RNA stable-isotope probing

Whiteley, Andrew S.; Thomson, Bruce C.; Lueders, Tillmann; Manefield, Mike

doi:10.1038/nprot.2007.115

Cited by 106 publications

(106 citation statements)

References 18 publications

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“…2) indicates that the labeling was intense. This finding is supported by estimated buoyant densities of 1.78-1.80 g/ml for the unlabeled and 1.82-1.85 g/ml for the labeled RNA, comparable with values reported for pure [ 12 C]rRNA and pure [ 13 C]rRNA, respectively (16). In addition, there was no delay: heavy RNA was found immediately after the end of the 5-h pulse labeling.…”

Section: Resultssupporting

confidence: 76%

Active root-inhabiting microbes identified by rapid incorporation of plant-derived carbon into RNA

Vandenkoornhuyse

Mahé

Ineson

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

203

149

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Plant roots harbor a large diversity of microorganisms that have an essential role in ecosystem functioning. To better understand the level of intimacy of root-inhabiting microbes such as arbuscular mycorrhizal fungi and bacteria, we provided 13 CO2 to plants at atmospheric concentration during a 5-h pulse. We expected microbes dependent on a carbon flux from their host plant to become rapidly labeled. We showed that a wide variety of microbes occurred in roots, mostly previously unknown. Strikingly, the greatest part of this unsuspected diversity corresponded to active primary consumers. We found 17 bacterial phylotypes co-occurring within roots of a single plant, including five potentially new phylotypes. Fourteen phylotypes were heavily labeled with the 13 C. Eight were phylogenetically close to Burkholderiales, which encompass known symbionts; the others were potentially new bacterial root symbionts. By analyzing unlabeled and 13 C-enriched RNAs, we demonstrated differential activity in C consumption among these root-inhabiting microbes. Arbuscular mycorrhizal fungal RNAs were heavily labeled, confirming the high carbon flux from the plant to the fungal compartment, but some of the fungi present appeared to be much more active than others. The results presented here reveal the possibility of uncharacterized root symbioses.ribosomal RNA ͉ stable isotope probing ͉ symbiosis ͉ arbuscular mycorrhiza ͉ endophytes P lants are the dominant primary producers in most terrestrial ecosystems. In the soil, they are escorted by a myriad of microorganisms living freely or in intimate interaction with their roots (1, 2). These microorganisms can be pathogenic, parasitic, saprotrophic, or mutualistic. Among the root symbionts, arbuscular mycorrhizal (AM) fungi are well known and have been observed colonizing the roots of most plant species in many ecosystems (3). Recent studies show that high diversity is the norm even where plant diversity is low (4-6). These AM fungi are biotrophs, unable to grow in the absence of a living plant, and often display a broad host range although there is growing evidence for differences in host preference (5-8). They have been demonstrated to improve plant mineral nutrition (3) and stress resistance (3). AM fungi are important for the global carbon cycle because up to 20% of photoassimilates can be translocated to them (9). We also know that the diversity of AM fungi can determine plant community structure and ecosystem productivity (10). The plant-bacteria symbioses are variably documented. The best studied symbiosis is the rhizobiumlegume interaction, but a single plant root can harbor a large variety of fungi (1) and bacteria (2), as well as several different archaea (2). So far we have no information about the functions of most of these root-living microbes. The strategy chosen herein, stable isotope probing (SIP)-RNA analysis, enabled us to highlight an unsuspected diversity of microbes living in roots. We identified microbes that are active and direct utilizers of photosynthetic carbon ...

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Section: Resultssupporting

confidence: 76%

Active root-inhabiting microbes identified by rapid incorporation of plant-derived carbon into RNA

Vandenkoornhuyse

Mahé

Ineson

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

203

149

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“…RNA stable isotope probing (RNA SIP) RNA SIP was performed per published protocol (Whiteley et al, 2007). A gradient solution (buoyant density of 1.793±0.002 g ml À 1 ) was prepared by mixing 4.61 ml caesiumtrifluoroacetate solution (buoyant density: 2.0 ± 0.05 g ml À 1 ; GE Healthcare, Buckinghamshire, UK), 0.175 ml formamide and a variable amount of gradient buffer (100 mM KCl, 100 mM Tris, 1 mM EDTA, pH 8).…”

Section: Extraction Of Nucleic Acidsmentioning

confidence: 99%

Methanogenic food web in the gut contents of methane-emitting earthwormEudrilus eugeniaefrom Brazil

et al. 2015

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The anoxic saccharide-rich conditions of the earthworm gut provide an ideal transient habitat for ingested microbes capable of anaerobiosis. It was recently discovered that the earthworm Eudrilus eugeniae from Brazil can emit methane (CH 4 ) and that ingested methanogens might be associated with this emission. The objective of this study was to resolve trophic interactions of bacteria and methanogens in the methanogenic food web in the gut contents of E. eugeniae. RNA-based stable isotope probing of bacterial 16S rRNA as well as mcrA and mrtA (the alpha subunit of methyl-CoM reductase and its isoenzyme, respectively) of methanogens was performed with [ 13 C]-glucose as a model saccharide in the gut contents. Concomitant fermentations were augmented by the rapid consumption of glucose, yielding numerous products, including molecular hydrogen (H 2 ), carbon dioxide (CO 2 ), formate, acetate, ethanol, lactate, succinate and propionate. Aeromonadaceaeaffiliated facultative aerobes, and obligate anaerobes affiliated to Lachnospiraceae, Veillonellaceae and Ruminococcaceae were associated with the diverse fermentations. Methanogenesis was ongoing during incubations, and 13 C-labeling of CH 4 verified that supplemental [ 13 C]-glucose derived carbon was dissimilated to CH 4 . Hydrogenotrophic methanogens affiliated with Methanobacteriaceae and Methanoregulaceae were linked to methanogenesis, and acetogens related to Peptostreptoccocaceae were likewise found to be participants in the methanogenic food web. H 2 rather than acetate stimulated methanogenesis in the methanogenic gut content enrichments, and acetogens appeared to dissimilate supplemental H 2 to acetate in methanogenic enrichments. These findings provide insight on the processes and associated taxa potentially linked to methanogenesis and the turnover of organic carbon in the alimentary canal of methane-emitting E. eugeniae.

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“…The RNA was further purified with a Qiagen (Hilden, Germany) AllPrep DNA/RNA Mini Kit and quantified with Ribogreen RNA-quantification kit (Invitrogen, Eugene, OR, USA). Portions of 530 ng total RNA were separated by density gradient ultracentrifugation and the gradients were fractionated and further processed as described by Whiteley et al (2007). The concentration of RNA in each fraction was determined using Ribogreenbased fluorimetry (Invitrogen).…”

Section: Sediment Incubationsmentioning

confidence: 99%

“…Substrate labeled with 13 C is fed to, for example, sediment incubations so that the RNA and DNA of microorganisms turning over this substrate become labeled with 13 C and the organisms can be identified subsequently by molecular methods (Whiteley et al, 2006;Whiteley et al, 2007;Pester and Loy, 2010). Particularly in SIP incubations with only one dominating electronaccepting process, selective isotopic enrichment of nucleic acids strongly suggests that the microorganisms harboring these heavy nucleic acids are the ones that catalyze this process.…”

Section: Introductionmentioning

confidence: 99%

Three manganese oxide-rich marine sediments harbor similar communities of acetate-oxidizing manganese-reducing bacteria

et al. 2012

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Dissimilatory manganese reduction dominates anaerobic carbon oxidation in marine sediments with high manganese oxide concentrations, but the microorganisms responsible for this process are largely unknown. In this study, the acetate-utilizing manganese-reducing microbiota in geographically well-separated, manganese oxide-rich sediments from Gullmar Fjord (Sweden), Skagerrak (Norway) and Ulleung Basin (Korea) were analyzed by 16S rRNA-stable isotope probing (SIP). Manganese reduction was the prevailing terminal electron-accepting process in anoxic incubations of surface sediments, and even the addition of acetate stimulated neither iron nor sulfate reduction. The three geographically distinct sediments harbored surprisingly similar communities of acetateutilizing manganese-reducing bacteria: 16S rRNA of members of the genera Colwellia and Arcobacter and of novel genera within the Oceanospirillaceae and Alteromonadales were detected in heavy RNA-SIP fractions from these three sediments. Most probable number (MPN) analysis yielded up to 10 6 acetate-utilizing manganese-reducing cells cm À 3 in Gullmar Fjord sediment. A 16S rRNA gene clone library that was established from the highest MPN dilutions was dominated by sequences of Colwellia and Arcobacter species and members of the Oceanospirillaceae, supporting the obtained RNA-SIP results. In conclusion, these findings strongly suggest that (i) acetatedependent manganese reduction in manganese oxide-rich sediments is catalyzed by members of taxa (Arcobacter, Colwellia and Oceanospirillaceae) previously not known to possess this physiological function, (ii) similar acetate-utilizing manganese reducers thrive in geographically distinct regions and (iii) the identified manganese reducers differ greatly from the extensively explored iron reducers in marine sediments.

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RNA stable-isotope probing

Cited by 106 publications

References 18 publications

Active root-inhabiting microbes identified by rapid incorporation of plant-derived carbon into RNA

Active root-inhabiting microbes identified by rapid incorporation of plant-derived carbon into RNA

Methanogenic food web in the gut contents of methane-emitting earthwormEudrilus eugeniaefrom Brazil

Three manganese oxide-rich marine sediments harbor similar communities of acetate-oxidizing manganese-reducing bacteria

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