Mutation of the promoter and LexA binding sites of cea, the gene encoding colicin E1.

Abstract

Three mutations were introduced into the cea promoter using oligonucleotide directed mutagenesis. The resulting mutant promoter has the Escherichia coli consensus sequences at its - 35 and - 10 positions, separated by the optimal spacing. In addition, a plasmid with a mutation in one of the two LexA repressor binding sites in the cea regulatory region was isolated that decreases homology with the consensus LexA binding site. The effects of these mutations on cea expression were studied in cea-lacZ protein fusions. The promoter-up mutant, when present in a multicopy plasmid, showed a shorter induction lag when compared to the wild-type cea gene, and there was less of an effect of the catabolite repression system on cea expression. However, when present in a single copy in the bacterial chromosome, catabolite repression and an induction delay were observed, despite the increased strength of the promoter. The operator mutant showed a slightly higher basal level of expression, but was still repressible. Induction occurred with a shortened lag period, but the effects were not as great as with the promoter mutant. These results support the idea that tight repression by LexA contributes to the delay in cea induction.