A fragment of Staphylococcus aureus DNA encoding the α-hemolysin determinant was cloned from strain Wood 46 by inserting Sau3A-generated genomic DNA fragments between the BamHI sites of the λ replacement vector L47.1. Phages expressing α-hemolysin were detected by overlaying plaques formed from several thousand independent recombinant phage with erythrocytes and looking for zones of hemolysis. One phage expressing α-hemolysin was purified and named λwα3. This was subsequently shown to contain a 10.2-kilobase pair insert of S. aureus DNA. A 7.6-kilobase pair HindIII fragment encoding the α-hemolysin was subcloned from λwα3 into the plasmid vector pACYC184 to form the hybrid plasmid pDU1148. Escherichia coli K-12 cells harboring pDU1148 synthesized a low level of α-hemolysin which remained associated with the cells and was not secreted into culture supernatants. When the same strain was stabbed onto blood agar plates, no zones of hemolysis were detected after overnight growth at 37°C but hemolysis developed if the plates were left at room temperature for 48 h. By introducing specific deletions or Tn5 insertions into plasmid pDI1148, the α-hemolysin gene was mapped to a region within a 3.3-kilobase pair EcoRI-HindIII fragment which was subcloned onto the vector plasmid pBR322. A specific enzyme-linked immunosorbent assay with peroxidase-labeled rabbit anti-α-hemolysin antibodies was used to measure the levels of α-hemolysin antigen expressed in E. coli K-12 cells harboring pDU1148 or a variety of pDU1148::Tn5 and pDU1148 deletion mutants.
|Number of pages||7|
|Journal||Infection and Immunity|
|Publication status||Published - 27 Oct 1983|