Bacillus subtilis responses to stress

1
Bacillus subtilis responses to stress
Competence development
Chemotaxis/motility
Sporulation
Degradation enzymes
2
Clp family in E. coli - degradation of many
regulatory proteins
3
ClpC function in B. subtilis
stress
clpC
ClpC
comK
Late competence genes
ComK
4
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
SigH expression in cell cycle
6
?H
?H
?H
?H
?H
?H
?H
?H
?H
?H
?H
?H
?H
Exponential growth
Sporulation
SigH half-life increases during sporulation
7
?H
?H
?H
??H
?H
?H
?H
?H
?H
?H
Exponential growth
Sporulation
SigH half-life increases during sporulation
8
?H
?H
?H
?H
?H
?H
?H
?H
Exponential growth
Sporulation
SigH half-life increases during sporulation
9
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
Transcriptional fusion used to detect clpC
expression
bgaB
clpC
?A
?B
heat shock response
Integrate into chromosome
Evaluate beta-galactosidase activity in various
situations
11
Induction of clpC expression
sporulation
T0
T4
wildtype
sigB null
spo0H null
clpC?S (deletion mutation)
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 sH accumulation
Possible causes of this result
Deficiency in active degradation of SigH
Deficiency in active transcription of spo0H
14
Degradation of SigH vs. Transcription of spo0H
spo0HbgaB fusion
bgaB
spo0H
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.