Revealing the Uncultivated Majority: Combining DNA Stable-Isotope Probing, Multiple Displacement Amplification and Metagenomic Analyses of Uncultivated Methylocystis in Acidic Peatlands

Environ Microbiol. 2008 Oct;10(10):2609-22. doi: 10.1111/j.1462-2920.2008.01683.x. Epub 2008 Jul 10.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alcohol Oxidoreductases / genetics
  • Bacterial Proteins / genetics
  • DNA Fingerprinting
  • DNA Probes
  • DNA, Bacterial / chemistry
  • DNA, Bacterial / genetics*
  • DNA, Bacterial / isolation & purification
  • DNA, Ribosomal / genetics
  • England
  • Genomic Library
  • Isotope Labeling
  • Methylocystaceae / classification*
  • Methylocystaceae / genetics
  • Methylocystaceae / isolation & purification*
  • Methylocystaceae / metabolism
  • Molecular Sequence Data
  • Multigene Family
  • Nucleic Acid Amplification Techniques / methods
  • Oxygenases / genetics
  • Phylogeny
  • RNA, Ribosomal, 16S / genetics
  • Sequence Analysis, DNA
  • Sequence Homology
  • Soil Microbiology*

Substances

  • Bacterial Proteins
  • DNA Probes
  • DNA, Bacterial
  • DNA, Ribosomal
  • RNA, Ribosomal, 16S
  • Alcohol Oxidoreductases
  • alcohol dehydrogenase (acceptor)
  • Oxygenases
  • methane monooxygenase

Associated data

  • GENBANK/EU362857
  • GENBANK/EU362858
  • GENBANK/EU362859
  • GENBANK/EU362860
  • GENBANK/EU362861
  • GENBANK/EU362862
  • GENBANK/EU362863