Whole cell immunomagnetic enrichment of environmental microbial consortia using rRNA-Targeted Magneto-FISH

Elizabeth Trembath-Reichert, Abigail Green-Saxena, Victoria J. Orphan

Research output: Chapter in Book/Report/Conference proceedingChapter

6 Scopus citations

Abstract

Magneto-FISH, in combination with metagenomic techniques, explores the middle ground between single-cell analysis and complex community characterization in bulk samples to better understand microbial partnerships and their roles in ecosystems. The Magneto-FISH method combines the selectivity of catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) with immunomagnetic capture to provide targeted molecular and metagenomic analysis of co-associated microorganisms in the environment. This method was originally developed by Pernthaler et al. (Pernthaler et al., 2008; Pernthaler & Orphan, 2010). It led to the discovery of new bacterial groups associated with anaerobic methane-oxidizing (ANME-2) archaea in methane seeps, as well as provided insight into their physiological potential using metagenomics. Here, we demonstrate the utility of this method for capturing aggregated consortia using a series of nested oligonucleotide probes of differing specificity designed to target either the ANME archaea or their Deltaproteobacteria partner, combined with 16S rRNA and mcrA analysis. This chapter outlines a modified Magneto-FISH protocol for large- and small-volume samples and evaluates the strengths and limitations of this method predominantly focusing on (1) the relationship between FISH probe specificity and sample selectivity, (2) means of improving DNA yield from paraformaldehyde-fixed samples, and (3) suggestions for adapting the Magneto-FISH method for other microbial systems, including potential for single-cell recovery.

Original languageEnglish (US)
Title of host publicationMicrobial Metagenomics, Metatranscriptomics, and Metaproteomics
PublisherAcademic Press Inc
Pages21-44
Number of pages24
ISBN (Print)9780124078635
DOIs
StatePublished - 2013
Externally publishedYes

Publication series

NameMethods in Enzymology
Volume531
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Keywords

  • ANME
  • CARD-FISH
  • Cell separation
  • Metagenomics
  • Microbial association
  • Symbiosis
  • Syntrophy

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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