Caractérisation d’un facteur de transcription hétérodimérique artificiel

Details

Supervisors

Soumillion, Patrice

Faculty

Faculté des sciences

Degree label

Master [120] en biochimie et biologie moléculaire et cellulaire, à finalité approfondie

Abstract

enNatural transformation is a biological mechanism that allows the uptake of exogenous DNA and its integration into the bacterial genome. This energetically costly process is finely regulated by the ComRS system in streptococci of the salivarius group, including S. thermophilus. The effector of this system is the ComR protein. Once bound to its activating peptide XIP, the mature form of ComS, this regulator forms homodimers that act as transcription factors. This complex activates the expression of competence genes, bacteriocins, and comS, thereby triggering a positive feedback loop. The wild-type homodimer is stabilized by two pairs of salt bridges at the dimerization interface: K87-D200 and K246-E282. Through protein engineering, two variants, ComR1 and ComR2, were designed to disrupt this symmetry. The ComR1 and ComR2 variants, carrying the D200K-K246E and K87D-E282K substitutions respectively, prevent homodimerization through charge repulsion. In addition, ComR2 derives from a variant called ComR “Sve-like”, which bears five additional mutations (R92G, V205A, S248G, S289K, and I290T) allowing it to specifically recognize the XIP of S. vestibularis instead of the endogenous XIPSth peptide. This asymmetric system enables precise control of activation, which is only possible in the presence of the two distinct peptides. The objective of this thesis is to study in vitro the ability of the different ComR/XIP combinations to form a DNA-binding complex, in order to confirm and complement the functional observations obtained in vivo. First, the WT and “Sve-like” ComR proteins were studied using electrophoretic mobility shift assays to observe the formation of the nucleoprotein complex, followed by the study of the ComR1 and ComR2 variants. Many efforts have been devoted to optimizing protein purification protocols in order to improve the likelihood of obtaining results. Our findings indicate that the ComR mutants show limited stability in vitro, preventing us from demonstrating the formation of a stable nucleoprotein complex composed of the five different partners (ComR1/XIPSth/ComR2/XIPSve/DNA) under the conditions tested. Nevertheless, this project allowed us to improve the efficiency of our ComR purification methods. A second aspect of the project concerns the development of a synthetic bacterial community, or SynCom, involving three S. thermophilus strains whose interdependence relies on the ComR1-ComR2/XIPSth-XIPSve system. When grown in a medium poor in free amino acids but enriched with casein, their survival depends on the establishment of cooperation. A prtS+ strain expresses the PrtS protease, which is able to hydrolyze casein into oligopeptides. The expression of prtS will be placed under the control of the PcomS promoter, and this strain will possess the asymmetric ComR1-ComR2 transcription factor but will be unable to produce the XIPs required for its activation. The two other prtS- strains will carry the endogenous and “Sve-like” ComRS systems, respectively, and will therefore each produce one of the two XIPs required to activate the asymmetric heterodimer. Thus, the prtS- strains depend on the oligopeptides produced by the prtS+ strain, while the latter depends on the XIPs produced by the two other strains to trigger its proteolytic activity. We identified a medium, named CasHMCW, that supports the growth of prtS+ strains while limiting that of prtS- strains. Although promising, the implementation of the system revealed a limitation related to insufficient XIPSve production to activate the asymmetric transcription factor and therefore induce casein digestion.