Cloning of a gene involved in regulation of exotoxin A expression in Pseudomonas aeruginosa

Abstract

We have cloned a gene from Pseudomonas aeruginosa that stimulates the expression of exotoxin A. A recombinant library of genomic DNA from strain PA103 constructed with a broad-host-range plasmid vector containing chromosomal insert fragments generated by Sau3A was used to transform the hypotoxigenic mutant strain PA103-29. A recombinant plasmid, pFHK6, was isolated from a PA103-29 transformant which displayed increased toxin production. From pFHK6, which contained a 20-kilobase-pair chromosomal insert, a 3-kilobase-pair XhoI fragment was isolated and subcloned into the plasmid cloning vector pVK101 to give pFHK10. In toxigenic P. aeruginosa strains containing pFHK10, toxin expression was increased 10-fold and high levels of iron in the culture medium only partially inhibited the overproduction. Expression studies suggested that pFHK10 did not contain the toxin structural gene. In addition, Southern analysis with the 3-kilobase-pair XhoI fragment suggested that the putative toxin regulatory gene is common among different strains of P. aeruginosa including previously reported nontoxigenic strains.