Molecular cloning and genetic analysis of the determinant for gamma-lysin, a two-component toxin of Staphyloccus aureus

The γ-lysin determinant of Staphylococcus aureus strain Smith 5R has been cloned in phage λ and plasmid vectors in Escherichia coli. Genetic evidence is presented which demonstrates that γ-lysin requires the co-operative action of two polypeptides expressed by the closely linked hlgA and hlgB genes. Recombinant expressed haemolytic activity in agarose medium but not in agar, a known property of γ-lysin. Haemolysis was inhibited by antiserum raised against the 32 kDa component of γ-lysin, but not by anti-α, anti-β, or anti-δ-serum. Subcloning and transposon Tn5 mutagenesis identified a 3.5 kb region which was necessary for γ-lysin expression in E. coli. Two genes (hlgA and hlgB) were mapped and their polypeptide products identified. Non-haemolytic Tn5 mutants fell into two groups based upon complementation tests done between extracts in vitro and also between extracts of mutants and components of γ-lysin purified from S. aureus culture supernates. Immunoblotting showed that some mutants in group A (defective in expression of hlgA) did not express a 32 kDa polypeptide which was synthesized by the parental haemolytic recombinant and by mutants in group B. Minicell analysis suggested that the products of the hlgB gene were proteins of 38 kDa and 36 kDa. The smaller molecule co-migrates with a protein in a fraction of the S. aureus culture supernate containing component B of γ-lysin. The 38 kDa polypeptide is probably an unprocessed precursor. Southern hybridization demonstrated that the hlgA and hlgB genes are closely linked in the chromosome of several strains of S. aureus.