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Determination of (p)ppGpp Levels During Stringent Response in Streptomyces coelicolor by Thin Layer Chromatography

薄层色谱法测定天蓝色链霉菌在应急反应过程中的(p)ppGpp水平

Smitha Sivapragasam Smitha Sivapragasam
Anne Grove Anne Grove

发布: 2016年11月05日第6卷第21期 DOI: 10.21769/BioProtoc.1995 浏览次数: 11450

评审: Valentine V TrotterDaan C. SwartsModesto Redrejo-Rodriguez

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Cover of Molecular Microbiology, featuring study using the protocol.

May 2016

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Abstract

The stringent response in bacteria is a stress response that is mediated by the signaling molecules guanosine tetraphosphate and pentaphosphate [(p)ppGpp], alarmones that are also directly related to virulence. Therefore, determination of (p)ppGpp levels is crucial for studying the stringent response. The protocol here outlines in a step-wise manner the detection of (p)ppGpp in the bacterium Streptomyces coelicolor during stringent response (Strauch et al., 1991) by thin layer chromatography (TLC). In the example shown here, stringent response is induced by addition of serine hydroxamate, an inhibitor of seryl tRNA synthetase. This protocol was first published in Molecular Microbiology (Sivapragasam and Grove, 2016).

Background

Thin layer chromatography has been used for analyzing (p)ppGpp levels during stringent response in various bacterial species for a long time, and it is a generally accepted method for this purpose. However, previously published protocols only summarized the main concepts, and it was challenging to identify a comprehensive protocol that comprised every step of the procedure. We present here a detailed protocol that has been optimized for studying stringent response in S. coelicolor. Steps unique to handling of S. coelicolor cultures have been identified, and the protocol can therefore be readily adapted to other bacterial species. The method relies on the use of TLC plates that incorporate polyethyleneimine (PEI), which is a strong basic anion exchanger. PEI is therefore the matrix of choice for separation of ionic compounds such as phosphorylated nucleosides (Calderón-Flores et al., 2005; Mechold et al., 2013; Strauch et al., 1991).

Materials and Reagents

  1. Polyethyleneimine (PEI)-cellulose TLC plates (Sigma-Aldrich, catalog number: Z122882 )
  2. 1.5 ml sterile microcentrifuge tubes with cap
  3. Aluminium foil
  4. Streptomyces coelicolor
  5. 32P-labelled orthophosphate (PerkinElmer, catalog number: NEX053H001MC )
    Note: Use of radioactive materials requires institutional authorization.
  6. Serine hydroxamate (Sigma-Aldrich, catalog number: S4503 )
  7. MilliQ quality water (dH2O)
  8. Formic acid (Thermo Fisher Scientific, Fisher Scientific, catalog number: A118P100 )
  9. 100% ethanol at room temperature
  10. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: 60218 )
  11. 3-morpholinopropane-1-sulfonic acid (MOPS) (AMRESCO, catalog number: 0670 )
  12. Sucrose (Thermo Fisher Scientific, Fisher Scientific, catalog number: BP220-1 )
  13. Magnesium sulfate heptahydrate (AMRESCO, catalog number: 0662 )
  14. Dextrose/glucose (VWR, catalog number: BDH9230 )
  15. BactoTM yeast extract (BD, catalog number: 212750 )
  16. BactoTM peptone (BD, catalog number: 211677 )
  17. BactoTM tryptone (BD, catalog number: 211705 )
  18. Potassium phosphate dibasic (K2HPO4) (Sigma-Aldrich, catalog number: P3786 )
  19. Casamino acids (BD, catalog number: 228830 )
  20. L-histidine monohydrochloride monohydrate (Sigma-Aldrich, catalog number: H8125 )
  21. L-tryptophan (Sigma-Aldrich, catalog number: T0254 )
  22. L-tyrosine (Sigma-Aldrich, catalog number: T3754 )
  23. L-proline (Sigma-Aldrich, catalog number: P0380 )
  24. Springs (stainless) for dispersing the mycelia (e.g., Grainger, catalog number: 1NCH7 ) (specific to dispersal of S. coelicolor)
  25. Dry ice
  26. Guanosine monophosphate (Sigma-Aldrich, catalog number: G8377 ) (optional)
  27. Guanosine triphosphate (Sigma-Aldrich, catalog number: G8877 ) (optional)
  28. Modified MOPS minimal media (for growing S. coelicolor, see Recipes)
  29. 1.5 M KH2PO4 (pH 3.4) (see Recipes)
  30. 13 M formic acid (see Recipes)
  31. 100 mM serine hydroxamate (see Recipes)
  32. ISP1 medium (for growing S. coelicolor, see Recipes)

Equipment

  1. 50 ml glass Erlenmeyer flasks
  2. TLC chamber with lid (Sigma-Aldrich, catalog number: Z126195 )
  3. Phosphorimager (GE Healthcare)
  4. Phosphor screens with storage cassettes (GE Healthcare)
  5. Refrigerated microcentrifuge with speed up to 16,000 x g and rotor that fits 1.5 ml centrifuge tubes
  6. Shaker-incubator with temperature control

Software

  1. ImageQuant software

Procedure

English
中文翻译

文章信息

版权信息

© 2016 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Sivapragasam, S. and Grove, A. (2016). Determination of (p)ppGpp Levels During Stringent Response in Streptomyces coelicolor by Thin Layer Chromatography. Bio-protocol 6(21): e1995. DOI: 10.21769/BioProtoc.1995.

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分类

微生物学 > 微生物生物化学 > 其它化合物

微生物学 > 微生物生理学 > 胁迫反应

生物化学 > 其它化合物 > 鸟苷五磷酸 

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