A complete collection of single-gene deletion mutants of Acinetobacter baylyi ADP1

We have constructed a collection of single-gene deletion mutants for all dispensable genes of the soil bacterium Acinetobacter baylyi ADP1. A total of 2594 deletion mutants were obtained, whereas 499 (16%) were not, and are therefore candidate essential genes for life on minimal medium. This essentiality data set is 88% consistent with the Escherichia coli data set inferred from the Keio mutant collection profiled for growth on minimal medium, while 80% of the orthologous genes described as essential in Pseudomonas aeruginosa are also essential in ADP1. Several strategies were undertaken to investigate ADP1 metabolism by (1) searching for discrepancies between our essentiality data and current metabolic knowledge, (2) comparing this essentiality data set to those from other organisms, (3) systematic phenotyping of the mutant collection on a variety of carbon sources (quinate, 2-3 butanediol, glucose, etc.). This collection provides a new resource for the study of gene function by forward and reverse genetic approaches and constitutes a robust experimental data source for systems biology approaches

Viral to metazoan marine plankton nucleotide sequences from the Tara Oceans expedition

A unique collection of oceanic samples was gathered by the Tara Oceans expeditions (2009-2013), targeting plankton organisms ranging from viruses to metazoans, and providing rich environmental context measurements. Thanks to recent advances in the field of genomics, extensive sequencing has been performed for a deep genomic analysis of this huge collection of samples. A strategy based on different approaches, such as metabarcoding, metagenomics, single-cell genomics and metatranscriptomics, has been chosen for analysis of size-fractionated plankton communities. Here, we provide detailed procedures applied for genomic data generation, from nucleic acids extraction to sequence production, and we describe registries of genomics datasets available at the European Nucleotide Archive (ENA, www.ebi.ac.uk/ena). The association of these metadata to the experimental procedures applied for their generation will help the scientific community to access these data and facilitate their analysis. This paper complements other efforts to provide a full description of experiments and open science resources generated from the Tara Oceans project, further extending their value for the study of the world's planktonic ecosystems

Restitution virtuelle. Une frontière entre (non) visible et sensible

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(RÉ)APPRENDRE À LIRE DES TEXTES DE JEUNES SCRIPTEURS ?

International audiencePartant du constat qu’une lecture coopérative des textes d’élèves se heurte à de nombreuses résistances, l’article rend compte d’une recherche collaborative menée en classe de 6e. Il s’efforce de montrer comment le professeur peut, sans renoncer à ses exigences ni alourdir le temps d’annotation, s’assumer comme sujet–lecteur et engager un dialogue avec des élèves-auteurs et des textes en devenir. Ce changement de posture passe par des gestes langagiers tissant une communauté de lecteurs. Il a des conséquences sur la conception même de la séquence ou les formes et fonctions de l’écrit sont réévalués : des écrits de travail prennent place dans le déploiement du travail d’écriture, installant des temps de réflexivité articulés à des activités orales et écrites. Une analyse de comptes rendus précédés par la rédaction de listes en fournit une illustration

Restitution virtuelle. Une frontière entre (non) visible et sensible

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Microbial urate catabolism: characterization of HpyO, a non-homologous isofunctional isoform of the flavoprotein urate hydroxylase HpxO

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Elucidation of the trigonelline degradation pathway reveals previously undescribed enzymes and metabolites

International audienceTrigonelline (TG; N-methylnicotinate) is a ubiquitous osmolyte. Although it is known that it can be degraded, the enzymes and metabolites have not been described so far. In this work, we challenged the laboratory model soil-borne, gram-negative bacterium Acinetobacter baylyi ADP1 (ADP1) for its ability to grow on TG and we identified a cluster of catabolic, transporter, and regulatory genes. We dissected the pathway to the level of enzymes and metabolites, and proceeded to in vitro reconstruction of the complete pathway by six purified proteins. The four enzymatic steps that lead from TG to methylamine and succinate are described, and the structures of previously undescribed metabolites are provided. Unlike many aromatic compounds that undergo hydroxylation prior to ring cleavage, the first step of TG catabolism proceeds through direct cleavage of the C5-C6 bound, catalyzed by a flavin-dependent, two-component oxygenase, which yields (Z)-2-((N-methylformamido)methylene)-5-hydroxy-butyrolactone (MFMB). MFMB is then oxidized into (E)-2-((N-methylformamido) methylene) succinate (MFMS), which is split up by a hydrolase into carbon dioxide, methylamine, formic acid, and succinate semialdehyde (SSA). SSA eventually fuels up the TCA by means of an SSA dehydrogenase, assisted by a Conserved Hypothetical Protein. The cluster is conserved across marine, soil, and plant-associated bacteria. This emphasizes the role of TG as a ubiquitous nutrient for which an efficient microbial catabolic toolbox is available. bacterial metabolism | functional genomics | LC/MS-Orbitrap | trigonelline | N-heterocycle degradation E xtensive and accurate bacterial genome annotation is critical for developing a comprehensive and detailed understanding of cellular physiology, and is therefore a major concern in biological research. As a result, 30-40% of genes of a typical ge-nome remain unannotated or associated with a putative function (1, 2). In many cases, function is extrapolated from a small number of characterized proteins (3). In this context, the need for a global effort of experimental assignation, validation, or correction of function is major. Experimental work guided by bioinformatics has proved to be an invaluable tool for assigning new functions (4-7). However, investigations on catabolic pathways are still often conducted in a few model organisms and with a restricted set of nutrients. Given the vast array of natural secondary metabolites and their underrepresentation in metabolic maps, the full range of transformations afforded by bacteria is clearly underestimated. Thus, simply varying the range of organisms tested and the set of nutrients remains a useful tactic for elucidating hidden latent microbial catabolic pathways and providing access to gene function (8-10). Trigonelline (TG; N-methylnicotinate) is a metabolite of nic-otinamide involved in plant cell cycle regulation and oxidative stress (11). Released by legume roots and seeds, such as in alfalfa , it activates nodulation genes in Rhizobium meliloti (12). It is one of the most widely distributed betaines in higher plants (12, 13), and it is also present with different concentration ranges in organisms such as reef-building corals (14, 15), algae (16), and marine plankton, in which it can reach the millimolar range (17, 18). It is likely released in the environment through the death of these organisms. As a consequence, numerous heterotrophic prokaryotes probably use this compound as a nutrient. Surprisingly , the soil bacterium R. meliloti RCR2011 is the only organism reported to use TG as a carbon, nitrogen, and energy source (19). Although an inducible genetic region involved in the degradation of this compound was specified (20, 21), investigations aimed at deciphering this metabolic pathway did not go further. Therefore, the complete set of genes and catabolites involved in this process is not reported. Acinetobacter baylyi ADP1 (ADP1) is a nutritionally versatile strictly aerobic bacterium capable of metabolizing a wide range of aromatic compounds (22). Its extraordinary competence for natural transformation and the ease with which it can be genetically engineered (23, 24) make ADP1 a key organism for the study of soil bacteria metabolism of natural compounds. In this work, we show that ADP1 can use TG as the sole source of carbon, nitrogen, and energy. A gene cluster responsible for TG degradation, which we called tgn (for trigonelline), was revealed Significance The experimental dissection of novel metabolic pathways, from genes and enzymes to metabolites, is a key issue for improving our knowledge of the enzymatic capabilities of the microbial world and providing accurate functional annotation of genomes. We used an integrative methodology combining the phenotyping of a complete genome-scale mutant collection of Acinetobacter baylyi ADP1 with an untargeted liquid chromatography/MS-based approach to uncover the degradation pathway of trigonelline (TG), a widespread osmolyte. We provide extensive information about this unusual N-heterocyclic aromatic degradation route that expands the metabolite repertoire. The occurrence of conserved gene clusters for TG dis-similation in soil, plant-associated, and marine bacteria underlines its environmental abundance

Synthesis of Mono- and Dihydroxylated Amino Acids with New α\alpha-Ketoglutarate-Dependent Dioxygenases: Biocatalytic Oxidation of C−-H Bonds

International audienceIron(II)/$\alpha$-ketoacid-dependent oxygenases ($\alpha$KAOs) are enzymesthat mainly catalyse hydroxylation reaction. By usinga genomic approach combining sequence comparison andprotein-domain sharing, a set of 131 $\alpha$KAO enzymes was prepared.The screening of various substrates revealed five new$\alpha$KAOs. Four $\alpha$KAOs were found to be active towards L-lysine,L-ornithine and L-arginine with total regio- and stereoselectivitiesand yielding the corresponding 3- or 4-hydroxyaminoacids. The enzymatic cascade reaction with two stereoselectiveregiodivergent $\alpha$ KAOs enabled the synthesis of 3,4-dihydroxy-L-lysine

Nitrilase Activity Screening on Structurally Diverse Substrates: Providing Biocatalytic Tools for Organic Synthesis

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