title: Tracking heavy water (D2O) incorporation for identifying and sorting active microbial cells.
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.
authors.affiliation: Division of Microbial Ecology, Department of Microbiology and Ecosystem Science and.
authors.forename: David
authors.initials: D
authors.lastname: Berry
authors.affiliation: Division of Microbial Ecology, Department of Microbiology and Ecosystem Science and.
authors.forename: Esther
authors.initials: E
authors.lastname: Mader
authors.affiliation: Division of Microbial Ecology, Department of Microbiology and Ecosystem Science and.
authors.forename: Tae Kwon
authors.initials: TK
authors.lastname: Lee
authors.affiliation: Division of Microbial Ecology, Department of Microbiology and Ecosystem Science and.
authors.forename: Dagmar
authors.initials: D
authors.lastname: Woebken
authors.affiliation: 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.
authors.forename: Yun
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Kroto Research Institute and.
authors.forename: Di
authors.initials: D
authors.lastname: Zhu
authors.affiliation: Division of Microbial Ecology, Department of Microbiology and Ecosystem Science and.
authors.forename: Marton
authors.initials: M
authors.lastname: Palatinszky
authors.affiliation: 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;
authors.forename: Arno
authors.initials: A
authors.lastname: Schintlmeister
authors.affiliation: Division of Microbial Ecology, Department of Microbiology and Ecosystem Science and.
authors.forename: Markus C
authors.initials: MC
authors.lastname: Schmid
authors.affiliation: Division of Microbial Ecology, Department of Microbiology and Ecosystem Science and.
authors.forename: Buck T
authors.initials: BT
authors.lastname: Hanson
authors.affiliation: Department of Ruminant Sciences, Institute of Animal Science, Agricultural Research Organization, Bet-Dagan 50250, Israel;
authors.forename: Naama
authors.initials: N
authors.lastname: Shterzer
authors.affiliation: Department of Ruminant Sciences, Institute of Animal Science, Agricultural Research Organization, Bet-Dagan 50250, Israel;
authors.forename: Itzhak
authors.initials: I
authors.lastname: Mizrahi
authors.affiliation: Department of Microbiology, Immunobiology, and Genetics, Max F. Perutz Laboratories, University of Vienna, A-1030 Vienna, Austria;
authors.forename: Isabella
authors.initials: I
authors.lastname: Rauch
authors.affiliation: Department of Microbiology, Immunobiology, and Genetics, Max F. Perutz Laboratories, University of Vienna, A-1030 Vienna, Austria;
authors.forename: Thomas
authors.initials: T
authors.lastname: Decker
authors.affiliation: Institute of Physical Chemistry and Abbe Center of Photonics, University of Jena, D-07743 Jena, Germany;
authors.forename: Thomas
authors.initials: T
authors.lastname: Bocklitz
authors.affiliation: 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.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Popp
authors.affiliation: Department of Chemistry, University of Sheffield, Sheffield S3 7HQ, United Kingdom;
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Gibson
authors.affiliation: Department of Chemistry, University of Sheffield, Sheffield S3 7HQ, United Kingdom;
authors.forename: Patrick W
authors.initials: PW
authors.lastname: Fowler
authors.affiliation: Kroto Research Institute and Department of Engineering Science, University of Oxford, Oxford OX1 3PJ, United Kingdom.
authors.forename: Wei E
authors.initials: WE
authors.lastname: Huang
authors.affiliation: 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.
authors.forename: Michael
authors.initials: M
authors.lastname: Wagner
mesh_terms: D000818:Animals; D001105:Archaea / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001419:Bacteria / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D018533:Biomass; D002432:Cecum / Q000382:microbiology; D019641:DNA, Archaeal / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D017666:Deuterium Oxide / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D051379:Mice; D008810:Mice, Inbred C57BL; D059013:Microbial Consortia / Q000235:genetics; D008828:Microbiological Techniques; D008969:Molecular Sequence Data; D052898:Optical Tweezers; D010802:Phylogeny; D013059:Spectrum Analysis, Raman
chemical_list: D019641:DNA, Archaeal; D004269:DNA, Bacterial; D017666:Deuterium Oxide
keywords: Raman microspectroscopy; carbohydrate utilization; ecophysiology; nitrifier; single-cell microbiology