Journal of Industrial Microbiology (1996) 16, 129-133 1996 Society for Industrial Microbiology 0169-4146/96/$12,00

Production of an extracellular polysaccharide by Agrobacterium sp DS3 NRRL 8-14297 isolated from soil

1 2 1 2 CT Hou , JA Ahlgren , W Brown and JJ Nicholson

1 Oil Chemical Research Unit; 2Biopolymer Research Unit, National Center for Agricultural Utilization Research, ARS, USDA, Peoria, IL, USA

A bacterium isolated from soil and identified as Agrobacterium sp produced a water-soluble extracellular polysacch­ aride capable of producing highly viscous solutions. Gas chromatographic analysis revealed a sugar composition of glucose, galactose and mannose in the molar ratio of 7.5 : 2.4 : 1, together with 3.7% (w/w) pyruvic acid. Methyl­ ation analyses showed the presence of (1 -> 3)-, (1 -> 4)- and (1 -> 6)-linked glucose, (1 -> 3)- and (1 -> 4, 1 -> 6)-linked galactose and a small portion of (1 -> 3)-linked mannose residues. Succinic acid was not present. The molecular weight of the polysaccharide was estimated by light scattering to be 2 x 106 Da. The viscosity of solutions containing the polysaccharide remained constant from pH 3 to 11, and decreased by 50% when heated from 5 to 55°C. Maximum yield of the polysaccharide, 20 g L-1, was reached in 48 h at 30°C incubation.

Keywords: extracellular polysaccharide; Agrobacterium; viscous polysaccharide

Introduction the respective molar ratios of 7.5: 2.4 : l. The sugar components and their ratio found in this polysaccharide are Microorganisms are known to accumulate various kinds of different from other known microbial polysaccharides [4]. polysaccharide inside or outside of the cells. Many polysac­ This paper describes the culture identification, purification charides produced by bacteria have characteristic rheologi­ of the polysaccharide, identification of sugar components cal and physiological properties that are different from and type of linkages, some physical properties and the opti­ those of natural gums and synthetic polymers. They are also mum conditions for production of the polysaccharide. susceptible to biodegradation in nature and are typically less harmful to the environment than many synthetic poly­ mers. Therefore, some microbial polysaccharides are pro­ duced on industrial scales and used as raw materials for Materials and methods food processing and for medical and industrial purposes. Xanthan produced by Xanthomonas campestris [23,24] and Microorganisms alginate produced by Pseudomonas aeruginosa [2] are used Microorganisms obtained from a variety of soil and water as food stabilizers. Dextran produced by Leuconostoc samples were screened for the ability to modify oleic or mesenteroides [19] is used as a blood plasma extender, linoleic acids or to produce unusual products [15,16]. Each while Scleroglucan, produced by Sclerotium glucanicum isolate from a single colony on TGY [5] agar plates was [9], is used for industrial gum. Curdans from Alcaligenes grown at 30°C aerobically in a 125-ml Erlenmeyer flask faecalis var myxogenes [10,11] and Agrobacterium radio­ shaken at 200 rpm containing 50 ml of SMD medium with bacter [20] are used for either food or non-food materials the following composition (per liter): dextrose, 20 g: as gelling agents. K2HP04, 5 g: yeast extract, 5 g: soybean meaL 5 g: We isolated a bacterium from soil which produces a FeS04 ·7H20. 0.5 g: ZnS04 , 0.014 g; MnS04 · H20, water-soluble extracellular polysaccharide. The bacterium 0.008 g; and nicotinic acid, 0.0 I g. Prior to autoclaving it. is a non-spore forming, Gram-negative rod and was ident­ the medium including dextrose was adjusted to pH 7.0 with ified as an Agrobacterium sp. Two types of polysaccharides dilute phosphoric acid. Cultures were maintained on agar produced by agrobacteria have been reported: a water­ (3%) slants of the same medium. Microbial isolates were soluble acidic polysaccharide like succinoglycan [13,14]: identified by using Biolog GN Microplates and analyzed and the water-insoluble f3-(l -> 3)-glucans (curdlan-type) with the con-esponding microstation (Biolog, Inc, Hayward, [21]. These polysaccharides were believed to promote nod­ CA, USA). ule invasion by nitrogen-fixing microorganisms [8]. The polysaccharide produced by Agrobacterium sp DS3 Chemicals described in this work was water-soluble and free of suc­ Oleic acid [purity >99% by gas chromatography (GC)] was cinic acid; aqueous solutions of the polysaccharide were purchased from Nuchek Prep Inc, Elysian, MN, USA. highly viscous. It contains glucose: galactose: mannose in Immobilized protease on 4% beaded agarose was obtained from Sigma (St Louis, MO, USA). Thin-layer precoated Correspondence: Dr CT Hou. National Center for Agricultural Utilization Kieselgel 60 F254 plates were obtained from EM Science Research. ARSfUSDA. 1815 N University Street. Peoria. IL 61604. USA (Chen-y HilL NJ, USA). All other chemicals were analytical Received 27 June 1995; accepted 23 November 1995 grade reagents from commercial sources. New polysaccharide from Agrobacterium CT Hou et at 130 Quantitation of polysaccharide Non-carbohydrate substituents Ten milliliters of culture broth were diluted with an equal For analysis of substituent organic acids, 100 mg of DS3 volume of distilled water and centrifuged at 11000 x g at polysaccharide was hydrolyzed with 10 ml 5% sulfuric acid 4°C for 20 min to remove bacterial cells. To the supernatant (95°C. 10 h). The hydrolyzate was extracted twice with an solution, two volumes of ethanol were added and the pre­ equal volume of diethyl ether. and then the combined ether cipitate was collected by centrifugation. The precipitate was layer was washed with water and evaporated to dryness. A washed with 30% ethanol and centrifuged. The washed pre­ portion of this residue was methylated with diazomethane: cipitate was taken up in water for quantitation of the poly­ the resulting fatty acid methyl esters were analyzed by GC saccharide by the phenol-sulfuric acid method [6] from a isothermally at 200°C as described previously [15.16]. calibration curve using glucose as a standard. The aqueous layer of the hydrolyzate was neutralized with barium hydroxide and, after centrifugation, the pre­ Isolation and purification of polysaccharide cipitate was discarded. The supernatant fluid was lyophil­ After the bacterial culture had grown for 48 h. an equal ized and the residue taken up in methanoL and methylated volume of IN NaOH was added to the viscous culture with diazomethane as previously described. The methyl broth, and the mixture was centrifuged at 11 000 x g for ester derivatives were used for the analysis of smaller mol­ 20 min to remove cells. The supernatant phase was neu­ ecular weight dibasic organic acids, such as succinic acid. tralized with 3 N HCI and then centrifuged to separate by GC on a crosslinked polyethylene glycol capillary col­ water-soluble from insoluble curdlan-like polysaccharides. umn (30 m x 0.53 mm i.d. x 1.0 fLm thickness: Hewlett The majority of the polysaccharide remained in the super­ Packard Innowax) and a temperature gradient program: natant phase. Two volumes of ethanol containing 2% KCI 45°C for I min: 5°C per min to 240°C: and held 5 min at were added to the supernatant fraction, and the polysacchar­ 240°C. Authentic dimethyl succinate has a retention time ide precipitate was collected by centrifugation. The precipi­ of 22 min. tate was washed consecutively by suspension followed by Pyruvic acid was quantified by the enzymatic assay centrifugation in 30% ethanol, acetone, and finally diethyl described by Duckworth and Yaphe [7]. ether. The resulting precipitate was dried to a white powder with a stream of nitrogen. Fifty milligrams of the polysac­ charide in 50 ml of water were incubated for 16 h with Measurement of viscosity and surface tension 100 mg of immobilized protease at 30°C with shaking. The The viscosity of the crude culture broth was measured with mixture was then dialyzed against deionized distilled water a rotation viscometer (SynChro-Lectric with the #3 spindle: for 24 h. The dialyzate was then precipitated with three vol­ Brookfield, Stoughton, MA. USA). Measurement was at umes of ethanol and centrifuged. The resulting precipitate 2S°C. and readings were taken after rotation for 2 min. The was again dried to a white powder. values obtained were averages of three measurements. To determine the effects of temperature and pH on the Thin-layer chromatography of sugar components viscosity of aqueous solutions of the polysaccharide, pur­ Following hydrolysis of the polysaccharide in 2 M trifluo­ ified polysaccharide was dissolved in buffer. and the vis­ roacetic acid (TFA) for I h at 120°C in a sealed tube, the cosity measured with a Brookfield Digital viscometer following solvents were used for separating component model DV-I equipped with a temperature-regulated small sugars on Kieselgel plates [18]: 2-propanol : acetone : sample adapter and the SC4-18/13R spindle. The tempera­ 0.1 M lactic acid (4: 4: 2, v/v) and acetone: \vater (90: 10, ture range evaluated was from 5 to SS°C: the system was v/v). The sugars were made visible by means of the allowed to equilibrate thermally between viscosity readings diphenylamine-aniline-phosphate spray reagent [1]. on the same sample. Solutions ranging from pH 3 to 11 were prepared from a stock polysaccharide solution and Gas chromatography of sugar components mixed with sodium acetate/sodium phosphate stocks To identify the native sugar components, the purified DS3 (100 mM each) titrated to the appropriate pH. The pH of polysaccharide was hydrolyzed with 2 ml 2 M TFA for I h the buffered polysaccharide solution was determined prior at 120°C in a sealed tube. After TFA was removed, the to viscosity measurement (12 rpm, 30°C). sugars were converted to the peracetylated aldononitrile The surface tension of the polysaccharide solution was (PAAN) derivatives [22] and analyzed by GC/MS incorpor­ measured with a Kruss Tensiometer (Hamburg, Germany) ating a Hewlett-Packard 5970B mass selective detector using small ring (K-813) at 25°C. operating at 70 eV and using a methylsilicone column (25 m x 0.022 i.d. x 0.1 fLm thickness: Hewlett Packard, Wilmington. DE, USA). The column temperature was held Molecular weight for 3 min at 160°C and then raised at 5°C per min to 185°C. The molecular weight of the polysaccharide was deter­ To identify the linkage sites in the polysaccharide, samples mined by multiangle light scattering (Dawn DSP model B of purified DS3 were permethylated [22] with sodium photometer: Wyatt Technology Corporation, Santa Barbara, methylsulfinyl methanide and methyl iodide in dimethyl CA, USA). Purified polysaccharide was dissolved in ultra­ sulfoxide. The methylated polysaccharide was then pure HPLC grade water. filtered through a 0.45-fLm pore hydrolyzed with TFA and the sugars converted to PAAN size filter. and placed in a sample cuvette. Data were col­ derivatives. For the separation of these derivatives, the GC lected at several polysaccharide concentrations and ana­ column temperature was held for 3 min at 130°C. raised at lyzed by means of the Aurora software program (Wyatt 5°C per min to 165°C. and then held for 10 min: helium Technology Corporation). The value determined was the was used as a can'ier gas for both analyses. average of molecular weights derived from the Zimm. New polysaccharide from Agrobacterium CT Hou et a{

20 250 12 1.5 131

18 11 16 200 10 14 1.0 -e •0- 012 ~ 150 ....<1 Q. 1\ :§" 840 0-'" ii\ ~ 10 .§. •.c g c iy "-.. 0- 'on I' ~.L-.. ~ "12 0'" 8 ~"" If -.A.-...... _.,_.,_.. _ .. _ ...... 0- 'a 100 o 4 u i 0.5 f·-~-·-~-~-·_--_ 8 600 I --~ >< 50 ~ 3 ! c: !1l -0c: _'_--,-----,--,---,---+4 0.0 ~ 360 5 10 6 TIme (Days) 2

Figure 1 Production of water-soluble extracellular polysaccharide by Agrobacleriwtl sp OS3, grown on SMO medium, 200 rpm, at 30°C. 120 .A.-.A., Cell density (00660nm); .-., viscosity; e--e, pH; '7----'7, product polysaccharide (g). 5.0 6.0 7.0 8.0 9.0 10.0 11.0 Retention time (min) Berry, and Debye plotting strategies that are part of the Aurora program, Figure 2 GCIMS of the peracetylated aldononitrile (PAAN) derivatives of sugars from strain OS3 polysaccharide. Column temperature was held for 3 min at 130°C. raised at 5°C per min to 165°C. and then heid for Results and discussion 10 min. Peak identifications; (l) methyl-2,4.6-tri-O-methyl-D-glucose PAAN; (2) methyl-2,4,6-tri-O-methyl-D-mannose PAAN; (3) methyl­ Identification of microorganism 2,4,6-tri-O-methyl-D-galactose PAAN; (4) methyl-2,3,6-tri-O-methyl-D­ Of many water and soil samples screened, only one culture, glucose PAAN; (5) methyl-2,3,4-tri-O-methyl-D-glucose PAAN; DS3, isolated from a dry soil sample collected at Peoria, (6) methyl-2,3-di-O-methyl-galactose PAAN. IL, produced a significant amount of extracellular polysac­ charide that was capable of producing viscous solutions, that addition of fatty acid was not required for the pro­ Strain DS3 is a Gram-negative, non-spore-forming rod duction of polysaccharide. We therefore investigated the (0.8 /.Lm x 2 /.Lm). Fifty-one of the ninety-six wells of a conditions for the optimum production of polysaccharide. Biolog GN microplate yielded positive results: dextrin, gly­ Strain DS3 was grown on SMD medium with I, 2, 3 cogen, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, or 5% (w/v) of glucose. After five days of incubation, the adonitol, L-arabinose, D-arabitol, cellobiose, D-fructose, L­ polysaccharide in the culture medium was isolated and ana­ fucose, D-galactose, gentiobiose, D-glucose, m-inositol, D­ lyzed. Polysaccharide yields for 1, 2, 3, and 5% glucose lactose, lactulose, maltose, D-mannitol, D-mannose, {3­ were 0.3, 0.34, 1.03, and 1.2 g per 50 ml, respectively. Five methyl-o-glucoside, D-psicose, D-raffinose, L-rhamnose, D­ percent glucose in SMD medium was used for sub­ sorbitol, sucrose, D-trehalose, o-turanose, xylitol, acetic sequent studies. acid, formic acid, o,L-Iactic acid, propionic acid, succinic The time course for the production of polysaccharide was acid, bromo succinic acid, alaninamide, L-alanine, L-alanyl­ also studied. Cell density, culture viscosity, pH and amount glycine, L-asparagine, L-aspartic acid, L-glutamic acid, gly­ of polysaccharide produced were followed for 9 days. Fig­ cyl-L-aspartic acid, glycyl-L-glutamic acid, L-omithine, L­ ure I shows that the concentration of polysaccharide proline, L-serine, L-threonine, inosine, uridine, glycerol, reached its maximum after 5 days of incubation, after glucose I-phosphate and glucose 6-phosphate. By compari­ which it remained relatively constant. There was not much son to known strains, the Biolog GN microstation identified change in pH of the culture broth during the course of strain DS3 as belonging to the genus Agrobacterium, and investigation. The viscosity of the culture broth increased having a 20% similarity to the closest species, A. rhizo­ genes. Therefore, strain DS3 is classified as Agrobacterium sp DS3 NRRL B-14297. Table 1 GCIMS analysis of PAAN derivatives of sugars from OS3

PAAN methyl ether Relative retention rvlolar ratio Production of polysaccharide time Production of extracellular polysaccharide by Agrobacter­ ium sp DS3 was observed originally with our routine fatty 2,4.6-tri D-glucose 1.00 8.7 acid bioconversion screen in which oleic or linoleic acid 2,4,6-tri D-mannose 1.05 1.0 served as the substrate [IS]. When the organism was grown 2.4.6-tri D-galactose 1.06 3.7 at 30°C on a TGY agar plate for several days, translucent 2,3.6-tri D-glucose 1.07 5.2 2.3,4-tri D-glucose 1.11 2.9 gelatinous colonies developed. It was also observed that 2,3-di D-galactose 1.36 2.5 the liquid culture medium became viscous during bacterial growth under aerobic conditions due to the formation of "Trivial names for the O-methyl sugars; eg 2,4,6-tri D-glucose is actually extracellular polysaccharide. Further investigation revealed 2,4,6-tri-O-methyl-D-glucose PAAN. New polysaccharide from Agrobacterium CT Hou et al 132 a", b '00 C,OOO

.. ..•. u u .... ., g 100 ..•...... '" so % 60 '" 0.. 0.. ~ ...• '" •..•.. • ...•. .s ...... •• .s ..•..• .s ...... •. >- ..• i:: ~o .'t:' .0 .•.....• .~ ..... 'iii o ~ '"o 10 (J (J .•..•..• .. '" '" 1il :> 20 :> 20 :>

a oL-~'o.l.-~2o.I.-~30.l.-~.o.l.-~so.l.-...... so 1l-~~-...... JL-~ ...... _ ...... ~...... , a.' 10 '00 pH Temperature (0C) Shear Rate (sec")

Figure 3 Rheological properties of the purified DS3 polysaccharide. (al Effect of pH on viscosity at 30c C. 12 rpm: (b) effect of temperature on viscosity at pH 7. 12 rpm: (c) effect of shear rate on viscosity. 30c C. pH 7.

sharply (to 240 mPa-sec) during the first 2 days of incu­ linked glucose: galactose is primarily (I -+ 3)-linked, but bation and then decreased significantly. Because the con­ some (I -+ 4, I -+ 6)-linked galactose is also present. Man­ centration of the polysaccharide remained constant. the nose is the least abundant component of the polysaccharide, decrease in solution viscosity suggests structural change(s) and is (I -+ 3)-linked. occurred in the polysaccharide. Zevenhuizen [25] identified an acidic polysaccharide similar to succinoglycan from Agrobacteriul/I tUlIlejaciens. Identification of sugar components of polysaccharide Hisamatsu et al [13,14] found that water-soluble. exocellu­ When an acid hydrolyzate of the purified polysaccharide lar polysaccharides from nine strains of AgrobacteriulIl was analyzed by thin-layer chromatography, three sugars contained succinic acid as well as pyruvic and acetic acids.

with Rf values identical to those of authentic D-glucose, Their structures, which were similar to succinoglycan, were D-galactose and D-mannose were detected with two differ­ composed of (I -+ 3), (I -+ 4), and (I -+ 6)-linked D-gluco­ ent solvent systems. With 2-propanol : acetone: 0.1 M lac­ syl and (I -+ 3)-linked galactosyl residues. Zevenhuizen

tic acid as the solvent system, the Rf values for D-glucose, [25,26] also reported that RhizobiulIl lIleliloti YE-2SL D-galactose, and D-mannose were 0.54, 0.41 and 0.57. secreted sucinnoglycans and a galactoglucan. Alcaligenes respectively. With acetone: water as the solvent system, faecalis var l/Iyxogenes IOC3 produces large amounts of a

their respective Rf values were 0.55, 0.50 and 0.59. When water-soluble ,B-o-glucan containing succinic acid peracetylated aldononitrile derivatives of these sugar (succinoglucan) and small amounts of an insoluble (I -+ 3)­ components were analyzed by Gc, three peaks with reten­ ,B-o-glucan (curdlan) [10,11,14]. tion times of 7.39 min, 7.74 min. and 7.11 min were found In contrast to several succinoglycans from Agrobacter­ which coincided with retention times of the derivatives ium [13,14], Alcaligenes [10,1 1,]4] and Rhizobium [3,16], from authentic o-glucose. o-galactose, and o-mannose, strain DS3 polysaccharide contains mannose and pyruvic respectively. From these results, it was confirmed that the acid, and lacks succinic acid. The ratio of sugar constituents polysaccharide consisted of three sugars. Their mole ratios (glucose: galactose: mannose = 7.5 : 2.4 : I) differs from are glc : gal: man = 7.5 : 2.4 : I. any known microbial polysaccharide. Although there are The pyruvic acid content was 3.7% by weight. To deter­ some structural similarities between DS3 polysaccharide mine whether other organic acid moieties existed in the and succinoglycans, in that all have (I -+ 3)-, (I -+ 4)-, and polysaccharide, an acid hydrolyzate of the polysaccharide (I -+ 6)-linked glucose and (I -+ 3)-linked galactose, the was extracted with diethyl ether. When methyl esters were ratios of these linkages are quite different. In addition, the prepared and analyzed by Gc, no fatty acids were detected. DS3 polysaccharide has (I -+ 4, I -+ 6)-linked galactose. This ruled out the existence of lipopolysaccharide. The possible existence of water-soluble organic acids such as Physical properties of the polysaccharide succinic acid was also studied with the acid hydrolyzate. The effects of temperature and pH on solution viscosity Results obtained from an HP-Innowax GC capillary column from the DS3 polysaccharide were investigated. Figure 3a showed that there were no succinic or other water-soluble shows that the solution viscosity remained relatively dibasic organic acids in the polysaccharide. unchanged from pH 3 to II. This stability of viscosity over a wide pH range is similar to that found for the succinogly­ Structure analysis can from Alcaligenes faecalis var l/Iyxogenes, which was Table I summarizes the methylation analysis of purified constant from pH 3 to 10 [12]. The solution viscosity of polysaccharide (Figure 2). The PAAN-hexose that most DS3 polysacchmide decreased with increasing temperature, likely contained the pyruvate group (a 2-0-methyl PAAN) and dropped by 40% as the temperature was increased from had a retention time of 12 min (result not shown). The 5 to 55°C (Figure 3b). The viscosities of succinoglycan sol­ results show that the most prominent sugar is (I -+ 3)­ utions were also reported to be temperature dependent. with linked glucose, followed in relative abundance by (I -+ 4)- the viscosity approaching that of water at 60°C [12]. New polysaccharide from Agrobacterium CT Hou et at 133 The effect of shear rate on solution viscosity was also pyranosyl units appended at intervals by (I - 6) linkages are produced examined. As shown in Figure 3c, viscosity decreased with by a variety of microorganisms. US Patent 330I 848. 10 Harada T. M Masada. K Fujimori and I Maeda. 1966. Production of increasing shear rate. Many other microbial polysacchar­ a firm. resilient gel-forming polysaccharide by a mutant of Alcaligenes ides show pseudoplastic viscosity behavior [17]. faecalis var mycwgenes IOC3. Agric Bioi Chem 30: 196-198. Surface tensions of polysaccharide solutions (0.75% and II Harada T. A l'vlisaki and H Saito. 1968. Curdlan: a bacterial gel-for­ 0.375% in water) were measured in order to determine ming f3-1.3-glucan. Arch Biochem Biophys 124: 292-298. 12 Harada T. M Terasaki and A Harada. 1993. In: Industrial Gums. Poly­ whether the polysaccharide had surface-active properties. saccharides and Their Derivatives (Whistler RL and IN BeMiller. eds). I Both were 43.12 dynes cm- , indicating that the polysacch­ pp 427-445. Academic Press. New York. aride has moderate surface tension-lowering activity (water 13 Hisamatsu M. J Abe. A Amemura and T Harada. 1980. Structural is 72.8 dynes cm-I at 18°C). elucidation on succinoglycan and related polysaccharides from Agro­ baclerium and Rhiz.obillln by fragmentation with two special beta-D­ glycanases and methylation analysis. Agric BioI Chem 44: 1049-1055. Determination of molecular weight 14 Hisamatsu lvl. K Sano. A Amemura and T Harada. 1978. Acidic poly­ A multiangle light scattering detector was used to measure saccharides containing succinic acid in various strains of Agrobacler­ the molecular weight of the polysaccharide in water. An ium. Carbohydr Res 61: 89-96. 6 15 Hou CT and MO Bagby. 1991. Production of a new compound. 7.10­ average value of 2 x 10 Da was determined from analyzing dihydroxy-8(E)-octadecenoic acid from oleic acid by Pseudomonas sp data collected at several biopolymer concentrations, which PR3. J Ind Microbiol 7: 123-130. is comparable to the values reported for other extracellular 16 Hou CT. MO Bagby. RD Plattner and S Koritala. 1991. A novel com­ heteropolysaccharides [17]. The large size of the biopo­ pound 7.1 0-dihydroxy-8(El-octadecenoic acid from oleic acid by bioconversion. J Am Oil Chem Soc 68: 99-101. lymer is consistent with the decrease in solution viscosity 17 Jeanes A and JE Pittsley. 1973. Viscosity profiles for aqueous disper­ as a result of shear thinning which is a typical phenomenon sions of extracellular anionic microbial polysaccharides. J Appl Polym seen with high-molecular-weight polysaccharides. Sci 17: 1621-1624. 18 Lewis BA and F Smith. 1969. In: Thin-Layer Chromatography, A Lab­ oratory Handbook. 2nd edn (Stahl S. ed). p 813. Springer-Verlag. New York. Acknowledgements 19 Murphy PT and RL Whistler. 1973. In: Industrial Gums: Polysacchar­ ides and Their Derivatives (Whistler RL. ed). pp 513-542. Academic We thank Dr 0 Labeda for identification of strain OS3. Press. New York. 20 Nakanishi 1. K Kimura. S Kushi and E Yamazaki. 1974. Complex formation of gel-forming bacterial (l - 3)-f3-o-g1ucans (curdlan-type References polysaccharides) with dyes in aqueous solution. Carbohydr Res 32: 47-52. Bailey RW and EJ Bourne. 1960. Color reactions given by sugars 21 Nakanishi 1. K Kimura, T Suzuki. M Ishikawa. I Banno. 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Constitution. M. ed). pp 578-611. vol 16. John Wiley & Sons. New York. Can J Chem 40: 2066-2071. 5 DlFCO Manual. 10th edn. DlFCO Laboratories. Detroit. Ml. 24 Sioneker JH and DG Orentas. 1962. Exocellular bacterial polysacchar­ 6 Dubois M. KA Gilles. JK Hamilton. PA Rebers and F Smith. 1956. ide from Xalllhomonas campeslris NRRL B-1459. Part II. Linkage of Colorimetric method for determination of sugars and related sub­ the pyruvic acid. Can J Chem 40: 2188-2189. stances. Anal Chem 28: 350-356. 25 Zevenhuizen LPTM. 1973. Methylation analysis of acidic exopolysac­ 7 Duckworth M and W Yaphe. 1970. Definitive assay for pyruvic acid charides of Rhizobium and Agrobaclerium. Carbohydr Res 26: 409­ in agar and other algal polysaccharides. Chem Ind (Lond) 23: 747-748. 419. 8 Glazebrook J. JW Reed. TL Reuber and GC Walker. 1990. Genetic 26 Zevenhuizen LPTM and P Faleschini. 1991. Effect of the concen­ analyses of Rhiz.obium meliloli exopolysaccharides. Int J Bioi Macro­ tration of sodium chloride in the medium on the relative properties of mol 12: 67-70. poly- and oligosaccharides excreted by Rhizobium meliloli strain YE­ 9 Halleck FE. 1967. New f3-o-(l - 3l-linked glucans with f3-o-g1uco- 2SL. Carbohydr Res 209: 203-209.