Characterization of uncultivated prokaryotes: Isolation and analysis of a 40-kilobase-pair genome fragment front a planktonic marine archaeon
Characterization of uncultivated prokaryotes: Isolation and analysis of a 40-kilobase-pair genome fragment front a planktonic marine archaeon
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DOI：
10.1128/jb.178.3.591-599.1996
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发表时间：
1996-02-01
影响因子：
3.2
通讯作者：
DeLong, EF
中科院分区：
文献类型：
--
作者：
Stein, JL;Marsh, TL;DeLong, EF
一种从天然组合中表征未培养原核生物的潜在方法涉及对直接从天然存在的微生物生物质中回收的 DNA 片段进行基因组分析。在这项研究中，我们试图从分布广泛且相对丰富但尚未培养的原核生物群体——浮游海洋古细菌中分离出大的基因组片段。 fosmid DNA 文库是从北太平洋东部 200 m 深度收集的海洋超微型浮游生物组合中制备的。我们鉴定了一个 38.5 kbp 的重组 fosmid 克隆，其中含有古细菌小亚基核糖体 DNA 基因。小亚基 rRNA 序列的系统发育分析证明了它与之前描述的浮游古菌的密切关系，后者形成了一个深深扎根于古菌领域的泉古菌分支的连贯群体。对古细菌 fosmid 克隆的亚克隆片段进行随机鸟枪测序，揭示了几个与古细菌同源物具有最高相似性的基因，包括大亚基核糖体 DNA 和翻译延伸因子 2 (EF2)。对浮游古菌 EF2 推断的氨基酸序列的分析支持其与古生菌的 Crenarchaeote 分支的关系。还鉴定了两个编码以前在古细菌中未发现的蛋白质的基因片段：RNA解旋酶（负责RNA二级结构的ATP依赖性改变）和谷氨酸半醛转氨酶（一种参与血红素生物合成初始步骤的酶）。总而言之，我们的结果表明，对从混合微生物组合中检索到的大 DNA 片段进行基因组分析可以为丰富但尚未培养的原核生物的生理潜力提供有用的视角。
One potential approach for characterizing uncultivated prokaryotes from natural assemblages involves genomic analysis of DNA fragments retrieved directly from naturally occurring microbial biomass. In this study, we sought to isolate large genomic fragments from a widely distributed and relatively abundant but as yet uncultivated group of prokaryotes, the planktonic marine Archaea. A fosmid DNA library was prepared from a marine picoplankton assemblage collected at a depth of 200 m in the eastern North Pacific. We identified a 38.5-kbp recombinant fosmid clone which contained an archaeal small subunit ribosomal DNA gene. Phylogenetic analyses of the small subunit rRNA sequence demonstrated its close relationship to that of previously described planktonic archaea, which form a coherent group rooted deeply within the Crenarchaeota branch of the domain Archaea. Random shotgun sequencing of subcloned fragments of the archaeal fosmid clone revealed several genes which bore highest similarity to archaeal homologs, including large subunit ribosomal DNA and translation elongation factor 2 (EF2). Analyses of the inferred amino acid sequence of archaeoplankton EF2 supported its affiliation with the Crenarchaeote subdivision of Archaea. Two gene fragments encoding proteins not previously found in Archaea were also identified: RNA helicase, responsible for the ATP-dependent alteration of RNA secondary structure, and glutamate semialdehyde aminotransferase, an enzyme involved in initial steps of heme biosynthesis. In total, our results indicate that genomic analysis of large DNA fragments retrieved from mixed microbial assemblages can provide useful perspective on the physiological potential of abundant but as yet uncultivated prokaryotes.