EAPSI: Defining Resource Preferences of Single Cells from Aquatic Microbial Communities using Raman Microspectroscopy

该研究项目将确定日本大阪湾和木子河中消耗氨基酸的微生物种群。这一目标将通过拉曼显微光谱来测量从环境中获取氨基酸并利用它们生长的细胞来实现。溶解氨基酸是水生微生物生产能量和生物量的重要有机物来源，因此，微生物对这种底物的周转和再循环是世界各地沿海边缘碳循环的关键步骤。这项研究将与日本神户关西学院大学的著名拉曼光谱生物和化学分析专家Shinsuke Shigeto博士合作进行。很少有实验室配备了用于扫描活生物细胞的拉曼显微光谱仪，因此这次合作是将项目PI和日本合作者的专业知识结合起来的独特机会。本研究项目将测量日本大阪湾和木子河天然微生物群落中单个浮游细菌细胞生物量中13c标记氨基酸（AAs）的含量。这一目标将通过稳定同位素探测、荧光原位杂交和拉曼显微光谱的结合来实现。用13c标记的AAs孵育群落后，优势种群的单个细胞生物量中同位素的掺入量将通过红移测量。苯丙氨酸在它们的拉曼光谱中的峰值。本研究将开发一种非破坏性技术，从利用特定底物的天然微生物群落中鉴定单细胞。原核生物16S rRNA基因扩增子也将在孵育前后测序，以将社区水平的数据与群体和单细胞水平的AA掺入数据联系起来。该奖项隶属于东亚和太平洋暑期研究所项目，由美国国家科学基金会和日本科学促进会共同资助，支持一名美国研究生进行暑期研究。

This research project will determine the microbial populations that are consuming amino acids in Osaka Bay and Muko River, Japan. This objective will be accomplished using Raman microspectroscopy to measure cells that have taken amino acids from the environment and used them for growth. Dissolved amino acids are an important organic matter source for both energy and biomass production in aquatic microorganisms, thus, turnover and recycling of this substrate by microbes is a critical step in the carbon cycle of coastal margins around the world. This research will be conducted in collaboration with Dr. Shinsuke Shigeto, a noted expert in biological and chemical analysis using Raman spectroscopy, at Kwansei Gakuin University in Kobe, Japan. Few laboratories are equipped with a Raman microspectrometer set up to scan living biological cells, thus this collaboration represents a unique opportunity to combine the expertise of the project PI and the Japanese collaborator. This research project will measure the incorporation of 13C-labeled amino acids (AAs) into the biomass of single bacterioplankton cells in natural microbial communities from Osaka Bay and the Muko River, Japan. This objective will be accomplished using a combination of stable isotope probing, fluorescence in-situ hybridization, and Raman microspectroscopy. After incubating communities with 13C-labeled AAs, incorporation of the isotope into biomass of individual cells from dominant populations will be measured by a ?red-shift? of the phenylalanine peak in their Raman spectra. This study will develop a non-destructive technique to identify single cells from a natural microbial community that utilize a specific substrate. Prokaryotic 16S rRNA gene amplicons will also be sequenced before and after incubations to correlate community-level data with population- and single-cell level AA incorporation data. This award under the East Asia and Pacific Summer Institutes program supports summer research by a U.S. graduate student and is jointly funded by NSF and the Japan Society for the Promotion of Science.