Preparation of Genomic DNA from a Single Species of Uncultured Magnetotactic Bacterium by Multiple-Displacement Amplification
Preparation of Genomic DNA from a Single Species of Uncultured Magnetotactic Bacterium by Multiple-Displacement Amplification
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DOI：
10.1128/aem.02124-09
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发表时间：
2010-01
影响因子：
4.4
通讯作者：
Atsushi Arakaki;Mie Shibusawa;Masahito Hosokawa;T. Matsunaga
中科院分区：
文献类型：
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作者：
Atsushi Arakaki;Mie Shibusawa;Masahito Hosokawa;T. Matsunaga
摘要趋磁菌是一个系统发育不同的类群，能够合成细胞内的磁性颗粒。尽管在环境中观察到了不同形态的趋磁细菌，但由于培养和培养的困难，目前纯培养的细菌菌株仅限于几个属。为了从未培养的趋磁细菌中获得遗传信息，本研究采用了一种包括细胞磁分离、流式细胞术和φ-29聚合酶多重置换扩增在内的基因组制备方法。使用一种纯培养的趋磁细菌“磁性磁螺杆菌AMB-1”评估了使用包含1到100个细胞的样本进行丙二醛反应的条件，该细菌的完整基因组序列是可用的。以100个细胞为模板，定量聚合酶链式反应(Q-PCR)证实基因扩增均一。然后将该方法应用于水环境中复杂细菌群落中未培养的趋磁细菌的基因组制备。用磁性细胞分离和流式细胞术制备了含有100个细胞的未培养趋磁球菌的样品，并将其作为丙二醛模板。对这100个细胞的丙二醛产物的16S rRNA序列分析表明，扩增的基因组DNA来自于一种单一的趋磁性细菌，该细菌与甲型变形杆菌中的趋磁性球菌有亲缘关系。磁分离、流式细胞仪和丙二醛的联合使用为获取环境样品中趋磁细菌的个体遗传信息提供了一种新的策略。
ABSTRACT Magnetotactic bacteria comprise a phylogenetically diverse group that is capable of synthesizing intracellular magnetic particles. Although various morphotypes of magnetotactic bacteria have been observed in the environment, bacterial strains available in pure culture are currently limited to a few genera due to difficulties in their enrichment and cultivation. In order to obtain genetic information from uncultured magnetotactic bacteria, a genome preparation method that involves magnetic separation of cells, flow cytometry, and multiple displacement amplification (MDA) using φ29 polymerase was used in this study. The conditions for the MDA reaction using samples containing 1 to 100 cells were evaluated using a pure-culture magnetotactic bacterium, “Magnetospirillum magneticum AMB-1,” whose complete genome sequence is available. Uniform gene amplification was confirmed by quantitative PCR (Q-PCR) when 100 cells were used as a template. This method was then applied for genome preparation of uncultured magnetotactic bacteria from complex bacterial communities in an aquatic environment. A sample containing 100 cells of the uncultured magnetotactic coccus was prepared by magnetic cell separation and flow cytometry and used as an MDA template. 16S rRNA sequence analysis of the MDA product from these 100 cells revealed that the amplified genomic DNA was from a single species of magnetotactic bacterium that was phylogenetically affiliated with magnetotactic cocci in the Alphaproteobacteria. The combined use of magnetic separation, flow cytometry, and MDA provides a new strategy to access individual genetic information from magnetotactic bacteria in environmental samples.