1 / 26

Stringent response in E. coli

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

didina
Download Presentation

Stringent response in E. coli

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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 relA spoT

  8. Bacillus subtilis Stress ppGpp STRINGENTRESPONSE E. coli relA spoT 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!!

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

  20. Try Something Else... Pspac relA

  21. 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

  24. 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

More Related