Expression of the Pseudomonas aeruginosa toxA positive regulatory gene (regA) in Escherichia coli

Abstract

The regA gene is a positive regulatory gene that regulates toxin A production in Pseudomonas aeruginosa at the transcriptional level. The product of the regA gene was examined in Escherichia coli with the expression vector pT7-7. A 1.3-kilobase AvaI-HindIII fragment containing the regA gene was cloned into the pT7-7 vector. A recombinant plasmid (pAH1) encoded a 29-kilodalton protein. The molecular weight of this protein correlated closely with the predicted molecular weight of the RegA protein. Production of the RegA protein in E. coli required both an E. coli promoter and an E. coli ribosome-binding site. Two in-frame deletion derivatives in which certain regions of the regA gene were expressed from the T7 promoter encoded 26- and 18-kilodalton fusion proteins, respectively. The RegA protein and the two fusion proteins were localized to the inner membrane of E. coli. Neither RegA protein nor the two fusion proteins showed DNA-binding activity to the 410-base-pair fragment containing the upstream region of toxA when synthesized in E. coli.