title: Reverse and Multiple Stable Isotope Probing to Study Bacterial Metabolism and Interactions at the Single Cell Level.
abstract: The interactions between microorganisms driven by substrate metabolism and energy flow are important to shape diversity, abundance, and structure of a microbial community. Single cell technologies are useful tools for dissecting the functions of individual members and their interactions in microbial communities. Here, we developed a novel Raman stable isotope probing (Raman-SIP), which uses Raman microspectroscopy coupled with reverse and D2O colabeling to study metabolic interactions in a two-species community consisting of Acinetobacter baylyi ADP1 and Escherichia coli DH5α-GFP. This Raman-SIP approach is able to detect carbon assimilation and general metabolic activity simultaneously. Taking advantage of Raman shift of single cell Raman spectra (SCRS) mediated by incorporation of stable-isotopic substrates, Raman-SIP with reverse labeling has been applied to detect initially13C-labeled bands of ADP1 SCRS reverting back to12C positions in the presence of12C citrate. Raman-SIP with D2O labeling has been employed to probe metabolic activity of single cells without the need of cell replication. Our results show that E. coli alone in minimal medium with citrate as the sole carbon source had no metabolic activity, but became metabolically active in the presence of ADP1. Mass spectrometry-based metabolite footprint analysis suggests that putrescine and phenylalanine excreted by ADP1 cells may support the metabolic activity of E. coli. This study demonstrates that Raman-SIP with reverse labeling would be a useful tool to probe metabolism of any carbon substrate, overcoming limitations when stable isotopic substrates are not readily available. It is also found that Raman-SIP with D2O labeling is a sensitive and reliable approach to distinguish metabolically active cells but not quiescent cells. This novel approach extends the application of Raman-SIP and demonstrates its potential application as a valuable strategic approach for probing cellular metabolism, metabolic activity, and interactions in microbial communities at the single cell level.
authors: Single Cell Center, CAS Key Laboratory of Biofuels and Shandong Key Laboratory of Energy Genetics, Chinese Academy of Sciences , Qingdao, 266101, P. R. China.
authors: Yun
authors: Y
authors: Wang
authors: Department of Engineering Science, University of Oxford , Parks Road, Oxford, OX1 3PJ, United Kingdom.
authors: Yizhi
authors: Y
authors: Song
authors: Department of Operative Dentistry and Endodontics, Guanghua School and Hospital of Stomatology, Sun Yat-sen University , Guangzhou, 510055, P. R. China.
authors: Yifan
authors: Y
authors: Tao
authors: School of Chemistry, Manchester Institute of Biotechnology, University of Manchester , 131 Princess Street, Manchester, M1 7DN, United Kingdom.
authors: Howbeer
authors: H
authors: Muhamadali
authors: School of Chemistry, Manchester Institute of Biotechnology, University of Manchester , 131 Princess Street, Manchester, M1 7DN, United Kingdom.
authors: Royston
authors: R
authors: Goodacre
authors: State Key Laboratory of Microbial Metabolism and School of Life Sciences & Biotechnology, Shanghai Jiao Tong University , Shanghai, 200240, P. R. China.
authors: Ning-Yi
authors: NY
authors: Zhou
authors: Department of Plant Sciences, University of Oxford , South Parks Road, Oxford OX1 3RB, United Kingdom.
authors: Gail M
authors: GM
authors: Preston
authors: Single Cell Center, CAS Key Laboratory of Biofuels and Shandong Key Laboratory of Energy Genetics, Chinese Academy of Sciences , Qingdao, 266101, P. R. China.
authors: Jian
authors: J
authors: Xu
authors: Department of Engineering Science, University of Oxford , Parks Road, Oxford, OX1 3PJ, United Kingdom.
authors: Wei E
authors: WE
authors: Huang
mesh_terms: D000150:Acinetobacter / Q000378:metabolism*; D002247:Carbon Isotopes; D019343:Citric Acid / Q000378:metabolism; D017666:Deuterium Oxide / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism*; D059010:Single-Cell Analysis / Q000379:methods*; D013059:Spectrum Analysis, Raman / Q000379:methods
chemical_list: D002247:Carbon Isotopes; D019343:Citric Acid; D017666:Deuterium Oxide