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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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?
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What controls the activity of ?G so that it is
active at the right time and place?
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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 require spoIIIA to be present in order
to be active?
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spoIIIA is required for ?G activity

• Methods
• Fusion of lacZ to sspE promoter
• (sspE is utilized by ?G polymerase
• during sporulation)

?-Gal synthesis
REDwild-type strain BLUEspoIIIA mutant
Time (Hours)
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However, the anti-sigma factor, SpoIIAB, may
antagonize ?G activity in the spoIIIA mutant!

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

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So, firstto test if SpoIIAB can interact
directly with ?G in vitro, chemical cross-linking
was used.
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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
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SpoIIAB CAN interact with ?G, but does SpoIIAB
antagonize ?G during sporulation??

• Test with mutant ?G that does not bind
  efficiently to SpoIIAB

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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

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In the presence of nucleotides and under optimal
binding conditions--
Wild-type ?G binding
?G mutant E115K no binding
SDS-Page
SDS-Page
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Confirmed the E155K mutation in ?G reduces its
affinity for binding to SpoIIAB and antagonizes
?G activity.

• How are SpoIIAB and spoIIIA related?

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Is there a mechanism that explains the late
activation of ?G as 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?

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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 mutant does not require SpoIIIA for its
activation.
Time (Hours)
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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