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 (α‐toxin) gene (plc) of Clostridium perfringenswas cloned into pUC19 and the effects of the upstream regions on expression of the plcgene were examined in Escherichia coliJM109. 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 plctranscript showed that the upstream region inhibited transcription from the plcpromoter. Nucleotide sequencing of this upstream region revealed that there are three periodically repeated (dA)5–6tracts between positions −66 and −40 of the plcgene. A fragment containing this sequence showed anomalously slow electrophoretic mobility at low temperature, indicating that the region immediately upstream of the plcpromoter 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 plcgene were examined. When the 77‐bp fragment containing the two (dA)5–6tracts was deleted, phospholipase Cproduction increased markedly. These results indicate that the intrinsic DNA curvature upstream of the plcpromoter is involved in the negative regulation of the plcgene transcription.