Abstract
3-Deoxy-D-manno-octulosonic acid (Kdo) is an eight-carbon sugar ubiquitous in Gram-negative bacterial lipopolysaccharides (LPS). Although its biosynthesis is well described, no protein has yet been identified as a Kdo hydrolase. However, Kdo hydrolase enzymatic activity has been detected in membranes of Helicobacter pylori and Francisella tularensis and may be responsible for the removal of side-chain Kdo from the LPS core saccharides. We now report the identification of genes encoding a Kdo hydrolase in F. tularensis Schu S4 and live vaccine strain strains, in H. pylori 26695 strain and in Legionella pneumophila Philadelphia 1 strain. We have renamed the genes kdhA for keto-deoxyoctulosonate hydrolase A. Deletion of kdhA abolished Kdo hydrolase activity in membranes of F. tularensis live vaccine strain. The F. tularensis kdhA mutant synthesized a core oligosaccharide containing a Kdo disaccharide with one of the Kdo residues being a terminal side chain. This side-chain Kdo monosaccharide was absent in the wild-type core oligosaccharide. Expression in Escherichia coli of recombinant KdhA from F. tularensis, H. pylori, and L. pneumophila resulted in a reduction of membrane-associated side-chain Kdo. The identification of this previously faceless enzyme will accelerate study of the biosynthetic basis and biologic impact for postbiosynthetic LPS structural modification.
Highlights
The eight-carbon sugar 3-deoxy-D-manno-octulosonic acid (Kdo)2 is an important component of cell wall polysaccharides of Gram-negative bacteria, of some green algae [1], and of most higher plants [2]
We report the identification of genes encoding a Kdo hydrolase in F. tularensis Schu S4 and live vaccine strain strains, in H. pylori 26695 strain and in Legionella pneumophila Philadelphia 1 strain
Kdo hydrolase activity has been described in oysters [10] and in Helicobacter pylori and Francisella tularensis membranes [11, 12], but the gene coding for this enzyme remains unknown
Summary
Construction of F. tularensis LVS ⌬kdhA Deletion Mutant— A ⌬kdhA deletion mutant in F. tularensis LVS was constructed by allelic exchange [20]. To determine the subcellular localization of KdhA protein, total membranes from LVS and ⌬kdhA mutant strains were resolved in 4 –20% Tris-glycine gradient gels (Bio-Rad) and immunoblotted with a rabbit polyclonal anti-KdhASchuS4 serum (1:10,000) or with mAb 2033 monoclonal antibody (1:5,000). Analysis of Glycosyl Residues by High-performance AnionExchange Chromatography (HPAEC)—Each LPS was dissolved in deionized water (15 mg/ml), acetic acid (Mallinckrodt Baker) was added to 1%, and the solution was heated at 90 °C for 2 h This procedure hydrolyzes the ketosidic bond between sidechain and inner Kdos and between inner Kdo and lipid A, which precipitates. Side-chain Kdo amount was measured in E. coli BL21 strains by treating membranes with 1% acetic acid at 90 °C for 2 h Kdo released by this mild hydrolysis was analyzed by HPAEC as described above.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
