Hutter Bernd, Fischer Christina, Jacobi Alexander, Schaab Christoph, Loferer Hannes
GPC Biotech AG, Fraunhoferstrasse 20, 82152 Martinsried/Munich, Germany.
Antimicrob Agents Chemother. 2004 Jul;48(7):2588-94. doi: 10.1128/AAC.48.7.2588-2594.2004.
In a recent project, we collected the transcriptional profiles of Bacillus subtilis 168 after treatment with a large set of diverse antibacterial agents. One result of the data analysis was the identification of marker genes that are indicative of certain compounds or compound classes. We cloned these promoter regions in front of a luciferase reporter gene and reintroduced the constructs individually into the B. subtilis chromosome. Strains were analyzed for their responsiveness after treatment with a set of 37 antibacterials. Twelve functional reporter strains were generated that were selectively and significantly upregulated by the compounds. The selectivity of the reporter strains ranged from generic pathways like protein biosynthesis, cell wall biosynthesis, and fatty acid biosynthesis to compound classes (quinolones and glycopeptides) and individual compounds (rifampin, cycloserine, and clindamycin). Five of the strains are amenable for high-throughput applications, e.g., pathway-specific screening. In summary, we successfully generated B. subtilis reporter strains that are indicative of the mechanisms of action of various classes of antibacterials. The set of reporter strains presented herein can be used for mode-of-action analyses and for whole-cell screening of compound libraries in a mode-of-action-specific manner.
在最近的一个项目中，我们收集了用大量不同抗菌剂处理后的枯草芽孢杆菌168的转录谱。数据分析的一个结果是鉴定出了指示某些化合物或化合物类别的标记基因。我们将这些启动子区域克隆到荧光素酶报告基因的前面，并将构建体分别重新导入枯草芽孢杆菌染色体中。用一组37种抗菌剂处理后，分析菌株的反应性。产生了12种功能性报告菌株，它们被这些化合物选择性地且显著地上调。报告菌株的选择性范围从蛋白质生物合成、细胞壁生物合成和脂肪酸生物合成等一般途径到化合物类别（喹诺酮类和糖肽类）以及个别化合物（利福平、环丝氨酸和克林霉素）。其中5种菌株适用于高通量应用，例如途径特异性筛选。总之，我们成功地构建了能够指示各类抗菌剂作用机制的枯草芽孢杆菌报告菌株。本文介绍的报告菌株集可用于作用机制分析以及以作用机制特异性方式对化合物文库进行全细胞筛选。
