Present addresses: Max Planck Institute for Terrestrial Microbiology, D-35043 Marburg, Germany., ‡Department of Biology, University of Waterloo, Waterloo, Ontario, N2L 3G1, Canada.
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Revealing the uncultivated majority: combining DNA stable-isotope probing, multiple displacement amplification and metagenomic analyses of uncultivated Methylocystis in acidic peatlands
Article first published online: 10 JUL 2008
DOI: 10.1111/j.1462-2920.2008.01683.x
© 2008 University of Warwick. Journal compilation © 2008 Society for Applied Microbiology and Blackwell Publishing Ltd
Additional Information
How to Cite
Chen, Y., Dumont, M. G., Neufeld, J. D., Bodrossy, L., Stralis-Pavese, N., McNamara, N. P., Ostle, N., Briones, M. J. I. and Murrell, J. C. (2008), Revealing the uncultivated majority: combining DNA stable-isotope probing, multiple displacement amplification and metagenomic analyses of uncultivated Methylocystis in acidic peatlands. Environmental Microbiology, 10: 2609–2622. doi: 10.1111/j.1462-2920.2008.01683.x
Publication History
- Issue published online: 10 SEP 2008
- Article first published online: 10 JUL 2008
- Received 25 February, 2008; accepted 7 May, 2008.
Summary
Peatlands represent an enormous carbon reservoir and have a potential impact on the global climate because of the active methanogenesis and methanotrophy in these soils. Uncultivated methanotrophs from seven European peatlands were studied using a combination of molecular methods. Screening for methanotroph diversity using a particulate methane monooxygenase-based diagnostic gene array revealed that Methylocystis-related species were dominant in six of the seven peatlands studied. The abundance and methane oxidation activity of Methylocystis spp. were further confirmed by DNA stable-isotope probing analysis of a sample taken from the Moor House peatland (England). After ultracentrifugation, 13C-labelled DNA, containing genomic DNA of these Methylocystis spp., was separated from 12C DNA and subjected to multiple displacement amplification (MDA) to generate sufficient DNA for the preparation of a fosmid metagenomic library. Potential bias of MDA was detected by fingerprint analysis of 16S rRNA using denaturing gradient gel electrophoresis for low-template amplification (0.01 ng template). Sufficient template (1–5 ng) was used in MDA to circumvent this bias and chimeric artefacts were minimized by using an enzymatic treatment of MDA-generated DNA with S1 nuclease and DNA polymerase I. Screening of the metagenomic library revealed one fosmid containing methanol dehydrogenase and two fosmids containing 16S rRNA genes from these Methylocystis-related species as well as one fosmid containing a 16S rRNA gene related to that of Methylocella/Methylocapsa. Sequencing of the 14 kb methanol dehydrogenase-containing fosmid allowed the assembly of a gene cluster encoding polypeptides involved in bacterial methanol utilization (mxaFJGIRSAC). This combination of DNA stable-isotope probing, MDA and metagenomics provided access to genomic information of a relatively large DNA fragment of these thus far uncultivated, predominant and active methanotrophs in peatland soil.