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Using click-chemistry for visualizing in situ changes of translational activity in planktonic marine bacteria

Leizeaga, Ainara LU ; Estrany, Margarita; Forn, Irene and Sebastián, Marta (2017) In Frontiers in Microbiology 8(DEC).
Abstract

A major challenge in microbial ecology is linking diversity and function to determine which microbes are actively contributing to processes occurring in situ. Bioorthogonal non-canonical amino acid tagging (BONCAT) is a promising technique for detecting and quantifying translationally active bacteria in the environment. This technique consists of incubating a bacterial sample with an analog of methionine and using click-chemistry to identify the cells that have incorporated the substrate. Here, we established an optimized protocol for the visualization of protein-synthesizing cells in oligotrophic waters that can be coupled with taxonomic identification using Catalyzed Reporter Deposition Fluorescent in Situ Hybridization. We also... (More)

A major challenge in microbial ecology is linking diversity and function to determine which microbes are actively contributing to processes occurring in situ. Bioorthogonal non-canonical amino acid tagging (BONCAT) is a promising technique for detecting and quantifying translationally active bacteria in the environment. This technique consists of incubating a bacterial sample with an analog of methionine and using click-chemistry to identify the cells that have incorporated the substrate. Here, we established an optimized protocol for the visualization of protein-synthesizing cells in oligotrophic waters that can be coupled with taxonomic identification using Catalyzed Reporter Deposition Fluorescent in Situ Hybridization. We also evaluated the use of this technique to track shifts in translational activity by comparing it with leucine incorporation, and used it to monitor temporal changes in both cultures and natural samples. Finally, we determined the optimal concentration and incubation time for substrate incorporation during BONCAT incubations at an oligotrophic site. Our results demonstrate that BONCAT is a fast and powerful semi-quantitative approach to explore the physiological status of marine bacteria.

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Please use this url to cite or link to this publication:
author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
BONCAT, Click-chemistry, Marine bacteria, Marine ecology, Single-cell microbiology
in
Frontiers in Microbiology
volume
8
issue
DEC
publisher
Frontiers
external identifiers
  • scopus:85036551312
ISSN
1664-302X
DOI
10.3389/fmicb.2017.02360
language
English
LU publication?
yes
id
3395ff2e-79fd-467a-bb55-4def853d6b8f
date added to LUP
2017-12-18 10:30:42
date last changed
2018-03-20 03:00:31
@article{3395ff2e-79fd-467a-bb55-4def853d6b8f,
  abstract     = {<p>A major challenge in microbial ecology is linking diversity and function to determine which microbes are actively contributing to processes occurring in situ. Bioorthogonal non-canonical amino acid tagging (BONCAT) is a promising technique for detecting and quantifying translationally active bacteria in the environment. This technique consists of incubating a bacterial sample with an analog of methionine and using click-chemistry to identify the cells that have incorporated the substrate. Here, we established an optimized protocol for the visualization of protein-synthesizing cells in oligotrophic waters that can be coupled with taxonomic identification using Catalyzed Reporter Deposition Fluorescent in Situ Hybridization. We also evaluated the use of this technique to track shifts in translational activity by comparing it with leucine incorporation, and used it to monitor temporal changes in both cultures and natural samples. Finally, we determined the optimal concentration and incubation time for substrate incorporation during BONCAT incubations at an oligotrophic site. Our results demonstrate that BONCAT is a fast and powerful semi-quantitative approach to explore the physiological status of marine bacteria.</p>},
  articleno    = {2360},
  author       = {Leizeaga, Ainara and Estrany, Margarita and Forn, Irene and Sebastián, Marta},
  issn         = {1664-302X},
  keyword      = {BONCAT,Click-chemistry,Marine bacteria,Marine ecology,Single-cell microbiology},
  language     = {eng},
  month        = {12},
  number       = {DEC},
  publisher    = {Frontiers},
  series       = {Frontiers in Microbiology},
  title        = {Using click-chemistry for visualizing in situ changes of translational activity in planktonic marine bacteria},
  url          = {http://dx.doi.org/10.3389/fmicb.2017.02360},
  volume       = {8},
  year         = {2017},
}