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Auteur Thomas, F.; Dittami, S.M.; Brunet, M.; Le Duff, N.; Tanguy, G.; Leblanc, C.; Gobet, A. doi  openurl
  Titre Evaluation of a new primer combination to minimize plastid contamination in 16S rDNA metabarcoding analyses of alga-associated bacterial communities Type Article scientifique
  Année (up) Publication Revue Abrégée Environ. Microbiol. Rep.  
  Volume Numéro Pages  
  Mots-Clés diversity; phylogenetic analysis; ribosomal-rna sequences; surfaces  
  Résumé Plant- and alga-associated bacterial communities are generally described via 16S rDNA metabarcoding using universal primers. As plastid genomes encode 16S rDNA related to cyanobacteria, these data sets frequently contain >90% plastidial sequences, and the bacterial diversity may be under-sampled. To overcome this limitation we evaluated in silico the taxonomic coverage for four primer combinations targeting the 16S rDNA V3-V4 region. They included a forward primer universal to Bacteria (S-D-Bact-0341-b-S-17) and four reverse primers designed to avoid plastid DNA amplification. The best primer combination (NOCHL) was compared to the universal primer set in the wet lab using a synthetic community and samples from three macroalgal species. The proportion of plastid sequences was reduced by 99%-100% with the NOCHL primers compared to the universal primers, irrespective of algal hosts, sample collection and extraction protocols. Additionally, the NOCHL primers yielded a higher richness while maintaining the community structure. As Planctomycetes, Verrucomicrobia and Cyanobacteria were underrepresented (70%-90%) compared to universal primers, combining the NOCHL set with taxon-specific primers may be useful for a complete description of the alga-associated bacterial diversity. The NOCHL primers represent an innovation to study algal holobionts without amplifying host plastid sequences and may further be applied to other photosynthetic hosts.  
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  ISSN 1758-2229 ISBN Médium  
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  Numéro d'Appel MARBEC @ isabelle.vidal-ayouba @ collection 2661  
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Auteur Auguet, J.C.; Casamayor, E.O. doi  openurl
  Titre A hotspot for cold crenarchaeota in the neuston of high mountain lakes Type Article scientifique
  Année (up) 2008 Publication Revue Abrégée Environ Microbiol  
  Volume 10 Numéro 4 Pages 1080-1086  
  Mots-Clés 16S/genetics Spain *Water Microbiology; Archaeal/genetics RNA; Biodiversity Crenarchaeota/*classification/genetics/*isolation & purification Fresh Water/*microbiology In Situ Hybridization; Fluorescence Indoles Phylogeny RNA; Ribosomal  
  Résumé We have surveyed the first 1 m of 10 oligotrophic high mountain lakes in the Central Pyrenees (Spain) for both abundance and predominant phylotypes richness of the archaeaplankton assemblage, using CARD-FISH and 16S rRNA gene sequencing respectively. Archaea inhabiting the air-water surface microlayer (neuston) ranged between 3% and 37% of total 4,6-diamidino-2-phenylindole (DAPI) counts and were mainly Crenarchaeota of a new freshwater cluster distantly related to the Marine Group 1.1a. Conversely, most of the Archaea from the underlying waters (the remaining first 1 m integrated) were mainly Euryarchaeota of three distantly related branches ranging between 0.4% and 27% of total DAPI counts. Therefore, a consistent qualitative and quantitative spatial segregation was observed for the two main archaeal phyla between neuston and underlying waters at a regional scale. We also observed a consistent pattern along the lakes surveyed between lake area, lake depth and water residence time, and the archaeal enrichment in the neuston: the larger the lake the higher the proportion of archaea in the neuston as compared with abundances from the underlying waters (n = 10 lakes; R(2) > 0.80; P < 0.001, in all three cases). This is the first report identifying a widespread non-thermophilic habitat where freshwater planktonic Crenarchaeota can be found naturally enriched. High mountain lakes offer great research opportunities to explore the ecology of one of the most enigmatic and far from being understood group of prokaryotes.  
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  Numéro d'Appel MARBEC @ isabelle.vidal-ayouba @ collection 1300  
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Auteur Auguet, J.C.; Montanie, H.; Hartmann, H.J.; Lebaron, P.; Casamayor, E.O.; Catala, P.; Delmas, D. doi  openurl
  Titre Potential effect of freshwater virus on the structure and activity of bacterial communities in the Marennes-Oleron Bay (France) Type Article scientifique
  Année (up) 2009 Publication Revue Abrégée Microb Ecol  
  Volume 57 Numéro 2 Pages 295-306  
  Mots-Clés 16S/genetics Seasons Seawater/microbiology/virology Viruses/*growth & development *Water Microbiology; Bacteria/genetics/*growth & development/*virology Biodiversity Colony Count; Bacterial/genetics France Fresh Water/virology Polymorphism; Microbial DNA Fingerprinting DNA; Ribosomal; Single-Stranded Conformational Population Dynamics RNA  
  Résumé Batch culture experiments using viral enrichment were conducted to test the response of a coastal bacterial community to autochthonous (i.e., co-existing) or allochthonous riverine viruses. The effects of viral infections on bacterial dynamics and activity were assessed by epifluorescence microscopy and thymidine incorporation, respectively, whereas the effect of viral infection on bacterial community composition was examined by polymerase chain reaction-single strand conformation polymorphism 16S ribosomal RNA fingerprinting. The percentages of high nucleic acid-containing cells, evaluated by flow cytometry, were significantly correlated (r2=0.91, n=12, p<0.0001) to bacterial production, making this value a good predictor of active cell dynamics along the study. While confinement and temperature were the two principal experimental factors affecting bacterial community composition and dynamics, respectively, additions of freshwater viruses had significant effects on coastal bacterial communities. Thus, foreign viruses significantly reduced net bacterial population increase as compared to the enrichment treated with inactivated virus. Moreover, freshwater viruses recurrently and specifically affected bacterial community composition, as compared to addition of autochthonous viruses. In most cases, the combined treatment viruses and freshwater dissolved organic matter helped to maintain or even enhance species richness in coastal bacterial communities in agreement to the 'killing the winner' hypothesis. Thus, riverine virus input could potentially influence bacterial community composition of the coastal bay albeit with modest modification of bulk bacterial growth.  
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  Numéro d'Appel MARBEC @ isabelle.vidal-ayouba @ collection 1301  
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Auteur Lliros, M.; Gich, F.; Plasencia, A.; Auguet, J.C.; Darchambeau, F.; Casamayor, E.O.; Descy, J.P.; Borrego, C. doi  openurl
  Titre Vertical distribution of ammonia-oxidizing crenarchaeota and methanogens in the epipelagic waters of Lake Kivu (Rwanda-Democratic Republic of the Congo) Type Article scientifique
  Année (up) 2010 Publication Revue Abrégée Appl Environ Microbiol  
  Volume 76 Numéro 20 Pages 6853-6863  
  Mots-Clés 16S/genetics Sequence Analysis; Ammonia/*metabolism Animals *Biodiversity Cluster Analysis Crenarchaeota/*classification/genetics/isolation & purification/*metabolism DNA Fingerprinting DNA; Archaeal/chemistry/genetics DNA; Archaeal/genetics RNA; DNA Sequence Homology; Fluorescence Methane/*metabolism Molecular Sequence Data Nucleic Acid Denaturation Oxidation-Reduction Phylogeny Polymerase Chain Reaction RNA; Nucleic Acid *Water Microbiology; Polyacrylamide Gel Genes; Ribosomal; Ribosomal/chemistry/genetics Democratic Republic of the Congo Electrophoresis; rRNA In Situ Hybridization  
  Résumé Four stratified basins in Lake Kivu (Rwanda-Democratic Republic of the Congo) were sampled in March 2007 to investigate the abundance, distribution, and potential biogeochemical role of planktonic archaea. We used fluorescence in situ hybridization with catalyzed-reported deposition microscopic counts (CARD-FISH), denaturing gradient gel electrophoresis (DGGE) fingerprinting, and quantitative PCR (qPCR) of signature genes for ammonia-oxidizing archaea (16S rRNA for marine Crenarchaeota group 1.1a [MCG1] and ammonia monooxygenase subunit A [amoA]). Abundance of archaea ranged from 1 to 4.5% of total DAPI (4',6-diamidino-2-phenylindole) counts with maximal concentrations at the oxic-anoxic transition zone ( approximately 50-m depth). Phylogenetic analysis of the archaeal planktonic community revealed a higher level of richness of crenarchaeal 16S rRNA gene sequences (21 of the 28 operational taxonomic units [OTUs] identified [75%]) over euryarchaeotal ones (7 OTUs). Sequences affiliated with the kingdom Euryarchaeota were mainly recovered from the anoxic water compartment and mostly grouped into methanogenic lineages (Methanosarcinales and Methanocellales). In turn, crenarchaeal phylotypes were recovered throughout the sampled epipelagic waters (0- to 100-m depth), with clear phylogenetic segregation along the transition from oxic to anoxic water masses. Thus, whereas in the anoxic hypolimnion crenarchaeotal OTUs were mainly assigned to the miscellaneous crenarchaeotic group, the OTUs from the oxic-anoxic transition and above belonged to Crenarchaeota groups 1.1a and 1.1b, two lineages containing most of the ammonia-oxidizing representatives known so far. The concomitant vertical distribution of both nitrite and nitrate maxima and the copy numbers of both MCG1 16S rRNA and amoA genes suggest the potential implication of Crenarchaeota in nitrification processes occurring in the epilimnetic waters of the lake.  
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  Numéro d'Appel MARBEC @ isabelle.vidal-ayouba @ collection 1303  
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Auteur Auguet, J.C.; Nomokonova, N.; Camarero, L.; Casamayor, E.O. doi  openurl
  Titre Seasonal changes of freshwater ammonia-oxidizing archaeal assemblages and nitrogen species in oligotrophic alpine lakes Type Article scientifique
  Année (up) 2011 Publication Revue Abrégée Appl Environ Microbiol  
  Volume 77 Numéro 6 Pages 1937-1945  
  Mots-Clés 16S/genetics Seasons Spain; Ammonia/*metabolism Archaea/classification/genetics/*metabolism Biodiversity Fresh Water Molecular Sequence Data Nitrogen/*metabolism Oxidoreductases/genetics Phylogeny Polymerase Chain Reaction RNA; Ribosomal  
  Résumé The annual changes in the composition and abundance of ammonia-oxidizing archaea (AOA) were analyzed monthly in surface waters of three high mountain lakes within the Limnological Observatory of the Pyrenees (LOOP; northeast Spain) using both 16S rRNA and functional (ammonia monooxygenase gene, amoA) gene sequencing as well as quantitative PCR amplification. The set of biological data was related to changes in nitrogen species and to other relevant environmental variables. The whole archaeal assemblage was dominated by phylotypes closely related to the crenarchaeal 1.1a group (58% +/- 18% of total 16S rRNA gene sequences), and consistent structural changes were detected during the study. Water temperature was the environmental variable that better explained spring, summer, and winter (ice-covered lakes) archaeal assemblage structure. The amoA gene was detected year round, and seasonal changes in amoA gene composition were well correlated with changes in the archaeal 16S rRNA gene pool. In addition, copy numbers of both the specific 1.1a group 16 rRNA and archaeal amoA genes were well correlated, suggesting that most freshwater 1.1a Crenarchaeota had the potential to carry out ammonia oxidation. Seasonal changes in the diversity and abundance of AOA (i.e., amoA) were better explained by temporal changes in ammonium, the substrate for nitrification, and mostly nitrite, the product of ammonia oxidation. Lacustrine amoA gene sequences grouped in coherent freshwater phylogenetic clusters, suggesting that freshwater habitats harbor typical amoA-containing ecotypes, which is different from soils and seas. We observed within the freshwater amoA gene sequence pool a high genetic divergence (translating to up to 32% amino acid divergence) between the spring and the remaining AOA assemblages. This suggests that different AOA ecotypes are adapted to different temporal ecological niches in these lakes.  
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  Numéro d'Appel MARBEC @ isabelle.vidal-ayouba @ collection 1304  
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