Identification of structural determinants involved in ComR activation and XIP specificity in salivarius streptococci

Natural competence is the genetically specified ability of some bacteria to take up extracellular DNA in order to provide an evolutionary advantage or as a means of genome plasticity/repair. It is a social behavior extensively studied in the Streptococcus genus, in which this transient and energy-intensive physiological process is linked, inter alia, to biofilm formation and bacteriocin production. In most species of this genus, competence development is tightly regulated by the pheromone quorum-sensing system ComRS, which activates the expression of the central regulator of competence, the sigma factor X (also known as σX/ComX/SigX), responsible for the expression of late competence genes, including the transformasome and the recombination machinery. ComS is the precursor of the competence pheromone peptide. After secretion and maturation, the matured ComS form (named XIP) is reinternalized and directly binds the transcriptional regulator ComR inducing its activation and enabling it in turn to stimulate the expression of comS and comX genes, marking the onset of competence development. The transcriptional regulator ComR belongs to the RRNPP superfamily of cytoplasmic sensors. It is characterized by an N-terminal helix-turn-helix (HTH) DNA-binding domain and a C-terminal alpha-helical domain containing 5 tetratricopeptide repeats (TPR) which interacts with the XIP pheromone required for its activation. Depending on the species, ComR proteins display strict or more permissive specificity accordinfg to their ability to be activated by heterologous peptides. Thus, some ComRs are able to recognize peptides of various sequences, while others can only be activated by their cognate peptides. The aim of this master's thesis project is to identify structural determinants involved in ComR activation and XIP specificity in two closely-related species of the salivarius group of streptococci, i.e. Streptococcus thermophilus (Sth), a model organism for the study of the ComRS system, and a species of medical interest, Streptococcus vestibularis (Sve), a commensal species that has been shown to adopt a pathogenic behavior opportunistically. In this project, we report that ComRSve is fully functional in S. thermophilus, but that the two highly similar ComRS systems do not cross-talk. We also report concurrently that S. vestibularis F0396 is competent under laboratory conditions and that its competence system is functional when XIPSve is provided in the culture medium. Finally, we generated ComRSth mutants (ComR*) with altered XIP recognition capacity, including some capable of moderate cross-activation with XIPSve. These results highlighted the importance of different regions of the ComR TPR domain involved in XIP specificity and its binding-induced activation mechanism, notably loop L6 and alpha-helices α7 and α12 of TPR-1 and TPR-4, respectively. Those results pave the way towards a detailed molecular understanding of ComR-XIP interactions responsible for ComR activation and XIP selectivity.