Variation in transformation frequency and competence gene expression among serotype 3 Streptococcus pneumoniae

Streptococcus pneumoniae is a human commensal and the causative agent of pneumococcal disease. Pneumococci are naturally competent – able to uptake exogenous DNA from the environment and incorporate it into their genome through homologous and non-homologous recombination. Recombination has significantly shaped the evolutionary history of S. pneumoniae, as it allows pneumococci to rapidly adapt to interventions such as antibiotic therapy or vaccine introduction. Recombination frequencies vary considerably across pneumococcal populations; yet the underlying mechanisms for these variations are not well understood. Entry and exit into competence, a state in which the cell can uptake DNA, is tightly regulated through transcriptional changes. We observed consistent differences in transformation frequencies among groups, which correlated with variation in differentially expressed genes during competence. While all strains exhibited a similar response to competence stimulating peptide (CSP) for early competence genes, we observed variation in expression of late competence genes, which encode the DNA uptake apparatus, DNA repair and recombination proteins needed for recombination. We also observed differences in expression of genes linked to bacteriocin production, which may partially explain observed population-level differences. Further genomic analysis suggests variation in promoter sequences governing late competence genes may be slowing transition from early to late components of the competence pathway. To elucidate differences in transformation frequency among strains as well as the underlying genetic mechanisms, we carried out in-vitro competence assays and measured gene expression changes during the competent state using RNA sequencing of strains belonging to Serotype 3 clonal complex (CC) 180 and a non-CC180 comparison. Cultures were treated with 200 ng/ml of competence stimulating peptide (CSP). Two volumes of RNAprotect bacteria reagent (Qiagen) were added to the cultures at 10, 15, 20 mins after CSP addition, vortexed and incubated at room temperature for 5 mins. These timepoints were selected as it has been shown that peak competence is achieved after 15 - 20 mins.