Revealing the uncultivated majority: combining DNA stable-isotope probing, multiple displacement amplification and metagenomic analyses of uncultivated Methylocystis in acidic peatlands

Chen, Yin, Dumont, Marc G., Neufeld, Josh D., Bodrossy, Levante, Stralis-Pavese, Nancy, McNamara, Niall P., Ostle, Nick, Briones, Maria J. I. and Murrell, 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, 10 (10). pp. 2609-2622. ISSN 1462-2920

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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, (13)C-labelled DNA, containing genomic DNA of these Methylocystis spp., was separated from (12)C 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:	Article
Uncontrolled Keywords:	sdg 13 - climate action ,/dk/atira/pure/sustainabledevelopmentgoals/climate_action
Faculty \ School:	Faculty of Science > School of Environmental Sciences
UEA Research Groups:	Faculty of Science > Research Centres > Centre for Ecology, Evolution and Conservation Faculty of Science > Research Groups > Environmental Biology Faculty of Science > Research Groups > Resources, Sustainability and Governance (former - to 2018) Faculty of Science > Research Groups > Marine and Atmospheric Sciences (former - to 2017)
Depositing User:	Rhiannon Harvey
Date Deposited:	27 Mar 2012 15:51
Last Modified:	24 Oct 2022 00:38
URI:	https://ueaeprints.uea.ac.uk/id/eprint/38524
DOI:	10.1111/j.1462-2920.2008.01683.x

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