4‐O‐(2‐Amino‐2‐deoxy‐α‐d‐glucopyranosyl)‐3‐deoxy‐d‐manno‐2‐octulosonic acid, a constituent of lipopolysaccharides of the genus Pectinatus

Abstract

A disaccharide containing GlcN and 3-deoxy-D-manno-2-octu~osonic acid (Kdo) was detected after acidic methanolysis and peracetylation of hydrofluoric-acid-treated smooth-type and roughtype lipopolysaccharide of Pectinatus cerevisiiphilus and Pectinatus frisingensis, which are strictly anaerobic bacteria capable of growing in packaged beer. The disaccharide was also found in alkalitreated lipopolysaccharide, but was not directly detectable from intact lipopolysaccharide. This suggested that the disaccharide carried a phosphate residue. The position of this phosphate was shown, by GLCMS of appropriately degraded and derivatized samples, to be 0 6 of the GlcN. Methylation analysis of the purified disaccharide revealed that GlcN was linked to position 4 of Kdo. The acetylated derivative of the disaccharide was isolated in pure form, and, by 'H-NMR and I3C-NMR spectroscopy, it was confirmed to possess the structure a-~-GlcpN-(1'+4)-Kdo. In the lipopolysaccharide the amino group of GlcN is free.The Gram-negative bacterial genus Pectinatus contains two species, I? cerevisiiphilus and I? frisingensis. Both species are strict anaerobes, and they occur as contaminants and spoilage organisms in packaged beer [ 1, 21. The cell-surface lipopolysaccharides of the type strains of Pectinatus were recently reported to possess exceptional heterogeneity, and in fact two distinct lipopolysaccharide populations could be extracted from both species [3]. One of these lipopolysaccharides was of low-molecular-mass (rough, R) type, whereas the other contained additional sugar chains resembling smooth ( S ) lipopolysaccharide. Chemical characterization of the lipopolysaccharides indicated possible differences in the core oligosaccharide regions of the R and S type, particularly in the substitution of the 3-deoxyoctulosonic-acid region.These findings prompted us to structurally study the core region of Pectinatus lipopolysaccharides. In the present communication we describe a novel saccharide structure in the lipopolysaccharides of Pectinatus. This saccharide consists of a 3-deoxy-D-manno-2-octulosonic acid (Kdo) carrying at its position 4 an a-pyranosidic GlcN, which is substituted at position 6' by phosphate. MATERIALS AND METHODS Reference compoundsMethyl 2-acetarnido-3,4,6-triri-O-acetyl-2-deoxy-a-~-glucopyranoside was prepared from methyl 2-acetamido-2-de- oxy-a-D-glucopyranoside (a-D-GlcpNAc-OMe, Sigma) by standard derivatization (peracetylation) and purification (silica gel HPLC) procedures. Methyl (methyl 4,5,7,8-tetra-O-acetyl-3-deoxy-a-~-manno-2-octulopyranos~d)onate (aKdop-OMe methyl ester) was kindly provided by Prof. Paul Kosma, Universitat fur Bodenkultur, Vienna, Austria. Lipopoly saccharidesLipopolysaccharides were extracted from P. cerevisiiphilus ATCC 29359= and I? frisingensis ATCC 33332T by the phenol/chloroform/petroleum-ether method (5 : 5 :8, by vol. modification [4]) as described previously [3]. By sequential precipitations with water and acetone, R-type and S-type lipopolysaccharides, respectively, were obtained fr...

Pectinatus, Megasphaera and Zymophilus

“…Several exceptional properties are assigned to Pectinatus LPSs, including the production of at least two distinct types of LPS by one strain (Helander et al, 1992), the presence of the phosphorylated disaccharide α-D-GlcpN-(1'-4)-Kdo (3-deoxy-D-manno-oct-2-ulopyranosonic acid) in the LPS core (Helander et al, 1993), the resistance of the lipid A-polysaccharide linkage to acid (Helander et al, 1994), and the predominance of furanosidic 6-deoxyhexoses in the O-specific chains (Senchenkova et al, 1995). The lipid A backbone of P. cerevisiiphilus and P. frisingensis is composed of the common bisphosphorylated β1'-6-linked glucosamine (GlcN) disaccharide, with almost quantitative substitution of the ester-linked phosphate by 4-amino-4-deoxyarabinose (Helander et al, 1994).…”

Lipopolysaccharides of anaerobic beer spoilage bacteria of the genusPectinatus– lipopolysaccharides of a Gram-positive genus

“…2) was, however, better amenable to structural analysis, and the structures of the LPS carbohydrate backbone of this species has been thoroughly elucidated [39]. The structural analyses revealed that the position of the disaccharide component consisting of GlcN and Kdo, previously reported for LPS of P. cerevisiiphilus and P. frisingensis type strains [38] is in the inner part of the core oligosaccharide, and confirmed the presence of the putative phosphate-linked Ara4N at position 6 of the GlcN residue. The larger major structure of the carbohydrate backbone consists of 24 monosaccharides (including the lipid A backbone), whereas the shorter is composed of 14 saccharide residues; in addition, minor components lacking a terminal glucose in the smaller (lipid A-proximal) fraction were identified.…”

“…4). These are likely to reside in the core oligosaccharide, but hitherto only one has been assigned ( [38,39]; see Chapter 4.3). Based on published literature of 31 P NMR signals in LPS [35][36][37], the signal at 3 ppm was assigned to the glycosidic monoester, and the diester signal at )1.38 ppm (b) was assigned to the ester-linked phosphate of lipid A at position 4 0 , carrying a stoichiometric substitution by L L -Arap4N.…”

“…The structure of the disaccharide fragment W-D was previously reported[38]. (a) Strains VTT E-82164 and VTT E-79100 T ; (b) strain VTT E-79104.…”

Structure of the exceptionally large nonrepetitive carbohydrate backbone of the lipopolysaccharide of Pectinatus frisingensis strain VTT E‐82164

“…The lipid A was shown to be quantitatively substituted at the 4′‐phosphate and partially at the glycosidic phosphate by 4‐amino‐4‐deoxy‐β‐ l ‐arabinose [6]. There are no structural data on Pectinatus core structures, except a report that LPS of both P. frisingesis and P. cerevisiiphilus contain a disaccharide structure, a phosphorylated GlcN linked to O4 of a Kdo residue, tentatively assigned to the core region [7].…”