Chen, Yin, Dumont, Marc G., Neufeld, Josh D., Bodrossy, Levente, Stralis-Pavese, Nancy, McNamara, Niall P., Ostle, Nick, Briones, Maria J. I. and Murrell, J. C. (J. Colin). (2008) Revealing the uncultivated majority: combining DNA stable-isotope probing, multiple displacement amplification and metagenomic analyses of uncultivated Methylocystis in acidic peatlands. Environmental Microbiology, Vol.10 (No.10). pp. 2609-2622. ISSN 1462-2912

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Abstract

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, C-13-labelled DNA, containing genomic DNA of these Methylocystis spp., was separated from C-12 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.

Item Type:	Journal Article
Subjects:	Q Science > QR Microbiology
Divisions:	Faculty of Science > Life Sciences (2010- ) > Biological Sciences ( -2010)
Journal or Publication Title:	Environmental Microbiology
Publisher:	Blackwell
ISSN:	1462-2912
Date:	October 2008
Volume:	Vol.10
Number:	No.10
Number of Pages:	14
Page Range:	pp. 2609-2622
Identification Number:	10.1111/j.1462-2920.2008.01683.x
Status:	Peer Reviewed
Publication Status:	Published
Access rights to Published version:	Restricted or Subscription Access
Funder:	Natural Environment Research Council (Great Britain) (NERC)
Grant number:	NE/C001 923/1
URI:	http://wrap.warwick.ac.uk/id/eprint/29380

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