Whole cell immunomagnetic enrichment of environmental microbial consortia using rRNA-targeted Magneto-FISH.
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.
Division of Geological and Planetary Sciences, California Institute of Technology, Pasadena, California, USA.
Elizabeth
E
Trembath-Reichert
Abigail
A
Green-Saxena
Victoria J
VJ
Orphan
D000693:Anaerobiosis; D001105:Archaea / Q000145:classification / Q000235:genetics; D019641:DNA, Archaeal / Q000145:classification / Q000235:genetics*; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D056186:Metagenomics; D059013:Microbial Consortia / Q000235:genetics*; D012336:RNA, Ribosomal, 16S / Q000235:genetics*
D019641:DNA, Archaeal; D012336:RNA, Ribosomal, 16S
ANME; CARD–FISH; Cell separation; Metagenomics; Microbial association; Symbiosis; Syntrophy