FliT Selectively Enhances Proteolysis of FlhC Subunit in FlhD4C2 Complex by an ATP-dependent Protease ClpXP [Enzymology]

October 2nd, 2014 by Sato, Y., Takaya, A., Mouslim, C., Hughes, K. T., Yamamoto, T.

We previously reported that the ClpXP ATP-dependent protease specifically recognizes and degrades flagellar master transcriptional activator complex, FlhD4C2, to negatively control flagellar biogenesis. The flagellum-related protein, FliT, is also a negative regulator of flagellar regulon by inhibiting the binding of FlhD4C2 to the promoter DNA. We have found a novel pathway of FliT inhibition of FlhD4C2 activity connected to ClpXP proteolysis. An in vitro degradation assay using purified proteins shows that FliT selectively increases ClpXP proteolysis of the FlhC subunit in the FlhD4C2 complex. FliT behaves specifically to ClpXP-dependent proteolysis of FlhC. An in vitro interaction assay detects the ternary complex of FliT-FlhD4C2-ClpX. FliT promotes the affinity of ClpX against FlhD4C2 complex, whereas FliT does not directly interact with ClpX. Thus FliT interacts with the FlhC in FlhD4C2 complex and increases the presentation of FlhC-recognition region to ClpX. The DNA-bound form of FlhD4C2 complex is resistant to ClpXP-proteolysis. We suggest that the role of FliT in negatively controlling the flagellar gene expression involves increasing free molecules of FlhD4C2 sensitive to ClpXP proteolysis by inhibiting the binding to the promoter DNA as well as enhancing the selective proteolysis of FlhC subunit by ClpXP.