Stringent response in E. coli

Some ppGpp activityrelA

Stringent response in E. coli

Some ppGpp activity

So there must be another gene also controllingppGpp levels...

relA

Stringent response in E. coli

Some ppGpp activity

So there must be another gene also controllingppGpp levels spoT

relA

Bacillus subtilis

Stress

Bacillus subtilis

Stress

ppGpp

Bacillus subtilis

Stress

ppGpp

STRINGENTRESPONSE

Bacillus subtilis

spoT

Stress

ppGpp

STRINGENTRESPONSE

relA

E. coli

Bacillus subtilis

spoT

relA

Stress

ppGpp

STRINGENTRESPONSE

relA

E. coli

B. subtilis

So...

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

So...

• What happens in B. subtilis when RelA is mutated?– Mutants retain some protein activity

• BR17 RelA has 2% of the activity of wt

Is it possible to obtain a RelA null mutant?

Knocking relA out

Cmr

relA

Knocking relA out

Cmr

relA

Knocking relA out

Cmr

relA

Knocking relA out

Cmr

relA

B. subtilis transformation

Cmr

relA

B. subtilis transformation

cellmembrane

Results

No double recombinants!!

Results

No double recombinants!!

Some single crossover

RelA vital??

Try Something Else...

relAPspac

Try Something Else...

relAPspac

IPTG

Try Something Else...

relAPspac

IPTG

Delete nativerelA

Results #2

OD600

Time

Wild type

relA-

Removal of 1/2 of culture tomedium w/o aa

Results #2

OD600

Time

Wild type

relA-

Removal of 1/2 of culture tomedium w/o aa

relA is essentialto stringent response

Also...

Amino acid/glucose starvation

+

[32P]

(p)ppGppaccumulation

No (p)ppGppaccumulation

relA-wt

Conclusions

• The relA/spoT homologue is the sole source of (p)ppGpp in B. subtilis

• It is essential for cell viability under nutrient stress