Identification of L-Iduronic Acid As a Constituent of the Major Extracellular Polysaccharide Produced by Butyriuibrio Fibrisoluens Strain X6C61

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. 4

ment. The anhydro form constitutes - 90% of the tanol and hydrolysates of EPS-II-EDCjNaBH4 • total idose, yet was not detected in EPS-II hydro Application of the method described by Leontein lyzates by TLC or GLC (as 2,3,4-tri-O-acetyl-1,6 et al. [11] was complicated by the failure of 1,6 anhydroidose) following reduction and acetyla anhydroidose to form glycosides with (-)-2-oc tion. These results confirm the absence of idose in tanol. Only the idose, representing about 10% of EPS-II. the total amount of idosejl,6-anhydroidose in the EPS-II contained uronic acid component(s), as acid hydrolysates, formed glycosides with (- )-2 determined by the harmine procedure [9]. The octanol, which, following acetylation, yielded GLC carboxyl group(s) of EPS-II was reduced with peaks coincident with those from similarly-treated

EDC and NaBH 4 (or NaBD4 ), converting the L-idose (data not shown). The acetylated di uronosyl constituent(s) to its corresponding neu astereomeric glycosides prepared from D-idose tral sugar(s). GLC analysis of the alditol acetates yielded peaks with retention times different from from reduced EPS-II (EPS-II-EDCjNaBH4 ) gave those obtained from EPS-II-EDCjNaBH4 . There peaks coincident with 2,3,4-tri-O-acetyl-1,6 fore, the idose in these preparations must have the anhydroidose and iditol hexaacetate in a 9 : 1 ratio, L-configuration, and, since no change in config in addition to the alditol acetates of rhamnose, uration occurs during carboxyl reduction, the galactose, and glucose. Also, TLC analysis of iduronic acid in the original polysaccharide must acid-hydrolyzed EPS-II-EDCjNaBH4 yielded also have the L-configuration. spots coincident with both idose and 1,6 These results conclusively show that L-iduronic anhydroidose, rhamnose, galactose, and glucose. acid is a constituent of the major EPS from B. GLC-MS analysis of the alditol acetates pre fibrisolvens strain X6C61. The iditol hexaacetate pared from EPS-II-EDCjNaBH4 was used to detected in EPS-II using the method of AI confirm the identities of both iditol hexaacetate bersheim et al. [6] must have come from the and 2,3,4-tri-O-acetyl-1,6-anhydroidose. The c.i. reduction of iduronolactone, which was probably mass spectrum of the former compound (retention formed from iduronic acid following acid hydroly time = 16.15 min) had an M + 1 peak at mjz 435, sis. Our results illustrate the potential for error and its e.i. mass spectrum was identical to a when a single method, in this case the Albersheim library spectrum of a hexitol hexaacetate (data not procedure.[6], is relied upon to analyze samples shown). Similarly, the c.i. mass spectrum of the more complex than those for which they were latter compound (retention time = 3.91 min) had originally intended. an M + 1 peak at mjz 289, and its e.i. mass L-Iduronic acid is a common component of spectrum was identical to a library spectrum of a several mammalian connective tissue polysac 2,3,4-tri-O-acetyl-l,6-anhydrohexose. Both the re charides, but the only prokaryotic polysaccharide tention times and mass spectra of these two com previously reported to contain iduronic acid is the pounds were identical to those obtained from sim 'type-specific' polysaccharide of Clostridium per ilarly-derivatized standard L-idose. fringens strain Hobbs 10 [15]. Tsuchihashi et al. GLC-MS investigations of the alditol acetates [16] have recently reported that L-iduronic acid is prepared from EPS-II-EDCjNaBD4 showed a net a constituent of the glycuronans produced by gain of 2 a.m.u. in the M + 1 peak of both 2,3,4 several different species of fungi. In all of these tri-O-acetyl-l,6-anhydroidose and iditol hexaace reports, and in the present case as well, the iduronic tate (data not shown). No deuterium was incor acid found has had the L-configuration. porated in the peaks corresponding to rhamnose, Of nearly 35 strains of B. fibrisolvens which galactose, or glucose. These data conclusively show have been screened for polysaccharide production, that iduronic acid, not idose, is a constituent of only strain X6C61 contains iduronic acid [4]. This EPS-II. could thus represent a useful biochemical marker The absolute configuration of the iduronic acid for following this strain or any recombinant strains was deduced by GLC analysis of the acetylated derived from X6C61 in the rumen or other en diastereomeric glycosides prepared from (-)-2-oc- vironments. 5

ACKNOWLEDGMENTS [4] Stack, R.J. (1988) Appl. Environ. Microbiol. (in press). [5] Stack. R.J. (1987) FEMS Microbiol. Lett. 48. 83-87. [6] Albersheim, P., Nevins, D.J., English. P.D. and Karr, A. The authors thank Ms. Linda Ericsson and Mr. (1967) Carbohydr. Res. 5, 340-345. Robert Woli for excellent technical assistance, and [7] Cotta, M.A. and Hespell, R.B. (1986) Appl. Environ. Dr. B. Lindberg for critiquing our preliminary Microbiol. 52, 51-58. data and providing helpful suggestions and com [8] Dische, E. (1962) in Methods in Carbohydrate Chemistry, ments. Vol 1. (Whistler, R.L. and Wolfrom, M.L., Eds.) pp. 477-512. Academic Press. New York. The mention of firm names or trade products [9] Wardi, A.H., Allen, W.S. and Varma, R. (1974) Anal. does not imply that they are endorsed or recom Biochem. 57. 268-273. mended by the U.S. Department of Agriculture [10] Taylor, R.L. and Conrad, H.E. (1972) Biochemistry 11, over other firms or similar products not men 1383-1388. tioned. [11] Leontein, K., Lindberg, B. and Lonngren, J. (1978) Carbohydr. Res. 62, 359-362. [12] Gauch, R., Leuenberger, U. and Baumgartner, E. (1979) J. Chromatogr. 174, 195-200. REFERENCES [13] Bounias, M. (1980) Anal. Biochem. 106, 291-295. [14] Angyal, S.J. and Dawes, K. (1968) Austr. J. Chern. 21, [1] Bryant. M.P. and Small, N. (1956) J. Bacteriol. 72, 16-21. 2747-2760. [2] Latham, MJ., Storry, J.E. and Sharpe, M.E. (1972) Appl. [15] Lee, L. and Cherniak, R. (1974) Carbohydr. Res. 33, Microbiol. 24, 871-877. 387-390. [3] Bryant, M.P. (1984) in Bergey's Manual of Systemic [16] Tsuchihashi, H., Yadomae, T. and Miyazaki, T. (1983) Bacteriology, 9th ed. (Krieg, N.R., Ed.) pp. 641-643. Carbohydr. Res. 122, 174-177. Williams and Wilkins, Baltimore. MD.