Sigma F -in forespore -pre-divisional synthesis -inactive until after division

1. Sigma F -in forespore -pre-divisional synthesis -inactive until after division

2. Sigma F SpoIIAB -dual function: -anti-sigma factor - inhibits sigma F -kinase - phosporylates SpoIIAA

3. Sigma F SpoIIAA -anti-anti-sigma factor -release sigma F from SpoIIAB complex -active in unphosphorylated state SpoIIAB

4. Sigma F SpoIIAA SpoIIE -phosphatase -activates SpoIIAA -indirectly activates sigma F -membrane protein -polar septum SpoIIAB

5. Sigma F SpoIIAA X P SpoIIE SpoIIAB

6. Sigma F SpoIIAA X P SpoIIE SpoIIAB

7. transcription Sigma F SpoIIAA X P SpoIIE SpoIIAB

8. = SpoIIE Mother cell Forespore Localization of SpoIIE in sporulating B.subtilis.

9. = SpoIIE Mother cell Forespore sigma-F active Localization of SpoIIE in sporulating B.subtilis.

10. So…How are the timing and selectivity of sigma-F activation determined??

11. 1. SpoIIE on both sides of septum -higher concentration in forespore 2. SpoIIE on both sides of septum -only active in forespore 3. SpoIIE only in forespore 4. Some combination of the above

12. Wu et al., 1998 SpoIIE initially localizes at 1 pole, comcomitant with septal formation. After division, SpoIIE just in forespore, and another E-ring forms at distal pole.

13. BUT…If SpoIIE can be found at both poles before division occurs, the whole idea falls apart...

14. So, where and when during polar division does SpoIIE accumulate? gfp spoIIE B. subtilis Time-lapse microscopy Growth stopped @ 75, 90, 105, 120, and 150 minutes after the initiation of sporulation. visualization

16. Conclusions... • Bipolar localization of SpoIIE protein can and does precede septation • SpoIIE associated with septum persists until sigma-F is activated

17. But this doesn’t tell us whether SpoIIE is found on one or both sides of the septum.

18. But this doesn’t tell us whether SpoIIE is found on one or both sides of the septum. So how is it that SpoIIE only activates sigma-F in the forespore?

19. Localization of SpoIIE post-division lysozyme ? spoIIE::gfp strain

20. Localization of SpoIIE post-division lysozyme ? spoIIE::gfp strain Majority of fluorescence from forespore… -SpoIIE localized to forespore side of septum?

21. Localization of SpoIIE post-division lysozyme ? spoIIE::gfp strain Majority of fluorescence from forespore… -SpoIIE localized to forespore side of septum? What about comparative size of mother cell?

22. Localization of SpoIIE post-division lysozyme ? spoIIE::gfp strain Majority of fluorescence from forespore… -SpoIIE localized to forespore side of septum? What about comparative size of mother cell? -integrated numbers...

23. Mother cell had equal amount of fluorescence!!!

24. Mother cell had equal amount of fluorescence!!! But one more consideration... :-(

25. Mother cell had equal amount of fluorescence!!! But one more consideration... :-( What about those SpoIIEs that were at the distal pole?

26. What about those SpoIIEs that were at the distal pole? Same assay Mutant strain that forms septum at each end Hypothesis: = no fluorescence in mother cell = flourescence in mother cell

27. Results of assay for fluorescence in mother cell, corrected... Mother cells had an average of 2.8 times less fluorescence than forespores. But that’s still some…

28. Conclusions... • SpoIIE can be found on both sides of the septum • Must be some other way of regulating sigma-F activation, not spatially