title: Identification of diazotrophic microorganisms in marine sediment via fluorescence in situ hybridization coupled to nanoscale secondary ion mass spectrometry (FISH-NanoSIMS).
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.
authors.affiliation: Division of Geological and Planetary Sciences, California Institute of Technology, Pasadena, California, USA.
authors.forename: Anne E
authors.initials: AE
authors.lastname: Dekas
authors.forename: Victoria J
authors.initials: VJ
authors.lastname: Orphan
mesh_terms: D001105:Archaea / Q000235:genetics / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D004783:Environmental Microbiology; D019015:Geologic Sediments / Q000032:analysis* / Q000382:microbiology; D017404:In Situ Hybridization, Fluorescence; D007553:Isotope Labeling / Q000379:methods; D008697:Methane / Q000378:metabolism; D009586:Nitrogen Fixation / Q000235:genetics*; D009587:Nitrogen Isotopes / Q000378:metabolism; D010802:Phylogeny; D012336:RNA, Ribosomal, 16S / Q000235:genetics / Q000378:metabolism; D018629:Spectrometry, Mass, Secondary Ion
chemical_list: D009587:Nitrogen Isotopes; D012336:RNA, Ribosomal, 16S; D008697:Methane