Sélection de clones résistants appartenant aux genres Kiebsiella, Serratia et Pseudomonas afin de suivre leur implantation dans un biofiltre

Des souches appartenant aux espèces : Klebsiella oxytoca, Serratia marcescens et Pseudomonas putida, isolées d'un biofiltre utilisé pour le traitement d'effluents urbains ont été choisies parmi une centaine d'autres pour être réimplantées dans un réacteur du même type. Dans le but de suivre leur fixation en réacteur ouvert, une méthode spécifique de sélection a été développée. Des clones de ces souches résistant naturellement à des antibiotiques (rifampicine, streptomycine, acide nalidixique) et à des substrats suicides (chlorate, bromoacétate, fluorouracile) ont été recherchés. Cette sélection a permis d'obtenir des clones de Klebsiella et de Serratia résistants à 2 g/l de streptomycine, 1 g/l de rifampicine et à 2 g/l de chlorate ainsi que des clones de Pseudomonas résistants à 0,5 g/l d'acide nalidixique et à 2 g/l de bromoacétate ou à 40 mgll de fluorouracile.Les clones résistants dont les caractéristiques de croissance et les activités enzymatiques sont identiques à celles de la souche sauvage et dont la stabilité génétique a été maintenue après de nombreux repiquages ont été retenus. Afin de valider notre méthode de reconnaissance, une numération de la flore indigène d'un effluent urbain a été réalisée sur les milieux spécifiques des clones résistants : seule une faible proportion de cette flore, à savoir 0,02 % est capable de s'y développer. Des essais préliminaires d'ensemencement du biofiltre avec les souches sélectionnées ont été réalisés, ils montrent que celles-ci s'implantent puisqu'elles sont retrouvées sur les grains de matériau de garnissage et que chacune d'elle représente 1 % de la flore totale.Comparison with free tell system, fixed process applied for biological wastewater treatment have been shown to offer numerous advantages. The Biocarbone process, an aerobic down flow immersed bed reactor (ODA patent n° 78-30246), has been selected for many industrial and municipal wastewater treatment facilities.From this type of aerobic fixed-bed reactor, made of expanded schist as a granular support and fed with clarified domestic wastewater, eigthy-eigth strains were isolated (ZINEBI et al., 1992). Three of the bacterial strains were chosen for their abilities to express high levels of glucidolytic, proteolytic or lipolytic activities and to grow on the granular support as microcolonies which developed into a film of organisms over the whole surface.Our objective was to initiate biofilm formation by feeding the clean support with thon selected strains named : Klebsiella oxytoca, 501; Serratia marcescens, 532 and Pseudomonas putida, 601. In order to follow attachment kinetics of these selected strains of this biofilter, and to verify their perenity within the biofilm in non sterile conditions (mixed with indigeneous flora from the influent), a specific labelling method was required.As antibiotic-resistant mutants are easily isolated and the resistances can often serve as convenient genetic markers for use in characterizing bacterial strains, a direct selection of tells acquiring resistance to various antibiotics (ampicillin, streptomycin, nalidixic acid and rifampicin) bas been performed. Selected antibiotic-resistant strains were further incubated in presence of growth inhibitors or suicide substrates in order to select again spontaneous arising mutants well characterized by two distinct markers. From the two bacteria belonging to the Enterobacteriaceae family, mutants having lost the nitrate reductase have been isolated under anaerobic growth conditions in the presence of chlorate. In the case of Pseudomonas strain, mutants resistant towards substrate halogen analogues were obtained.Colonies resistant to antibiotics and resistant to lethal substrates were isolated : thus, colonies of Klebsiella resistant to streptomycin at 2 g/l, to rifampicin al 1 g/l and chlorate al 2 g/l ; colonies of Serratia resistant to streptomycin at 2 g/l or to rifampicine at 1 g/l and chlorate at 2 g/l and colonies of Pseudomonas resistant to nalidixic acid at 0.5 g/l and to bromoacetate at 2 g/l or to fluorouracil at 40 mg/l, were obtained. We have selected : trains showing the same doubling time as well as the same final population titan the parental strains when growths were performed with or without the markers. The three strains retained were : Klebsiellaoxytoca, 501 R1S2Cl2 which grew on the Mac Conkey medium added with 1 g/l of rifampicin, 2 g/l at streptomycin and 2 g/l of chlorate; Serratia marcescens, 532 S2Cl2 (on Mac Conkey plus 2 g/l of streptomycin and 2 g/l of chlorate) and Pseudomonas putida, 601 NB2 (on King plus 0,5 g/l of nalidixic acid and 2 g/l of bromoacetate). These specific media for the detection of selectionned clones were selective toward a fixed indigenous flora since only 0,02 % of total heterotrophic population can grow.A column filled with grains of « Biodagen » either colonized by natural, microbial populations or with clean grains of « Biodagen » was fed with a population of the : train Klebsiella 501 R1S2Cl2. The strain colonized virgin « Biodagen » and maintained population of 4.106 CFU per grain for 9 days with new material and 105 CFU for 7 days with precolonized material.Experiment with a mixed population resulting from the three identified microbial species have been conducted with clean grains of « Biodagen », a whole population of 107 CFU per grain was obtained after two days and each identified strain corresponded to 1 % of the entire bacterial population. The relative concentrations of the three : trains did not decrease feeding the column with a mixture of the three : trains and of wastewater but slightly decreased when the column was fed with wastewater only

Protein crystallography with a micrometre-sized synchrotron-radiation beam

For the first time, protein microcrystallography has been performed with a focused synchrotron-radiation beam of 1 µm using a goniometer with a sub-micrometre sphere of confusion. The crystal structure of xylanase II has been determined with a flux density of about 3 × 1010 photons s−1 µm−2 at the sample

Unconventional Repertoire Profile Is Imprinted during Acute Chikungunya Infection for Natural Killer Cells Polarization toward Cytotoxicity

Chikungunya virus (CHIKV) is a worldwide emerging pathogen. In humans it causes a syndrome characterized by high fever, polyarthritis, and in some cases lethal encephalitis. Growing evidence indicates that the innate immune response plays a role in controlling CHIKV infection. We show here that CHIKV induces major but transient modifications in NK-cell phenotype and function soon after the onset of acute infection. We report a transient clonal expansion of NK cells that coexpress CD94/NKG2C and inhibitory receptors for HLA-C1 alleles and are correlated with the viral load. Functional tests reveal cytolytic capacity driven by NK cells in the absence of exogenous signals and severely impaired IFN-γ production. Collectively these data provide insight into the role of this unique subset of NK cells in controlling CHIKV infection by subset-specific expansion in response to acute infection, followed by a contraction phase after viral clearance

Combined inactivation of the Clostridium cellulolyticum lactate and malate dehydrogenase genes substantially increases ethanol yield from cellulose and switchgrass fermentations

<p>Abstract</p> <p>Background</p> <p>The model bacterium <it>Clostridium cellulolyticum </it>efficiently degrades crystalline cellulose and hemicellulose, using cellulosomes to degrade lignocellulosic biomass. Although it imports and ferments both pentose and hexose sugars to produce a mixture of ethanol, acetate, lactate, H₂and CO₂, the proportion of ethanol is low, which impedes its use in consolidated bioprocessing for biofuels production. Therefore genetic engineering will likely be required to improve the ethanol yield. Plasmid transformation, random mutagenesis and heterologous expression systems have previously been developed for <it>C. cellulolyticum</it>, but targeted mutagenesis has not been reported for this organism, hindering genetic engineering.</p> <p>Results</p> <p>The first targeted gene inactivation system was developed for <it>C. cellulolyticum</it>, based on a mobile group II intron originating from the <it>Lactococcus lactis </it>L1.LtrB intron. This markerless mutagenesis system was used to disrupt both the paralogous <smcaps>L</smcaps>-lactate dehydrogenase (<it>Ccel_2485; ldh</it>) and <smcaps>L</smcaps>-malate dehydrogenase (<it>Ccel_0137; mdh</it>) genes, distinguishing the overlapping substrate specificities of these enzymes. Both mutations were then combined in a single strain, resulting in a substantial shift in fermentation toward ethanol production. This double mutant produced 8.5-times more ethanol than wild-type cells growing on crystalline cellulose. Ethanol constituted 93% of the major fermentation products, corresponding to a molar ratio of ethanol to organic acids of 15, versus 0.18 in wild-type cells. During growth on acid-pretreated switchgrass, the double mutant also produced four times as much ethanol as wild-type cells. Detailed metabolomic analyses identified increased flux through the oxidative branch of the mutant's tricarboxylic acid pathway.</p> <p>Conclusions</p> <p>The efficient intron-based gene inactivation system produced the first non-random, targeted mutations in <it>C. cellulolyticum</it>. As a key component of the genetic toolbox for this bacterium, markerless targeted mutagenesis enables functional genomic research in <it>C</it>. <it>cellulolyticum </it>and rapid genetic engineering to significantly alter the mixture of fermentation products. The initial application of this system successfully engineered a strain with high ethanol productivity from cellobiose, cellulose and switchgrass.</p

Quantitative proteomic analysis of the influence of lignin on biofuel production by Clostridium acetobutylicum ATCC 824

Background: Clostridium acetobutylicum has been a focus of research because of its ability to produce high-value compounds that can be used as biofuels. Lignocellulose is a promising feedstock, but the lignin–cellulose–hemicellulose biomass complex requires chemical pre-treatment to yield fermentable saccharides, including cellulose-derived cellobiose, prior to bioproduction of acetone–butanol–ethanol (ABE) and hydrogen. Fermentation capability is limited by lignin and thus process optimization requires knowledge of lignin inhibition. The effects of lignin on cellular metabolism were evaluated for C. acetobutylicum grown on medium containing either cellobiose only or cellobiose plus lignin. Microscopy, gas chromatography and 8-plex iTRAQ-based quantitative proteomic technologies were applied to interrogate the effect of lignin on cellular morphology, fermentation and the proteome. Results: Our results demonstrate that C. acetobutylicum has reduced performance for solvent production when lignin is present in the medium. Medium supplemented with 1 g L−1 of lignin led to delay and decreased solvents production (ethanol; 0.47 g L−1 for cellobiose and 0.27 g L−1 for cellobiose plus lignin and butanol; 0.13 g L−1 for cellobiose and 0.04 g L−1 for cellobiose plus lignin) at 20 and 48 h, respectively, resulting in the accumulation of acetic acid and butyric acid. Of 583 identified proteins (FDR < 1 %), 328 proteins were quantified with at least two unique peptides. Up- or down-regulation of protein expression was determined by comparison of exponential and stationary phases of cellobiose in the presence and absence of lignin. Of relevance, glycolysis and fermentative pathways were mostly down-regulated, during exponential and stationary growth phases in presence of lignin. Moreover, proteins involved in DNA repair, transcription/translation and GTP/ATP-dependent activities were also significantly affected and these changes were associated with altered cell morphology. Conclusions: This is the first comprehensive analysis of the cellular responses of C. acetobutylicum to lignin at metabolic and physiological levels. These data will enable targeted metabolic engineering strategies to optimize biofuel production from biomass by overcoming limitations imposed by the presence of lignin

Magnetohydrodynamics of stably stratified regions in planets and stars

International audienceStably-stratified layers are present in stellar interiors (radiative zones) as well as planetary interiors (recent observations and theoretical studies of the Earth's magnetic field seem to indicate the presence of a thin, stably-stratified layer at the top of the liquid outer core). We present direct numerical simulations of this region, which is modeled as an ax-isymmetric spherical Couette flow for a stably-stratified fluid embedded in a dipolar magnetic field. For strong magnetic fields, a super-rotating shear layer, rotating nearly 40% faster than the inner region, is generated in the stably stratified region. In the Earth context, and contrary to what was previously believed, we show that this super-rotation may extend toward the Earth magnetostrophic regime if the density stratification is sufficiently large. The corresponding differential rotation triggers mag-netohydrodynamic instabilities and waves in the stratified region, which feature growth rates comparable to the observed timescale for geomag-netic secular variations and jerks. In the stellar context, we perform a linear analysis which shows that similar instabilities are likely to arise, and we argue that it may explain the observed magnetic desert among massive and intermediate mass stars