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

一个多效性突变，sin(flaD1)，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，我们采用肝素-琼脂糖柱层析和高效液相色谱（TSK凝胶G3000SWXL）纯化了sin（flaD）蛋白。凝胶移位实验表明，纯化的蛋白与aprE基因结合作为对照，而不与cwlB操纵子结合。另一方面，degS（Hy）和degU（Hy）也是影响降解酶产生、细胞成丝、运动和能力的多效性基因。为了研究其对cwlB操纵子表达的影响，我们测定了其转录点和转录量。结果表明，突变完全抑制Pd启动子的转录，但不抑制Pa启动子的转录。由于SigD蛋白的数量可能决定了其作用，因此我们构建了SigD - lacz融合菌株。对于具有sigD-lacZ基因的degU32（Hy）菌株，未发现β -半乳糖苷酶活性。因此，cwlB操纵子受影响sigD表达的sin（flaD）基因调控。DegU-phosphate可能是sigD表达的调节因子。

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.