Identification of β‐haemolysin‐encoding genes in S treptococcus anginosus

S treptococcus anginosus is an emerging pathogen, but little is known about its virulence factors. To detect the genes responsible for β‐haemolysis we performed genomic mutagenesis of the β‐haemolytic S . anginosus type strain ATCC 12395 using the vector p G host9:IS S1 . Integration site analysis of 15 non‐haemolytic mutants identified a gene cluster with high homology to the genes of the streptolysin S ( SLS ) encoding sag gene cluster of S . pyogenes . The gene cluster harbours 10 open reading frames displaying significant similarities to the S . pyogenes genes sagA‐sagI , with the identities on protein level ranging from 38 to 87%. Complementation assays of S . anginosus sagB and sagD integration mutants with the respective genes confirmed their importance for β‐haemolysin production and suggest the presence of post‐translational modifications in S . anginosus SLS similar to SLS of S . pyogenes . Characterization of the S . anginosus haemolysin in comparison to the S . pyogenes SLS showed that the haemolysin is surface bound, but in contrast to S . pyogenes neither fetal calf serum nor RNA was able to stabilize the haemolysin of S . anginosus in culture supernatants. Inhibition of β‐haemolysis by polyethylene glycol of different sizes was carried out, giving no evidence of a pore‐forming haemolytic mechanism. Analysis of a whole genome shotgun sequence of S treptococcus constellatus , a closely related streptococcal species that belongs to the S . anginosus group, revealed a similar sag gene cluster. Employing a genomic mutagenesis strategy we were able to determine an SLS encoding gene cluster in S . anginosus and demonstrate its importance for β‐haemolysin production in S . anginosus .