Isolation and preparation of bacterial cell walls for compositional analysis by ultra performance liquid chromatography.
Isolation and preparation of bacterial cell walls for compositional analysis by ultra performance liquid chromatography.
复制标题
DOI：
10.3791/51183
复制
发表时间：
2014-01-15
期刊：
影响因子：
--
通讯作者：
Huang KC
中科院分区：
文献类型：
--
作者：
Desmarais SM;Cava F;de Pedro MA;Huang KC
细菌细胞壁在生长和分裂过程中对细胞形状的决定至关重要，并在几个大气压量级的膨胀压力下保持细胞的机械完整性。在不同形状和大小的细菌王国中，细胞壁是由肽聚糖组成的，肽聚糖是一种由短肽交联的糖链大分子网络。肽聚糖对细菌生理的核心重要性是其作为抗生素靶点的基础，并推动了基因、结构和细胞生物学的研究，研究它在生长和分裂过程中是如何强大地组装的。尽管如此，仍然需要广泛的研究来充分表征肽聚糖合成中的关键酶活性和细菌细胞壁的化学组成。高效液相色谱法（HPLC）是一种强大的分析方法，用于定量在各种环境和遗传条件下生长的细菌壁的化学成分差异，但其通量往往受到限制。在这里，我们提出了一个简单的程序，通过高效液相色谱和超高效液相色谱（UPLC）分离和制备细菌细胞壁，用于肽聚糖的生物分析，UPLC是高效液相色谱的延伸，利用泵提供高达15000 psi的超高压，而高效液相色谱为6000 psi。结合这里介绍的细菌细胞壁制备，小体积样品进样器、高采样率的检测器、更小的样品体积和更短的UPLC运行时间将使大多数生物实验室的肽聚糖组成和基本细菌细胞生物学的新发现具有高分辨率和通量，并且可以使用超离心机和UPLC。
The bacterial cell wall is critical for the determination of cell shape during growth and division, and maintains the mechanical integrity of cells in the face of turgor pressures several atmospheres in magnitude. Across the diverse shapes and sizes of the bacterial kingdom, the cell wall is composed of peptidoglycan, a macromolecular network of sugar strands crosslinked by short peptides. Peptidoglycan's central importance to bacterial physiology underlies its use as an antibiotic target and has motivated genetic, structural, and cell biological studies of how it is robustly assembled during growth and division. Nonetheless, extensive investigations are still required to fully characterize the key enzymatic activities in peptidoglycan synthesis and the chemical composition of bacterial cell walls. High Performance Liquid Chromatography (HPLC) is a powerful analytical method for quantifying differences in the chemical composition of the walls of bacteria grown under a variety of environmental and genetic conditions, but its throughput is often limited. Here, we present a straightforward procedure for the isolation and preparation of bacterial cell walls for biological analyses of peptidoglycan via HPLC and Ultra Performance Liquid Chromatography (UPLC), an extension of HPLC that utilizes pumps to deliver ultra-high pressures of up to 15000 psi, compared with 6000 psi for HPLC. In combination with the preparation of bacterial cell walls presented here, the low-volume sample injectors, detectors with high sampling rates, smaller sample volumes, and shorter run times of UPLC will enable high resolution and throughput for novel discoveries of peptidoglycan composition and fundamental bacterial cell biology in most biological laboratories with access to an ultracentrifuge and UPLC.
登录
查看更多内容
DOI：
10.1038/nrmicro2677
发表时间：
2011-12-28
期刊：
Nature reviews. Microbiology
影响因子：
--
作者：
Typas A;Banzhaf M;Gross CA;Vollmer W
通讯作者：
Vollmer W
影响因子：
3.2
作者：
BOYLEN, CW;ENSIGN, JC
通讯作者：
ENSIGN, JC
影响因子：
3.6
作者：
Desmarais SM;De Pedro MA;Cava F;Huang KC
通讯作者：
Huang KC
影响因子：
4.4
作者：
Wilson, ID;Nicholson, JK;Plumb, RS
通讯作者：
Plumb, RS
影响因子：
3.2
作者：
Joseleau-Petit, Daniele;Liebart, Jean-Claude;D'Ari, Richard
通讯作者：
D'Ari, Richard