Physiological and genetic characterization of uncultured methanogenic archaea via an ultra-high sensitive fluorescence in situ hybridization technique

Research Project

Project/Area Number	25650138
Research Category	Grant-in-Aid for Challenging Exploratory Research
Allocation Type	Multi-year Fund
Research Field	Biodiversity/Systematics
Research Institution	Japan Agency for Marine-Earth Science and Technology
Principal Investigator	IMACHI Hiroyuki 国立研究開発法人海洋研究開発機構, 深海・地殻内生物圏研究分野, 主任研究員 (20361933)
Co-Investigator(Renkei-kenkyūsha)	KUBOTA Kengo 東北大学, 工学研究科, 准教授 (80455807)
Research Collaborator	SAITO Yayoi
Project Period (FY)	2013-04-01 – 2016-03-31
Project Status	Completed (Fiscal Year 2015)
Budget Amount *help	¥4,160,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥960,000) Fiscal Year 2015: ¥650,000 (Direct Cost: ¥500,000、Indirect Cost: ¥150,000) Fiscal Year 2014: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000) Fiscal Year 2013: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
Keywords	FISH法 / メタン菌 / mcrA遺伝子 / 生態
Outline of Final Research Achievements	The investigation of uncultured methanogens is a significant challenge in comprehensive understanding global carbon cycle. In this study, we aimed to gain physiological and genetic information of taxonomically new methanogens by obtaining genomic sequences and isolate. For this purpose, we first performed mcrA and 16S rRNA gene-based clone analyses on methanogenic sludges to explore taxonomically novel methanogens. Through the analyses, we found a novel methanogen group, which may represent the eighth order of methanogen. To obtain genetic information of the methanogen, we tried to do specific sorting of the methanogen cells using a combination of an ultra-high sensitive fluorescence in situ hybridization (FISH) technique (i.e., two-pass tyramide signal amplification FISH with polynucleotide probes) and fluorescence-activated cell sorting. However, unfortunately, the targeted methanogen cells could not be detected by the FISH technique even after several cell wall treatments.