SUMMARY: The γ-lysin determinant of strain Smith 5R has been cloned in phage Δ and plasmid vectors in . Genetic evidence is presented which demonstrates that γ-lysin requires the co-operative action of two polypeptides expressed by the closely linked and genes. Recombinants 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-γ-lysin senlm. Subcloning and transposon Tn5 mutagenesis identified a 3-5 kb region which was necessary for γ-lysin expression in . Two genes ( and ) were mapped and their polyoeptide products identified. Non-haemolytic Tn5 mutants fell into two groups based upon complementation tests done between extracts of mutants in vitro and also between extracts of mutants and components of -lysin purified from culture supernates. Immunoblotting showed that some mutants in group A (defective in expression of ) 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 gene were proteins of 38 kDa and 36 kDa. The smaller molecule co-migrates with a protein in a fraction of the culture supernate containing component B of -lysin. The 38 kDa polypeptide is probably an unprocessed precursor. Southern hybridization demonstrated that the and genes are closely linked in the chromosome of several strains of .


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