Bacillus subtilis responses to stress

Competence development

Chemotaxis/motility

Degradation enzymes

Sporulation

Environmental stresses

Heat shock stress clpC

Clp family in E. coli - degradation of many regulatory proteins

clpC

comKClpC

ComKLate

competence genes

stress

ClpC function in

B. subtilis

SigH is also required for the transcription of many sporulation genes

RNA polymerase sigma factor H is essential for normal competence development

H H H H H H

H H

H

H H H

Exponential growth Sporulation

SigH expression in cell cycle

H

H H H H H H

H H

H

H H H

Exponential growth Sporulation

SigH half-life increases during sporulation

H

H H H H

H H

H H H

Exponential growth Sporulation

SigH half-life increases during sporulation

H

H H

H H

H H

Exponential growth Sporulation

SigH half-life increases during sporulation

H H

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

bgaBclpCA B

heat shockresponse

Integrate into chromosome

Evaluate beta-galactosidase activity in various situations

Transcriptional fusion used to detect clpC expression:

Induction of clpC expression

T0 T4

sporulation

wildtype

sigB null

spo0H null

T3.5

T3.5

>T4

clpCS (deletion mutation)

>T4

What needs to happen in order for ClpC to be involved in SigH expression?

ClpC should have an effect on SigH accumulation

ClpC has to be expressed when SigH is at the onset of sporulation

Western blot analysis of σH accumulation

T-0.5 T1 T4T0 T3T2 T-0.5 T1 T4T0 T3T2

clpC+ clpCS

Possible causes of this result:

Deficiency in active degradation of SigH

Deficiency in active transcription of spo0H

Degradation of SigH vs. Transcription of spo0H

bgaBspo0H

spo0H::bgaB fusion

No difference between spo0H expression in wildtype and clpC deletion mutant strains.

50% of SigH disappeared after 12 minutes in wildtype.

At 12 min, 85% of SigH was still present in clpCS.

SigH half-life analysis:

Because this occurred in the clpCC deletion mutant, it appears that ClpC is somehow involved in

the degradation of H.

Accumulation of SigH is due to a deficiency in its degradation.