Pyruvic‐acid‐containing polysaccharide in the cell wall of Bacillus polymyxa AHU 1385

Abstract

Three acidic polymer fractions with molecular masses of about 16 kDa, 35 kDa and 70 kDa were isolated from lysozyme digests of N-acetylated cell walls of Bacilluspolymyxa AHU 1385 by ion-exchange chromatography and gel chromatography. These fractions, containing mannosamine, glucosamine and pyruvic acid in a molar ratio of about 1 : 1 : 1 together with glycopeptide components, were characterized as polysaccharide-linked glycopeptides with one, two and more polysaccharide chains. On the other hand, treatment of the cell walls with glycine/HCl buffer, pH 2.5, at 100°C for 10 min followed by separation of water-soluble products on ion-exchange chromatography gave three polysaccharide fractions, PS-I -111, whch contained different amounts of pyruvic acid (0, 0.6 and 0.9 residue/mannosamine residue) along with equimolar amounts of mannosamine and glucosamine. Pyruvate-free polysaccharides similar to PS-I were also obtained from PS-11, PS-I11 and polysaccharide-linked glycopeptides by treatment with 10mM HCl at 100°C for 1 h. Results of analyses of these polysaccharide preparations by 'H-NMR and I3C-NMR measurement and methylation, together with data from characterization of fragments obtained by hydrogen fluoride hydrolysis, lead to the most likely structure, +3)[4,6-0-(1-carboxyethylidene)]ManNAc(~1-+4)GlcNAc(~1--f, for the acidic polysaccharide of this strain.Pyruvic acid has been found in a ketal form in exocellular acidic polysaccharides of different bacteria [l] and cell wall teichoic acid of Brevibacterium iodium [2]. In connection with a study on distribution of mannosaminuronic acid and mannosamine among cell walls of gram-positive bacteria [3], the cell wall polysaccharide of Bacillus polymyxa AHU 1385 was shown to contain pyruvic acid as an acidic component, in addition to mannosamine and glucosamine. The present paper reports the structure of this polysaccharide, in which the pyruvic acid is attached to N-acetylmannosamine to form a six-membered ketal ring. MATERIALS AND METHODS Preparation of pyruvic-acid-containing polysaccharide and their glycopeptide complexesThe methods used for cultivation of B. polymyxa AHU 1385 (kindly given by Dr S. Takao, Hokkaido University) and for preparation and N-acetylation of cell walls were the same as those described in a previous paper [3]. For isolation of polysaccharide-linked glycopeptides, the N-acetylated cell walls (400 mg) were exhaustively digested with lysozyme (4 mg), and the polymer fraction obtained from the digest by dialysis and gel chromatography on Sephadex G-50 was subjected to chromatography on a DEAE-cellulose column (2 x 8 cm) equilibrated with 5 mM Tris/HCI buffer, pH 7.2 [4]. The column was eIuted with the same buffer, followed by a linear gradient of 0-0.4 M NaCl in the same buffer. A polymer fraction eluted without salt and fractions eluted with 0.20 M and with 0.35 M NaCl were separately purified by gel chromatography on a Sephacryl S-200 column (1 x 100 cm) in 50 mM (NH4)2C03 and used as the polysaccharide-linked glycopeptides, PS-GP-I (30 mg)...

Are the Surface Layer Homology Domains Essential for Cell Surface Display and Glycosylation of the S-Layer Protein from Paenibacillus alvei CCM 2051T?

“…Pyruvylation of ManNAc residues present in the backbone structure seems to be a feature of many Bacillus SCWPs (9,16,46,47), possibly pinpointing an ancestral origin of this epitope that is necessary for the interaction between the SCWP and SLH domains (10,38). There is a strong correlation between the presence of the csaB gene encoding a putative pyruvyltransferase catalyzing the addition of pyruvate residues onto ManNAc and the nonco- valent anchoring mechanism via SLH domains (11).…”

The Mycobacterium avium Complex gtfTB Gene Encodes a Glucosyltransferase Required for the Biosynthesis of Serovar 8-Specific Glycopeptidolipid

“…M. smegmatis also produces glycolipids containing 4,6-O-(1-carboxyethylidene)-3-O-methyl-Glc as a sugar moiety (25,34), which suggests that both homologues participate in the synthesis of these glycolipids. Sugar residues with a 4,6-O-(1-carboxyethylidene) group substitution have been found in carbohydrates such as extracellular polysaccharide and N-linked glycan, which are produced by some bacteria and yeasts (1,15,20,22,26). It has been shown that an increase in 4,6-O-(1-carboxyethylidene)-containing sugar residues leads to enhanced viscosity of extracellular polysaccharide from Xanthomonas sp., which alters the cell surface properties related to cellular attachment and protection from environmental stress (10).…”

Characterizing non-hydrolyzing Neisseria meningitidis serogroup A UDP-N-acetylglucosamine (UDP-GlcNAc) 2-epimerase using UDP-N-acetylmannosamine (UDP-ManNAc) and derivatives

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