Infectious Diseases, Novartis Institutes for BioMedical Research, Emeryville, CA, USA.
hemotune AG, Schlieren, Switzerland.
Methods Mol Biol. 2022;2548:267-278. doi: 10.1007/978-1-0716-2581-1_16.
Metabolic labeling of lipopolysaccharides (LPS) with the exogenous azido analog of 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) or Kdo-N allows for both live-cell and molecular analysis of the outer membrane composition and biosynthesis in different Gram-negative bacteria. Here, we describe Kdo-N incorporation into bacterial cells, followed by click labeling with a fluorescent dye. The fluorescently labeled LPS can be analyzed from lysed cells by SDS-PAGE and from intact cells by microscopy and flow cytometry. These methods have been applied to the Gram-negative bacteria Escherichia coli and Klebsiella pneumoniae, which possess the sialic acid transporter NanT that is also capable of transporting exogenous Kdo and Kdo analogs into the cytoplasm for incorporation into nascent LPS.
利用 3-去氧-D-甘露-2-庚酮糖酸（Kdo）或 Kdo-N 的外源叠氮类似物对脂多糖（LPS）进行代谢标记，可用于对不同革兰氏阴性菌的外膜组成和生物合成进行活细胞和分子分析。在这里，我们描述了 Kdo-N 掺入细菌细胞，然后用荧光染料进行点击标记。荧光标记的 LPS 可以通过 SDS-PAGE 从裂解细胞中进行分析，也可以通过显微镜和流式细胞术从完整细胞中进行分析。这些方法已应用于革兰氏阴性菌大肠杆菌和肺炎克雷伯菌，它们都具有唾液酸转运蛋白 NanT，该蛋白还能够将外源性 Kdo 和 Kdo 类似物转运到细胞质中，用于掺入新生的 LPS 中。
