权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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.

反应调节剂 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了一个双组分系统。已知 DegU 的非磷酸化和磷酸化形式可激活枯草芽孢杆菌中的靶基因转录。尽管磷酸化 DegU (DegU-P) 调节超过 120 个基因，但除 comK 之外，未磷酸化 DegU 的靶标尚不清楚。我们发现fla/che（鞭毛和趋化性）操纵子受到未磷酸化的DegU的正向调节。这种效应在带有功能性 swrAA 基因（fla/che 的正调节因子）的菌株中最为显着。未磷酸化的 DegU 与 flalche 调节区中的两个区域结合，其中包含类似于 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 SwrAA 对 fla/che 的正向调节需要 DegU 结合。通过足迹和突变分析对 DegU-P 调节的基因 sacB（左聚蔗糖酶基因）进行分析表明，DegU-P 与 DegU 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，而未磷酸化的 DegU 则不然。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 还是 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。这通过未磷酸化的 DegU 与 sacB 启动子中的 IR 的结合得到证实。这项研究表明 DegU 通过与启动子区域的结合对 flgB 和 sacB 进行正向调节。我们证明 DegU-P 更喜欢与 sacB 启动子中的 DR 结合，而不是与 IR 结合。