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Appl. Environ. Microbiol. doi:10.1128/AEM.02800-06
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Selective Phylogenetic Analysis (SePA) Targeting 16S rRNA Genes of Hyperthermophilic Archaea in the Deep-Subsurface Hot Biosphere

Department of Geosciences, Faculty of Science, Shizuoka University, 836 Oya, Suruga-ku, Shizuoka 422-8529, Department of Earth and Planetary Science, Faculty of Science, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Department of Geosciences, Osaka City University, 3-3-138, Sugimoto, Sumiyoshi-ku, Osaka 558-8585, and Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan

^* To whom correspondence should be addressed. Email: shkimur{at}ipc.shizuoka.ac.jp.

	Abstract

International drilling projects have been expanded for the study of microbial communities in the deep-subsurface hot biosphere. Core samples obtained by deep drilling are commonly contaminated with mesophilic microorganisms in the drilling fluid, making it difficult to examine the microbial community by 16S rRNA gene clone library analysis. To eliminate mesophilic contamination, we previously developed a new method (selective phylogenetic analysis: SePA) based on the strong correlation between the guanine and cytosine (GC) contents of the 16S rRNA gene and the optimal growth temperature of prokaryotes, and we verified the method's effectiveness (H. Kimura, M. Sugihara, K. Kato, and S. Hanada, Appl. Environ. Microbiol. 72:21-27, 2006). In the present study we ascertain SePA's ability to eliminate contamination by archaeal ribosomal RNA genes, using deep-sea hydrothermal fluid (117°C) and surface seawater (29.9°C) as substitutes for deep-subsurface geothermal samples and drilling fluid, respectively. Archaeal 16S rRNA gene fragments, PCR-amplified from the surface seawater, were denatured at 82°C and completely digested by exonuclease I (Exo I), while those from the deep-sea hydrothermal fluid remained intact after denaturation at 84°C because of their high GC contents. An examination using mixtures of DNAs from the two environmental samples showed that denaturation at 84°C and digestion with Exo I completely eliminated archaeal 16S rRNA genes from the surface seawater. Our method was quite useful for the culture-independent community analysis of hyperthermophilic archaea in core samples recovered from deep-subsurface geothermal environments.