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dc.contributor.authorProchnow, Hans
dc.contributor.authorFetz, Verena
dc.contributor.authorHotop, Sven-Kevin
dc.contributor.authorGarcía-Rivera, Mariel A
dc.contributor.authorHeumann, Axel
dc.contributor.authorBrönstrup, Mark
dc.date.accessioned2019-03-01T13:41:34Z
dc.date.available2019-03-01T13:41:34Z
dc.date.issued2019-02-05
dc.identifier.citationAnal Chem. 2019 Feb 5;91(3):1863-1872. doi: 10.1021/acs.analchem.8b03586. Epub 2018 Nov 11en_US
dc.identifier.issn1520-6882
dc.identifier.pmid30485749
dc.identifier.doi10.1021/acs.analchem.8b03586
dc.identifier.urihttp://hdl.handle.net/10033/621709
dc.description.abstractInfections by Gram-negative pathogens represent a major health care issue of growing concern due to a striking lack of novel antibacterial agents over the course of the last decades. The main scientific problem behind the rational optimization of novel antibiotics is our limited understanding of small molecule translocation into, and their export from, the target compartments of Gram-negative species. To address this issue, a versatile, label-free assay to determine the intracellular localization and concentration of a given compound has been developed for Escherichia coli and its efflux-impaired ΔTolC mutant. The assay applies a fractionation procedure to antibiotic-treated bacterial cells to obtain periplasm, cytoplasm, and membrane fractions of high purity, as demonstrated by Western Blots of compartment-specific marker proteins. This is followed by an LC-MS/MS-based quantification of antibiotic content in each compartment. Antibiotic amounts could be converted to antibiotic concentrations by assuming that an E. coli cell is a cylinder flanked by two half spheres and calculating the volumes of bacterial compartments. The quantification of antibiotics from different classes, namely ciprofloxacin, tetracycline, trimethoprim, and erythromycin, demonstrated pronounced differences in uptake quantities and distribution patterns across the compartments. For example, in the case of ciprofloxacin, a higher amount of compound was located in the cytoplasm than in the periplasm (592 ± 50 pg vs 277 ± 13 pg per 3.9 × 10en_US
dc.language.isoenen_US
dc.publisherACS Publicationsen_US
dc.rightsAttribution-NonCommercial-ShareAlike 4.0 International*
dc.rights.urihttp://creativecommons.org/licenses/by-nc-sa/4.0/*
dc.titleSubcellular Quantification of Uptake in Gram-Negative Bacteria.en_US
dc.typeArticleen_US
dc.contributor.departmentHZI, Helmholtz Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig Germany.en_US
dc.identifier.journalAnalytical chemistryen_US
dc.source.journaltitleAnalytical chemistry


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