Genetic locus for streptolysin S production by group A streptococcus

Group A streptococcus (CAS) is an important human pathogen that causes phar yngitis and invasive infections, including necrotizing fasciitis, Streptoly sin S (SLS) is the cytolytic factor that creates the zone of beta-hemolysis surrounding GAS colonies grown on blood agar, We recently reported the dis covery of a potential genetic determinant involved in SLS production, sagA, encoding a small peptide of 53 amino acids (S, D. Betschel, S, M, Borgia, N, L, Barg, D, E. Low, and J. C. De Azavedo, Infect. Immun, 66:1671-1679, 1 998), Using transposon mutagenesis, chromosomal walking steps, and data fro m the GAS genome sequencing project (www.genome.ou.edu/strep.html), we have now identified a contiguous nine-gene locus (sagA to sagI) involved in SLS production. The sag locus Is conserved among GAS strains regardless of M p rotein type, Targeted plasmid integrational mutagenesis of each gene in the sag operon resulted in an SLS-negative phenotype, Targeted integrations (i ) upstream of the sagA promoter and (ii) downstream of a terminator sequenc e after sagI did not affect SLS production, establishing the functional bou ndaries of the operon, A she-independent terminator sequence between sagA a nd sagB appears to regulate the amount of sagA transcript produced versus t ranscript for the entire operon, Reintroduction of the nine-gene sag locus on a plasmid vector restored SLS activity to the nonhemolytic sagA knockout mutant, Finally, heterologous expression of the intact sag operon conferre d the SLS beta-hemolytic phenotype to the nonhemolytic Lactococcus lactis, We conclude that gene products of the GAS sag operon are both necessary and sufficient for SLS production. Sequence homologies of sag operon gene prod ucts suggest that SLS is related to the bacteriocin family of microbial tox ins.