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A novel high-throughput cell-based assay aimed at identifying inhibitors of DNA metabolism in bacteria

机译:一种新颖的基于细胞的高通量测定法,旨在鉴定细菌中DNA代谢的抑制剂

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摘要

Bacterial biosensor strains can be useful tools for thediscovery andcharacterization of antibacterial compounds.Aplasmid-based reporter vector containing a transcriptional fusionbetween the recA promoterandgreen fluorescence protein genewas introduced into an Escherichia coli δtolC strain to create a biosensor strain that selectively senses inhibitors of DNA metabolism via the SOS response. The strain was used to develop a high-throughput assay to identify new inhibitors of DNA metabolism. Screening of the Astra- Zeneca compound library with this strain identified known inhibitors of DNA metabolism, as well as novel chemotypes. The cellular target of one novel series was elucidated as DNA gyrase through genetic characterization of laboratory-generated resistant mutants followed by 50% inhibitory concentration measurements in a DNA gyrase activity assay. These studies validated the use of this antibiotic biosensor strain to identify novel selective inhibitors of DNA metabolism by high-throughput screening.
机译:细菌生物传感器菌株可作为发现和表征抗菌化合物的有用工具。将包含recA启动子和绿色荧光蛋白基因之间转录融合体的基于质粒的报告载体导入大肠杆菌δtolC菌株中,以创建一种生物传感器菌株,该菌株通过DNA选择性检测DNA代谢的抑制剂。 SOS响应。该菌株用于开发高通量测定法,以鉴定DNA代谢的新抑制剂。用该菌株筛选Astra-Zeneca化合物文库,鉴定出已知的DNA代谢抑制剂以及新的化学型。通过对实验室产生的抗性突变体进行遗传表征,然后在DNA旋转酶活性测定中进行50%抑制浓度测量,将一个新系列的细胞靶标阐明为DNA旋转酶。这些研究验证了这种抗生素生物传感器菌株在通过高通量筛选中鉴定DNA代谢的新型选择性抑制剂的用途。

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