Sigma F

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
SoHow 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
BUTIf 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 _at_ 75, 90, 105, 120, and 150
minutes after the initiation of sporulation.
visualization
15
(No Transcript)
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 doesnt tell us whether SpoIIE is found
on one or both sides of the septum.
18
But this doesnt 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
?
spoIIEgfp strain
20
Localization of SpoIIE post-division
lysozyme
?
spoIIEgfp strain
Majority of fluorescence from forespore -SpoIIE
localized to forespore side of septum?
21
Localization of SpoIIE post-division
lysozyme
?
spoIIEgfp 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
?
spoIIEgfp 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 thats 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