Using stable isotope probing to obtain a targeted metatranscriptome of aerobic methanotrophs in lake sediment

Dumont, Marc G.; Pommerenke, Bianca; Casper, Peter

doi:10.1111/1758-2229.12078

Cited by 69 publications

(44 citation statements)

References 44 publications

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“…The sequences of the non-rRNAs were searched against the sequences in the NR database (25) using the BLASTX algorithm (23) with an E-value cutoff of 1 ϫ 10 Ϫ5 , and the best hits were subjected to analysis with Metagenome Analyzer (MEGAN) (29), a program for taxonomic analysis which has been widely used to assign taxa to sequences obtained from a metagenome (8,9,11,30) or a metatranscriptome (17,22,31,32).…”

Section: Rumen Content Samplingmentioning

confidence: 99%

Metatranscriptomic Analyses of Plant Cell Wall Polysaccharide Degradation by Microorganisms in the Cow Rumen

Dai

Tian

et al. 2015

Appl Environ Microbiol

194

178

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gThe bovine rumen represents a highly specialized bioreactor where plant cell wall polysaccharides (PCWPs) are efficiently deconstructed via numerous enzymes produced by resident microorganisms. Although a large number of fibrolytic genes from rumen microorganisms have been identified, it remains unclear how they are expressed in a coordinated manner to efficiently degrade PCWPs. In this study, we performed a metatranscriptomic analysis of the rumen microbiomes of adult Holstein cows fed a fiber diet and obtained a total of 1,107,083 high-quality non-rRNA reads with an average length of 483 nucleotides. Transcripts encoding glycoside hydrolases (GHs) and carbohydrate binding modules (CBMs) accounted for ϳ1% and ϳ0.1% of the total non-rRNAs, respectively. The majority (ϳ98%) of the putative cellulases belonged to four GH families (i.e., GH5, GH9, GH45, and GH48) and were primarily synthesized by Ruminococcus and Fibrobacter. Notably, transcripts for GH48 cellobiohydrolases were relatively abundant compared to the abundance of transcripts for other cellulases. Two-thirds of the putative hemicellulases were of the GH10, GH11, and GH26 types and were produced by members of the genera Ruminococcus, Prevotella, and Fibrobacter. Most (ϳ82%) predicted oligosaccharide-degrading enzymes were GH1, GH2, GH3, and GH43 proteins and were from a diverse group of microorganisms. Transcripts for CBM10 and dockerin, key components of the cellulosome, were also relatively abundant. Our results provide metatranscriptomic evidence in support of the notion that members of the genera Ruminococcus, Fibrobacter, and Prevotella are predominant PCWP degraders and point to the significant contribution of GH48 cellobiohydrolases and cellulosome-like structures to efficient PCWP degradation in the cow rumen. In nature, the cow rumen represents a highly specialized bioreactor wherein plant cell wall polysaccharides (PCWPs) are efficiently deconstructed. The extraordinary efficiency results from the concerted action of various enzymes produced by rumen-resident bacteria, archaea, fungi, and protozoa. Three rumen bacteria, i.e., Ruminococcus flavefaciens, Ruminococcus albus, and Fibrobacter succinogenes, which can be isolated and cultivated in the laboratory, have been thought to serve a predominant role in the degradation of cellulosic PCWPs in this niche (1, 2). However, metagenomic quantitations based on 16S rRNA gene analysis indicate that these three species of bacteria account for only less than 5% of the total rumen microorganisms (3). In addition, Koike et al. estimated that ϳ77% of the rumen microorganisms attached to solid fibers are uncultured, as their 16S rRNA gene sequences share less than 97% similarity with those of known isolates (4).To bypass the cultivation step, metagenomic approaches involving the direct analysis of total DNA sequences have been extensively used to investigate the PCWP-degrading gastrointestinal microbes in a variety of herbivores, such as termite hindguts (5); cow (6-8), yak (9), and Svalbard reindeer (10) ...

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Section: Rumen Content Samplingmentioning

confidence: 99%

Metatranscriptomic Analyses of Plant Cell Wall Polysaccharide Degradation by Microorganisms in the Cow Rumen

Dai

Tian

et al. 2015

Appl Environ Microbiol

194

178

View full text Add to dashboard Cite

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“…A number of RNA-SIP studies have addressed aerobic methanotrophs and methylotrophs in different habitats. Recent work has investigated aerobic methanotroph diversity in the sediment of an oligotrophic German lake [5,6 ], revealing that mostly type I methanotrophs, closely related to phylotypes also found in other lakes, were active in methane turnover. The labelled metatranscriptome provided a wealth of detail on expressed metabolic pathways active in methane and nitrogen cycling [6 ].…”

Section: Applications Of Rna-sipmentioning

confidence: 99%

“…Recent work has investigated aerobic methanotroph diversity in the sediment of an oligotrophic German lake [5,6 ], revealing that mostly type I methanotrophs, closely related to phylotypes also found in other lakes, were active in methane turnover. The labelled metatranscriptome provided a wealth of detail on expressed metabolic pathways active in methane and nitrogen cycling [6 ]. Other studies have pulsed methanotroph communities in soil with 13 C-acetate instead of 13 C-methane [9,12], showing that distinct uncultured lineages of type II methanotrophs assimilated carbon from acetate, proving them to be facultative methanotrophs.…”

Section: Applications Of Rna-sipmentioning

confidence: 99%

“…Although the methodologies are at hand and the nextgeneration sequencing of amplicons from RNA gradients is now routine [14,15 ,25,28,29 ,31 ,36 ,37], only one study to date has retrieved a labelling-assisted targeted transcriptome [6 ]. Although this was mostly a proofof-concept experiment with aerobic methanotrophs, it clearly shows that the metatranscriptome of target populations in environmental samples can be selectively recovered.…”

Section: Future Directionsmentioning

confidence: 99%

“…Dumont and colleagues [5] compared the results of labelling methanotrophs with 13 CH 4 in lake sediment by targeting 16S rRNA and pmoA markers by both DNA-SIP and RNA-SIP and showed that the labelling of pmoA transcripts was more rapid than that of pmoA genes. This study laid the foundation for the combination of SIP with next-generation sequencing-based metatranscriptomics [6 ]. This emerging approach to target specific microbial transcriptomes has the potential to alleviate some of the most fundamental limitations of non-targeted 'omics [7] in complex systems.…”

Section: Introductionmentioning

confidence: 99%

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RNA-stable isotope probing: from carbon flow within key microbiota to targeted transcriptomes

Lueders

Dumont

Bradford

et al. 2016

Current Opinion in Biotechnology

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Stable isotope probing of RNA has enthused researchers right from its first introduction in 2002. The concept of a labelling-based detection of process-targeted microbes independent of cellular replication or growth has allowed for a much more direct handle on functionally relevant microbiota than by labelling of other biomarkers. This has led to a widespread application of the technology, and breakthroughs in our understanding of carbon flow in natural microbiomes, autotrophic and heterotrophic physiologies, microbial food webs, host-microbe interactions and environmental biotechnology. Recent studies detecting labelled mRNA demonstrate that RNA-SIP is not limited to the analysis of rRNA, but is currently developing towards an approach for accessing targeted transcriptomes. In combination with next-generation sequencing and other methodological advances, RNA-SIP will continue to deliver invaluable insights into the functioning of microbial communities.

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Proteomic evidence of methanotrophy in methane‐enriched hypolimnetic lake water

Ullrich

Casper

Otto

et al. 2016

Limnology & Oceanography

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Freshwaters have recently been recognized as important sources of methane emitted to the atmosphere, and microbial methane oxidation at the oxic/anoxic interface is a key process controlling these emissions. We applied proteomics to determine enzyme expression patterns of methanotrophs in response to methane enrichment of lake water. In a small‐scale incubation experiment with natural bacterial communities we compared enzymes involved in methane metabolism between control and methane‐enriched hypolimnetic water simulating high (∼1 mM) and low (∼0.001 mM) methane concentrations at oxyclines in lakes. Methane was effectively consumed when the supply was high, reducing oxygen levels from 0.40 mM (12.9 mg L−1) to 0.09 mM (3.0 mg L−1), well below those in the controls. The dominant key enzyme of microbial methane oxidation, particulate methane monooxygenase, was identified in both enriched and control flasks, whereas enzymes potentially involved in methane metabolism via the RuMP pathway and serine cycle were essentially restricted to the enriched flasks. All enzymes had best sequence matches with type I methanotrophs, whereas no indication of type II or type X methanotrophs was found, even though four enzymes of the serine cycle were identified. Overall, our proteomic analysis provides convincing evidence that a suite of genes required for methanotrophy are quickly expressed when the presence of both methane and oxygen creates conditions characteristic of oxyclines in lakes.

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Using stable isotope probing to obtain a targeted metatranscriptome of aerobic methanotrophs in lake sediment

Cited by 69 publications

References 44 publications

Metatranscriptomic Analyses of Plant Cell Wall Polysaccharide Degradation by Microorganisms in the Cow Rumen

Metatranscriptomic Analyses of Plant Cell Wall Polysaccharide Degradation by Microorganisms in the Cow Rumen

RNA-stable isotope probing: from carbon flow within key microbiota to targeted transcriptomes

Proteomic evidence of methanotrophy in methane‐enriched hypolimnetic lake water

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