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.

ForesporeMothercell

= SpoIIE

Localization of SpoIIE in sporulating B.subtilis.

Forespore sigma-F active

Mothercell

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 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.

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!!!

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?

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