The final proteolytic step in transmembrane signaling of multiple RsgI anti-σ factors in Clostridium thermocellum.

In Clostridium thermocellum, there are nine RsgI factors responsible for sensing different types of substrates and regulating the transcription and expression of cellulosome genes. Within the signaling pathway of RsgI, the membrane protease RseP cleaves RsgI in its transmembrane helix, thus releasing the N-terminal fragment of RsgI from the membrane. This released RsgI N-terminal fragment is subsequently recognized and degraded by a cytoplasmic protease complex consisting of an AAA+ ATPase and ClpP protease. Previous research showed that the ClpXP complex, comprising ClpX and ClpP, is capable of recognizing and degrading the N-terminal fragment of RsgI6. However, due to the low conservation of the transmembrane helical region of RsgI, it remains unclear whether other RsgIs are similarly recognized and degraded by the same unfoldase. In this study, we employed in vitro protease assays to examine the recognition and degradation of the N-terminal fragment of each RsgI by various ClpP-unfoldase complexes. Results confirm that ClpXP is responsible for degrading the N-terminal fragments of all RsgI proteins in C. thermocellum, suggesting a degree of sequence promiscuity in substrate recognition by ClpXP. ClpXP can recognize multiple XAA sites in the transmembrane helix region of RsgI. Moreover, we unexpectedly discovered that the cytoplasmic domain influences the degradation of RsgI2-NF by ClpXP in our in vitro assay. This study provides new insights into understanding the complex regulatory mechanisms of cellulosome genes and the role of AAA+ proteases in C. thermocellum, thereby offering critical clues for unraveling the internal regulatory networks of bacteria.