title: Structure and biosynthesis of free lipid A molecules that replace lipopolysaccharide in Francisella tularensis subsp. novicida.
abstract: Francisella tularensis subsp. novicida U112 phospholipids, extracted without hydrolysis, consist mainly of phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, and two lipid A species, designated A1 and A2. These lipid A species, present in a ratio of 7:1, comprise 15% of the total phospholipids, as judged by 32Pi labeling. Although lipopolysaccharide is detectable in F. tularensis subsp. novicida U112, less than 5% of the total lipid A is covalently linked to it. A1 and A2 were analyzed by electrospray ionization and matrix-assisted laser desorption ionization mass spectrometry, gas chromatography/mass spectrometry, and NMR spectroscopy. Both compounds are disaccharides of glucosamine, acylated with primary 3-hydroxystearoyl chains at positions 2, 3, and 2' and a secondary palmitoyl residue at position 2'. Minor isobaric species and some lipid A molecules containing a 3-hydroxypalmitoyl chain in place of 3-hydroxystearate are also present. The 4'- and 3'-positions of A1 and A2 are not derivatized, and 3-deoxy-d-manno-octulosonic acid (Kdo) is not detectable. The 1-phosphate groups of both A1 and A2 are modified with an alpha-linked galactosamine residue, as shown by NMR spectroscopy and gas chromatography/mass spectrometry. An alpha-linked glucose moiety is attached to the 6'-position of A2. The lipid A released by mild acid hydrolysis of F. tularensis subsp. novicida lipopolysaccharide consists solely of component A1. F. tularensis subsp. novicida mutants lacking the arnT gene do not contain a galactosamine residue on their lipid A. Formation of free lipid A in F. tularensis subsp. novicida might be initiated by an unusual Kdo hydrolase present in the membranes of this organism.
authors.affiliation: Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: Xiaoyuan
authors.initials: X
authors.lastname: Wang
authors.forename: Anthony A
authors.initials: AA
authors.lastname: Ribeiro
authors.forename: Ziqiang
authors.initials: Z
authors.lastname: Guan
authors.forename: Sara C
authors.initials: SC
authors.lastname: McGrath
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Cotter
authors.forename: Christian R H
authors.initials: CR
authors.lastname: Raetz
mesh_terms: D000468:Alkalies / Q000737:chemistry; D002236:Carbohydrate Conformation; D005604:Francisella tularensis / Q000737:chemistry* / Q000378:metabolism*; D005688:Galactosamine / Q000737:chemistry / Q000378:metabolism; D006868:Hydrolysis; D007477:Ions / Q000737:chemistry; D008050:Lipid A / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism
chemical_list: D000468:Alkalies; D007477:Ions; D008050:Lipid A; D010743:Phospholipids; D005688:Galactosamine