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 from a pss(-) pmrA(C) mutant of E. coli and assign its structure as undecaprenyl phosphate-alpha-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-alpha-l-Ara4N (M(r) = 978.41). (31)P 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 (1)H 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 IV(A) to lipid II(A), which is substituted with a single l-Ara4N moiety. The identification of undecaprenyl phosphate-alpha-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.
Trent
M S
MS
Department of Biochemistry and the Duke NMR Spectroscopy Center, Duke University Medical Center, Durham, North Carolina 27710, USA.
Ribeiro
A A
AA
Doerrler
W T
WT
Lin
S
S
Cotter
R J
RJ
Raetz
C R
CR
D000606:Amino Sugars / Q000302:isolation & purification* / Q000494:pharmacology*; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics*; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000737:chemistry*; D002467:Cell Nucleus / Q000378:metabolism; D002474:Cell-Free System; D002845:Chromatography; D003636:DEAE-Cellulose / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D004983:Ethanolamines / Q000737:chemistry* / Q000494:pharmacology; D006868:Hydrolysis; D008050:Lipid A / Q000737:chemistry* / Q000378:metabolism*; D008055:Lipids / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009154:Mutation; D009227:Myristic Acids / Q000494:pharmacology; D019308:Palmitic Acid / Q000494:pharmacology; D019897:Periplasm / Q000737:chemistry*; D010758:Phosphorus / Q000737:chemistry; D011113:Polymyxins / Q000494:pharmacology*; D011485:Protein Binding; D011487:Protein Conformation; D017368:Protein Prenylation; D012486:Salmonella typhimurium / Q000378:metabolism*; D012824:Silicic Acid / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000606:Amino Sugars; D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D002241:Carbohydrates; D004983:Ethanolamines; D008050:Lipid A; D008055:Lipids; D009227:Myristic Acids; D011113:Polymyxins; C081902:pmrA protein, Bacteria; D012824:Silicic Acid; C017418:alpha-hydroxymyristic acid; D010758:Phosphorus; D019308:Palmitic Acid; C040134:4-amino-4-deoxyarabinose; C005448:phosphorylethanolamine; D003636:DEAE-Cellulose