Cloning, nucleotide sequence, and expression in Escherichia coli of the exotoxin A structural gene of Pseudomonas aeruginosa

Abstract

A 2760-base pair DNA segment of the Pseudomonas aeruginosa strain PA103 chromosome encoding the exotoxin A (ETA) structural gene has been cloned in Escherichia coli and the nucleotide sequence has been determined. Analysis of the 5'- and 3'-flanking regions indicate that ETA is translated from a monocistronic message. Comparison of the deduced NH2-terminal amino acid sequence with that determined by sequence analysis of the secreted protein indicates that ETA is made as a 638 amino acid precursor from which a highly hydrophobic leader peptide of 25 amino acids is removed during the secretion process. Data are presented that indicate a COOH-terminal location of the ADP-ribosylation activity of the molecule. Expression of the ETA coding sequence in E. coli under control of the E. coli trp promoter, but not the ETA promoter, results in the production of enzymatically and immunologically active protein. However, most of this material appears to be neither processed nor secreted. Comparison of the ETA amino acid and nucleotide sequences to those of diphtheria toxin revealed no significant homologies, indicating that these functionally similar toxins evolved from different genes.