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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
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.
So, where and when during polar division does SpoIIE accumulate?
gfp spoIIE
B. subtilis
Growth stopped @ 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
?
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...
:-(What about those SpoIIEs that were at the distal pole?
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…