The structure of the O‐specific polysaccharide of Citrobacter O16 containing glycerol phosphate

Abstract

The O-specific polysaccharide, obtained by mild acid degradation of Citrobacrer 016 lipopolysaccharide, consists of D-glucose, D-galactose, 2-acetamido-2-deoxy-~-galactose, glycerol and phosphate in the ratios 2 : 2:2 : 1 :l. Selective cleavage of the polysaccharide was carried out by Smith degradation, N-deacetylation-deamination and dephosphorylation with 48 % hydrofluoric acid, which was accompanied by unexpected splitting of one of the glycosidic linkages. The structures of the oligosaccharides thus obtained were established using 'H-and I3C-NMR spectroscopy, including one-dimensional NOE, two-dimensional rotating-frame NOE, homonuclear and heteronuclear 'qC,lH correlation spectroscopy, and, for the Smith degradation product, positive-and negative-ion-mode fast-atom-bombardment MS and MSMS with collision-induced dissociation. On the basis of these data and the results of methylation analysis, it was concluded that the O-specific polysaccharide has the following repeating unit structure :Like many other enterobacteria, Citrobacter freundii is a serologically highly heterogeneous species [ 11. A number of O-antigens of Citrobacter have been structurally elucidated [2-71 and shown to be neutral glycans or hexosaminoglycans. Some of them are related to the O-antigens of Salmonella [2, 71; others are distinguished by the presence of 4-deoxy-L-arabinose [3-51 which has not been found in other bacterial polysaccharides.We report now the structure of the first acidic O-antigen of Citrobacter 016 containing glycerol phosphate. Fax: f7095 1355328. Abbreviations. 2,5anhTal-ol, 2,5-anhydrotalitol; GalNAc-ol, 2-acetamido-2-deoxygalactitol; CID, collision-induced dissociation; COSY, correlation spectroscopy; D, dimensional; FAB, fast-atombombardment; ROESY, rotating-frame NOE spectroscopy. MATERIALS AND METHODS Miscellaneous methodsGel-permeation chromatography was carried out on a column (70x3 cm) of Sephadex G-50 in pyridine/acetic acid pH 5.5 or a column (80X1.6 cm) of TSK HW 40 (S) in water and monitored with a Technicon sugar analyzer or a Knauer differential refractometer, respectively. HPLC was performed on a column (30X0.8 cm) with Silasorb SPH C18 eluted with water and monitored with a Knauer differential refractometer. Anion-exchange chromatography was achieved on a colu r n (15X0.6 cm) of Durmm DAx4 resin in 0.3 M sodium borate pH 7.0 and monitored with a Technicon sugar analyzer. GLC was carried out using a Hewlett-Packard 5890 instrument equipped with a glass capillary column (25 mX0.2 mm) coated with Ultra 1 stationary phase in a temperature gradient of 180-290 "C in sugar analysis or 150-250°C in methylation analysis at a rate of lO"C/min. GLCfMS was run on the same instrument equipped with a Hewlett-Packard 5970 MS detector under the same chromatographic conditions.

Structures of the O‐specific Polysaccharide Chains of Pectinatus cerevisiiphilus and Pectinatus frisingensis Lipopolysaccharides

“…The coupling constants determined for the two predominant forms (P-pyranose, J,,2 1.5,Jz.3 3.9, J , 3.3, J4,5 9.7 and J5,6 6.3 Hz, and p-furanose, J,,2 4.4, Jz,, 6.5, J1. 4 3.4, J4,5 6.3 and J5,6 6.7 Hz) fitted well with the published data for altrose [9, 101. As judged by the negative value of the specific optical rotation, 6-deoxyaltrose has the L configuration.…”

“…2 3 ppm, 6,31.45 ppm). Selective spin decoupling [2], one-dimensional NOE 131, two-dimensional rotating-frame NOE spectroscopy (ROESY) [4], two-dimensional correlation spectroscopy (COSY) [3 J and heteronuclear lTC,'H-COSY [3] were performed as described.Miscellaneous methods. Optical rotations were measured with a Jasco DIP 360 polarimeter in water at 20°C.…”

The Lipid A from Vibrio fischeri Lipopolysaccharide

“…There are precedents in the literature for the incorporation of GroP on the O-antigen region of LPS (27)(28)(29)(30) but no previous reports of such modifications on lipid A. To our knowledge, no lysophosphatidic acids or phosphatidic acids have ever been detected on LPS before.…”

Phosphate release and heavy metal accumulation by biofilm-immobilized and chemically-coupled cells of acitrobacter sp. pre-grown in continuous culture

“…EPM contains substantial mobile phosphate (by 31 P NMR), which provides nucleation foci for metal precipitation (Macaskie, 1995a). There are some studies describing the nature and role of phosphorus within bacterial exopolymers (e.g., Kocharova et al, 1994;Strain et al, 1983a,b), but given the possible role of phosphate as a complexing ligand for heavy metals, this, and the relative amount and structure of EPM in the planktonic and biofilm cells, warrant more detailed study. However, by this argument the enzyme of the biofilm cells should have been more, not less, resistant to uranyl toxicity.…”