Using stable isotope probing to obtain a targeted metatranscriptome of aerobic methanotrophs in lake sediment
Summary In this study, we demonstrate the possibility of obtaining a targeted metatranscriptome from a functional group of microorganisms using a stable isotope probing (SIP) approach. Methanotrophs in lake sediment were labelled using 13CH4, and both labelled and unlabelled‐RNA were isolated and se...
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| Published in: | Environmental microbiology reports Vol. 5; no. 5; pp. 757 - 764 |
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| Main Authors: | , , |
| Format: | Journal Article |
| Language: | English |
| Published: |
United States
Blackwell Publishing Ltd
01-10-2013
John Wiley & Sons, Inc |
| Subjects: | |
| Online Access: | Get full text |
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| Summary: | Summary
In this study, we demonstrate the possibility of obtaining a targeted metatranscriptome from a functional group of microorganisms using a stable isotope probing (SIP) approach. Methanotrophs in lake sediment were labelled using 13CH4, and both labelled and unlabelled‐RNA were isolated and sequenced by 454 pyrosequencing. The unlabelled metatranscriptome had a large diversity of bacterial, archaeal, eukaryotic and viral sequences as expected from a diverse sediment community. In contrast, the labelled‐RNA metatranscriptome was dominated by methanotroph sequences, particularly from Methylococcaceae. Transcripts of the methane monooxygenase genes pmoCAB were the most abundant in this metatranscriptome, and the pathway of methane oxidation to CO2 could be traced, as well as many steps in the ribulose monophosphate pathway for carbon assimilation. A high abundance of mRNA transcripts for proteins related to motility was detected, suggesting an importance for methanotrophs in lake sediments. This combination of SIP and metatranscriptomics should be broadly applicable, and will enhance the detection and identification of mRNA from target organisms. |
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| Bibliography: | istex:47CE928E054D82216FD68625073676271144AD7D ArticleID:EMI412078 Fig. S1. Terminal restriction fragment length polymorphism (T-RFLP) analysis of pmoA transcripts to test for a bias during mRNA amplification. RNA was collected from CsTFA gradients and directly converted to cDNA and analysed by T-RFLP (A,B) or first amplified to aRNA using the ExpressArtTM mRNA amplification system and then converted to cDNA and analysed by T-RFLP (C,D). The analysis was performed with the labelled (A,C) and unlabelled (B,D) RNA. The most abundant T-RF was 445 bp. Minor T-RFs were also recovered after mRNA amplification at similar initial and final relative abundances.Fig. S2. Depiction of a pmoCAB operon obtained by assembling reads (with > 80% sequence identity and a minimum of 20 nucleotide overlap) using the SeqMan software program (DNAStarTM). A total of 528 sequences were assigned to the depicted contig. The top panel shows an alignment of the non-coding regions (5′ to 3′) of the contig against the corresponding region from the Methylovulum miyakonense H12 sequence (GenBank accession number AB501288) using EMBOSS (Rice et al., 2000); the top strand is the contig sequence and the bottom strand the M. miyakonense sequence. The bottom panel shows the BLASTX alignment of the sequences against those of M. miyakonense. The alternative amino acid residues at ambiguous sites (X) are shown.Appendix S1. Supplementary methods. ark:/67375/WNG-B6HDM41R-P ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
| ISSN: | 1758-2229 1758-2229 |
| DOI: | 10.1111/1758-2229.12078 |