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

A Targetron-Recombinase System for Large-Scale Genome Engineering of Clostridia

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Physiology, Genomics, and Pathway Engineering of an Ethanol-Tolerant Strain of Clostridium phytofermentans

International audienceNovel processing strategies for hydrolysis and fermentation of lignocellulosic biomass in a single reactor offer large potential cost savings for production of biocommodities and biofuels. One critical challenge is retaining high enzyme production in the presence of elevated product titers. Toward this goal, the cellulolytic, ethanol-producing bacterium Clostridium phytofermentans was adapted to increased ethanol concentrations. The resulting ethanol-tolerant (ET) strain has nearly doubled ethanol tolerance relative to the wild-type level but also reduced ethanol yield and growth at low ethanol concentrations. The genome of the ET strain has coding changes in proteins involved in membrane biosynthesis, the Rnf complex, cation homeostasis, gene regulation, and ethanol production. In particular, purification of the mutant bifunctional acetaldehyde coenzyme A (CoA)/alcohol dehydrogenase showed that a G609D variant abolished its activities, including ethanol formation. Heterologous expression of Zymomonas mobilis pyruvate decarboxylase and alcohol dehydrogenase in the ET strain increased cellulose consumption and restored ethanol production, demonstrating how metabolic engineering can be used to overcome disadvantageous mutations incurred during adaptation to ethanol. We discuss how genetic changes in the ET strain reveal novel potential strategies for improving microbial solvent tolerance

Functional Diversity of Carbohydrate-Active Enzymes Enabling a Bacterium to Ferment Plant Biomass

International audienceMicrobial metabolism of plant polysaccharides is an important part of environmental carbon cycling, human nutrition, and industrial processes based on cellulosic bioconversion. Here we demonstrate a broadly applicable method to analyze how microbes catabolize plant polysaccharides that integrates carbohydrate-active enzyme (CAZyme) assays, RNA sequencing (RNA-seq), and anaerobic growth screening. We apply this method to study how the bacterium Clostridium phytofermentans ferments plant biomass components including glucans, mannans, xylans, galactans, pectins, and arabinans. These polysaccharides are fermented with variable efficiencies, and diauxies prioritize metabolism of preferred substrates. Strand-specific RNA-seq reveals how this bacterium responds to polysaccharides by up-regulating specific groups of CAZymes, transporters, and enzymes to metabolize the constituent sugars. Fifty-six up-regulated CAZymes were purified, and their activities show most polysaccharides are degraded by multiple enzymes, often from the same family, but with divergent rates, specificities, and cellular localizations. CAZymes were then tested in combination to identify synergies between enzymes acting on the same substrate with different catalytic mechanisms. We discuss how these results advance our understanding of how microbes degrade and metabolize plant biomass

Global repositioning of transcription start sites in a plant-fermenting bacterium

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Genome-Wide TSS Distribution in Three Related Clostridia with Normalized Capp-Switch Sequencing

International audienceTranscription initiation is a tightly regulated process that is crucial for many aspects of prokaryotic physiology. High-throughput transcription start site (TSS) mapping can shed light on global and local regulation of transcription initiation, which in turn may help us understand and predict microbial behavior. In this study, we used Capp-Switch sequencing to determine the TSS positions in the genomes of three model solventogenic clostridia: Clostridium acetobutylicum ATCC 824, C. beijerinckii DSM 6423, and C. beijerinckii NCIMB 8052. We first refined the approach by implementing a normalization pipeline accounting for gene expression, yielding a total of 12,114 mapped TSSs across the species. We further compared the distributions of these sites in the three strains. Results indicated similar distribution patterns at the genome scale, but also some sharp differences, such as for the butyryl-CoA synthesis operon, particularly when comparing C. acetobutylicum to the C. beijerinckii strains. Lastly, we found that promoter structure is generally poorly conserved between C. acetobutylicum and C. beijerinckii. A few conserved promoters across species are discussed, showing interesting examples of how TSS determination and comparison can improve our understanding of gene expression regulation at the transcript level. IMPORTANCE Solventogenic clostridia have been employed in industry for more than a century, initially being used in the acetone-butanol-ethanol (ABE) fermentation process for acetone and butanol production. Interest in these bacteria has recently increased in the context of green chemistry and sustainable development. However, our current understanding of their genomes and physiology limits their optimal use as industrial solvent production platforms. The gene regulatory mechanisms of solventogenesis are still only partly understood, impeding efforts to increase rates and yields. Genome-wide mapping of transcription start sites (TSSs) for three model solventogenic Clostridium strains is an important step toward understanding mechanisms of gene regulation in these industrially important bacteria

Tuning of Gene Expression in Clostridium phytofermentans Using Synthetic Promoters and CRISPRi

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ABC Transporters Required for Hexose Uptake by Clostridium phytofermentans

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Evolution of a Biomass-Fermenting Bacterium To Resist Lignin Phenolics

International audienceIncreasing the resistance of plant-fermenting bacteria to lignocellulosic inhibitors is useful to understand microbial adaptation and to develop candidate strains for consolidated bioprocessing. Here, we study and improve inhibitor resistance in Clostridium phytofermentans (also called Lachnoclostridium phytofermentans), a model anaerobe that ferments lignocellulosic biomass. We survey the resistance of this bacterium to a panel of biomass inhibitors and then evolve strains that grow in increasing concentrations of the lignin phenolic, ferulic acid, by automated, long-term growth selection in an anaerobic GM3 automat. Ultimately, strains resist multiple inhibitors and grow robustly at the solubility limit of ferulate while retaining the ability to ferment cellulose. We analyze genome-wide transcription patterns during ferulate stress and genomic variants that arose along the ferulate growth selection, revealing how cells adapt to inhibitors through changes in gene dosage and regulation, membrane fatty acid structure, and the surface layer. Collectively, this study demonstrates an automated framework for in vivo directed evolution of anaerobes and gives insight into the genetic mechanisms by which bacteria survive exposure to chemical inhibitors.IMPORTANCE Fermentation of plant biomass is a key part of carbon cycling in diverse ecosystems. Further, industrial biomass fermentation may provide a renewable alternative to fossil fuels. Plants are primarily composed of lignocellulose, a matrix of polysaccharides and polyphenolic lignin. Thus, when microorganisms degrade lignocellulose to access sugars, they also release phenolic and acidic inhibitors. Here, we study how the plant-fermenting bacterium Clostridium phytofermentans resists plant inhibitors using the lignin phenolic, ferulic acid. We examine how the cell responds to abrupt ferulate stress by measuring changes in gene expression. We evolve increasingly resistant strains by automated, long-term cultivation at progressively higher ferulate concentrations and sequence their genomes to identify mutations associated with acquired ferulate resistance. Our study develops an inhibitor-resistant bacterium that ferments cellulose and provides insights into genomic evolution to resist chemical inhibitors

mRNA expression of all 171 CAZymes during growth on pectins A–C, hemicelluloses D–E, glucans F–H, and raw corn stover I relative to expression on glucose.

<p>Expression was quantified as log₂(RPKM) with significantly differentially expressed genes on a given polysaccharide shown as triangles and unchanged genes as circles. The 56 purified CAZymes are red and others are blue.</p