Substrate-specific clades of active marine methylotrophs associated with a phytoplankton bloom in a temperate coastal environment

Marine microorganisms that consume one-carbon (C1) compounds are poorly described, despite their impact on global climate via an influence on aquatic and atmospheric chemistry. This study investigated marine bacterial communities involved in the metabolism of C1 compounds. These communities were of relevance to surface seawater and atmospheric chemistry in the context of a bloom that was dominated by phytoplankton known to produce dimethylsulfoniopropionate. In addition to using 16S rRNA gene fingerprinting and clone libraries to characterize samples taken from a bloom transect in July 2006, seawater samples from the phytoplankton bloom were incubated with 13C-labeled methanol, monomethylamine, dimethylamine, methyl bromide, and dimethyl sulfide to identify microbial populations involved in the turnover of C1 compounds, using DNA stable isotope probing. The [13C]DNA samples from a single time point were characterized and compared using denaturing gradient gel electrophoresis (DGGE), fingerprint cluster analysis, and 16S rRNA gene clone library analysis. Bacterial community DGGE fingerprints from 13C-labeled DNA were distinct from those obtained with the DNA of the nonlabeled community DNA and suggested some overlap in substrate utilization between active methylotroph populations growing on different C1 substrates. Active methylotrophs were affiliated with Methylophaga spp. and several clades of undescribed Gammaproteobacteria that utilized methanol, methylamines (both monomethylamine and dimethylamine), and dimethyl sulfide. rRNA gene sequences corresponding to populations assimilating 13C-labeled methyl bromide and other substrates were associated with members of the Alphaproteobacteria (e.g., the family Rhodobacteraceae), the Cytophaga-Flexibacter-Bacteroides group, and unknown taxa. This study expands the known diversity of marine methylotrophs in surface seawater and provides a comprehensive data set for focused cultivation and metagenomic analyses in the future

Analyses moléculaires de la diversité et des fonctions de micro-organismes incultivés des sources hydrothermales profondes

Nos connaissances de la diversité des communautés microbiennes marines ont longtemps été restreintes aux informations précieuses, mais incomplètes, issues de l utilisation de méthodes de culture. Lors de cette étude, des techniques moléculaires (PCR, clonage, séquençage, hybridation, construction d une banque métagénomique) et des marqueurs génétiques [ARNr 16S, gènes codants pour des enzymes spécifiques de la voie inverse du cycle tricarboxylique (acIB, oorA)] ont été utilisés pour contourner les limites inhérentes aux méthodes de culture et accéder a la diversité spécifique ainsi qu aux fonctions des micro-organismes incultivés des sources hydrothermales profondes. Ce travail de recherche a permis de : -confirmer l importance écologique des epsilon-Proteobacteria dans l écosystème hydrothermal profond, notamment dans les processus de colonisation de surfaces vierges exposées aux fluides hydrothermaux. de mettre au point une sonde oligonucléotidique spécifique des epsilon-Proteobacteria permettant de les quantifier et de les observer dans un échantillon environnemental. de montrer la présence de bactéries Arcobacter-like vraisemblablement sulfo-oxydantes et qui seraient impliquées dans la formation d un biofilm filamenteux contenant essentiellement du soufre. Et d obtenir via l approche métagénomique, les premiers fragments génomiques d Archea incultivées hydrothermales. Grâce à l utilisation combinée de différentes approches moléculaires, ce travail a apporté un nouvel éclairage sur la diversité des communautés microbiennes de l écosystème hydrothermal marin profond.Our knowledge of the diversity of marine microbial communities has long been restricted to the precious but incomplete information generated by the culture-based methods. In this study, molecular techniques (PCR, cloning, sequencing, hybridization, metagenomics library construction and genetic markers [16S rRNA, genes coding for enzymes specific to the reverse tricarboxylic acid cycle (acIB, oorA)] were used to circumvent the limits inherent to cultivation methods, and to obtain a more realistic view of the specific and functional diversity of the deep-sea hydrothermal vent microbial communities. This research allowed (i) to confirm the ecological significance of free-living epsilon-Proteobacteria at deep-sea hydrothermal vents, especially during in-situ colonization experiments (this study provides the first example of the prevalence and ecological significance of free-living Arcobacter-like at deep-sea hydrothermal vents, which are supposed to be sulfo-oxidizing bacteria involved in filamentous sulphur formation, (ii) to design and validate a 16S rRNA oligonucleotide probe targeting most of the epsilon-Proteobacteria found in hydrothermal systems, (iii) to obtain the physiology of yet uncultured groups of archea from deep-sea hydrothermal vents using metagenomics. By the combined use of variety of molecular approaches this work enlarges our view of the diversity of microbial communities in deep-sea hydrothermal ventsBREST-BU Droit-Sciences-Sports (290192103) / SudocPLOUZANE-Bibl.La Pérouse (290195209) / SudocSudocFranceF

Analyses moléculaires de la diversité et des fonctions de micro-organismes incultivés des sources hydrothermales profondes

Nos connaissances de la diversité des communautés microbiennes marines ont longtemps été restreintes aux informations précieuses, mais incomplètes, issues de l utilisation de méthodes de culture. Lors de cette étude, des techniques moléculaires (PCR, clonage, séquençage, hybridation, construction d une banque métagénomique) et des marqueurs génétiques [ARNr 16S, gènes codants pour des enzymes spécifiques de la voie inverse du cycle tricarboxylique (acIB, oorA)] ont été utilisés pour contourner les limites inhérentes aux méthodes de culture et accéder a la diversité spécifique ainsi qu aux fonctions des micro-organismes incultivés des sources hydrothermales profondes. Ce travail de recherche a permis de : -confirmer l importance écologique des epsilon-Proteobacteria dans l écosystème hydrothermal profond, notamment dans les processus de colonisation de surfaces vierges exposées aux fluides hydrothermaux. de mettre au point une sonde oligonucléotidique spécifique des epsilon-Proteobacteria permettant de les quantifier et de les observer dans un échantillon environnemental. de montrer la présence de bactéries Arcobacter-like vraisemblablement sulfo-oxydantes et qui seraient impliquées dans la formation d un biofilm filamenteux contenant essentiellement du soufre. Et d obtenir via l approche métagénomique, les premiers fragments génomiques d Archea incultivées hydrothermales. Grâce à l utilisation combinée de différentes approches moléculaires, ce travail a apporté un nouvel éclairage sur la diversité des communautés microbiennes de l écosystème hydrothermal marin profond.Our knowledge of the diversity of marine microbial communities has long been restricted to the precious but incomplete information generated by the culture-based methods. In this study, molecular techniques (PCR, cloning, sequencing, hybridization, metagenomics library construction and genetic markers [16S rRNA, genes coding for enzymes specific to the reverse tricarboxylic acid cycle (acIB, oorA)] were used to circumvent the limits inherent to cultivation methods, and to obtain a more realistic view of the specific and functional diversity of the deep-sea hydrothermal vent microbial communities. This research allowed (i) to confirm the ecological significance of free-living epsilon-Proteobacteria at deep-sea hydrothermal vents, especially during in-situ colonization experiments (this study provides the first example of the prevalence and ecological significance of free-living Arcobacter-like at deep-sea hydrothermal vents, which are supposed to be sulfo-oxidizing bacteria involved in filamentous sulphur formation, (ii) to design and validate a 16S rRNA oligonucleotide probe targeting most of the epsilon-Proteobacteria found in hydrothermal systems, (iii) to obtain the physiology of yet uncultured groups of archea from deep-sea hydrothermal vents using metagenomics. By the combined use of variety of molecular approaches this work enlarges our view of the diversity of microbial communities in deep-sea hydrothermal ventsBREST-BU Droit-Sciences-Sports (290192103) / SudocPLOUZANE-Bibl.La Pérouse (290195209) / SudocSudocFranceF

Novel uncultured Epsilonproteobacteria dominate a filamentous sulphur mat from the 13°N hydrothermal vent field, East Pacific Rise

The definitive version is available at ww3.interscience.wiley.com. En libre-accès sur Archimer : http://archimer.ifremer.fr/doc/2006/publication-2110.pdfInternational audienceRapid growth of microbial sulphur mats have repeatedly been observed during oceanographic cruises to various deep-sea hydrothermal vent sites. The microorganisms involved in the mat formation have not been phylogenetically characterized, although the production of morphologically similar sulphur filaments by a Arcobacter strain coastal marine has been documented. An in situ collector deployed for 5 days at the 13°N deep-sea hydrothermal vent site on the East Pacific Rise (EPR) was rapidly colonized by a filamentous microbial mat. Microscopic and chemical analyses revealed that the mat consisted of a network of microorganisms embedded in a mucous sulphur-rich matrix. Molecular surveys based on 16S rRNA gene and aclB genes placed all the environmental clone sequences within the Epsilonproteobacteria. Although few 16S rRNA gene sequences were affiliated with that of cultured organisms, the majority was related to uncultured representatives of the Arcobacter group (≤95% sequence similarity). A probe designed to target all of the identified lineages hybridized with more than 95% of the mat community. Simultaneous hybridizations with the latter probe and a probe specific to Arcobacter spp. confirmed the numerical dominance of Arcobacter-like bacteria. This study provides the first example of the prevalence and ecological significance of free-living Arcobacter at deep-sea hydrothermal vents

Uncultured Archaea in a hydrothermal microbial assemblage: phylogenetic diversity and characterization of a genome fragment from a euryarchaeote

The definitive version is available at ww3.interscience.wiley.comInternational audienceThe polychaete Alvinella pompejana lives in organic tubes on the walls of active hydrothermal chimneys along the East Pacific Rise. To examine the diversity of the archaeal community associated with the polychaete tubes, we constructed libraries by direct PCR amplification and cloning of 16S rRNA genes. Almost half of the sequences of the 16S rRNA gene libraries clustered with uncultured archaeal groups. In an effort to access genomic information from uncultured archaeal members we further constructed a fosmid library from the same DNA source. One of the clones, Alv-FOS5, was sequenced completely. Its sequence analysis revealed an incomplete rRNA operon and 32 predicted ORFs. Seventeen of these ORFs have been assigned putative functions, including transcription and translation, cellular processes and signalling, transport systems and metabolic pathways. Phylogenetic analyses of the 16S rRNA gene suggested that Alv-FOS5 formed a new lineage related to members of Deep-Sea Hydrothermal Vent Euryarchaeota group II. Phylogenetic analyses of predicted proteins revealed the existence of likely cases of horizontal gene transfer, both between Crenarchaeota and Euryarchaeota and between Archaea and Bacteria. This study is the first step in using genomics to reveal the physiology of an as yet uncultured group of archaea from deep-sea hydrothermal vents

Thermophilic Lifestyle for an Uncultured Archaeon from Hydrothermal Vents: Evidence from Environmental Genomics

We present a comparative analysis of two genome fragments isolated from a diverse and widely distributed group of uncultured euryarchaea from deep-sea hydrothermal vents. The optimal activity and thermostability of a DNA polymerase predicted in one fragment were close to that of the thermophilic archaeon Thermoplasma acidophilum, providing evidence for a thermophilic way of life of this group of uncultured archaea