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Title: Tracking heavy water (D2O) incorporation for identifying and sorting active microbial cells

Here, microbial communities are essential to the function of virtually all ecosystems and eukaryotes, including humans. However, it is still a major challenge to identify microbial cells active under natural conditions in complex systems. Here in this study, we developed a new method to identify and sort active microbes on the single-cell level in complex samples using stable isotope probing with heavy water (D2O) combined with Raman microspectroscopy. Incorporation of D2O-derived D into the biomass of autotrophic and heterotrophic bacteria and archaea could be unambiguously detected via C-D signature peaks in single-cell Raman spectra, and the obtained labeling pattern was confirmed by nanoscale-resolution secondary ion MS. In fast-growing Escherichia coli cells, label detection was already possible after 20 min. For functional analyses of microbial communities, the detection of D incorporation from D2O in individual microbial cells via Raman microspectroscopy can be directly combined with FISH for the identification of active microbes. Applying this approach to mouse cecal microbiota revealed that the host-compound foragers Akkermansia muciniphila and Bacteroides acidifaciens exhibited distinctive response patterns to amendments of mucin and sugars. By Raman-based cell sorting of active (deuterated) cells with optical tweezers and subsequent multiple displacement amplification and DNA sequencing, novel cecal microbesmore » stimulated by mucin and/or glucosamine were identified, demonstrating the potential of the nondestructive D2O-Raman approach for targeted sorting of microbial cells with defined functional properties for single-cell genomics.« less
Authors:
 [1] ;  [1] ;  [1] ;  [1] ;  [2] ;  [3] ;  [1] ;  [1] ;  [1] ;  [1] ;  [4] ;  [4] ;  [1] ;  [1] ;  [5] ;  [6] ;  [3] ;  [3] ;  [7] ;  [1]
  1. Univ. of Vienna, Vienna (Austria)
  2. Chinese Academy of Sciences (CAS), Shandong (China); Univ. of Sheffield, Sheffield (United Kingdom)
  3. Univ. of Sheffield, Sheffield (United Kingdom)
  4. Agricultural Research Org., Bet-Dagan (Israel)
  5. Univ. of Jena, Jena (Germany)
  6. Univ. of Jena, Jena (Germany); Institute of Photonic Technology, Jena (Germany)
  7. Univ. of Sheffield, Sheffield (United Kingdom); Univ. of Oxford, Oxford (United Kingdom)
Publication Date:
Grant/Contract Number:
AC02-05CH11231
Type:
Published Article
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
Additional Journal Information:
Journal Volume: 112; Journal Issue: 2; Journal ID: ISSN 0027-8424
Publisher:
National Academy of Sciences, Washington, DC (United States)
Research Org:
Univ. of Vienna, Vienna (Austria)
Sponsoring Org:
USDOE Office of Science (SC)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; ecophysiology; single-cell microbiology; carbohydrate utilization; nitrifier; Raman microspectroscopy
OSTI Identifier:
1235155
Alternate Identifier(s):
OSTI ID: 1342520