Selective phylogenetic analysis targeting 16S rRNA genes of hyperthermophilic archaea in the deep-subsurface hot biosphere
Selective phylogenetic analysis targeting 16S rRNA genes of hyperthermophilic archaea in the deep-subsurface hot biosphere
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DOI：
10.1128/aem.02800-06
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发表时间：
2007-04-01
影响因子：
4.4
通讯作者：
Hanada, Satoshi
中科院分区：
文献类型：
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作者：
Kimura, Hiroyuki;Ishibashi, Jun-Ichiro;Hanada, Satoshi
研究地下深层热生物圈微生物群落的国际钻探项目已经扩大。深部钻井获得的岩心样品普遍受到钻井液中的中温微生物的污染，难以通过16S rRNA基因克隆文库分析来检测微生物群落。为了消除嗜温生物污染，我们之前基于16S rRNA基因的鸟嘌呤加胞嘧啶（G+C）含量与原核生物的最佳生长温度之间的强相关性开发了一种新方法（选择性系统发育分析[SePA]），并验证了该方法的有效性（H. Kimura, M. Sugihara, K. Kato, and S. Hanada, Appl. Environ. Microbiol. 72:21-27, 2006)。在本研究中，我们分别使用深海热液（117摄氏度）和表层海水（29.9摄氏度）作为深层地下地热样品和钻井液的替代品，确定了SePA消除古菌rRNA基因污染的能力。从表层海水中 PCR 扩增出的古菌 16S rRNA 基因片段在 82 摄氏度下变性，并用核酸外切酶 I (Exo 1) 完全消化，而来自深海热液的基因片段由于其高 G+C 含量，在 84 摄氏度变性后仍保持完整。使用来自两个环境样本的 DNA 混合物进行的检查表明，84 摄氏度的变性和 Exo I 消化完全消除了表层海水中的古菌 16S rRNA 基因。我们的方法对于从深层地下地热环境中回收的岩芯样本中的超嗜热古菌的非培养物群落分析非常有用。
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 degrees C) and surface seawater (29.9 degrees 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 degrees C and completely digested with exonuclease I (Exo 1), while gene fragments from the deep-sea hydrothermal fluid remained intact after denaturation at 84 degrees C because of their high G+C contents. An examination using mixtures of DNAs from the two environmental samples showed that denaturation at 84 degrees 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.