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

B. subtilis re、响应调节子 DegU 与其靶基因启动子之间的相互作用分析

基本信息

批准号：
18580082
负责人：
OGURA Mitsuo
金额：
$ 2.57万
依托单位：
Tokai University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18580082/
关键词：
B. subtilis Two-component system Chemotaxis DNA binding Phosnhorvlation Gene expression 転写因子リン酸化

项目摘要

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.

响应调节剂DEGU及其同源性组氨酸激酶DEGS构成了革兰氏阳性的土壤细菌枯草菌中的两个组成系统。已知未磷酸化和磷酸化形式可以激活枯草芽孢杆菌中的靶基因转录。尽管磷酸化的DEGU（DEGU-P）调节了一百二十多个基因，但未磷酸化的degu的靶标是未知的。我们发现FLA/CHE（鞭毛和趋化性）操纵子由未磷酸化的DEG阳性调节。该作用在带有功能性SWRAA基因的菌株中最突出，该菌株是Fla/che.unphosphorypated的degu的阳性调节剂，结合了flalche调节区域的两个区域，其中包含倒置的重复样序，类似于Comk启动子中倒置的重复（IR）。突变分析表明，SWRAA对FLA/CHE的阳性调节需要DEGU结合。通过足迹和突变分析对DEGU-P调节的基因SACB（左旋糖基因）的分析表明，DEGU-P与Degu-Replas识别基序的直接重复（DR）结合，该基序已被证明在体内具有功能性，而未磷酸化的DEGU则没有。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更改为IR时，通过观察到DEG独立的表达来证实该假设，这表明未磷酸化的DEGU通过新创建的IR调节SACB启动子。通过在SACB启动子中未磷酸化的DEGU与IR的结合证实了这一点。这项研究表明，DEGU通过与启动子区域的结合对FLGB和SACB进行了积极调节。我们证明了DEGU-P更喜欢与DR结合，但在SACB启动子中不与IR结合。