Diverse regulatory pathways modulate bet hedging of competence induction in epigenetically-differentiated phase variants of Streptococcus pneumoniae

Despite enabling Streptococcus pneumoniae to acquire antibiotic resistance and evade vaccine-induced immunity, transformation occurs at variable rates across pneumococci. Phase variants of isolate RMV7, distinguished by altered methylation patterns driven by the translocating variable restriction-modification (tvr) locus, differed significantly in their transformation efficiencies and biofilm thicknesses. These differences were replicated when the corresponding tvr alleles were introduced into an RMV7 derivative lacking the locus. RNA-seq identified differential expression of the type 1 pilus, causing the variation in biofilm formation, and inhibition of competence induction in the less transformable variant, RMV7domi. This was partly attributable to RMV7domi's lower expression of ManLMN, which promoted competence induction through importing N-acetylglucosamine. This effect was potentiated by analogues of some proteobacterial competence regulatory machinery. Additionally, one of RMV7domi's phage-related chromosomal island was relatively active, which inhibited transformation by increasing expression of the stress response proteins ClpP and HrcA. However, HrcA increased competence induction in the other variant, with its effects depending on Ca2+ supplementation and heat shock. Hence the heterogeneity in transformation efficiency likely reflects the diverse signalling pathways by which it is affected. This regulatory complexity will modulate population-wide responses to synchronising quorum sensing signals to produce co-ordinated yet stochastic bet hedging behaviour.Graphical Abstract