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Structural Analysis of the Extracellular Polysaccharide Produced by Sphaerotilus Natans

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Structural Analysis of the Extracellular Polysaccharide Produced by Sphaerotilus Natans Biosci. Biotechnol. Biochem., 66 (7), 1546–1551, 2002 Structural Analysis of the Extracellular Polysaccharide Produced by Sphaerotilus natans Minoru TAKEDA,† Shogo NOMOTO, and Jun-ichi KOIZUMI Division of Materials Science and Chemical Engineering, Faculty of Engineering, Yokohama National University, Yokohama, Kanagawa 240-8501, Japan Received February 13, 2002; Accepted March 18, 2002 An extracellular polysaccharide (EPS) was recovered natans are covered with a cohering slime layer, espe- and puriˆed from the culture ‰uid of a sheathed bacteri- cially when grown under eutrophic conditions.9) Not um, Sphaerotilus natans. Glucose, rhamnose, and only ˆlamentation by the sheath but also the highly aldobiouronic acid were detected in the acid hydrolysate hydroscopic property of the surface of the ˆlaments of EPS by thin-layer chromatography (TLC). The due to EPS might lead to bulking of the activated aldobiouronic acid was found to be composed of sludge abundant in S. natans. Gaudy and Wolfe ana- glucuronic acid and rhamnose by TLC and gas-liquid lyzed the chemical composition of the slime layer and chromatography analyses of the corresponding neutral concluded that it is an EPS composed of fucose, disaccharide. The structure of EPS was identiˆed by galactose, glucose, and glucuronic acid in molar methylation linkage analysis and nuclear magnetic ratios of 1.0:0.77:0.77:0.80.10) However, a detailed resonance. Additionally, partial acid hydrolysates of structural analysis of EPS has not been done. In this EPS were prepared and put through fast atom bom- paper, we demonstrate that EPS of S. natans is a new bardment-mass spectrometry to determine the sugar gellan-like acidic polysaccharide constructed from a sequence of EPS. The resulting data showed that EPS repeating unit containing D-glucose, L-rhamnose, produced by S. natans is a new gellan-like polysaccha- and D-glucuronic acid; thus, our conclusion is incon- ride constructed from a tetrasaccharide repeating unit, sistent with the former report. We believe that the as shown below. knowledge of EPS produced by S. natnas should be ª4)-a-D-Glc p-(1ª2)-b-D-GlcA p-(1ª2)-a-L-Rha p- corrected. (1ª3)-b-L-Rha p-(1ª Materials and Methods Key words: Sphaerotilus natans; extracellular poly- saccharide; structural analysis Isolation of extracellular polysaccharide. Sphaerotilus natans IFO13543T was cultured for EPS Sphaerotilus natans is one of the sheathed ˆlamen- production. Cultivation and isolation of EPS from tous b-proteobacteria often found in polluted the culture ‰uid were done by the procedure de- streams and in activated sludge suŠering from a poor scribed previously.11) Anion exchange and gel ˆltra- settling problem, called bulking.1,2) The sheath is con- tion chromatography were done to conˆrm the structed from 70z (w Ww) polysaccharide, which is homogeneity of EPS. A column of Resource Q (1 ml; composed of glucose and (N-acetyl)galactosamine in Amersham Pharmacia Biotech, Uppsala, Sweden) molarratiosof1:4,and30z (w Ww) peptide rich in equilibrated with 66 mM phosphate buŠer (pH 7) was cysteine.3) Recently, a gene essential for sheath for- used for anion exchange chromatography. The mation in S. natans was identiˆed.4) The most closely column was washed with a linear gradient of NaCl phylogenetically related taxon to S. natans is the ge- (0–1 M) in the same buŠer, and the elution of EPS nus Leptothrix, which is also capable of forming a was monitored by absorbance at 210 nm. Gel sheath and is a typical inhabitant of metal-rich ˆltration chromatography was done using a TSKgel 5,6) streams. The sheath of L. cholodnii (formerly L. G4000PWXL (Tosoh, Tokyo, Japan) column. The discophora SP-6)7) is a complex of a heteropolysac- mobile phase was distilled water, and the elution of charide (composed of amino sugars and uronic acids) polysaccharide was monitored by a refractive index and a cysteine-rich peptide similar to the sheath of S. detector. Molecular mass calibration of the column natans.8) One of the distinguishing phenotypic char- was done with a series of dextrans of diŠerent degrees acteristics between S. natans and L. discophora is of polymerization. whether the secretion of extracellular polysaccharide (EPS) out of the sheath occures.2) The sheaths of S. Sugar composition and methylation linkage ana- † To whom correspondence should be addressed. Tel: +81-45-339-4266; Fax: +81-45-339-4267; Email: mtake＠ynu.ac.jp Structure of the Polysaccharide from Sphaerotilus natans 1547 lyses. Preliminary sugar composition analysis was were recorded on a Br äuker AMX 400 or a JEOL done by thin-layer chromatography (TLC) by the JNM ECP 500 spectrometers at the probe tempera- method described previously.3) Measurement of ture of 609C. Before the NMR experiments, the sam- 12) sugars was done by the alditol acetate method. The ples were lyophilized thrice with D2O(99.9z). Sodi- hydrolysis was done in 2 M tri‰uoroacetic acid at um 4,4-dimethyl-4-silopentane-1-sulphonate (DSS) 1009C for 2 h, and the hydrolysate was put through was used as the internal reference at dH 0. DQF- TLC and alditol acetate derivatization. Identiˆcation COSY and NOESY were recorded at 500 MHz. 1D of the alditol acetate derivatives was done by gas- and TOCSY (tm 106) was recorded at 400 MHz. liquid chromatography (GLC) using a Shimadzu (Kyoto, Japan) GC-15A chromatograph equipped Results with a TC1701 column (GL sciences, Tokyo, Japan). Enantiomeric conˆgurations of monosaccharides Sugar composition of EPS were identiˆed by the formation of the („)-2-butyl EPS of S. natans was recovered from the culture glycosides according to the method of Gerwig ‰uid. The puriˆed EPS was found to be homogene- et al.13,14) Methylation linkage analysis was done us- ous by anion exchange chromatography. On gel ing lithium methylsulˆnyl carbanion as a catalyst.15) ˆltration HPLC, EPS eluted as a single peak but in Characterization of the partially methylated alditol the void volume, suggesting that the molecules tend acetates was done by GLC-mass spectrometry (JMS- to associate with each other in water. By TLC analy- AX500; JEOL, Tokyo, Japan) equipped with a Su- sis of the acid hydrolysate of EPS, glucose, rham- pelco SBP-1 column in the EI mode. nose, and an unidentiˆed sugar were detected. Molar ratios of glucose and rhamnose were determined to Reduction of uronic acid residues. Uronic acid be 1.0:1.0 by GLC analysis. When rEPS was put residues of EPS were reduced to the corresponding through TLC, only glucose and rhamnose were de- neutral sugar residues with NaBH4 in the presence of tected and the unidentiˆed sugar spot was no longer carbodiimide by the method of Taylor and Conrad16) observed. Almost equimolal glucose and rhamnose to obtain carboxy-reduced EPS (rEPS). Deuterium- were found by GLC analysis of the rEPS hydroly- substituted rEPS (drEPS) was also prepared by sate. It is assumed that the unidentiˆed sugar ob- reduction with NaBD4 for identiˆcation of the neu- served on TLC is an aldobiouronic acid composed of tral sugar derived from uronic acid. Conversion of glucuronic acid and rhamnose. To conˆrm this the aldobiouronic acid to the corresponding natural hypothesis, the aldobiouronic acid was separated disaccharide was done with methyl esteriˆcation from the hydrolysate of EPS with an anion exchange followed by LiBH4 according to the method of resin, reduced to the corresponding natural disaccha- Maekawa and Koshijima.17) The aldobiouronic acid ride, hydrolyzed, and analyzed by TLC. Glucose and was recovered from the acid hydrolysate of EPS rhamnose were detected, revealing that EPS is an using a column (1 by 6 cm) packed with AG1-X8, acidic polysaccharide composed of glucose, acetate form (Bio-Rad, Hercules, CA, USA). The glucuronic acid, and rhamnose and that the column was washed with distilled water and 30z glucuronic acid residue binds at its anomeric position acetic acid, in this order. The ˆnal eluate was reco- to a rhamnose residue. An enantiomeric conˆgura- vered and evaporated to obtain the aldobiouronic tion experiment indicated that EPS contains D-glu- acid. cose (D-Glc), D-glucuronic acid (D-GlcA) and L- rhamnose (L-Rha). Fast atom bombardment-mass spectrometry (FAB-MS). EPS was partially hydrolyzed in 0.1 M Methylation analysis and GLC-MS spectrometric tri‰uoroacetic acid at 1009C for 2 h. The partial examination hydrolysate was obtained by evaporation and labeled Results of methylation analysis and GLC-MS spec- with 4-aminobenzoic acid octylester (ABOE) using trometric examination are summarized in Table 1. an ABOE labeling kit (Seikagaku Corporation, The partially methylated alditol acetates derived Tokyo, Japan). ABOE-derivatized oligosaccharides from EPS were 3,4-Me2-Rha, 2,4-Me2-Rha, and (ABOE-oligosaccharides) were separated and puri- 2,3,6-Me3-Glc in molar ratios of 0.89:1.11:1.00. ˆed by reverse-phase HPLC according to the From drEPS, 3,4-Me2-Rha, 2,4-Me2-Rha, 3,4,6-Me3- manufacturer's instruction. After demineralization Glc(dideuterated), and 2,3,6-Me3-Glc were detected with Toyopack ODS-M (Tosoh), ABOE-oligosaccha- in molar ratios of 0.89:0.94:0.82:1.00. These results rides were put through positive ion FAB-MS, which suggested that EPS was composed of equimolal 3- was carried out on a JEOL JMS-AX500 mass spec- linked rhamnose( p), 2-linked rhamnose( p), 4-linked trometer with thioglycerol as the matrix. glucose( p), and 2-linked glucuronic acid( p). In addi- tion, only 3,4-Me2-Rha( p) was obtained from the Nuclear magnetic resonance spectrometry (NMR). aldobiouronic acid, indicating that glucuronic acid 1-Dimensional (1D) and 2D 1H NMR experiments residues are (1ª2) linked to rhamnose residues. It 1548 M. TAKEDA et al. Table 1.
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