Accumulation of a polyisoprene-linked amino sugar in polymyxin-resistant Salmonella typhimurium and Escherichia coli - Structural characterization and transfer to lipid A in the periplasm

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 fr om a pss(-) pmrA(C) mutant of E. coli and assign its structure as undecapre nyl phosphate-alpha -L-Ara4N. Approximately 0.2 mg of homogeneous material was isolated from an 8-liter culture by solvent extraction, followed by chr omatography on DEAE-cellulose, C18 reverse phase resin, and silicic acid. M atrix-assisted laser desorption ionization/time of flight mass spectrometry in the negative mode yielded a single species [M - H]- at m/z 977.5, consi stent with undecaprenyl phosphate-alpha -L-Ara4N (M-r = 978.41). P-31 NMR s pectroscopy showed a single phosphorus atom at -0.44 ppm characteristic of a phosphodiester linkage. Selective inverse decoupling difference spectrosc opy demonstrated that the undecaprenyl phosphate group is attached to the a nomeric carbon of the L-Ara4N unit. One- and two-dimensional H-1 NMR studie s confirmed the presence of a polyisoprene chain and a sugar moiety with ch emical shifts and coupling constants expected for an equatorially substitut ed 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 l ipid IIA, which is substituted with a single L-Ara4N moiety. The identifica tion of undecaprenyl phosphate-alpha -L-Ara4N implies that L-Ara4N transfer to lipid A occurs in the periplasm of polymyxin-resistant strains, and est ablishes a new enzymatic pathway by which Gram-negative bacteria acquire an tibiotic resistance.