Accessing the genomic information of unculturable oceanic picoeukaryotes by combining multiple single cells

Mangot, Jean-François et al.-- 12 pages, 7 figures, 2 tables, supplementary information https://dx.doi.org/10.1038/srep41498Pico-sized eukaryotes play key roles in the functioning of marine ecosystems, but we still have a limited knowledge on their ecology and evolution. The MAST-4 lineage is of particular interest, since it is widespread in surface oceans, presents ecotypic differentiation and has defied culturing efforts so far. Single cell genomics (SCG) are promising tools to retrieve genomic information from these uncultured organisms. However, SCG are based on whole genome amplification, which normally introduces amplification biases that limit the amount of genomic data retrieved from a single cell. Here, we increase the recovery of genomic information from two MAST-4 lineages by co-assembling short reads from multiple Single Amplified Genomes (SAGs) belonging to evolutionary closely related cells. We found that complementary genomic information is retrieved from different SAGs, generating co-assembly that features >74% of genome recovery, against about 20% when assembled individually. Even though this approach is not aimed at generating high-quality draft genomes, it allows accessing to the genomic information of microbes that would otherwise remain unreachable. Since most of the picoeukaryotes still remain uncultured, our work serves as a proof-of-concept that can be applied to other taxa in order to extract genomic data and address new ecological and evolutionary questionsThis work was supported by the US NSF grants DEB-1031049 and OCE-821374 (to M.E.S.), by the ANR French projects Oceanomics (ANR-11-BTBR-0008, to C.V.), France Génomique (ANR-10-INBS-09, to P.W.), and Prometheus (ANR-09-PCS-GENM_217, to O.J.), by the EU project SINGEK (H2020-MSCA-ITN-2015-675752, to R.M.), and by the Spanish project MEFISTO (CTM2013-43767-P, MINECO). J.-F.M. was supported by a Marie Curie Intra-European Fellowship (PIEF-GA-2012-331190, EU). R.L. was supported by Juan de la Cierva (JCI-2010-06594, MINECO) and Ramón y Cajal fellowships (RYC-2013-12554, MINECO)Peer Reviewe

Phylogenetic Affiliation of SSU rRNA Genes Generated by Massively Parallel Sequencing: New Insights into the Freshwater Protist Diversity

International audienceRecent advances in next-generation sequencing (NGS) technologies spur progress in determining the microbial diversity in various ecosystems by highlighting, for example, the rare biosphere. Currently, high-throughput pyrotag sequencing of PCR-amplified SSU rRNA gene regions is mainly used to characterize bacterial and archaeal communities, and rarely to characterize protist communities. In addition, although taxonomic assessment through phylogeny is considered as the most robust approach, similarity and probabilistic approaches remain the most commonly used for taxonomic affiliation. In a first part of this work, a tree-based method was compared with different approaches of taxonomic affiliation (BLAST and RDP) of 18S rRNA gene sequences and was shown to be the most accurate for near full-length sequences and for 400 bp amplicons, with the exception of amplicons covering the V5-V6 region. Secondly, the applicability of this method was tested by running a full scale test using an original pyrosequencing dataset of 18S rRNA genes of small lacustrine protists (0.2-5 mm) from eight freshwater ecosystems. Our results revealed that i) fewer than 5% of the operational taxonomic units (OTUs) identified through clustering and phylogenetic affiliation had been previously detected in lakes, based on comparison to sequence in public databases; ii) the sequencing depth provided by the NGS coupled with a phylogenetic approach allowed to shed light on clades of freshwater protists rarely or never detected with classical molecular ecology approaches; and iii) phylogenetic methods are more robust in describing the structuring of under-studied or highly divergent populations. More precisely, new putative clades belonging to Mamiellophyceae, Foraminifera, Dictyochophyceae and Euglenida were detected. Beyond the study of protists, these results illustrate that the tree-based approach for NGS based diversity characterization allows an in-depth description of microbial communities including taxonomic profiling, community structuring and the description of clades of any microorganisms (protists, Bacteria and Archaea)

Isolation and characterization of mesotrione-degrading Bacillus sp. from soil

International audienceDissipation kinetics of mesotrione, a new triketone herbicide, sprayed on soil from Limagne (Puy-de-Dôme, France) showed that the soil microflora were able to biotransform it. Bacteria from this soil were cultured in mineral salt solution supplemented with mesotrione as sole source of carbon for the isolation of mesotrione-degrading bacteria. The bacterial community structure of the enrichment cultures was analyzed by temporal temperature gradient gel electrophoresis (TTGE). The TTGE fingerprints revealed that mesotrione had an impact on bacterial community structure only at its highest concentrations and showed mesotrione-sensitive and mesotrione-adapted strains. Two adapted strains, identified as Bacillus sp. and Arthrobacter sp., were isolated by colony hybridization methods. Biodegradation assays showed that only the Bacillus sp. strain was able to completely and rapidly biotransform mesotrione. Among several metabolites formed, 2-amino-4-methylsulfonylbenzoic acid (AMBA) accumulated in the medium. Although sulcotrione has a chemical structure closely resembling that of mesotrione, the isolates were unable to degrade i

Diel variation of seawater volatile organic compounds, DMSP-related compounds, and microbial plankton inside and outside a tropical coral reef ecosystem

Biogenic volatile organic compounds (VOCs) play key roles in coral reef ecosystems, where, together with dimethylated sulfur compounds, they are indicators of ecosystem health and are used as defense strategies and infochemicals. Assessment and prediction of the exchange rates of VOCs between the oceans and atmosphere, with implications for atmospheric reactivity and climate, are hampered by poor knowledge of the regulating processes and their temporal variability, including diel cycles. Here, we measured the variation over 36h of the concentrations of DMSPCs (dimethylsulfoniopropionate (DMSP)-related compounds, namely DMSP, dimethylsulfoxide, acrylate, dimethylsulfide, and methanethiol as dimethyl disulfide) and VOCs (COS, CS2, isoprene, the iodomethanes CH3I and CH2ClI, and the bromomethanes CHBr3 and CH2Br2), in surface waters inside the shallow, northern coral-reef lagoon of Mo’orea (French Polynesia) and 4 km offshore, in the tropical open ocean. Comparisons with concurrent measurements of sea surface temperature, solar radiation, biogeochemical variables (nutrients, organic matter), and the abundances and taxonomic affiliations of microbial plankton were conducted with the aim to explain interconnections between DMSPCs, VOCs, and their environment across diel cycles. In open ocean waters, deeper surface mixing and low nutrient levels resulted in low phytoplankton biomass and bacterial activity. Consequently, the diel patterns of VOCs were more dependent on photochemical reactions, with daytime increases for several compounds including dissolved dimethylsulfoxide, COS, CS2, CH3I, and CH2ClI. A eukaryotic phytoplankton assemblage dominated by dinoflagellates and haptophytes provided higher cell-associated DMSP concentrations, yet the occurrence of DMSP degradation products (dimethylsulfide, dimethyl disulfide) was limited by photochemical loss. Conversely, in the shallow back reef lagoon the proximity of seafloor sediments, corals and abundant seaweeds resulted in higher nutrient levels, more freshly-produced organic matter, higher bacterial activity, and larger algal populations of Mamiellales, diatoms and Cryptomonadales. Consequently, DMSP and dimethylsulfoxide concentrations were lower but those of most VOCs were higher. A combination of photobiological and photochemical processes yielded sunny-daytime increases and nighttime decreases of dimethylsulfide, dimethyl disulfide, COS, isoprene, iodomethanes and bromomethanes. Our results illustrate the important role of solar radiation in DMSPC and VOC cycling, and are relevant for the design of sampling strategies that seek representative and comparable measurements of these compounds

Spatial and temporal dynamic of eukaryotic picoplankton in lakes : molecular approaches by FISH and pyrosequencing

Ces travaux de recherche, menés par une approche écosystémique et par la mise en œuvre de plusieurs méthodes moléculaires (TSA-FISH, qPCR et pyroséquençage), ont eu pour objectif (i) d'acquérir des données quantitatives concernant divers groupes eucaryotes unicellulaires présents dans la fraction picoplanctonique lacustre, (ii) d'explorer la dynamique spatiale et temporelle de ces groupes picoeucayotes tant en terme d'abondance que de diversité phylogénétique, et ceci à différentes échelles d'observation, et (iii) d' apporter une profondeur d'analyse supplémentaire de la diversité et de la structure des picoeucaryotes par l'utilisation de séquençage massif. L'utilisation de sondes oligonucléotidiques spécifiques a mis en évidence l'importance quantitative d'organismes photosynthétiques appartenant aux Chrysophycées, Chlorophycées, ainsi que la présence récurrente de groupes potentiellement parasites-saprophytes (Perkinsozoa, Fungi, LKM11). L'étude de la répartition spatiale (verticale) et géographique (trois lacs) de ces groupes par cette même approche a mis en lumière des différences d'abondance selon le statut trophique et la profondeur. Ainsi, la présence dans des proportions significatives d'organismes pigmentés (Chlorophycées, Haptophycées) en zone hypolimnique non éclairée nous a conduit à émettre l'hypothèse de l'importance de la mixotrophie au sein de la fraction picoplanctonique. Par ailleurs, l'utilisation du séquençage à haut débit a permis de révéler une importante diversité (1017 OTUs) 10 à 100 fois supérieure à celle décrite classiquement (clonage-séquençage de l'ADNr 18S). Son application à une échelle temporelle fine (2/3 jours) a permis de mettre en évidence d'importants et continuels remaniements au sein de la communauté picoeucaryote impliquant principalement les 21 OTUs appartenant au “core taxa” (>1% des séquences), et des taxa présentant des abondances intermédiaires (0,01-1% des séquences). A contrario, l'assemblage des taxa rares ( 1% of sequences), and taxa with intermediate abundances (0.01 to 1% of sequences).Conversely, the rare taxa community (<0.01%), which represented more than half of the describeddiversity, is in turn relatively stable over time.The data acquired contribute to the debate about thepatterns of microbial diversity. They suggest the need to better integrate the concept of functionaldiversity in these reflections

Review of the Perkinsozoa, a well-known marine protozoan flagellate parasite group, newly discovered in lacustrine systems

International audienceThe recurrent detection of parasitic zoospores among aquatic heterotrophic flagellates (HFs) has recently modified our view of how the microbial loop is organized, and called into question the role of eukaryotic parasites in the aquatic trophic food web. The Perkinsozoa group, already known to play a significant role as parasite in marine systems, is of special interest here, since it has recently been detected in several lakes by constructing clone libraries. In marine systems, this group is known to consist solely of intracellular parasites of molluscs or phytoplanktonic species, but their hosts in freshwater environments are still unknown, and little is yet known about their functional importance in planktonic systems. This review summarizes the main information currently available about Perkinsozoa through a description of their phylogenetic position, their life cycles, and regulatory factors, and the consideration of the specificities of their hosts in marine systems, and the few data recently acquired in lakes

Accessing the ecology of uncultured picoeukaryotes through a high-throughput automatic cell enumeration approach

VII European Congress of Protistology (ECP) in partnership with The International Society of Protistologists (ISOP), 5-10 September 2015, Sevilla, SpainSmall planktonic protists (i.e. picoeukaryotes, cells of 0.8-3 µm) have fundamental roles in the functioning of marine ecosystems, both as primary producers and as microbial grazers, and are likely the most abundant eukaryotes on Earth. Over the last fifteen years, technological progresses in molecular ecology and environmental sequencing have substantially boosted our understanding of these marine microbes, unveiling an unexpected diversity and notably the existence of new uncultured clades. These methodological improvements have notably offered the possibility to analyse simultaneously a substantial number of samples, allowing a more accurate description of their diversity in time and space. Faced with this growing amount of data available on microbial diversity, it is now challenging to design parallel molecular tools combined with microscopy to acccess their quantititive importance in environmental systems. However, this quantitative approach is relatively expensive, time-consuming and observant-dependant. Furthermore, their tiny size, their lack of specific morphological traits and the changing number of 18S rDNA copies between taxa among the picosize protistan community do not allow the use of classical microscopy and SSU-based molecular approaches (qPCR) to access their abundance in a large numbers of samples. Within this study we tested a newly developed automatic image acquisition and subsequently cell enumeration system of picoeukaryotic organisms. Two uncultured groups of picoeukaryotes differing by their cell size, MAST-4 (2 µm) and MAST-1C (5 µm), have been considered here to optimize this high-throughput quantitative approach for picoeukarytic cells. After targeting these groups by TSA-FISH using specific oligonucleotide probes, microscopic images were acquired fully automatically and cells enumerated using the program ACMEtool 2.0. In general, direct microscopic cell counts were in agreement with our automatic cell counting approach. The automated method can process a larger number of fields of view (FOVs) and consequently analyzes more cells, so it provides an estimate that is closer to true cell abundance. We were further able to test this automatic microscope and cell enumeration system on a spatial survey with a wide geographical coverage, about 100 stations from a circumglobal expedition, by depicting the spatial distribution of the two above-mentioned MAST groupsPeer Reviewe

Rarity in aquatic microbes: placing protists on the map

Special issue on Microbial diversity, adaptation and evolution.-- 11 pages, 1 figure, 1 tableMost microbial richness at any given time tends to be represented by low-abundance (rare) taxa, which are collectively referred to as the “rare biosphere”. Here we review works on the rare biosphere using high-throughput sequencing (HTS), with a particular focus on unicellular eukaryotes or protists. Evidence thus far indicates that the rare biosphere encompasses dormant as well as metabolically active microbes that could potentially play key roles in ecosystem functioning. Rare microbes appear to have biogeography, and sometimes the observed patterns can be similar to what is observed among abundant taxa, suggesting similar community-structuring mechanisms. There is limited evidence indicating that the rare biosphere contains taxa that are phylogenetically distantly related to abundant counterparts; therefore, the rare biosphere may act as a reservoir of deep-branching phylogenetic diversity. The potential role of the rare biosphere as a bank of redundant functions that can help to maintain continuous ecosystem function following oscillations in taxonomic abundances is hypothesized as its main ecological role. Future studies focusing on rare microbes are crucial for advancing our knowledge of microbial ecology and evolution and unveiling their links with ecosystem functionRL was supported by Juan de la Cierva (JCI-2010-06594, Spanish Ministry of Economy and Competitiveness, Spain) and Ramón y Cajal fellowships (RYC-2013-12554, Spanish Ministry of Economy and Competitiveness, Spain). JFM was supported by a Marie Curie Intra-European Fellowship (PIEF-GA-2012-331190, EU)Peer Reviewe

Dynamique spatio-temporelle du picoplancton eucaryote lacustre (approches moléculaires par FISH et pyrosequençage)

Ces travaux de recherche, menés par une approche écosystémique et par la mise en œuvre de plusieurs méthodes moléculaires (TSA-FISH, qPCR et pyroséquençage), ont eu pour objectif (i) d'acquérir des données quantitatives concernant divers groupes eucaryotes unicellulaires présents dans la fraction picoplanctonique lacustre, (ii) d'explorer la dynamique spatiale et temporelle de ces groupes picoeucayotes tant en terme d'abondance que de diversité phylogénétique, et ceci à différentes échelles d'observation, et (iii) d' apporter une profondeur d'analyse supplémentaire de la diversité et de la structure des picoeucaryotes par l'utilisation de séquençage massif. L'utilisation de sondes oligonucléotidiques spécifiques a mis en évidence l'importance quantitative d'organismes photosynthétiques appartenant aux Chrysophycées, Chlorophycées, ainsi que la présence récurrente de groupes potentiellement parasites-saprophytes (Perkinsozoa, Fungi, LKM11). L'étude de la répartition spatiale (verticale) et géographique (trois lacs) de ces groupes par cette même approche a mis en lumière des différences d'abondance selon le statut trophique et la profondeur. Ainsi, la présence dans des proportions significatives d'organismes pigmentés (Chlorophycées, Haptophycées) en zone hypolimnique non éclairée nous a conduit à émettre l'hypothèse de l'importance de la mixotrophie au sein de la fraction picoplanctonique. Par ailleurs, l'utilisation du séquençage à haut débit a permis de révéler une importante diversité (1017 OTUs) 10 à 100 fois supérieure à celle décrite classiquement (clonage-séquençage de l'ADNr 18S). Son application à une échelle temporelle fine (2/3 jours) a permis de mettre en évidence d'importants et continuels remaniements au sein de la communauté picoeucaryote impliquant principalement les 21 OTUs appartenant au core taxa (>1% des séquences), et des taxa présentant des abondances intermédiaires (0,01-1% des séquences). A contrario, l'assemblage des taxa rares ( 1% of sequences), and taxa with intermediate abundances (0.01 to 1% of sequences).Conversely, the rare taxa community (<0.01%), which represented more than half of the describeddiversity, is in turn relatively stable over time.The data acquired contribute to the debate about thepatterns of microbial diversity. They suggest the need to better integrate the concept of functionaldiversity in these reflections.SAVOIE-SCD - Bib.électronique (730659901) / SudocGRENOBLE1/INP-Bib.électronique (384210012) / SudocGRENOBLE2/3-Bib.électronique (384219901) / SudocSudocFranceF