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 puriêd 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 identiêd 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 polysaccharide; 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 identiêd.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 identiêd 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 puriêd 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 unidentiêd 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 unidentiêd 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 unidentiêd 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. Methylation Linkage Analysis of EPS, drEPS, and Aldobiourinic Acid

a Molar ratios Methylated sugar Relative retention timeb (as alditol acetate) EPS drEPS aldobiourinic acid

3,4-Me2-Rha 0.70 0.89 0.89 1.00

2,4-Me2-Rha 0.72 1.11 0.94 —

2,3,6-Me3-Glc 1.00 1.00 1.00 —

3,4,6-Me3-Glc (dideuterated) 0.96 — 0.82 —

a 3,4-Me2-Rha＝1,2,5-tri-O-acetyl-3,4-di–O–methylrhamnitol, etc. b Relative to 2,3,6-Me3-Glc.

Table 2. Assignment of the Positive Ions Obtained in FAB-MS Experiment with ABOE Derivatives

Observed Derivative ions Proposed sequence (m Wz) I 398 [Rha-ABOE+H]+ 420 [Rha-ABOE+Na]+ 574 [GlcA-Rha-ABOE+H]+ 736 [Glc-GlcA-Rha-ABOE+H]+ 758 [Glc-GlcA-Rha-ABOE+Na]+ 904 [Rha-Glc-GlcA-Rha-ABOE+Na]+

II 414 [Glc-ABOE+H]+ 582 [Rha-Glc-ABOE+Na]+ 728 [Rha-Rha-Glc-ABOE+Na]+ 1066 [Glc-GlcA-Rha-Rha-Glc-ABOE+Na]+ 1212 [Rha-Glc-GlcA-Rha-Rha-Glc-ABOE+Na]+

III 398 [Rha-ABOE+H]+ 420 [Rha-ABOE+Na]+ 566 [Rha-Rha-ABOE+Na]+ 904 [Glc-GlcA-Rha-Rha-ABOE+Na]+ 1050 [Rha-Glc-GlcA-Rha-Rha-ABOE+Na]+

IV 398 [Rha-ABOE+H]+ + Fig. 1. HPLC Chromatogram of the ABOE Derivatives of 566 [Rha-Rha-ABOE+Na] + Partially Hydrolyzed EPS. 742 [GlcA-Rha-Rha-ABOE+Na]

HPLC was done on a column of TSKgel ODS-80TM (Tosoh). The ABOE derivatives were eluted with 0.1 M ammonium acetate buŠer (pH 4.0) mixed with varied concentrations of acetonitrile (linear gradient from 29 to 39 volz acetonitrile for found by methylation analysis. The collected data for 50min).The‰owratewassetat1.0mlWmin and detection of the derivative I showed that it has a sequence of Rha- ABOE derivatives was done by monitoring the absorbance at Glc-GlcA-Rha. From the data for derivative II, a se- 305 nm. quence of Rha-Glc-GlcA-Rha-Rha-Glc was suggested. A sequence of Rha-Glc-GlcA-Rha-Rha-Glc was obtained from the data for derivative III. The data was also found here that EPS is a straight-chained for derivative IV clearly demonstrated a sequence of heteropolysaccharide. GlcA-Rha-Rha. From these results, it is assumable that EPS is constructed of a repeating unit of Glc- FAB-MS analysis GlcA-Rha-Rha. Considering the sugar linkage desig- A partial hydrolysate of EPS was labeled with nated by methylation analysis, a repeating unit of the ABOE and separated by reverse-phase HPLC following structure was suggested. (Fig. 1). Based on retention time, peaks at 39 and ª4)-D-Glc p-(1ª2)-D-GlcA p-(1ª2)-L-Rha p-(1ª3)- 48 min were identiˆed as the derivatives from glucose L-Rha p-(1ª and rhamnose, respectively. Substances with peaks appearing between 16 to 24 min (derivatives I, II, III, and IV) were recovered and put through FAB-MS NMR studies of EPS analysis. The primary structure of these ABOE The 1D 1H NMR spectrum for EPS (Fig. 2) showed derivatives was deˆned as shown in Table 2, based on four anomeric protons, conˆrming that EPS is com- the molecular mass and fragment ions detected by posed of a repeating unit of tetrasaccharide. The su- FAB-MS in consideration of a sequence of GlcA-Rha gar residues in the unit are tentatively designated as Structure of the Polysaccharide from Sphaerotilus natans 1549

Fig. 3. Part of the NOESY (tm 500) Spectrum for EPS. The cross peaks identiˆed are indicated by A1WD2 (a cross peak between A1 and D2), etc.

Table 3. 1H NMR Chemical Shifts (ppm) of EPS

Residue H-1 H-2 H-3 H-4 H-5 H-6 CH3 A 5.36 3.64 3.85 3.67 4.08 NDa — Fig. 2. 400 MHz 1D 1H NMR Spectrum for EPS. B 5.30 4.14 3.94 3.44 3.85 — 1.28 (7.58)b C 4.87 4.16 3.63 ND 3.44 — 1.31 (5.61) D 4.71 (7.29) 3.52 3.59 3.82 3.72 — — residues A, B, C, and D. Signals due to the anomeric a ND, not determined. protons of the residues (A1, B1, C1, and D1) are b coupling constants (Hz) are included in parentheses. shown in Fig. 2. Note that only D1 is a doublet signal. Based on these signals, A2, B2, C2, and D2 were then assigned by COSY experiment. In Fig. 2, the ing the result of methylation analysis. Chemical

CH3 proton signals of Rha residues were clearly ob- shifts of the protons deˆned in this study are listed in served at 1.28–1.32 ppm. These signals were assigned Table 3. Depending on all the data described above, tobeB6andC6byCOSYandTOCSYexperiments. we concluded that EPS secreted by S. natans is con- Therefore, residues B and C were Rha residues. More structed from the following tetrasaccharide repeating assignments were made by TOCSY experiment. The unit. NOESY spectrum of the anomeric proton region is ª4)-a-D-Glc p-(1ª2)-b-D-GlcA p-(1ª2)-a-L-Rha p- shown in Fig. 3. Besides a cross peak between C1 and (1ª3)-b-L-Rha p-(1ª C2 (C1WC2), cross peaks of C1WC3 and C1WC5 indicating strong 1,3 and 1,5 diaxial interactions are observed for the anomeric proton resonance of Discussion residue C; whereas only the B1WB2 cross peak was observed for the anomeric proton resonance of We demonstrated that EPS of S. natans is com- residue B. Thus, residues B and C were assigned to a- posed of glucose, glucuronic acid, and rhamnose in Rha and b-Rha, respectively. An NOE (nuclear Over- molecularratiosof1:1:2.Incontrast,Gaudyand hauser eŠect) was observed between B1 and C3 in- Wolfe reported that EPS contains fucose, glucose, dicating a sequence of a-Rha-(1ª3)-b-Rha. A cross galactose, and glucuronic acid.10) Initially, we at- peak was observed between the doublet anomeric tempted to trace their procedure, i.e., recover EPS proton signal (D1) and B2. It is likely that residue D from the cell surface by vigorous washing with dis- is b-GlcA because a sequence of GlcA-(1ª2)-Rha tilled water.10) However, it was impossible to remove was shown by sugar composition analysis, methyla- the cells and the sheaths contaminating the slimy tion linkage analysis, and an FAB-MS experiment. In EPS. It is likely that EPS is tightly associated with addition to the D1WB2 cross peak, strong cross peaks the sheaths and free cells. Therefore, we decided to of D1WD3 and D1WD5 were observed for the anomer- recover the EPS that is over-produced and released ic proton resonance of residue D. These two peaks into the culture ‰uid during shaking cultivation. It is demonstrate the presence of strong 1,3 and 1,5 diaxi- true that diŠerences in growth conditions aŠect the al interactions, conˆrming that residue D is the b composition of bacterial EPS,18) but the purity of anomer of GlcA. Residue A was considered to be a- EPS the former researchers prepared is much more Glc because A1 is a singlet signal, and a sequence of questionable. We are afraid that the composition of Glc-GlcA was indicated by methylation linkage anal- EPS has been misunderstood. ysis and FAB-MS experiment. The A1WD2 cross peak The structure of EPS from S. natans is similar to proved that a-Glc is 1ª2 linked to b-GlcA, support- that of gellan. Gellan, which is secreted by an a- 1550 M. TAKEDA et al. peoteobacteria, Sphingomonas paucimobilis,isan 4) Suzuki, T., Kanagawa, T., and Kamagata, Y., Iden- acidic polysaccharide mainly composed of glucose, tiˆcation of a gene essential for sheathed structure glucuronic acid, and rhamnose.19) The repeating unit formation in Sphaerotilus natans, a ˆlamentous of this linear polysaccharide is the tetrasaccharide sheathed bacterium. Appl. Environ. Microbiol., 68, shown below.20,21) 365–371 (2002). 5) Siering, P. L. and Ghiorse, W. C., Phylogeny of the ª3)-b-D-Glc p-(1ª4)-b-D-GlcA p-(1ª4)-b-D-Glc p- Sphaerotilus-Leptothrix group inferred from mor- (1ª4)-a-L-Rha p-(1ª phological comparisons, genomic ˆngerprinting, and 16S ribosomal DNA sequence analyses. Int. J. Syst. Gellan forms gels in the presence of either Bacteriol., 46, 173–182 (1996). + + monovalent or divalent cations such as K ,Na, 6) Sly, L. I., Hodgkinson, M. C., and Arunpairojana, Ca2+,andMg2+.22) The gels are very clear and are V., Deposition of manganese in a drinking water formed over a wide pH range at lower polysaccharide distribution system. Appl. Environ. Microbiol., 56, concentration compared to conventional gelling 628–639 (1990). agents.23) Despite the resemblance in chemical struc- 7) Spring, S., K äampfer, P., Ludwig, W., and Schleifer, ture, gelation of 1z EPS sol was not observed when K.-H., Polyphasic characterization of the genus Lep- tothrix: new descriptions of Leptothrix mobilis sp. KCl, NaCl, CaCl2,orMgCl2 was added at a concen- nov. and Leptothrix discophora sp.nov.mon.rev. tration of 1 mM. Additionally, the viscosity of EPS and emended description of Leptothrix cholodnii sol was much less than that of gellan. These results emend. System. Appl. Microbiol., 19, 634–643 indicated that EPS is not suitable for a food additive (1996). as a gelling agent. 8) Emerson, D. and Ghiorse, W. 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