PCR-generated artefact from 16S rRNA gene-specific primers

Osborne, Catherine A.; Galić, Maja; Sangwan, Parveen; Janssen, Peter H.

doi:10.1016/j.femsle.2005.05.043

Cited by 74 publications

(41 citation statements)

References 17 publications

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“…Polymerase chain reactions (PCR) for TRFLP analysis targeted bacterial and archaeal 16S rRNA genes as well as the methanogenic marker gene mcrA, coding for the methyl coenzyme M reductase subunit A, and the methanotrophic marker gene pmoA, coding for the methane monooxygenase subunit A. Primer combinations used in the study were: 27f/907r for Bacteria (Osborne et al 2005;Muyzer et al 1995) with FAMlabeled forward primer; 109f/934r for Archaea (Großkopf et al 1998) with FAM-labeled reverse primer; MCRf/MCRr (Springer et al 1995) for methanogens with FAM-labeled forward primer; A189f/mb661r (Costello and Lidstrom 1999) for methanotrophs with FAM-labeled forward primer. PCR was carried out in a total volume of 50 μL, containing 200 μM desoxynucleotriphosphates (Fermentas), 1× GoFlexiGreen Buffer (Sigma-Aldrich), 10 μg Bovine Serum Albumin (BSA; Roche), 1 U GoTaq DNA Polymerase (Sigma-Aldrich), 0.5 μM of each primer pair, 1.5 mM MgCl 2 (Promega) and 20 ng template DNA.…”

Section: Molecular Analysesmentioning

confidence: 99%

Bromeliad tanks are unique habitats for microbial communities involved in methane turnover

2016

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Background and aims Tank bromeliads collect organic matter and rainwater (= tank slurry) between their densely arranged leaf axils for their nutrient demand. Diverse communities of microorganisms inhabit these tanks and are responsible for the breakdown of organic matter. Anaerobic degradation results in the release of substantial amounts of methane. We hypothesized that each individual bromeliad harbors its own microbial community, which is affected by chemical tank-slurry properties. We further hypothesized that methanotrophic bacteria inhabit bromeliad tank slurries, potentially able to oxidize the produced CH 4 . Methods We investigated communities of Bacteria, Archaea, methanogenic and methanotrophic microorganisms measuring their abundance (qPCR) and composition (TRFLP) within eight bromeliad tanks of the species Werauhia gladioliflora sampled in a Costa Rican lowland forest. Tank slurries were analyzed for pH, carbon, nitrogen, oxygen and fatty acid concentrations. Methane oxidation rates were determined in five bromeliad tank slurries.Results Our results showed that microbial communities differed between plants and were affected by chemical tank slurry properties. Further, not only methanogenic archaea but also methanotrophic bacteria were detected in the tanks of all bromeliad plants, the latter being potentially able to aerobically oxidize between 25 and 62 μg CH 4 gdw. Conclusion Our results indicate that every bromeliad tank is a unique island with respect to its resident microbial community. The presence of methanogens and active methanotrophs in all tank slurries further indicates the potential for both methane formation and methane oxidation.

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Section: Molecular Analysesmentioning

confidence: 99%

Bromeliad tanks are unique habitats for microbial communities involved in methane turnover

2016

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“…Clone libraries were created by substituting the FAM27f primer with the unlabeled 27f primer in the PCR used to generate T-RFLP profiles and cloned, screened, and sequenced as described previously (26). Sequences were edited and in silico restriction digests were carried out using Sequencher version 4.1.…”

Section: Buffer [Ph 80])mentioning

confidence: 99%

Detection of a Reproducible, Single-Member Shift in Soil Bacterial Communities Exposed to Low Levels of Hydrogen

Osborne

Peoples

Janssen

2010

Appl Environ Microbiol

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Soil is exposed to hydrogen when symbiotic rhizobia in legume root nodules cannot recycle the hydrogen that is generated during nitrogen fixation. The hydrogen emitted is most likely taken up by free-living soil bacteria that use hydrogen as an energy source, though the bacteria that do this in situ remain unclear. In this study, we investigated the effect of hydrogen exposure on the bacteria of two different soils in a microcosm setup designed to simulate hydrogen-emitting root nodules. Although the size and overall composition of the soil bacterial community did not significantly alter after hydrogen exposure, one ribotype increased in relative abundance within each soil. This single-ribotype shift was identified by generating multiple terminal restriction fragment length polymorphism (T-RFLP) profiles of 16S rRNA genes from each soil sample, with gene sequence confirmation to identify terminal restriction fragments. The increased abundance of a single ribotype after hydrogen exposure, within an otherwise similar community, was found in replicate samples taken from each microcosm and was reproducible across replicate experiments. Similarly, only one member of the soil bacterial community increased in abundance in response to hydrogen exposure in soil surrounding the root nodules of field-grown soybean (Glycine max). The ribotypes that increased after hydrogen exposure in each soil system tested were all from known hydrogen-oxidizing lineages within the order Actinomycetales. We suggest that soil actinomycetes are important utilizers of hydrogen at relevant concentrations in soil and could be key contributors to soil's function as a sink in the global hydrogen cycle.

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“…The 16S rRNA gene was amplified using the Y1f and Y3r primers (CHEN et al, 2000) in a thermocycler (Aeris TM Thermal Cycler, Esco ® Micro Pte, Singapore). The amplification products were subjected to DNA sequencing by the Sanger method with the primers 27f and 1492r (OSBORNE et al, 2005) in an automatic sequencer (ABI 3500 Genetic Analyzer, Applied Biosystems, Carlsbad, CA, USA).…”

Section: Os Esporos De Bactérias Estão Amplamente Difundidos No Ambiementioning

confidence: 99%

Spoilage potential of Paenibacillussp. in Brazilian raw milk

2016

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PCR-generated artefact from 16S rRNA gene-specific primers

Cited by 74 publications

References 17 publications

Bromeliad tanks are unique habitats for microbial communities involved in methane turnover

Bromeliad tanks are unique habitats for microbial communities involved in methane turnover

Detection of a Reproducible, Single-Member Shift in Soil Bacterial Communities Exposed to Low Levels of Hydrogen

Spoilage potential of Paenibacillussp. in Brazilian raw milk

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