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首页> 外文期刊>Applied and Environmental Microbiology >Preparation of Genomic DNA from a Single Species of Uncultured Magnetotactic Bacterium by Multiple-Displacement Amplification
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Preparation of Genomic DNA from a Single Species of Uncultured Magnetotactic Bacterium by Multiple-Displacement Amplification

机译：通过多种置换扩增从单种未培养趋磁细菌中制备基因组DNA

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摘要

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.
机译：趋磁细菌包含能够合成细胞内磁性颗粒的系统发育多样性基团。尽管已在环境中观察到各种形态的趋磁细菌，但由于其富集和培养困难，目前在纯培养中可获得的细菌菌株仅限于少数属。为了从未培养的趋磁细菌中获得遗传信息，本研究使用了一种基因组制备方法，该方法涉及细胞的磁分离，流式细胞术和使用φ29聚合酶的多位移扩增（MDA）。使用可得到完整基因组序列的纯培养趋磁细菌“ Magnetospirillum magneticum AMB-1”评估使用1至100个细胞的样品进行MDA反应的条件。当将100个细胞用作模板时，通过定量PCR（Q-PCR）证实了均匀的基因扩增。然后将该方法用于从水生环境中复杂细菌群落中培养的趋磁细菌的基因组制备。通过磁性细胞分离和流式细胞术制备了包含100个未培养的趋磁球菌细胞的样品，并将其用作MDA模板。对这100个细胞的MDA产物的16S rRNA序列分析表明，扩增的基因组DNA来自单一物种的趋磁细菌，在系统上与 Alphaproteobacteria 中的趋磁球菌相关。磁分离，流式细胞术和MDA的组合使用提供了一种新策略，可从环境样品中的趋磁细菌中获取单个遗传信息。

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