Sigma factors in sporulation Sporulation regulated sigma factor

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?

Sigma G Kellner, 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?

What controls the activity of G so that it is active at the right time and place?

Backtrack to E Background and Conclusions (Trempy et al; Stragier et al) • spo. IIGA + sig. E expression in vegetative cells gives E • Sporulation-dependent expression requires Spo. IIE • Spo. IIE also required for sporulation-dependent division • F required for activity of E in mother cell! Does G require spo. IIIA to be present in order to be active?

spo. IIIA is required for G activity Methods: • Fusion of lac. Z to ssp. E promoter -Gal synthesis (ssp. E is utilized by G polymerase during sporulation) RED=wild-type strain BLUE=spo. IIIA mutant Time (Hours)

However, the anti-sigma factor, G Spo. IIAB, may antagonize activity in the spo. IIIA mutant! (Kirchman et al. , 1993; Rather et al. , 1990; Foulger et al. , 1993)

So, first…to test if Spo. IIAB can G interact directly with in vitro, chemical cross-linking was used.

In the presence of nucleotides and under optimal binding conditions-- Spo. IIAB + E. coli =no binding SDS-Page Spo. IIAB + E. coli w/ cloned G =binding SDS-Page

G , Spo. IIAB CAN interact with G but does Spo. IIAB antagonize during sporulation? ? …Test with mutant G that does not bind efficiently to Spo. IIAB

E 155 K Mutation 1. In F, substitution of lys for glu reduces affinity of the sigma factor for Spo. IIAB 2. F and G are similar 3. Homologous mutation in G may also prevent binding to Spo. IIAB (Glu Lys) 4. chemical cross-linking was used to determine if the G mutant will bind to Spo. IIAB

In the presence of nucleotides and under optimal binding conditions-- G mutant E 115 K =no binding SDS-Page Wild-type G =binding SDS-Page

Confirmed: the E 155 K mutation in G reduces its affinity for binding to Spo. IIAB and G antagonizes activity. How are Spo. IIAB and spo. IIIA related?

Is there a mechanism that explains the late activation of G as dependant on another protein? • Spo. IIAB inhibits G activity • G mutant does not bind to Spo. IIAB What would happen if the G mutant was combined with a spo. IIIA mutant? How would G activity be effected?

Activity of G during sporulation using the ssp. E-lac. Z reporter fusion -Gal synthesis In spo. IIIA mutant: Wt G E 155 K mutant G is active in a spo. IIIA mutant!! spo. IIIA relieves the inhibition of G activity by Spo. IIAB E 155 K mutant does not require Spo. IIIA for its activation. Time (Hours)

Conclusions • Spo. IIAB inhibits G activity early after its synthesis • Spo. IIIA functions to relieve inhibition of G activity by Spo. IIAB • Relief occurs after engulfment of the forespore