Orientation and expression of the cloned hemolysin gene of Pseudomonas aeruginosa.

The structural gene for Pseudomonas aeruginosa hemolysin, carried on recombinant plasmid pSL2 and cloned in Escherichia coli, was analyzed by insertional and deletional mutagenesis. Expression of the hemolysin was blocked by insertion of transposon Tn5 into different locations. Two of the mutants allowed detectable synthesis of truncated hemolysin polypeptides of two different sizes and thus defined the structural gene. The location of the hemolysin gene in the recombinant plasmid, and the direction of transcription, were further established by nuclease BAL 31 digestion, and by construction of gene fusions between hemolysin and beta-galactosidase. Evidently, the tet promoter contributed to the majority of the expression of cloned hemolysin gene, but the Pseudomonas promoter was present in the cloned DNA and was functional in E. coli since inactivation of the tet promoter either by Tn5 insertion or by deletion decreased synthesis of the 80-kDal hemolysin but did not fully abolish it.