Nanoplastic production procedure for scientific purposes: PP, PVC, PE-LD, PE-HD, and PS

International audienceStudies on the environmental impact of nanoplastics face challenges in plastic analysis and a scarcity of nanoplastic materials necessary for the development of analytical techniques and experiments on biota impact. Here we provide detailed procedures for obtaining nanoparticles suspended in water for the most commonly used polymers: Polypropylene (PP), Polyvinylchloride (PVC), Low-and High-Density Polyethylene (PE-LD, PE-HD), and Polystyrene (PS). We dissolved larger size material to reprecipitate nanoparticles. For all plastic types, we obtained nanoparticles with a size between 50 and 300 nm, and a mainly spherical morphology. We verified that no irreversible agglomeration or coalescence of the particles occurred after 5 days of storage. The concentrations obtained in the final carrier solution were of the order of 10 9 particles mL-1. To prevent the persistence of reagents in the final carrier solution, a filtration step was implemented at the end of the process. The method proved unsuitable for Polyethylene Terephthalate (PET)

Genomics study of diatom-bacteria interactions

Les diatomées sont des algues microscopiques qui contribuent à hauteur de 25% environ à la production primaire planétaire. Les diatomées sont très souvent entourées d’une flore bactérienne, avec laquelle de nombreuses interactions ont été documentées. Les génomes de diatomées contiennent par ailleurs de nombreux gènes dont l’origine prédite est bactérienne.Nous avons étudié Asterionella formosa, une diatomée pennée présente dans de nombreux lacs et cours d’eau à l'aide de données omiques. L’utilisation de la métagénomique a permis de reconstruire 30 génomes bactériens, utilisés pour prédire d'éventuelles interactions avec la diatomée. Le séquençage de la sous-unité 16S de l’ARN ribosomique a montré que les différentes espèces bactériennes avaient une abondance variable au cours des phases de croissance de la diatomée, et que certaines étaient plus souvent au contact de la diatomée que libres dans le milieu. Le génome d'A. formosa a ensuite été séquencé à l'aide de la technologie Pacbio et comparé à ceux d'espèces proches. Enfin, l’impact des bactéries sur les diatomées a été abordé sous l’angle de l’évolution et des transferts horizontaux de gènes, qui ont été prédits à partir des données transcriptomiques d’une centaine de diatomées marines.Ce travail représente une première étape dans l'étude de la communauté bactérienne associée à A. formosa. Des expériences complémentaires incluant l’utilisation de transcriptomique ou métabolomique sont maintenant envisageables. Les données collectées et/ou analysées dans ce travail contribuent d'ores et déjà à l’effort global de caractérisation génomique des diatomées.Diatoms are ubiquitous microalgae that contribute approximately 25% to the primary production worldwide. Many interactions, either positive, neutral or negative, have been documented between diatoms and bacteria. Diatom genomes also harbor numerous genes of putative bacterial origin.We are studying Asterionella formosa, a freshwater pennate diatom. We characterized the community using a combination of omics and laboratory techniques. We reconstructed of the genome of the diatom as well as 30 individual genomes from co-cultured bacterial species and investigated metabolisms that could support diatom-bacteria interactions. 16S rRNA sequencing revealed that the abundance of some bacterial species was highly variable over the course of A. formosa growth. Some species seemed preferentially attached to the diatom while others were mainly free-living. Then, the reference sequence of the A. formosa genome was improved by additional long-read (Pacbio) sequencing. Last, relationships between diatoms and bacteria were investigated at a broader evolutionary scale, by looking at horizontal gene transfers using transcriptomic data of a hundred marine diatoms.This work is a first step in the study of the dynamic and complex bacterial community associated with the diatom A. formosa. The accurate identification and the reconstruction of the genome of these bacteria will enable further in silico predictions based on metabolic networks and new omics experiments using transcriptomic or metabolomic. This work already contributes to a global effort to study diatoms by the means of genomics

Giant viruses at the core of microscopic wars with global impacts

International audienceThe unicellular eukaryotes (also called protists) that inhabit the contemporary oceans have large impacts on major biogeochemical cycles. Populations of oceanic protists are to a large extent regulated by their viral parasites, especially nucleocytoplasmic large DNA viruses (NCLDVs). NCLDVs can themselves be the prey of smaller viruses called virophages and can also be infected by transposable elements termed transpovirons. These entangled parasitisms have fostered the emergence of sophisticated infection and defence strategies. In addition persistent contact has facilitated the exchange of genes between different parties. Recent advances shed light on the strategies that govern such microbial wars. Endogenous virophage-like elements found in the genome of a marine alga could for instance provide the host acquired immunity against NCLDVs. In return, it was recently speculated that virophage sequences can be hijacked by NCLDVs and used as genetic weapons against virophages

Draft Genome Sequences of 15 Bacterial Species Constituting the Stable Defined Intestinal Microbiota of the GM15 Gnotobiotic Mouse Model

International audienceThe GM15 community is a bacterial consortium used to generate a novel standardized mouse model with a simplified controlled intestinal microbiota recapitulating the specific opportunistic pathogen-free (SOPF) mouse phenotype and the potential to ensure an increased reproducibility and robustness of preclinical studies by limiting the confounding effect of microbiota composition fluctuation. T he intestinal microbiota is a complex and dynamic ecosystem largely composed of bacteria whose activity greatly impacts the health and diseases of the host (1). Associating mice with stable defined bacterial consortia reduces the complexity of the microbiota and overcomes limitations related to the variability between individuals and animal facilities (2, 3). Therefore, gnotobiotics contribute to standardization and experimental reproducibility and are a powerful tool for testing causality in host-microbiome studies (4-6). Thus, we have developed a simplified mouse microbiota that is representative of the fecal microbiota found in C57BL/6J mice on the functional level and derived a standardized gnotobiotic mouse model called GM15, which has been bred successfully for over eight generations in the gnotobiology unit of BIOASTER. All animal procedures were approved by the French Ministry of Higher Education, Research and Innovation (MESR) and the ANSES/ENVA/UPEC ethics committee (Autorisation de Projet Utilisant des Animaux à des Fins Scientifiques [APAFIS] no. 4529-2016022616404045v3, 785-2015042819315178v2, and 18918-2019020118003843v3) and were conducted in accordance with national French and European legislation on the protection of animals used for scientific purposes. We report here the draft genome sequences of 9 bacterial strains isolated from the intestinal microbiota of C57BL/6J specific-opportunistic-pathogen-free (SOPF) mice (Charles River Laboratories, France), 2 bacterial strains isolated from C57BL/6J axenic mice recolonized with feces of the altered Schaedler flora (ASF) mouse model (Taconic, USA), and 4 bacterial strains obtained from the DSMZ collection. Then, the colonization of the axenic C57BL/6J mice with these 15 bacterial isolates resulted in the GM15 mice. Fresh cecal contents and fecal pellets of mice were resuspended (1/10 [wt/vol]) in reduced broth medium for direct dilution plating onto agar plates (same medium as the broth) and growth at 37°C under an anaerobic atmosphere (90% N 2 , 5% H 2 , 5% CO 2). Lactobacillus johnsonii MD006 was isolated on MRS agar. Lactobacillus murinus MD040 and Parabacteroides goldsteinii MD072 were isolated on Columbia nalidixic acid (CNA) agar with 5% sheep blood. Bacteroides acidifaciens MD185 and Lachnospiraceae sp. strain MD308 were isolated on Gifu anaerobic medium (GAM) agar. Bacteroides caecimuris MD237 and Lactobacillus reuteri MD207 were isolated on GAM agar supplemented , respectively, with 32 g/ml vancomycin and 32 g/ml erythromycin. Lachno-spiraceae sp. strains MD335 and MD329 were isolated on M2GSC (modified Med2 of Citation Elie C, Mathieu A, Saliou A, Villain A, Darnaud M, Leulier F, Tamellini A. 2020. Draft genome sequences of 15 bacterial species constituting the stable defined intestinal microbiota of the GM15 gnotobiotic mouse model. Microbiol Resou

Inorganic carbon uptake in a freshwater diatom, Asterionella formosa (Bacillariophyceae): from ecology to genomics

Inorganic carbon availability can limit primary productivity and control species composition of freshwater phytoplankton. This is despite the presence of CO2-concentrating mechanisms (CCMs) in some species that maximize inorganic carbon uptake. We investigated the effects of inorganic carbon on the seasonal distribution, growth rates and photosynthesis of a freshwater diatom, Asterionella formosa, and the nature of its CCM using genomics. In a productive lake, the frequency of A. formosa declined with CO2 concentration below air-equilibrium. In contrast, CO2 concentrations at 2.5-times air-equilibrium did not increase growth rate, cell C-quota or the ability to remove inorganic carbon. A pH-drift experiment strongly suggested that HCO3− as well as CO2 could be used. Calculations combining hourly inorganic carbon concentrations in a lake with known CO2 and HCO3− uptake kinetics suggested that rates of photosynthesis of A. formosa would be approximately carbon saturated and largely dependent on CO2 uptake when CO2 was at or above air-equilibrium. However, during summer carbon depletion, HCO3− would be the major form of carbon taken up and carbon saturation will fall to around 30%. Genes encoding proteins involved in CCMs were identified in the nuclear genome of A. formosa. We found carbonic anhydrases from subclasses α, β, γ and θ, as well as solute carriers from families 4 and 26 involved in HCO3− transport, but no periplasmic carbonic anhydrase. A model of the components of the CCM and their location in A. formosa showed that they are more similar to Phaeodactylum tricornutum than to Thalassiosira pseudonana, two marine diatoms

Quiescence unveils a novel mutational force in fission yeast

International audienceTo maintain life across a fluctuating environment, cells alternate between phases of cell division and quiescence. During cell division, the spontaneous mutation rate is expressed as the probability of mutations per generation (Luria and Delbrü ck, 1943; Lea and Coulson, 1949), whereas during quiescence it will be expressed per unit of time. In this study, we report that during quiescence, the unicellular haploid fission yeast accumulates mutations as a linear function of time. The novel mutational landscape of quiescence is characterized by insertion/deletion (indels) accumulating as fast as single nucleotide variants (SNVs), and elevated amounts of deletions. When we extended the study to 3 months of quiescence, we confirmed the replication-independent mutational spectrum at the whole-genome level of a clonally aged population and uncovered phenotypic variations that subject the cells to natural selection. Thus, our results support the idea that genomes continuously evolve under two alternating phases that will impact on their size and composition

RSH enzyme diversity for (p)ppGpp metabolism in Phaeodactylum tricornutum and other diatoms

International audienceThe nucleotides guanosine tetraphosphate and pentaphosphate (together known as (p)ppGpp or magic spot) are produced in plant plastids from GDP/GTP and ATP by RelA-SpoT homologue (RSH) enzymes. In the model plant Arabidopsis (p)ppGpp regulates chloroplast transcription and translation to affect growth, and is also implicated in acclimation to stress. However, little is known about (p)ppGpp metabolism or its evolution in other photosynthetic eukaryotes. Here we studied (p)ppGpp metabolism in the marine diatom Phaeodactylum tricornutum. We identified three expressed RSH genes in the P. tricornutum genome, and determined the enzymatic activity of the corresponding enzymes by heterologous expression in bacteria. We showed that two P. tricornutum RSH are (p)ppGpp synthetases, despite substitution of a residue within the active site believed critical for activity, and that the third RSH is a bifunctional (p)ppGpp synthetase and hydrolase, the first of its kind demonstrated in a photosynthetic eukaryote. A broad phylogenetic analysis then showed that diatom RSH belong to novel algal RSH clades. Together our work significantly expands the horizons of (p)ppGpp signalling in the photosynthetic eukaryotes by demonstrating an unexpected functional, structural and evolutionary diversity in RSH enzymes from organisms with plastids derived from red algae

Golden magic: RSH enzymes for (p)ppGpp metabolism in the diatom PhaeodactylumPhaeodactylum tricornutumtricornutum

The nucleotides guanosine tetraphosphate and pentaphosphate (together known as (p)ppGpp or magic spot) are produced in plant plastids from GDP/GTP and ATP by RelA-SpoT homologue (RSH) enzymes. In the model plant Arabidopsis (p)ppGpp regulates chloroplast transcription and translation to affect growth, and is also implicated in acclimation to stress. However, little is known about (p)ppGpp metabolism or its evolution in other photosynthetic eukaryotes. Here we studied (p)ppGpp metabolism in the golden-coloured marine diatom Phaeodactylum tricornutum. We identified three expressed RSH genes in the P. tricornutum genome, and determined the enzymatic activity of the corresponding enzymes by heterologous expression in bacteria. We showed that two P. tricornutum RSH are (p)ppGpp synthetases, despite substitution of a residue within the active site believed critical for activity, and that the third RSH is a bifunctional (p)ppGpp synthetase and hydrolase, the first of its kind demonstrated in a photosynthetic eukaryote. A broad phylogenetic analysis then showed that diatom RSH belong to novel algal RSH clades. Together our work significantly expands the horizons of (p)ppGpp signalling in the photosynthetic eukaryotes by demonstrating an unexpected functional, structural and evolutionary diversity in RSH enzymes from organisms with plastids derived from red alga

Demography and Intercontinental Spread of the USA300 Community-Acquired Methicillin-Resistant Staphylococcus aureus Lineage.

International audienceCommunity-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) was recognized worldwide during the 1990s; in less than a decade, several genetically distinct CA-MRSA lineages carrying Panton-Valentine leukocidin genes have emerged on every continent. Most notably, in the United States, the sequence type 18-IV (ST8-IV) clone known as USA300 has become highly prevalent, outcompeting methicillin-susceptible S. aureus (MSSA) and other MRSA strains in both community and hospital settings. CA-MRSA bacteria are much less prevalent in Europe, where the European ST80-IV European CA-MRSA clone, USA300 CA-MRSA strains, and other lineages, such as ST22-IV, coexist. The question that arises is whether the USA300 CA-MRSA present in Europe (i) was imported once or on very few occasions, followed by a broad geographic spread, anticipating an increased prevalence in the future, or (ii) derived from multiple importations with limited spreading success. In the present study, we applied whole-genome sequencing to a collection of French USA300 CA-MRSA strains responsible for sporadic cases and micro-outbreaks over the past decade and United States ST8 MSSA and MRSA isolates. Genome-wide phylogenetic analysis demonstrated that the population structure of the French isolates is the product of multiple introductions dating back to the onset of the USA300 CA-MRSA clone in North America. Coalescent-based demography of the USA300 lineage shows that a strong expansion occurred during the 1990s concomitant with the acquisition of the arginine catabolic mobile element and antibiotic resistance, followed by a sharp decline initiated around 2008, reminiscent of the rise-and-fall pattern previously observed in the ST80 lineage. A future expansion of the USA300 lineage in Europe is therefore very unlikely. To trace the origin, evolution, and dissemination pattern of the USA300 CA-MRSA clone in France, we sequenced a collection of strains of this lineage from cases reported in France in the last decade and compared them with 431 ST8 strains from the United States. We determined that the French CA-MRSA USA300 sporadic and micro-outbreak isolates resulted from multiple independent introductions of the USA300 North American lineage. At a global level, in the transition from an MSSA lineage to a successful CA-MRSA clone, it first became resistant to multiple antibiotics and acquired the arginine catabolic mobile element and subsequently acquired resistance to fluoroquinolones, and these two steps were associated with a dramatic demographic expansion. This expansion was followed by the current stabilization and expected decline of this lineage. These findings highlight the significance of horizontal gene acquisitions and point mutations in the success of such disseminated clones and illustrate their cyclic and sporadic life cycle

Complete mitochondrial genome sequence of the freshwater diatom Asterionella formosa

International audienceWe report the complete mitochondrial genome sequence of the freshwater diatom Asterionella formosa. The large 61.9 kb circular sequence encodes 34 proteins and 25 tRNAs that are universally conserved in other sequenced diatoms. We fully resolved a unique 24 kb region containing highly conserved repeated sequence units, possibly collocating with an origin of replicatio