CLONING AND SEQUENCING OF THE STAPHYLOCOCCAL EXFOLIATIVE TOXIN B GENE (STAPHYLOCOCCUS AUREUS)

Staphylococcus aureus exfoliative toxin B (ETB) is a causative agent of the staphylococcal scalded skin syndrome (SSSS). Besides ETB, the phage group II SSSS clinical isolates also produce a bacteriocin (Bac) and are resistant to the bacteriocin (Bac('r)). Curing experiments designed to eliminate the 37.5 kb plasmid pRW001 from one SSSS isolate, UT0007, resulted in the loss of the ETB and Bac phenotypes as well as resistance to Cd(NO(,3))(,2) (Cd('r)). Subsequent transduction and transformation experiments showed that pRW001 carries the structural genes for: (i) ETB synthesis, (ii) Bac synthesis, (iii) Bac('r) and (iv) Cd('r). The structural genes for ETB and Cd('r) have been cloned onto pDH5060 from HindIII fragments of pRW001 and transformed into the phage group III strain S. aureus, RN4220. The RN4220 ETB clone produced extracellular ETB and demonstrated biological activity in neonatal mice. The ETB gene was only expressed in Escherichia coli when cloned into the positive selection-expression vector pSCC31.;The entire DNA sequence of the ETB gene has been determined. The translated ETB sequence is in close agreement with the published amino terminal sequence and total amino acid composition of the protein. In addition, a signal peptide was demonstrated for the ETB molecule. Further analysis of the ETB DNA sequence revealed that the promoter for the toxin gene does not correspond to the consensus regulatory signals found in other bacteria.