Determination of (p)ppGpp Levels During Stringent Response in Streptomyces coelicolor by Thin Layer Chromatography
Determination of (p)ppGpp Levels During Stringent Response in Streptomyces coelicolor by Thin Layer Chromatography
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DOI：
10.21769/bioprotoc.1995
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发表时间：
2016
期刊：
影响因子：
--
通讯作者：
Smitha Sivapragasam;A. Grove
中科院分区：
文献类型：
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作者：
Smitha Sivapragasam;A. Grove
[摘要]细菌的严格反应是一种应激反应，由信号分子鸟苷四磷酸和五磷酸[（p）ppGpp]介导，这些信号分子也与毒力直接相关。因此，测定（p）ppGpp水平对于研究严格响应至关重要。这里的方案以逐步的方式概述了在严格响应期间在细菌天蓝色链霉菌中检测（p）ppGpp（Strauch等，1991）通过薄层色谱法（TLC）。在此处所示的实施例中，通过添加丝氨酸异羟肟酸盐（一种丝氨酰tRNA合成酶抑制剂）诱导严格反应。该方案首次发表于《分子微生物学》（Sivapragasam和格罗夫，2016）。[背景]薄层色谱法长期以来一直用于分析各种细菌物种在严格响应期间的（p）ppGpp水平，并且它是用于该目的的普遍接受的方法。然而，以前发表的协议只总结了主要概念，它是具有挑战性的，以确定一个全面的协议，包括每一个步骤的程序。我们在这里提出了一个详细的协议，已优化研究严格的反应，在S。天蓝色。处理S的独特步骤。已经鉴定了腔肠菌培养物，因此该方案可以容易地适用于其它细菌物种。该方法依赖于TLC板的使用，该TLC板结合了聚乙烯亚胺（PEI），这是一种强碱性阴离子交换剂。因此，PEI是用于分离离子化合物如磷酸化核苷的选择基质
[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