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

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

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

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

Sigma F SpoIIAB SpoIIAA SpoIIE P X

Sigma F SpoIIAB SpoIIAA SpoIIE P X

Sigma F SpoIIAB SpoIIAA SpoIIE P transcription X

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

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

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

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

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.

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

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

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

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?

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

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

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?

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...

Mother cell had equal amount of fluorescence!!!

But one more consideration... :-(

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

Mutant strain that forms septum at each end Same assay Hypothesis: = no fluorescence in mother cell = flourescence in mother cell

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…

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