Role of the Upstream Region Containing an Intrinsic DNA Curvature in the Negative Regulation of the Phospholipase C Gene of Clostridium perfringens

The phospholipase C (a-toxin) gene (pic) of Clostridium perfringens was cloned into pUC19 and the effects of the upstream regions on expression of the pic gene were examined in Escherichia coli JM109. When the 0.7-kb region just upstream of the putative — 35 site of the gene was deleted, production of phospholipase C increased approximately 10-fold. Northern blot hybridization analysis of the pic transcript showed that the upstream region inhibited transcription from the pic promoter. Nucleotide sequencing of this upstream region revealed that there are three periodically repeated (dA)5_6 tracts between positions —66 and —40 of the pic gene. A fragment containing this sequence showed anomalously slow electrophoretic mobility at low temperature, indicating that the region immediately upstream of the pic promoter is a locus of sequence directed DNA-bending. Nested deletions of the upstream region were created from its 5′ end by exonuclease III and the effects of deletions on the expression of the pic gene were examined. When the 77-bp fragment containing the two (dA)5_e tracts was deleted, phospholipase C production increased markedly. These results indicate that the intrinsic DNA curvature upstream of the pic promoter is involved in the negative regulation of the pic gene transcription.