TY - JOUR
T1 - Tracking heavy water (D2O) incorporation for identifying and sorting active microbial cells
AU - Berry, David
AU - Mader, Esther
AU - Lee, Tae Kwon
AU - Woebken, Dagmar
AU - Wang, Yun
AU - Zhu, Di
AU - Palatinszky, Marton
AU - Schintlmeister, Arno
AU - Schmid, Markus C.
AU - Hanson, Buck T.
AU - Shterzer, Naama
AU - Mizrahi, Itzhak
AU - Rauch, Isabella
AU - Decker, Thomas
AU - Bocklitz, Thomas
AU - Popp, Jürgen
AU - Gibson, Christopher M.
AU - Fowler, Patrick W.
AU - Huang, Wei E.
AU - Wagner, Michael
PY - 2015/1/13
Y1 - 2015/1/13
N2 - 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 nanoscaleresolution 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 Ramanbased 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 singlecell genomics.
AB - 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 nanoscaleresolution 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 Ramanbased 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 singlecell genomics.
KW - Carbohydrate utilization
KW - Ecophysiology
KW - Nitrifier
KW - Raman microspectroscopy
KW - Single-cell microbiology
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U2 - 10.1073/pnas.1420406112
DO - 10.1073/pnas.1420406112
M3 - Article
C2 - 25550518
AN - SCOPUS:84920944435
SN - 0027-8424
VL - 112
SP - E194-E203
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 2
ER -