THE STRUCTURES OF OLIGOSACCHARIDE BISPHOSPHATES ISOLATED FROM THE LIPOPOLYSACCHARIDE OF A RECOMBINANT ESCHERICHIA-COLI STRAIN EXPRESSING THE GENE GSEA [3-DEOXY-D-MANNO-OCTULOPYRANOSONIC ACID (KDO) TRANSFERASE] OF CHLAMYDIA-PSITTACI 6BC

The lipopolysaccharide from the recombinant strain Escherichia coli F515-140 containing the cloned gene gseA [3-deoxy-D-manno-octulopyranosonic acid (Kdo) transferase] from Chlamydia psittaci 6BC was isolated and sequentially de-O-acylated and de-N-acylated. The products were separated by high-performance anion-exchange chromatography into three fractions, two of which contained a single compound. Their structures were elucidated by high-field NMR spectroscopy as alpha-Kdo-(2-->4)-alpha-Kdo-(2-->6)-beta-D-GlcN-(1-->6)-alpha-D-GlcN 1,4'-P-2 (compound 1) (tetrasaccharide bisphosphate) [Holst, O., Broer, W., Thomas-Gates, J. E., Mamat, U. and Brade, H. (1993) Eur. J. Biochem. 214, 703-710] and alpha-Kdo-(2-->4)-[alpha-Kdo-(2--> 8)-]-alpha-Kdo-(2-->4)-alpha-Kdo-(2-->6)-beta-D-GlcN-(1-->6)-alpha-D-GlcN 1,4'-P-2 (compound 4) (hexasaccharide bisphosphate). The third fraction comprised two pentasaccharide bisphosphates, which could be separated by affinity chromatography using an immobilized monoclonal antibody specific for the trisaccharide alpha-Kdo-(2-->8)-alpha-Kdo-(2-->4)-alpha-Kdo. The bound fraction was identified as alpha-Kdo-(2-->8)-alpha-Kdo-(2-->4)-alpha-Kdo- (2-->6)-beta-D-GlcN-(1-->6)-alpha-D-GlcN 1,4'-P-2 (compound 2) [Hoist, O., Broer, W., Thomas-Gates, J. E., Mamat, U. and Brade, H. (1993) Eur. J. Biochem. 214, 703-710], whereas the unbound fraction was identified as alpha-Kdo-(2-->4)-alpha-Kdo-(2-->4)-alpha-Kdo-(2-->6)-beta-D-GlcN-(1-->6)-alpha-D-GlcN 1,4'-P-2 (compound 3). This novel Kdo tetrasaccharide extends our knowledge on multifunctional Kdo transferases.