Structural characterization of the outer core and the O-chain linkage region of lipopolysaccharide from Pseudomonas aeruginosa serotype O5

The point of attachment of the O-chain in the outer core region of Pseudomo nas aeruginosa serotype O5 lipopolysaccharide (LPS) was determined followin g a detailed analysis of the extended core oligosaccharide, containing one trisaccharide O-chain repeating unit, present in both the wild-type strain PAO1 and O-chain deficient mutant strains AK1401 and PAO-rfc. The structure of the extended core oligosaccharide was determined by various mass spectr ometric methods as well as one-dimensional and two-dimensional NMR spectros copy. Furthermore, the one-dimensional analogues of NOESY and TOCSY experim ents were applied to confirm the structure of the outer core region in the O-chain polysaccharide. In both the extended core oligosaccharide and the c ore of the smooth LPS, a loss of one of the beta-glucosyl residues and the translocation of the alpha-rhamnosyl residue, followed by the attachment of the first O-chain repeating unit was observed. This process is complicated and could involve two distinct rhamnosyltransferases, one with alpha-1,6-l inkage specificity and another with alpha-1,3-linkage specificity. It is al so plausible that an alpha-1,3 rhamnosyltransferase facilitates the additio n of the 'new' alpha-rhamnosyl residue that will act as a receptor for the attachment of the single O-antigen repeating unit in the LPS of the semi-ro ugh mutant. The 2-amino-2-deoxy-fucosyl residue of the first O-chain repeat ing unit directly attached to the core was found to have a beta-anomeric co nfiguration instead of an alpha configuration, characteristic for this resi due as a component of the O-chain polysaccharide. The results of this study provide the first example of the mechanistic implications of the structure of the outer core region in a fully assembled O-chain containing LPS, diff ering from the O-chain deficient rough LPS.