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

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

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

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

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

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

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

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

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

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

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 genes @ 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

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

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

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.

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.