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Sigma factors in sporulation

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Sigma factors in sporulation. Sporulation: regulated sigma factor cascade. … but what regulates the sigma factors?. Not transcriptional control What kind of post-transcriptional control? How are events coordinated? Checkpoints?.

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Presentation Transcript
slide2

Sigma factors in sporulation

Sporulation: regulated sigma factor cascade

… but what regulates the sigma factors?

  • Not transcriptional control
  • What kind of post-transcriptional control?
  • How are events coordinated? Checkpoints?
slide3

Sigma GKellner, E.M., Decatur, A., and Moran, C. (1996). Two-Stage Regulation of an Anti-Sigma Factor Determines Developmental Fate During Bacterial Endospore Formation. Molecular Microbiology, 21(5), 913-924.

  • G is active only in the forespore (Driks and Losick, 1991)
  • G does not become active until later during sporulation, after engulfment of the forespore(Partridge and Errington, 1993)
  • G is present before it is activated

Why and How?

slide5

Backtrack to E

  • Background and Conclusions (Trempy et al; Stragier et al)
    • spoIIGA + sigE expression in vegetative cells gives E
    • Sporulation-dependent expression requires SpoIIE
    • SpoIIE also required for sporulation-dependent division
    • F required for activity of E in mother cell!

Does G requirespoIIIA to be present

in order to be active?

slide6

spoIIIA is required for G activity

  • Methods:
  • Fusion of lacZ to sspE promoter
  • (sspE is utilized by Gpolymerase
  • during sporulation)

-Gal synthesis

RED=wild-type strain

BLUE=spoIIIA mutant

Time (Hours)

however the anti sigma factor spoiiab may antagonize g activity in the spoiiia mutant

However, the anti-sigma factor, SpoIIAB, may antagonize Gactivity in the spoIIIA mutant!

(Kirchman et al., 1993; Rather et al., 1990; Foulger et al., 1993)

so first to test if spoiiab can interact directly with g in vitro chemical cross linking was used

So, first…to test if SpoIIAB can interact directly with Gin vitro, chemical cross-linking was used.

slide9

In the presence of nucleotides and under optimal binding conditions--

SpoIIAB + E. coli

=no binding

SpoIIAB + E. coli w/

cloned G

=binding

SDS-Page

SDS-Page

spoiiab can interact with g but does spoiiab antagonize g during sporulation

SpoIIAB CAN interact with G, but does SpoIIAB antagonize G during sporulation??

…Test with mutant G that does not bind efficiently to SpoIIAB

e155k mutation
E155K Mutation
  • In F, substitution of lys for glu reduces affinity of the sigma factor for SpoIIAB
  • F and G are similar
  • Homologous mutation in G may also prevent binding to SpoIIAB (GluLys)
  • chemical cross-linking was used to determine if the G mutant will bind to SpoIIAB
slide12

In the presence of nucleotides and under optimal binding conditions--

Wild-type G

=binding

G mutant

E115K

=no binding

SDS-Page

SDS-Page

slide13

Confirmed: the E155K mutation in G reduces its affinity for binding to SpoIIAB and antagonizes G activity.

How are SpoIIAB and spoIIIA related?

is there a mechanism that explains the late activation of g as dependant on another protein
Is there a mechanism that explains the late activation of Gas dependant on another protein?
  • SpoIIAB inhibits G activity
  • G mutant does not bind to SpoIIAB

What would happen if the G mutant was combined with a spoIIIA mutant? How would G activity be effected?

activity of g during sporulation using the sspe lacz reporter fusion
Activity of G during sporulation using the sspE-lacZ reporter fusion

In spoIIIA mutant:

Wt G

E155K G

E155K mutant G is active

in a spoIIIA mutant!!

spoIIIA relieves the inhibition of

G activity by SpoIIAB

-Gal synthesis

E155K mutantdoes not require

SpoIIIA for its activation.

Time (Hours)

conclusions
Conclusions
  • SpoIIAB inhibits G activity early after its synthesis
  • SpoIIIA functions to relieve inhibition of G activity by SpoIIAB
  • Relief occurs after engulfment of the forespore