Cloning, and expression in Escherichia coli K-12, of the chromosomal hemolysin (phospholipase C) determinant of Pseudomonas aeruginosa.

Abstract

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.

• Authors: K Coleman, G Dougan, J P Arbuthnott
• Journal: Journal of bacteriology (J Bacteriol) Vol. 153 Issue 2 Pg. 909-15 (Feb 1983) ISSN: 0021-9193 [Print] United States
• PMID: 6401709 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Hemolysin Proteins
• Phospholipases
• Type C Phospholipases

Topics

• Cloning, Molecular
• Escherichia coli (enzymology, genetics)
• Genes, Bacterial
• Hemolysin Proteins (genetics)
• Hemolysis
• Phospholipases (genetics)
• Plasmids
• Pseudomonas aeruginosa (enzymology, genetics)
• Type C Phospholipases (genetics, metabolism)