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Junier, Pilar
Nom
Junier, Pilar
Affiliation principale
Fonction
Professeure assistante
Email
pilar.junier@unine.ch
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Résultat de la recherche
Voici les éléments 1 - 10 sur 14
- PublicationAccès librePhysical Isolation of Endospores from Environmental Samples by Targeted Lysis of Vegetative Cells(2016)
; ; ; Endospore formation is a survival strategy found among some bacteria from the phylum Firmicutes. During endospore formation, these bacteria enter a morpho-physiological resting state that enhances survival under adverse environmental conditions. Even though endospore-forming Firmicutes are one of the most frequently enriched and isolated bacterial groups in culturing studies, they are often absent from diversity studies based on molecular methods. The resistance of the spore core is considered one of the factors limiting the recovery of DNA from endospores. We developed a method that takes advantage of the higher resistance of endospores to separate them from other cells in a complex microbial community using physical, enzymatic and chemical lysis methods. The endospore-only preparation thus obtained can be used for re-culturing or to perform downstream analysis such as tailored DNA extraction optimized for endospores and subsequent DNA sequencing. This method, applied to sediment samples, has allowed the enrichment of endospores and after sequencing, has revealed a large diversity of endospore-formers in freshwater lake sediments. We expect that the application of this method to other samples will yield a similar outcome. - PublicationMétadonnées seulementMLgsc: A Maximum-Likelihood General Sequence Classifier(2015)
; ; We present software package for classifying protein or nucleotide sequences to user-specified sets of reference sequences. The software trains a model using a multiple sequence alignment and a phylogenetic tree, both supplied by the user. The latter is used to guide model construction and as a decision tree to speed up the classification process. The software was evaluated on all the 16S rRNA gene sequences of the reference dataset found in the GreenGenes database. On this dataset, the software was shown to achieve an error rate of around 1% at genus level. Examples of applications based on the nitrogenase subunit NifH gene and a protein-coding gene found in endospore-forming Firmicutes is also presented. The programs in the package have a simple, straightforward command-line interface for the Unix shell, and are free and open-source. The package has minimal dependencies and thus can be easily integrated in command-line based classification pipelines. - PublicationAccès libreGenome Sequence of Anoxybacillus geothermalis Strain GSsed3, a Novel Thermophilic Endospore-Forming Species(2015)
; ; ; ; - PublicationAccès libreGenome Sequence of Aeribacillus pallidus Strain GS3372, an Endospore-Forming Bacterium Isolated in a Deep Geothermal Reservoir(2015)
; ; ; - PublicationAccès libreGenome Sequence of Bacillus alveayuensis Strain 24KAM51, a Halotolerant Thermophile Isolated from a Hydrothermal Vent(2015)
; ; ; - PublicationMétadonnées seulement
- PublicationAccès libreDiversity of endospore-forming bacteria in sediment as a proxy for environmental lake history(2013)
Cette thèse présente la recherche sur la détection et la diversité des bactéries sporulantes et leur usage comme indicateurs des conditions écologiques du lac Léman (France-Suisse) au cours des 100 dernières années. Les endospores sont des structures résistantes qui sont produites par un groupe de bactéries dans des conditions de stress. Les endospores dormantes sont déposées avec le sédiment et sont des capsules biologiques qui reflètent les conditions environnementales au moment de leur sédimentation.
Les approches métagénomiques sont des études de séquençage du métagénome entier qui se trouve dans un échantillon environnemental. Une approche ciblée est réalisée uniquement sur une partie de la communauté (fraction ciblée). Ces approches métagénomiques ciblées augmentent la couverture de séquençage et également la résolution de détection de taxons. Elles résolvent donc les problèmes connus des approches métagénomiques globales. Deux méthodes de métagénomique ciblée ont été développées au cours de cette thèse pour étudier la diversité des bactéries sporulantes dans les sédiments.
La première méthode est basée sur des amorces moléculaires dessinée pour amplifier un fragment du gène spo0A, gène spécifique aux bactéries sporulantes codant pour le facteur de transcription de la sporulation. De plus, une méthode optimisée d'extraction d'ADN pour les bactéries sporulantes a été développée. En appliquant ces méthodes, la diversité des cellules végétatives des bactéries sporulantes ainsi que les endospores dans le sédiment peut être étudiée.
La deuxième méthode de métagénomique ciblée est une méthode de traitement avec chaleur et agents chimiques pour détruire les cellules végétatives, qui sont fragiles, comparé aux endospores qui résistent au traitement. Avec cette méthode, la diversité seule des endospores peut être évaluée. Le traitement pour détruire les cellules végétatives est efficace, 90% des séquences détectées sont classifiées comme bactéries sporulantes. Avec une approche globale, seulement 10% des séquences détectées sont classifiées comme bactéries sporulantes. De plus, la résolution a été augmentée, en détectant jusqu'à 10 fois plus de taxon. La meilleure résolution permet de détecter 34 genres de bactéries sporulantes non révélés avec l'approche globale, dont certain genres qui ont été définis comme étant asporogénique, comme Ethanoligenens et Trichococcus. D'autres séquences ont pu être classifiées au niveau de l'espèce comme Bacillus longiquaesitum ou Clostridium bowmanii.
Les méthodes métagénomiques ciblées ont été appliquées à une carotte de sédiment du Lac Léman, qui couvre la période de 1921 à 2010. Une diversité extraordinaire de bactéries sporulantes a été observée dans ces sédiments. Il y a des fluctuations importantes dans la diversité au cours des 100 dernières années. Prioritairement les changements de diversité peuvent être liée à l'eutrophisation du lac de 1960 à 1990, mais aussi au métabolisme du soufre, la charge de matière organique d'origine terrestre ou à des évènements climatiques. La communauté bactérienne dans la période eutrophe a changé vers une dominance de bactéries anaérobies comme les Clostridia, qui reflètent les conditions anoxiques de la surface du sédiment pendant cette période.
Les avantages du traitement pour séparer les endospores des cellules végétatives sont que ces deux fractions peuvent être analysées individuellement. Avec ces différentiations, des genres actifs dans le sédiment comme Clostridium et Heliobacterium peuvent être relevés. Par contre, il y a toute une fraction diversifiée d'endospores dormantes qui est présente en faible nombre, jusqu'à ce que les conditions environnementales changent en leur faveur ou elles deviennent dominantes. Des exemples de ce phénomène ont été observés pour les genres Desulfotomaculum, Sporomusa et Brevibacillus.
Les nouvelles méthodes de métagénomique ciblée qui ont été développées dans le cadre de cette thèse sont des améliorations importantes pour étudier la diversité des bactéries sporulantes à haute résolution. Les données acquises pendant cette recherche amènent de nouvelles informations sur le rôle des bactéries sporulantes dans les sédiments et en général sur les bactéries des sédiments lacustres. Cette étude est aussi la première étude sur la diversité des bactéries sporulantes par des méthodes de séquençage et la première étude d'utilisation des endospores comme indicateur paléolimnologique., Freshwater systems are of high ecological and economical importance but at the same time subjected to anthropogenic pressure such as input of pollutants, overfishing, changes in climate regime, and eutrophication (high nutrient input). For environmental management and the implementation of conservation measures, the dynamics of freshwater ecosystems need to be known and biological reference conditions have to be established against which future changes can be measured. Baseline knowledge about biodiversity and ecosystem responses to environmental perturbations in lakes can be obtained from the sediments that provide an ideal environmental archive of past conditions.
This thesis presents research on the diversity detection of bacterial endospores, and its use as proxy to reconstruct the environmental history of the last 100 years of Lake Geneva at the border of France and Switzerland.
Endospores are resistant structures formed when bacteria are under stress. Once these endospores are deposited in the sediment they remain dormant and serve as natural biological time capsules, archiving the conditions at the time of sedimentation. To infer the diversity of endospores, two specific methods for targeted metagenomics were developed and validated in sediments. Metagenomics is a sequencing approach of the entire genetic pool directly retrieved from an environmental sample. Similarly, targeted metagenomics is based on a targeted genetic pool, for example a sub-community of the sample. Targeted metagenomics increases the sequencing coverage and resolution of detection, circumventing common problems of traditional metagenomics studies. For this work the endospore-forming bacterial community was targeted.
The first targeting method was based on a molecular marker for endospore-forming bacteria, the global transcription regulator of sporulation (spo0A). After an optimized DNA extraction method for endospores in sediment, where biomass was separated from the sediment particles (indirect DNA extraction), and the spo0A gene fragment was amplified and sequenced. With this method, the diversity of the endospore-forming bacteria (vegetative cells and endospores) in sediments was determined.
The second targeting method consisted of a treatment to separate the endospores from vegetative cells, prior to DNA extraction and sequencing. The goal of the treatment was to destroy vegetative cells that are generally more fragile, while leaving the more resistant endospores intact. With this method, the diversity of only the endospores in sediment was detected. The treatment to separate endospores was successful, as shown by an enrichment of endospore-forming bacteria from 10% abundance in the global approach to over 90% abundance in the targeted metagenome. Also the resolution was improved to up to 10-fold increase in detected endospore-forming taxa. The better resolution led to the detection of 34 genera unique to the targeted metagenome, including some supposedly asporogenic groups like Ethanoligenens and Trichococcus and high numbers of sequences that could be classified to a species level such as Bacillus longiquaesitum or Clostridium bowmanii.
The application of targeted metagenomics to a sediment core retrieved from Lake Geneva spanning a time period from 1921 to 2010, revealed substantial diversity of endospore-forming bacteria in sediments. The diversity fluctuated significantly in the last 100 years, reflecting the eutrophication period from 1960 to 1990 as well as sulphate metabolism, input of terrestrial organic matter, and specific climate events. The shift in the community composition during eutrophication was linked to a dominance of anaerobic Clostridia-like members that reflect anoxic sediment conditions during this time.
The advantage of the treatment is that the communities in vegetative cell state can be differentiated from the communities present as dormant endospores. Using this differentiation we report activity of selected endospore-forming bacteria in sediment, for example members from genus Clostridium and Heliobacterium at the sediment surface.
In contrast, a small fraction of dormant endospores present at high diversity represent the microbial seed bank. This group of bacteria is inactive for long periods of time but selected members can propagate and become dominant if the environmental conditions change to their favour, as was observed in this study for Desulfotomaculum, Sporomusa or Brevibacillus.
The novel targeted metagenomics approaches developed here provide a significant experimental improvement to explore the diversity of endospore-forming bacteria at high resolution. The data provides knowledge on the role of endospore-forming bacteria in freshwater sediment and on freshwater sediment bacteria in general. It is also the first report of metagenomics to reveal the diversity of endospores in sediment and the use of endospores as paleolimnological proxies. - PublicationAccès libreBacterial communities in trace metal contaminated lake sediments are dominated by endospore-forming bacteria
; ; ; - PublicationAccès libreStage 0 sporulation gene A as a molecular marker to study diversity of endospore-forming Firmicutes
; ; ; In this study, we developed and validated a cultureindependent method for diversity surveys to specifically detect endospore-forming Firmicutes. The global transcription regulator of sporulation (spo0A) was identified as a gene marker for endosporeforming Firmicutes. To enable phylogenetic classification, we designed a set of primers amplifying a 602 bp fragment of spo0A that we evaluated in pure cultures and environmental samples. The amplification was positive for 35 strains from 11 genera, yet negative for strains from Alicyclobacillus and Sulfobacillus. We also evaluated various DNA extraction methods because endospores often result in reduced yields. Our results demonstrate that procedures utilizing increased physical force improve DNA extraction. An optimized DNA extraction method on biomass pre-extracted from the environmental sample source (indirect DNA extraction) followed by amplification with the aforementioned primers for spo0A was then tested in sediments from two different sources. Specifically, we validated our cultureindependent diversity survey methodology on a set of 8338 environmental spo0A sequences obtained from the sediments of Lakes Geneva (Switzerland) and Baikal (Russia). The phylogenetic affiliation of the environmental sequences revealed a substantial number of new clades within endospore-formers. This novel culture-independent approach provides a significant experimental improvement that enables exploration of the diversity of endospore-forming Firmicutes. - PublicationAccès libreUnder-detection of endospore-forming Firmicutes in metagenomic data
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