1. Stringent response in E. coli Some ppGpp activity relA

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

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

4. Bacillus subtilis Stress

5. Bacillus subtilis Stress ppGpp

6. Bacillus subtilis Stress ppGpp STRINGENT RESPONSE

7. Bacillus subtilis spoT Stress ppGpp STRINGENT RESPONSE relA E. coli

8. Bacillus subtilis spoT relA Stress ppGpp STRINGENT RESPONSE relA E. coli 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 Cm r relA

13. Knocking relA out Cm r relA

14. Knocking relA out Cm r relA

15. Knocking relA out Cm r relA

16. B. subtilis transformation Cm r relA

17. B. subtilis transformation cell membrane

18. Results No double recombinants!!

19. Results No double recombinants!! Some single crossover RelA vital??

20. Try Something Else... relA P spac

21. Try Something Else... relA P spac IPTG

22. Try Something Else... relA P spac IPTG Delete native relA

23. Results #2 OD 600 Time Wild type relA- Removal of 1/2 of culture to medium w/o aa

24. Results #2 OD 600 Time Wild type relA- Removal of 1/2 of culture to medium w/o aa relA is essential to stringent response

25. Also... Amino acid/glucose starvation + [ 32 P] (p)ppGpp accumulation No (p)ppGpp accumulation relA- wt

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