FEMS Microbiology Letters 51 (1988) 1-6 Published by Elsevier FEM 03178 Identification of L-iduronic acid as a constituent of the major extracellular polysaccharide produced by Butyriuibrio fibrisoluens strain X6C61 Robert J. Stack, Ronald D. Plattner and Gregory L. Cote Northern Regional Research Cen:er,. Agricultural Research Service, u.s. Department ofAgriculture, 181:J N. University St., Peoria, 11., U.S.A. Received 8 February 1988 Accepted 12 February 1988 Key words: L-iduronic acid; Iduronolactone; Butyriuibrio fibrisolvens; Rumen; Extracellular polysaccharide 1. SUMMARY 2. INTRODUCTION Butyrivibrio fibrisolvens strain X6C61 produces Butyrivibrio fibrisolvens is one of the most fre­ two extracellular polysaccharides (EPS-I and EPS­ quently isolated species of ruminal bacteria [1,2]. II) separable by anion-exchange chromatography. There are, at present, a large number of isolates The neutral sugar constituents of EPS-I were iden­ that fit the species description, with correspond­ tified by gas-liquid chromatography (GLC) as the ingly wide range of reported metabolic activities alditol acetates of rhamnose, mannose, galactose, [3]. glucose, and an unidentified component. These Stack [4] has recently reported that many strains results were confirmed using thin-layer chro­ of B. fibrisolvens produce EPS containing unusual matography (TLC). Neutral sugar analysis of monosaccharide constituents. For example, B. EPS-II, which eluted from DEAE-Sephadex at 0.4 fibrisolvens strain CF3 produces an EPS which M NaCl, yielded the alditol acetates of rhamnose, contains L-altrose [5], the first reported occurrence galactose, glucose, and idose. However, idose was of this hexose in nature. However, analysis of not found when hydrolysates of EPS-II were L-altrose-containing EPS by conventional alditol analysed by TLC. Further investigations showed acetate procedures was ambiguous, due to the that the iditol hexaacetate detected via GLC was acid-catalyzed formation of 1,6-anhydroaltrose. an artifact of the commonly-used procedures for Following reduction and acetylation, both altritol neutral sugar analysis. This compound was instead hexaacetate and 2,3,4-tri-O-acetyl-1,6­ generated from L-iduronic acid, as shown by anhydroaltrose were produced. These two com­ GLC-MS studies. pounds yielded GLC peaks coincident with the alditol acetates of mannose and fucose, respec-' tively [5]. Correspondence to: R.J. Stack, Northern Regional Research Center, Agricultural Research Service, U.S. Department of Nonetheless, GLC analysis of alditol acetate Agriculture, 1815 N. University St., Peoria, IL 61604, U.S.A. derivatives remains a useful method for the de- 0378-1097/88/$03.50 .:g 1988 Federation of European Microbiological Societies 2 termination of the neutral sugar composltlOn of aminopropyl)carbodiimide (EDC) and reduced polysaccharides [6]. However, uronic acids usually with either sodium borohydride (NaBH 4 ) or can not be identified or quantitated by these pro­ sodium borodeuteride (NaBD4 ) using a modifi­ cedures, and are generally determined by other cation of the procedure described by Taylor and methods. Conrad [10]. EPS-II (25 mg) was dissolved in 5 rnl During the course of our studies on extracellu­ H 20 and solid EDC (60 mg) was slowly added lar polysaccharide (EPS) produced by various while the pH was maintained at 4.75 with dilute strains of B. fibrisoluens, one strain, X6C61, pro­ HCl (10-25 mM). The EDC-activated carboxyl duced an EPS which yielded iditol hexaacetate group was reduced with either 2 M NaBH4 or 2 M upon hydrolysis, reduction, and acetylation NaBD4 (10 rnI) over several hours, while the pH according to the method of Albersheirn et al. [6]. was maintained at 7.0-7.2 with 1-2 M HCI. The While these data would seem to indicate that the reaction mixture was acidified to pH 2 with 12 M EPS of B. fibrisoluens strain X6C61 contains idose, HCl, and quickly returned to pH 7 with 10 M a more thorough investigation has revealed that it NaOH. These preparations were designated as contains L-iduronic acid instead. EPS-II-EDC/NaBH4 (or EPS-II-EDC/NaBD4 ) and were dialyzed against water at 4 0 C and lyophilized. 3. MATERIALS AND METHODS 3.4. Determination of the absolute configuration of 3.1. Organism and growth conditions iduronic acid B. fibrisoluens strain X6C61, used in all studies, The absolute configuration of the iduronic acid was kindly provided by N.O. van Gylswyk, Na­ in EPS-II was inferred from the configuration of tional Chemical Research Laboratory, Pretoria, the idose in EPS-II-EDC/NaBH • This was de­ Republic of South Africa. It was isolated from a 4 termined by analyzing the acetylated di­ roll-tube containing 3% xylan-agar which had been astereomeric glycosides prepared from (-)-2-oc­ inoculated from the rumen of a sheep fed corn tanol and hydrolyzates of EPS-II-EDC/NaBH , stover. Cultures were grown on the chemically 4 as described by Leontein et al. [11]. defined medium of Cotta and Hespell [7], as previ­ ously described [5]. 3.5. Miscellaneous techniques 3.2. Polysaccharide purification Neutral sugar analyses were done according to Crude EPS was obtained from culture super­ Albersheirn et al. [6], as previously described [5]. natants as previously described [5]. Crude EPS TLC separation of EPS hydrolysates were per­ (50-100 mg) was dissolved in 10-20 rnl of 10 mM formed on K5 silica gel plates (Whatman, Inc., potassium phosphate buffer pH 7.0, applied to a Clifton, NJ) using acetonitrile/water (9: 1) as the 2.5 X 8 cm column of DEAE-Sephadex A-25 solvent [12]. Carbohydrates were visualized on (Pharmacia, Piscataway, NJ) which had been equi­ developed plates using the N-(1-naphthyl) librated with the same buffer, and eluted with a ethylenediamine dihydrochloride (Aldrich Chem­ linear gradient of buffered sodium chloride (0-2.0 ical Co., Milwaukee, WI) spray reagent described M, 800 rnI). 10 rnl fractions were collected and by Bounias [13]. An idose/1,6-anhydroidose aliquots of each fraction were analyzed for neutral standard for TLC was prepared by heating 5 carbohydrate content via anthrone [8] and for mg/rnl L-idose (Sigma, St. Louis, MO) in 2 M uronic acids via the harmine procedure [9]. Pooled trifluoroacetic acid (TFA) for 1 h at 100 0 C. Re­ fractions (designated EPS-I and EPS-II) were di­ duction and acetylation of this mixture afforded alyzed against water at 4 0 C and lyophilized. an iditol hexaacetate/2,3,4-tri-O-acetyl-1,6­ anydroidose standard for GLC and GLC-MS. 3.3. Uronic acid reduction Electron impact and chemical ionization mass The uronic acid(s) in EPS-II were reacted with spectra of alditol acetates were obtained as previ­ the water-soluble diimide 1-ethyl-3-(3-dimethyl- ously described [5]. Total carbohydrate was mea- 3 sured by the anthrone procedure [8] using glucose 1.0 2.0 as a standard. 0.9 1.8 3.0 - 0.8 1.6 2.5 t, 0.7 '0 1.4 o N ~ 0.6 <0 1.2 2.0 c: 4. RESULTS AND DISCUSSION < < g ~ ~ 0.5 "''" 1.0 c: "' 1.5 w "' lii u The yield of crude EPS from 500 ml cultures of ~ 0.4 0.8 c: < (/)'" 0 " () B. fibrisoluens strain X6C61 generally ranged from -2 0.3 "§ 0.6 1.0 :; u e w 70 to 80 mg, as determined by the anthrone proce­ ::> 0.2 0.4 ~'" z - 0.5 dure. DEAE-Sephadex chromatography separated 0.1 0.2 the crude EPS into two components, as shown in I I Fig. 1. Approximately 10% of the total carbohy­ 10 20 30 40 50 60 70 80 90 100 drate recovered from the column passed through Fraction Number without binding - these fractions were pooled and Fig. 1. Separation of crude EPS into two components by designated as EPS-I. No hexuronic acid(s) was anion-exchange chromatography on DEAE-Sephadex A-25. 0, detected in EPS-I using the harmine procedure [9]. total carbohydrate; *, uronic acids; •. NaCl gradient. The neutral sugars of EPS-I were identified by GLC as the alditol acetates of rhamnose, man­ nose, galactose, and glucose (Table 1). An ad­ iditol hexaacetate by GLC after hydrolysis, reduc­ ditional GLC peak, partially resolved from tion, and acetylation of EPS-II led us to initially rhamnitol pentaacetate, was obtained from EPS-I. report that idose was a constituent of the EPS TLC analysis of acid-hydrolyzed EPS-I gave re­ from strain X6C61 (Stack, R.J. and Cote, G.L., sults consistent with the above. The purification (1986) Abstr. XIII Int. Carbohydr. Symp., B123, and identification of the unknown component in p. 250). However, idose was not found when acid­ EPS-I is presently underway. hydrolyzed EPS-II was subsequently analyzed by Most of the crude EPS (approx. 90%) eluted TLC. These results suggested that the iditol from DEAE-Sephadex between 0.3 and 0.5 M hexaacetate identified by GLC might represent an NaCl, and was designated as EPS-IL The alditol artifact of the Albersheim et al. procedure [6]. acetates of rhamnose, galactose, glucose, and idose Angyal and Dawes [14] have reported that idose were identified by GLC. The identification of is converted to 1,6-anhydroidose upon acid treat- Table 1 Monosaccharide content of various EPS fractions of B. /ibrisolvens strain X6C61 Polysaccharide preparations were hydrolyzed, reduced, and acetylated. Resulting alditol acetates were analyzed by gas-liquid chromatography/mass spectrometry. EDC-EPS refers to EPS-II-EDCjNaBH4 . Unknown is 4-0-(1-carboxyethyl)-D-galactose; proof of structure to be published elsewhere. Compound Retention Relative amount (galactose = 1.00) time (min) Total EPS EPS-I EPS-II EDC-EPS l,6-Anhydroidose 3.91 0.35 Rhamnose 4.64 0.85 1.71 a 0.70 0.71 Mannose 11.80 trace 0.12 Galactose 12.90 1.00 1.00 1.00 1.00 Glucose 14.02 0.99 0.67 0.97 0.99 Inositol (internal standard) 15.02 Idose 16.15 0.25 0.14 0.04 Unknown 31.85 0.64 a Sometimes detected as a double peak, see text.