Structural Basis for the Regulation Mechanism of the Tyrosine Kinase CapB from Staphylococcus aureus

Bacteria were thought to be devoid of tyrosine-phosphorylating enzymes. However, several tyrosine kinases without similarity to their eukaryotic counterparts have recently been identified in bacteria. They are involved in many physiological processes, but their accurate functions remain poorly understood due to slow progress in their structural characterization. They have been best characterized as copolymerases involved in the synthesis and export of extracellular polysaccharides. These compounds play critical roles in the virulence of pathogenic bacteria, and bacterial tyrosine kinases can thus be considered as potential therapeutic targets. Here, we present the crystal structures of the phosphorylated and unphosphorylated states of the tyrosine kinase CapB from the human pathogen Staphylococcus aureus together with the activator domain of its cognate transmembrane modulator CapA. This first high-resolution structure of a bacterial tyrosine kinase reveals a 230-kDa ring-shaped octamer that dissociates upon intermolecular autophosphorylation. These observations provide a molecular basis for the regulation mechanism of the bacterial tyrosine kinases and give insights into their copolymerase function

Biodisponobilité du cuivre dans la rhizosphère de différentes plantes cultivées. Cas de sols viticoles contaminés par de fongicides

@L'accumulation de Cu constatée dans les couches superficielles des sols viticoles, suite aux apports répétés de fongicides cupriques, peut être à l'origine d'une contamination des plantes cultivées dans ces sols . Dans ce contexte, notre objectif a été d'évaluer les facteurs du sol et de la rhizosphère contrôlant la biodisponibilité de Cu pour différentes espèces cultivées sur des sols acides et calcaires viticoles du Languedoc Roussillon . Pour cela, des cultures-tests (biotests), conduites au laboratoire, ont été réalisées dans des dispositifs particuliers offrant un accès aisé aux racines et au sol rhizosphérique. Les teneurs de Cu mesurées dans les parties aériennes n'ont jamais atteint les seuils critiques de phytotoxicité définis dans la littérature. Les plus fortes teneurs en Cu ont été mesurées dans les racines et majoritairement dans l'espace intercellulaire (apoplasme). Contrairement aux teneurs en Cu dans les parties aériennes,..AIX-MARSEILLE3-BU Sc.St Jérô (130552102) / SudocSudocFranceF

De la biodégradation à la conception d'un bio-capteur (exemple du 3,5-dinitrotrifluorométhylbenzène)

Le 3,5-dinitrotrifluorométhylbenzène 3,5-DNBTF est un composé utilisé par le CEA. Sa détection spécifique constitue une priorité sur les plans industriel et environnemental. Dans cette perspective, nous avons étudié la conception d'un biocapteur spécifique de ce composé. Nous avons tout d'abord mis au point des méthodes d'analyse du 3,5-DNBTF et de ses dérivés en solution. Étant donné la très faible solubilité dans l'eau du composé étudié, nous avons été amené à mettre au point une méthode HPLC couplé à la spectrométrie de masse pour identifier les dérivés et métabolites du 3,5-DNBTF. Afin d'orienter le choix des réactions enzymatiques susceptibles d'affecter le 3,5-DNBTF, nous avons exploré sa réactivité chimique. Seuls les groupements nitro se sont avérés réactifs, et conduisent par réduction au 3,5-diaminotrifluorométhylbenzene (3,5-DABTF). Cette molécule est le siège d'une réactivité photochimique originale. En effet, l'irradiation à 300nm du 3,5-DABTF dans l'eau conduit à la formation de l'acide 3,5-diaminobenzoïque, mais également à la formation de différents polymères, qui se forment grâce à des liaisons amides entre les amines et les intermédiaires de défluorination. L'étude de la dégradation microbiologique du 3,5-DNBTF a permis la sélection d'une souche de Bacillus (Bacillus LMA), seule capable de réduire les deux groupements nitro du 3,5-DNBTF avec une grande efficacité. La purification, couplée à la caractérisation par spectrométrie de masse, a permis d'identifier une nitroréductase de Bacillus subtilis. Celle-ci a été clonée et sur-exprimée chez E.coli avec succès, permettant ainsi un accès facile à des quantités importantes de protéine active. La double nitro-réduction du 3,5-DNBTF, catalysée par un microorganisme entier ou par la nitroréductase purifiée, a été couplée à la transformation photochimique de la diamine correspondante. Ceci est à la base de la conception du biocapteur. Plusieurs moyens de détection ont été évalués, la formation du 3,5-DABA et la possibilité d'y associer une émission de fluorescence, la chute du pH au cours de la réaction, la production d'ions fluorure ou la coloration de la solution après irradiation. L'évaluation d'un prototype expérimental a été engagée.The 3,5-dinitrotrifluoromethylbenzene (3,5-DNBTF) is currently evaluated by the (Commissariat a l'Energie Atomique). The challenge was the conception of a specific biosensor, based on enzymatic or microbial interactions. This will help monitoring of the compound for either environmental protection and security. Our first investigations were focused on the analytical methods for the detection of 3,5-DNBTF metabolites and derivatives in solution. Taken into account thee weak solubility of these molecules in water, we used a sensitive chromatographic HPLC technique coupled to mass spectrometry on APCI ionization, to assign the structure of newly observed compounds. We first investigate the chemical reactivity of 3,5-DNBTF. Only nitro substituants can be transformed to their respective diamino derivatives through catalytic hydrogenation. This molecule give rise to original photochemical reactivity. Indeed, the photo-irradiation at 310nm of the 3,5-DABTF in water lead to 3,5-diaminobenzoïc acid, as well as various polymers diriving from the interaction between 3,5-DABTF and some defluorination intermediates. A screnning was conducted on the biodegradation of 3,5-DNBTF, that allows the selection of a bacterial strain of Bacillus (Bacillus LMA), that was the solely strain to reduce both nitro groups of the 3,5-DNBTF with high efficiency. The purification of the nitroréductase involved in this reaction was investigated by classical chromatographic method, as well as an affinity chromatography process. This latter was based on a specific matrice coupled with a substrate analog, 3,5-dinitrobenzoic acid (3,5-DNBA). Mass spectrometry of the protein spots lead to the identification of a putative nitroreductase from Bacillus subtilis168. The corresponding gene was cloned and has been over-expressed in E.coli successfully. The presence of a His-tag on the protein lead to a large quantities of purified active protein. The total nitro-reduction of 3,5-DNBTF, catalyzed by a whole microorganism or by the purified nitroreductase, has been coupled to the photochemical transformation of the corresponding diamine. This is the basis of our biosensor. The detection was oriented against Several parameters: the formation of the 3,5-DABA, that could be fluorescent under particular conditions, the decrease in pH during the reaction, the production of fluoride ions and the dark colour associated to particular, polymers formed during irradiation. Last phases in biosensor construction are under study.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Fe-deficiency increases Cu acquisition by wheat cropped in a Cu-contaminated vineyard soil

International audienc

Copper bioavailability and rhizosphere pH changes as affected by nitrogen supply for tomato and oilseed rape cropped on an acidic and a calcareous soil

International audienc

Copper availability and bioavailability are controlled by rhizosphere pH in rape grown in an acidic Cu-contaminated soil

Correspondance auteur: P. Hinsinger e-mail: [email protected] audienceWe evaluated how root-induced changes in rhizosphere pH varied and interacted with Cu availability and bioavailability in an acidic soil. Rape was grown on a Cu-contaminated acidic soil, which had been limed at 10 rates. Soil Cu bioavailability was not influenced by liming. However, liming significantly decreased CaCl2-extracted Cu for pH between 3.7 and 5.1. Little effect was found for pH above 5.1. For soil pH < 4.4, CaCl2-Cu contents were smaller in rhizosphere than uncropped soil. Rhizosphere alkalisation occurred at pH < 4.8, while acidification occurred at greater pH. This explained the changes of CaCl2-Cu in the rhizosphere at low pH and the absence of pH dependency of Cu bioavailability to rape. In addition, apoplastic Cu in roots increased with increasing soil pH, most probably as a result of increased dissociation and affinity of cell wall compounds for C

NADH oxidase activity of Bacillus subtilis nitroreductase NfrA1: Insight into its biological role

AbstractNfrA1 nitroreductase from the Gram-positive bacterium Bacillus subtilis is a member of the NAD(P)H/FMN oxidoreductase family. Here, we investigated the reactivity, the structure and kinetics of NfrA1, which could provide insight into the unclear biological role of this enzyme. We could show that NfrA1 possesses an NADH oxidase activity that leads to high concentrations of oxygen peroxide and an NAD+ degrading activity leading to free nicotinamide. Finally, we showed that NfrA1 is able to rapidly scavenge H2O2 produced during the oxidative process or added exogenously.Structured summaryMINT-7990140: nfrA1 (uniprotkb:P39605) and nfrA1 (uniprotkb:P39605) bind (MI:0407) by X-ray crystallography (MI:0114

The Reductive Dehydroxylation Catalyzed by IspH, a Source of Inspiration for the Development of Novel Anti-Infectives

International audienceThe non-mevalonate or also called MEP pathway is an essential route for the biosynthesis of isoprenoid precursors in most bacteria and in microorganisms belonging to the Apicomplexa phylum, such as the parasite responsible for malaria. The absence of this pathway in mammalians makes it an interesting target for the discovery of novel anti-infectives. As last enzyme of this pathway, IspH is an oxygen sensitive [4Fe-4S] metalloenzyme that catalyzes 2H+/2e- reductions and a water elimination by involving non-conventional bioinorganic and bioorganometallic intermediates. After a detailed description of the discovery of the [4Fe-4S] cluster of IspH, this review focuses on the IspH mechanism discussing the results that have been obtained in the last decades using an approach combining chemistry, enzymology, crystallography, spectroscopies, and docking calculations. Considering the interesting druggability of this enzyme, a section about the inhibitors of IspH discovered up to now is reported as well. The presented results constitute a useful and rational help to inaugurate the design and development of new potential chemotherapeutics against pathogenic organisms