Modification of the lipid A portion of LPS with cationic monosaccharides provides resistance to polymyxins, which are often employed as a last resort to treat multidrug-resistant bacterial infections. Here, we describe the use of fluorescent polyisoprenoids, liquid chromatography-mass spectrometry, and bacterial genetics to probe the activity of membrane-localized proteins that utilize the 55-carbon lipid carrier bactoprenyl phosphate (BP). We have discovered that a substantial background reaction occurs when B-strain E. coli cell membrane fractions are supplemented with exogenous BP. This reaction involves proteins associated with the arn operon, which is necessary for the covalent modification of lipid A with the cationic 4-aminoarabinose (Ara4N). Using a series of arn operon gene deletion mutants, we identified that the modification was dependent on ArnC, which is responsible for forming BP-linked Ara4N, or ArnT, which transfers Ara4N to lipid A. Surprisingly, we found that the majority of the Ara4N-modified isoprenoid was due to the reverse reaction catalyzed by ArnT and demonstrate this using heat-inactivated membrane fractions, isolated lipopolysaccharide fractions, and analyses of a purified ArnT. This work provides methods that will facilitate thorough and rapid investigation of bacterial outer membrane remodeling and the evaluation of polyisoprenoid precursors required for covalent glycan modifications.

中文翻译：

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脂多糖是外源性聚异戊二烯磷酸酯的 4-氨基阿拉伯糖供体，通过酶 ArnT 的逆反应

用阳离子单糖修饰脂质 LPS 的一部分可提供对多粘菌素的抗性，多粘菌素通常被用作治疗耐多药细菌感染的最后手段。在这里，我们描述了使用荧光聚异戊二烯、液相色谱-质谱法和细菌遗传学来探测利用 55 碳脂质载体磷酸乳酸杆菌 (BP) 的膜定位蛋白的活性。我们已经发现，当 B 菌株大肠杆菌细胞膜组分补充有外源性 BP时，会发生显着的背景反应。该反应涉及与arn操纵子相关的蛋白质，这是用阳离子 4-氨基阿拉伯糖 (Ara4N) 对脂质 A 进行共价修饰所必需的。使用一系列arn操纵子基因缺失突变体，我们发现修饰依赖于 ArnC，它负责形成 BP 连接的 Ara4N或ArnT，它将 Ara4N 转移到脂质 A。令人惊讶的是，我们发现大部分 Ara4N 修饰的类异戊二烯是由于ArnT 催化的逆反应，并使用热灭活的膜组分、分离的脂多糖组分和纯化的 ArnT 分析证明了这一点。这项工作提供了有助于对细菌外膜重塑进行彻底和快速研究以及对共价聚糖修饰所需的聚异戊二烯前体进行评估的方法。