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

Hatzenpichler, Roland and Connon, Stephanie A. and Goudeau, Danielle and Malmstrom, Rex R. and Woykeb, Tanja and Orphan, Victoria J. (2016) Visualizing in situ translational activity for identifying and sorting slow-growing archaeal−bacterial consortia. Proceedings of the National Academy of Sciences of the United States of America, 113 (28). E4069-E4078. ISSN 0027-8424. PMCID PMC4948357.

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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.

Item Type:Article
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URLURL TypeDescription Information CentralArticle
Hatzenpichler, Roland0000-0002-5489-3444
Orphan, Victoria J.0000-0002-5374-6178
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
Funding AgencyGrant Number
FWF Der WissenschaftsfondsJ 3162-B20
Center for Dark Energy Biosphere InvestigationsUNSPECIFIED
Gordon and Betty Moore FoundationGBMF3780
Department of Energy (DOE)DE-PS02-09ER09-25
JGI Director Discretionary Project AwardUNSPECIFIED
Department of Energy (DOE)DE-AC02-05CH11231
Subject Keywords:activity-based cell sorting; BONCAT; click chemistry; ecophysiology; single-cell microbiology
Issue or Number:28
PubMed Central ID:PMC4948357
Record Number:CaltechAUTHORS:20160628-092522305
Persistent URL:
Official Citation:Roland Hatzenpichler, Stephanie A. Connon, Danielle Goudeau, Rex R. Malmstrom, Tanja Woyke, and Victoria J. Orphan Visualizing in situ translational activity for identifying and sorting slow-growing archaeal−bacterial consortia PNAS 2016 113 (28) E4069-E4078; published ahead of print June 28, 2016, doi:10.1073/pnas.1603757113
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:68706
Deposited By: Tony Diaz
Deposited On:29 Jun 2016 17:39
Last Modified:09 Mar 2020 13:18

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