Cloning, and expression in Escherichia coli K-12, of the chromosomal hemolysin (phospholipase C) determinant of Pseudomonas aeruginosa.
A hemolysin determinant was cloned from Pseudomonas aeruginosa PA103 by inserting Sau3a-generated DNA fragments between the BamHI sites of the lambda replacement vector WL47.1. A 9.5-kilobase HindIII fragment encoding the hemolysin was subcloned from this phage and inserted into the plasmid vector pHC79 to generate the recombinant plasmid pKC95. Escherichia coli K-12 strains harboring pKC95 exhibited zones of hemolysis after several days of growth on blood agar plates. Hemolysis was shown to be due to phospholipase C activity by using the chromogenic substrate p-nitrophenylphosphorylcholine. Deletion mutants of pKC95 were isolated, and polypeptides expressed from these plasmids were examined by using the E. coli minicell system. A polypeptide of 78,000 daltons was associated with phospholipase C activity. The hemolytic activity was cell associated when expressed in E. coli.
D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005798:Genes, Bacterial; D006460:Hemolysin Proteins / Q000235:genetics; D006461:Hemolysis; D010740:Phospholipases / Q000235:genetics*; D010957:Plasmids; D011550:Pseudomonas aeruginosa / Q000201:enzymology / Q000235:genetics*; D010738:Type C Phospholipases / Q000235:genetics* / Q000378:metabolism
D006460:Hemolysin Proteins; D010740:Phospholipases; D010738:Type C Phospholipases