Isolation and Characterization of Structural Components of Bacillus cereus AHU 1356 Cell Walls

Self Cite

Structural studies were carried out on the acidic polysaccharide fraction obtained from lysozyme digest of the cell walls of Bacillus subtilis AHU 1031. The polysaccharide fraction contained N-acetylmannosaminuronic acid (ManNAcA), N-acetylglucosamine (GlcNAc), glucose, glycerol and phosphorus in a molar ratio of 2 :2 :4:1 :I, together with glycopeptide components. The results of analyses involving Smith degradation, chromium trioxide oxidation, methylation and proton magnetic resonance spectroscopy led to the conclusion that the backbone chain of the polysaccharide has the repeating unit +6)Glc(a1+3/4)ManNAcA(~1+4)GlcNAc(~1+. About 50 of the N-acetylglucosamine residues in the backbone chain seem to be substituted at C-3 by the glycosidic branches, glycerol phospho-6-glucose, while the other half seem to be substituted by glucose.D-Mannosaminuronic acid has been found in the acidic wall polysaccharide of Micrococcus luteus [I, 21, the capsular polysaccharide of Vibrio parahaemolyticus [3,4], the surface antigen of Staphylococcus aureus T [5], the K-7 antigen of Escherichia coli [6,7] and the pseudomurein of Halococcus morrahuae [8]. This saccharide has also been shown to occur in Salmonella montevideo [9] and Shigella sonnei [lo] as an essential constituent of enterobacterial common antigens, but information on the occurrence of this hexosaminuronic acid in nonpathogenic bacteria is still limited.In our previous study, mannosaminuronic acid was found in the cell walls of four strains of Bacillus megaterium and one strain each of Bacillus subtilis and Bacillus polymyxa [ll]. Strong acid hydrolysis of these cell wall preparations gave a fragment denoted as compound X, which was tentatively characterized as a disaccharide mannosaminuronosyl-glucosamine. Furthermore, the membranes of' these bacterial strains were shown to catalyze incorporation of N-acetylglucosamine from UDP-N-acetylglucosamine into polymers in the presence of UDP-N-acetylmannosaminuronic acid and UDP-glucose. These facts prompted us to elucidate the structure of the mannosaminuronic-acid-containing polymers in the bacillus strains. The present paper reports the isolation and structure of the mannosaminuronic-acid-containing polysaccharide from B. subtilis AHU 1031. MATERIALS AND METHODS Isolation of polysaccharide fractionsThe methods used for the culture of Bacillus subtilis AHU 1031 (kindly supplied by Dr S. Takao, Hokkaido University) and for the isolation and N-acetylation of the cell walls of this strain were those described previously [11]. The N-acetylated cell walls (2.3 g) were exhaustively digested Abbreviations. ManNAc, N-acetylmannosamine; ManNAcA, Nacetylmannosaminuronic acid ; water-soluble carbodiimide, 1 -ethyl-3-(3-dimethylaminopropyl)-carbodiimide; TU-GP, teichuronic-acidlinked glycopeptide.Enzyme. Lysozyme (EC 3.2.1.17).with 50mg lysozyme, and the digest was fractionated by gel filtration through Sephadex G-50 after dialysis [12]. The polymer fraction excluded from the gel was applied on a DEAE-cellulose column, and the colum...

Section: Characterization Of Oligosaccharidessupporting

confidence: 73%

“…with 50mg lysozyme, and the digest was fractionated by gel filtration through Sephadex G-50 after dialysis [12]. The polymer fraction excluded from the gel was applied on a DEAE-cellulose column, and the column was eluted with a linear gradient of NaCl in 5 mM Tris/HCl, pH 7.2.…”

Section: Isolation Of Polysaccharide Fractionsmentioning

confidence: 99%

The primary structure of teichuronic acid in Bacillus subtilis AHU 1031

Yoneyama

Araki

Ito

1984

Self Cite

“…Hexose was determined by the phenol/H2S04 method [22] with rhamnose as a standard and also determined by gasliquid chromatography as trimethylsilyl derivatives or alditol acetates after hydrolysis of samples in 1 M HCl at 100°C for 1 h. Hexosamine and amino acid were determined with a Shibata AA-autoanalyzer after hydrolysis of samples in 4 M HCl at 100°C for 4 h [23]. Total hexosamine was determined The O-polysaccharide fraction obtained as described in Materials and Methods was treated with 0.1 M NaOH and 0.2 M NaBH4 at 37°C for 24 h. After neutralization, the product was applied on a Sephadex G-50 column (2.6 x 100 cm) in 50 mM (NH4)2C03.…”

Section: Other Methods and Materialsmentioning

confidence: 99%

Characterization of a polysaccharide component of lipopolysaccharide from Pseudomonas aeruginosa IID 1008 (ATCC 27584) as D‐rhamnan

Yokota

Kaya

Sawada

et al. 1987

Self Cite

Structural studies were carried out on a rhamnose‐rich polysaccharide isolated from the O‐polysaccharide fraction of lipopolysaccharide in Pseudomonas aeruginosa IID 1008 (ATCC 27584) after destruction of the major O‐specific chain by alkaline treatment. The isolated polysaccharide contained rhamnose, 3‐O‐methyl‐6‐deoxyhexose, glucose, xylose, alanine, galactosamine and phosphorus in a molar ratio of 67:6.9:4.3:2.1:1.1:1.0:4.1. Data from analysis involving Smith degradation, methylation, 1H‐NMR spectroscopy and optical rotation measurement showed that the polysaccharide was built up of three moieties, a rhamnan chain composed of about 70 D‐rhamnose residues, the core chain and an oligosaccharide chain comprising 3‐O‐methyl‐6‐deoxyhexose, xylose, rhamnose and probably glucose. The repeating unit of the rhamnan chain was indicated to have the following structure: →3)D‐Rha(α1 → 3)D‐Rha(α1 → 2)D‐Rha(α1 →. This structure is identical with that proposed previously for the repeating unit of the side chain of lipopolysaccharide from plant pathogenic bacteria Pseudomonas syringae pv. morsprunorum C28 [Smith, A. R. W., Zamze, S. E., Munro, S. M., Carter, K. J. and Hignett, R. C. (1985) Eur. J. Biochem. 149, 73–78].

“…Total hexosamine was determined by the method of Tsuji et al [8] after N-deacetylation of samples by acid hydrolysis (2 M HCI, 100°C, 1 h); pyruvic acid, by the enzymatic method using L-lactate dehydrogenase [9] after treatment of samples with mild acid (0.01 M HC1, 1OO"C, 1 h) followed by neutralization with 0.1 M NaOH; reducing groups, by the method of Park and Johnson [lo]. Hexosamine and hexosaminitol were analyzed by gas-liquid chromatography after acid hydrolysis (4 M HCl, 100°C, 4 h) of samples followed by N-acetylation and trimethylsilylation [Ill. Amino acids, amino sugars and muramic acid 6-phosphate were determined with an amino acid analyzer as described previously [12]. NaIO, oxidation and Smith degradation were carried out under the conditions similar to those described previously [4].…”

Section: Other Materials and Analytical Methodsmentioning

confidence: 99%

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

Kojima

Kaya

Araki

et al. 1988

Self Cite

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)...