Analysis of interaction between B. subtilis re, sponse regulaator DegU and its target gene promoters

在革兰氏阳性土壤细菌枯草芽孢杆菌中，反应调节剂DegU及其同源组氨酸激酶DegS构成双组分系统。已知DegU的非磷酸化和磷酸化形式激活B中的靶基因转录。枯草杆菌。尽管磷酸化DegU（DegU-P）调节超过120个基因，但除了comK之外，未磷酸化DegU的靶点是未知的。我们发现，弗拉/che（鞭毛和趋化性）操纵子正调控的非磷酸化DegU。这种效应在带有功能性swrAA基因的菌株中最为突出，swrAA基因是弗拉/che的正调控因子。未磷酸化的DegU结合到flalche调控区的两个区域，该区域含有类似于comK启动子中的反向重复序列（IR）的反向重复序列。突变分析表明，弗拉/che的正调控SwrAA需要DegU结合。通过足迹和突变分析对DegU-P调节的基因sacB（果聚糖蔗糖酶基因）进行的分析显示，DegU-P结合到DegU识别基序的直接重复（DR），这已被证明在体内是功能性的，而未磷酸化的DegU没有。这些结果有力地表明，DegU结合基序的排列决定了未磷酸化的DegU或DegU-P是否与sacB启动子结合。当sacB-lacZ融合体中的DR变为IR时，通过观察degS独立表达证实了这一假设，表明未磷酸化的DegU通过新创建的IR调节sacB启动子。这项研究表明，DegU正调控flgB和sacB通过其结合的启动子区域。我们证明DegU-P在sacB启动子中更倾向于结合DR而不是IR。

The response regulator DegU and its cognate histidine kinase DegS constitute a two-component system in the Gram-positive soil bacterium Bacillus subtilis. Unphosphorylated and phosphorylated forms of DegU are known to activate target gene transcription in B. subtilis. Although phosphorylated DegU (DegU-P) regulates more than one hundred and twenty genes, the targets of unphosphorylated DegU are unknown, except for comK. We found that the fla/che (flagella and chemotaxis) operon is positively regulated by unphosphorylated DegU. The effect was most prominent in a strain bearing the functional swrAA gene, a positive regulator of fla/che.Unphosphorylated DegU bound to two regions in the flalche regulatory region containing an inverted repeat-like sequence that resembles the inverted repeat (IR) in the comK promoter. Mutational analysis revealed that positive regulation of fla/che by SwrAA requires DegU-binding. An analysis of the DegU-P-regulated gene sacB (levansucrase gene) by footprint and mutational analyses revealed that DegU-P bound to a direct repeat (DR) of the DegU-recognition motifs, which has been shown to be functional in vivo, while unphosphorylated DegU did not. These results strongly suggest that the arrangement of the DegU-binding motifs determines whether unphosphorylated DegU or DegU-P binds to the sacB promoter. The hypothesis was confirmed by observing degS-independent expression when the DR in the sacB-lacZ fusion was changed to an IR, suggesting that unphosphorylated DegU regulates the sacB promoter through the newly created IR. This was confirmed by binding of unphosphorylated DegU to the IR in the sacB promoter. This study demonstrated that DegU positively regulates flgB and sacB through its binding to the promoter regions. We demonstrated that DegU-P prefers binding to DR but not to IR in the sacB promoter.