The competence system of Streptococcus anginosus and its use for genetic engineering.

Streptococcus anginosus is considered a human commensal but improvements in species identification in recent years have highlighted its role as an emerging pathogen. However, our knowledge about the pathogenicity mechanisms in this species is scarce. One reason for this is the lack of published genetic manipulation techniques in the S. anginosus group. To establish a novel mutation technique we investigated the competence system of S. anginosus and created a Cre-recombinase-based mutation method that allows the generation of markerless gene deletions in S. anginosus. In silico analysis of the competence system demonstrated that S. anginosus encodes homologues for the vast majority of genes that are known to be essential for the transformation of S. pneumoniae. Analysis of transformation kinetics confirmed that S. anginosus SK52 possesses an S. pneumoniae-like competence development with a rapid increase of competence after treatment with Competence Stimulating Peptide (CSP), reaching a maximum transformation efficiency of 0.24% ± 0.08%. The combination of CSP-induced transformation and the Cre-lox system allows the efficient and fast creation of markerless gene deletions and will facilitate the investigation of the pathogenicity of S. anginosus on a genetic level.

Identification of ß-haemolysin-encoding genes in Streptococcus anginosus.

Streptococcus anginosus is an emerging pathogen, but little is known about its virulence factors. To detect the genes responsible for ß-haemolysis we performed genomic mutagenesis of the ß-haemolytic S. anginosus type strain ATCC 12395 using the vector pGhost9:ISS1. 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 ß-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 ß-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 Streptococcus 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 ß-haemolysin production in S. anginosus.