Lipopolysaccharide Is a 4-Aminoarabinose Donor to Exogenous Polyisoprenyl Phosphates through the Reverse Reaction of the Enzyme ArnT.
Lipopolysaccharide Is a 4-Aminoarabinose Donor to Exogenous Polyisoprenyl Phosphates through the Reverse Reaction of the Enzyme ArnT.
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DOI：
10.1021/acsomega.1c04036
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发表时间：
2021-10-05
期刊：
影响因子：
4.1
通讯作者：
Troutman JM
中科院分区：
文献类型：
--
作者：
Scarbrough BA;Eade CR;Reid AJ;Williams TC;Troutman JM
用阳离子单糖修饰 LPS 的脂质 A 部分可提供对多粘菌素的抵抗力，多粘菌素通常被用作治疗多重耐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 bactoprenyl 磷酸盐 (BP) 的膜定位蛋白的活性。我们发现，当向 B 株大肠杆菌细胞膜组分中添加外源 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) 共价修饰脂质 A 所必需的。使用一系列 arn 操纵子基因缺失突变体，我们发现这种修饰依赖于 ArnC，ArnC 负责形成 BP 连接的 Ara4N 或 ArnT，将 Ara4N 转移到脂质 A。令人惊讶的是，我们发现大多数 Ara4N 修饰的类异戊二烯是由于 ArnT 催化的逆反应造成的，并使用热灭活的膜组分、分离的脂多糖组分、以及纯化的 ArnT 的分析。这项工作提供了有助于彻底、快速研究细菌外膜重塑和评估共价聚糖修饰所需的聚异戊二烯前体的方法。
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.
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DOI：
10.1007/978-1-62703-245-2_15
发表时间：
2013
期刊：
Methods in molecular biology (Clifton, N.J.)
影响因子：
--
作者：
Hankins JV;Madsen JA;Needham BD;Brodbelt JS;Trent MS
通讯作者：
Trent MS
影响因子：
3.6
作者：
Gunn, JS;Lim, KB;Miller, SI
通讯作者：
Miller, SI
DOI：
10.1099/13500872-145-5-1263
发表时间：
1999-05-01
期刊：
MICROBIOLOGY-UK
影响因子：
--
作者：
Guan, S;Bastin, DA;Verma, NK
通讯作者：
Verma, NK
DOI：
10.1073/pnas.1220220110
发表时间：
2013-05-07
影响因子：
11.1
作者：
Gantt, Richard W.;Peltier-Pain, Pauline;Thorson, Jon S.
通讯作者：
Thorson, Jon S.
影响因子：
6.8
作者：
Kahler, Charlene M.;Sarkar-Tyson, Mitali;Vrielink, Alice
通讯作者：
Vrielink, Alice