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

响应调节因子DegU及其同源组氨酸激酶DegS在革兰氏阳性土壤细菌枯草芽孢杆菌中构成双组分系统。已知未磷酸化和磷酸化形式的DegU可以激活枯草芽孢杆菌的靶基因转录。尽管磷酸化的DegU （DegU- p）调控了超过120个基因，但除comK外，未磷酸化的DegU的靶基因是未知的。我们发现fla/che（鞭毛和趋化性）操纵子受到未磷酸化的DegU的正调控。这种效应在携带功能性swrAA基因的菌株中最为突出，swrAA基因是fla/che的正调节因子。未磷酸化的DegU结合到flalche调控区的两个区域，其中包含一个类似于comK启动子中的倒置重复序列（IR）的倒置重复序列。突变分析显示，SwrAA对fla/che的正向调控需要degu结合。通过足迹分析和突变分析对DegU- p调控基因sacB（左旋蔗糖酶基因）进行分析发现，DegU- p与DegU识别基序的直接重复（DR）结合，该基序在体内已被证明具有功能，而未磷酸化的DegU则没有功能。这些结果强烈表明，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融合中的DR变为IR时，观察到degs不依赖的表达证实了这一假设，表明未磷酸化的DegU通过新形成的IR调节sacB启动子。未磷酸化的DegU与sacB启动子中的IR结合证实了这一点。本研究表明，DegU通过与启动子区结合，正向调节flgB和sacB。我们证明DegU-P更倾向于与DR结合，而不是与sacB启动子中的IR结合。

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.