title: Expression and secretion of the cloned Pseudomonas aeruginosa exotoxin A by Escherichia coli.
abstract: The exotoxin A gene from Pseudomonas aeruginosa PAK was expressed in Escherichia coli from recombinant plasmids when transcription was initiated from a promoter in the cloning vector. The exotoxin A polypeptide synthesized was found to have an electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels of 66,000 daltons, identical in size to the mature exotoxin A made by P. aeruginosa. Analysis of the location of exotoxin A in various bacterial compartments by immunoblotting revealed that exotoxin A was exported by E. coli into its periplasmic space. Several functional assays, including analyses of disulfide bond formation, potentiation of ADP-ribosyltransferase activity, and HeLa cell cytotoxicity, were used to establish that the conformation of exotoxin A isolated from the E. coli periplasmic space is identical to that of exotoxin exported by P. aeruginosa to its extracellular space. Previous studies with recombinant plasmids expressing exotoxin A from P. aeruginosa PA103 (G. D. Gray, D. Smith, J. Baldridge, R. Markins, M. Vasil, E. Chen, and M. Heyneker, Proc. Natl. Acad. Sci. USA 81:2645-2649, 1984) showed a complete lack of processing and export of pre-exotoxin A in E. coli, differing from results reported here. These discrepancies may be explained by observed differences in the sequence of signal peptides encoded by the exotoxin A genes of PAK and PA103 strains of P. aeruginosa.
mesh_terms: D036002:ADP Ribose Transferases; D000595:Amino Acid Sequence; D001427:Bacterial Toxins; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005098:Exotoxins / Q000096:biosynthesis / Q000235:genetics*; D005786:Gene Expression Regulation; D005796:Genes; D005798:Genes, Bacterial; D007118:Immunoassay; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D021382:Protein Sorting Signals; D011550:Pseudomonas aeruginosa / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
chemical_list: D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D005098:Exotoxins; D021382:Protein Sorting Signals; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A