Genetic basis for the β-haemolytic/cytolytic activity of group B Streptococcus

Group B streptococci (GBS) express a β-haemolysin/cytolysin that contributes to disease pathogenesis. We report an independent discovery and extension of a genetic locus encoding the GBS β-haemolysin/cytolysin activity. A plasmid library of GBS chromosomal DNA was cloned into Escherichia coli , and a transformant was identified as β-haemolytic on blood agar. The purified plasmid contained a 4046 bp insert of GBS DNA encoding two complete open reading frames (ORFs). A partial upstream ORF ( cyl B) and the first complete ORF ( cyl E) represent the 3' end of a newly reported genetic locus ( cyl ) required for GBS haemolysin/cytolysin activity . ORF cyl E is predicted to encode a 78.3 kDa protein without GenBank homologies. The GBS DNA fragment also includes a previously unreported ORF, cyl F, with homology to bacterial aminomethyltransferases, and the 5' end of cyl H, with homology to 3-ketoacyl-ACP synthases. Southern analysis demonstrated that the cyl locus was conserved among GBS of all common serotypes. Targeted plasmid integrational mutagenesis was used to disrupt cyl B, cyl E, cyl F and cyl H in three wild-type GBS strains representing serotypes Ia, III and V. Targeted integrations in cyl B, cyl F and cyl H retaining wild-type haemolytic activity were identified in all strains. In contrast, targeted integrations in cyl E were invariably non-haemolytic and non-cytolytic, a finding confirmed by in frame allelic exchange of the cyl E gene. The haemolytic/cytolytic activity of the cyl E allelic exchange mutants could be restored by reintroduction of cyl E on a plasmid vector. Inducible expression of cyl E, cyl F and cyl EF demonstrated that it is CylE that confers haemolytic activity in E. coli . We conclude that cyl E probably represents the structural gene for the GBS haemolysin/cytolysin, a novel bacterial toxin.