IDENTIFICATION OF A DEGRADATION SIGNAL IN THE ANTI-SIGMA FACTOR CSFB REQUIRED FOR THE SWITCH TO LATE GENE EXPRESSION DURING BACILLUS SUBTILIS SPORULATION
In response to adverse environmental conditions, the gram-positive bacterium Bacillus subtilis undergoes an adaptive process known as sporulation, culminating in the production of a metabolically dormant, extraordinarily resistant cell type called a spore. Sporulation is tightly regulated by alternating early and late sigma factors, which direct the expression of developmental genes. The anti-sigma factor CsfB plays an important role in the σF to σG switch in the developing spore by inhibiting σG at early times while σF must be active. Unpublished work from the Camp laboratory revealed that unlike GFP alone, a GFP-CsfB fusion protein degraded quickly during sporulation. Moreover, this degradation is known to depend, at least partially, upon the protease ClpCP. To identify the signal sequence on CsfB that allows for its recognition by the protease, I constructed GFP-CsfB fusion protein variants either harboring or lacking parts of the CsfB C-terminus. B. subtilis strains expressing these constructs were screened for changes to protein levels during sporulation by both western blot and fluorescence microscopy. My data revealed that the last 3 residues (LYS) are sufficient but not necessary for degradation. Further inspection of various GFP-CsfB variants shows the presence of a second internal degron that is also sufficient but not necessary for degradation. Next, I attempted to construct a variant of GFP-CsfB that lacked both degrons but retains its ability to inhibit σG. Finally, I tested the dependence of each degron upon the ClpCP protease by knocking out the clpC gene and observing the degradation pattern of various GFP-CsfB variants. Data indicated that degradation via the C-terminal degron and the internal degron both depend upon the ClpCP protease. These results will help elucidate the importance of proper CsfB degradation during σG regulation, and more broadly, proper cell differentiation in Bacillus subtilis sporulation.
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