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  • Publication
    Accès libre
    Community analysis of betaproteobacterial ammonia-oxidizing bacteria using the amoCAB operon
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    Kim, Ok-Sun
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    Ahn, Tae-Seok
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    Imhoff, Johannes F
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    Witzel
    The genes and intergenic regions of the amoCAB operon were analyzed to establish their potential as molecular markers for analyzing ammonia-oxidizing betaproteobacterial (beta-AOB) communities. Initially, sequence similarity for related taxa, evolutionary rates from linear regressions, and the presence of conserved and variable regions were analyzed for all available sequences of the complete amoCAB operon. The gene amoB showed the highest sequence variability of the three amo genes, suggesting that it might be a better molecular marker than the most frequently used amoA to resolve closely related AOB species. To test the suitability of using the amoCAB genes for community studies, a strategy involving nested PCR was employed. Primers to amplify the whole amoCAB operon and each individual gene were tested. The specificity of the products generated was analyzed by denaturing gradient gel electrophoresis, cloning, and sequencing. The fragments obtained showed different grades of sequence identity to amoCAB sequences in the GenBank database. The nested PCR approach provides a possibility to increase the sensitivity of detection of amo genes in samples with low abundance of AOB. It also allows the amplification of the almost complete amoA gene, with about 300 bp more sequence information than the previous approaches. The coupled study of all three amo genes and the intergenic spacer regions that are under different selection pressure might allow a more detailed analysis of the evolutionary processes, which are responsible for the differentiation of AOB communities in different habitats.
  • Publication
    Accès libre
    Genome Sequence of Kosakonia radicincitans Strain YD4, a Plant Growth-Promoting Rhizobacterium Isolated from Yerba Mate (Ilex paraguariensis St. Hill.)
    Bergottini, Veronica M
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    Johnson, Shannon
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    Chain, Patrick S
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    Otegui, Monica B
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    Zapata, Pedro D
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    Kosakonia radicincitans strain YD4 is a rhizospheric isolate from yerba mate (Ilex paraguariensis St. Hill.) with plant growth-promoting effects on this crop. Genes involved in different plant growth-promoting activities are present in this genome, suggesting its potential as a bioinoculant for yerba mate.
  • Publication
    Accès libre
    Bacterial communities in trace metal contaminated lake sediments are dominated by endospore-forming bacteria
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    Masson, Matthieu
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    Wunderlin, Tina
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    Kohler-Milleret, Roxane
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    Gascon Diez, Elena
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    Loizeau, Jean-Luc
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    Tercier-Waeber, Mary-Lou
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    Lake sediments in areas close to the outlet of wastewater treatment plants are sinks for pollutants. Bacterial communities in sediments are likely affected by the released effluents, but in turn they might modify the distribution and bioavailability of pollutants. On the shore of Lake Geneva, Switzerland, wastewater from the City of Lausanne is treated and discharged into the lake via an outlet pipe in the Vidy Bay. The objectives of this study were to assess (1) the impact of the treated wastewater release on the bacterial communities in the Vidy Bay sediments and (2) the potential link between bacterial communities and trace metal sediment content. Bacterial community composition and abundance were assessed in sediments collected in three areas with different levels of contamination. The main factors affecting bacterial communities were inferred by linking biological data with chemical analyses on these sediments. Near to the outlet pipe, large quantities of bacterial cells were detected in the three upper most cm (3.2 × 109 cells assessed by microscopy and 1.7 × 1010 copies of the 16S rRNA gene assessed by quantitative PCR, per gram of wet sediment), and the dominant bacterial groups were those typically found in activated sludge (e.g. Acidovorax defluivii and Hydrogenophaga caeni). Three samples in an area further away from the outlet and one sample close to it were characterized by 50 % of endospore-forming Firmicutes (Clostridium spp.) and a clear enrichment in trace metal content. These results highlight the potential role of endospore-forming Firmicutes on transport and deposition of trace metals in sediments.
  • Publication
    Accès libre
    Endospore-forming bacteria as new proxies to assess impact of eutrophication in Lake Geneva (Switzerland–France)
    Wunderlin, Tina
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    Corella, Juan Pablo
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    Loizeau, Jean-Luc
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    Girardclos, Stéphanie
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    Measurements of chemical composition and biological parameters of sediment cores are used as proxies for changes in past environmental conditions and more recently the human impact on ecosystem health. In this study, endospore-forming bacteria are proposed as a new biological proxy for such paleoecological reconstructions. A sediment core providing a record for the past 90 years (137Cs and magnetic susceptibility dating) was retrieved from the Rhone Delta of Lake Geneva. X-ray fluorescence was analyzed at a 0.2-cm resolution, while DNA extracts, elemental geochemistry and grain size were obtained at 4-cm intervals. The total number of bacteria and endospore-forming bacteria were quantified by qPCR using the 16S rRNA gene and the endosporulation-specific spo0A gene. Furthermore, a spo0A fragment was subjected to amplicon sequencing to define OTUs (operational taxonomic units) and the phylogenetic affiliation of the endospore formers. The results showed that despite the fact that the quantity of extracted DNA decreased with the age of the sediment, the abundance of endospore-forming bacteria remained constant. However, the diversity of this group of bacteria changed significantly, reflecting the eutrophication of the lake from 1960 to 1990. The shift in community composition was linked to the dominance of anaerobic clostridia-like endospore formers. This trend has reversed in the last 10 years of the record, suggesting a recovery after perturbation. This study shows that the abundance and diversity of endospore-forming bacteria can be used as proxies to reconstruct lake history. We hereby successfully introduce a new strategy for paleoecology that could also be applied to ocean sediments and long sediment cores.
  • Publication
    Accès libre
    Genome Sequence of Aeribacillus pallidus Strain GS3372, an Endospore-Forming Bacterium Isolated in a Deep Geothermal Reservoir
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    Jaussi, Marion
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    Wunderlin, Tina
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    Regenspurg, Simona
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    Li, Po-E
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    Lo, Chien-Chi
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    Johnson, Shannon
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    McMurry, Kim
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    Gleasner, Cheryl D
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    Vuyisich, Momchilo
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    Chain, Patrick S
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    The genome of strain GS3372 is the first publicly available strain of Aeribacillus pallidus. This endospore-forming thermophilic strain was isolated from a deep geothermal reservoir. The availability of this genome can contribute to the clarification of the taxonomy of the closely related Anoxybacillus, Geobacillus, and Aeribacillus genera.
  • Publication
    Accès libre
    TRiFLe, a Program for In Silico Terminal Restriction Fragment Length Polymorphism Analysis with User-Defined Sequence Sets
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    Witzel, Karl-Paul
    We describe TRiFLe, a freely accessible computer program that generates theoretical terminal restriction fragments (T-RFs) from any user-supplied sequence set tailored to a particular group of organisms, sequences from clone libraries, or sequences from specific genes. The program allows a rapid identification of the most polymorphic enzymes, creates a collection of T-RFs for the data set, and can potentially identify specific T-RFs in T-RF length polymorphism (T-RFLP) patterns by comparing theoretical and experimental results. TRiFLE was used for analyzing T-RFLP data generated for the amoA and pmoA genes. The peaks identified in the T-RFLP patterns show an overlap of ammonia- and methane-oxidizing bacteria in the metalimnion of a subtropical lake.
  • Publication
    Accès libre
    Under-detection of endospore-forming Firmicutes in metagenomic data
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    Wunderlin, Tina
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    Lo, Chien-Chi
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    Li, Po-E
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    Chain, Patrick S
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    Microbial diversity studies based on metagenomic sequencing have greatly enhanced our knowledge of the microbial world. However, one caveat is the fact that not all microorganisms are equally well detected, questioning the universality of this approach. Firmicutes are known to be a dominant bacterial group. Several Firmicutes species are endospore formers and this property makes them hardy in potentially harsh conditions, and thus likely to be present in a wide variety of environments, even as residents and not functional players. While metagenomic libraries can be expected to contain endospore formers, endospores are known to be resilient to many traditional methods of DNA isolation and thus potentially undetectable. In this study we evaluated the representation of endospore-forming Firmicutes in 73 published metagenomic datasets using two molecular markers unique to this bacterial group (spo0A and gpr). Both markers were notably absent in well-known habitats of Firmicutes such as soil, with spo0A found only in three mammalian gut microbiomes. A tailored DNA extraction method resulted in the detection of a large diversity of endospore-formers in amplicon sequencing of the 16S rRNA and spo0A genes. However, shotgun classification was still poor with only a minor fraction of the community assigned to Firmicutes. Thus, removing a specific bias in a molecular workflow improves detection in amplicon sequencing, but it was insufficient to overcome the limitations for detecting endospore-forming Firmicutes in whole-genome metagenomics. In conclusion, this study highlights the importance of understanding the specific methodological biases that can contribute to improve the universality of metagenomic approaches.
  • Publication
    Accès libre
    The genome of the Gram-positive metal- and sulfate-reducing bacterium Desulfotomaculum reducens strain MI-1
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    Podell, Sheila
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    Sims, David R
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    Detter, John C
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    Lykidis, Athanasios
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    Han, Cliff S
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    Wigginton, Nicholas S
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    Gaasterland, Terry
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    Bernier-Latmani, Rizlan
    Spore-forming, Gram-positive sulfate-reducing bacteria (SRB) represent a group of SRB that dominates the deep subsurface as well as niches in which resistance to oxygen and dessication is an advantage. Desulfotomaculum reducens; strain MI-1 is one of the few cultured representatives of that group with a complete genome sequence available. The metabolic versatility of this organism is reflected in the presence of genes encoding for the oxidation of various electron donors, including three- and four-carbon fatty acids and alcohols. Synteny in genes involved in sulfate reduction across all four sequenced Gram-positive SRB suggests a distinct sulfate-reduction mechanism for this group of bacteria. Based on the genomic information obtained for sulfate reduction in D. reducens;, the transfer of electrons to the sulfite and APS reductases is proposed to take place via the quinone pool and heterodisulfide reductases respectively. In addition, both H2-evolving and H2-consuming cytoplasmic hydrogenases were identified in the genome, pointing to potential cytoplasmic H2 cycling in the bacterium. The mechanism of metal reduction remains unknown.
  • Publication
    Accès libre
    Quantification of Endospore-Forming Firmicutes by Quantitative PCR with the Functional Gene spo0A
    Bacterial endospores are highly specialized cellular forms that allow endospore-forming Firmicutes (EFF) to tolerate harsh environmental conditions. EFF are considered ubiquitous in natural environments, in particular, those subjected to stress conditions. In addition to natural habitats, EFF are often the cause of contamination problems in anthropogenic environments, such as industrial production plants or hospitals. It is therefore desirable to assess their prevalence in environmental and industrial fields. To this end, a high-sensitivity detection method is still needed. The aim of this study was to develop and evaluate an approach based on quantitative PCR (qPCR). For this, the suitability of functional genes specific for and common to all EFF were evaluated. Seven genes were considered, but only spo0A was retained to identify conserved regions for qPCR primer design. An approach based on multivariate analysis was developed for primer design. Two primer sets were obtained and evaluated with 16 pure cultures, including representatives of the genera Bacillus, Paenibacillus, Brevibacillus, Geobacillus, Alicyclobacillus, Sulfobacillus, Clostridium, and Desulfotomaculum, as well as with environmental samples. The primer sets developed gave a reliable quantification when tested on laboratory strains, with the exception of Sulfobacillus and Desulfotomaculum. A test using sediment samples with a diverse EFF community also gave a reliable quantification compared to 16S rRNA gene pyrosequencing. A detection limit of about 104 cells (or spores) per gram of initial material was calculated, indicating this method has a promising potential for the detection of EFF over a wide range of applications.
  • Publication
    Accès libre
    Genome Sequence of Bacillus alveayuensis Strain 24KAM51, a Halotolerant Thermophile Isolated from a Hydrothermal Vent
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    Wunderlin, Tina
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    Johnson, Shannon
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    McMurry, Kim
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    Gleasner, Cheryl D
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    Lo, Chien-Chi
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    Li, Po-E
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    Vuyisich, Momchilo
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    Chain, Patrick S
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    Bacillus alveayuensis strain 24KAM51 was isolated from a marine hydrothermal vent in Milos, Greece. Its genome depicts inter-esting features of halotolerance and resistance to heavy metals.