Cloning and expression in Escherichia coli and Staphylococcus aureus of the beta-lysin determinant from Staphylococcus aureus: evidence that bacteriophage conversion of beta-lysin activity is caused by insertional inactivation of the beta-lysin determinant

Abstract

The beta-lysin determinant (Hlb) from Staphylococcus aureus CN6708 was cloned in Escherichia coli K-12 using the bacteriophage replacement vector λL47.1. The Hlb determinant was localised to a 1250 base pair DNA sequence by cloning fragments from a Hlb + recombinant phage into the plasmid vectors pACYC184 and pBR322 in E. coli K-12, and by the subsequent construction and analysis of several sub-clones, in vitro deletion and Tn5 insertion mutations. E. coli cells harbouring Hlb + plasmids expressed readily detectable levels of β-lysin and sphingomyelinase activity, which were located in the cytoplasm. Two polypeptides of molecular weight 38000 and 33000 which were encoded by the Hlb determinant were detected in E. coli minicells, but only the 33000 dalton protein was detected in immunoblotting experiments with specific anti-β-lysin serum. Hybridisation analysis with probes made from the cloned Hlb determinant and from DNA of the staphylokinase-converting phage φ13, indicated that bacteriophage conversion of S. aureus to loss of β-lysin activity is due to insertion of φ13 DNA into or adjacent to the β-lysin determinant. A shuttle plasmid was used to transfer the cloned Hlb determinant into a β-lysin negative strain of S. aureus where the wild-type chromosomal determinant was inactivated by lysogenic conversion. Beta-lysin activity was readily detected in supernatants of S. aureus harbouring the cloned determinant.