Function Analysis Of ClpS And Its Binding Protein In Mycobacterium Smegmatis

Adaptor protein ClpS plays an important role in the Gram-negative bacterium such as E. coli, in which system ClpS is an essential regulator of prokaryotic ATP-dependent protease ClpAP. ClpS protein could deliver certain protein substrates with specific amino acid sequences to ClpAP for protein degradation. On the other hand, ClpS could also function as the inhibitor of the ClpAP-mediated protein degradation for other proteins. However, the biological function and potential binding proteins of ClpS protein in the Gram-positive system have not been studied.Here, we constructed the clpS-overexpression Mycobacterium smegmatis strain,and showed for the first time that overexpression of ClpS increases the resistance of M. smegmatis to rifampicin that is one of the most widely usedantibiotic drugs in treatment of tuberculosis. First of all, using quantitative proteomic technology, we systematically analyzed effects of ClpS overespression on changes in M. smegmatis proteome, and proposed that the increasedrifampicin resistance was caused by ClpS-regulated drug sedimentation and drug metabolism. Our results indicated that the changes in degradation related proteins enhances drug resistance and quantitative proteomic analysis is an important tool for understanding molecular mechanisms responsible for bacteria drug resistance. Second, we further identified some potential ClpS-binding proteins in vivo using Fc-III tag, among which we demonstrated the high binding affinity of a new unknown protein MSMEG₄909 with our bait protein ClpS.Furthermore, we systematically analyzed effects of MSMEG₄909 overespression on changes in M. smegmatis proteome, and also tried to identify potential binding proteins of MSEMG₄909 both in vivo and in vitro.Taken together, we systematically studied the biological function of ClpS and its binding protein MSEMG₄909 in M. smegmatis. Our research provides a new perspective to understand the functions and action mechanisms of the adaptor protein ClpS’s new function in the Gram-positive system.