Abstract
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.
Highlights
High performance liquid chromatography (HPLC) enables quantification of bacterial cell-wall composition, yet systematic studies across strains, species, and chemical perturbations are lacking
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
UPLC Accurately Quantifies Muropeptide Abundance from Extremely Low Volumes—To test the resolution of UPLC for muropeptide analysis, we first sought to identify elution conditions that maximize resolution and speed compared with HPLC
Summary
HPLC enables quantification of bacterial cell-wall composition, yet systematic studies across strains, species, and chemical perturbations are lacking. 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. We 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. We present a UPLC protocol for muropeptide analyses and report its application to three Gram-negative model organisms (E. coli, Vibrio cholerae, and P. aeruginosa) to compare peptidoglycan variability both across species and across common laboratory strains, over a range of morphologies and cell sizes. We use UPLC to demonstrate that the cell widening caused by treatment with sublethal doses of A22 is not coupled to changes in either the abundances of any muropeptide species or peptidoglycan density
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