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

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

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

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New Insights into the Alternative d-Glucarate Degradation Pathway*S⃞

Although the d-glucarate degradation pathway is well characterized in Escherichia coli, genetic and biochemical information concerning the alternative pathway proposed in Pseudomonas species and Bacillus subtilis remains incomplete. Acinetobacter baylyi ADP1 is a Gram-negative soil bacterium possessing the alternative pathway and able to grow using d-glucarate as the only carbon source. Based on the annotation of its sequenced genome (1), we have constructed a complete collection of singlegene deletion mutants (2). High throughput profiling for growth on a minimal medium containing d-glucarate as the only carbon source for ∼2450 mutants led to the identification of the genes involved in d-glucarate degradation. Protein purification after recombinant production in E. coli allowed us to reconstitute the enzymatic pathway in vitro. We describe here the kinetic characterization of d-glucarate dehydratase, d-5-keto-4-deoxyglucarate dehydratase, and of cooperative α-ketoglutarate semialdehyde dehydrogenase. Transcription and expression analyses of the genes involved in d-glucarate metabolism within a single organism made it possible to access information regarding the regulation of this pathway for the first time

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

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