Gerth U, Krüger E, Derré I, Msadek T, Hecker M
Institut für Mikrobiologie und Molekularbiologie, Ernst-Moritz-Arndt-Universität, Greifswald, Germany.
Mol Microbiol. 1998 May;28(4):787-802. doi: 10.1046/j.1365-2958.1998.00840.x.
The Bacillus subtilis clpP gene, encoding the proteolytic component of the Clp or Ti protease, was cloned and sequenced. The amount of clpP-specific mRNA increased after heat shock, salt and ethanol stress, as well as after treatment with puromycin. Two transcriptional start sites upstream of the clpP structural gene were identified, preceded by sequences resembling the consensus sequences of promoters recognized by sigmaA and sigmaB transcriptional factors of the B. subtilis RNA polymerase respectively. Transcription initiation occurred predominantly at the putative sigmaA-dependent promoter in exponentially growing cells and was induced under stress conditions. After exposure to stress, initiation of transcription also increased at the sigmaB-dependent promoter, but to a lesser extent, indicating that clpP belongs to a double promoter-controlled subgroup of class III general stress genes in B. subtilis. In a sigB mutant strain, clpP remained heat and stress inducible at the sigmaA-dependent promoter. BgaB-reporter gene fusions, carrying either the sigmaA- or the sigmaB-dependent promoter, showed a higher bgaB induction at the sigmaA-dependent promoter, whereas a significantly lower level of induction was measured at the sigmaB-dependent promoter. The sigmaA-dependent promoter appeared to be crucial for the heat-inducible transcription of clpP. A CIRCE (controlling inverted repeat of chaperone expression) element, the characteristic regulation target of class I heat shock genes such as dnaK and groESL, was not found between the transcriptional and translational start sites. Mutants lacking either the proteolytic component ClpP or the regulatory ATPase component ClpX were phenotypically distinct from the wild type. Both mutants produced chains of elongated cells and exhibited severely impaired growth under stress conditions and starvation. Comparison of two-dimensional protein gels from wild-type cells with those from clpP and clpX mutant cells revealed several changes in the protein pattern. Several proteins, such as GroEL, PpiB, PykA, SucD, YhfP, YqkF, YugJ and YvyD, which were found preferentially in higher amounts in both clpP and clpX mutants, might be potential substrates for the ClpXP protease.
克隆并测序了编码Clp或Ti蛋白酶蛋白水解成分的枯草芽孢杆菌clpP基因。热休克、盐和乙醇胁迫以及嘌呤霉素处理后，clpP特异性mRNA的量增加。在clpP结构基因上游鉴定出两个转录起始位点，其前面的序列分别类似于枯草芽孢杆菌RNA聚合酶的sigmaA和sigmaB转录因子识别的启动子共有序列。转录起始主要发生在指数生长细胞中假定的依赖sigmaA的启动子处，并在胁迫条件下被诱导。暴露于胁迫后，依赖sigmaB的启动子处的转录起始也增加，但程度较小，表明clpP属于枯草芽孢杆菌III类一般胁迫基因的双启动子控制亚组。在sigB突变菌株中，clpP在依赖sigmaA的启动子处仍可被热和胁迫诱导。携带依赖sigmaA或依赖sigmaB启动子的BgaB报告基因融合体在依赖sigmaA的启动子处显示出更高的bgaB诱导，而在依赖sigmaB的启动子处测得的诱导水平明显较低。依赖sigmaA的启动子似乎对clpP的热诱导转录至关重要。在转录和翻译起始位点之间未发现CIRCE（伴侣蛋白表达的控制反向重复序列）元件，CIRCE元件是dnaK和groESL等I类热休克基因的特征性调控靶点。缺乏蛋白水解成分ClpP或调节性ATP酶成分ClpX的突变体在表型上与野生型不同。两种突变体均产生细长细胞链，并在胁迫条件和饥饿状态下表现出严重受损的生长。对野生型细胞与clpP和clpX突变体细胞的二维蛋白质凝胶进行比较，发现蛋白质图谱有几个变化。在clpP和clpX突变体中均优先发现几种蛋白质（如GroEL、PpiB、PykA、SucD、YhfP、YqkF、YugJ和YvyD）的含量较高，这些蛋白质可能是ClpXP蛋白酶的潜在底物。
