This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kimura, H. Articles by Hanada, S. Search for Related Content PubMed PubMed Citation Articles by Kimura, H. Articles by Hanada, S. Agricola Articles by Kimura, H. Articles by Hanada, S.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, January 2006, p. 21-27, Vol. 72, No. 1
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.1.21-27.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Selective Phylogenetic Analysis Targeted at 16S rRNA Genes of Thermophiles and Hyperthermophiles in Deep-Subsurface Geothermal Environments

Hiroyuki Kimura,^1,2 Maki Sugihara,¹ Kenji Kato,² and Satoshi Hanada^1*

Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8566,¹ Department of Biology and Geosciences, Faculty of Science, Shizuoka University, Shizuoka, Shizuoka 422-8529, Japan²

Received 25 April 2005/ Accepted 17 September 2005

Deep-subsurface samples obtained by deep drilling are likely to be contaminated with mesophilic microorganisms in the drilling fluid, and this could affect determination of the community structure of the geothermal microflora using 16S rRNA gene clone library analysis. To eliminate possible contamination by PCR-amplified 16S rRNA genes from mesophiles, a combined thermal denaturation and enzyme digestion method, based on a strong correlation between the G+C content of the 16S rRNA gene and the optimum growth temperatures of most known prokaryotic cultures, was used prior to clone library construction. To validate this technique, hot spring fluid (76°C) and river water (14°C) were used to mimic a deep-subsurface sample contaminated with drilling fluid. After DNA extraction and PCR amplification of the 16S rRNA genes from individual samples separately, the amplified products from river water were observed to be denatured at 82°C and completely digested by exonuclease I (Exo I), while the amplified products from hot spring fluid remained intact after denaturation at 84°C and enzyme digestion with Exo I. DNAs extracted from the two samples were mixed and used as a template for amplification of the 16S rRNA genes. The amplified rRNA genes were denatured at 84°C and digested with Exo I before clone library construction. The results indicated that the 16S rRNA gene sequences from the river water were almost completely eliminated, whereas those from the hot spring fluid remained.

---

* Corresponding author. Mailing address: 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. Phone: 81-29-861-6591. Fax: 81-29-861-6587. E-mail: s-hanada{at}aist.go.jp

---

Applied and Environmental Microbiology, January 2006, p. 21-27, Vol. 72, No. 1
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.1.21-27.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Kimura, H., Ishibashi, J.-I., Masuda, H., Kato, K., Hanada, S. (2007). Selective Phylogenetic Analysis Targeting 16S rRNA Genes of Hyperthermophilic Archaea in the Deep-Subsurface Hot Biosphere. Appl. Environ. Microbiol. 73: 2110-2117 [Abstract] [Full Text]