Bacterial Cell Wall Precursor Phosphatase Assays Using Thin-layer Chromatography (TLC) and High Pressure Liquid Chromatography (HPLC).
Bacterial Cell Wall Precursor Phosphatase Assays Using Thin-layer Chromatography (TLC) and High Pressure Liquid Chromatography (HPLC).
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DOI：
10.21769/bioprotoc.2761
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发表时间：
2018-03-20
期刊：
影响因子：
0.8
通讯作者：
Vollmer W
中科院分区：
文献类型：
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作者：
Pazos M;Otten C;Vollmer W
肽聚糖包裹细菌细胞质膜以保护细胞免于由于膨压而溶解。肽聚糖合成的最后步骤需要称为脂质II的膜锚定底物，其中肽聚糖亚基通过焦磷酸部分与载体脂质十一异戊二烯连接。脂质II是糖肽类抗生素和几种抗微生物肽的靶标，并且通过参与细菌竞争的“攻击”酶降解以诱导裂解。在这里，我们描述了两个协议，使用薄层色谱法（TLC）和高压液相色谱法（HPLC），分别测定的消化脂质II的磷酸酶，如大肠杆菌素M或LXG毒素蛋白泰尔克从中间链球菌。TLC方法还可以监测十一异戊二烯基（焦）磷酸的消化，而HPLC方法允许分离脂质II的二-、单-或未磷酸化的二糖五肽产物。
Peptidoglycan encases the bacterial cytoplasmic membrane to protect the cell from lysis due to the turgor. The final steps of peptidoglycan synthesis require a membrane-anchored substrate called lipid II, in which the peptidoglycan subunit is linked to the carrier lipid undecaprenol via a pyrophosphate moiety. Lipid II is the target of glycopeptide antibiotics and several antimicrobial peptides, and is degraded by ‘attacking’ enzymes involved in bacterial competition to induce lysis. Here we describe two protocols using thin-layer chromatography (TLC) and high pressure liquid chromatography (HPLC), respectively, to assay the digestion of lipid II by phosphatases such as Colicin M or the LXG toxin protein TelC from Streptococcus intermedius. The TLC method can also monitor the digestion of undecaprenyl (pyro)phosphate, whereas the HPLC method allows to separate the di-, mono- or unphosphorylated disaccharide pentapeptide products of lipid II.