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首页> 外文期刊>Journal of bacteriology >Functional Analysis of the Stability Determinant AlfB of pBET131, a Miniplasmid Derivative of Bacillus subtilis (natto) Plasmid pLS32
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Functional Analysis of the Stability Determinant AlfB of pBET131, a Miniplasmid Derivative of Bacillus subtilis (natto) Plasmid pLS32

机译：枯草芽孢杆菌（纳豆）质粒pLS32的微质粒衍生物pBET131的稳定性决定子AlfB的功能分析

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Bacillus subtilis plasmid pBET131 is a derivative of pLS32, which was isolated from a natto strain of Bacillus subtilis. The DNA region in pBET131 that confers segregational stability contains an operon consisting of three genes, of which alfA, encoding an actin-like ATPase, and alfB are essential for plasmid stability. In this work, the alfB gene product and its target DNA region were studied in detail. Transcription of the alf operon initiated from a σA-type promoter was repressed by the alfB gene product. Overproduction of AlfA was inhibitory to cell growth, suggesting that the repression of the alf operon by AlfB is important for maintaining appropriate levels of AlfA. An electrophoretic mobility shift assay and footprinting analysis with purified His-tagged AlfB showed that it bound to a DNA region containing three tandem repeats of 8-bp AT-rich sequence (here designated parN), which partially overlaps the ?35 sequence of the promoter. A sequence alteration in the first or third repeat did not affect the AlfB binding and plasmid stability, whereas that in the second repeat resulted in inhibition of these phenomena. The repression of alfA-lacZ expression was observed in the constructs carrying a mutation in either the first or third repeat, but not in the second repeat, indicating a correlation between plasmid stability, AlfB binding, and repression. It was also demonstrated by the yeast two-hybrid system that AlfA and AlfB interact with each other and among themselves. From these results, it was concluded that AlfB participates in partitioning pBET131 by forming a complex with AlfA and parN, the mode of which is typified by the type II partition mechanism.
机译：枯草芽孢杆菌质粒pBET131是pLS32的衍生物，是从枯草芽孢杆菌的纳豆菌株中分离出来的。赋予分离稳定性的pBET131 DNA区域包含一个操纵子，该操纵子由三个基因组成，其中 alfA 编码肌动蛋白样ATPase，而 alfB 对质粒稳定性至关重要。在这项工作中，对 alfB 基因产物及其靶DNA区域进行了详细研究。 alfB 基因产物可抑制由σ A 型启动子启动的 alf 操纵子的转录。 AlfA的过量生产抑制细胞生长，这表明AlfB抑制 alf 操纵子对于维持适当的AlfA水平很重要。电泳迁移率变动分析和纯化的带有His标签的AlfB的足迹分析表明，它与包含三个串联重复序列的DNA区域结合在一起，这些重复序列包含三个串联的8 bp AT富集序列（此处称为 parN ），部分重叠启动子的〜35序列。在第一或第三重复序列中的序列改变不影响AlfB结合和质粒稳定性，而在第二重复序列中的序列改变导致对这些现象的抑制。在第一个或第三个重复序列中携带突变的构建体中观察到了 alfA-lacZ 表达的抑制，但在第二个重复序列中均没有突变，表明质粒稳定性，AlfB结合和抑制之间存在相关性。酵母双杂交系统还证明了AlfA和AlfB相互之间以及彼此之间相互作用。从这些结果可以得出结论，AlfB通过与AlfA和 parN 形成复合物参与了pBET131的划分，复合物的模式以II型分配机制为代表。

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