Molecular Microbiology (2001) 39(2), 236±247 Genetic basis for the b-haemolytic/cytolytic activity of group B Streptococcus

Group B streptococci (GBS) express a b-haemolysin/ cytolysin that contributes to disease pathogenesis. We report an independent discovery and extension of a genetic locus encoding the GBS b-haemolysin/cytoly-sin activity. A plasmid libraryofGBS chromosomalDNA was cloned into Escherichia coli, and a transformant was identified as b-haemolytic on blood agar. The purifiedplasmid containeda 4046 bp insert of GBS DNA encoding two complete open reading frames (ORFs). A partial upstream ORF (cylB) and the first complete ORF (cylE) represent the 3 0 end of a newly reported genetic locus (cyl) required for GBS haemolysin/cytolysin activity. ORF cylE is predicted to encode a 78.3 kDa protein without GenBank homologies. The GBS DNA fragment also includes a previously unreported ORF, cylF, with homology to bacterial aminomethyltrans-ferases, and the 5 0 end of cylH, with homology to 3-ketoacyl-ACP synthases. Southern analysis demon-strated that the cyl locus was conserved among GBS of all common serotypes. Targeted plasmid integrational mutagenesis was used to disrupt cylB, cylE, cylF and cylH in three wild-type GBS strains representing serotypes Ia, III and V. Targeted integrations in cylB, cylF and cylH retaining wild-type haemolytic activity were identified in all strains. In contrast, targeted integrations in cylE were invariably non-haemolytic and non-cytolytic, a finding confirmed by in frame allelic exchange of the cylE gene. The haemolytic/cytolytic activity of the cylE allelic exchange mutants could be restored by reintroduction of cylE on a plasmid vector. Inducible expression of cylE, cylF and cylEF demon-strated that it is CylE that confers haemolytic activity in E. coli. We conclude that cylE probably represents the structural gene for the GBS haemolysin/cytolysin, a novel bacterial toxin