Desmarais Samantha M, Tropini Carolina, Miguel Amanda, Cava Felipe, Monds Russell D, de Pedro Miguel A, Huang Kerwyn Casey
From the Departments of Bioengineering and.
From the Departments of Bioengineering and the Biophysics Program, Stanford University, Stanford, California 94305.
J Biol Chem. 2015 Dec 25;290(52):31090-100. doi: 10.1074/jbc.M115.661660. Epub 2015 Oct 14.
The bacterial cell wall is a network of glycan strands cross-linked by short peptides (peptidoglycan); it is responsible for the mechanical integrity of the cell and shape determination. Liquid chromatography can be used to measure the abundance of the muropeptide subunits composing the cell wall. Characteristics such as the degree of cross-linking and average glycan strand length are known to vary across species. However, a systematic comparison among strains of a given species has yet to be undertaken, making it difficult to assess the origins of variability in peptidoglycan composition. We present a protocol for muropeptide analysis using ultra performance liquid chromatography (UPLC) and demonstrate that UPLC achieves resolution comparable with that of HPLC while requiring orders of magnitude less injection volume and a fraction of the elution time. We also developed a software platform to automate the identification and quantification of chromatographic peaks, which we demonstrate has improved accuracy relative to other software. This combined experimental and computational methodology revealed that peptidoglycan composition was approximately maintained across strains from three Gram-negative species despite taxonomical and morphological differences. Peptidoglycan composition and density were maintained after we systematically altered cell size in Escherichia coli using the antibiotic A22, indicating that cell shape is largely decoupled from the biochemistry of peptidoglycan synthesis. High-throughput, sensitive UPLC combined with our automated software for chromatographic analysis will accelerate the discovery of peptidoglycan composition and the molecular mechanisms of cell wall structure determination.
细菌细胞壁是由短肽交联的聚糖链网络（肽聚糖）；它负责细胞的机械完整性和形状确定。液相色谱可用于测量构成细胞壁的胞壁肽亚基的丰度。已知交联程度和平均聚糖链长度等特征在不同物种间存在差异。然而，尚未对给定物种的菌株进行系统比较，这使得评估肽聚糖组成变异性的来源变得困难。我们提出了一种使用超高效液相色谱（UPLC）进行胞壁肽分析的方案，并证明UPLC实现了与高效液相色谱（HPLC）相当的分离度，同时进样体积要求低几个数量级，洗脱时间仅为其几分之一。我们还开发了一个软件平台来自动识别和定量色谱峰，结果表明该平台相对于其他软件具有更高的准确性。这种结合实验和计算的方法表明，尽管存在分类学和形态学差异，但来自三种革兰氏阴性菌的菌株的肽聚糖组成大致保持不变。在使用抗生素A22系统改变大肠杆菌细胞大小时，肽聚糖组成和密度保持不变，这表明细胞形状在很大程度上与肽聚糖合成的生物化学过程解耦。高通量、灵敏的UPLC与我们用于色谱分析的自动化软件相结合，将加速肽聚糖组成的发现以及细胞壁结构确定的分子机制研究。
