Identification of diazotrophic microorganisms in marine sediment via fluorescence in situ hybridization coupled to nanoscale secondary ion mass spectrometry (FISH-NanoSIMS)

Methods Enzymol. 2011:486:281-305. doi: 10.1016/B978-0-12-381294-0.00012-2.

Abstract

Growing appreciation for the biogeochemical significance of uncultured microorganisms is changing the focus of environmental microbiology. Techniques designed to investigate microbial metabolism in situ are increasingly popular, from mRNA-targeted fluorescence in situ hybridization (FISH) to the "-omics" revolution, including metagenomics, transcriptomics, and proteomics. Recently, the coupling of FISH with nanometer-scale secondary ion mass spectrometry (NanoSIMS) has taken this movement in a new direction, allowing single-cell metabolic analysis of uncultured microbial phylogenic groups. The main advantage of FISH-NanoSIMS over previous noncultivation-based techniques to probe metabolism is its ability to directly link 16S rRNA phylogenetic identity to metabolic function. In the following chapter, we describe the procedures necessary to identify nitrogen-fixing microbes within marine sediment via FISH-NanoSIMS, using our work on nitrogen fixation by uncultured deep-sea methane-consuming archaea as a case study.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Archaea / genetics
  • Archaea / metabolism
  • Centrifugation, Density Gradient
  • Environmental Microbiology
  • Geologic Sediments / analysis*
  • Geologic Sediments / microbiology
  • In Situ Hybridization, Fluorescence*
  • Isotope Labeling / methods
  • Methane / metabolism
  • Nitrogen Fixation / genetics*
  • Nitrogen Isotopes / metabolism
  • Phylogeny
  • RNA, Ribosomal, 16S / genetics
  • RNA, Ribosomal, 16S / metabolism
  • Spectrometry, Mass, Secondary Ion*

Substances

  • Nitrogen Isotopes
  • RNA, Ribosomal, 16S
  • Methane