期刊
ENVIRONMENTAL MICROBIOLOGY
卷 16, 期 8, 页码 2568-2590出版社
WILEY
DOI: 10.1111/1462-2920.12436
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资金
- Beckman Institute
- Gordon & Betty Moore Foundation
- Caltech's Division of Geological and Planetary Sciences
- Erwin Schrodinger Postdoctoral Fellowship of the Austrian Science Fund (FWF) [J 3162-B20]
- Swiss National Science Foundation [PBEZP2_142903]
- Gordon and Betty Moore Foundation [GBMF3780]
- Department of Energy [DE-PS02-09ER09-25]
- National Institutes of Health [NIH R01 GM062523]
- Swiss National Science Foundation (SNF) [PBEZP2_142903] Funding Source: Swiss National Science Foundation (SNF)
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 ((NH3)-N-15 assimilation) for individual cells within a sediment-sourced enrichment culture showed concordance between AHA-positive cells and N-15 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.
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