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High-throughput, Highly Sensitive Analyses of Bacterial Morphogenesis Using Ultra Performance Liquid Chromatography
Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
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2015 (Engelska)Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 290, nr 52, s. 31090-31100Artikel i tidskrift (Refereegranskat) Published
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Abstract [en]

The bacterial cell wall is a network of glycan strands crosslinked 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.

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American Society for Biochemistry and Molecular Biology, 2015. Vol. 290, nr 52, s. 31090-31100
Nationell ämneskategori
Biomedicinsk laboratorievetenskap/teknologi Medicinsk bioteknologi (med inriktning mot cellbiologi (inklusive stamcellsbiologi), molekylärbiologi, mikrobiologi, biokemi eller biofarmaci)
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URN: urn:nbn:se:umu:diva-114616DOI: 10.1074/jbc.M115.661660ISI: 000367199000037PubMedID: 26468288OAI: oai:DiVA.org:umu-114616DiVA, id: diva2:900905
Tillgänglig från: 2016-02-05 Skapad: 2016-01-25 Senast uppdaterad: 2018-06-07Bibliografiskt granskad

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Cava, Felipe

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Molekylär Infektionsmedicin, Sverige (MIMS)Institutionen för molekylärbiologi (Medicinska fakulteten)
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Journal of Biological Chemistry
Biomedicinsk laboratorievetenskap/teknologiMedicinsk bioteknologi (med inriktning mot cellbiologi (inklusive stamcellsbiologi), molekylärbiologi, mikrobiologi, biokemi eller biofarmaci)

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