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Applied and Environmental Microbiology, April 2007, p. 2110-2117, Vol. 73, No. 7
0099-2240/07/$08.00+0     doi:10.1128/AEM.02800-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Selective Phylogenetic Analysis Targeting 16S rRNA Genes of Hyperthermophilic Archaea in the Deep-Subsurface Hot Biosphere{triangledown}

Hiroyuki Kimura,1* Jun-Ichiro Ishibashi,2 Harue Masuda,3 Kenji Kato,1 and Satoshi Hanada4

Department of Geosciences, Faculty of Science, Shizuoka University, 836 Oya, Suruga-ku, Shizuoka 422-8529,1 Department of Earth and Planetary Science, Faculty of Science, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581,2 Department of Geosciences, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi-ku, Osaka 558-8585,3 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, Japan4

Received 30 November 2006/ Accepted 20 January 2007

International drilling projects for the study of microbial communities in the deep-subsurface hot biosphere have been expanded. 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 organism contamination, we previously developed a new method (selective phylogenetic analysis [SePA]) based on the strong correlation between the guanine-plus-cytosine (G+C) contents of the 16S rRNA genes and the optimal growth temperatures 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 ascertained SePA's ability to eliminate contamination by archaeal rRNA 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 with exonuclease I (Exo I), while gene fragments from the deep-sea hydrothermal fluid remained intact after denaturation at 84°C because of their high G+C 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 culture-independent community analysis of hyperthermophilic archaea in core samples recovered from deep-subsurface geothermal environments.

* Corresponding author. Mailing address: Department of Geosciences, Faculty of Science, Shizuoka University, 836 Oya, Suruga-ku, Shizuoka 422-8529, Japan. Phone: 81-54-238-4784. Fax: 81-54-238-0491. E-mail: shkimur{at}ipc.shizuoka.ac.jp.

{triangledown} Published ahead of print on 2 February 2007.

Applied and Environmental Microbiology, April 2007, p. 2110-2117, Vol. 73, No. 7
0099-2240/07/$08.00+0     doi:10.1128/AEM.02800-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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