title: Interaction of aminoglycosides with the outer membranes and purified lipopolysaccharide and OmpF porin of Escherichia coli.
abstract: The mechanism of uptake of aminoglycosides across the outer membrane of Escherichia coli was reevaluated. Porin-deficient mutants showed no alteration in gentamicin or kanamycin susceptibility. Furthermore, the influence of kanamycin on intrinsic tryptophan fluorescence of porin OmpF (Y. Kobayashi, and T. Nakae, Eur. J. Biochem. 151:231-236, 1985) was shown to be strongly influenced by protein concentration and EDTA. This led to the hypothesis that aminoglycoside-mediated increases and decreases in intrinsic tryptophan fluorescence were due to aggregation-disaggregation of OmpF mediated by interaction at a divalent cation binding site on OmpF. Gentamicin, kanamycin, and polymyxin B increased E. coli outer membrane permeability to the hydrophobic fluorescent compound 1-N-phenyl-naphthylamine (NPN) and the peptidoglycan-degrading enzyme lysozyme. Addition of Mg2+ blocked these permeabilizing activities. Furthermore, gentamicin and polymyxin B bound to Mg(2+)-binding sites on E. coli lipopolysaccharide, as determined in dansyl polymyxin displacement experiments. A polymyxin-resistant, lipopolysaccharide-altered pmr mutant of E. coli had a fourfold-lower MIC of gentamicin and kanamycin and was more poorly permeabilized to 1-N-phenylnaphthylamine than was its parent strain. These data were consistent with uptake of aminoglycosides across the E. coli outer membrane by the self-promoted uptake mechanism.
authors.affiliation: Department of Microbiology, University of British Columbia, Vancouver, Canada.
authors.forename: R E
authors.initials: RE
authors.lastname: Hancock
authors.forename: S W
authors.initials: SW
authors.lastname: Farmer
authors.forename: Z S
authors.initials: ZS
authors.lastname: Li
authors.forename: K
authors.initials: K
authors.lastname: Poole
mesh_terms: D000900:Anti-Bacterial Agents / Q000378:metabolism / Q000494:pharmacology*; D001425:Bacterial Outer Membrane Proteins / Q000187:drug effects* / Q000378:metabolism; D003470:Culture Media; D004926:Escherichia coli / Q000187:drug effects / Q000378:metabolism*; D005839:Gentamicins / Q000494:pharmacology; D007612:Kanamycin / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000378:metabolism*; D011112:Polymyxin B / Q000494:pharmacology; D018272:Porins
chemical_list: D000900:Anti-Bacterial Agents; D001425:Bacterial Outer Membrane Proteins; D003470:Culture Media; D005839:Gentamicins; D008070:Lipopolysaccharides; D018272:Porins; D007612:Kanamycin; D011112:Polymyxin B