1. Bacillus subtilis responses to stress
Competence development
Environmental stresses
Chemotaxis/motility
Sporulation
Degradation enzymes

2. Clp family in E. coli - degradation of many regulatory proteins
Heat shock stress
clpC
Clp family in E. coli - degradation of many regulatory proteins

3. ClpC function in B. subtilis Late competence genes
stress
clpC
ClpC
Stress (specifically heat shock) induces clpC, which produces a negative regulator of comK, which is a positive regulator of late-competence genes. Members of the Ecoli Clp family are involved in degradation of regulatory proteins and ClpC is thought to be a molecular chaperone although no evidence existed for protease activity at the time this paper was written.
comK
Late competence genes
ComK

4. SigH is also required for the transcription of many sporulation genes
RNA polymerase sigma factor H is essential for normal competence development
SigH is also required for the transcription of many sporulation genes

5. H H H H H H H H H H H H H Exponential growth Sporulation
Sigma H activity increases at the onset of sporulation
SigH expression in cell cycle

6. SigH half-life increases during sporulation
Exponential growth
Sporulation
Sigma H activity increases at the onset of sporulation
SigH half-life increases during sporulation

7. SigH half-life increases during sporulation
Exponential growth
Sporulation
Halflife of sigma H increases several-fold during sporulation (stays around longer)
SigH half-life increases during sporulation

8. SigH half-life increases during sporulation
Exponential growth
Sporulation
Halflife of sigma H increases several-fold during sporulation (stays around longer)
SigH half-life increases during sporulation

9. ClpC has to be expressed when SigH is at the onset of sporulation
What needs to happen in order for ClpC to be involved in SigH expression?
ClpC has to be expressed when SigH is at the onset of sporulation

10. A B Transcriptional fusion used to detect clpC expression: clpC bgaB
heat shock
response
Integrate into chromosome
Transcriptional fusion created to allow detection of clpC expression – attach to BgaB gene, which allows for the measuring of thermostable beta-galactosidase (necessary because of the temperature changes used in the study)
Evaluate beta-galactosidase activity
in various situations

11. Induction of clpC expression
sporulation T0 T4
wildtype
T3.5
sigB null
T3.5
spo0H null
Wt with fusion – clpC expression began at onset of sporulation, ended around T3.5
sigB null mutant, because clpC is transcribed partly by sigB – expression didn’t differ significantly from wt
spo0H null mutant, b/c many beginning of sporulation are dependent on spo0H function – clpC expression began at onset of sporulation but continued past T3.5, and even past T4 approaching T5.
Conclusions: no sigmaB effect, therefore must be sigmaA regulated
repression of clpC after T3.5 apparently requires sigmaH activity
Does clpC have an effect on sporulation?
YES – in the deletion mutant, sporulation frequency drops to 10% at 37 deg C and 0.05% at 45 deg C.
Deletion mutant’s effect on expression in fusion – clpC induced at onset of sporulation, continued after T4 (as with spo0H null mutant)
>T4
clpCS
(deletion mutation)
>T4

12. What needs to happen in order for ClpC to be involved in SigH expression?
ClpC has to be expressed when SigH is at the onset of sporulation
ClpC should have an effect on SigH accumulation

13. Western blot analysis of σH accumulation
clpC+
clpCS
Wildtype – cellular accumulation of sigH increased to T2, then decreased.
Deletion mutant – sigH accumulated up to T1, but did not decrease after at all.
Possible causes of this result:
Deficiency in active degradation of SigH
Deficiency in active transcription of spo0H

14. 50% of SigH disappeared after 12 minutes in wildtype.
Degradation of SigH vs. Transcription of spo0H
spo0H::bgaB fusion
spo0H
bgaB
No difference between spo0H expression in wildtype and clpC deletion mutant strains.
SigH half-life analysis:
50% of SigH disappeared after 12 minutes in wildtype.
At 12 min, 85% of SigH was still present in clpCS.

15. Accumulation of SigH is due to a deficiency in its degradation.
Because this occurred in the clpCC deletion mutant, it appears that ClpC is somehow involved in the degradation of H.