Improvement of the ComRS quorum-sensing system for competence and predation in streptococci

Details

Supervisors

Hols, Pascal

Faculty

Faculté des sciences

Degree label

Master [120] en biochimie et biologie moléculaire et cellulaire

Abstract

To achieve natural transformation, bacteria need to enter in a transient physiological state called competence. This state has the major drawback to be highly energy-consuming and therefore needs to be tightly regulated. In many streptococci species, this regulation is carried out by the ComRS quorum-sensing system. This system involves the peptide pheromone XIP that interacts with the effector ComR. This interaction led to the formation of the ComR-XIP complex that binds to different promoters to activate their transcription. Among the controlled genes, the transcriptional activation of comX, which encodes an alternative sigma factor (σX), allows the expression of all the genes involved in the transformation process. Besides, the ComR-XIP complex upregulates the production of bacteriocins, which ensures the presence of DNA in the environment during transformation. Interestingly, the regulation of the entry into the competence state by this system shows a bimodal behavior. This regulatory system was discovered by the host laboratory for salivarius streptococci (e.g. Streptococcus thermophilus, S. salivarius). Several molecular studies have allowed to better characterize this system, leading to the discovery of key residues involved in ComR-XIP binding and complex activation. Moreover, they have given clues regarding how the transcriptional regulator selects its cognate pheromone. During these studies, some XIP variants enhancing competence and bacteriocin production were discovered. Those peptides shared substitutions at XIP-5 position, which had not been previously reported to be essential for ComR activation. The main goal of this master thesis was the study of the molecular mechanisms that improves competence by using different pheromone variants in combination with ComR mutants. This part of the thesis revealed the interconnected role of four residues (ComR-F171, ComR-Y174, XIP-1 and XIP-5) in complex activation and in enhanced phenotype provided by the XIPSth-A5M variant. Additionally, it was observed that hydrophobicity and length/bulkiness of the lateral chain of XIP-5 induce an increase in competence activation. It was also observed that the presence of a methionine at this position seems to provide a more specific interaction, such as recently reported for Aro-Met-Aro interactions in many proteins. In parallel, effects of this improvement on the competence bimodal behavior was investigated through the use of a GFP reporter and epifluorescence microscopy. During these experiments, the deleterious effect of competence overactivation by XIPSth-A5M was highlighted. Finally, how this improvement affects the production of bacteriocins was tackled. To this end, experiments using different reporter systems (fluorescence and light emission) were performed to study the expression of bacteriocin genes (Pslvx as proxy). Those experiments have uncovered that the expression of XIPSth-A5M allows a constitutive production of bacteriocins, which was sufficient to inhibit the growth of Lactococcus lactis and S. vestibularis. Additionally, these investigations showed that the PSlvX promoter displays a bimodal activity and is active upstream of competence genes.