Structure and Biosynthesis of Free Lipid A Molecules That Replace Lipopolysaccharide in Francisella tularensis subsp. novicida

• Published in: Biochemistry (Easton) 2006-12, Vol.45 (48), p.14427-14440
• Main Authors: Wang, Xiaoyuan, Ribeiro, Anthony A, Guan, Ziqiang, McGrath, Sara C, Cotter, Robert J, Raetz, Christian R. H
• Format: Article
• Language: English
• Subjects: Alkalies - chemistry; Carbohydrate Conformation; Francisella tularensis; Francisella tularensis - chemistry; Francisella tularensis - metabolism; Galactosamine - chemistry; Galactosamine - metabolism; Hydrolysis; Ions - chemistry; Lipid A - biosynthesis; Lipid A - chemistry; Lipid A - isolation & purification; Magnetic Resonance Spectroscopy; Mass Spectrometry; Phospholipids - chemistry; Phospholipids - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi061767s

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 α-linked galactosamine residue, as shown by NMR spectroscopy and gas chromatography/mass spectrometry. An α-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.