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In situ visualization of newly synthesized proteins in environmental microbes using amino acid tagging and click chemistry

Hatzenpichler, Roland and Scheller, Silvan and Tavormina, Patricia L. and Babin, Brett M. and Tirrell, David A. and Orphan, Victoria J. (2014) In situ visualization of newly synthesized proteins in environmental microbes using amino acid tagging and click chemistry. Environmental Microbiology, 16 (8). pp. 2568-2590. ISSN 1462-2912. PMCID PMC4122687. https://resolver.caltech.edu/CaltechAUTHORS:20140303-103306009

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Abstract

Here we describe the application of a new click chemistry method for fluorescent tracking of protein synthesis in individual microorganisms within environmental samples. This technique, termed bioorthogonal non-canonical amino acid tagging (BONCAT), is based on the in vivo incorporation of the non-canonical amino acid L-azidohomoalanine (AHA), a surrogate for L-methionine, followed by fluorescent labeling of AHA containing cellular proteins by azide-alkyne click chemistry. BONCAT was evaluated with a range of phylogenetically and physiologically diverse archaeal and bacterial pure cultures and enrichments, and used to visualize translationally active cells within complex environmental samples including an oral biofilm, freshwater, and anoxic sediment. We also developed combined assays that couple BONCAT with rRNA-targeted FISH, enabling a direct link between taxonomic identity and translational activity. Using a methanotrophic enrichment culture incubated under different conditions, we demonstrate the potential of BONCAT-FISH to study microbial physiology in situ. A direct comparison of anabolic activity using BONCAT and stable isotope labeling by nanoSIMS (^(15)NH_4^+ assimilation) for individual cells within a sediment sourced enrichment culture showed concordance between AHA positive cells and ^(15)N enrichment. BONCAT-FISH offers a fast, inexpensive, and straightforward fluorescence microscopy method for studying the in situ activity of environmental microbes on a single cell level.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1111/1462-2920.12436DOIArticle
http://onlinelibrary.wiley.com/doi/10.1111/1462-2920.12436/abstractPublisherArticle
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4122687/PubMed CentralArticle
ORCID:
AuthorORCID
Hatzenpichler, Roland0000-0002-5489-3444
Scheller, Silvan0000-0002-0667-9224
Babin, Brett M.0000-0002-4133-6665
Tirrell, David A.0000-0003-3175-4596
Orphan, Victoria J.0000-0002-5374-6178
Alternate Title:Visualizing new proteins in microbes by click chemistry
Additional Information:© 2014 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Accepted manuscript online: 26 Feb 2014; Manuscript Accepted: 18 Feb 2014; Manuscript Received: 20 Dec 2013. This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/1462-2920.12436. We thank Katherine Dawson and Hiroyuki Imachi for advice on anaerobic culturing, Yunbin Guan for assistance with nanoSIMS analyses, John D. Bagert for discussions on click chemistry, Grayson Chadwick for calculating genomic Met contents, and Jennifer Glass for helpful comments on an early version of this manuscript. We acknowledge the Caltech Proteome Exploration Laboratory (PEL) staff for analyzing mass spectrometry samples and their technical assistance with sample preparation and interpretation of results. The PEL is supported by the Beckman Institute and the Gordon & Betty Moore Foundation. Roland Hatzenpichler was supported via an O.K. Earl Postdoctoral Scholarship awarded by Caltech’s Division of Geological and Planetary Sciences as well as an Erwin Schrӧdinger Postdoctoral Fellowship of the Austrian Science Fund (FWF), J 3162-B20. Silvan Scheller was supported by the Swiss National Science Foundation (grant PBEZP2_142903). Funding for this project was provided by the Gordon and Betty Moore Foundation through Grant GBMF3780 to VJO, grant from the Department of Energy (DE-PS02-09ER09-25) to VJO, and by a National Institutes of Health grant NIH R01 GM062523 to DAT. Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
Funders:
Funding AgencyGrant Number
FWF Der WissenschaftsfondsJ 3162-B20
Swiss National Science Foundation (SNSF)PBEZP2_142903
Gordon and Betty Moore FoundationGBMF3780
Department of Energy (DOE)DE-PS02-09ER09-25
NIHR01 GM062523
Issue or Number:8
PubMed Central ID:PMC4122687
Record Number:CaltechAUTHORS:20140303-103306009
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20140303-103306009
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:44082
Collection:CaltechAUTHORS
Deposited By: Ruth Sustaita
Deposited On:26 Mar 2014 21:35
Last Modified:03 Oct 2019 23:47

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