PMID- 24571640
OWN - NLM
STAT- MEDLINE
DCOM- 20140930
LR  - 20211021
IS  - 1462-2920 (Electronic)
IS  - 1462-2912 (Print)
IS  - 1462-2912 (Linking)
VI  - 16
IP  - 8
DP  - 2014 Aug
TI  - In situ visualization of newly synthesized proteins in environmental microbes 
      using amino acid tagging and click chemistry.
PG  - 2568-90
LID - 10.1111/1462-2920.12436 [doi]
AB  - 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 
      labelling 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 ribosomal RNA (rRNA)-targeted fluorescence in situ 
      hybridization (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 labelling by nano-scale secondary ion mass spectrometry 
      ((15)NH(3) 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.
CI  - (c) 2014 The Authors. Environmental Microbiology published by Society for Applied 
      Microbiology and John Wiley & Sons Ltd.
FAU - Hatzenpichler, Roland
AU  - Hatzenpichler R
AD  - Divisions of Geological and Planetary Sciences, California Institute of 
      Technology, Pasadena, CA, 91125, USA.
FAU - Scheller, Silvan
AU  - Scheller S
FAU - Tavormina, Patricia L
AU  - Tavormina PL
FAU - Babin, Brett M
AU  - Babin BM
FAU - Tirrell, David A
AU  - Tirrell DA
FAU - Orphan, Victoria J
AU  - Orphan VJ
LA  - eng
GR  - R01 GM062523/GM/NIGMS NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, Non-P.H.S.
DEP - 20140402
PL  - England
TA  - Environ Microbiol
JT  - Environmental microbiology
JID - 100883692
RN  - 0 (Alkynes)
RN  - 0 (Archaeal Proteins)
RN  - 0 (Azides)
RN  - 0 (Bacterial Proteins)
RN  - 0 (azidohomoalanine)
RN  - AE28F7PNPL (Methionine)
RN  - OF5P57N2ZX (Alanine)
SB  - IM
MH  - Alanine/*analogs & derivatives/chemistry/metabolism
MH  - Alkynes/chemistry
MH  - Archaea/chemistry/metabolism
MH  - Archaeal Proteins/biosynthesis/*chemistry
MH  - Azides/chemistry
MH  - Bacteria/chemistry/metabolism
MH  - Bacterial Proteins/biosynthesis/*chemistry
MH  - Biofilms/growth & development
MH  - *Click Chemistry
MH  - Fresh Water/microbiology
MH  - Geologic Sediments/microbiology
MH  - Humans
MH  - In Situ Hybridization, Fluorescence
MH  - Methionine/chemistry/metabolism
MH  - Microscopy, Fluorescence
MH  - Mouth/microbiology
MH  - Staining and Labeling
PMC - PMC4122687
MID - NIHMS573585
EDAT- 2014/02/28 06:00
MHDA- 2014/10/01 06:00
PMCR- 2014/12/05
CRDT- 2014/02/28 06:00
PHST- 2013/12/20 00:00 [received]
PHST- 2014/02/14 00:00 [revised]
PHST- 2014/02/18 00:00 [accepted]
PHST- 2014/02/28 06:00 [entrez]
PHST- 2014/02/28 06:00 [pubmed]
PHST- 2014/10/01 06:00 [medline]
PHST- 2014/12/05 00:00 [pmc-release]
AID - 10.1111/1462-2920.12436 [doi]
PST - ppublish
SO  - Environ Microbiol. 2014 Aug;16(8):2568-90. doi: 10.1111/1462-2920.12436. Epub 
      2014 Apr 2.