title: Revealing the uncultivated majority: combining DNA stable-isotope probing, multiple displacement amplification and metagenomic analyses of uncultivated Methylocystis in acidic peatlands.
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.
authors.affiliation: Department of Biological Sciences, the University of Warwick, Coventry, CV4 7AL, UK.
authors.forename: Yin
authors.initials: Y
authors.lastname: Chen
authors.forename: Marc G
authors.initials: MG
authors.lastname: Dumont
authors.forename: Josh D
authors.initials: JD
authors.lastname: Neufeld
authors.forename: Levente
authors.initials: L
authors.lastname: Bodrossy
authors.forename: Nancy
authors.initials: N
authors.lastname: Stralis-Pavese
authors.forename: Niall P
authors.initials: NP
authors.lastname: McNamara
authors.forename: Nick
authors.initials: N
authors.lastname: Ostle
authors.forename: Maria J I
authors.initials: MJ
authors.lastname: Briones
authors.forename: J Colin
authors.initials: JC
authors.lastname: Murrell
mesh_terms: D000429:Alcohol Oxidoreductases / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics; D016172:DNA Fingerprinting; D015342:DNA Probes; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D004275:DNA, Ribosomal / Q000235:genetics; D004739:England; D015698:Genomic Library; D007553:Isotope Labeling; D042083:Methylocystaceae / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D005810:Multigene Family; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D010105:Oxygenases / Q000235:genetics; D010802:Phylogeny; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D017422:Sequence Analysis, DNA; D017385:Sequence Homology; D012988:Soil Microbiology
chemical_list: D001426:Bacterial Proteins; D015342:DNA Probes; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S; D000429:Alcohol Oxidoreductases; C019641:alcohol dehydrogenase (acceptor); D010105:Oxygenases; C021555:methane monooxygenase