The Yin-Yang of the Green Fluorescent Protein:Impact on Saccharomyces cerevisiae stress resistance

International audienceAlthough fluorescent proteins are widely used as biomarkers (Yin), no study focuses on their influence on the microbial stress response. Here, the Green Fluorescent Protein (GFP) was fused to two proteins of interest in Saccharomyces cerevisiae. Pab1p and Sur7p, respectively involved in stress granules structure and in Can1 membrane domains. These were chosen since questions remain regarding the understanding of the behavior of S. cerevisiae facing different heat kinetics or oxidative stresses. The main results showed that Pab1p-GFP fluorescent mutant displayed a higher resistance than that of the wild type under a heat shock. Moreover, fluorescent mutants exposed to oxidative stresses displayed changes in the cultivability compared to the wild type strain. In silico approaches showed that the presence of the GFP did not influence the structure and so the functionality of the tagged proteins meaning that changes in yeast resistance were certainly related to GFP ROS-scavenging ability (Yang)

La phytase de Debaryomyces castellii CBS 2923 (surexpression, propriétés catalytiques et structure)

Résumé en françaisRésumé en anglaisMONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

Design of chromogenic probes for efficient screening and evaluation of feruloyl esterase-like activities

International audienc

Survie et/ou réactivation de microorganismes du sol sous hautes pressions gazeuses

National audienceLe sol constitue une réserve considérable de microorganismes représentatifs de nombreuses communautés cellulaires. Les recherches reposent sur l’hypothèse qu’il existe, dans les échantillons de sol, des communautés microbiennes adaptées à des conditions physiques (pression, température) très différentes de celles rencontrées dans nos laboratoires : soit parce que les conditions de milieu ont changé (sol), soit parce que les conditions de prélèvement sont très différentes des conditions de culture (grands fonds marins). Dans cette optique, l’utilisation des hautes pressions gazeuses (200-1000 bars dans un premier temps) est envisagée afin d’essayer de réactiver des microorganismes dormants que l’on ne sait pas cultiver. La découverte et la culture de ces microorganismes pourrait permettre de découvrir des formes microbiennes avec des adaptations qu’il serait possible d’utiliser à des fins de connaissance sur la plasticité cellulaire ou d’exploiter industriellement certains éléments spécifiques du métabolisme (enzymes, produits). L’objectif de la présente étude est de valider l’utilisation de procédés originaux de culture sous Hautes Pressions. La maîtrise de ces procédés devrait permettre d’approcher les conditions optimales de culture par l’utilisation d’enceintes Hautes Pressions spécifiques : (1) Enceintes « aveugles » : elles permettent de placer des échantillons sous pression et de maintenir cette pression constante au cours de l’incubation mais ne permettent pas de visualiser la croissance des microorganismes lors du traitement hyperbare. Elles possèdent un volume utile d’environ 100 cm3 permettant l’introduction de 3 petites boîtes de Petri (4 cm de diamètre) et résistent à des pressions allant de 250 à 2000 bars selon le modèle de l’enceinte. (2) Enceintes « optiques » ou cellules de visualisation : elles permettent de visualiser les échantillons et de suivre, en temps réel, la croissance microbienne au cours du traitement hyperbare. Elles peuvent être utilisées pour réaliser des cultures sous pression gazeuse isostatique en milieu solide ou sous pression hydrostatique en milieu liquide. La présence d’une double enveloppe permet de réguler la température d’incubation. Cette cellule de visualisation peut résister à des pressions allant jusqu’à 4000 bars. Dans un premier temps, la levure Saccharomyces cerevisiae a été utilisée comme microorganisme modèle. Son taux de croissance a été mesuré entre 1 et 500 bars dans ces deux types d’enceintes. Une étude comparative a été menée afin de retenir l’enceinte la plus adaptée au suivi de la croissance microbienne. Dans un second temps, des échantillons de différents sols (terrestre et marin) ont été récupérés afin d’identifier les populations microbiennes piézotolérantes et/ou piézophiles éventuelles. Les échantillons de sol terrestre proviennent du domaine de Bretenières (I.N.R.A., Dijon) et les échantillons de sol marin ont été prélevés dans deux carottes profondes du Bassin Indien Central (Océan Indien) à plus de 5000 m de profondeur et ont été gracieusement fournis par la carothèque océanique du Muséum National d’Histoire Naturelle

High gas pressure survival/reactivation of soil microorganisms

International audienceDeep sea sediments constitute a considerable reserve of microorganisms belonging to different microbial communities. Our researches aimed to better understand cellular mechanisms related to cellular plasticity involved in resistance of such microbial communities to extreme conditions and more particularly to high level of pressure (> 50 MPa). Obviously, the first step is to isolate microorganisms present in deep sea sediments and then cultivate. The comparison of the cultivation of such microorganisms under atmospheric conditions and under pressure conditions will afford a possible reactivation of specific piezotolerants and/or piezophiles organisms from dormancy. The aim of the present study is to validate the use of original processes of culture under high gaseous pressures (50 MPa). (1) « Blind » chambers, of about 100 cm3, are designed to maintain microorganisms spread on solid medium contained in 3 small Petri Dishes (4 cm diameter) at 25-200 MPa. Observation in real time of colonies is not possible. (2) « Optical » or microscopic chambers are designed to resist up to 400 MPa and to visualize the microbial growth during hyperbaric treatment in real time. They can be used to cultivate and visualize microorganisms under gaseous isostatic pressure on solid medium or under hydrostatic pressure in liquid medium. The yeast Saccharomyces cerevisiae was used as a model of microorganism. Results show that its growth rate, measured between 1 and 50 MPa, decreased when the pressure level increased. The “optical” chamber is more adapted to the follow-up of the microbial growth because the growth rate can be calculated without decompression of the chamber and cells can be followed-up individually. The “blind” chamber is more adapted to cultivate samples in order to isolate piezotolerants and/or piezophiles microorganisms. Thus, they will be used to cultivate and isolate microorganisms present in deep sea sediments (-5000 m under Indian Ocean surface)

La biopréservation: une stratégie d’avenir pour la conservation des aliments

N° d'article 201511121820La demande grandissante des consommateurs pour des produits alimentaires à teneur réduite en conservateurs pousse les industriels de l’agroalimentaire à rechercher des méthodes de conservation alternatives. Dans ce contexte, la bioprotection s’impose comme un procédé à fort potentiel. Cette technique consiste à inoculer sur les aliments des cultures de bactéries protectrices. Celles-ci, par l’intermédiaire de plusieurs mécanismes, vont inhiber la croissance de bactéries pathogènes et d’altération telles que Listeria monocytogenes et Salmonella. Cependant, cette technique n’a pas d’effet sur les spores bactériennes. Ainsi, l’objet central du projet de recherche ANR BlacHP vise à inactiver ces formes résistantes, notamment en combinant la biopréservation avec un procédé de traitement par hautes pressions hydrostatiques

Extremely rapid acclimation of Escherichia coli to high temperature over a few generations of a fed-batch culture during slow warming

International audienceThis study aimed to demonstrate that adequate slow heating rate allows two strains of Escherichia coli rapid acclimation to higher temperature than upper growth and survival limits known to be strain‐dependent. A laboratory (K12‐TG1) and an environmental (DPD3084) strain of E. coli were subjected to rapid (few seconds) or slow warming (1°C 12 h−1) in order to (re)evaluate upper survival and growth limits. The slow warming was applied from the ancestral temperature 37°C to total cell death 46–54°C: about 30 generations were propagated. Upper survival and growth limits for rapid warming (46°C) were lower than for slow warming (46–54°C). The thermal limit of survival for slow warming was higher for DPD3084 (50–54°C). Further experiments conducted on DPD3084, showed that mechanisms involved in this type of thermotolerance were abolished by a following cooling step to 37°C, which allowed to imply reversible mechanisms as acclimation ones. Acquisition of acclimation mechanisms was related to physical properties of the plasma membrane but was not inhibited by unavoidable appearance of aggregated proteins. In conclusion, E.coli could be rapidly acclimated within few generations over thermal limits described in the literature. Such a study led us to propose that rapid acclimation may give supplementary time to the species to acquire a stable adaptation through a random mutation

Regioselective chemo-enzymatic syntheses of ferulate conjugates as chromogenic substrates for feruloyl esterases

International audienceGenerally, carbohydrate-active enzymes are studied using chromogenic substrates that provide quick and easy color-based detection of enzyme-mediated hydrolysis. In the case of feruloyl esterases, commercially available chromogenic ferulate derivatives are both costly and limited in terms of their experimental application. In this study, we describe solutions for these two issues, using a chemoenzymatic approach to synthesize different ferulate compounds. The overall synthetic routes towards commercially available 5-bromo-4-chloro-3-indolyl and 4-nitrophenyl O-5-feruloyl-α-l-arabinofuranosides 1a and 1b were significantly shortened (7-8 steps reduced to 4-6) and transesterification yields enhanced (from 46 to 73% for 1a and 47 to 86 % for 1b). This was achieved using enzymatic (immobilized Lipolase 100T from Thermomyces lanuginosus) transesterification of unprotected vinyl ferulate to the primary hydroxyl group of α‐l‐arabinofuranosides. Moreover, a novel feruloylated-butanetriol 4-nitrocatechol-1-yl analog 12, containing a cleavable hydroxylated linker was also synthesized in 29% overall yield in 3 steps (convergent synthesis). The latter route combined regioselective functionalization of 4-nitrocatechol and enzymatic transferuloylation. The use of 12 as a substrate to characterize type A feruloyl esterase from Aspergillus niger reveals the advantages of this substrate for the characterizations of feruloyl esterases

Management of Listeria monocytogenes on Surfaces via Relative Air Humidity: Key Role of Cell Envelope

International audienceAlthough relative air humidity (RH) strongly influences microbial survival, its use for fighting surface pathogens in the food industry has been inadequately considered. We asked whether RH control could destroy Listeria monocytogenes EGDe by envelope damage. The impact of dehydration in phosphate-buffered saline (PBS) at 75%, 68%, 43% and 11% RH on the bacterial envelope was investigated using flow cytometry and atomic force microscopy. Changes after rehydration in the protein secondary structure and peptidoglycan were investigated by infrared spectroscopy. Complementary cultivability measurements were performed by running dehydration–rehydration with combinations of NaCl (3–0.01%), distilled water, city water and PBS. The main results show that cell membrane permeability and cell envelope were greatly altered during dehydration in PBS at 68% RH followed by rapid rehydration. This damage led cells to recover only 67% of their initial volume after rehydration. Moreover, the most efficient way to destroy cells was dehydration and rehydration in city water. Our study indicates that rehydration of dried, sullied foods on surfaces may improve current cleaning procedures in the food industry