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Published July 12, 2016 | Published + Supplemental Material
Journal Article Open

Visualizing in situ translational activity for identifying and sorting slow-growing archaeal−bacterial consortia

Abstract

To understand the biogeochemical roles of microorganisms in the environment, it is important to determine when and under which conditions they are metabolically active. Bioorthogonal noncanonical amino acid tagging (BONCAT) can reveal active cells by tracking the incorporation of synthetic amino acids into newly synthesized proteins. The phylogenetic identity of translationally active cells can be determined by combining BONCAT with rRNA-targeted fluorescence in situ hybridization (BONCAT-FISH). In theory, BONCAT-labeled cells could be isolated with fluorescence-activated cell sorting (BONCAT-FACS) for subsequent genetic analyses. Here, in the first application, to our knowledge, of BONCAT-FISH and BONCAT-FACS within an environmental context, we probe the translational activity of microbial consortia catalyzing the anaerobic oxidation of methane (AOM), a dominant sink of methane in the ocean. These consortia, which typically are composed of anaerobic methane-oxidizing archaea (ANME) and sulfate-reducing bacteria, have been difficult to study due to their slow in situ growth rates, and fundamental questions remain about their ecology and diversity of interactions occurring between ANME and associated partners. Our activity-correlated analyses of >16,400 microbial aggregates provide the first evidence, to our knowledge, that AOM consortia affiliated with all five major ANME clades are concurrently active under controlled conditions. Surprisingly, sorting of individual BONCAT-labeled consortia followed by whole-genome amplification and 16S rRNA gene sequencing revealed previously unrecognized interactions of ANME with members of the poorly understood phylum Verrucomicrobia. This finding, together with our observation that ANME-associated Verrucomicrobia are found in a variety of geographically distinct methane seep environments, suggests a broader range of symbiotic relationships within AOM consortia than previously thought.

Additional Information

© 2016 National Academy of Sciences. Edited by Edward F. DeLong, University of Hawaii at Manoa, Honolulu, HI, and approved May 25, 2016 (received for review March 7, 2016). Published online before print June 28, 2016. We thank Alexis Pasulka and Kat Dawson for shipboard sample processing, Silvan Scheller and Kat Dawson for measurements of AOM rates and methane concentrations, Hang Yu for performing cline assays, Connor Skennerton for help during sampling of sediment incubations, David Case for discussions on tag sequence analyses, and Shawn McGlynn for discussions on storage compounds. David Case, Kat Dawson, and Elizabeth Wilbanks are acknowledged for critical comments on the manuscript. We thank The Biological Imaging Facility of California Institute of Technology for access to their confocal microscope. We thank the crew and pilots of R/V Atlantis Cruises AT-15-68 and AT-18-10 to Hydrate Ridge (supported by National Science Foundation Grant OCE-0825791) and the R/V Western Flyer Cruise to Santa Monica Basin run by the Monterey Bay Aquarium Research Institute. R.H. was supported by an Erwin Schrödinger Postdoctoral Fellowship from the Austrian Science Fund (FWF) (project no. J 3162-B20), and a postdoctoral fellowship from the Center for Dark Energy Biosphere Investigations (C-DEBI). Funding for this project was provided by Gordon and Betty Moore Foundation Grant GBMF3780 (to V.J.O.), Department of Energy (DOE) Grant DE-PS02-09ER09-25 (to V.J.O.), and a JGI Director Discretionary Project Award (to R.H. and V.J.O.). The work conducted by the DOE Joint Genome Institute, a DOE Office of Science User Facility, is supported under Contract DE-AC02-05CH11231. This is C-DEBI Contribution 330. Author contributions: R.H. and V.J.O. designed research; R.H., S.A.C., and D.G. performed research; R.R.M., T.W., and V.J.O. contributed new reagents/analytic tools; R.H. and D.G. analyzed data; and R.H. and V.J.O. wrote the paper with input from all authors. The authors declare no conflict of interest. This article is a PNAS Direct Submission. Data deposition: The sequences reported in this paper have been deposited in the National Center for Biotechnology Information GenBank database (accession nos. KT945170–KT945234 and KU564217–KU564240) and Sequence Read Archive (accession no. SRP066109). This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1603757113/-/DCSupplemental.

Attached Files

Published - PNAS-2016-Hatzenpichler-E4069-78.pdf

Supplemental Material - pnas.1603757113.sd01.xlsx

Supplemental Material - pnas.1603757113.sd02.xlsx

Supplemental Material - pnas.1603757113.sd03.xlsx

Supplemental Material - pnas.201603757SI.pdf

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