IADR Abstract Archives
Reciprocal Posttranslational Control of Streptococcus mutans LRS and Natural Competence
Objectives: The development of natural competence provides bacteria with a mechanism to repair damaged genes or acquire new advantageous traits, such as antibiotic resistance. In the human cariogenic species Streptococcus mutans, natural competence development is critically dependent upon the natural competence-specific sigma factor ComX. BrsRM, a member of the recently described LytTR Regulatory Systems (LRS), was previously investigated for its role in bacteriocin production and the activation of cell death in S. mutans. Recently, we also observed potent stimulation of S. mutans natural competence via the BrsRM LRS. Our objective was to determine the mechanism of this ability.
Methods: Mass spectrometry analysis of BrsR coimmunoprecipitates (Co-IP) was employed to identify candidate protein-protein interaction partners. Candidate interactions were further verified using specific Co-IP assays. Genetic regulatory mechanisms were characterized using a combination of luciferase assays, western blots, and electrophoretic mobility shift assays (EMSA).
Results: Mass spectrometry analyses indicated that BrsR directly interacts with a variety of late competence proteins as well as ComX. A subset of 6 natural competence-related proteins were chosen from the list of potential interactors and confirmed via direct Co-IP studies with BrsR. Due to protein-protein interactions, BrsR increases the abundance of the ComX protein by inhibiting ComX degradation. This stimulates late competence protein production and the induction of natural competence. Upon their production, late competence proteins like DprA directly bind to BrsR to antagonize its autoregulatory activity.
Conclusions: BrsR activates the S. mutans natural competence system through its ability to directly stabilize ComX. The late competence proteins produced as a result of ComX stabilization can then inhibit BrsR autoregulation as a mechanism to reset the circuit. This posttranslational regulatory mechanism is a new paradigm for S. mutans natural competence development, which could conceivably be employed by other protein regulators of natural competence that act via unknown mechanisms.
Methods: Mass spectrometry analysis of BrsR coimmunoprecipitates (Co-IP) was employed to identify candidate protein-protein interaction partners. Candidate interactions were further verified using specific Co-IP assays. Genetic regulatory mechanisms were characterized using a combination of luciferase assays, western blots, and electrophoretic mobility shift assays (EMSA).
Results: Mass spectrometry analyses indicated that BrsR directly interacts with a variety of late competence proteins as well as ComX. A subset of 6 natural competence-related proteins were chosen from the list of potential interactors and confirmed via direct Co-IP studies with BrsR. Due to protein-protein interactions, BrsR increases the abundance of the ComX protein by inhibiting ComX degradation. This stimulates late competence protein production and the induction of natural competence. Upon their production, late competence proteins like DprA directly bind to BrsR to antagonize its autoregulatory activity.
Conclusions: BrsR activates the S. mutans natural competence system through its ability to directly stabilize ComX. The late competence proteins produced as a result of ComX stabilization can then inhibit BrsR autoregulation as a mechanism to reset the circuit. This posttranslational regulatory mechanism is a new paradigm for S. mutans natural competence development, which could conceivably be employed by other protein regulators of natural competence that act via unknown mechanisms.
