Autoregulation of the Bacillus subtilis response regulator gene degU is coupled with the proteolysis of DegU-P by ClpCP
Autoregulation of the Bacillus subtilis response regulator gene degU is coupled with the proteolysis of DegU-P by ClpCP
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DOI：
10.1111/j.1365-2958.2010.07047.x
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发表时间：
2010-03-01
影响因子：
3.6
通讯作者：
Tsukahara, Kensuke
中科院分区：
文献类型：
--
作者：
Ogura, Mitsuo;Tsukahara, Kensuke
反应调节因子DegU及其同源激酶DegS构成枯草芽孢杆菌中的双组分系统，其调节许多细胞过程，包括外切蛋白酶产生和感受态发育。利用DNA足迹分析、凝胶迁移分析和P3 degU-lacZ融合突变分析，我们发现磷酸化的DegU（DegU-P）与P3 degU启动子中共有的DegU结合序列的两个直接重复序列（DR 1和DR 2）结合。染色体DR 2的改变严重降低了degU的表达，表明其在degU的正性自身调节中的重要性。DegU蛋白水平的观察表明DegU被降解。在编码各种ATP依赖性蛋白酶的基因的破坏突变体中DegU的蛋白质印迹分析强烈表明ClpCP降解DegU。此外，当从头蛋白质合成被阻断时，DegU在野生型中迅速降解，但在clpC和clpP菌株中不降解，并且具有突变的磷酸化位点的DegU非常稳定。这些结果表明ClpCP优先降解DegU-P，但不降解未磷酸化的DegU。我们证实，DegU-P的降解优先使用在体外ClpCP降解系统。此外，突变分析表明，N-末端区域的DegU是重要的蛋白水解。
P>The response regulator DegU and its cognate kinase DegS constitute a two-component system in Bacillus subtilis that regulates many cellular processes, including exoprotease production and competence development. Using DNA footprint assay, gel shift assay and mutational analyses of P3degU-lacZ fusions, we showed that phosphorylated DegU (DegU-P) binds to two direct repeats (DR1 and DR2) of the consensus DegU-binding sequence in the P3degU promoter. The alteration of chromosomal DR2 severely decreased degU expression, demonstrating its importance in positive autoregulation of degU. Observation of DegU protein levels suggested that DegU is degraded. Western blot analysis of DegU in disruption mutants of genes encoding various ATP-dependent proteases strongly suggested that ClpCP degrades DegU. Moreover, when de novo protein synthesis was blocked, DegU was rapidly degraded in the wild-type but not in the clpC and clpP strains, and DegU with a mutated phosphorylation site was much stable. These results suggested preferential degradation of DegU-P by ClpCP, but not of unphosphorylated DegU. We confirmed that DegU-P was degraded preferentially using an in vitro ClpCP degradation system. Furthermore, a mutational analysis showed that the N-terminal region of DegU is important for proteolysis.