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Proteomic Stable Isotope Probing Reveals Biosynthesis Dynamics of Slow Growing Methane Based Microbial Communities

Marlow, Jeffrey J. and Skennerton, Connor T. and Li, Zhou and Chourey, Karuna and Hettich, Robert L. and Pan, Chongle and Orphan, Victoria J. (2016) Proteomic Stable Isotope Probing Reveals Biosynthesis Dynamics of Slow Growing Methane Based Microbial Communities. Frontiers in Microbiology, 7 . Art. No. 563. ISSN 1664-302X. PMCID PMC4850331.

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Marine methane seep habitats represent an important control on the global flux of methane. Nucleotide-based meta-omics studies outline community-wide metabolic potential, but expression patterns of environmentally relevant proteins are poorly characterized. Proteomic stable isotope probing (proteomic SIP) provides additional information by characterizing phylogenetically specific, functionally relevant activity in mixed microbial communities, offering enhanced detection through system-wide product integration. Here we applied proteomic SIP to ^(15)NH_4^+ and CH_4 amended seep sediment microcosms in an attempt to track protein synthesis of slow-growing, low-energy microbial systems. Across all samples, 3495 unique proteins were identified, 11% of which were ^(15)N-labeled. Consistent with the dominant anaerobic oxidation of methane (AOM) activity commonly observed in anoxic seep sediments, proteins associated with sulfate reduction and reverse methanogenesis—including the ANME-2 associated methylenetetrahydromethanopterin reductase (Mer)—were all observed to be actively synthesized (^(15)N-enriched). Conversely, proteins affiliated with putative aerobic sulfur-oxidizing epsilon- and gammaproteobacteria showed a marked decrease over time in our anoxic sediment incubations. The abundance and phylogenetic range of ^(15)N-enriched methyl-coenzyme M reductase (Mcr) orthologs, many of which exhibited novel post-translational modifications, suggests that seep sediments provide niches for multiple organisms performing analogous metabolisms. In addition, 26 proteins of unknown function were consistently detected and actively expressed under conditions supporting AOM, suggesting that they play important roles in methane seep ecosystems. Stable isotope probing in environmental proteomics experiments provides a mechanism to determine protein durability and evaluate lineage-specific responses in complex microbial communities placed under environmentally relevant conditions. Our work here demonstrates the active synthesis of a metabolically specific minority of enzymes, revealing the surprising longevity of most proteins over the course of an extended incubation experiment in an established, slow-growing, methane-impacted environmental system.

Item Type:Article
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URLURL TypeDescription CentralArticle
Skennerton, Connor T.0000-0003-1320-4873
Orphan, Victoria J.0000-0002-5374-6178
Additional Information:Copyright © 2016 Marlow, Skennerton, Li, Chourey, Hettich, Pan and Orphan. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Received: 21 February 2016; Accepted: 04 April 2016; Published: 29 April 2016. THIS ARTICLE IS PART OF THE RESEARCH TOPIC Studies on Life at the Energetic Edge – from Laboratory Experiments to Field-Based Investigations. Author Contributions: JM, CS, RH, and VO designed the study; JM performed incubation set-up, microbiological and geochemical analyses; CS constructed the metagenomic databases and developed the computational architecture for protein analysis; KC developed protein extraction procedures; ZL and CP performed proteomic SIP computational analysis; JM analyzed proteomic findings from an environmental microbiological perspective and wrote the manuscript with input from all authors. Funding: This work was supported by the US Department of Energy, Office of Science, Office of Biological Environmental Research, under award numbers DE-SC0004949 and DE-SC0010574, and the Life Underground NASA Astrobiology Institute (NNA13AA92A) (to VO). JM was supported by a National Energy Technology Laboratory Methane Hydrate Research Fellowship funded by the National Research Council of the National Academies. The funders had no role in study design, data collection and interpretation, or the manuscript preparation and submission process. Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Acknowledgments: We thank the Captains, Crew, Alvin group, Jason group, and Science party members from RV Atlantis legs AT-15-68, and AT-18-10. David Case and Stephanie Connon assisted with iTAG data collection and interpretation; Shawn McGlynn performed the nanoSIMS analysis, and Joshua Steele, Rachel Poretsky, and Shulei Sun collected and processed earlier iterations of the metagenomic database. We thank Peter Girguis and the ARPA-E REMOTE team for helpful comments during the preparation of this manuscript. This research used resources of the Oak Ridge Leadership Computing Facility. Oak Ridge National Laboratory is supported by the Office of Science of the U.S. Department of Energy.
Funding AgencyGrant Number
Department of Energy (DOE)DE-SC0004949
Department of Energy (DOE)DE-SC0010574
National Energy Technology Laboratory Methane Hydrate Research FellowshipUNSPECIFIED
National Research Council of the National AcademiesUNSPECIFIED
Subject Keywords:proteomics, stable isotope probing, methane seeps, anaerobic oxidation of methane, environmental microbiology
PubMed Central ID:PMC4850331
Record Number:CaltechAUTHORS:20160601-152032127
Persistent URL:
Official Citation:Marlow JJ, Skennerton CT, Li Z, Chourey K, Hettich RL, Pan C and Orphan VJ (2016) Proteomic Stable Isotope Probing Reveals Biosynthesis Dynamics of Slow Growing Methane Based Microbial Communities. Front. Microbiol. 7:563. doi: 10.3389/fmicb.2016.00563
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:67556
Deposited By: George Porter
Deposited On:02 Jun 2016 15:23
Last Modified:03 Oct 2019 10:06

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