Sigma factors in sporulation

2. Sigma factors in sporulation Sporulation: regulated sigma factor cascade … but what regulates the sigma factors? • Not transcriptional control • What kind of post-transcriptional control? • How are events coordinated? Checkpoints?

3. Sigma GKellner, E.M., Decatur, A., and Moran, C. (1996). Two-Stage Regulation of an Anti-Sigma Factor Determines Developmental Fate During Bacterial Endospore Formation. Molecular Microbiology, 21(5), 913-924. • G is active only in the forespore (Driks and Losick, 1991) • G does not become active until later during sporulation, after engulfment of the forespore(Partridge and Errington, 1993) • G is present before it is activated Why and How?

4. What controls the activity of G so that it is active at the right time and place?

5. Backtrack to E • Background and Conclusions (Trempy et al; Stragier et al) • spoIIGA + sigE expression in vegetative cells gives E • Sporulation-dependent expression requires SpoIIE • SpoIIE also required for sporulation-dependent division • F required for activity of E in mother cell! Does G requirespoIIIA to be present in order to be active?

6. spoIIIA is required for G activity • Methods: • Fusion of lacZ to sspE promoter • (sspE is utilized by Gpolymerase • during sporulation) -Gal synthesis RED=wild-type strain BLUE=spoIIIA mutant Time (Hours)

7. However, the anti-sigma factor, SpoIIAB, may antagonize Gactivity in the spoIIIA mutant! (Kirchman et al., 1993; Rather et al., 1990; Foulger et al., 1993)

8. So, first…to test if SpoIIAB can interact directly with Gin vitro, chemical cross-linking was used.

9. In the presence of nucleotides and under optimal binding conditions-- SpoIIAB + E. coli =no binding SpoIIAB + E. coli w/ cloned G =binding SDS-Page SDS-Page

10. SpoIIAB CAN interact with G, but does SpoIIAB antagonize G during sporulation?? …Test with mutant G that does not bind efficiently to SpoIIAB

11. E155K Mutation • In F, substitution of lys for glu reduces affinity of the sigma factor for SpoIIAB • F and G are similar • Homologous mutation in G may also prevent binding to SpoIIAB (GluLys) • chemical cross-linking was used to determine if the G mutant will bind to SpoIIAB

12. In the presence of nucleotides and under optimal binding conditions-- Wild-type G =binding G mutant E115K =no binding SDS-Page SDS-Page

13. Confirmed: the E155K mutation in G reduces its affinity for binding to SpoIIAB and antagonizes G activity. How are SpoIIAB and spoIIIA related?

14. Is there a mechanism that explains the late activation of Gas dependant on another protein? • SpoIIAB inhibits G activity • G mutant does not bind to SpoIIAB What would happen if the G mutant was combined with a spoIIIA mutant? How would G activity be effected?

15. Activity of G during sporulation using the sspE-lacZ reporter fusion In spoIIIA mutant: Wt G E155K G E155K mutant G is active in a spoIIIA mutant!! spoIIIA relieves the inhibition of G activity by SpoIIAB -Gal synthesis E155K mutantdoes not require SpoIIIA for its activation. Time (Hours)

16. Conclusions • SpoIIAB inhibits G activity early after its synthesis • SpoIIIA functions to relieve inhibition of G activity by SpoIIAB • Relief occurs after engulfment of the forespore