Abstract
The extraction, purification and structural characterization of two lipid A precursors (Ia and Ib) differing only in one hexadecanoic acid are described. Both precursors were synthesized at elevated temperatures by a new mutant of Salmonella typhimurium (mutant Ts5) which is conditionally defective in synthesis of the 3-deoxy-d-manno-octulosonic acid region of lipopolysaccharides.
Both precursors were purified by repeated phenol/chloroform/petroleum ether (PCP) extractions followed by thin layer chromatography. Teh precursor preparation was free of lipopolysaccharides and phospholipids and contained less than 0.1% protein. Structural analysis which included chemical degradation procedures as well as positive ion laser desorption (LDMS) mass spectroscopy of dephosphorylated lipid A precursors showed together that precursor Ia represents a diphosphorylated glucosamine disaccharide containing two ester, two amide-linked residues of 3-hydroxytetradecanoic acid and lacks the ester-linked dodecanoic, tetradecanoic and hexadecanoic acid as well as 3-deoxy-d-manno-octulosonic acid. Precursor Ib has the same basic structure as precursor Ia, but contains in addition one mol of hexadecanoic acid per mol disaccharide which is linked to the 3-hydroxy group of the amide-bound 3-hydroxy-tetradecanoic acid of the reducing, terminal glucosamine residue.
The structure of precursor Ib supports the conclusion that hexadecanoic acid incorporation occurs at an early stage in lipid A biosynthesis prior to the attachment of 3-deoxy-d-manno-octulosonic acid and/or other polar substituents.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Abbreviations
- LDMS:
-
laser desorption mass spectrometry
- KDO:
-
3-Deoxy-d-manno-octulosonic acid
- Ts5:
-
Salmonella typhimurium mutant Ts5
- PCP:
-
phenol/chloroform/petroleum ether
- H2F2 :
-
hydrogen fluoride
References
Galanos C, Lüderitz O, Westphal O (1969) A new method for the extraction of R lipopolysaccharides. Eur J Biochem 9:245–249
Hansen-Hagge T, Lehmann V, Lüderitz O (1984) Free flow electrophoresis as a tool for enrichment of mutants with temperature dependent lethal mutations in lipid A synthesis. Eur J Biochem (accepted)
Lehmann V (1977) Isolation, purification and properties of an intermediate in 3-deoxy-d-manno-octulosonic acid lipid A biosynthesis. Eur J Biochem 75:257–266
Lehmann V, Rupprecht E, Osborn MJ (1977) Isolation of mutants conditionally blocked in the biosynthesis of the 3-deoxy-d-manno-octulosonic-acid lipid A part of lipopolysaccharides derived from Salmonella typhimurium. Eur J Biochem 76:41–49
Loeb T, Zinder ND (1961) A bacteriophage containing ribonucleic acid (RNA). Proc Natl Acad Sci USA 47:282–289
Lowry OH, Roberts NR, Leiner KY, Wu ML, Farr AL (1954) The quantitative histochemistry of brain. I. Chemical methods. J Biol Chem 207:1–17
Nishijima M, Raetz CRH (1981) Characterization of two membrane associated glycolipids from an Escherichia coli mutant deficient in phosphatidylglycerol. J Biol Chem 256:10690–10696
Qureshi N, Takayama K, Heller D, Fenselau C (1983) Position of ester groups in the lipid A backbone of lipopolysaccharides obtained from Salmonella typhimurium. J Biol Chem 258:12947–12951
Rick PD, Osborn MJ (1972) Isolation of a mutant of Salmonella typhimurium dependent on d-arabinose-5-phosphate for growth and synthesis of 3-deoxy-d-manno-octulosonate (ketodeoxyoctonate). Proc Natl Acad Sci, USA, 69:3756–3760
Rick PD, Young DA (1982) Isolation and characterization of a temperatursensitive lethal mutant of Salmonella typhimurium that is conditionally defective in 3-deoxy-d-manno-octulosonate-8-phosphate synthesis. J Bacteriol 150:447–455
Rick PD, Flung LWM, Ho C, Osborn MJ (1977) Lipid A mutants of Salmonella typhimurium. Purification and characterization of a lipid A precursor produced by a mutant in 3-deoxy-d-mannooctulosonate-8-phosphate synthetase. J Biol Chem 252:4904–4912
Rietschel ET, Galanos C, Lüderitz O, Westphal O (1982) The chemistry and biology of lipopolysaccharides and their lipid A component. In: Webb DR (ed) Immunopharmacology and the regulation of leukocyte function. Marcel Dekker, Inc, New York Basel, 9:183–229
Seydel U, Lindner B, Zähringer U, Rietschel ET, Kusomoto S, Shiba T (1984a) Laser desorptions mass spectrometry of synthetic lipid A like compounds. Biomed Mass Spectrom 11:132–141
Seydel U, Lindner B, Wollenweber HW, Rietschel ET (1984b) Structural studies on the lipid A component of enterobacterial lipopolysaccharides by laser desorption mass spectrometry. Eur J Biochem (accepted)
Strominger JL, Park LT, Thompson RE (1959) Composition of the cell wall of Staphylococcus aureus: its relation to the mechanism of action of penicillin. J Biol Chem 234:3263–3268
Waravdekar VS, Saslaw LD (1959) A sensitive method for the estimation of 2-deoxysugars with the use of the malondialde-hydethiobarbituric acid. J Biol Chem 234:1945–1950
Wollenweber HW, Broady KW, Lüderitz O, Rietschel ET (1982) The chemical structure of lipid A: Demonstration of amide-linked 3-acyloxyacyl residues in Salmonella minnesota Re lipopolysaccharide. Eur J Biochem 124:191–198
Author information
Authors and Affiliations
Additional information
This work is dedicated to Prof. Dr. Drews, Freiburg, on the occasion of his 60th birthday
Rights and permissions
About this article
Cite this article
Hansen-Hagge, T., Lehmann, V., Seydel, U. et al. Isolation and structural analysis of two lipid A precursors from a KDO deficient mutant of Salmonella typhimurium differing in their hexadecanoic acid content. Arch. Microbiol. 141, 353–358 (1985). https://doi.org/10.1007/BF00428849
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00428849