σ54 (σL) plays a central role in carbon metabolism in the industrially relevant Clostridium beijerinckii

International audiencethe solventogenic C. beijerinckii DSM 6423, a microorganism that naturally produces isopropanol and butanol, was previously modified by random mutagenesis. In this work, one of the resulting mutants was characterized. this strain, selected with allyl alcohol and designated as the AA mutant, shows a dominant production of acids, a severely diminished butanol synthesis capacity, and produces acetone instead of isopropanol. Interestingly, this solvent-deficient strain was also found to have a limited consumption of two carbohydrates and to be still able to form spores, highlighting its particular phenotype. sequencing of the AA mutant revealed point mutations in several genes including CIBE_0767 (sigL), which encodes the σ 54 sigma factor. Complementation with wild-type sigL fully restored solvent production and sugar assimilation and Rt-qpCR analyses revealed its transcriptional control of several genes related to solventogensis, demonstrating the central role of σ 54 in C. beijerinckii DSM 6423. Comparative genomics analysis suggested that this function is conserved at the species level, and this hypothesis was further confirmed through the deletion of sigL in the model strain C. beijerinckii NCIMB 8052. In the context of worldwide energy transition, research for alternatives to fossil fuels has become a priority. In particular, the replacement of petrochemistry by a low carbon emission industry has been a major challenge as our global consumption of petrochemicals keeps on increasing 1. The valorization of plant biomass to synthesize ethanol by microbial fermentation has already been pioneered for biofuel production 2 and could therefore be applied to bio-based chemistry 3. A few strains from the Clostridium genus are naturally able to produce isopropanol and butanol 4,5 , two compounds that could be used as biochemical and biofuel, respectively. However, those organisms are not producing these metabolites in quantities compatible with an economically viable industrial process 6. However, with the increasing availability of efficient genetic tools in Clostridia 7 , metabolic engineering approaches could be undertaken to enhance solvent productivity. Clostridium beijerinckii DSM 6423 (NRRL B-593) is the only natural isopropanol-butanol producing strain whose genome and transcriptome have been investigated 8. It may therefore be the best candidate for genetic engineering, although its particular physiology is still poorly understood. For this purpose, gaining additional knowledge on metabolism regulation in this strain would greatly benefit future optimization efforts. In particular, identifying the molecular effectors controlling solvent production may provide valuable insights to define adequate genetic engineering strategies. As no genetic toolbox was available for this particular strain, Máté de Gerando and coworkers performed random mutagenesis coupled with genome shuffling to increase isopropanol productivity by selecting isopropanol-tolerant strains 9. In this work, random mutagenesis followed by allyl alcohol selection also generated an interesting mutant, further referred to as AA mutant. This strain mainly produces acids, shows no isopropanol production and a strongly attenuated butanol synthesis capacity. These results are consistent with those obtained in Clostridium acetobutylicum DSM 1792, in which mutants obtained in the presence of allyl alcohol-precursor of the highly toxic acrolein molecule in the reaction catalyzed by alcohol dehydrogenases-permitted the selection of butanol-deficient strains 10. Nevertheless, in both cases the key mutated genes causing these phenotypes have not been clearly identified

Monitored eCLIP: high accuracy mapping of RNA-protein interactions

International audienceCLIP-seq methods provide transcriptome-wide snapshots of RNA-protein interactions in live cells. Reverse transcriptases stopping at cross-linked nucleotides sign for RNA-protein binding sites. Reading through cross-linked positions results in false binding site assignments. In the 'monitored enhanced CLIP' (meCLIP) method, a barcoded biotiny-lated linker is ligated at the 5 end of cross-linked RNA fragments to purify RNA prior to the reverse transcription. cDNAs keeping the barcode sequence correspond to reverse transcription read-throughs. Read through occurs in unpredictable proportions, representing up to one fourth of total reads. Filtering out those reads strongly improves reliability and precision in protein binding site assignment

A fluorescent reporter system for anaerobic thermophiles

Owing to their inherent capacity to make invisible biological processes visible and quantifiable, fluorescent reporter systems have numerous applications in biotechnology. For classical fluorescent protein systems (i.e., GFP and derivatives), chromophore maturation is O2-dependent, restricting their applications to aerobic organisms. In this work, we pioneered the use of the oxygen-independent system FAST (Fluorescence Activating and absorption Shifting tag) in the thermophilic anaerobe Thermoanaerobacter kivui. We developed a modular cloning system that was used to easily clone a library of FAST expression cassettes in an E. coli—Thermoanaerobacter shuttle plasmid. FAST-mediated fluorescence was then assessed in vivo in T. kivui, and we observed bright green and red fluorescence for cells grown at 55°C. Next, we took advantage of this functional reporter system to characterize a set of homologous and heterologous promoters by quantifying gene expression, expanding the T. kivui genetic toolbox. Low fluorescence at 66°C (Topt for T. kivui) was subsequently investigated at the single-cell level using flow cytometry and attributed to plasmid instability at higher temperatures. Adaptive laboratory evolution circumvented this issue and drastically enhanced fluorescence at 66°C. Whole plasmid sequencing revealed the evolved strain carried functional plasmids truncated at the Gram-positive origin of replication, that could however not be linked to the increased fluorescence displayed by the evolved strain. Collectively, our work demonstrates the applicability of the FAST fluorescent reporter systems to T. kivui, paving the way for further applications in thermophilic anaerobes

Differentiation of calcified regions and iron deposits in the ageing brain on conventional structural MR images:Calcium and Iron on Conventional MRI

Purpose: In the human brain, minerals such as iron and calcium accumulate increasingly with age. They typically appear hypointense on T2*‐weighted MRI sequences. This study aims to explore the differentiation and association between calcified regions and noncalcified iron deposits on clinical brain MRI in elderly, otherwise healthy subjects. Materials and Methods: Mineral deposits were segmented on co‐registered T1‐ and T2*‐weighted sequences from 100 1.5 Tesla MRI datasets of community‐dwelling individuals in their 70s. To differentiate calcified regions from noncalcified iron deposits we developed a method based on their appearance on T1‐weighted images, which was validated with a purpose‐designed phantom. Joint T1‐ and T2*‐weighted intensity histograms were constructed to measure the similarity between the calcified and noncalcified iron deposits using a Euclidean distance based metric. Results: We found distinct distributions for calcified regions and noncalcified iron deposits in the cumulative joint T1‐ and T2*‐weighted intensity histograms across all subjects (correlations ranging from 0.02 to 0.86; mean = 0.26 ± 0.16; t = 16.93; P < 0.001) consistent with differences in iron and calcium signal in the phantom. The mean volumes of affected tissue per subject for calcified and noncalcified deposits were 236.74 ± 309.70 mm3 and 283.76 ± 581.51 mm3; respectively. There was a positive association between the mineral depositions (β = 0.32, P < 0.005), consistent with existing literature reports. Conclusion: Calcified mineral deposits and noncalcified iron deposits can be distinguished from each other by signal intensity changes on conventional 1.5T T1‐weighted MRI and are significantly associated in brains of elderly, otherwise healthy subjects

Early treatment versus expectative management of patent ductus arteriosus in preterm infants

_Background:_ Much controversy exists about the optimal management of a patent ductus arteriosus (PDA) in preterm infants, especially in those born at a gestational age (GA) less than 28weeks. No causal relationship has been proven between a (haemodynamically significant) PDA and neonatal complications related to pulmonary hyperperfusion and/or systemic hypoperfusion. Although studies show conflicting results, a common understanding is that medical or surgical treatment of a PDA does not seem to reduce the risk of major neonatal morbidities and mortality. As the PDA might have closed spontaneously, treated children are potentially exposed to iatrogenic adverse effects. A conservative approach is gaining interest worldwide, although convincing evidence to support its use is lacking. _Methods:_ This multicentre, randomised, non-inferiority trial is conducted in neonatal intensive care units. The study population consists of preterm infants (GA1.5mm. Early treatment (between 24 and 72h postnatal age) with the cyclooxygenase inhibitor(COXi) ibuprofen (IBU) is compared with an expectative management (no intervention intended to close a PDA). The primary outcome is the composite of mortality, and/or necrotising enterocolitis (NEC) Bell stage ≥ IIa, and/or bronchopulmonary dysplasia (BPD) defined as the need for supplemental oxygen, all at a postmenstrual age (PMA) of 36weeks. Secondary outcome parameters are short term sequelae of cardiovascular failure, comorbidity and adverse events assessed during hospitalization and long-term neurodevelopmental outcome assessed at a corrected age of 2 years. Consequences regarding health economics are evaluated by cost effectiveness analysis and budget impact analysis. _Discussion:_ As a conservative approach is gaining interest, we investigate whether in preterm infants, born at a GA less than 28weeks, with a PDA an expectative management is non-inferior to early treatment with IBU regarding to the composite outcome of mortality and/or NEC and/or BPD at a PMA of 36weeks

Le rôle des représentations et de la confiance en soi dans l'apprentissage de l'expression orale en langue étrangère

La confrontation des résultats du questionnaire et de nos hypothèses permet de tirer trois enseignements : l’influence directe de la perception des expériences d’apprentissage sur la confiance en sa capacité d’expression orale est limitée. Cette perception est imbriquée dans un ensemble de représentations plus large dont l’influence semble plus significative. On peut préciser néanmoins que plus la méthode d’apprentissage s’attachera à construire un pont solide entre l’acquisition d’un niveau tremplin et sa réutilisation en situation-objectif, plus elle sera reconnue et appréciée par les apprenants. Il existe un triangle d’interactions fort entre la confiance en son expression orale, le niveau de langue estimé et la gestion émotionnelle des représentations. L’acquisition rapide d’un niveau linguistique tremplin et la construction d’un lien fort avec sa réutilisation en contexte-objectif constitueront alors un critère émotionnel et motivationnel déterminant de la réussite d’un apprentissage. La création de ce lien fort passera par la mise en place d’activités et d’un contexte de travail exigeants et favorisant des activités interactives et au plus proche de la réalité linguistique à venir pour les apprenants. Ces analyses demandent néanmoins à être contextualisées et ne peuvent en l’état prêter à généralisation : elles sont tirées de verbalisations d’un panel d’expatriés en immersion linguistique dont les expériences et le vécu sont spécifiques et difficilement extrapolables

Maurice Schumann, un élu du Nord, entre démocratie chrétienne et gaullisme (1945-1998)

À la Libération, le pays découvre l’homme dont la voix anonyme a entretenu l’espérance pendant les années noires de l’occupation, comme porte-parole du général de Gaulle. À l’initiative de Georges Bidault, il est élu président national du Mouvement Républicain populaire (MRP) lors de son congrès constitutif, en novembre 1944. Ce nouveau parti appartient à la famille démocrate d’inspiration chrétienne, à laquelle Maurice Schumann appartenait dès avant-guerre, comme membre de la Jeune Républiqu..

Clostridium beijerinckii DSM 6423, an emerging platform strain for solvent bioproduction

Clostridium beijerinckii DSM 6423 est une souche bactérienne atypique capable de fermenter une variété de substrats et de produire naturellement un mélange de solvants composé d’isopropanol, de butanol et d’éthanol (IBE). Malgré ce potentiel, des limitations subsistent pour son utilisation dans un procédé de synthèse d’isopropanol biosourcé. En particulier, sa faible sélectivité envers cet alcool et sa faible tolérance au mélange de solvants réduisent la quantité de produits récupérables. Ces défauts pourraient être corrigés par des modifications ciblées du génome de cette souche, mais ceci passe par le développement d’outils génétiques, jusqu’à présent inexistants pour cette bactérie, et une meilleure compréhension de son métabolisme. Dans ce contexte, une première thèse réalisée à IFPEN a permis d’établir sa carte génétique et d’isoler de premiers mutants présentant une tolérance accrue à l’isopropanol.Dans la continuité de ces premiers résultats, les travaux de thèse présentés dans ce manuscrit se sont d’abord attachés à développer des outils de génétique ciblée performants. L’utilisation de plasmides Dam- Dcm- a permis pour la première fois de transformer ce microorganisme avec succès, ouvrant la voie au développement d’un outil basé sur la technologie CRISPR-Cas9, qui révolutionne l’ingénierie génétique en modifiant les génomes de manière itérative et sans laisser de trace. Comme preuve de concept, les 2 gènes CIBE_0562 (upp) et CIBE_3859 (catB) ont été inactivés. Cette deuxième modification a rendu ce micro-organisme sensible au thiamphénicol et compatible avec un système CRISPR à deux plasmides développé précédemment à IFPEN pour modifier le génome d’une souche solvantogène de référence, Clostridium acetobutylicum. Après suppression du plasmide endogène pNF2, les efficacités de transformation de la souche ont été drastiquement accrues, nous permettant ainsi d’obtenir une souche plateforme ∆catB ∆pNF2 qui pourra constituer le point de départ de la construction d’une souche performante pour la production d’isopropanol.Parallèlement au développement de ces outils génétiques, ma thèse s’est également consacrée à la caractérisation du microorganisme par plusieurs approches. La première a consisté à déterminer l’impact de différents substrats, modèles ou industriels, sur les performances et le métabolisme de la souche. Après sélection de trois substrats (glucose, saccharose, mélasse de betterave), un procédé de fermentation continue a été mis au point pour isoler les deux états physiologiques caractéristiques d’une fermentation IBE, puis couplé à une analyse multi-omiques. Les résultats préliminaires, s’appuyant sur les données obtenues par protéomique, soulignent l’impact très important de l’utilisation de substrats industriels sur la croissance.Deuxièmement, une approche transcriptomique, le Capp-Switch sequencing, a été employée pour cartographier, à l’échelle du génome, l’ensemble des sites d’initiation de la transcription (TSS) pour la souche DSM 6423 et deux autres souches solvantogènes de référence. Une analyse comparative des TSS a notamment révélé une régulation différentielle de l’opéron de synthèse du butyryl-CoA entre les espèces C. beijerinckii et C. acetobutylicum.Bien que non prévu au début de ce travail, le séquençage d’un mutant obtenu lors de travaux précédents et incapable de produire de l’isopropanol (souche AA), puis la reconstruction de son phénotype dans une souche modèle de C. beijerinckii, a par ailleurs révélé le rôle fondamental du facteur σ54 pour le contrôle des voies de synthèse des solvants et d’assimilation de sucres alternatifs.En conclusion, en approfondissant nos connaissances sur C. beijerinckii DSM 6423 et en permettant des modifications ciblées de son génome, ma thèse s’inscrit dans un ensemble de travaux menés à IFPEN visant à faire de cette souche un organisme modèle pour la fermentation IBE, permettant à terme la bioproduction d’isopropanol à échelle industrielle.Clostridium beijerinckii DSM 6423 is an atypical bacterial strain which can ferment a wide array of substrates and naturally produce a solvent mixture composed of isopropanol, butanol and ethanol (IBE). Despite its potential, the prospect of using this microorganism for a biobased isopropanol synthesis process remains limited. In particular, its poor selectivity to this alcohol and its weak tolerance to the solvent mixture reduce isopropanol recoverability, although these drawbacks might be alleviated by targeted genome editing approaches. For this matter, developing genetic tools, that were until this work inexistent for this bacterium, and increasing our knowledge of its metabolism are important steps. In this context, a first IFPEN thesis did the ground work by drawing its genetic map and isolating isopropanol-tolerant mutants.Based on this context, my thesis first aimed at developing efficient targeted genetic tools for this relevant strain. Use of Dam- Dcm- plasmids permitted for the first time the successful transformation of this microorganism, enabling the development of a reusable, scarless, genome editing tool based on the CRISPR-Cas9 technology. As a proof of concept, the 2 genes CIBE_0562 (upp) and CIBE_3859 (catB) were inactivated. This second modification made the strain sensitive to thiamphenicol and allowed the use of a dual-plasmid CRISPR system that had previously been engineered in IFPEN to modify the genome of a reference solventogenic species: Clostridium acetobutylicum. After the deletion of the pNF2 endogenous plasmid, transformation efficiencies were drastically increased, resulting in a ∆catB ∆pNF2 platform strain that may be used in the future to construct an efficient strain for isopropanol production.In addition to the development of these genetic tools, my thesis also aimed at characterizing the microorganism by different approaches. Firstly, we studied the impact of several model or industrial carbon sources on the strain performances and on its metabolism. Focusing on three substrates (glucose, sucrose, beetroot molasse), a continuous fermentation process was next designed to isolate the two IBE-characteristic physiological states and coupled to a multi-omic analysis. Proteomics preliminary results highlight the severe impacts of industrial substrates on growth.Secondly, the Capp-Switch sequencing transcriptomic approach was used to map transcription start sites (TSS) at the genome scale for the DSM 6423 strain and two other reference solventogenic strains. A comparative TSS analysis notably revealed a differential regulation of the butyryl-CoA synthesis operon between the C. beijerinckii and C. acetobutylicum species.Although initially not planned at the beginning of this work, the sequencing of the AA strain, one of the mutants that were obtained in the first thesis and that is unable to produce isopropanol, as well as a targeted genome approach reproducing its phenotype in a C. beijerinckii model strain, also revealed the crucial role of σ54 in controlling solvent synthesis and alternative sugar uptake pathways.To conclude, by deepening our knowledge on C. beijerinckii DSM 6423 and by allowing targeted genome editing approaches, my thesis is involved in a series of work conducted at IFPEN that aim at making this strain an IBE fermentation model organism, in order to use it for the bioproduction of isopropanol at the industrial scale