JOURNAL OF BACTERIOLOGY, JUlY 1991, P. 4271-4276 0021-9193/91/144271-06$02.00/0 Copyright C) 1991, American Society for Microbiology
Vol. 173, No. 14
A Novel Membrane-Bound Glucosyltransferase from Bradyrhizobium japonicum JODI L. COHEN' AND KAREN J. MILLER'12,3* Department of Food Science' and Graduate Programs in Plant Physiology2 and Genetics,3 The Pennsylvania State University, University Park, Pennsylvania 16802 Received 28 February 1991/Accepted 3 May 1991
Bacteria within the family Rhizobiaceae are distinguished by their ability to infect higher plants. The cell envelope carbohydrates of these bacteria are believed to be involved in the plant infection process. One class of cell envelope carbohydrate, the cyclic f0-1,2-glucans, is synthesized by species within two genera of this family, Agrobacterium and Rhizobium. In contrast, species of the genus Bradyrhizobium, a third genus within this family, appear to lack the capacity for cyclic P1-1,2-glucan biosynthesis. Instead, these bacteria synthesize cyclic glucans containing ,-1,6 and 0-1,3 glycosidic linkages (K. J. Miller, R. S. Gore, R. Johnson, A. J. Benesi, and V. N. Reinhold, J. Bacteriol. 172:136-142, 1990). We now report the initial characterization of a novel membrane-bound glucosyltransferase activity from Bradyrhizobiumjaponicum USDA 110. Analysis of the product of this glucosyltransferase activity revealed the following: the presence of 0-1,3 and 0-1,6 glycosidic linkages, an average molecular weight of 2,100, and no detectable reducing terminal residues. The glucosyltransferase activity was found to have an apparent Km of 50 ,uM for UDP-glucose, and activity was stimulated optimally by Mn2+ ions. On the basis of the structural properties of the in vitro glucan product, it is possible that this membrane-bound glucosyltransferase activity may be responsible for the biosynthesis of cyclic 0I-1,6-0-1,3-glucans by this organism. It generally is believed that the cell envelope carbohydrates of bacteria within the family Rhizobiaceae provide important functions during the plant infection process. These molecules include periplasmic glucans, extracellular polysaccharides, capsular polysaccharides, and lipopolysaccharides (for reviews, see references 12 and 19), as well as a recently discovered class of glucosamine oligosaccharides (18). The periplasmic cyclic P-1,2-glucans appear to be synthesized by all species of Rhizobium and Agrobacterium (13, 14, 16, 17, 33-35), and recent studies have provided evidence that these molecules function during plant infection as well as during bacterial adaptation to hypoosmotic stress (4, 8-11, 23, 26, 36). With respect to their possible functions during legume nodulation, it appears that the cyclic ,B-1,2glucans may function during plant attachment as well as during infection thread initiation. In our recent analysis of the cell-associated oligosaccharides of bacterial strains within the closely related genus Bradyrhizobium, we were unable to detect the presence of cyclic 13-1,2-glucans (22). However, we have shown that species of Bradyrhizobium instead synthesize a different class of cyclic glucans that are linked by ,B-1,6 and 1-1,3 glycosidic bonds (22). We now report the initial characterization of a glucosyltransferase activity within membrane preparations from Bradyrhizobium japonicum USDA 110. The in vitro product of this glucosyltransferase activity shares structural features with the cyclic P-1,6-,B-1,3-glucans isolated from growing cultures of this organism, thus indicating a role for this activity in cyclic glucan biosynthesis.
Fixation and Soybean Genetics Laboratory, Agricultural Research Service, Beltsville, Md. Six-liter cultures were grown in YM medium (22) and incubated at 30°C with vigorous aeration on a rotary shaker. Growth was monitored at 650 nm. Preparation of the membrane fraction. Cultures were harvested during mid-logarithmic growth (cell density of approximately 50 ,ug of total cell protein per ml of culture) by centrifugation at 8,000 x g for 20 min at 5°C. Cell pellets were washed twice with 25 ml of YM salts (22), and pellets were frozen at -20°C. Frozen pellets were thawed on ice, washed twice with ice-cold buffer A [50 mM 3-(N-morpholino)propanesulfonic acid (pH 7.2) containing 6 mM MgSO4, 5 mM 2-mercaptoethanol, and 1 mM dithiothreitol], and resuspended in 1/120 of the original volume of the culture in the same buffer. The cell suspension was cooled in a salt-ice bath and subjected to sonic irradiation (Vibra-cell, model 250W; Sonics and Materials, Inc., Danbury, Conn.) by using a standard horn (0.5-in. [ca. 1-cm] diameter). Samples were treated for a total of 6 min with 1-s bursts, interrupted by 1-s periods of cooling. After sonication, unbroken cells were removed by centrifugation at 3,000 x g for 10 min at 5°C. The supernatant was then centrifuged at 100,000 x g for 1 h at 5°C in order to pellet the membrane fraction. The supernatant (nonmembrane fraction) was removed and was stored in aliquots at -20°C. The membrane pellet was resuspended in buffer A in a volume corresponding to 1/3,000 of the original culture and stored in aliquots at -20°C. Preparation of radiolabeled UDP-glucose. Radiolabeled UDP-glucose (UDP-[1-3H]glucose, 10.4 Ci/mmol) was purchased from New England Nuclear or was prepared enzymatically from D-[6-3H]glucose (New England Nuclear, 38 Ci/mmol) and purified according to procedures previously described (32). Assay for glucosyltransferase activity. The standard reaction mixture contained 3-(N-morpholino)propanesulfonic acid (50 mM, pH 7.2), MgCl2 (10 mM), 2-mercaptoethanol (3
MATERIALS AND METHODS Bacterial strain and growth conditions. B. japonicum USDA 110 was provided by R. F. Griffin of the Nitrogen *
Corresponding author. 4271
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mM), UDP-[3H]glucose (0.8 mM, 1,400 cpm/nmol), and membrane fraction (30 jig of protein) in a total volume of 100 RI. After incubation for 30 min at 37°C, 0.6 ml of 40% (vol/vol) ethanol was added to stop the reaction. The suspension was vortexed and then centrifuged for 5 min at 12,500 x g. A portion of the supernatant (0.5 ml) was applied to a DEAE-cellulose column (Whatman DE-52, 1-ml bed volume) previously equilibrated with 10 mM Tris-HCl buffer (pH 7.4) containing 7% (vol/vol) 1-propanol. The column then was washed with 1.5 ml of 25% (vol/vol) ethanol, and the flowthrough and wash volumes were combined and collected as the neutral fraction (under these conditions, UDP-glucose is adsorbed onto the column). An aliquot (0.5 ml) of the neutral fraction was counted in 5 ml of Liquiscint scintillation solution (National Diagnostics) with a Beckman model LS1701 liquid scintillation spectrophotometer. Identification of radiolabeled glucose. Aliquots of the radiolabeled neutral product (40,000 to 70,000 cpm, 1,600 cpm/ nmol) were subjected to complete acid hydrolysis by treatment with 1 N HCl for 4 h at 100°C (0.5-ml total volume per hydrolysis). After hydrolysis, the preparations were cooled, diluted with 1.5 ml of water, and neutralized with 0.8 g of AG 501-X8 ion-exchange resin (Bio-Rad). The neutralized hydrolysates were dried under nitrogen at 37°C and subsequently were analyzed for [3H]glucose content by the following three methods: thin-layer chromatography, the hexokinase method (32), and reduction with sodium borohydride. Thin-layer chromatography method for glucose analysis. Aliquots (5,000 cpm) of the neutralized hydrolysate were resuspended in water and applied to aluminum-backed silica gel 60 thin-layer chromatography plates (EM Science). The samples were chromatographed on thin-layer chromatography plates in one dimension by using three successive ascents. All three ascents were performed with the same solvent system. Two different solvent systems were used for these analyses: solvent system A consisted of butanolpyridine-water (75:15:10, vol/vol/vol), and solvent system B consisted of ethyl acetate-pyridine-water (1.6:4.5:6.4, vol/ vol/vol). Carbohydrate standards (glucose, galactose, fructose, mannose, and ribose [10 ,ug]) were detected by charring at 170°C after spraying plates with cupric acetate (3%, wt/vol) in sulfuric acid (8%, wt/vol). Radioactivity was detected by cutting thin-layer chromatography lanes into 0.5-cm bands, each of which was directly added to a scintillation vial containing 10 ml of scintillation solution. Hexokinase method. Free glucose present within complete acid hydrolysates of the neutral product was detected with hexokinase (Type V from baker's yeast; Sigma Chemical Co.) as previously described (32). Reduction with sodium borohydride. The presence of free glucose within complete acid hydrolysates was also assayed by reduction with sodium borohydride followed by analysis for glucitol by paper electrophoresis. Aliquots of the neutralized hydrolysate (40,000 cpm) were incubated with 1 mg of sodium borohydride in 40 RI of water for 30 min at 22°C. The reaction was stopped by the addition of 15 ,lI of glacial acetic acid. Next, the mixtures were dried under a stream of nitrogen at 37°C and resuspended with 25 RI of water. The reduced products were analyzed by paper electrophoresis in 0.1 M sodium molybdate buffer, pH 5 (27), using a SemiMicro II chamber (Gelman Sciences). Samples (25 ,lI) were applied to Whatman no. 3 chromatography paper strips (16 by 6 cm), and electrophoresis was performed at 50 V for 3 h. Glucose and glucitol standards (10 ,ug) were detected by the silver nitrate method (20). Radioactive spots were located by
J. BACTERIOL.
cutting the paper strips into 0.5-cm bands, each of which was directly added to a scintillation vial containing 5 ml of scintillation solution.
Glycosidic linkage analysis. The glycosidic linkage composition of the neutral product was determined by analyzing the disaccharides generated after partial acid hydrolysis of the native material. Samples of radiolabeled neutral product (60,000 cpm, 1,200 cpm/nmol) were hydrolyzed in 0.2 M trifluoroacetic acid for 3 h at 100°C in a total volume of 0.5 ml. The hydrolysates were then cooled, diluted with 0.5 ml of water, and neutralized with 0.2 g of AG 501-X8 resin. Next, the partial hydrolysates were fractionated by highperformance liquid chromatography on an Aminex CSA carbohydrate analysis column (300 by 7.8 mm; Bio-Rad). Radioactive material eluting at the retention time corresponding to that of the disaccharide fraction was pooled for further analysis by glucosidase treatment and thin-layer chromatography as described below. Glucosidase treatment. Samples of the radiolabeled disaccharide fraction (1,500 cpm) were digested either with 1 U of P-glucosidase (Boehringer Mannheim) in 50 mM sodium acetate buffer, pH 5.3 (at 37°C for 1 h in a final volume of 1 ml), or with 1 U of a-glucosidase (Boehringer Mannheim) in 50 mM potassium phosphate buffer, pH 6.0 (at 22°C for 1 h in a final volume of 1 ml). After incubation, the mixtures were applied to a Bio-Gel-P2 column (1 by 100 cm). The column was eluted with 7% (vol/vol) 1-propanol, and fractions (1.5 ml) were collected at room temperature at a flow rate of 5 ml/h. Disaccharides were resolved from glucose on this column, thus providing an assay for the release of radiolabeled glucose upon glucosidase digestion. Thin-layer chromatography of disaccharides. In addition to glucosidase treatment, samples of the radiolabeled disaccharide fraction (4,000 cpm) were applied to an aluminumbacked silica gel 60 (EM Science) thin-layer chromatography plate. The plate was developed in one dimension by using two ascents with solvent system A. The relative mobilities of standards (sophorose, gentiobiose, cellobiose, and laminaribiose [10 ,ug]) were revealed by charring with cupric acetate-sulfuric acid reagent (see above). Radioactivity was detected by scintillation counting as described above. RESULTS Evidence for glucosyltransferase activity within membrane fractions from B. japonicum USDA 110. When membrane preparations from B. japonicum USDA 110 were incubated with UDP-[3H]glucose, a radiolabeled neutral fraction was obtained which did not adsorb to DEAE-cellulose. The production of this neutral material was linearly dependent upon both time (up to 60 min) and membrane protein concentration (up to 60 ,ug of total membrane protein). The rate of product formation by membrane fractions (1.6 nmol of UDP-glucose converted to neutral product per min per mg of membrane protein under standard assay conditions) was found to be similar to values previously reported for the membrane-associated glucosyltransferase activities of species of Agrobacterium and Rhizobium (2). After chromatography of the neutral fraction on a Sephadex G50 column, a single peak of radiolabeled material was detected (Fig. 1). The elution volume of this peak was slightly greater than that of cyclic P-1,2-glucan standard obtained from Agrobacterium tumefaciens. Further analysis by gel filtration chromatography on Bio-Gel-P4 (Fig. 2) revealed that the size of the neutral product corresponded to
VOL. 173, 1991
700 600
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FIG. 1. Gel-filtration chromatography of the neutral product. An aliquot (3,400 cpm) of the neutral product synthesized in vitro by the membrane fraction from B. japonicum USDA 110 was chromatographed on a Sephadex G-50 column. The column (1 by 56 cm) was eluted at room temperature with 0.15 M ammonium acetate (pH 7) containing 7% (vol/vol) 1-propanol at a flow rate of 15 ml/h. Fractions (1 ml) were collected, and an aliquot of each was assayed for radioactivity. The arrows indicate the void volume and the peak elution volume for cyclic 0-1,2-glucan standard (prepared from A. tumefaciens C58).
glucan of 2,100 Da (i.e., a glucan containing an of approximately 13 glucose residues). The neutral product is composed solely of glucose. When the neutral product was subjected to complete acid hydrolysis, subsequent analysis demonstrated that glucose was the only detectable radiolabeled monosaccharide. Thin-layer chromatographic analysis of the hydrolysate revealed only one radiolabeled compound which comigrated with glucose in solvent systems A and B (data not shown). In addition, essentially all (97%) of the radioactivity present within the hydrolysate was converted from a neutral form to an anionic form after treatment with hexokinase (it is noted, however, that hexokinase from yeast also phosphorylates fructose and mannose). Furthermore, glucitol was the only product detected by paper electrophoresis after the incubation of the neutralized hydrolysate with sodium borohydride. The neutral product lacks a reducing terminal glucose residue. As described above, the neutral glucan product was estimated to be composed of 13 glucose residues. If this product represents a linear glucan containing one reducing glucose residue per mole, then reduction of the native molecule with sodium borohydride should result in the conversion of approximately 8% of the total glucose residues to glucitol. However, when the native glucan was treated with sodium borohydride prior to acid hydrolysis, little, if any, glucitol was detected within the hydrolysate (0.7% of the total). Thus, the neutral product lacks a reducing terminal glucose residue. The neutral glucan product is linked by P-1,3 and 13-1,6 glycosidic bonds. In order to examine the nature of the glycosidic linkages present within the bradyrhizobial neutral glucan product, the native glucan was partially hydrolyzed a
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log MW
Volume eluted (ml)
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A NOVEL GLUCOSYLTRANSFERASE FROM B. JAPONICUM
FIG. 2. Size determination of the neutral product. The size of the neutral product was determined by using a Bio-Gel-P4 column. An aliquot (30,000 cpm) of the neutral product was applied to the column (1 by 100 cm), and the column was eluted at room temperature with 0.15 M ammonium acetate (pH 7) in 7% (vol/vol) 1-propanol. Fractions (1.5 ml) were collected at a flow rate of 3 ml/h, and aliquots (0.5 ml) of each fraction were assayed for radioactivity. The column was calibrated with the following glucan standards: A, cyclohexaamylose (molecular weight, 973); B, cyclooctaamylose (molecular weight, 1,297); C, cyclic P-1,6-P-1,3-glucan standard from B. japonicum USDA 110 (average molecular weight, 1,863 [22]); D, cyclic P-1,2-glucan standard from A. tumefaciens C58 (average molecular weight, 3,080 [24]). The carbohydrate standards were detected by the phenol method (22). Ka, represents the fraction of the stationary gel volume which is available for diffusion of a given solute species. Kav was calculated as Ve - VJ4Vt - VO, where Ve is the elution volume, VO is the void volume (determined with blue dextran), and V, is the total volume of the packed bed. The molecular weight of the neutral product (filled triangle) was estimated by using the standard curve of Ka, versus the log of the molecular weight of the glucan standards.
average
and the disaccharide fraction was isolated (see Materials and Methods). Initial characterization of the disaccharide fraction was performed by glucosidase treatment. When the disaccharide fraction was treated with 3-glucosidase, approximately 80% of the total radioactivity was liberated as free glucose. In contrast, no detectable glucose was released from the disaccharide fraction upon treatment with a-glucosidase. Thus, these results provide evidence that the neutral glucan product is composed solely of P glycosidic linkages. The nature of the Pi glycosidic linkages subsequently was examined by thin-layer chromatography. Thin-layer chromatographic analysis revealed the presence of two disaccharide components within the partial hydrolysate of the neutral glucan product (Fig. 3). These components were shown to comigrate with laminaribiose (3-0-,-D-glucopyranosyl-D-glucose; 90% of the total disaccharide fraction) and gentiobiose (6-O-p-D-glucopyranosylD-glucose; 9% of the total disaccharide fraction). Thus, the neutral glucan product is composed of P-1,3 and P-1,6 glycosidic linkages in an apparent ratio of 10:1. Properties of the enzymatic activity. The Km for UDPglucose was determined in the standard assay system by varying the UDP-glucose concentration between 10 to 200 ,iM. As shown in Fig. 4, the Km for UDP-glucose was found to be 50 ,uM. The glucosyltransferase activity was found to be greatly stimulated by the addition of manganese, with an optimum concentration for activity of about 10 mM (Fig. 5). When
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COHEN AND MILLER
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FIG. 3. Thin-layer chromatographic analysis of the disaccharide fraction isolated from partial acid hydrolysates of the neutral glucan product. The disaccharide fraction from partial acid hydrolysates was isolated by high-performance liquid chromatography and subsequently analyzed by thin-layer chromatography as described in Materials and Methods. The brackets indicate the positions of migration for the following disaccharide standards: A, gentiobiose (6O---D-glucopyranosyl-D-glucose); B, sophorose (2-O-3-D-glucopyranosyl-D-glucose) and cellobiose (4-O-p-D-glucopyranosyl-Dglucose); C, laminaribiose (3-0-,-D-glucopyranosyl-D-glucose).
other divalent cations were examined at a final concentration of 10 mM, magnesium was also found to stimulate glucosyltransferase activity (Table 1). However, stimulation by magnesium was only about 45% of the level found for manganese at this concentration. The bradyrhizobial glucosyltransferase was active in the
FIG. 5. Effect of manganese on glucosyltransferase activity. Membrane fractions were washed and resuspended in buffer B as described in the footnote to Table 1. The concentration of MnCl2 was varied as indicated. The assay was performed under standard conditions; however, no magnesium was added. Results are expressed as nanomoles of UDP-glucose converted to neutral product per minute per milligram of total membrane protein.
pH range 6.2 to 7.8, with an optimum of about pH 7 when 3-(N-morpholino)propanesulfonic acid buffer adjusted with potassium hydroxide was used (data not shown). Because the glucosyltransferase system of Escherichia coli responsible for ,-1,2-glucan biosynthesis (during membrane-derived oligosaccharide backbone biosynthesis) previously has been shown to require both a membrane fraction and a cytosolic protein (29, 30, 32), it was of interest to examine whether the activity of the bradyrhizobial glucosyltransferase was influenced by the addition of the nonmembrane cell fraction (see Materials and Methods). However, when the nonmembrane cell fraction from B. japonicum USDA 110 (10 ,ug of total protein) was added to the assay mixture (containing 10 ,ug of total membrane protein), a dramatic inhibition of glucosyltransferase activity was observed (data not shown).
2.0'
DISCUSSION We have identified a novel glucosyltransferase activity within membrane preparations from B. japonicum USDA 110. The neutral glucan product formed was found to contain both P-1,3 and ,-1,6 glycosidic linkages in a ratio of approx-
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TABLE 1. Stimulation of glucosyltransferase activity by manganese and magnesiuma
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t UDPG I, p>M FIG. 4. Km determination for UDP-glucose. The concentration of the substrate (S), UDP-[3H]glucose, in the standard assay mixture was varied from 10 to 200 ,uM (880 to 104,000 cpm/nmol). Velocity (V) is expressed as nanomoles of UDP-glucose converted to neutral product per minute per milligram of total membrane protein. A double-reciprocal plot is shown in the inset. S,
MgSO4 ....................................... MgC12 .......................................
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.......................................
0.37
3.82 1.74 1.60 0.66 0.03
a Membrane fractions were washed twice with buffer B [50 mM 3-(Nmorpholino)propanesulfonic acid (pH 7.2) containing 5 mM 2-mercaptoethanol and 1 mM dithiothreitolJ and resuspended in the same buffer. The indicated salts were added to the assay mixture at a final concentration of 10 mM in an otherwise standard assay.
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A NOVEL GLUCOSYLTRANSFERASE FROM B. JAPONICUM
imately 10:1. On the basis of an average molecular weight of 2,100 (corresponding to 13 glucose residues), it would appear that the neutral glucan product contains approximately one ,B-1,6 glycosidic linkage per molecule. The lack of a detectable reducing terminus within the neutral glucan product suggests that the glucan may be cyclic in character. This result is intriguing in view of the fact that we previously have shown that B.japonicum USDA 110 synthesizes cyclic, branched 1-glucans containing both P-1,6 and 1-1,3 glycosidic linkages (22). Thus, it is possible that the glucosyltransferase activity described herein may be responsible for the biosynthesis of these cyclic 1-1,6-13-1,3glucans. However, there are two important distinctions between the product of the in vitro glucosyltransferase activity and the cell-associated cyclic 1-glucans isolated from growing cultures of B. japonicum USDA 110. First, the cyclic 1-glucans extracted from cultures of B. japonicum USDA 110 are slightly smaller (1,620 to 2,106 Da [22]) than the in vitro product (2,100 Da) of the glucosyltransferase activity (Fig. 2). Second, the relative proportion of 1-1,3 to 13-1,6 linkages is different for the two glucans. As described above, analysis of the disaccharide fraction of partial acid hydrolysates of the radiolabeled neutral glucan product revealed a ratio of 10 ,B-1,3 glycosidic linkages per 1 13-1,6 glycosidic linkage. In contrast, the ratio within cyclic 13glucans produced in vivo is 0.6 1-1,3 glycosidic linkages per 1 1-1,6 glycosidic linkage (22). One possible explanation for this apparent discrepancy is that the glycosidic linkage composition of the disaccharide fraction isolated from partial acid hydrolysates may not be truly representative since rate constants for the acid hydrolysis of glycosidic bonds may vary greatly (25). It is also possible that certain regulatory factors may be limiting or absent in the in vitro assay system, leading to products with structural features different from those of products synthesized in vivo. Similar results have previously been reported during the in vitro study of the glucosyltransferase system of E. coli responsible for membrane-derived oligosaccharide biosynthesis (32). With respect to the Km for UDP-glucose and the stimulation of activity by manganese and magnesium, the glucosyltransferase activity of B. japonicum USDA 110 would appear to be similar to the membrane-bound glucosyltransferase activities of the closely related genera Agrobacterium and Rhizobium (2, 38). However, the products of the membrane-bound activities of the organisms of the latter two genera represent a mixture of cyclic glucans containing 14 to 25 glucose residues linked solely by 13-1,2 glycosidic bonds (2, 38). In contrast, it is noted that essentially no 13-1,2-linked glucose was detected within the neutral fraction synthesized by membrane preparations derived from B. japonicum USDA 110 (Fig. 3). Our results would also appear to be in contrast to those reported by Dedonder and Hassid (6), who found that membrane preparations from Rhizobium japonicum contained a glucosyltransferase activity which synthesized a 13-1,2-linked glucan from UDP-glucose. However, on the basis of the growth rate of the bacterium examined by Dedonder and Hassid, it is likely that this organism represented a fast-growing strain within the Rhizobium genus and was not a true slow-growing strain of the presently recognized Bradyrhizobium genus. Our results indicate that species of Bradyrhizobium apparently lack the capacity for cyclic 13-1,2-glucan biosynthesis. In fact, this conclusion is consistent with our previous analysis of the cell-associated glucans of species of Bradyrhizobium (22), which demonstrated an absence of detectable levels of cyclic 1-1,2-glucans. Also consistent
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with this conclusion is the report by Dylan and coworkers (10) that genomic DNA from B. japonicum USDA 110 lacks significant homology with the chvA and chvB genes from Agrobacterium spp. and the ndvA and ndvB genes from Rhizobium spp. The NdvA and ChvA proteins appear to provide functions during cyclic 13-1,2-glucan export (3, 7, 28). The NdvB and ChvB proteins are high-molecular-weight membrane proteins (molecular weight, 319,000) which have been shown to form a covalently linked intermediate with the cyclic 13-1,2-glucan during biosynthesis (15, 37, 39). It may be noted that our attempts to detect the formation of a protein-linked intermediate during in vitro glucan biosynthesis with membrane fractions from B. japonicum USDA 110 have been unsuccessful (5). By using methods described previously by Zorreguieta and Ugalde (39), we were unable to detect the presence of a radiolabeled protein-linked intermediate within B. japonicum membrane preparations. However, in control experiments with membrane preparations derived from Rhizobium meliloti 1021, a radiolabeled protein-linked intermediate (apparent Mr, 235,000) was readily detected. Furthermore, we were unable to detect by Coomassie blue staining of polyacrylamide gels the presence of a protein within B. japonicum USDA 110 membrane preparations which migrated with an apparent molecular weight of 235,000 (the NdvB protein within R. meliloti 1021 membrane preparations is readily detected by Coomassie blue staining of polyacrylamide gels). Because the cyclic 13-1,2-glucans of Rhizobium spp. appear to have important roles in bacterial osmotic adaptation and legume nodulation (9-11, 36), it is interesting that these molecules are not synthesized by species of Bradyrhizobium. However, it is possible that the cyclic 1-1,613-1,3-glucans of Bradyrhizobium spp. may represent functional analogs of the cyclic 1-1,2-glucans. Indeed, this notion is supported by the recent demonstration that the biosynthesis of both classes of glucans is strictly osmoregulated (21, 23, 31) and that both classes of glucans are localized predominantly in the periplasmic compartment (1, 21, 23). In order to gain further insight regarding the possible functions of the cyclic 13-1,6-P-1,3-glucans of Bradyrhizobium spp., future studies will be directed towards the isolation of mutants specifically blocked for the biosynthesis of these glucans. Such mutants will also be required in order to elucidate whether the glucosyltransferase activity described herein provides an essential function during cyclic 1-1,61-1,3-glucan biosynthesis. ACKNOWLEDGMENT This research was supported by National Science Foundation grant DCB-8803247 awarded to K.J.M. REFERENCES 1. Abe, M., A. Amemura, and S. Higashi. 1982. Studies on cyclic beta-1,2-glucan obtained from periplasmic space of Rhizobium trifolii cells. Plant Soil 64:315-324. 2. Amemura, A. 1984. Synthesis of (1-2)-beta-D-glucan by cell-free extracts of Agrobacterium radiobacter IFO 12665bl and Rhizobium phaseoli AHU 1133. Agric. Biol. Chem. 48:1809-1817. 3. Cangelosi, G. A., G. Martinetti, J. A. Leigh, C. C. Lee, C. Theines, and E. W. Nester. 1989. Role of Agrobacterium tumefaciens ChvA protein in export of P-1,2-glucan. J. Bacteriol. 171:1609-1615. 4. Cangelosi, G. A., G. Martinetti, and E. W. Nester. 1990. Osmosensitivity phenotypes of Agrobacterium tumefaciens mutants that lack periplasmic ,-1,2-glucan. J. Bacteriol. 172:21722174. 5. Cohen, J. L., and K. J. Miller. Unpublished data.
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6. Dedonder, R. A., and W. Z. Hassid. 1964. The enzymatic synthesis of a (,-1,2-)-linked glucan by an extract of Rhizobium japonicum. Biochim. Biophys. Acta 90:239-248. 7. de lannino, N. I., and R. A. Ugalde. 1989. Biochemical characterization of avirulent Agrobacterium tumefaciens chvA mutants: synthesis and excretion of P-(1-2)glucan. J. Bacteriol. 171:2842-2849. 8. Douglas, L. J., R. J. Staneloni, R. A. Rubin, and E. W. Nester. 1985. Identification and genetic analysis of an Agrobacterium tumefaciens chromosomal virulence region. J. Bacteriol. 161: 850-860. 9. Dylan, T., D. R. Helinski, and G. S. Ditta. 1990. Hypoosmotic adaptation in Rhizobium meliloti requires P-(1-2)-glucan. J. Bacteriol. 172:1400-1408. 10. Dylan, T., L. lelpi, S. Stanfield, L. Kashyap, C. Douglas, M. Yanofsky, E. Nester, D. R. Helinski, and G. Ditta. 1986. Rhizobium meliloti genes required for nodule development are related to chromosomal virulence genes in Agrobacterium tumefaciens. Proc. Natl. Acad. Sci. USA 83:4403-4407. 11. Dylan, T., P. Nagpal, D. R. Helinski, and G. S. Ditta. 1990. Symbiotic pseudorevertants of Rhizobium meliloti ndv mutants. J. Bacteriol. 172:1409-1417. 12. Halverson, L. J., and G. Stacey. 1986. Signal exchange in plant-microbe interactions. Microbiol. Rev. 50:193-225. 13. Hisamatsu, M., A. Amemura, K. Koizumi, T. Utamura, and Y. Okada. 1983. Structural studies on cyclic (1-2)-beta-D-glucans (cyclosophoraose) produced by Agrobacterium and Rhizobium. Carbohydr. Res. 121:31-40. 14. Hisamatsu, M., A. Amemura, T. Matsuo, H. Matsuda, and T. Harada. 1982. Cyclic (1-2)-beta-D-glucan and the octasaccharide repeating-unit of succinoglycan produced by Agrobacterium. J. Gen. Microbiol. 128:1873-1879. 15. Ielpi, L., T. Dylan, G. S. Ditta, D. R. Helinski, and S. W. Stanfield. 1990. The ndvB locus of Rhizobium meliloti encodes a 319-kDa protein involved in the production of beta-(1-2)-glucan. J. Biol. Chem. 265:2843-2851. 16. Koizumi, K., Y. Okada, S. Horiyama, and T. Utamura. 1983. Separation of cyclic (1-2)-beta-D-glucans (cyclosophoraose) produced by Agrobacterium and Rhizobium, and determination of the degree of polymerization by high-performance liquid chromatography. J. Chromatogr. 265:89-96. 17. Koizumi, K., Y. Okada, T. Utamura, M. Hisamatsu, and A. Amemura. 1984. Further studies on the separation of cyclic (1-2)-beta-D-glucans (cyclosophoraoses) produced by Rhizobium meliloti IFO 13336, and determination of their degrees of polymerization by high-performance liquid chromatography. J. Chromatogr. 299:215-224. 18. Lerouge, P., P. Roche, C. Faucher, F. Mailiet, G. Truchet, J. C. Prome, and J. Denarie. 1990. Symbiotic host-specificity of Rhizobium meliloti is determined by a sulphated and acylated glucosamine oligosaccharide signal. Nature (London) 344:781784. 19. Long, S. R. 1989. Rhizobium-legume nodulation: life together in the underground. Cell 56:203-214. 20. Mayer, F. C., and J. Larner. 1959. Substrate cleavage point of the alpha and beta-amylases. J. Am. Chem. Soc. 81:188-198. 21. Miller, K. J., and R. S. Gore. Unpublished data. 22. Miller, K. J., R. S. Gore, R. Johnson, A. J. Benesi, and V. N. Reinhold. 1990. Cell-associated oligosaccharides of Bradyrhizo-
J. BACTERIOL.
bium spp. J. Bacteriol. 172:136-142. 23. Miller, K. J., E. P. Kennedy, and V. N. Reinhold. 1986. Osmotic adaptation by Gram-negative bacteria: possible role for periplasmic oligosaccharides. Science 231:48-51. 24. Miller, K. J., V. N. Reinhold, A. C. Weissborn, and E. P. Kennedy. 1987. Cyclic glucans produced by Agrobacterium tumefaciens are substituted with sn-1-phosphoglycerol residues. Biochim. Biophys. Acta 901:112-118. 25. Pazur, J. H. 1986. Neutral polysaccharides, p. 55-96. In M. F. Chaplin and J. F. Kennedy (ed.), Carbohydrate analysis, a practical approach. IRL Press, Inc., Oxford. 26. Puvanesarajah, V., F. M. Schell, G. Stacey, C. J. Douglas, and E. W. Nester. 1985. Role for 2-linked-,3-D-glucan in the virulence of Agrobacterium tumefaciens. J. Bacteriol. 164:102-106. 27. Sandermann, H., Jr., and R. F. H. Dekker. 1979. Beta-1,2glucosyl transfer by membrane preparations from Acetobacter xylinum. FEBS Lett. 107:237-240. 28. Stanfield, S. W., L. Ielpi, D. O'Brochta, D. R. Helinski, and G. S. Ditta. 1988. The ndvA gene product of Rhizobium meliloti is required for P-(1-2)glucan production and has homology to the ATP-binding export protein HlyB. J. Bacteriol. 170:3523-3530. 29. Therisod, H., and E. P. Kennedy. 1987. The function of acyl carrier protein in the synthesis of membrane-derived oligosaccharides does not require its phosphopantetheine prosthetic group. Proc. Natl. Acad. Sci. USA 84:8235-8238. 30. Therisod, H., A. C. Weissborn, and E. P. Kennedy. 1986. An essential function for acyl carrier protein in the biosynthesis of membrane-derived oligosaccharides of Escherichia coli. Proc. Natl. Acad. Sci. USA 83:7236-7240. 31. Tully, R. E., D. L. Keister, and K. C. Gross. 1990. Fractionation of the 3-linked glucans of Bradyrhizobium japonicum and their response to osmotic potential. Appl. Environ. Microbiol. 56: 1518-1522. 32. Weissborn, A. C., and E. P. Kennedy. 1984. Biosynthesis of membrane-derived oligosaccharides. J. Biol. Chem. 259:1264412651. 33. York, W. S., M. McNeil, A. G. Darvill, and P. Albersheim. 1978. Beta-2-linked glucans secreted by fast-growing species of Rhizobium. J. Bacteriol. 142:243-248. 34. Zevenhuizen, L. P. T. M. 1981. Cellular glycogen, beta-1,2glucan, poly-beta-hydroxybutyric acid and extracellular polysaccharides in fast-growing species of Rhizobium. Antonie van Leeuwenhoek J. Microbiol. Serol. 47:481-497. 35. Zevenhuizen, L. P. T. M., and H. J. Scholten-Koerselman. 1979. Surface carbohydrates of Rhizobium. I. Beta-1,2-glucans. Antonie van Leeuwenhoek J. Microbiol. Serol. 45:165-175. 36. Zorreguieta, A., S. Cavaignac, R. A. Geremia, and R. A. Ugalde. 1990. Osmotic regulation of P(1-2) glucan synthesis in members of the family Rhizobiaceae. J. Bacteriol. 172:4701-4704. 37. Zorreguieta, A., R. A. Geremia, S. Cavaignac, G. A. Cangelosi, E. W. Nester, and R. A. Ugalde. 1988. Identification of the product of an Agrobacterium tumefaciens chromosomal virulence gene. Mol. Plant Microbe Interact. 1:121-127. 38. Zorreguieta, A., M. E. Tomalsky, and R. J. Staneloni. 1985. The enzymatic synthesis of beta-1,2 glucans. Arch. Biochem. Biophys. 238:368-372. 39. Zorreguieta, A., and R. A. Ugalde. 1986. Formation in Rhizobium and Agrobacterium spp. of a 235-kilodalton protein intermediate in P-D-(1-2) glucan synthesis. J. Bacteriol. 167:947-951.
Vol. 173, No. 14
A Novel Membrane-Bound Glucosyltransferase from Bradyrhizobium japonicum JODI L. COHEN' AND KAREN J. MILLER'12,3* Department of Food Science' and Graduate Programs in Plant Physiology2 and Genetics,3 The Pennsylvania State University, University Park, Pennsylvania 16802 Received 28 February 1991/Accepted 3 May 1991
Bacteria within the family Rhizobiaceae are distinguished by their ability to infect higher plants. The cell envelope carbohydrates of these bacteria are believed to be involved in the plant infection process. One class of cell envelope carbohydrate, the cyclic f0-1,2-glucans, is synthesized by species within two genera of this family, Agrobacterium and Rhizobium. In contrast, species of the genus Bradyrhizobium, a third genus within this family, appear to lack the capacity for cyclic P1-1,2-glucan biosynthesis. Instead, these bacteria synthesize cyclic glucans containing ,-1,6 and 0-1,3 glycosidic linkages (K. J. Miller, R. S. Gore, R. Johnson, A. J. Benesi, and V. N. Reinhold, J. Bacteriol. 172:136-142, 1990). We now report the initial characterization of a novel membrane-bound glucosyltransferase activity from Bradyrhizobiumjaponicum USDA 110. Analysis of the product of this glucosyltransferase activity revealed the following: the presence of 0-1,3 and 0-1,6 glycosidic linkages, an average molecular weight of 2,100, and no detectable reducing terminal residues. The glucosyltransferase activity was found to have an apparent Km of 50 ,uM for UDP-glucose, and activity was stimulated optimally by Mn2+ ions. On the basis of the structural properties of the in vitro glucan product, it is possible that this membrane-bound glucosyltransferase activity may be responsible for the biosynthesis of cyclic 0I-1,6-0-1,3-glucans by this organism. It generally is believed that the cell envelope carbohydrates of bacteria within the family Rhizobiaceae provide important functions during the plant infection process. These molecules include periplasmic glucans, extracellular polysaccharides, capsular polysaccharides, and lipopolysaccharides (for reviews, see references 12 and 19), as well as a recently discovered class of glucosamine oligosaccharides (18). The periplasmic cyclic P-1,2-glucans appear to be synthesized by all species of Rhizobium and Agrobacterium (13, 14, 16, 17, 33-35), and recent studies have provided evidence that these molecules function during plant infection as well as during bacterial adaptation to hypoosmotic stress (4, 8-11, 23, 26, 36). With respect to their possible functions during legume nodulation, it appears that the cyclic ,B-1,2glucans may function during plant attachment as well as during infection thread initiation. In our recent analysis of the cell-associated oligosaccharides of bacterial strains within the closely related genus Bradyrhizobium, we were unable to detect the presence of cyclic 13-1,2-glucans (22). However, we have shown that species of Bradyrhizobium instead synthesize a different class of cyclic glucans that are linked by ,B-1,6 and 1-1,3 glycosidic bonds (22). We now report the initial characterization of a glucosyltransferase activity within membrane preparations from Bradyrhizobium japonicum USDA 110. The in vitro product of this glucosyltransferase activity shares structural features with the cyclic P-1,6-,B-1,3-glucans isolated from growing cultures of this organism, thus indicating a role for this activity in cyclic glucan biosynthesis.
Fixation and Soybean Genetics Laboratory, Agricultural Research Service, Beltsville, Md. Six-liter cultures were grown in YM medium (22) and incubated at 30°C with vigorous aeration on a rotary shaker. Growth was monitored at 650 nm. Preparation of the membrane fraction. Cultures were harvested during mid-logarithmic growth (cell density of approximately 50 ,ug of total cell protein per ml of culture) by centrifugation at 8,000 x g for 20 min at 5°C. Cell pellets were washed twice with 25 ml of YM salts (22), and pellets were frozen at -20°C. Frozen pellets were thawed on ice, washed twice with ice-cold buffer A [50 mM 3-(N-morpholino)propanesulfonic acid (pH 7.2) containing 6 mM MgSO4, 5 mM 2-mercaptoethanol, and 1 mM dithiothreitol], and resuspended in 1/120 of the original volume of the culture in the same buffer. The cell suspension was cooled in a salt-ice bath and subjected to sonic irradiation (Vibra-cell, model 250W; Sonics and Materials, Inc., Danbury, Conn.) by using a standard horn (0.5-in. [ca. 1-cm] diameter). Samples were treated for a total of 6 min with 1-s bursts, interrupted by 1-s periods of cooling. After sonication, unbroken cells were removed by centrifugation at 3,000 x g for 10 min at 5°C. The supernatant was then centrifuged at 100,000 x g for 1 h at 5°C in order to pellet the membrane fraction. The supernatant (nonmembrane fraction) was removed and was stored in aliquots at -20°C. The membrane pellet was resuspended in buffer A in a volume corresponding to 1/3,000 of the original culture and stored in aliquots at -20°C. Preparation of radiolabeled UDP-glucose. Radiolabeled UDP-glucose (UDP-[1-3H]glucose, 10.4 Ci/mmol) was purchased from New England Nuclear or was prepared enzymatically from D-[6-3H]glucose (New England Nuclear, 38 Ci/mmol) and purified according to procedures previously described (32). Assay for glucosyltransferase activity. The standard reaction mixture contained 3-(N-morpholino)propanesulfonic acid (50 mM, pH 7.2), MgCl2 (10 mM), 2-mercaptoethanol (3
MATERIALS AND METHODS Bacterial strain and growth conditions. B. japonicum USDA 110 was provided by R. F. Griffin of the Nitrogen *
Corresponding author. 4271
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COHEN AND MILLER
mM), UDP-[3H]glucose (0.8 mM, 1,400 cpm/nmol), and membrane fraction (30 jig of protein) in a total volume of 100 RI. After incubation for 30 min at 37°C, 0.6 ml of 40% (vol/vol) ethanol was added to stop the reaction. The suspension was vortexed and then centrifuged for 5 min at 12,500 x g. A portion of the supernatant (0.5 ml) was applied to a DEAE-cellulose column (Whatman DE-52, 1-ml bed volume) previously equilibrated with 10 mM Tris-HCl buffer (pH 7.4) containing 7% (vol/vol) 1-propanol. The column then was washed with 1.5 ml of 25% (vol/vol) ethanol, and the flowthrough and wash volumes were combined and collected as the neutral fraction (under these conditions, UDP-glucose is adsorbed onto the column). An aliquot (0.5 ml) of the neutral fraction was counted in 5 ml of Liquiscint scintillation solution (National Diagnostics) with a Beckman model LS1701 liquid scintillation spectrophotometer. Identification of radiolabeled glucose. Aliquots of the radiolabeled neutral product (40,000 to 70,000 cpm, 1,600 cpm/ nmol) were subjected to complete acid hydrolysis by treatment with 1 N HCl for 4 h at 100°C (0.5-ml total volume per hydrolysis). After hydrolysis, the preparations were cooled, diluted with 1.5 ml of water, and neutralized with 0.8 g of AG 501-X8 ion-exchange resin (Bio-Rad). The neutralized hydrolysates were dried under nitrogen at 37°C and subsequently were analyzed for [3H]glucose content by the following three methods: thin-layer chromatography, the hexokinase method (32), and reduction with sodium borohydride. Thin-layer chromatography method for glucose analysis. Aliquots (5,000 cpm) of the neutralized hydrolysate were resuspended in water and applied to aluminum-backed silica gel 60 thin-layer chromatography plates (EM Science). The samples were chromatographed on thin-layer chromatography plates in one dimension by using three successive ascents. All three ascents were performed with the same solvent system. Two different solvent systems were used for these analyses: solvent system A consisted of butanolpyridine-water (75:15:10, vol/vol/vol), and solvent system B consisted of ethyl acetate-pyridine-water (1.6:4.5:6.4, vol/ vol/vol). Carbohydrate standards (glucose, galactose, fructose, mannose, and ribose [10 ,ug]) were detected by charring at 170°C after spraying plates with cupric acetate (3%, wt/vol) in sulfuric acid (8%, wt/vol). Radioactivity was detected by cutting thin-layer chromatography lanes into 0.5-cm bands, each of which was directly added to a scintillation vial containing 10 ml of scintillation solution. Hexokinase method. Free glucose present within complete acid hydrolysates of the neutral product was detected with hexokinase (Type V from baker's yeast; Sigma Chemical Co.) as previously described (32). Reduction with sodium borohydride. The presence of free glucose within complete acid hydrolysates was also assayed by reduction with sodium borohydride followed by analysis for glucitol by paper electrophoresis. Aliquots of the neutralized hydrolysate (40,000 cpm) were incubated with 1 mg of sodium borohydride in 40 RI of water for 30 min at 22°C. The reaction was stopped by the addition of 15 ,lI of glacial acetic acid. Next, the mixtures were dried under a stream of nitrogen at 37°C and resuspended with 25 RI of water. The reduced products were analyzed by paper electrophoresis in 0.1 M sodium molybdate buffer, pH 5 (27), using a SemiMicro II chamber (Gelman Sciences). Samples (25 ,lI) were applied to Whatman no. 3 chromatography paper strips (16 by 6 cm), and electrophoresis was performed at 50 V for 3 h. Glucose and glucitol standards (10 ,ug) were detected by the silver nitrate method (20). Radioactive spots were located by
J. BACTERIOL.
cutting the paper strips into 0.5-cm bands, each of which was directly added to a scintillation vial containing 5 ml of scintillation solution.
Glycosidic linkage analysis. The glycosidic linkage composition of the neutral product was determined by analyzing the disaccharides generated after partial acid hydrolysis of the native material. Samples of radiolabeled neutral product (60,000 cpm, 1,200 cpm/nmol) were hydrolyzed in 0.2 M trifluoroacetic acid for 3 h at 100°C in a total volume of 0.5 ml. The hydrolysates were then cooled, diluted with 0.5 ml of water, and neutralized with 0.2 g of AG 501-X8 resin. Next, the partial hydrolysates were fractionated by highperformance liquid chromatography on an Aminex CSA carbohydrate analysis column (300 by 7.8 mm; Bio-Rad). Radioactive material eluting at the retention time corresponding to that of the disaccharide fraction was pooled for further analysis by glucosidase treatment and thin-layer chromatography as described below. Glucosidase treatment. Samples of the radiolabeled disaccharide fraction (1,500 cpm) were digested either with 1 U of P-glucosidase (Boehringer Mannheim) in 50 mM sodium acetate buffer, pH 5.3 (at 37°C for 1 h in a final volume of 1 ml), or with 1 U of a-glucosidase (Boehringer Mannheim) in 50 mM potassium phosphate buffer, pH 6.0 (at 22°C for 1 h in a final volume of 1 ml). After incubation, the mixtures were applied to a Bio-Gel-P2 column (1 by 100 cm). The column was eluted with 7% (vol/vol) 1-propanol, and fractions (1.5 ml) were collected at room temperature at a flow rate of 5 ml/h. Disaccharides were resolved from glucose on this column, thus providing an assay for the release of radiolabeled glucose upon glucosidase digestion. Thin-layer chromatography of disaccharides. In addition to glucosidase treatment, samples of the radiolabeled disaccharide fraction (4,000 cpm) were applied to an aluminumbacked silica gel 60 (EM Science) thin-layer chromatography plate. The plate was developed in one dimension by using two ascents with solvent system A. The relative mobilities of standards (sophorose, gentiobiose, cellobiose, and laminaribiose [10 ,ug]) were revealed by charring with cupric acetate-sulfuric acid reagent (see above). Radioactivity was detected by scintillation counting as described above. RESULTS Evidence for glucosyltransferase activity within membrane fractions from B. japonicum USDA 110. When membrane preparations from B. japonicum USDA 110 were incubated with UDP-[3H]glucose, a radiolabeled neutral fraction was obtained which did not adsorb to DEAE-cellulose. The production of this neutral material was linearly dependent upon both time (up to 60 min) and membrane protein concentration (up to 60 ,ug of total membrane protein). The rate of product formation by membrane fractions (1.6 nmol of UDP-glucose converted to neutral product per min per mg of membrane protein under standard assay conditions) was found to be similar to values previously reported for the membrane-associated glucosyltransferase activities of species of Agrobacterium and Rhizobium (2). After chromatography of the neutral fraction on a Sephadex G50 column, a single peak of radiolabeled material was detected (Fig. 1). The elution volume of this peak was slightly greater than that of cyclic P-1,2-glucan standard obtained from Agrobacterium tumefaciens. Further analysis by gel filtration chromatography on Bio-Gel-P4 (Fig. 2) revealed that the size of the neutral product corresponded to
VOL. 173, 1991
700 600
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FIG. 1. Gel-filtration chromatography of the neutral product. An aliquot (3,400 cpm) of the neutral product synthesized in vitro by the membrane fraction from B. japonicum USDA 110 was chromatographed on a Sephadex G-50 column. The column (1 by 56 cm) was eluted at room temperature with 0.15 M ammonium acetate (pH 7) containing 7% (vol/vol) 1-propanol at a flow rate of 15 ml/h. Fractions (1 ml) were collected, and an aliquot of each was assayed for radioactivity. The arrows indicate the void volume and the peak elution volume for cyclic 0-1,2-glucan standard (prepared from A. tumefaciens C58).
glucan of 2,100 Da (i.e., a glucan containing an of approximately 13 glucose residues). The neutral product is composed solely of glucose. When the neutral product was subjected to complete acid hydrolysis, subsequent analysis demonstrated that glucose was the only detectable radiolabeled monosaccharide. Thin-layer chromatographic analysis of the hydrolysate revealed only one radiolabeled compound which comigrated with glucose in solvent systems A and B (data not shown). In addition, essentially all (97%) of the radioactivity present within the hydrolysate was converted from a neutral form to an anionic form after treatment with hexokinase (it is noted, however, that hexokinase from yeast also phosphorylates fructose and mannose). Furthermore, glucitol was the only product detected by paper electrophoresis after the incubation of the neutralized hydrolysate with sodium borohydride. The neutral product lacks a reducing terminal glucose residue. As described above, the neutral glucan product was estimated to be composed of 13 glucose residues. If this product represents a linear glucan containing one reducing glucose residue per mole, then reduction of the native molecule with sodium borohydride should result in the conversion of approximately 8% of the total glucose residues to glucitol. However, when the native glucan was treated with sodium borohydride prior to acid hydrolysis, little, if any, glucitol was detected within the hydrolysate (0.7% of the total). Thus, the neutral product lacks a reducing terminal glucose residue. The neutral glucan product is linked by P-1,3 and 13-1,6 glycosidic bonds. In order to examine the nature of the glycosidic linkages present within the bradyrhizobial neutral glucan product, the native glucan was partially hydrolyzed a
3.3
3.5
log MW
Volume eluted (ml)
that of
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B1-2 I
void
I
A NOVEL GLUCOSYLTRANSFERASE FROM B. JAPONICUM
FIG. 2. Size determination of the neutral product. The size of the neutral product was determined by using a Bio-Gel-P4 column. An aliquot (30,000 cpm) of the neutral product was applied to the column (1 by 100 cm), and the column was eluted at room temperature with 0.15 M ammonium acetate (pH 7) in 7% (vol/vol) 1-propanol. Fractions (1.5 ml) were collected at a flow rate of 3 ml/h, and aliquots (0.5 ml) of each fraction were assayed for radioactivity. The column was calibrated with the following glucan standards: A, cyclohexaamylose (molecular weight, 973); B, cyclooctaamylose (molecular weight, 1,297); C, cyclic P-1,6-P-1,3-glucan standard from B. japonicum USDA 110 (average molecular weight, 1,863 [22]); D, cyclic P-1,2-glucan standard from A. tumefaciens C58 (average molecular weight, 3,080 [24]). The carbohydrate standards were detected by the phenol method (22). Ka, represents the fraction of the stationary gel volume which is available for diffusion of a given solute species. Kav was calculated as Ve - VJ4Vt - VO, where Ve is the elution volume, VO is the void volume (determined with blue dextran), and V, is the total volume of the packed bed. The molecular weight of the neutral product (filled triangle) was estimated by using the standard curve of Ka, versus the log of the molecular weight of the glucan standards.
average
and the disaccharide fraction was isolated (see Materials and Methods). Initial characterization of the disaccharide fraction was performed by glucosidase treatment. When the disaccharide fraction was treated with 3-glucosidase, approximately 80% of the total radioactivity was liberated as free glucose. In contrast, no detectable glucose was released from the disaccharide fraction upon treatment with a-glucosidase. Thus, these results provide evidence that the neutral glucan product is composed solely of P glycosidic linkages. The nature of the Pi glycosidic linkages subsequently was examined by thin-layer chromatography. Thin-layer chromatographic analysis revealed the presence of two disaccharide components within the partial hydrolysate of the neutral glucan product (Fig. 3). These components were shown to comigrate with laminaribiose (3-0-,-D-glucopyranosyl-D-glucose; 90% of the total disaccharide fraction) and gentiobiose (6-O-p-D-glucopyranosylD-glucose; 9% of the total disaccharide fraction). Thus, the neutral glucan product is composed of P-1,3 and P-1,6 glycosidic linkages in an apparent ratio of 10:1. Properties of the enzymatic activity. The Km for UDPglucose was determined in the standard assay system by varying the UDP-glucose concentration between 10 to 200 ,iM. As shown in Fig. 4, the Km for UDP-glucose was found to be 50 ,uM. The glucosyltransferase activity was found to be greatly stimulated by the addition of manganese, with an optimum concentration for activity of about 10 mM (Fig. 5). When
4274
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COHEN AND MILLER
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FIG. 3. Thin-layer chromatographic analysis of the disaccharide fraction isolated from partial acid hydrolysates of the neutral glucan product. The disaccharide fraction from partial acid hydrolysates was isolated by high-performance liquid chromatography and subsequently analyzed by thin-layer chromatography as described in Materials and Methods. The brackets indicate the positions of migration for the following disaccharide standards: A, gentiobiose (6O---D-glucopyranosyl-D-glucose); B, sophorose (2-O-3-D-glucopyranosyl-D-glucose) and cellobiose (4-O-p-D-glucopyranosyl-Dglucose); C, laminaribiose (3-0-,-D-glucopyranosyl-D-glucose).
other divalent cations were examined at a final concentration of 10 mM, magnesium was also found to stimulate glucosyltransferase activity (Table 1). However, stimulation by magnesium was only about 45% of the level found for manganese at this concentration. The bradyrhizobial glucosyltransferase was active in the
FIG. 5. Effect of manganese on glucosyltransferase activity. Membrane fractions were washed and resuspended in buffer B as described in the footnote to Table 1. The concentration of MnCl2 was varied as indicated. The assay was performed under standard conditions; however, no magnesium was added. Results are expressed as nanomoles of UDP-glucose converted to neutral product per minute per milligram of total membrane protein.
pH range 6.2 to 7.8, with an optimum of about pH 7 when 3-(N-morpholino)propanesulfonic acid buffer adjusted with potassium hydroxide was used (data not shown). Because the glucosyltransferase system of Escherichia coli responsible for ,-1,2-glucan biosynthesis (during membrane-derived oligosaccharide backbone biosynthesis) previously has been shown to require both a membrane fraction and a cytosolic protein (29, 30, 32), it was of interest to examine whether the activity of the bradyrhizobial glucosyltransferase was influenced by the addition of the nonmembrane cell fraction (see Materials and Methods). However, when the nonmembrane cell fraction from B. japonicum USDA 110 (10 ,ug of total protein) was added to the assay mixture (containing 10 ,ug of total membrane protein), a dramatic inhibition of glucosyltransferase activity was observed (data not shown).
2.0'
DISCUSSION We have identified a novel glucosyltransferase activity within membrane preparations from B. japonicum USDA 110. The neutral glucan product formed was found to contain both P-1,3 and ,-1,6 glycosidic linkages in a ratio of approx-
1.6
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TABLE 1. Stimulation of glucosyltransferase activity by manganese and magnesiuma
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t UDPG I, p>M FIG. 4. Km determination for UDP-glucose. The concentration of the substrate (S), UDP-[3H]glucose, in the standard assay mixture was varied from 10 to 200 ,uM (880 to 104,000 cpm/nmol). Velocity (V) is expressed as nanomoles of UDP-glucose converted to neutral product per minute per milligram of total membrane protein. A double-reciprocal plot is shown in the inset. S,
MgSO4 ....................................... MgC12 .......................................
CaCl2 ....................................... ZnCl2
.......................................
0.37
3.82 1.74 1.60 0.66 0.03
a Membrane fractions were washed twice with buffer B [50 mM 3-(Nmorpholino)propanesulfonic acid (pH 7.2) containing 5 mM 2-mercaptoethanol and 1 mM dithiothreitolJ and resuspended in the same buffer. The indicated salts were added to the assay mixture at a final concentration of 10 mM in an otherwise standard assay.
VOL. 173, 1991
A NOVEL GLUCOSYLTRANSFERASE FROM B. JAPONICUM
imately 10:1. On the basis of an average molecular weight of 2,100 (corresponding to 13 glucose residues), it would appear that the neutral glucan product contains approximately one ,B-1,6 glycosidic linkage per molecule. The lack of a detectable reducing terminus within the neutral glucan product suggests that the glucan may be cyclic in character. This result is intriguing in view of the fact that we previously have shown that B.japonicum USDA 110 synthesizes cyclic, branched 1-glucans containing both P-1,6 and 1-1,3 glycosidic linkages (22). Thus, it is possible that the glucosyltransferase activity described herein may be responsible for the biosynthesis of these cyclic 1-1,6-13-1,3glucans. However, there are two important distinctions between the product of the in vitro glucosyltransferase activity and the cell-associated cyclic 1-glucans isolated from growing cultures of B. japonicum USDA 110. First, the cyclic 1-glucans extracted from cultures of B. japonicum USDA 110 are slightly smaller (1,620 to 2,106 Da [22]) than the in vitro product (2,100 Da) of the glucosyltransferase activity (Fig. 2). Second, the relative proportion of 1-1,3 to 13-1,6 linkages is different for the two glucans. As described above, analysis of the disaccharide fraction of partial acid hydrolysates of the radiolabeled neutral glucan product revealed a ratio of 10 ,B-1,3 glycosidic linkages per 1 13-1,6 glycosidic linkage. In contrast, the ratio within cyclic 13glucans produced in vivo is 0.6 1-1,3 glycosidic linkages per 1 1-1,6 glycosidic linkage (22). One possible explanation for this apparent discrepancy is that the glycosidic linkage composition of the disaccharide fraction isolated from partial acid hydrolysates may not be truly representative since rate constants for the acid hydrolysis of glycosidic bonds may vary greatly (25). It is also possible that certain regulatory factors may be limiting or absent in the in vitro assay system, leading to products with structural features different from those of products synthesized in vivo. Similar results have previously been reported during the in vitro study of the glucosyltransferase system of E. coli responsible for membrane-derived oligosaccharide biosynthesis (32). With respect to the Km for UDP-glucose and the stimulation of activity by manganese and magnesium, the glucosyltransferase activity of B. japonicum USDA 110 would appear to be similar to the membrane-bound glucosyltransferase activities of the closely related genera Agrobacterium and Rhizobium (2, 38). However, the products of the membrane-bound activities of the organisms of the latter two genera represent a mixture of cyclic glucans containing 14 to 25 glucose residues linked solely by 13-1,2 glycosidic bonds (2, 38). In contrast, it is noted that essentially no 13-1,2-linked glucose was detected within the neutral fraction synthesized by membrane preparations derived from B. japonicum USDA 110 (Fig. 3). Our results would also appear to be in contrast to those reported by Dedonder and Hassid (6), who found that membrane preparations from Rhizobium japonicum contained a glucosyltransferase activity which synthesized a 13-1,2-linked glucan from UDP-glucose. However, on the basis of the growth rate of the bacterium examined by Dedonder and Hassid, it is likely that this organism represented a fast-growing strain within the Rhizobium genus and was not a true slow-growing strain of the presently recognized Bradyrhizobium genus. Our results indicate that species of Bradyrhizobium apparently lack the capacity for cyclic 13-1,2-glucan biosynthesis. In fact, this conclusion is consistent with our previous analysis of the cell-associated glucans of species of Bradyrhizobium (22), which demonstrated an absence of detectable levels of cyclic 1-1,2-glucans. Also consistent
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with this conclusion is the report by Dylan and coworkers (10) that genomic DNA from B. japonicum USDA 110 lacks significant homology with the chvA and chvB genes from Agrobacterium spp. and the ndvA and ndvB genes from Rhizobium spp. The NdvA and ChvA proteins appear to provide functions during cyclic 13-1,2-glucan export (3, 7, 28). The NdvB and ChvB proteins are high-molecular-weight membrane proteins (molecular weight, 319,000) which have been shown to form a covalently linked intermediate with the cyclic 13-1,2-glucan during biosynthesis (15, 37, 39). It may be noted that our attempts to detect the formation of a protein-linked intermediate during in vitro glucan biosynthesis with membrane fractions from B. japonicum USDA 110 have been unsuccessful (5). By using methods described previously by Zorreguieta and Ugalde (39), we were unable to detect the presence of a radiolabeled protein-linked intermediate within B. japonicum membrane preparations. However, in control experiments with membrane preparations derived from Rhizobium meliloti 1021, a radiolabeled protein-linked intermediate (apparent Mr, 235,000) was readily detected. Furthermore, we were unable to detect by Coomassie blue staining of polyacrylamide gels the presence of a protein within B. japonicum USDA 110 membrane preparations which migrated with an apparent molecular weight of 235,000 (the NdvB protein within R. meliloti 1021 membrane preparations is readily detected by Coomassie blue staining of polyacrylamide gels). Because the cyclic 13-1,2-glucans of Rhizobium spp. appear to have important roles in bacterial osmotic adaptation and legume nodulation (9-11, 36), it is interesting that these molecules are not synthesized by species of Bradyrhizobium. However, it is possible that the cyclic 1-1,613-1,3-glucans of Bradyrhizobium spp. may represent functional analogs of the cyclic 1-1,2-glucans. Indeed, this notion is supported by the recent demonstration that the biosynthesis of both classes of glucans is strictly osmoregulated (21, 23, 31) and that both classes of glucans are localized predominantly in the periplasmic compartment (1, 21, 23). In order to gain further insight regarding the possible functions of the cyclic 13-1,6-P-1,3-glucans of Bradyrhizobium spp., future studies will be directed towards the isolation of mutants specifically blocked for the biosynthesis of these glucans. Such mutants will also be required in order to elucidate whether the glucosyltransferase activity described herein provides an essential function during cyclic 1-1,61-1,3-glucan biosynthesis. ACKNOWLEDGMENT This research was supported by National Science Foundation grant DCB-8803247 awarded to K.J.M. REFERENCES 1. Abe, M., A. Amemura, and S. Higashi. 1982. Studies on cyclic beta-1,2-glucan obtained from periplasmic space of Rhizobium trifolii cells. Plant Soil 64:315-324. 2. Amemura, A. 1984. Synthesis of (1-2)-beta-D-glucan by cell-free extracts of Agrobacterium radiobacter IFO 12665bl and Rhizobium phaseoli AHU 1133. Agric. Biol. Chem. 48:1809-1817. 3. Cangelosi, G. A., G. Martinetti, J. A. Leigh, C. C. Lee, C. Theines, and E. W. Nester. 1989. Role of Agrobacterium tumefaciens ChvA protein in export of P-1,2-glucan. J. Bacteriol. 171:1609-1615. 4. Cangelosi, G. A., G. Martinetti, and E. W. Nester. 1990. Osmosensitivity phenotypes of Agrobacterium tumefaciens mutants that lack periplasmic ,-1,2-glucan. J. Bacteriol. 172:21722174. 5. Cohen, J. L., and K. J. Miller. Unpublished data.
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6. Dedonder, R. A., and W. Z. Hassid. 1964. The enzymatic synthesis of a (,-1,2-)-linked glucan by an extract of Rhizobium japonicum. Biochim. Biophys. Acta 90:239-248. 7. de lannino, N. I., and R. A. Ugalde. 1989. Biochemical characterization of avirulent Agrobacterium tumefaciens chvA mutants: synthesis and excretion of P-(1-2)glucan. J. Bacteriol. 171:2842-2849. 8. Douglas, L. J., R. J. Staneloni, R. A. Rubin, and E. W. Nester. 1985. Identification and genetic analysis of an Agrobacterium tumefaciens chromosomal virulence region. J. Bacteriol. 161: 850-860. 9. Dylan, T., D. R. Helinski, and G. S. Ditta. 1990. Hypoosmotic adaptation in Rhizobium meliloti requires P-(1-2)-glucan. J. Bacteriol. 172:1400-1408. 10. Dylan, T., L. lelpi, S. Stanfield, L. Kashyap, C. Douglas, M. Yanofsky, E. Nester, D. R. Helinski, and G. Ditta. 1986. Rhizobium meliloti genes required for nodule development are related to chromosomal virulence genes in Agrobacterium tumefaciens. Proc. Natl. Acad. Sci. USA 83:4403-4407. 11. Dylan, T., P. Nagpal, D. R. Helinski, and G. S. Ditta. 1990. Symbiotic pseudorevertants of Rhizobium meliloti ndv mutants. J. Bacteriol. 172:1409-1417. 12. Halverson, L. J., and G. Stacey. 1986. Signal exchange in plant-microbe interactions. Microbiol. Rev. 50:193-225. 13. Hisamatsu, M., A. Amemura, K. Koizumi, T. Utamura, and Y. Okada. 1983. Structural studies on cyclic (1-2)-beta-D-glucans (cyclosophoraose) produced by Agrobacterium and Rhizobium. Carbohydr. Res. 121:31-40. 14. Hisamatsu, M., A. Amemura, T. Matsuo, H. Matsuda, and T. Harada. 1982. Cyclic (1-2)-beta-D-glucan and the octasaccharide repeating-unit of succinoglycan produced by Agrobacterium. J. Gen. Microbiol. 128:1873-1879. 15. Ielpi, L., T. Dylan, G. S. Ditta, D. R. Helinski, and S. W. Stanfield. 1990. The ndvB locus of Rhizobium meliloti encodes a 319-kDa protein involved in the production of beta-(1-2)-glucan. J. Biol. Chem. 265:2843-2851. 16. Koizumi, K., Y. Okada, S. Horiyama, and T. Utamura. 1983. Separation of cyclic (1-2)-beta-D-glucans (cyclosophoraose) produced by Agrobacterium and Rhizobium, and determination of the degree of polymerization by high-performance liquid chromatography. J. Chromatogr. 265:89-96. 17. Koizumi, K., Y. Okada, T. Utamura, M. Hisamatsu, and A. Amemura. 1984. Further studies on the separation of cyclic (1-2)-beta-D-glucans (cyclosophoraoses) produced by Rhizobium meliloti IFO 13336, and determination of their degrees of polymerization by high-performance liquid chromatography. J. Chromatogr. 299:215-224. 18. Lerouge, P., P. Roche, C. Faucher, F. Mailiet, G. Truchet, J. C. Prome, and J. Denarie. 1990. Symbiotic host-specificity of Rhizobium meliloti is determined by a sulphated and acylated glucosamine oligosaccharide signal. Nature (London) 344:781784. 19. Long, S. R. 1989. Rhizobium-legume nodulation: life together in the underground. Cell 56:203-214. 20. Mayer, F. C., and J. Larner. 1959. Substrate cleavage point of the alpha and beta-amylases. J. Am. Chem. Soc. 81:188-198. 21. Miller, K. J., and R. S. Gore. Unpublished data. 22. Miller, K. J., R. S. Gore, R. Johnson, A. J. Benesi, and V. N. Reinhold. 1990. Cell-associated oligosaccharides of Bradyrhizo-
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