Construction of a Deep-rough Mutant of Burkholderia cepacia ATCC 25416 and Characterization of Its Chemical and Biological Properties

• Published in: The Journal of biological chemistry Vol. 278; no. 3; pp. 1647 - 1655
• Main Authors: Gronow, Sabine, Noah, Christian, Blumenthal, Antje, Lindner, Buko, Brade, Helmut
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 17-01-2003; American Society for Biochemistry and Molecular Biology
• Subjects: Base Sequence; Burkholderia cepacia - chemistry; Burkholderia cepacia - enzymology; Burkholderia cepacia - genetics; Burkholderia cepacia - physiology; Cloning, Molecular; DNA Primers; Electrophoresis, Polyacrylamide Gel; Glycosyltransferases - genetics; Humans; Macrophages - metabolism; Macrophages - microbiology; Molecular Sequence Data; Mutation; Oligosaccharides - chemistry; Spectrometry, Mass, Electrospray Ionization; Transferases - genetics; Tumor Necrosis Factor-alpha - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M206942200

Burkholderia cepacia is a bacterium with increasing importance as a pathogen in patients with cystic fibrosis. The deep-rough mutant Ko2b was generated from B. cepacia type strain ATCC 25416 by insertion of a kanamycin resistance cassette into the gene waaC encoding heptosyltransferase I. Mass spectrometric analysis of the de-O-acylated lipopolysaccharide (LPS) of the mutant showed that it consisted of a bisphosphorylated glucosamine backbone with two 3-hydroxyhexadecanoic acids in amide-linkage, 4-amino-4-deoxyarabinose (Ara4N) residues on both phosphates, and a core oligosaccharide of the sequence Ara4N-(1 → 8)d-glycero-d-talo-oct-2-ulosonic acid (Ko)-(2 → 4)3-deoxy-d-manno-oct-2-ulosonic acid (Kdo). The mutant allowed investigations on the biosynthesis of the LPS as well as on its role in human infection. Mutant Ko2b showed no difference in its ability to invade human macrophages as compared with the wild type. Furthermore, isolated LPS of both strains induced the production of tumor necrosis factor α from macrophages to the same extent. Thus, the truncation of the LPS did not decrease the biological activity of the mutant or its LPS in these aspects.