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- 저자 : Palatinszky, Marton (검색결과 1 건)
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Berry, David,Mader, Esther,Lee, Tae Kwon,Woebken, Dagmar,Wang, Yun,Zhu, Di,Palatinszky, Marton,Schintlmeister, Arno,Schmid, Markus C.,Hanson, Buck T.,Shterzer, Naama,Mizrahi, Itzhak,Rauch, Isabella,De National Academy of Sciences 2015 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF Vol.112 No.2
<P><B>Significance</B></P><P>Measuring activity patterns of microbes in their natural environment is essential for understanding ecosystems and the multifaceted interactions of microorganisms with eukaryotes. In this study, we developed a technique that allows fast and nondestructive activity measurements of microbial communities on a single-cell level. Microbial communities were amended with heavy water (D<SUB>2</SUB>O), a treatment that does not change the available substrate pool. After incubation, physiologically active cells are rapidly identified with Raman microspectroscopy by measuring cellular D incorporation. Using this approach, we characterized the activity patterns of two dominant microbes in mouse cecum samples amended with different carbohydrates and discovered previously unidentified bacteria stimulated by mucin and/or glucosamine by combining Raman microspectroscopy and optical tweezer-based sorting.</P><P>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 (D<SUB>2</SUB>O) combined with Raman microspectroscopy. Incorporation of D<SUB>2</SUB>O-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 <I>Escherichia coli</I> cells, label detection was already possible after 20 min. For functional analyses of microbial communities, the detection of D incorporation from D<SUB>2</SUB>O 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 <I>Akkermansia muciniphila</I> and <I>Bacteroides acidifaciens</I> 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 D<SUB>2</SUB>O-Raman approach for targeted sorting of microbial cells with defined functional properties for single-cell genomics.</P>
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