Abstract
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 lower expression of ManLMN in RMV7domi, which promoted competence induction through importing N-acetylglucosamine. This effect was potentiated by orthologues of the gram-negative competence regulatory machinery. Furthermore, a phage-related chromosomal island was more active in RMV7domi, 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 or 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.
Competing Interest Statement
NJC has received an investigator-initiated award from GlaxoSmithKline, consulted for Pfizer, and been invited to speak at an event organised by MSD.
Footnotes
- Phase variation in type 1 pilus expression (Fig. 3): in the original version of the manuscript, we found that the RMV7domi and RMV7rare variants differed in their ability to form a biofilm. We have now added data to the manuscript showing that the knock out of the tvr locus, followed by the insertion of the tvrrare and tvrdomi alleles, restored the divergence in biofilm formation associated with the two distinct methylation patterns. The formation of a thicker biofilm correlated with the higher expression of the rlrA type 1 pilus islet. Knocking out the pilus genes eliminated this difference between the variants; complementing the knock out restored the difference, demonstrating phase variation in pilus expression was responsible for the difference in biofilm formation. - Phase variation in suppression of competence induction by ClpP (Fig. 4): we expanded our screen for differential expression of stress-induced genes associated with PRCI activity to include ClpP. The clpP gene varied in expression both between RMV7wt and a derivative lacking the PRCI, and between the two phase variants RMV7domi and RMV7rare. ClpP is known to repress competence in S. pneumoniae R6. Our experiments confirmed that ClpP also repressed competence in RMV7, and had a bigger effect in RMV7domi, in which it was more highly expressed, likely because of the higher PRCI activity in this variant. - The conflict between quorum sensing and bet hedging (Fig. 5, Fig. 6, Fig. S47): by comparing the frequencies of single and double mutations following transformation with two independently-acquired markers, we were able to infer that the regulatory mechanisms affect the induction of competence, rather than reducing the transformation efficiencies of all pneumococci uniformly. Studies of competence gene transcription in mutant genotypes, confirmed ManLMN and HrcA both changed transformation efficiency through different mechanisms that affected the activity of the competence machinery in an individual cell, without affecting quorum sensing. This has enabled us to outline four ways in which the properties of the signalling pathways we have identified enable cells to respond stochastically even to a uniform quorum sensing signal. Hence transformation is coordinated by intercellular signalling, but its distribution across the population is shaped by bet hedging. This has informed our modifications to the title and Discussion of the paper, which now focus on how multiple signalling mechanisms modulate the induction of competence.