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).
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