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

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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.