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Polymyxin-resistant mutants of Escherichia coli and Salmonella typhimurium accumulate a novel minor lipid that can donate 4-amino-4-deoxy-L-arabinose units (L-Ara4N) to lipid A. We now report the purification of this lipid from a pss- pmrAC mutant of E. coli and assign its structure as undecaprenyl phosphate-α-L-Ara4N. Approximately 0.2 mg of homogeneous material was isolated from an 8-liter culture by solvent extraction, followed by chromatography on DEAE-cellulose, C18 reverse phase resin, and silicic acid. Matrix-assisted laser desorption ionization/time of flight mass spectrometry in the negative mode yielded a single species [M - H]- at m/z 977.5, consistent with undecaprenyl phosphate-α-L-Ara4N (Mr = 978.41). 31P NMR spectroscopy showed a single phosphorus atom at -0.44 ppm characteristic of a phosphodiester linkage. Selective inverse decoupling difference spectroscopy demonstrated that the undecaprenyl phosphate group is attached to the anomeric carbon of the L-Ara4N unit. One- and two-dimensional 1H NMR studies confirmed the presence of a polyisoprene chain and a sugar moiety with chemical shifts and coupling constants expected for an equatorially substituted arabinopyranoside. Heteronuclear multiple-quantum coherence spectroscopy analysis demonstrated that a nitrogen atom is attached to C-4 of the sugar residue. The purified donor supports in vitro conversion of lipid IVA to lipid IIA, which is substituted with a single L-Ara4N moiety. The identification of undecaprenyl phosphate-α -L-Ara4N implies that L-Ara4N transfer to lipid A occurs in the periplasm of polymyxin-resistant strains, and establishes a new enzymatic pathway by which Gram-negative bacteria acquire antibiotic resistance.

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Journal of Biological Chemistry

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