Stringent response in E. coli

1
Stringent response in E. coli

relA
Some ppGpp activity
2
Stringent response in E. coli

relA
Some ppGpp activity
So there must be another gene also
controlling ppGpp levels...
3
Stringent response in E. coli

relA
Some ppGpp activity
So there must be another gene also
controlling ppGpp levels
spoT
4
Bacillus subtilis
Stress
5
Bacillus subtilis
Stress
ppGpp
6
Bacillus subtilis
Stress
ppGpp
STRINGENTRESPONSE
7
Bacillus subtilis
Stress
ppGpp
STRINGENTRESPONSE
E. coli
spoT
relA
8
Bacillus subtilis
Stress
ppGpp
STRINGENTRESPONSE
E. coli
spoT
relA
relA
B. subtilis
9
So...

• What happens in B. subtilis when RelA is mutated?

10
So...

• What happens in B. subtilis when RelA is mutated?
• Mutants retain some protein activity
• BR17 RelA has 2 of the activity of wt

11

• Is it possible to obtain a RelA null mutant?

12
Knocking relA out
Cmr
relA
13
Knocking relA out
Cmr
relA
14
Knocking relA out
Cmr
relA
15
Knocking relA out
Cmr
relA
16
B. subtilis transformation
Cmr
relA
17
B. subtilis transformation
cell membrane
18
Results

• No double recombinants!!

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Results

• No double recombinants!!
• Some single crossover
• RelA vital??

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Try Something Else...
Pspac
relA
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Try Something Else...
Pspac
relA
IPTG
22
Try Something Else...
Pspac
relA
Delete native relA
IPTG
23
Results 2
Wild type
relA-
OD600
Removal of 1/2 of culture to medium w/o aa
Time
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Results 2
Wild type
relA-
OD600
relA is essential to stringent response
Removal of 1/2 of culture to medium w/o aa
Time
25
Also...

• Amino acid/glucose starvation
• 32P

relA-
wt
No (p)ppGpp accumulation
(p)ppGpp accumulation
26
Conclusions

• The relA/spoT homologue is the sole source of
  (p)ppGpp in B. subtilis
• It is essential for cell viability under nutrient
  stress