Ogura Mitsuo, Ohsawa Taku, Tanaka Teruo
Institute of Oceanic Research and Development, Tokai University, Orido-Shimizu, Shizuoka, Japan.
Biosci Biotechnol Biochem. 2008 Jan;72(1):186-96. doi: 10.1271/bbb.70548. Epub 2008 Jan 7.
The Bacillus subtilis yrkP gene encodes a response regulator of a two-component regulatory system of unknown function. A previous DNA microarray experiment suggested that multicopy yrkP greatly enhanced the expression of yrkN, the ykcBC operon, and yrkO, which encodes a putative transporter. Here, lacZ fusion analysis confirmed these results and also revealed that YrkP autoregulates the putative yrkPQR operon, indicating that yrkPQR and yrkO form a divergon structure. In addition, real-time PCR analysis revealed that transcription of yrkO, yrkN, and ykcBC was significantly reduced in the yrkP strain. Hence, YrkP positively regulates the expression of these genes. Gel retardation analyses showed that YrkP bound to the promoter regions of yrkO, yrkN, and ykcB, albeit with lower binding affinities to the latter two promoters. The in vitro binding of YrkP to the promoter region of the yrkPQR and yrkO divergon was then analyzed by DNase I footprinting analysis. This revealed that YrkP recognizes three regions containing single-motifs or a direct repeat of the ten-base sequence [T/G]TCA[T/C]AAATT. lacZ fusion analysis of deleted and mutagenized promoter regions of yrkO and yrkPQR divergon confirmed that the three YrkP-binding regions are needed for the YrkP-mediated activation of yrkO and/or yrkPQR.
枯草芽孢杆菌的yrkP基因编码一种功能未知的双组分调节系统的应答调节因子。先前的DNA微阵列实验表明，多拷贝的yrkP极大地增强了yrkN、ykcBC操纵子以及编码推定转运蛋白的yrkO的表达。在此，β-半乳糖苷酶融合分析证实了这些结果，并且还揭示YrkP可自动调节推定的yrkPQR操纵子，这表明yrkPQR和yrkO形成了一个反向操纵子结构。此外，实时PCR分析显示，在yrkP菌株中，yrkO、yrkN和ykcBC的转录显著降低。因此，YrkP正向调节这些基因的表达。凝胶阻滞分析表明，YrkP与yrkO、yrkN和ykcB的启动子区域结合，尽管与后两个启动子的结合亲和力较低。随后通过DNase I足迹分析对YrkP与yrkPQR和yrkO反向操纵子启动子区域的体外结合进行了分析。结果表明，YrkP识别三个区域，这些区域包含单基序或十碱基序列[T/G]TCA[T/C]AAATT的直接重复序列。对yrkO和yrkPQR反向操纵子缺失和诱变启动子区域的β-半乳糖苷酶融合分析证实，YrkP介导的yrkO和/或yrkPQR激活需要这三个YrkP结合区域。
