The ClpC M-Domain Facilitates in vivo Function of Sporulation Specific Adaptor Protein MdfA in Bacillus subtilis

Abstract

Dormancy is a strategy observed across all domains of life. Bacillus subtilis is a model organism that produces spores that are metabolically dormant. To become metabolically dormant, the B. subtilis cell engages in the process of sporulation, which allows bacteria to go on to proliferate even after extermination events that affect a bacteria population. Protein degradation done by AAA+ proteases is a key process in sporulation that prepares the developing spore to become the mature, metabolically inactive spore. Understanding the interactions that trigger the formation and activation of AAA+ proteases within the developing spore is also key to deepening the broader scientific community’s understanding of protease function as a whole. Within B. subtilis, the AAA+ protease ClpCP is responsible for cell maintenance, competence development, and spore dormancy progression. The unfoldase component, ClpC, is responsible for recognizing the degron tags of substrates as well as adaptor proteins. Adaptor proteins are proteins that regulate and facilitate interactions between AAA+ proteases and the substrates they target, allowing controlled, event-related protease activity. Novel sporulation specific adaptor protein MdfA is found only in the developing spore, and has recently been shown to activate the ClpCP complex to degrade metabolic enzymes, directly leading to the progression of metabolic dormancy. Current MdfA-ClpC models indicate ClpC and MdfA interact via the ClpC N-Domain with growing evidence of a secondary interaction on the ClpC M-Domain. The goal of this project was to determine if the ClpC the M-Domain facilitates the in vivo function MdfA during the events of sporulation in Bacillus subtilis. To accomplish this, we selected a number of mutations to be made along the ClpC M-Domain and in MdfA. We then measured functions in B. subtilis that indicate the MdfA-ClpCP complex was formed during sporulation, as well as outside of sporulation. Through this, we learned that mutations to mdfA could disrupt in vivo function of MdfA during sporulation, but we were unable to demonstrate the same for mutations made to the clpC M-Domain due to pleiotropic effects. However, when measuring in vivo function outside of sporulation, mutations to mdfA or the clpC M-Domain were enough to prevent the formation of MdfA-ClpCP. Furthermore, we were able to restore function by making complementary mutations to both mdfA and clpC, demonstrating the importance of the ClpC M-Domain in this interaction.