| Analysis of Gram-negative Bacteria Peptidoglycan by Ultra-performance Liquid Chromatography
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Author:
Date:
2020-10-05
[Abstract] Bacteria are surrounded by a protective peptidoglycan cell wall. Provided that this structure and the enzymes involved are the preferred target for our most successful antibiotics, determining its structural and chemical complexity is of the highest interest. Traditionally, high-performance liquid chromatography (HPLC) analyses have been performed, but these methods are very time consuming in terms of sample preparation and chromatographic separation. Here we describe an optimized method for preparation of Gram-negative bacteria peptidoglycan and its subsequent analysis by ultra-performance liquid chromatography (UPLC). The use of UPLC in peptidoglycan analyses provides a dramatic reduction of the sample volume and hands-on time required and, furthermore, permits in-line mass spectrometry ...
[摘要] [摘要]细菌被保护性肽聚糖细胞壁包围。如果这种结构和涉及的酶是我们最成功的抗生素的首选靶标，那么确定其结构和化学复杂性便是最重要的。传统上，已经进行了高效液相色谱（HPLC）分析，但是这些方法在样品制备和色谱分离方面非常耗时。在这里我们描述了一种优化的制备方法 革兰氏阴性细菌肽聚糖及其随后的超高效液相色谱（UPLC）分析。在肽聚糖分析中使用UPLC可以大大减少所需的样品量和动手时间，此外，还可以对U PLC解析的多肽进行在线质谱（MS），从而有助于对其进行鉴定。这种方法提高了我们执行高通量分析以更好地了解细胞壁生物学的能力。
[背景]细菌由肽聚糖（PG）的细胞壁所包围，除了到结构的作用，传达细胞形状和保护细菌免受外部损害，作为抗生物，化学屏障iCal和物理应力。murein囊或PG是细胞壁的主要成分。革兰氏阴性细菌在周质空间中呈现单层（Gan等，2008），而它构成了一个厚的网状结构，在革兰氏阳性细菌中具有多个堆积和交联的层（Pasquina-Lemonche等，2020）。Ť他细胞壁是交联的聚糖链的三维网状结构包围所述电池主体（Glauner等人，1988; Typas ...
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| Bacterial Cell Wall Precursor Phosphatase Assays Using Thin-layer Chromatography (TLC) and High Pressure Liquid Chromatography (HPLC)
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Author:
Date:
2018-03-20
[Abstract] 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 ...
[摘要] 肽聚糖包裹细菌细胞质膜以保护细胞免于因膨胀而导致的溶解。 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连接至载体脂质十一碳烯醇。 脂质II是糖肽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”酶来降解以诱导裂解。 在这里，我们分别描述了两种使用薄层色谱法（TLC）和高压液相色谱法（HPLC）的方案来测定磷脂酶如Colicin M或来自中间链球菌的LXG毒素蛋白TelC对脂质II的消化，的。 TLC方法也可以监测十一异戊二烯基（pyro）磷酸盐的消化，而HPLC方法允许分离脂质II的二 - ，单 - 或非磷酸化二糖五肽产物。
【背景】肽聚糖（PG）球囊是一种必需的细菌大分子，它可以保护细胞免受由于其膨胀引起的破裂并保持细胞的形状（Vollmer和Bertsche，2008; Typas等人，2012）。 PG由通过短肽连接的聚糖链组成。来自不同物种的PG在肽的结构和二级修饰的存在方面有所不同（Vollmer等人，2008）。 ...
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