaillat, Laurent Ouerdane, Shuanglong Wang, Clémentine Fojcik, Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Microbiologie Environnementale et Moléculaire (MEM), Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) (BIAM), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-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)-Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-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), Institut des sciences analytiques et de physico-chimie pour l'environnement et les materiaux (IPREM), Université de Pau et des Pays de l'Adour (UPPA)-Centre National de la Recherche Scientifique (CNRS), MICrobiologie de l'ALImentation au Service de la Santé (MICALIS), Institut National de la Recherche Agronomique (INRA)-AgroParisTech, Biologie végétale et microbiologie environnementale - UMR7265 (BVME), Interactions Protéine Métal (IPM), Bioénergie et Microalgues (EBM), Laboratoire d'ingénierie des systèmes macromoléculaires (LISM), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Umeå University, CEA program ToxNuc-E, HélioBiotec platform, ANR-14-CE09-0007,ANIBAL,Un nouveau metallophore dérivé de la nicotianamine chez des bactéries pathogènes(2014), 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)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Chimie Analytique Bio-Inorganique et Environnement (LCABIE), Laboratory for Molecular Infection Medicine Sweden, Department of Molecular Biology, Umeå Centre of Microbial Research, Umeå, Sweden, French Atomic Energy CommissionCEA program ToxNuc-E HelioBiotec platform - European Regional Development Fund Région Provence-Alpes-Côte d'AzurHelioBiotec platform - French Ministry of Research HelioBiotec platform - 'Commissariat a l'Energie Atomique et aux Energies Alternatives' Région Provence-Alpes-Côte d'AzurLaboratory for Molecular Infection Medicine Sweden Knut & Alice Wallenberg FoundationSwedish Research CouncilEuropean CommissionKempe foundation China Scholarship Council, Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Lobinski, Ryszard, and Appel à projets générique - Un nouveau metallophore dérivé de la nicotianamine chez des bactéries pathogènes - - ANIBAL2014 - ANR-14-CE09-0007 - Appel à projets générique - VALID
Subjects: 0301 basic medicine, Operon, [SDV]Life Sciences [q-bio], transferase, Dehydrogenase, Nicotianamine synthase, chemistry.chemical_compound, iron, Transferase, pathogenicity, Nicotianamine, oxidoreductase, azetidine 2 carboxylic acid, Amino Acid Isomerases, chemistry.chemical_classification, Multidisciplinary, metallophore, nicotianamine synthase, zinc, Imidazoles, Cobalt, [SDV] Life Sciences [q-bio], Biochemistry, Metabolome, Oxidoreductases, Azetidinecarboxylic Acid, Staphylococcus aureus, enzymology, 030106 microbiology, Biology, Biosynthesis, microbial activity, nicotine derivative, 03 medical and health sciences, nickel, Oxidoreductase, [CHIM]Chemical Sciences, Histidine, Alkyl and Aryl Transferases, nicotianamine, mutagenicity, Gene Expression Regulation, Bacterial, histidine, heavy metal, operon, Biosynthetic Pathways, staphylopine, 030104 developmental biology, chemistry, Gene Expression Regulation, copper, biology.protein, phytochemistry, imidazole derivative
Abstract: A new metal scavenger for bacteria All cells must find a way to acquire trace metals. Bacteria and plants scavenge iron, for instance, by synthesizing and releasing iron-chelating compounds called siderophores. Ghssein et al. describe three enzymes in Staphylococcus aureus that are responsible for the biosynthesis of another type of metallophore (see the Perspective by Nolan). Metabolomics and a range of biochemical assays show that this compound, named staphylopine, is involved in the uptake of a range of metals, depending on the growth environment. The genes required for staphylopine biosynthesis are conserved across a number of pathogenic bacteria and are similar to those for a broad-spectrum metallophore produced by plants. Science , this issue p. 1105 ; see also p. 1055
Published: 2016

26. Magnetosomes, Biogenic Magnetic Nanomaterials for Brain Molecular Imaging with 17.2 T MRI Scanner

Author: Laurence Motte, Benjamin Marty, Franck Lethimonnier, Sandra Prévéral, Christopher T. Lefèvre, Nicolas Ginet, Yoann Lalatonne, Géraldine Adryanczyk-Perrier, Françoise Geffroy, David Pignol, Michel Pean, Marianne Boucher, Sébastien Mériaux, Daniel Garcia, Service NEUROSPIN (NEUROSPIN), Université Paris-Saclay-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), Chimie, Structures et Propriétés de Biomatériaux et d'Agents Thérapeutiques (CSPBAT), Université Paris 13 (UP13)-Institut Galilée-Université Sorbonne Paris Cité (USPC)-Institut de Chimie du CNRS (INC)-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)), 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, 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: Materials science, Brain vasculature, Magnetotactic bacteria, Magnetosome, Biomedical Engineering, Pharmaceutical Science, Contrast Media, 02 engineering and technology, 010402 general chemistry, 01 natural sciences, Nanomaterials, Biomaterials, chemistry.chemical_compound, Magnetics, Mice, Nuclear magnetic resonance, In vivo, medicine, Animals, [CHIM]Chemical Sciences, Magnetospirillum, Magnetite Nanoparticles, ComputingMilieux_MISCELLANEOUS, Magnetite, medicine.diagnostic_test, Brain, Magnetic resonance imaging, Dextrans, 021001 nanoscience & nanotechnology, Magnetic Resonance Imaging, Ferrosoferric Oxide, 0104 chemical sciences, 3. Good health, Molecular Imaging, Nanostructures, chemistry, Nanoparticles, Magnetosomes, Molecular imaging, 0210 nano-technology, Biomedical engineering
Abstract: The fast development of sensitive molecular diagnostic tools is currently paving the way for a personalized medicine. A new class of ultrasensitive magnetic resonance imaging (MRI) T₂-contrast agents based on magnetosomes, magnetite nanocrystals biomineralized by magnetotactic bacteria, is proposed here. The contrast agents can be injected into the blood circulation and detected in the picomolar range. Purified magnetosomes are water-dispersible and stable within physiological conditions and exhibit at 17.2 T a transverse relaxivity r₂ four times higher than commercial ferumoxide. The subsequent gain in sensitivity by T₂(*) -weighted imaging at 17.2 T of the mouse brain vasculature is evidenced in vivo after tail vein injection of magnetosomes representing a low dose of iron (20 μmoliron kg(-1)), whereas no such phenomenon with the same dose of ferumoxide is observed. Preclinical studies of human pathologies in animal models will benefit from the combination of high magnetic field MRI with sensitive, low dose, easy-to-produce biocompatible contrast agents derived from bacterial magnetosomes.
Published: 2015

27. RNA Sequencing and Proteogenomics Reveal the Importance of Leaderless mRNAs in the Radiation-Tolerant Bacterium Deinococcus deserti

Author: Jean Armengaud, Bernard Fernandez, Stéphane Cruveiller, David Roche, David Pignol, Monika Ludanyi, Laurence Blanchard, David Vallenet, Arjan de Groot, 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), Microbiologie Environnementale et Moléculaire (MEM), Génomique métabolique (UMR 8030), Genoscope - Centre national de séquençage [Evry] (GENOSCOPE), Université Paris-Saclay-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-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 d'Évry-Val-d'Essonne (UEVE)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Biochimie des Systèmes Perturbés (LBSP), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Laboratoire Innovations technologiques pour la Détection et le Diagnostic (LI2D), Service de Pharmacologie et Immunoanalyse (SPI), Médicaments et Technologies pour la Santé (MTS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Paris-Saclay-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)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Médicaments et Technologies pour la Santé (MTS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), ANR-07-BLAN-0106,DEINOCOCCUS,Structure of the nucleoïd and radioresistance of Deinococcus radiodurans and Deinococcus deserti(2007), 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), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université d'Évry-Val-d'Essonne (UEVE)
Subjects: Proteomics, desiccation tolerance, protein protection, [SDV]Life Sciences [q-bio], Molecular Sequence Data, Biology, genome evolution, medicine.disease_cause, Radiation Tolerance, transcription start sites, Transcriptome, Evolution, Molecular, Eukaryotic translation, Start codon, Bacterial Proteins, Transcription (biology), Genetics, medicine, Deinococcus, small peptides, RNA, Messenger, Gene, Ecology, Evolution, Behavior and Systematics, protein translation initiation, ComputingMilieux_MISCELLANEOUS, Base Sequence, Sequence Analysis, RNA, Deinococcus deserti, Proteogenomics, biology.organism_classification, Adaptation, Physiological, RNA, Bacterial, Research Article
Abstract: Deinococcus deserti is a desiccation- and radiation-tolerant desert bacterium. Differential RNA sequencing (RNA-seq) was performed to explore the specificities of its transcriptome. Strikingly, for 1,174 (60%) mRNAs, the transcription start site was found exactly at (916 cases, 47%) or very close to the translation initiation codon AUG or GUG. Such proportion of leaderless mRNAs, which may resemble ancestral mRNAs, is unprecedented for a bacterial species. Proteomics showed that leaderless mRNAs are efficiently translated in D. deserti. Interestingly, we also found 173 additional transcripts with a 5'-AUG or 5'-GUG that would make them competent for ribosome binding and translation into novel small polypeptides. Fourteen of these are predicted to be leader peptides involved in transcription attenuation. Another 30 correlated with new gene predictions and/or showed conservation with annotated and nonannotated genes in other Deinococcus species, and five of these novel polypeptides were indeed detected by mass spectrometry. The data also allowed reannotation of the start codon position of 257 genes, including several DNA repair genes. Moreover, several novel highly radiation-induced genes were found, and their potential roles are discussed. On the basis of our RNA-seq and proteogenomics data, we propose that translation of many of the novel leaderless transcripts, which may have resulted from single-nucleotide changes and maintained by selective pressure, provides a new explanation for the generation of a cellular pool of small peptides important for protection of proteins against oxidation and thus for radiation/desiccation tolerance and adaptation to harsh environmental conditions.
Published: 2014

28. Structure and evolution of the magnetochrome domains: no longer alone

Author: Pascal Arnoux, Nicolas Ginet, Christopher T. Lefèvre, Marina I. Siponen, and David Pignol
Subjects: Microbiology (medical), Genetics, cytochrome, Magnetotactic bacteria, PDZ domain, Magnetosome, lcsh:QR1-502, magnetotactic bacteria, Biology, Microbiology, lcsh:Microbiology, Hypothesis and Theory Article, Evolution, Molecular, iron, Tandem repeat, magnetochrome, Cytoplasm, redox reactions, Organelle, evolution, Biophysics, magnetosome, Biogenesis, Membrane invagination
Abstract: Magnetotactic bacteria (MTB) can swim along Earth's magnetic field lines, thanks to the alignment of dedicated cytoplasmic organelles. These organelles, termed magnetosomes, are proteolipidic vesicles filled by a 35–120 nm crystal of either magnetite or greigite. The formation and alignment of magnetosomes are mediated by a group of specific genes, the mam genes, encoding the magnetosome-associated proteins. The whole process of magnetosome biogenesis can be divided into four sequential steps; (i) cytoplasmic membrane invagination, (ii) magnetosomes alignment, (iii) iron crystal nucleation and (iv) species-dependent mineral size and shape control. Since both magnetite and greigite are a mix of iron (III) and iron (II), iron redox state management within the magnetosome vesicle is a key issue. Recently, studies have started pointing out the importance of a MTB-specific c-type cytochrome domain found in several magnetosome-associated proteins (MamE, P, T, and X). This magnetochrome (MCR) domain is almost always found in tandem, and this tandem is either found alone (MamT), in combination with a PDZ domain (MamP), a domain of unknown function (MamX) or with a trypsin combined to one or two PDZ domains (MamE). By taking advantage of new genomic data available on MTB and a recent structural study of MamP, which helped define the MCR domain boundaries, we attempt to retrace the evolutionary history within and between the different MCR-containing proteins. We propose that the observed tandem repeat of MCR is the result of a convergent evolution and attempt to explain why this domain is rarely found alone.
Published: 2014

29. Reductive activation in periplasmic nitrate reductase involves chemical modifications of the Mo-cofactor beyond the first coordination sphere of the metal ion

Author: Sandrine Grosse, Emilien Etienne, Pascal Arnoux, Monique Sabaty, Julien Jacques, David Pignol, Bruno Guigliarelli, Frédéric Biaso, Patrick Bertrand, Bénédicte Burlat, Christophe Léger, Vincent Fourmond, Bioénergétique et Ingénierie des Protéines (BIP ), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Microbiologie Environnementale et Moléculaire (MEM), 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), ANR-11-BSV5-0005,MC2,Structure et Fonction du Cofacteur à Molybdène(2011), 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: Iron-Sulfur Proteins, Models, Molecular, Coenzymes, Photochemistry, Ligands, 01 natural sciences, Biochemistry, law.invention, chemistry.chemical_compound, law, Pterin, Electron paramagnetic resonance, 0303 health sciences, biology, Pteridines, Temperature, Protein film voltammetry, Periplasm, Molybdenum cofactor, Oxidation-Reduction, EPR spectroscopy, Coordination sphere, Biophysics, Rhodobacter sphaeroides, 010402 general chemistry, Nitrate reductase, Redox, Electron transfer, 03 medical and health sciences, Metalloproteins, [CHIM]Chemical Sciences, 030304 developmental biology, Ions, Molybdenum, [PHYS.PHYS]Physics [physics]/Physics [physics], Electron Spin Resonance Spectroscopy, Electrochemical Techniques, Cell Biology, biology.organism_classification, 0104 chemical sciences, Pterins, Pyranopterin, Enzyme Activation, Crystallography, Kinetics, chemistry, Spin Labels, Molybdenum Cofactors
Abstract: International audience; In Rhodobacter sphaeroides periplasmic nitrate reductase NapAB, the major Mo(V) form (the “high g” species) in air-purified samples is inactive and requires reduction to irreversibly convert into a catalytically competent form (Fourmond et al., J. Phys. Chem., 2008). In the present work, we study the kinetics of the activation process by combining EPR spectroscopy and direct electrochemistry. Upon reduction, the Mo (V) “high g” resting EPR signal slowly decays while the other redox centers of the protein are rapidly reduced, which we interpret as a slow and gated (or coupled) intramolecular electron transfer between the [4Fe–4S] center and the Mo cofactor in the inactive enzyme. Besides, we detect spin–spin interactions between the Mo(V) ion and the [4Fe–4S]1 + cluster which are modified upon activation of the enzyme, while the EPR signatures associated to the Mo cofactor remain almost unchanged. This shows that the activation process, which modifies the exchange coupling pathway between the Mo and the [4Fe–4S]1 + centers, occurs further away than in the first coordination sphere of the Mo ion. Relying on structural data and studies on Mo-pyranopterin and models, we propose a molecular mechanism of activation which involves the pyranopterin moiety of the molybdenum cofactor that is proximal to the [4Fe–4S] cluster. The mechanism implies both the cyclization of the pyran ring and the reduction of the oxidized pterin to give the competent tricyclic tetrahydropyranopterin form.
Published: 2014

30. Cu binding by the Escherichia coli metal-efflux accessory protein RcnB

Author: Agnès Rodrigue, David Pignol, Manon Gault, Pascal Arnoux, Camille Blériot, Marie-Andrée Mandrand-Berthelot, Erwan Gueguen, Microbiologie, adaptation et pathogénie (MAP), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), Laboratoire d'ingénierie des systèmes macromoléculaires (LISM), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Microbiologie Environnementale et Moléculaire (MEM), 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), Biologie végétale et microbiologie environnementale - UMR7265 (BVME), Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Aix Marseille Université (AMU), 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: Mutant, Biophysics, medicine.disease_cause, Biochemistry, Divalent, Biomaterials, 03 medical and health sciences, Copper Transport Proteins, Nickel, Mutant protein, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Escherichia coli, medicine, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Cation Transport Proteins, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, chemistry.chemical_classification, Metal ion homeostasis, 0303 health sciences, 030306 microbiology, Chemistry, Escherichia coli Proteins, Metals and Alloys, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Cobalt, Periplasmic space, Ligand (biochemistry), [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Chemistry (miscellaneous), Efflux, Copper
Abstract: Divalent cations play fundamental roles in biological systems where they act as structural and reactive determinants. Their high reactivity with biomolecules has forced living cells to evolve specific pathways for their in vivo handling. For instance the excess of metal can be expelled by dedicated efflux systems. The E. coli RcnA efflux pump expels both Ni and Co. This pump functions together with the periplasmic protein RcnB to maintain metal ion homeostasis. To gain insights into the efflux mechanism, metal binding properties of RcnB were investigated. Initial screening of metal ions by fluorescence quenching revealed Cu as a potential ligand for RcnB. Non-denaturing mass spectrometry and ITC experiments revealed the binding of one Cu ion per monomer with a micromolar affinity. This set of in vitro techniques was broadened by in vivo experiments that showed the accuracy of Cu binding by RcnB. RcnB implication in Cu detoxification was questioned and growth experiments as well as transcriptional analysis excluded a role for RcnB in Cu adaptation. Finally a mutant in a conserved methionine residue (Met86) displayed altered Cu binding. This mutant protein when tested for its Ni and Co resistance capacity was unable to complement an rcn mutant. Taken together these data show that RcnB is a new Cu-binding protein that is strikingly involved in a Ni/Co efflux system.
Published: 2014

31. Structural insight into magnetochrome-mediated magnetite biomineralization

Author: Wei-Jia Zhang, Michelle C. Y. Chang, Stephanie R. Jones, Marc Widdrat, Marina I. Siponen, Damien Faivre, David Pignol, Pierre Legrand, Pascal Arnoux, 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), Synchrotron SOLEIL (SSOLEIL), Centre National de la Recherche Scientifique (CNRS), Department of Biomaterials [Potsdam], Max Planck Institute of Colloids and Interfaces, Max-Planck-Gesellschaft-Max-Planck-Gesellschaft, Lawrence Berkeley National Laboratory [Berkeley] (LBNL), Laboratoire de chimie bactérienne (LCB), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Microbiologie Environnementale et Moléculaire (MEM), 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)), 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: Greigite, Models, Molecular, Magnetotactic bacteria, Iron, [SDV]Life Sciences [q-bio], PDZ domain, Magnetosome, Static Electricity, 02 engineering and technology, Ferric Compounds, 03 medical and health sciences, Ferrihydrite, chemistry.chemical_compound, Bacterial Proteins, MAMP, Conserved Sequence, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, Magnetite, 0303 health sciences, Multidisciplinary, Bacteria, 021001 nanoscience & nanotechnology, Ferrosoferric Oxide, Protein Structure, Tertiary, Crystallography, chemistry, Genes, Bacterial, Magnetosomes, Protein Multimerization, 0210 nano-technology, Oxidoreductases, Oxidation-Reduction, Biomineralization
Abstract: The magnetosome-associated protein mamP is an iron oxidase that reveals a unique arrangement of a self-plugged PDZ domain fused to two magnetochrome domains, defining a new class of c-type cytochrome exclusively found in magnetotactic bacteria. Magnetotactic bacteria use a specialized organelle known as the magnetosome, a biomineralized crystal of magnetite (Fe(II)Fe(III)2O4) or greigite (Fe(II)Fe(III)2S4), to sense and align along the Earth's magnetic field. This paper presents the X-ray crystal structure of the magnetosome-associated protein MamP, revealing a unique arrangement of a self-plugged PDZ domain fused to two magnetochrome domains. The authors also establish that MamP is an iron oxidase that contributes to the formation of iron(III) ferrihydrite, and is therefore important for mechanisms of iron management during magnetosome biogenesis. Magnetotactic bacteria align along the Earth’s magnetic field using an organelle called the magnetosome, a biomineralized magnetite (Fe(ii)Fe(iii)2O4) or greigite (Fe(ii)Fe(iii)2S4) crystal embedded in a lipid vesicle. Although the need for both iron(ii) and iron(iii) is clear, little is known about the biological mechanisms controlling their ratio1. Here we present the structure of the magnetosome-associated protein MamP and find that it is built on a unique arrangement of a self-plugged PDZ domain fused to two magnetochrome domains, defining a new class of c-type cytochrome exclusively found in magnetotactic bacteria. Mutational analysis, enzyme kinetics, co-crystallization with iron(ii) and an in vitro MamP-assisted magnetite production assay establish MamP as an iron oxidase that contributes to the formation of iron(iii) ferrihydrite eventually required for magnetite crystal growth in vivo. These results demonstrate the molecular mechanisms of iron management taking place inside the magnetosome and highlight the role of magnetochrome in iron biomineralization.
Published: 2013

32. Isolation and Characterization of Environmental Bacteria Capable of Extracellular Biosorption of Mercury

Author: Séverine Zirah, Carine Lombard, Jean-Michel Guigner, Paul Soreau, Florence Brian-Jaisson, Manon Vandervennet, Fabienne François, Jean Peduzzi, Grégory Martino, Anne-Laure Molinier, Sylvie Rebuffat, Daniel Garcia, David Pignol, Molécules de Communication et Adaptation des Micro-organismes (MCAM), Muséum national d'Histoire naturelle (MNHN)-Centre National de la Recherche Scientifique (CNRS), Institut de minéralogie, de physique des matériaux et de cosmochimie (IMPMC), Muséum national d'Histoire naturelle (MNHN)-Institut de recherche pour le développement [IRD] : UR206-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Aix Marseille Université (AMU), Biologie cellulaire et moléculaire des plantes et des bactéries (BCMPB), Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université de la Méditerranée - Aix-Marseille 2, and Université de la Méditerranée - Aix-Marseille 2-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)
Subjects: DNA, Bacterial, Molecular Sequence Data, chemistry.chemical_element, Microbial Sensitivity Tests, 010501 environmental sciences, DNA, Ribosomal, 01 natural sciences, Applied Microbiology and Biotechnology, Bacterial cell structure, 03 medical and health sciences, Bioremediation, Extracellular polymeric substance, Dry weight, RNA, Ribosomal, 16S, Environmental Microbiology, Cluster Analysis, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Effluent, Phylogeny, ComputingMilieux_MISCELLANEOUS, 0105 earth and related environmental sciences, 2. Zero hunger, 0303 health sciences, Bacteria, Ecology, biology, 030306 microbiology, Spectrum Analysis, Polysaccharides, Bacterial, Biosorption, Sequence Analysis, DNA, biology.organism_classification, Geomicrobiology, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, 6. Clean water, Mercury (element), chemistry, 13. Climate action, Environmental chemistry, Mercuric Chloride, Environmental Pollutants, Food Science, Biotechnology
Abstract: Accumulation of toxic metals in the environment represents a public health and wildlife concern. Bacteria resistant to toxic metals constitute an attractive biomass for the development of systems to decontaminate soils, sediments, or waters. In particular, biosorption of metals within the bacterial cell wall or secreted extracellular polymeric substances (EPS) is an emerging process for the bioremediation of contaminated water. Here the isolation of bacteria from soil, effluents, and river sediments contaminated with toxic metals permitted the selection of seven bacterial isolates tolerant to mercury and associated with a mucoid phenotype indicative of the production of EPS. Inductively coupled plasma-optical emission spectroscopy and transmission electron microscopy in conjunction with X-ray energy dispersive spectrometry revealed that bacteria incubated in the presence of HgCl 2 sequestered mercury extracellularly as spherical or amorphous deposits. Killed bacterial biomass incubated in the presence of HgCl 2 also generated spherical extracellular mercury deposits, with a sequestration capacity (40 to 120 mg mercury per g [dry weight] of biomass) superior to that of live bacteria (1 to 2 mg mercury per g [dry weight] of biomass). The seven strains were shown to produce EPS, which were characterized by Fourier transform-infrared (FT-IR) spectroscopy and chemical analysis of neutral-carbohydrate, uronic acid, and protein contents. The results highlight the high potential of Hg-tolerant bacteria for applications in the bioremediation of mercury through biosorption onto the biomass surface or secreted EPS.
Published: 2012

33. Heterologous expression of membrane proteins: choosing the appropriate host

Author: Sylvain Boutigny, Elisabeth Darrouzet, Daniel Salvi, Daphné Seigneurin-Berny, Sébastien Dementin, Annie Frelet-Barrand, Thierry Desnos, Eva Pebay-Peyroula, Norbert Rolland, Monique Sabaty, Jean-Baptiste Rioux, Florent Bernaudat, Jacques Joyard, Patrick Hivin, Nathalie Pochon, David Pignol, Pierre Richaud, Thierry Vernet, Institut de biologie structurale (IBS - UMR 5075), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), 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 Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de physiologie cellulaire végétale (LPCV), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Signalisation de l'Adaptation des Végétaux à l'Environnement (SAVE), 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), Microbiologie Environnementale et Moléculaire (MEM), Laboratoire d'Ingénierie Cellulaire et de Biologie (LICB), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Laboratoire des Echanges Membranaires et Signalisation (LEMS), Université de la Méditerranée - Aix-Marseille 2-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Transporteurs en Imagerie et Radiothérapie en Oncologie (TIRO - UMR E4320), Service Hospitalier Frédéric Joliot (SHFJ), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-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-UMR E4320 (TIRO-MATOs), Université Nice Sophia Antipolis (... - 2019) (UNS), Université Côte d'Azur (UCA)-Université Côte d'Azur (UCA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Nice Sophia Antipolis (... - 2019) (UNS), Université Côte d'Azur (UCA)-Université Côte d'Azur (UCA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Nice Sophia Antipolis (... - 2019) (UNS), Université Côte d'Azur (UCA)-Université Côte d'Azur (UCA), Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA), 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), Transporteurs et Imagerie, Radiothérapie en Oncologie et Mécanismes biologiques des Altérations du Tissu Osseux (TIRO-MATOs - UMR E4320), UMR E4320 (TIRO-MATOs), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Côte d'Azur (UCA)-Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Côte d'Azur (UCA)-Service Hospitalier Frédéric Joliot (SHFJ), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay, This work has been supported by the CEA (Commissariat a l'Energie Atomique et aux Energies Alternatives, funding of the CEA-PM project and PostDoc fellowships to F. Bernaudat, A. Frelet-Barrand, P. Hivin and S. Dementin), by the CNRS (Centre National de la Recherche Scientifique, Post-Doc fellowship to A. Frelet-Barrand) and by the University of Grenoble 1. This work was partly supported by the European grant LSMH-CT-EUR-INTAFAR 2004-512138. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript., Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Plant Environmental Physiology and Stress Signaling (PEPSS), Université Paris-Saclay-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)-UMR E4320 (TIRO-MATOs), Université Nice Sophia Antipolis (1965 - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Côte d'Azur (UCA)-Université Nice Sophia Antipolis (1965 - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Côte d'Azur (UCA), and Bernaudat, Florent
Subjects: Arabidopsis thaliana, Gene Expression, plant, Plant Science, medicine.disease_cause, Bioinformatics, Polymerase Chain Reaction, Biochemistry, law.invention, expression system, Transmembrane Transport Proteins, law, Molecular Cell Biology, Cloning, Molecular, Baculovirus, bacteria, membrane, Cellular proteins, Plant Proteins, bactérie, 0303 health sciences, Multidisciplinary, biology, Genetically Modified Organisms, Recombinant Proteins, Cell biology, Blot, Lactococcus lactis, expression host, Recombinant DNA, Cytochemistry, Medicine, bacterial proteins, dna primers, membrane proteins, plant proteins, multidisciplinary sciences, science and technology, Research Article, Biotechnology, Science, Blotting, Western, Rhodobacter sphaeroides, Spodoptera, Cell Line, 03 medical and health sciences, Bacterial Proteins, medicine, Escherichia coli, Nicotiana benthamiana, Animals, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Biology, Transgenic Plants, 030304 developmental biology, DNA Primers, Cloning, Base Sequence, 030306 microbiology, Cell Membrane, Membrane Proteins, Proteins, insecta, biology.organism_classification, Transmembrane Proteins, Membrane protein, Plant Biotechnology, Heterologous expression, protein, mamalia, overexpression
Abstract: International audience; BACKGROUND: Membrane proteins are the targets of 50% of drugs, although they only represent 1% of total cellular proteins. The first major bottleneck on the route to their functional and structural characterisation is their overexpression; and simply choosing the right system can involve many months of trial and error. This work is intended as a guide to where to start when faced with heterologous expression of a membrane protein. METHODOLOGY/PRINCIPAL FINDINGS: The expression of 20 membrane proteins, both peripheral and integral, in three prokaryotic (E. coli, L. lactis, R. sphaeroides) and three eukaryotic (A. thaliana, N. benthamiana, Sf9 insect cells) hosts was tested. The proteins tested were of various origins (bacteria, plants and mammals), functions (transporters, receptors, enzymes) and topologies (between 0 and 13 transmembrane segments). The Gateway system was used to clone all 20 genes into appropriate vectors for the hosts to be tested. Culture conditions were optimised for each host, and specific strategies were tested, such as the use of Mistic fusions in E. coli. 17 of the 20 proteins were produced at adequate yields for functional and, in some cases, structural studies. We have formulated general recommendations to assist with choosing an appropriate system based on our observations of protein behaviour in the different hosts. CONCLUSIONS/SIGNIFICANCE: Most of the methods presented here can be quite easily implemented in other laboratories. The results highlight certain factors that should be considered when selecting an expression host. The decision aide provided should help both newcomers and old-hands to select the best system for their favourite membrane protein.
Published: 2011

34. Single-step production of a recyclable nanobiocatalyst for organophosphate pesticides biodegradation using functionalized bacterial magnetosomes

Author: David Pignol, Romain Pardoux, Catherine Brutesco, Daniel Garcia, Géraldine Adryanczyk, and Nicolas Ginet
Subjects: Immobilized enzyme, Magnetotactic bacteria, Hydrolases, Applied Microbiology, Magnetosome, Materials Science, lcsh:Medicine, Biochemistry, Microbiology, Biomaterials, Environmental Biotechnology, Nanotechnology, Enzyme kinetics, Magnetospirillum, Pesticides, lcsh:Science, Biology, Multidisciplinary, biology, Chemistry, Enzyme Classes, lcsh:R, Agriculture, Biodegradation, biology.organism_classification, Combinatorial chemistry, Enzymes, Biodegradation, Environmental, Bionanotechnology, Pesticide degradation, Biocatalysis, Magnetic nanoparticles, Nanoparticles, lcsh:Q, Magnetosomes, Agrochemicals, Bioremediation, Research Article, Biotechnology
Abstract: Enzymes are versatile catalysts in laboratories and on an industrial scale; improving their immobilization would be beneficial to broadening their applicability and ensuring their (re)use. Lipid-coated nano-magnets produced by magnetotactic bacteria are suitable for a universally applicable single-step method of enzyme immobilization. By genetically functionalizing the membrane surrounding these magnetite particles with a phosphohydrolase, we engineered an easy-to-purify, robust and recyclable biocatalyst to degrade ethyl-paraoxon, a commonly used pesticide. For this, we genetically fused the opd gene from Flavobacterium sp. ATCC 27551 encoding a paraoxonase to mamC, an abundant protein of the magnetosome membrane in Magnetospirillum magneticum AMB-1. The MamC protein acts as an anchor for the paraoxonase to the magnetosome surface, thus producing magnetic nanoparticles displaying phosphohydrolase activity. Magnetosomes functionalized with Opd were easily recovered from genetically modified AMB-1 cells: after cellular disruption with a French press, the magnetic nanoparticles are purified using a commercially available magnetic separation system. The catalytic properties of the immobilized Opd were measured on ethyl-paraoxon hydrolysis: they are comparable with the purified enzyme, with K(m) (and k(cat)) values of 58 µM (and 178 s(-1)) and 43 µM (and 314 s(-1)) for the immobilized and purified enzyme respectively. The Opd, a metalloenzyme requiring a zinc cofactor, is thus properly matured in AMB-1. The recycling of the functionalized magnetosomes was investigated and their catalytic activity proved to be stable over repeated use for pesticide degradation. In this study, we demonstrate the easy production of functionalized magnetic nanoparticles with suitably genetically modified magnetotactic bacteria that are efficient as a reusable nanobiocatalyst for pesticides bioremediation in contaminated effluents.
Published: 2011

35. Dependence of catalytic activity on driving force in solution assays and protein film voltammetry: insights from the comparison of nitrate reductase mutants

Author: Christophe Léger, Pascal Arnoux, Emilien Etienne, Sébastien Dementin, Vincent Fourmond, Bruno Guigliarelli, Bénédicte Burlat, Monique Sabaty, Patrick Bertrand, David Pignol, Bioénergétique et Ingénierie des Protéines (BIP ), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Microbiologie Environnementale et Moléculaire (MEM), 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), Institut de biologie et chimie des protéines [Lyon] (IBCP), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-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), and Azzopardi, Laure
Subjects: Models, Molecular, Kinetics, Inorganic chemistry, Electron donor, Rhodobacter sphaeroides, Overpotential, 010402 general chemistry, Nitrate reductase, 01 natural sciences, Biochemistry, Redox, Nitrate Reductase, 03 medical and health sciences, chemistry.chemical_compound, Catalytic Domain, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Electrochemistry, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, 0303 health sciences, biology, Chemistry, Spectrum Analysis, Active site, biology.organism_classification, 0104 chemical sciences, Solutions, Mutation, Periplasm, biology.protein, Biophysics, Biocatalysis, Mutant Proteins, Electrode potential
Abstract: Rhodobacter sphaeroides periplasmic nitrate reductase (Rs NapAB) is one of the enzymes whose assays give odd results: in spectrophotometric assays with methyl viologen as the electron donor, the activity increases as the reaction progresses, whereas the driving force provided by the soluble redox partner decreases; in protein film voltammetry (PFV), whereby the enzyme directly exchanges electrons with an electrode, the activity of NapAB decreases at large overpotential, whereas a monotonic increase is expected [Elliott, S. J., et al. (2002) Biochim. Biophys. Acta 1555, 54-59]. The relations between these phenomena and the catalytic mechanism are still debated. By studying NapAB mutants, we found that the peculiar dependences of electrochemical and solution activities on driving force are greatly affected by substituting certain amino acids that are located in the vicinity of the active site (M153, Q384, R392); this led us to establish and discuss the relation between the experimental parameters of the electrochemical and spectrophotometric assays: we show that the rate of reduction of the enzyme (which depends on the electrode potential or on the concentration of reduced MV) modulates the activity of the enzyme, but the "solution potential" does not. Our results also support the view that the complex profiles of activity versus potential are fingerprints of the active site chemistry, rather than direct consequences of changes in the redox states of relays that are remote from the active site.
Published: 2010

36. Crystallographic snapshots of iterative substrate translocations during nicotianamine synthesis in Archaea

Author: Pascal Arnoux, Stéphane Mari, David Pignol, Cyril Dreyfus, David Lemaire, Microbiologie Environnementale et Moléculaire (MEM), Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) (BIAM), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-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)-Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-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), Biochimie et Physiologie Moléculaire des Plantes (BPMP), Université de Montpellier (UM)-Centre international d'études supérieures en sciences agronomiques (Montpellier SupAgro)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro)-Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS), 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)), Institut National de la Recherche Agronomique (INRA)-Centre international d'études supérieures en sciences agronomiques (Montpellier SupAgro)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro), 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), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)-Centre international d'études supérieures en sciences agronomiques (Montpellier SupAgro)-Institut National de la Recherche Agronomique (INRA)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro), and Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)
Subjects: Models, Molecular, 0106 biological sciences, Protein Folding, S-Adenosylmethionine, MESH: Sequence Homology, Amino Acid, Azetidine, MESH: Protein Structure, Secondary, Crystal structure, MESH: Amino Acid Sequence, NICOTIANAMINE, METAL HOMEOSTASIS, REACTION MECHANISM, BIOLOGIE DU DEVELOPPEMENT, BIOLOGIE MOLECULAIRE, Crystallography, X-Ray, 01 natural sciences, Mass Spectrometry, Protein Structure, Secondary, Substrate Specificity, Nicotianamine synthase, structure moléculaire, chemistry.chemical_compound, MESH: Protein Structure, Tertiary, Transferase, Nicotianamine, MESH: Cyclization, 0303 health sciences, Multidisciplinary, Molecular Structure, biology, plants, MESH: Models, Chemical, MESH: Archaeal Proteins, Biological Sciences, Small molecule, MESH: Azetidinecarboxylic Acid, synthase, Azetidinecarboxylic Acid, MESH: Models, Molecular, Methanobacteriaceae, MESH: Mutation, metal, Stereochemistry, Archaeal Proteins, MESH: Protein Folding, Molecular Sequence Data, MESH: Molecular Structure, cloning, arabidopsis-thaliana, MESH: Alkyl and Aryl Transferases, Catalysis, physiologie végétale, 03 medical and health sciences, nickel, [SDV.BV]Life Sciences [q-bio]/Vegetal Biology, Amino Acid Sequence, transport membranaire, MESH: Hydrogen Bonding, Binding site, 030304 developmental biology, MESH: Mass Spectrometry, Alkyl and Aryl Transferases, Binding Sites, MESH: Molecular Sequence Data, Sequence Homology, Amino Acid, MESH: Methanobacteriaceae, iron uptake, ACL, Active site, crystal-structure, MESH: S-Adenosylmethionine, Hydrogen Bonding, reaction mechanism converting-enzyme-inhibitor, MESH: Catalysis, MESH: Crystallography, X-Ray, Protein Structure, Tertiary, arabidopsis, Crystallography, Models, Chemical, chemistry, MESH: Binding Sites, Cyclization, Mutation, transport, biology.protein, metal homeostasis, MESH: Substrate Specificity, 010606 plant biology & botany
Abstract: Nicotianamine (NA), a small molecule ubiquitous in plants, is an important divalent metal chelator and the main precursor of phytosiderophores. Nicotianamine synthase (NAS) is the enzyme catalyzing NA synthesis by the condensation of three aminopropyl moieties of S-adenosylmethionine (SAM) and the cyclization of one of them to form an azetidine ring. Here we report five crystal structures of an archaeal NAS from Methanothermobacter thermautotrophicus , either free or in complex with its product(s) and substrate(s). These structures reveal a two-domains fold arrangement of MtNAS, a small molecule related to NA (named here thermoNicotianamine or tNA), and an original mechanism of synthesis in a buried reaction chamber. This reaction chamber is open to the solvent through a small inlet, and a single active site allows the selective entrance of only one substrate at a time that is then processed and translocated stepwise.
Published: 2009

37. Correcting for Electrocatalyst Desorption and Inactivation in Chronoamperometry Experiments

Author: Pierre-Pol Liebgott, Pascal Arnoux, Thomas Lautier, Patrick Bertrand, Fanny Leroux, Christophe Léger, Sébastien Dementin, Carole Baffert, Vincent Fourmond, Phillippe Soucaille, Marc Rousset, David Pignol, Isabelle Meynial-Salles, Bioénergétique et Ingénierie des Protéines (BIP ), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Institut de Microbiologie de la Méditerranée (IMM), Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), Microbiologie Environnementale et Moléculaire (MEM), Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) (BIAM), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-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)-Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-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), Institut National de la Recherche Agronomique (INRA), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-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)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), and Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA))
Subjects: Hydrogenase, substrate-binding, Chimie analytique, Inorganic chemistry, Analytical chemistry, Context (language use), 010402 general chemistry, Electrocatalyst, 01 natural sciences, Redox, active-site, Analytical Chemistry, 03 medical and health sciences, Electron transfer, electrocatalyst, 030304 developmental biology, 0303 health sciences, carbon nanotubes, Chemistry, Electric Conductivity, Electrochemical Techniques, Chronoamperometry, chronoamperometric, periplasmic nitrate reductase, protein film voltammetry, nife hydrogenase, clostridium-acetobutylicum, horseradish-peroxidase, nitrite reductase, catalytic cycle, électrocatalyseurs, Enzymes, 0104 chemical sciences, électrode, [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, Catalytic cycle, Biocatalysis, Adsorption, Cyclic voltammetry, expérimentation, Artifacts, Software
Abstract: International audience; Chronoamperometric experiments with adsorbed electrocatalysts are commonly performed either for analytical purposes or for studying the catalytic mechanism of a redox enzyme. In the context of amperometric sensors, the current may be recorded as a function of time while the analyte concentration is being increased to determine a linearity range. In mechanistic studies of redox enzymes, chronoamperometry proved powerful for untangling the effects of electrode potential and time, which are convoluted in cyclic voltammetric measurements, and for studying the energetics and kinetics of inhibition. In all such experiments, the fact that the catalyst's coverage and/or activity decreases over time distorts the data. This may hide meaningful features, introduce systematic errors, and limit the accuracy of the measurements. We propose a general and surprisingly simple method for correcting for electrocatalyst desorption and inactivation, which greatly increases the precision of chronoamperometric experiments. Rather than subtracting a baseline, this consists in dividing the current, either by a synthetic signal that is proportional to the instant electroactive coverage or by the signal recorded in a control experiment. In the latter, the change in current may result from film loss only or from film loss plus catalyst inactivation. We describe the different strategies for obtaining the control signal by analyzing various data recorded with adsorbed redox enzymes: nitrate reductase, NiFe hydrogenase, and FeFe hydrogenase. In each case we discuss the trustfulness and the benefit of the correction. This method also applies to experiments where electron transfer is mediated, rather than direct, providing the current is proportional to the time-dependent concentration of catalyst.
Published: 2009

38. 'Major Mo(V) EPR signature of Rhodobacter sphaeroides periplasmic nitrate reductase arising from a dead-end species that activates upon reduction. Relation to other molybdoenzymes from the DMSO reductase family.'

Author: Christophe Léger, Bruno Guigliarelli, Patrick Bertrand, Vincent Fourmond, Pascal Arnoux, Bénédicte Burlat, Sébastien Dementin, Monique Sabaty, Séverine Boiry, David Pignol, Institut de Microbiologie de la Méditerranée (IMM), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Bioénergétique et Ingénierie des Protéines (BIP ), Institut de biologie et chimie des protéines [Lyon] (IBCP), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Microbiologie Environnementale et Moléculaire (MEM), Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) (BIAM), 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)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Biologie végétale et microbiologie environnementale - UMR7265 (BVME), Aix Marseille Université (AMU), inconnu temporaire UPEMLV, Inconnu, Hôpital de la Timone [CHU - APHM] (TIMONE), 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), and Azzopardi, Laure
Subjects: Iron-Sulfur Proteins, Stereochemistry, Rhodobacter sphaeroides, 010402 general chemistry, Nitrate reductase, Nitrate Reductase, 01 natural sciences, Cofactor, [PHYS] Physics [physics], law.invention, 03 medical and health sciences, chemistry.chemical_compound, [SCCO]Cognitive science, law, Electrochemistry, Materials Chemistry, Physical and Theoretical Chemistry, Microwaves, Electron paramagnetic resonance, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, Molybdenum, [PHYS]Physics [physics], 0303 health sciences, DMSO reductase, biology, Chemistry, Electron Spin Resonance Spectroscopy, Molybdopterin, [SCCO] Cognitive science, Periplasmic space, biology.organism_classification, 0104 chemical sciences, Surfaces, Coatings and Films, Biochemistry, Catalytic cycle, Mutation, Potentiometry, biology.protein, Adsorption, Oxidoreductases, Oxidation-Reduction, Algorithms
Abstract: Enzymes of the DMSO reductase family use a mononuclear Mo-bis(molybdopterin) cofactor (MoCo) to catalyze a variety of oxo-transfer reactions. Much functional information on nitrate reductase, one of the most studied members of this family, has been gained from EPR spectroscopy, but this technique is not always conclusive because the signature of the MoCo is heterogeneous, and which signals correspond to active species is still unsure. We used site-directed mutagenesis, EPR and protein film voltammetry to demonstrate that the MoCo in R. sphaeroides periplasmic nitrate reductase (NapAB) is subject to an irreversible reductive activation process whose kinetics we precisely define. This activation quantitatively correlates with the disappearance of the so-called "Mo(V) high-g" EPR signal, but this reductive process is too slow to be part of the normal catalytic cycle. Therefore, in NapAB, this most intense and most commonly observed signature of the MoCo arises from a dead-end, inactive state that gives a catalytically competent species only after reduction. This activation proceeds, even without substrate, according to a reduction followed by an irreversible nonredox step, both of which are pH independent. An apparently similar process occurs in other nitrate reductases (both assimilatory and membrane bound), and this also recalls the redox cycling procedure, which activates periplasmic DMSO reductases and simplifies their spectroscopic signatures. Hence we propose that heterogeneity at the active site and reductive activation are common properties of enzymes from the DMSO reductase family. Regarding NapAB, the fact that we could detect no Mo EPR signal upon reoxidizing the fully reduced enzyme suggests that the catalytically active form of the Mo(V) is thermodynamically unstable, as is the case for other enzymes of the DMSO reductase family. Our original approach, which combines spectroscopy and protein film voltammetry, proves useful for discriminating the forms of the active site on the basis of their catalytic properties. This could be applied to other enzymes for which the question arises as to the catalytic relevance of certain long-lived, spectroscopically characterized species.
Published: 2008

39. Evolution of a bacteriophytochrome from light to redox sensor

Author: Mila Kojadinovic, David Pignol, André Verméglio, Joël Fardoux, Jean-Marc Adriano, Marianne Jaubert, Sébastien Zappa, Eric Giraud, Laure Hannibal, and Laurie Vuillet
Subjects: redox sensing, Light, Molecular Sequence Data, Light-Harvesting Protein Complexes, Biology, Models, Biological, General Biochemistry, Genetics and Molecular Biology, Article, Light-harvesting complex, Evolution, Molecular, chemistry.chemical_compound, bacteriophytochrome, Bacterial Proteins, Amino Acid Sequence, Kinase activity, Photosynthesis, Promoter Regions, Genetic, Molecular Biology, Transcription factor, Phylogeny, Biliverdin, General Immunology and Microbiology, Phytochrome, General Neuroscience, Chromophore, biology.organism_classification, Recombinant Proteins, Oxygen, Rhodopseudomonas, Biochemistry, chemistry, Biophysics, light harvesting complexes, Rhodopseudomonas palustris, Signal transduction, Oxidation-Reduction, Signal Transduction, Transcription Factors
Abstract: Bacteriophytochromes are red/far-red photoreceptors that bacteria use to mediate sensory responses to their light environment. Here, we show that the photosynthetic bacterium Rhodopseudomonas palustris has two distinct types of bacteriophytochrome-related protein (RpBphP4) depending upon the strain considered. The first type binds the chromophore biliverdin and acts as a light-sensitive kinase, thus behaving as a bona fide bacteriophytochrome. However, in most strains, RpBphP4 does not to bind this chromophore. This loss of light sensing is replaced by a redox-sensing ability coupled to kinase activity. Phylogenetic analysis is consistent with an evolutionary scenario, where a bacteriophytochrome ancestor has adapted from light to redox sensing. Both types of RpBphP4 regulate the synthesis of light harvesting (LH2) complexes according to the light or redox conditions, respectively. They modulate the affinity of a transcription factor binding to the promoter regions of LH2 complex genes by controlling its phosphorylation status. This is the first complete description of a bacteriophytochrome signal transduction pathway involving a two-component system.
Published: 2007

40. Legumes Symbioses: Absence of Nod Genes in Photosynthetic Bradyrhizobia

Author: Béatrice Segurens, Paul G. Richardson, Jean-Christophe Avarre, Bernard Dreyfus, Eddie Cytryn, Joël Fardoux, William L. Franck, Woo Suk Chang, Lionel Moulin, Zoé Rouy, Yves Prin, Philippe Normand, David Vallenet, Fabienne Cartieaux, Carole Dossat, Gilles Béna, Elizabeth Saunders, Claudine Médigue, Eric Giraud, Damien Simon, Marianne Jaubert, Michael J. Sadowsky, Aurélie Lajus, David Bruce, Laura Hannibal, André Verméglio, Laurie Vuillet, Mila Kojadinovic, Stéphane Cruveiller, Sophie Mangenot, David W. Emerich, David Pignol, Gary Stacey, Valérie Barbe, Laboratoire des symbioses tropicales et méditerranéennes (UMR LSTM), Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Université Montpellier 1 (UM1)-Institut de Recherche pour le Développement (IRD)-Institut National de la Recherche Agronomique (INRA)-Université Montpellier 2 - Sciences et Techniques (UM2)-Université de Montpellier (UM)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro), Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro), Génomique métabolique (UMR 8030), Genoscope - Centre national de séquençage [Evry] (GENOSCOPE), 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-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-Centre National de la Recherche Scientifique (CNRS)-Université d'Évry-Val-d'Essonne (UEVE), Centre National de Séquençage, Université d'Évry-Val-d'Essonne (UEVE), department of soil, Biotechnology institute-Saint Mary's University of Minnesota, Laboratoire d'Intégration des Systèmes et des Technologies (LIST), Direction de Recherche Technologique (CEA) (DRT (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Structure et évolution des génomes (SEG), CNS-Université d'Évry-Val-d'Essonne (UEVE)-Centre National de la Recherche Scientifique (CNRS), National Center of Soybean Biotechnology, University of Missouri [Columbia] (Mizzou), University of Missouri System-University of Missouri System, Department of Energy / Joint Genome Institute (DOE), Los Alamos National Laboratory (LANL), Laboratoire d'Ecologie Microbienne - UMR 5557 (LEM), Institut National de la Recherche Agronomique (INRA)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Ecole Nationale Vétérinaire de Lyon (ENVL), Commisariat à lEnergie Atomique ((CEA)), Université de la Méditerranée - Aix-Marseille 2, Université Paris-Saclay-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 d'Évry-Val-d'Essonne (UEVE)-Centre National de la Recherche Scientifique (CNRS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay, University of Missouri [Columbia], Centre National de la Recherche Scientifique (CNRS)-Ecole Nationale Vétérinaire de Lyon (ENVL)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Recherche Agronomique (INRA)-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS), Laboratoire des symbioses tropicales et méditerranéennes ( LSTM ), Centre de Coopération Internationale en Recherche Agronomique pour le Développement ( CIRAD ) -Université Montpellier 1 ( UM1 ) -Institut National de la Recherche Agronomique ( INRA ) -Université Montpellier 2 - Sciences et Techniques ( UM2 ) -Université de Montpellier ( UM ) -Institut national d’études supérieures agronomiques de Montpellier ( Montpellier SupAgro ), Génomique métabolique ( UMR 8030 ), Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université d'Évry-Val-d'Essonne ( UEVE ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ), Université d'Évry-Val-d'Essonne ( UEVE ), Laboratoire d'Intégration des Systèmes et des Technologies ( LIST ), Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay, Structure et évolution des génomes ( SEG ), CNS-Université d'Évry-Val-d'Essonne ( UEVE ) -Centre National de la Recherche Scientifique ( CNRS ), University of Missouri-Columbia, Department of Energy / Joint Genome Institute ( DOE ), Los Alamos National Laboratory ( LANL ), Ecologie microbienne ( EM ), Centre National de la Recherche Scientifique ( CNRS ) -Ecole Nationale Vétérinaire de Lyon ( ENVL ) -Université Claude Bernard Lyon 1 ( UCBL ), Université de Lyon-Université de Lyon-Institut National de la Recherche Agronomique ( INRA ) -VetAgro Sup ( VAS ), Commisariat à lEnergie Atomique ( (CEA) ), Laboratoire d'Intégration des Systèmes et des Technologies (LIST (CEA)), and Université de Lyon-Université de Lyon-Ecole Nationale Vétérinaire de Lyon (ENVL)-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Lipopolysaccharides, Root nodule, Cytokinins, Bradyrhizobium japonicum, nodosité racinaire, [SDE.BE.ECO]Environmental Sciences/Biodiversity and Ecology/domain_sde.be.eco, Plant Roots, F30 - Génétique et amélioration des plantes, Nod factor, Bradyrhizobium, Photosynthesis, Genetics, 0303 health sciences, Multidisciplinary, biology, Plant Stems, Formation de nodosités, food and beverages, Fabaceae, Genomics, [ SDE.BE.ECO ] Environmental Sciences/Biodiversity and Ecology/domain_sde.be.eco, [ SDV.BBM.GTP ] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Root Nodules, Plant, Signal Transduction, Séquence nucléotidique, Rhizobiaceae, Molecular Sequence Data, Symbiose, N-Acetylglucosaminyltransferases, Rhizobia, Amidohydrolases, 03 medical and health sciences, Symbiosis, Bacterial Proteins, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Botany, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Rhizobactérie, [ SDV.BBM ] Life Sciences [q-bio]/Biochemistry, Molecular Biology, 030304 developmental biology, [ SDE.BE ] Environmental Sciences/Biodiversity and Ecology, Génome, 030306 microbiology, Mutant, P34 - Biologie du sol, Biologie moléculaire, biology.organism_classification, Genes, Bacterial, Purines, Mutation, [SDE.BE]Environmental Sciences/Biodiversity and Ecology, Acyltransferases, Genome, Bacterial
Abstract: Leguminous plants (such as peas and soybeans) and rhizobial soil bacteria are symbiotic partners that communicate through molecular signaling pathways, resulting in the formation of nodules on legume roots and occasionally stems that house nitrogen-fixing bacteria. Nodule formation has been assumed to be exclusively initiated by the binding of bacterial, host-specific lipochito-oligosaccharidic Nod factors, encoded by the nodABC genes, to kinase-like receptors of the plant. Here we show by complete genome sequencing of two symbiotic, photosynthetic, Bradyrhizobium strains, BTAi1 and ORS278, that canonical nodABC genes and typical lipochito-oligosaccharidic Nod factors are not required for symbiosis in some legumes. Mutational analyses indicated that these unique rhizobia use an alternative pathway to initiate symbioses, where a purine derivative may play a key role in triggering nodule formation.
Published: 2007

41. Crystal structures of an Extracytoplasmic Solute Receptor from a TRAP transporter in its open and closed forms reveal a helix-swapped dimer requiring a cation for alpha-keto acid binding

Author: Sophie Gonin, Jérôme Lavergne, David Pignol, Pascal Arnoux, Bénédicte Pierru, Béatrice Alonso, Monique Sabaty, Microbiologie Environnementale et Moléculaire (MEM), Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) (BIAM), 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)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Service de Biochimie et Toxicologie Nucléaire (SBTN), 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)), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)
Subjects: Conformational change, Stereochemistry, Dimer, MESH: Biological Transport, MESH: Keto Acids, MESH: Protein Structure, Secondary, ATP-binding cassette transporter, MESH: Carrier Proteins, Rhodobacter sphaeroides, Crystallography, X-Ray, Protein Structure, Secondary, MESH: Membrane Transport Proteins, 03 medical and health sciences, chemistry.chemical_compound, Bacterial Proteins, Structural Biology, Cations, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Binding site, MESH: Cations, lcsh:QH301-705.5, MESH: Bacterial Proteins, 030304 developmental biology, MESH: Crystallization, 0303 health sciences, biology, Chemistry, Membrane transport protein, 030302 biochemistry & molecular biology, Membrane Transport Proteins, RNA-Binding Proteins, Biological Transport, Periplasmic space, MESH: Rhodobacter sphaeroides, MESH: Transcription Factors, MESH: Crystallography, X-Ray, Keto Acids, Transport protein, Crystallography, MESH: RNA-Binding Proteins, lcsh:Biology (General), MESH: Dimerization, biology.protein, Carrier Proteins, Crystallization, Dimerization, Alpha helix, Transcription Factors, Research Article
Abstract: Background The import of solutes into the bacterial cytoplasm involves several types of membrane transporters, which may be driven by ATP hydrolysis (ABC transporters) or by an ion or H+ electrochemical membrane potential, as in the tripartite ATP-independent periplasmic system (TRAP). In both the ABC and TRAP systems, a specific periplasmic protein from the ESR family (Extracytoplasmic Solute Receptors) is often involved for the recruitment of the solute and its presentation to the membrane complex. In Rhodobacter sphaeroides, TakP (previously named SmoM) is an ESR from a TRAP transporter and binds α-keto acids in vitro. Results We describe the high-resolution crystal structures of TakP in its unliganded form and as a complex with sodium-pyruvate. The results show a limited "Venus flytrap" conformational change induced by substrate binding. In the liganded structure, a cation (most probably a sodium ion) is present and plays a key role in the association of the pyruvate to the protein. The structure of the binding pocket gives a rationale for the relative affinities of various ligands that were tested from a fluorescence assay. The protein appears to be dimeric in solution and in the crystals, with a helix-swapping structure largely participating in the dimer formation. A 30 Å-long water channel buried at the dimer interface connects the two ligand binding cavities of the dimer. Conclusion The concerted recruitment by TakP of the substrate group with a cation could represent a first step in the coupled transport of both partners, providing the driving force for solute import. Furthermore, the unexpected dimeric structure of TakP suggests a molecular mechanism of solute uptake by the dimeric ESR via a channel that connects the binding sites of the two monomers.
Published: 2007

42. 'Access to the Active Site of Periplasmic Nitrate Reductase: Insights from Site-Directed Mutagenesis and Zinc Inhibition Studies'

Author: Pascal Arnoux, Christophe Léger, Monique Sabaty, Bruno Guigliarelli, Bénédicte Burlat, Sandrine Grosse, David Pignol, Bettina Frangioni, Sébastien Dementin, Bioénergétique et Ingénierie des Protéines (BIP ), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Microbiologie Environnementale et Moléculaire (MEM), 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), Interactions Protéine Métal (IPM), Aix Marseille Université (AMU), Centre de biophysique moléculaire (CBM), Université d'Orléans (UO)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-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), and Université d'Orléans (UO)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)
Subjects: [SDV]Life Sciences [q-bio], Rhodobacter sphaeroides, 010402 general chemistry, Nitrate reductase, 01 natural sciences, Biochemistry, Nitrate Reductase, Cofactor, 03 medical and health sciences, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Binding site, Site-directed mutagenesis, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, Molybdenum, 0303 health sciences, DMSO reductase, Binding Sites, Nitrates, biology, Chemistry, Active site, Periplasmic space, biology.organism_classification, 0104 chemical sciences, Zinc, Mutation, Periplasm, biology.protein, Mutagenesis, Site-Directed, Oxidation-Reduction, Protein Binding
Abstract: The periplasmic nitrate reductase (NapAB), a member of the DMSO reductase superfamily, catalyzes the first step of the denitrification process in bacteria. In this heterodimer, a di-heme NapB subunit is associated to the catalytic NapA subunit that binds a [4Fe-4S] cluster and a bis(molybdopterin guanine dinucleotide) cofactor. Here, we report the kinetic characterization of purified mutated heterodimers from Rhodobacter sphaeroides. By combining site-directed mutagenesis, redox potentiometry, EPR spectroscopy, and enzymatic characterization, we investigate the catalytic role of two conserved residues (M153 and R392) located in the vicinity of the molybdenum active site. We demonstrate that M153 and R392 are involved in nitrate binding: the Vm measured on the M153A and R392A mutants are similar to that measured on the wild-type enzyme, whereas the Km for nitrate is increased 10-fold and 200-fold, respectively. The use of an alternative enzymatic assay led us to discover that NapAB is uncompetitively inhibited by Zn2+ ions (Ki' = 1 microM). We used this property to further probe the active site access in the mutant enzymes. It is proposed that R392 acts as a filter by preventing a direct reduction of the Mo atom by small reducing molecules and partially protecting the active site against zinc inhibition. In addition, we show that M153 is a key residue mediating this inhibition likely by coordinating Zn2+ ions via its sulfur atom. This residue is not conserved in the DMSO reductase superfamily while it is conserved in the periplasmic nitrate reductase family. Zinc inhibition is therefore likely to be specific and restricted to periplasmic nitrate reductases.
Published: 2007

43. 'Effects of slow substrate binding and release in redox enzymes: theory and application to periplasmic nitrate reductase.'

Author: Christophe Léger, Bruno Guigliarelli, David Pignol, Bettina Frangioni, Patrick Bertrand, Sébastien Dementin, Monique Sabaty, Pascal Arnoux, Bioénergétique et Ingénierie des Protéines (BIP ), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Microbiologie Environnementale et Moléculaire (MEM), Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) (BIAM), 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)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-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)
Subjects: [SDV]Life Sciences [q-bio], Inorganic chemistry, Rhodobacter sphaeroides, Flavin group, 010402 general chemistry, Nitrate Reductase, 01 natural sciences, Redox, Catalysis, Cofactor, 03 medical and health sciences, Bacterial Proteins, Materials Chemistry, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Physical and Theoretical Chemistry, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, 0303 health sciences, Binding Sites, Nitrates, biology, Chemistry, Active site, Substrate (chemistry), Periplasmic space, 0104 chemical sciences, Surfaces, Coatings and Films, Models, Chemical, Periplasm, biology.protein, Biophysics, Oxidation-Reduction, Electrode potential
Abstract: For redox enzymes, the technique called protein film voltammetry makes it possible to determine the entire profile of activity against driving force by having the enzyme exchanging directly electrons with the rotating-disc electrode onto which it is adsorbed. Both the potential location of the catalytic response and its detailed shape report on the sequence of catalytic events, electron transfers and chemical steps, but the models that have been used so far to decipher this signal lack generality. For example, it was often proposed that substrate binding to multiple redox states of the active site may explain that turnover is greater in a certain window of electrode potential, but no fully analytical treatment has been given. Here, we derive (i) the general current equation for the case of reversible substrate binding to any redox states of a two-electron active site (as exemplified by flavins and Mo cofactors), (ii) the quantitative conditions for an extremum in activity to occur, and (iii) the expressions from which the substrate-concentration dependence of the catalytic potential can be interpreted to learn about the kinetics of substrate binding and how this affects the reduction potential of the active site. Not only does slow substrate binding and release make the catalytic wave shape highly complex, but we also show that it can have important consequences which will escape detection in traditional experiments: the position of the wave (this is the driving force that is required to elicit catalysis) departs from the reduction potential of the active site even at the lowest substrate concentration, and this deviation may be large if substrate binding is irreversible. This occurs in the reductive half-cycle of periplasmic nitrate reductase where irreversibility lowers the driving force required to reduce the active site under turnover conditions and favors intramolecular electron transfer from the proximal [4Fe4S]+ cluster to the active site Mo(V).
Published: 2007

44. A New Type of Bacteriophytochrome Acts in Tandem with a Classical Bacteriophytochrome to Control the Antennae Synthesis in Rhodopseudomonas palustris

Author: Laure Hannibal, David Pignol, Jean-Marc Adriano, Eric Giraud, Sébastien Zappa, Joël Fardoux, Catherine Berthomieu, Pierre Bouyer, Laurie Vuillet, André Verméglio, Biologie cellulaire et moléculaire des plantes et des bactéries (BCMPB), Université de la Méditerranée - Aix-Marseille 2-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), and CEA - Cadarache, Laboratoire de Bioénergétique Cellulaire (UMR 6191)
Subjects: food.ingredient, Time Factors, Photoisomerization, Light, Biochemical Phenomena, [SDV]Life Sciences [q-bio], Molecular Sequence Data, Photosynthetic Reaction Center Complex Proteins, Biophysics, Photochemistry, Biochemistry, Biophysical Phenomena, Light-harvesting complex, 03 medical and health sciences, food, Spectroscopy, Fourier Transform Infrared, Amino Acid Sequence, Cysteine, Phosphorylation, Molecular Biology, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, 0303 health sciences, Phytochrome, biology, Sequence Homology, Amino Acid, 030302 biochemistry & molecular biology, Cell Biology, Rhodopseudomonas, Chromophore, biology.organism_classification, Recombinant Proteins, Protein Structure, Tertiary, Light intensity, Response regulator, Phenotype, Spectrometry, Fluorescence, Models, Chemical, Spectrophotometry, Mutation, Electrophoresis, Polyacrylamide Gel, Rhodopseudomonas palustris
Abstract: Phytochromes are chromoproteins found in plants and bacteria that switch between two photointerconvertible forms via the photoisomerization of their chromophore. These two forms, Pr and Pfr, absorb red and far- red light, respectively. We have characterized the biophysical and biochemical properties of two bacteriophytochromes, RpBphP2 and RpBphP3, from the photosynthetic bacterium Rhodopseudomonas palustris. Their genes are contiguous and localized near the pucBAd genes encoding the polypeptides of the light harvesting complexes LH4, whose synthesis depends on the light intensity. At variance with all ( bacterio) phytochromes studied so far, the light-induced isomerization of the chromophore of RpBphP3 converts the Pr form to a form absorbing at shorter wavelength around 645 nm, designated as Pnr for near red. The quantum yield for the transformation of Pr into Pnr is about 6-fold smaller than for the reverse reaction. Both RpBphP2 and RpBphP3 auto-phosphorylate in their dark-adapted Pr forms and transfer their phosphate to a common response regulator Rpa3017. Under semi-aerobic conditions, LH4 complexes replace specifically the LH2 complexes in wild-type cells illuminated by wavelengths comprised between 680 and 730 nm. In contrast, mutants deleted in each of these two bacteriophytochromes display no variation in the composition of their light harvesting complexes whatever the light intensity. From both the peculiar properties of these bacteriophytochromes and the phenotypes of their deletion mutants, we propose that they operate in tandem to control the synthesis of LH4 complexes by measuring the relative intensities of 645 and 710 nm lights.
Published: 2005

45. Identification of the Ricin Lipase Site and Implication in Cytotoxicity

Author: David Pignol, Juliette Morlon-Guyot, Bruno Beaumelle, Mohamed Helmy, Sophie Lombard-Frasca, Gérard Pieroni, Dynamique moléculaire des interactions membranaires (DMIM), Centre National de la Recherche Scientifique (CNRS)-Université Montpellier 2 - Sciences et Techniques (UM2), Nutrition humaine et lipides : Biodisponibilité, métabolisme et régulation, Université de la Méditerranée - Aix-Marseille 2-Institut National de la Recherche Agronomique (INRA)-Université de Provence - Aix-Marseille 1-IFR125-Institut National de la Santé et de la Recherche Médicale (INSERM), CEA Cadarache, Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Université Montpellier 2 - Sciences et Techniques (UM2)-Centre National de la Recherche Scientifique (CNRS), and Herrada, Anthony
Subjects: Models, Molecular, endocrine system, Protein Conformation, RT B-chain, Ricin, Endocytosis, tetramethylrhodamine isothiocyanate, Biochemistry, 03 medical and health sciences, chemistry.chemical_compound, rRTA, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Lipase, ITs, Cytotoxicity, Molecular Biology, 030304 developmental biology, 0303 health sciences, biology, Ribosome-inactivating protein, TRITC, 030302 biochemistry & molecular biology, Lectin, recombinant RTA, Cell Biology, Molecular biology, carbohydrates (lipids), Enzyme Activation, Cytosol, Kinetics, Protein Subunits, immunotoxins, RTA, chemistry, RTB, RIP, biology.protein, Abrin, ribosome-inactivating protein, ricin toxin A-chain
Abstract: International audience; Ricin is a heterodimeric plant toxin and the prototype of type II ribosome-inactivating proteins. Its B-chain is a lectin that enables cell binding. After endocytosis, the A-chain translocates through the membrane of intracellular compartments to reach the cytosol where its N-glycosidase activity inactivates ribosomes, thereby arresting protein synthesis. We here show that ricin possesses a functional lipase active site at the interface between the two subunits. It involves residues from both chains. Mutation to alanine of catalytic serine 221 on the A-chain abolished ricin lipase activity. Moreover, this mutation slowed down the A-chain translocation rate and inhibited toxicity by 35%. Lipase activity is therefore required for efficient ricin A-chain translocation and cytotoxicity. This conclusion was further supported by structural examination of type II ribosome-inactivating proteins that showed that this lipase site is present in toxic (ricin and abrin) but is altered in nontoxic (ebulin 1 and mistletoe lectin I) members of this family.
Published: 2003

46. Neutron crystallographic evidence of lipase-colipase complex activation by a micelle

Author: Juan A. Hermoso, Catherine Chapus, Simon Penel, Juan C. Fontecilla-Camps, Michel Roth, David Pignol, Laboratoire de Biométrie et Biologie Evolutive - UMR 5558 (LBBE), Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Université de Lyon-Institut National de Recherche en Informatique et en Automatique (Inria)-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Models, Molecular, [SDV.OT]Life Sciences [q-bio]/Other [q-bio.OT], Protein Conformation, Swine, Neutron diffraction, Colipase, Micelle, General Biochemistry, Genetics and Molecular Biology, Protein Structure, Secondary, 03 medical and health sciences, Protein structure, Hydrolase, Animals, Colipases, Horses, Lipase, Molecular Biology, Ternary complex, Micelles, 030304 developmental biology, Neutrons, 0303 health sciences, Binding Sites, Crystallography, General Immunology and Microbiology, biology, General Neuroscience, 030302 biochemistry & molecular biology, Active site, Enzyme Activation, Models, Chemical, biology.protein, Research Article
Abstract: The concept of lipase interfacial activation stems from the finding that the catalytic activity of most lipases depends on the aggregation state of their substrates. It is thought that activation involves the unmasking and structuring of the enzyme's active site through conformational changes requiring the presence of oil-in-water droplets. Here, we present the neutron structure of the activated lipase-colipase-micelle complex as determined using the D2O/H2O contrast variation low resolution diffraction method. In the ternary complex, the disk-shaped micelle interacts extensively with the concave face of colipase and the distal tip of the C-terminal domain of lipase. Since the micelle- and substrate-binding sites concern different regions of the protein complex, we conclude that lipase activation is not interfacial but occurs in the aqueous phase and is mediated by colipase and a micelle.
Published: 1997

47. Crystal structure of bovine procarboxypeptidase A-S6 subunit III, a highly structured truncated zymogen E

Author: David Pignol, T. Michon, Christine Gaboriaud, Catherine Chapus, Brigitte Kerfelec, Juan-Carlos Fontecilla-Camps, and Institut National de la Recherche Agronomique (INRA)
Subjects: Models, Molecular, Proteases, Stereochemistry, Protein Conformation, Protein subunit, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Crystallography, X-Ray, General Biochemistry, Genetics and Molecular Biology, Serine, 03 medical and health sciences, Motion, Protein structure, Multienzyme Complexes, Zymogen, Animals, Molecular Biology, Ternary complex, Pancreas, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, Serine protease, 0303 health sciences, Enzyme Precursors, General Immunology and Microbiology, biology, General Neuroscience, 030302 biochemistry & molecular biology, Serine Endopeptidases, Hydrogen Bonding, Carboxypeptidase, Enzyme Activation, Biochemistry, biology.protein, Cattle, Research Article
Abstract: Subunit III, a defective serine endopeptidase lacking the typical N-terminal hydrophobic dipeptide is secreted by the pancreas of ruminant species as part of the bovine ternary complex procarboxypeptidase A-S6. Two monoclinic crystal forms were obtained and subsequently used to solve its X-ray structure. The highest resolution model of subunit III was refined at 1.7 A resolution to a crystallographic R-factor of 18.4%, with r.m.s. bond deviations from ideality of 0.012 A. About 80% of the model presents the characteristic architecture of trypsin-like proteases. The remaining zones, however, have well-defined, unique conformations. The regions from residues 70 to 80 and from 140 to 155 present maximum distances of 16 and 18 A relative to serine proteases and zymogens. Comparisons with the structures of porcine elastase 1 and chymotrypsinogen A indicate that the specific binding pocket of subunit III adopts a zymogen-like conformation and thus provide a basis for its inactivity. In general, the structural analysis of subunit III strongly suggests that it corresponds to a truncated version of a new class of highly structured elastase-like zymogen molecules. Based on the structures of subunit III and elastase 1, it is concluded that large concerted movements are necessary for the activation of zymogen E.
Published: 1994

48. Major Mo(V) EPR Signature of Rhodobacter sphaeroidesPeriplasmic Nitrate Reductase Arising from a Dead-End Species That Activates upon Reduction. Relation to Other Molybdoenzymes from the DMSO Reductase Family.

Author: Vincent Fourmond, Bénédicte Burlat, Sébastien Dementin, Pascal Arnoux, Monique Sabaty, Séverine Boiry, Bruno Guigliarelli, Patrick Bertrand, David Pignol, and Christophe Léger
Published: 2008
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49. Effects of Slow Substrate Binding and Release in Redox Enzymes: Theory and Application to Periplasmic Nitrate Reductase.

Author: Patrick Bertrand, Bettina Frangioni, Sébastien Dementin, Monique Sabaty, Pascal Arnoux, Bruno Guigliarelli, David Pignol, and Christophe Léger
Published: 2007
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50. Bacteriophytochrome and regulation of the synthesis of the photosynthetic apparatus in Rhodopseudomonas palustris: pitfalls of using laboratory strains.

Author: Eric Giraud, Sébastien Zappa, Marianne Jaubert, Laure Hannibal, Joël Fardoux, Jean-Marc Adriano, Pierre Bouyer, Bernard Genty, David Pignol, and André Verméglio
Published: 2004

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