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
相关论文
共 86 条
[51]   Insights into the genome of large sulfur bacteria revealed by analysis of single filaments [J].
Mussmann, Marc ;
Hu, Fen Z. ;
Richter, Michael ;
de Beer, Dirk ;
Preisler, Andre ;
Jorgensen, Bo B. ;
Huntemann, Marcel ;
Gloeckner, Frank Oliver ;
Amann, Rudolf ;
Koopman, Werner J. H. ;
Lasken, Roger S. ;
Janto, Benjamin ;
Hogg, Justin ;
Stoodley, Paul ;
Boissy, Robert ;
Ehrlich, Garth D. .
PLOS BIOLOGY, 2007, 5 (09) :1923-1937
[52]   PROFILING OF COMPLEX MICROBIAL-POPULATIONS BY DENATURING GRADIENT GEL-ELECTROPHORESIS ANALYSIS OF POLYMERASE CHAIN REACTION-AMPLIFIED GENES-CODING FOR 16S RIBOSOMAL-RNA [J].
MUYZER, G ;
DEWAAL, EC ;
UITTERLINDEN, AG .
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 1993, 59 (03) :695-700
[53]   Marine methylotrophs revealed by stable-isotope probing, multiple displacement amplification and metagenomics [J].
Neufeld, Josh D. ;
Chen, Yin ;
Dumont, Marc G. ;
Murrell, J. Colin .
ENVIRONMENTAL MICROBIOLOGY, 2008, 10 (06) :1526-1535
[54]   Stable-isotope probing implicates Methylophaga spp and novel Gammaproteobacteria in marine methanol and methylamine metabolism [J].
Neufeld, Josh D. ;
Schafer, Hendrik ;
Cox, Michael J. ;
Boden, Rich ;
McDonald, Ian R. ;
Murrell, J. Colin .
ISME JOURNAL, 2007, 1 (06) :480-491
[55]   Methodological considerations for the use of stable isotope probing in microbial ecology [J].
Neufeld, Josh D. ;
Dumont, Marc G. ;
Vohra, Jyotsna ;
Murrell, J. Colin .
MICROBIAL ECOLOGY, 2007, 53 (03) :435-442
[56]   DNA stable-isotope probing [J].
Neufeld, Josh D. ;
Vohra, Jyotsna ;
Dumont, Marc G. ;
Lueders, Tillmann ;
Manefield, Mike ;
Friedrich, Michael W. ;
Murrell, J. Colin .
NATURE PROTOCOLS, 2007, 2 (04) :860-866
[57]   Distribution of methanotrophs in managed and highly disturbed watersheds [J].
Ogram, Andrew ;
Castro, Hector ;
Stanley, Elizabeth ;
Chen, Weiwei ;
Prenger, Joseph .
ECOLOGICAL INDICATORS, 2006, 6 (04) :631-643
[58]   Targeted access to the genomes of low-abundance organisms in complex microbial communities [J].
Podar, Mircea ;
Abulencia, Carl B. ;
Walcher, Marion ;
Hutchison, Don ;
Zengler, Karsten ;
Garcia, Joseph A. ;
Holland, Trevin ;
Cotton, David ;
Hauser, Loren ;
Keller, Martin .
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 2007, 73 (10) :3205-3214
[59]   Methanotrophy below pH1 by a new Verrucomicrobia species [J].
Pol, Arjan ;
Heijmans, Klaas ;
Harhangi, Harry R. ;
Tedesco, Dario ;
Jetten, Mike S. M. ;
den Camp, Huub J. M. Op .
NATURE, 2007, 450 (7171) :874-U17
[60]   Stable-isotope probing as a tool in microbial ecology [J].
Radajewski, S ;
Ineson, P ;
Parekh, NR ;
Murrell, JC .
NATURE, 2000, 403 (6770) :646-649