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

被引:78
作者
Chen, Yin [1 ]
Dumont, Marc G. [1 ]
Neufeld, Josh D. [1 ]
Bodrossy, Levente [2 ]
Stralis-Pavese, Nancy [2 ]
McNamara, Niall P. [3 ]
Ostle, Nick [3 ]
Briones, Maria J. I. [4 ]
Murrell, J. Colin [1 ]
机构
[1] Univ Warwick, Dept Biol Sci, Coventry CV4 7AL, W Midlands, England
[2] Osterreich Forschungszent Seibersdorf GmbH, Dept Bioresources, A-2444 Seibersdorf, Austria
[3] Lancaster Environm Ctr, Ctr Ecol & Hydrol, Lancaster LA1 4AP, England
[4] Univ Vigo, Fac Biol, Dept Ecol & Biol Anim, Vigo 36310, Spain
基金
英国自然环境研究理事会;
关键词
D O I
10.1111/j.1462-2920.2008.01683.x
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
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.
引用
收藏
页码:2609 / 2622
页数:14
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