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

Abstract

Summary 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.

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