Beta-2-Linked Glucans Secreted by Fast-Growing Species of

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1 JOURNAL OF BACTERIOLOGY, Apr. 1980, p /80/ /06$02.00/0 Vol. 142, No. 1 Beta-2-Linked Glucans Secreted by Fast-Growing Species of Rhizobium WILLIAM S. YORK, MICHAEL McNEIL, ALAN G. DARVILL, AND PETER ALBERSHEIM* Department of Chemistry, University of Colorado, Boulder, Colorado Fast-growing species of Rhizobium were found to secrete low-molecular-weight /3-2-linked glucans when cultured in synthetic liquid medium. These glucans are quite similar to /3-2-linked glucans produced by species of Agrobacterium. No reducing terminus was detected in these glucans. The taxonomically related genera Rhizobium and Agrobacterium are also related in their ability to infect and stimulate tissue proliferation in dicotyledonous plants (11). The proliferation of host tissue associated with infection by these bacteria is a localized phenomenon and appears to result from transformation of host cells (11). Nodules are the consequence of infection by Rhizobium (5), whereas tumors are the result of infection by Agrobacterium (11). Rhizobium and Agrobacterium are uniquely related to another trait, that is, their ability to synthesize and secrete low-molecular-weight,b- 2-linked glucans. Several Agrobacterium species and R. japonicum, a slow-growing species of Rhizobium, have previously been shown to produce,b-2-linked glucans (7, 9). We now report that similar or identical polysaccharides are present in the culture fluid of three fast-growing Rhizobium species, namely, R. leguminosarum, R. phaseoli, and R. trifolii. This suggests that production of,b-2-linked glucans may be a general property of members of the genera Agrobacterium and Rhizobium (7, 9, 11). No organism outside these genera is known to synthesize such glucans. The infected tissues of the host are probably exposed to the /8-2-linked glucans produced by the bacteria, as these glucans are secreted. It remains to be determined whether /8-2-linked glucans play a significant role in the interaction of these bacteria and their hosts. MATERIALS AND METHODS Identity and culture of bacterial strains. R. leguminosarum 128c53, R. phaseoli 127K14, and R. trifolii 0403 were obtained from R. Carlson and C. Napoli (4). A. tumefaciens was obtained from the American Type Culture Collection. One-liter cultures were grown in defined synthetic medium as previously described (2). Bacteria were removed from 4-day-old cultures by centrifugation at 16,000 x g for 15 min, followed by two filtrations of the supernatant solution through a double layer of Whatman GF/A glass-fiber paper. Liquid chromatography. Solutions to be desalted were concentrated to a volume of 30 ml by rotary 243 evaporation and applied to a 4.5 by 45 cm Sephadex G column (Pharnacia Fine Chemicals, Inc.). The column had been equilibrated with and was eluted with distilled water at room temperature. Eluant fractions (10 ml) were assayed for hexose colorimetrically (8) and for salt by conductivity. Samples to be chromatographed on Sephadex G were dissolved in 2 ml of water. The samples were applied to a 3 by 50 cm column which was eluted with water at room temperature. Fractions (4 ml) were collected and assayed for hexose (8) and by absorbance at 280 nm for protein. Samples (2 mg) to be chromatographed on Bio-Gel P-4 (Bio-Rad Laboratories) were dissolved in 0.5 ml of 50 mm sodium acetate, ph 5.2. The samples were applied to a 1.5 by 29 cm column that had been equilibrated with and was eluted with the same buffer. Fractions (1 ml) were collected and colorimetrically assayed for hexose (8). Samples to be chromatographed on Bio-Gel P-2 were dissolved in 0.7 ml of water. The samples were applied to a 1.5 by 87 cm column heated to 55 C. The column was eluted with water. Fractions (1.25 ml) were collected and assayed for hexose (8). The void and totally included volumes of the gel filtration columns were determined by simultaneous chromatography, under the conditions described above, of high-molecular-weight dextran and glucose. Carbohydrate analyses. The neutral sugar compositions of the glucans were determined by the alditol acetate method of Albersheim et al. (1). Hexose content was determined colorimetrically by the anthrone method (8). Methylation of the glucans was accomplished by means of a modification (6) of the method of Hakomori (10), except that 0.2 mmol of potassium methyl sulfinyl carbanion and 0.2 mmol of methyl iodide were added alternately at 6-h intervals to a 0.5-ml dimethyl sulfoxide solution which contained 0.3 mg of glucan. The fourth and final addition of methyl iodide was a fivefold excess as compared with the methyl sulfinyl carbanion, and the reaction mixture was stirred overnight. Glycosyl linkage compositions were determined by syxithesis and analysis of the partially methylated, partially acetylated alditol derivatives as described (6). These derivatives were quantitated by gas chromatography (6) and were identified by gas chromatography retention times and by gas chromatography-mass spectroscopy (6).

2 244 YORK ET AL. Reduction of glucan prior to methylation. Glucan samples (0.3 mg) were reduced in 0.25 ml.of 10- mg/ml sodium borodeuteride (NaBD4) in 1 M NH40H for 2 h at room temperature. The reactions were terminated by the addition of 0.15 ml of acetic acid. Solvent and borate were removed as previously described (1). The products were methylated as described above. Assay for reducible glucose. Samples (0.1 mg) were reduced in 0.25 ml of 1 M NH40H containing 10- mg/ml NaBD4 for 75 min at room temperature and then for 15 min at 500C. Glacial acetic acid (0.15 ml) was added, and the solution was pased through a 0.5- ml Rexyn 101 column (H+ form). Borate was removed as described (1). The residue was hydrolyzed at 121 C in 2 N trifluoroacetic acid for 1 h, evaporated to dryness, and acetylated as described (1). Glucose pentaacetate (two isomers) and glucitol hexaacetate were identified and quantitated by gas chromatography at 22{1 C in a 25-meter capillary column containing SE- 30 (LKB, Broma, Sweden). Uronic acid determination. Uronic acids were quantitated by means of the m-hydroxybiphenyl assay of Blumenkrantz and Asboe-Hansen (3). Partial acid hydrolysis. A 3.5-mg sample of the,8-2-linked glucan from R. leguminosarum was hydrolyzed for 1 h in 30 ml of 2 N trifluoroacetic acid at 800C. The water and trifluoroacetic acid were removed by rotary evaporation. RESULTS Purification of the,8-2-linked glucans. Beta-2-linked glucans have not previously been detected in the culture fluid of fast-growing species of Rhizobium, perhaps because of the presence in the culture fluids of copious amounts of high-molecular-weight acidic polysaccharides (unpublished data of this laboratory). These acidic polysaccharides form viscous solutions at concentrations greater than 1 mg/ml. The high viscosity of the extracellular fluids of the fastgrowing species of Rhizobium have interfered with chromatographic fractionation of the components of these solutions. It proved difficult to obtain the,b-2-linked glucan produced by these species when the culture filtrates had been concentrated by precipitation with ethanol, as this resulted in precipitation of the,8-2-linked glucan in a complex mixture containing acidic polysaccharides. However, when the conditions were carefully controlled (see below), the acidic polysaccharides could be preferentially precipitated, leaving at least a portion of the,b-2-linked glucans in the supernatant solution. The 1-liter culture filtrates were concentrated to 300 ml by rotary evaporation. Ethanol (300 ml) was added, and the solution was stirred with a glass rod to remove a fibrillar precipitate, consisting primarily of acidic polysaccharide, which wound around the rod. The supematant solution was further concen- J. BACTERIOL. trated by rotary evaporation to 50 ml. Ethanol (400 ml) was added, and the resulting precipitate was again removed by stirring with a glass rod. The supematant solution was concentrated by rotary evaporation to a volume of 30 ml. Colorimetric assays (3, 8) of this solution indicated the presence of both hexose (5 to 30 mg) and uronic acid (1 to 5 mg). The concentrated polysaccharide-containing solution was desalted by chromatography on Sephadex G-25. The carbohydrate-containing G-25 fractions were pooled and lyophilized; the resulting residue was dissolved at 2 mg/ml in 10 mm potassium phosphate, ph 7.0. This solution was added to a slurry consisting of approximately 200 ml of preswollen DEAE-cellulose (Whatman DE-52) which had been equilibrated with and suspended in the phosphate buffer (200 ml). After 30 min of stirring, the DEAE-cellulose was removed by filtration on a coarse sinteredglass funnel. The DEAE-cellulose was washed two times with 200 ml of buffer. The wash and supernatant solutions were combined and concentrated to a volume of 30 ml by rotary evaporation. The concentrated solution contained hexose (1 to 10 mg) but no detectable uronic acids. The solution was desalted by chromatography on G-25. The salt-free carbohydrate-containing G-25 fractions were pooled and lyophilized to dryness. The residue was dissolved in water and chromatographed on Sephadex G-50 (Fig. 1). The major carbohydrate peak was identified as,t/-2- linked glucan. The 83-2-linked glucan produced by A. tumefaciens was isolated, for comparative purposes, from culture filtrates of this bacterium. The procedure used was the same as that described above, except that ethanol precipitates were removed by centrifugation at 16,000 x g rather than by stirring with a glass rod. Anomeric configuration. The optical rotation, [aid, of the 2-linked glucan from R. leguminosarum was determined to be -11, confirming that this polymer is predominantly or completely,8-linked. Carbohydrate analyses. The neutral sugar compositions of the /3-2-linked glucan preparations were determined by formation and analysis of the alditol acetate derivatives. Glucose was the only sugar detected in significant amounts. The,B-2-linked glucan from R. leguminosarum was assayed colorimetrically and found to be 95 ±5% hexose. It is known that /3-2-linked glucans are resistant to complete methylation (9). This has been suggested to be due to steric inaccessibility of certain hydroxyl groups after partial methylation of the glucosyl residues (13). The glucan

3 VOL. V,-2-LINKED 142, 1980 GLUCANS SECRETED BY RHIZOBIUM 245 was methylated by an exhaustive procedure aimed at achieving as complete methylation as possible (see Materials and Methods). The glycosyl linkage composition of the glucans is presented in Table 1. The glucans were found to be composed predominantly of 2-linked glucosyl residues; significant amounts of terminal glucosyl residues were also detected. Very small and variable amounts of 3-, 4-, 6-, 2,3-, 2,4-, 2,6-, and 2,3,4-linked glucosyl residues were detected. Because of the resistance of,b-2-linked glucans to FRACTION NUMBER FIG. 1. Sephadex G-50 chromatography of the /3-2- linked glucan produced by R. leguminosarum. Arrows indicate the void (Vo) and totally included (V1) volumes of the column. A2so and A62o, absorbance at 280 and 620 nm. TABLE 1. Glycosyl linkage composition of/3-2- linked glucans from Agrobacterium and Rhizobium Bacterial source Residuea A. tume- R. eg- R. phas- R. trifofaciens Usaro sarum eoli Iii Terminal glucosyl linked glucosyl linked glucosyl... Tr TR 1.1 Tr 4-linked glucosyl linked glucosyl ,3- and 2,4-linked glu cosyl... 2,6-linked glucosyl ,3,4-linked glucosyl Tr Tr Tr Tr a Nornalized percent. Tr, Trace. b The 2,3-linked and 2,4-linked glucosyl residues cochromatograph; individual quantitation was not achieved. methylation, all of these latter derivatives may be artifacts. This possibility is made likely by realizing that there is considerably less than one residue of each of these derivatives per glucan. It is also possible that some of these derivatives are not artifacts, but arise by some random branching or random cyclization of the glucans. Molecular-weight determinations. The molecular weight of the /3-2-linked glucans was estimated by observation of their behavior during gel filtration chromatography. All of the /3-2- linked glucans appeared to be size homogeneous and to have a degree of polymerization of 20 or less. The Sephadex G-50 elution profile of the,b-2-linked glucan from R. leguminosarum is illustrated in Fig. 1. The G-50 elution profiles of the /3-2-linked glucans produced by R. leguminosarum, R. phaseoli, R. trifolii, and A. tumefaciens were indistinguishable. These glucans eluted from the G-50 column at the volume expected of a typical polypeptide of molecular weight 3,000. The R. leguminosarum glucan eluted from a Bio-Gel P-4 column at the volume expected of a typical polypeptide of molecular weight 3,500. Assessing the reliability of our molecular-weight estimations based on gel filtration is made difficult by the twisted conformation of /-2-linked glucans (12, 13) and the lack of a well-characterized standard with similar conformation. Nonreducing nature of the,b-2-linked glucans. Beta-2-linked glucans from R. leguminosarum and R. phaseoli were subjected to sodium borodeuteride reduction prior to methylation in an unsuccessful attempt to detect and quantitate the glucose residue expected to be present at the reducing terminus of each molecule. Assuming that the reducing terminus of each molecule is a glucose residue with the aldehyde available for reduction, this procedure should have resulted in the formation, after methylation, of a unique derivative (having an 0-methyl group attached to a deuterium labeled C-1). No such C-1 0-methyl derivative was detected after this procedure. This suggests that there is no free reducing terminus, or that the reducing terminus is resistant to reduction. Colorimetric assays for reducing sugars were found to be unreliable when applied to /8-2- linked glucans. The response of sophorose, a disaccharide composed of a glucosyl residue /8- linked to C-2 of a reducing glucose, was unaccountably low and variable. Colorimetric assays for reducing sugars were not, for this reason, employed in the analysis of the /8-2-linked glucans. An assay was developed to quantitate the reducing glucose residues of the /-2-linked glucans. This assay is based on the sodium boro-

4 246 YORK ET AL. hydride conversion ofreducing sugars to alditols. Carbohydrate samples were reduced, hydrolyzed, and acetylated. The two anomers of glucose pentaacetate and glucitol hexaacetate were detected and quantitated by subsequent gas chromatography. Sophorose was found, as expected, to be 50% reducing glucose by this method. The,B-2-linked glucans produced by A. tumefaciens and by R. leguminosarum, R. phaseoli, and R. trifolii were found to contain less than 0.3% reducing glucose. This number may be compared to the predicted value of 5% reducing glucose if each glucan chain of 20 residues possessed a glucose residue at its reducing ter- Minus. The absence of a glucose residue at the reducing terminus of the,8-2-linked glucans suggested that this residue might itself be a glycoside; it could be glycosidically attached to another glucosyl residue in the 2-linked glucan or it could be attached to an aglycon. It was thought that the glycosidic linkage, of what might normally be the reducing glucose, could be more acid labile than the other glucosidic linkages of the glucans. Some support for this possibility was obtained by the following experiment. A sample of the /-2-linked glucan produced by R. keguminosarum was subjected to mild acid hydrolysis, and the hydrolysis products were chromatographed on Bio-Gel P-2 (Fig. 2). Approximately 40% of the partially hydrolyzed glucan eluted at the same volume as the unhydrolyzed glucan. This glucan, which had not detectably changed in molecular weight, contained about 5% reducible (i.e., reducing terminal) glucose as determined by the sodium borohydride method (Table 2). Analysis of this material, after reduction and pernethylation, revealed the presence of 1,3,4,5,6-penta-0-methyl-glucitol. This indicated that mild acid hydrolysis had generated a polysaccharide with a molecular weight which, by P-2 chromatography, was not detectably different from that of the unhydrolyzed /1-2-linked glucan, but which possessed a reducible 2-linked glucose residue. This result suggests, but does not prove, that the glucans possess a unique glucosidic linkage that is particularly acid labile. If the glucans are large circular molecules, the hydrolysis of any glucosidic linkage could yield the observed result. The short oligoglucosides generated by the partial acid hydrolysis are apparently free from the structural feature responsible for our inability to detect reducing terminal residues in the native glucans (Table 2). DISCUSSION The /1-2-linked glucans isolated from culture filtrates of each of three fast growing species of I FRA CTI ON NUMBER FIG. 2. Biogel P-2 chromatography of the native (top) and thepartially hydrolyzed (bottom) /B-2-linked glucan produced by R. leguminosarum. Samples (0.1 ml, 0; and 0.5 ml, *) were assayed colorimetrically for hexose. The arrows indicate the void volume of the column (Vo) and the elution volumes ofsophorose and glucose. Fractions a, b, c, and d were tentatively identified, on the basis of the content of reducing glucose residuespresent in the fractions (presented in Table 2), as glucose and the 2-linked glucosyl dimer, trimer, and tetramer, respectively. A62,, absorbance at 620 nm. TABLE 2. Reducible glucose in the native and the partially hydrolyzed,8-2-linked glucan from Rhizobium leguminosarum Carbohydrate fraction %IReducible giucose < Native glucan... Sophorose... Partial hydrolysate... P-2 fractionsa J. BACTERIOL d.25 c.31 b.46 a.92 " See Fig. 2. a

5 VOL. V-2-LINKED 142, 1980 GLUCANS SECRETED BY RHIZOBIUM 247 Rhizobium are indistinguishable from the f8-2- linked glucan produced by A. tumefaciens. Yields of about 15 mg of glucan per liter of culture, accounting for 5 to 10% of the extracellular polysaccharides, were obtained. Each glucan is composed predominantly of glucose, each has the same elution volume and narrow peak width when submitted to gel filtration chromatography, each lacks a reducible glucose residue at the reducing terminus, and the glucosidic linkage compositions of the glucans are not significantly different (Table 1). Interpretation of data obtained by methylation analysis of the f8-2-linked glucans is difficult. The appearance of branched residues, albeit less than one residue of any type per glucan molecule, may reflect the true structure of the glucans. On the other hand, the apparently branched glucosyl residues may result from undermethylation, as they were detected in variable amounts in methylation analyses of the same and different glucan preparations. If the branched glucosyl residues do not result from undermethylation, the presence of these residues would indicate that the glucans possess structural heterogeneity, although the gel filtration profiles indicate that the glucans are homogeneous with regard to radius of gyration. The molecular weight of the,b-2-linked glucans, as estimated by gel filtration, indicates that the glucans are composed of approximately 20 residues. Unfortunately, this value cannot be verified by nonreducing terminal glucosyl residue analysis because of the uncertainty as to whether the glucans are branched. It is also not possible to estimate the molecular weight of the native glucans by quantitation of the reducing terminus. Partial acid hydrolysis of the glucans does produce a fragment or altered structure which is not detectably different in size from the native glucan. This altered glucan possessed 5% reducible glucose, indicating that the molecule was composed, in agreement with the gel filtration estimate, of 20 residues (see Table 2). This value is based on the assumption that this fraction contains no glucan molecules in their native, nonreducing state. An alternative interpretation of these data is that the glucan molecules are composed, on average, of 11 glucosyl residues and are unbranched, and that about 55% of the molecules in fraction "e" are in their native, nonreducible state. This interpretation assumes that all apparent branching is due to undermethylation. There are two explanations for the absence of a reducing terminus which we have not been able to discriminate between. The 8i-2-linked glucans may be covalent circles, with every glucosyl residue linked through its C-1 to another glucosyl residue. Alternatively, the glucosyl residue at the reducing terminus of the glucans could be glycosidically linked to an acid-labile nonsugar residue, i.e., an aglycon. However, no such aglycon has thus far been detected in association with,b-2-linked glucans. Proton nuclear magnetic resonance spectra of the glucan secreted by R. keguminosarum contained no signals which could not be attributed to the protons of the glucosyl residues. Gas chromatography on Porapak Q of crude acid hydrolysis mixtures of the /i-2-linked glucans isolated from several different bacteria did not result in the detection of any acid-labile, volatile components such as methanol or ethanol. A macrocyclic structure for an unbranched B8-2-linked glucan would appear to be unlikely because these glucans possess a very restricted conformation which would, seemingly, not allow a circular or macrocyclic structure (12,13). However, if the branch points detected by methylation analysis are real, not only could the existence of an excess (based on 20 residues per molecule) of nonreducing terminal glucosyl residues be explained, but the molecules would contain a more flexible glucosyl residue, making a circular structure possible. We conclude that the,b-2-linked glucans secreted by Rhizobium and Agrobacterium species are structurally challenging molecules which may have unknown but intriguing biological functions. ACKNOWLEDGMENTS This is number VIII in a series of publications by this laboratory on "Host-Symbiont Interactions." This work was supported by grants from the U.S. Department of Agriculture ( ) and the Department of Energy (EY-76- S ). We thank Ruth Shkoller for assistance in culturing bacteria and Martin Ashley of the University of Colorado Chemistry Department and James Frye of the Colorado State University Regional NMR Center for 'H-nuclear magnetic resonance spectra of the,6-2-linked glucans. LITERATURE CITED 1. Albersheim, P., D. J. Nevins, P. D. English, and A. Karr A method for the analysis of sugars in plant cell-wall polysaccharides by gas-liquid chromatography. Carbohydr. Res. 5: Bergeson, F The growth of Rhizobium in synthetic media. Aust. J. Biol. Sci. 14: Blumenkrantz, N., and G. Asboe-Hansen A new method for quantitative determination of uronic acids. Anal. Biochem. 54: Carlson, R. W., R. E. Sanders, C. Napoli, and P. Albersheim Host-symbiont interactions. III. Purification and partial characterization of Rhizobium lipopolysaccharides. Plant Physiol. 62: Dart, P Infection and development of leguminous nodules, p In R. W. F. Hardy and W. S. Silver (ed.), A treatise on dinitrogen fixation. John Wiley & Sons, Inc., New York. 6. Darvill, A., M. McNeil, and P. Albersheim The

6 248 YORK ET AL. structure of plant cell walls. VIII. A new pectic polysaccharide. Plant Physiol. 62: Dedonder, R. A., and W. Z. Hasid The enzymatic synthesis of a,-1,2-o-linked glucan by an extract of Rhizobium japonicum. Biochim. Biophys. Acta 90: Dische, Z Color reaction of carbohydrates. Methods Carbohydr. Chem. 1: Gori, P. A. J., J. F. T. Spencer, and D. W. S. Westlake The structure and resistance to methylation of 1-2-f.glucans from species of Agrobacteria. Can. J. Chem. 39: Hakomori, S A rapid permethylation of glycolipids and polysaccharides catalyzed by methylsulfmnyl carbanion in dimethyl sulfoxide. J. Biochem. 56: J. BACTERIOL. 11. Lippincott, J. A., and B. B. Lippincott The genus Agrobacterium and plant tumorigenesis. Annu. Rev. Microbiol. 29: Rees, D. A., and W. E. Scott Polysaccharide conformation. VI. Computer model building for linear and branched pyranoglucans. Correlations with biological function. Preliminary assessment of interresidue forces in aqueous solution. Further interpretation of optical rotation in terms of chain conformation. J. Chem. Soc. B, p Rees, D. A., and R. J. Skerrett Conformational analysis of polysaccharides. IV. Long-range contacts in some,-glucans by model building in the computer and the influence of oligosaccharide conformation on optical rotation. J. Chem. Soc. B, p Downloaded from on November 28, 2018 by guest