title: High-throughput, Highly Sensitive Analyses of Bacterial Morphogenesis Using Ultra Performance Liquid Chromatography.
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.
authors.affiliation: From the Departments of Bioengineering and.
authors.forename: Samantha M
authors.initials: SM
authors.lastname: Desmarais
authors.affiliation: From the Departments of Bioengineering and the Biophysics Program, Stanford University, Stanford, California 94305.
authors.forename: Carolina
authors.initials: C
authors.lastname: Tropini
authors.affiliation: From the Departments of Bioengineering and.
authors.forename: Amanda
authors.initials: A
authors.lastname: Miguel
authors.affiliation: the Department of Molecular Biology and Laboratory for Molecular Infection Medicine Sweden, Umeå Centre for Microbial Research, Umeå University, Umeå, 90187 Sweden.
authors.forename: Felipe
authors.initials: F
authors.lastname: Cava
authors.affiliation: From the Departments of Bioengineering and the Bio-X Program, Stanford University, Stanford, California 94305, and.
authors.forename: Russell D
authors.initials: RD
authors.lastname: Monds
authors.affiliation: the Universidad Autonoma de Madrid, Campus de Cantoblanco, 28049 Madrid, Spain.
authors.forename: Miguel A
authors.initials: MA
authors.lastname: de Pedro
authors.affiliation: From the Departments of Bioengineering and the Biophysics Program, Stanford University, Stanford, California 94305, the Bio-X Program, Stanford University, Stanford, California 94305, and Microbiology and Immunology, Stanford University School of Medicine, Stanford, California 94305, kchuang@stanford.edu.
authors.forename: Kerwyn Casey
authors.initials: KC
authors.lastname: Huang
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D010457:Peptidoglycan / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D010457:Peptidoglycan
keywords: cell wall; cytoskeleton; high-performance liquid chromatography (HPLC); morphogenesis; muropeptide; peptidoglycan