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Magneto-FISH

Magneto-FISH: Capture and metagenomic analysis of diverse syntrophic partnerships from deep-sea methane vents. (Post-doc Annelie Pernthaler)

The anaerobic cycling of carbon frequently relies on closely coupled, interdependent microbial associations. The prevalence and biogeochemical importance of these associations emphasizes the need for effective methodologies that enable the study of novel microbial processes and interspecies relationships in situ. To this end, culture-independent environmental metagenomic studies have expanded our understanding of the phylogenetic and metabolic diversity on Earth. An important caveat of metagenomic studies is the ability to interpret the data in the context of organism identity, which increases in difficulty with increasing community complexity. Direct sequencing of complex soils and sediments using high throughput sequencing methods (shotgun cloning or clone-free pyrosequencing) yield important overviews of the metabolic potential of the entire community, however information regarding specific groups of microorganisms and interspecies associations is often lacking. Methods such as flow cytometry or microfluidics, which enable the selective extraction of individual microorganisms from native microbial communities prior to genome sequencing, have shown promise for interpreting genome data in the context of specific microorganisms. The ability to identify and characterize the metabolic potential of multi-species syntrophic partnerships in situ presents a greater challenge and requires new methodological strategies that are both compatible with metagenomic analysis and can be applied in complex environmental samples such as anoxic soils and sediments where these interspecies associations are likely to occur.

In response to this need, we developed a culture-independent method called magneto-FISH that enables the capture and assessment of novel interspecies associations and corresponding metabolic properties of these organisms directly from complex environments. Magneto-FISH allows for the targeted magnetic capture of whole microorganisms and cell aggregates using 16S rRNA oligonucleotide probes to the exclusion of other microorganisms within the environmental sample.  The capture of phylogenetically 'tagged' microorganisms and physically associated microorganisms, increases the probability of identifying potentially hidden microbial associations in situ and yields a low complexity sample for environmental metagenomics. In a proof-of-concept study, we used magneto-FISH for targeted metagenomic investigations of in situ syntrophic assemblages involved in the anaerobic oxidation of methane. Metagenomic studies of ANME archaea to date have primarily focused on methane and energy metabolism of the archaea, however the underlying nutrition and the intricacies of these interspecies associations are still unresolved. Using the phylogenetic selectiveness of the 16S rRNA targeted magneto-FISH assay, we examining the molecular underpinnings of the syntrophic lifestyle between the uncultured methane oxidizing ANME-2c ecotype and their diverse co-associated bacterial partners. We are currently using the information obtained through metagenomic analysis of the ANME consortium for testing new hypotheses relating to interspecies interactions and environmental adaptation of these uncultured, globally important microbial partnerships.

 

"PNAS supplemental Pernthaler et al. 454 reads (fasta format)"

NSF MO/MIP "Collaborative Research: Examination of diverse anaerobic methane oxidizing Archaea and associated syntrophic relationships using high resolution molecular and isotopic methods"  In collaboration with Christopher House, Penn State.