Location of glycosidases and two xylanases in strictly anaerobic rumen fungi

International audienc

Comparaison du peuplement fogique du rumen, du duodénum et des fèces de mouton

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Full capacity of recombinant Escherichia coli heat-stable enterotoxin fusion proteins for extracellular secretion, antigenicity, disulfide bond formation, and activity.

International audienceWe have successfully used the major subunit ClpG of Escherichia coli CS31A fimbriae as an antigenic and immunogenic exposure-delivery vector for various heterologous peptides varying in nature and length. However, the ability of ClpG as a carrier to maintain in vitro and in vivo the native biological properties of passenger peptide has not yet been reported. To address this possibility, we genetically fused peptides containing all or part of the E. coli human heat-stable enterotoxin (STh) sequence to the amino or carboxyl ends of ClpG. Using antibodies to the ClpG and STh portions for detecting the hybrids; AMS (4-acetamido-4'-maleimidylstilbene-2, 2'-disulfonate), a potent free thiol-trapping reagent, for determining the redox state of STh in the fusion; and the suckling mouse assay for enterotoxicity, we demonstrated that all ClpG-STh proteins were secreted in vitro and in vivo outside the E. coli cells in a heat-stable active oxidized (disulfide-bonded) form. Indeed, in contrast to many earlier studies, blocking the natural NH(2) or COOH extremities of STh had, in all cases, no drastic effect on cell release and toxin activity. Only antigenicity of STh C-terminally extended with ClpG was strongly affected in a conformation-dependent manner. These results suggest that the STh activity was not altered by the chimeric structure, and therefore that, like the natural toxin, STh in the fusion had a spatial structure flexible enough to be compatible with secretion and enterotoxicity (folding and STh receptor recognition). Our study also indicates that disulfide bonds were essential for enterotoxicity but not for release, that spontaneous oxidation by molecular oxygen occurred in vitro in the medium, and that the E. coli cell-bound toxin activity in vivo resulted from an effective export processing of hybrids and not cell lysis. None of the ClpG-STh subunits formed hybrid CS31A-STh fimbriae at the cell surface of E. coli, and a strong decrease in the toxin activity was observed in the absence of CS31A helper proteins. In fact, chimeras translocated across the outer membrane as a free folded monomer once they were guided into the periplasm by the ClpG leader peptide through the CS31A-dependent secretory pathway. In summary, ClpG appears highly attractive as a carrier reporter protein for basic and applied research through the engineering of E. coli for culture supernatant delivery of an active cysteine-containing protein, such as the heat-stable enterotoxin

Caractérisation de champignons polycentriques du rumen observés in vivo

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Microstructure de la mayonnaise = Microstructure of mayonnaise

Mayonnaise is a very popular sauce widely consumed all over the world. Crucial properties of mayonnaise for consumers such as stability, texture, colour and aspect depend on microstructure of the emulsion. The aim of this study was to characterize the microstructure of mayonnaise.Mayonnaise was produced in an agitated vessel (Rayneri Turbotest 33P/750 equipped with a defloculeuse turbine) controlled in velocity (300-3300 rot/min) and temperature. Microstructure was studied using Laser granulometry, scanning electron microscopy (SEM) and environmental microscopy.For given operating conditions, the comparison of Sauter mean diameter measured by Laser granulometry in two dilution media (water and SDS) has shown that the size of particle is higher in water (7.3 µm) than in SDS (2.2 µm) leading to the hypothesis of aggregation of oil droplets. The observation of images obtained by scanning electron microscopy has confirmed the presence of aggregates and has shown that they were arranged along a fractal pattern.La mayonnaise est une sauce très populaire consommée dans de nombreux pays. Pour le consommateur, des propriétés importantes telles que la stabilité, la texture, la couleur et l'aspect dépendent de la microsctructure de l'émulsion. L'objectif de l'étude était de caractériser la microstructure de la mayonnaise. La mayonnaise a été produite dans une cuve agitée (Rayneri Turbotest 33P/750 muni d'une turbine defloculeuse) contrôlée en vitesse (300-3300 tours/min) et température. Trois méthodes ont été utilisées pour étudier la microstructure : la granulométrie Laser, la microscopie électronique à balayage (MEB) et la microscopie environnementale. Pour des conditions opératoires fixées, la comparaison du diamètre moyen de Sauter mesuré par granulométrie Laser dans deux milieux diluants (eau et SDS) a montré que la taille des particules est plus élevée dans l'eau (7.3 µm) que dans le SDS (2.2 µm), conduisant à l'hypothèse d'une agrégation des gouttelettes d'huile. L'observation des images obtenues en MEB a confirmé la présence d'agrégats et a montré une organisation suivant une structure fractale

Isolation of the wheat aleurone layer for 2D electrophoresis and proteomics analysis

Whereas the endosperm of bread wheat has been studied for many years for obvious ecoomic reasons, studies of the aleurone layer of the seed only started recently after the discovery of its nutritional and health benefits. In this paper, we describe two different techniques to isolate either the peripheral layers including the aleurone layer or only the aleurone layer (AL) which can be used for 2D electrophoresis and proteomic analysis. The two techniques provided similar 2D electrophoresis profiles although the time needed for dissection of the kernel and isolation of the cell layer was different. The two 2D protein profiles shared more than 80% identity and enabled us to observe approximately 700 spots in the aleurone layer. Two bread wheat cultivars, Chinese Spring and Recital, were used and the two techniques revealed that their AL shared at least 70% of common spots. Several spots not present in AL and coming from peripheral layers were identified using mass spectrometry and database interrogation. These dissection techniques will enable proteomic analysis of AL which can be used for genetic analysis of its components, for investigating the AL response to fungi attack and helpful for improving nutritional and health value of wheat

Surface properties and behaviour on abiotic surfaces of Staphylococcus carnosus, a genetically homogeneous species

This work aimed to characterize the surface properties of Staphylococcus carnosus and the influence of different media on their ability to adhere and grow on industrial supports. As their colonization could be dependant of the strain, the genetic diversity of the strains was studied. The diversity of 13 strains analysed by pulsed-field gel electrophoresis revealed that the S. carnosus strains formed a homogeneous genetic group. Their surface properties, characterized by studying their affinity to solvents, were hydrophilic with a strong negative surface charge. The S. carnosus strain CIT 833 hardly adhered to polytetrafluoroethylene (PTFE) and stainless steel chips. Tryptic soy broth (TSB) was the most favourable medium for growth on stainless steel support while TSB/NaCl was better for growth on PTFE. Scanning electron microscopy (sem) showed that this strain weakly colonized both supports and did not form cell aggregates. Indeed, the strain did not synthesize polysaccharides. These results showed that S. carnosus adhered on different abiotic surfaces which are used in food factories but was not able to accumulate on these surfaces. The inability of S. carnosus to form biofilm could explain why S. carnosus is rarely isolated in meat processing environment. © 2006 Elsevier Ltd. All rights reserved.Fil: Planchon, S.. Institut National de la Recherche Agronomique; FranciaFil: Gaillard Martinie, B.. Institut National de la Recherche Agronomique; FranciaFil: Leroy, S.. Institut National de la Recherche Agronomique; FranciaFil: Bellon Fontaine, M.N.. Institut National de la Recherche Agronomique; FranciaFil: Fadda, Silvina G.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Talon, R.. Institut National de la Recherche Agronomique; Franci