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Proc Natl Acad Sci U S A. 2015 Jan 13;112(2):E194-203. doi: 10.1073/pnas.1420406112. Epub 2014 Dec 30.

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

Author information

1
Division of Microbial Ecology, Department of Microbiology and Ecosystem Science and.
2
Single-Cell Center, Chinese Academy of Sciences Key Laboratory of Biofuels and Shandong Key Laboratory of Energy Genetics, Qingdao Institute of BioEnergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong 266101, China; Kroto Research Institute and.
3
Kroto Research Institute and.
4
Division of Microbial Ecology, Department of Microbiology and Ecosystem Science and Large Instrument Facility for Advanced Isotope Research, Faculty of Life Sciences, University of Vienna, A-1090 Vienna, Austria;
5
Department of Ruminant Sciences, Institute of Animal Science, Agricultural Research Organization, Bet-Dagan 50250, Israel;
6
Department of Microbiology, Immunobiology, and Genetics, Max F. Perutz Laboratories, University of Vienna, A-1030 Vienna, Austria;
7
Institute of Physical Chemistry and Abbe Center of Photonics, University of Jena, D-07743 Jena, Germany;
8
Institute of Physical Chemistry and Abbe Center of Photonics, University of Jena, D-07743 Jena, Germany; Institute of Photonic Technology, D-07745 Jena, Germany; and.
9
Department of Chemistry, University of Sheffield, Sheffield S3 7HQ, United Kingdom;
10
Kroto Research Institute and Department of Engineering Science, University of Oxford, Oxford OX1 3PJ, United Kingdom.
11
Division of Microbial Ecology, Department of Microbiology and Ecosystem Science and Large Instrument Facility for Advanced Isotope Research, Faculty of Life Sciences, University of Vienna, A-1090 Vienna, Austria; wagner@microbial-ecology.net.

Abstract

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. 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 microbes 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.

KEYWORDS:

Raman microspectroscopy; carbohydrate utilization; ecophysiology; nitrifier; single-cell microbiology

PMID:
25550518
PMCID:
PMC4299247
DOI:
10.1073/pnas.1420406112
[Indexed for MEDLINE]
Free PMC Article

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