The cytotoxin of Pseudomonas aeruginosa: Cytotoxicity requires proteolytic activation

The primary structure of a cytotoxin from Pseudomonas aeruginosa was determined by sequencing of the structural gene. The cytotoxin (31,700 Mr) lacks an N-terminal signal sequence for bacterial secretion but contains a pentapeptide consensus sequence commonly found in prokaryotic proteins which function in a TonB-dependent manner. The cytotoxin gene has a [G+C]-content of 53.8% which is considerably lower than generally observed for genes from Pseudomonas aeruginosa. The cytotoxin gene was exclusively detected in strain 158 but not in three other clinical isolates, as determined by Southern and Northern hybridization. The latter technique revealed that the toxin is translated from monocistronic mRNA. The promoter of the cytotoxin is inactive in Escherichia coli. Upon site-directed modification of the 5′-noncoding region by the polymerase chain reaction the gene was expressed under control of the trcpromoter. The gene product obtained in Escherichia coli was nontoxic. Toxicity was induced by subsequent treatment with trypsin. [(35)S]methionine-labeled cytotoxin with high specific radioactivity was obtained by in vitro transcription/translation. Like [(125)I] labeled material from Pseudomonas aeruginosa this polypeptide bound to membrane preparations from Ehrlich ascites cells, as evidenced by sedimentation through a sucrose gradient at neutral pH.