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，反应调节因子DEGU和它的同源组氨酸蛋白激酶DEGS构成了一个双组分系统。已知非磷酸化和磷酸化形式的DECU可以激活枯草杆菌中的靶基因转录。虽然磷酸化的DECU-P调控了120多个基因，但除了comK外，非磷酸化的DECU的靶标尚不清楚。我们发现Fla/ChE操纵子(鞭毛和趋化性)操纵子受非磷酸化的Degu正向调节。这种影响在携带功能性swrAA基因的菌株中最为明显，swrAA基因是FLA/CHE的正向调节因子。未磷酸化的DECU与flalche调节区的两个区域结合，包含一个类似于comK启动子中的反向重复(IR)的反向重复序列。突变分析表明，SwrAA对Fla/ChE的正调节需要去谷氨酸结合。通过足迹和突变分析对DECU-P调节基因SACB(左旋糖苷酶基因)的分析表明，DECU-P与DECU识别基序的直接重复(DR)结合，这已被证明在体内具有功能，而未磷酸化的DECU不能。这些结果有力地表明，DECU结合基序的排列决定了未磷酸化的DECU或DECU-P是否与SACB启动子结合。当SACB-LacZ融合中的DR改变为IR时，观察到DGS非依赖性的表达，证实了这一假说，表明未磷酸化的DECU通过新创建的IR调节SACB启动子。通过SACB启动子中未磷酸化的DECU与IR的结合证实了这一点。本研究表明，DEGU通过与启动子区域结合，正向调节flgB和SACB。我们证明了在SACB启动子中，Degu-P更喜欢与DR结合，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.