Transcriptional regulation of the autolysin operon in Bacillus subutilis and target sites of the pleiotropic genes

一种多效性突变sin（flaD 1）抑制了自溶素（cwlB）操纵子的转录。为了确定Sin（flaD）是否直接影响cwlB操纵子，我们通过使用肝素-琼脂糖柱层析和HPLC（TSK凝胶G3000 SWXL）纯化Sin（FlaD）蛋白。凝胶迁移实验表明，纯化的蛋白与作为对照的aprE基因结合，而不与cwlB操纵子结合。另一方面，degS（Hy）和degU（Hy）也是多效性基因，其影响降解酶的产生、细胞表达、运动性和感受态。为了研究cwlB操纵子表达的影响，我们测定了cwlB操纵子的转录点和转录量。结果表明，突变完全抑制从Pd启动子的转录，但不是从Pa启动子。由于效果可能取决于SigD蛋白的量，我们构建了具有sigD-lacZ融合的菌株。对于具有sigD-lacZ基因的degU 32（Hy）菌株，没有发现β-半乳糖苷酶活性，因此，cwlB操纵子受sin（flaD）基因调控，而sin（flaD）基因影响sigD表达。DegU-磷酸似乎是sigD表达的调节剂。

A pleiotropic mutation, sin(flaD1), repressed transcrlption of the autolysin (cwlB) operon. To determine whether or not sin(flaD) directly affects the cwlB operon, we purified Sin(FlaD) protein by using heparin-agarose column chromatography and HPLC (TSK gel G3000SWXL). Gel shift experiment indicated that the purified protein was bound with the aprE gene as a control, but not with the cwlB operon. On the other hand, degS(Hy) and degU(Hy) are also pleiotropic genes which affect degradative enzyme production, cell filamentation, motility, and competence. To investigate the effect on the expression of cwlB operon, we detemined the transcriptional points and amounts. The results indicate that the mutations completely depress transcription from the Pd promoter, but not from the Pa promoter. Since the effect may depend on the amount of SigD protein, we constructed strains having the sigD-lacZ fusion. For a degU32(Hy) strain having the sigD-lacZ gene, none of beta-galactosidase activity was found. Therefore, the cwlB operon is regulated by sin(flaD) gene which affects sigD expression. It seems likely that DegU-phosphate is a regulator for the sigD expression.