JOURNAL OF BAC-ERIOLOGY, May 1993, p. 2490-2500 Vol. 175, No. 9 0021-9193/93/092490-11$02.00/0 Copyright X 1993, American Society for Sequential Assembly and Polymerization of the Polyprenol- Linked Pentasaccharide Repeating Unit of the Xanthan in Xanthomonas campestris

LUIS IELPI, ROBERTO 0. COUSOt AND MARCELO A. DANKERT* Instituto de Investigaciones Bioquimicas "Fundaci6n Campomar, " Facultad de Ciencias Exactas y Naturales and Consejo Nacional de Investigaciones Cientificas y Tecnicas, Av. Patricias Argentinas 435, 1405 Buenos Aires, Argentina Received 13 October 1992/Accepted 17 February 1993

Lipid-linked intermediates are involved in the synthesis of the exopolysaccharide xanthan produced by the bacterium Xanthomnonas campestris (L. Ielpi, R. 0. Couso, and M. A. Dankert, FEBS Lett. 130:253-256, 1981). In this study, the stepwise assembly of the repeating pentasaccharide unit of xanthan is described. EDTA-treated X. campestris cells were used as both enzyme preparation and lipid-P acceptor, and UDP-Glc, GDP-Man, and UDP- were used as donors. A linear pentasaccharide unit is assembled on a polyprenol-P lipid carrier by the sequential addition of -1-P, glucose, , glucuronic acid, and mannose. The in vitro synthesis of pentasaccharide-P-P-polyprenol was also accompanied by the incorporation of radioactivity into a polymeric product, which was characterized as xanthan, on the basis of gel filtration and permethylation studies. Results from two-stage reactions showed that essentially pentasaccharide-P-P- polyprenol is polymerized. In addition, the direction of chain elongation has been studied by in vivo experiments. The polymerization of lipid-linked repeat units occurs by the successive transfer of the growing chain to a new pentasaccharide-P-P-polyprenol. The reaction involves C-1 of glucose at the reducing end of the polyprenol-linked growing chain and C-4 of glucose at the nonreducing position of the newly formed polyprenol-linked pentasaccharide, generating a branched polymer with a side chain.

Microbial exopolysaccharides are produced during the MATERIALS AND METHODS growth of various genera of and yeasts, and after the pioneer work of the group at the Northern Regional Re- Chemicals. UDP-[14C]Glc (196 Ci/mol), UDP-[14C]glucu- search Laboratory, Peoria, Ill., many of these products have ronic acid (UDP-[14C]GlcA) (268 Ci/mol), and GDP- been shown to have a wide variety of applications as [14C]Man (216 Ci/mol) were prepared as described previ- ously (7, 9). [14C]Glc-(j3-1,4)-[ 4C]Glc (, C2), [14C] thickeners and emulsifiers in activities as diverse as the food, [14 pharmaceutical, and oil industries (23). One of these prod- Man-(ax-1,3)-Glc-(P-1,4)-Glc (trisaccharide X3); C]GlcA- ucts is , an exopolysaccharide liberated into the (13-1,2)-Man-(oa-1,3)-Glc-(1-1,4)-Glc ( X4), and culture medium by the plant pathogen Xanthomonas Glc-(P-1,6)-Glc-(ot-1,4)_[14C]GlcA-(P-1,2)-Man-(ot-1,3)-Glc- campestris. The structure proposed for this polysaccharide (P-1,4)-Glc (hexasaccharide X6) as well as their respective indicates that it can be considered a substituted cyclic phosphate esters in position 1,2 of the reducing (Fig. 1): the main chain is a 3-1,4- to which a trisac- glucose, C2Pc, X3Pc, X4Pc, and X6Pc, were prepared by charide branch, consisting of mannose-(P-1,4)-glucuronic mild acid or alkaline treatment, respectively, of the corre- acid-(ot-1,2)-mannose, is a-1,3 linked every two . In sponding prenyl-phosphosugars, obtained with anAcetobac- addition, the internal mannoses are 0 acetylated at position ter xylinum system as in previous work (6, 8). Glucuronyl- 6, and every two external mannoses carry a ketal pyruvate (,-1,2)-mannose 14C labeled in either sugar was obtained by bridging C-4 and C-6 (17, 21), but other proportions have partial of the respective hexasaccharide X6 simi- also been described (24). larly labeled, as already described (8). Tetra-O- and tri-O- Several genetic loci are involved in the biosynthesis of methyl-Glc derivatives used as standards were prepared xanthan gum. These include at least four DNA regions from the corresponding . Di-O- and mono-O- located in the chromosome of X. campestris (2, 10, 11, 13, methyl-substituted glucoses were purchased from Supelco 18, 26, 27). Particularly important to the study of the Inc., Bellefonte, Pa. 2,3,4-Tri-O-methyl- and 3,4,6-tri-O- functions of those genes is to understand in detail the methyl-Man were kindly provided by A. J. Parodi of this synthesis of this polymer. In this report, we describe the institute. Other chemicals were commercial samples. stepwise assembly of the xanthan gum repeat unit linked to Enzyme preparations. Cells from X. campestris NRRL a polyprenol acceptor through a diphosphate bridge and the B-1459 were grown as reported before (4) and harvested by subsequent polymerization process which produces the poly- centrifugation at late logarithmic phase. The enzyme prepa- saccharide. Preliminary results have been reported (14). ration consisted of the pellet resuspended in 0.01 M EDTA- Tris buffer (pH 8.2), frozen and thawed several times (EDTA cells) (14). Occasionally, the enzyme was dialyzed against 50 mM Tris-HCl buffer (pH 7.8)-10 mM EDTA-10 mM mer- captoethanol overnight. * Corresponding author. Assay procedure. (i) One-step incubations. The standard t Deceased on 15 August 1992. We dedicate this paper to his incubation mixture contained 70 mM Tris-HCl buffer (pH memory. 8.2), 8 mM MgCl2, EDTA cells (about 0.6 to 0.8 mg of 2490 VOL. 175, 1993 XANTHAN GUM BIOSYNTHESIS 2491 A B IH " CH20H,2H

COOH Ip,

,0cz/'---_ 0-6,0C-CCH221)0

FIG. 1. Basic structure of xanthan gum showing a branched (A) or linear (B) repeating unit. I and II are the two possible alternatives for the trisaccharide moiety of the repeating unit. The broken lines at the pyruvyl-mannose linkage indicate that not all terminal mannoses are substituted. protein), and UDP-[4CjGlc (15.7 puM) or UDP-[14C]GlcA phy and electrophoresis techniques were as described pre- (17.1 puM) or GDP-[ 4C]Man (15.7 puM), as indicated in each viously (6, 9). The following solvents were used: solvent A, case. The unlabeled sugar nucleotides, when noted, were (96%)-ammonium hydroxide (7:3); solvent B, isopro- added in the following concentrations: UDP-Glc and UDP- panol-acetic acid-water (27:4:9); solvent C, pyridine-acetic GlcA, 285 ,uM each; GDP-Man, 142 FM. The reactions were acid-water (1:0.04:9), pH 6.5; solvent D, butanol-pyridine- performed in a total volume of 70 Al for 30 min at 12 or 20'C, water (6:4:3); solvent E, sodium molybdate (0.1 M), pH 5.0. as indicated, and were stopped by adding 0.5 ml of 70 mM Thin-layer chromatography was carried out on silica gel 60 Tris-HCl buffer (pH 8.2) containing 30 mM EDTA. The plates (0.25 mm; Merck) with the following solvents: solvent mixtures were centrifuged at 6,000 x g for 5 min, and the F, acetone-benzene-ammonium hydroxide-water (200:50: pellets were resuspended and washed twice with Tris-HCl 1.35:1); and solvent G, acetone-benzene-ammonium hydrox- buffer without EDTA. The supernatants were combined and ide-water (200:50:1.5:3). Radioactivity was detected with a lyophilized to determine polysaccharide formation by gel Packard radiochromatogram scanner, model 7201 (Packard filtration. The washed cell pellets were then extracted three Instruments Co., Rockville, Md.). When indicated, the silica times (0.15 ml each) with -methanol-water (1:2: was scratched off the plates in bands 0.5 cm wide, and the 0.3, by volume) (1203 solvent) (14). This extract, which powder was counted for radioactivity. On thin-layer plates, contains the polyprenol-linked [ "C], will be saccharides were detected with 5% concentrated sulfuric referred to as 1203 extract. Aliquots were counted for acid in ethanol, and plates were heated to 140'C for 5 min radioactivity. (19). (ii) Two-step incubations. The first step was a standard Enzymatic treatments. Treatments with alkaline phos- incubation scaled up two- to fivefold, as indicated in each phatase (from Escherichia coli), with 0-glucuronidase (from case. The incubation mixtures were processed as described bovine liver), or with inorganic pyrophosphatase were per- above, and the supernatants that contained the excess sugar formed as already reported (9). The reaction mixture for nucleotides and the possible which formed degradation with a-mannosidase (from jack beans) contained were discarded. For the second incubation, the washed cell 50 mM sodium citrate buffer (pH 4.5), 2,500 cpm of labeled pellet was resuspended in the original volume of 70 mM substrate, and 2.4 U of enzyme in a total volume of 50 pAl. Tris-HCl buffer (pH 8.2) containing 8 mM MgCl2. Aliquots Incubations were carried out at 250C for 8 h. Reactions were (70 Al) were reincubated for 30 min with the additions and at ended by adding 1 volume of ethanol, and the supernatants the temperatures indicated in each case. The reactions were were desalted with Amberlite MB-3 (acetate form) and stopped with EDTA and processed as described for the analyzed by paper chromatography with solvent B. Under standard assay. these conditions, p-nitrophenyl P-mannose was not de- Chemical treatments. Methylation of saccharides and graded even after 18 h of incubation. Alkaline phosphatase, polysaccharides was carried out by the method of Hakomori 3-glucuronidase, and a-mannosidase were purchased from as described by Couso et al. (6) and analyzed by thin-layer Sigma Chemical Co., St. Louis, Mo. Inorganic pyrophos- chromatography with solvents D and E (5). The reduction of phatase was obtained by the method of Kunitz as described the carboxyl group of glucuronic acid of the pentasaccharide previously (8). to the and Smith degradation were performed as reported previously (8). RESULTS Mild acid hydrolysis was carried out at pH 2 (0.01 M HCl) and 100'C for 10 min (8). Cyclic phosphoric esters were Synthesis of polyprenol-linked pentasaccharide. Prepara- opened and diphosphoric esters were cleaved by treatment tions of EDTA X. campestris cells were incubated with at pH 1 (0.1 M HCl) and 100'C for 10 min. Partial and total UDP-Glc, GDP-Man, and UDP-GlcA, one of them after acid hydrolyses were performed with 1 M HCl at 100'C for 1 natively 14C labeled in the sugar moiety, and then the or 16 h, respectively, in sealed tubes. preparations were centrifuged to separate the cell pellet from Chromatography and electrophoresis. Paper chromatogra- the incubation supernatant. In the latter fraction, in addition 2492 IELPI ET AL. J. BACTERIOL.

TABLE 1. Incorporation of radioactivity into polysaccharide and liposoluble material? ['4Cjhexose incorporated Labeled donor (pmol mg of protein-') 1203 extract Polysaccharide UDP-[14C]Glc 71 44 GDP-[14C]Man 142 94 UDP-[14C]GlcA 65 81 a Standard incubations were performed at 20'C in the presence of the three sugar donors, one labeled as indicated. The mixtures were processed as ._ described in Materials and Methods. The amounts of synthesized polysaccha- u ride were calculated from the data in Fig. 7.

0 .Q._ to the excess radioactive sugar nucleotide, a "4C-labeled m component of high molecular weight could be detected (Table 1). It has been tentatively identified as the polysac- charide xanthan gum (14) and is discussed in more detail below. The radioactivity associated to the cell pellet was ex- tracted with 1203 solvent (Table 1). It consisted mainly of the polyprenol intermediates that are described in this report. Distance from origin (cm) The 1203 extracts, with the material labeled in either sugar, FIG. 3. Peak II analysis. Peak II was obtained as described in the were analyzed in several ways. DEAE column chromatog- legend to Fig. 2 and submitted to the following treatments: (A) mild raphy showed slightly different patterns, depending on the acid hydrolysis followed by paper electrophoresis with buffer C; (B) enzyme preparation and the labeled sugar used. For in- compound with RUMP = 0.6 from panel A chromatographed with stance, Figure 2 shows an experiment with UDP-[14C]GlcA solvent B; (C) paper chromatography with solvent A; (D) compound with Rf = 0.25 from panel C eluted and submitted to paper in which three peaks can be observed. Peak I, which eluted electrophoresis with buffer C. Where indicated, AMP, UMP, glucu- at 0.65 M ammonium acetate, was recently characterized as ronic acid, glucose, and maltooligosaccharides (M2 to M6) were a lipid-bound galacturonide (1). Its function is unknown and added as internal standards; ["4C-GlcA]X4 and ["4C-GIcA]X6 oli- it does not seem to participate in the reactions described in gosaccharides from A. xylinum were run as external standards. this work. It was seldom produced with the enzyme prepa- rations employed. Peak III, which was eluted at 1.1 M ammonium acetate, could be obtained with any sugar label and was the only one formed when the incubations were carried out with unla- beled sugar nucleotides and [14C]phosphoenolpyruvate. It has been characterized as the pyruvylated pentasaccharide derivative (15), and in the absence of phosphoenolpyruvate 1500 its formation was found to depend on the enzyme prepara- tion used. Its formation was very likely due to the presence of endogenous phosphoenolpyruvate in the EDTA cells and was highly reduced when the enzyme preparations had been dialyzed. The main product, usually the only one, was peak II; it 1000 could be obtained with any of the sugar nucleotides labeled. The high ammonium acetate concentration (0.95 M) needed 0.~~~~~~~~~~~~~ for its elution was consistent with the presence of a glucu- ronic acid residue in addition to the diphosphate bridge -.2. expected for a polyprenol intermediate. Under similar con- ditions cellobiose diphosphate polyprenol is eluted at 0.4 M ammonium acetate (14). Peak II did not migrate when submitted to paper electro- phoresis with buffer C, but if previously treated with mild acid, all radioactivity migrated (RUMP = 0.6) between the glucuronic acid-containing tetrasaccharide X4 (RUMP = 0.7) and the hexasaccharide X6 (RUMP = 0.5) from A. xylinum 60 100 150 200 used as standards (Fig. 3A), suggesting the liberation of an of intermediate size. A similar pattern was Fraction Number obtained by paper chromatography with solvent B in which FIG. 2. DEAE-cellulose column chromatography of [14C]GlcA- labeled glycolipid compounds. 1203 extract (28,000 cpm) obtained as peak II oligosaccharide ran between X4 and X6 oligosaccha- described in Table 1, line 3, was poured into a DEAE-cellulose rides (Fig. 3B). Paper chromatography of peak II with the column (1 by 60 cm) in 99% methanol and eluted with a linear mild alkaline solvent A showed a single component with an gradient of ammonium acetate in 99% methanol (0 to 2 M) in a total R of 0.25 (Fig. 3C). In this alkaline solvent prenyl-diphos- volume of 400 ml, as reported previously (9). Aliquots (0.25 ml) of phosugars are degraded to the respective oligosaccharide each fraction (3 ml) were counted for radioactivity. cyclic phosphate esters, provided the hydroxyl group at C-2 VOL. 175, 1993 XANTHAN GUM BIOSYNTHESIS 2493 and the phosphate bridge are in the cis configuration (8). The X6 GIcA above-mentioned compound (Rf = 0.25) migrated faster (RUMP = 1.0) than the pentasaccharide (RUMP = 0.6) upon B.D. Raf G lc paper electrophoresis with buffer C (Fig. 3D). But if previ- Sta Suc COC12 I ously treated with mild acid and alkaline phosphatase, its L-1IV mobility decreased to RUMP = 0.6, indicating that a phos- phoric cyclic ester was present in the product of mild alkali loooF degradation, which had been removed by the combined acid E and enzymatic treatments. These results reinforced the assumption that the pentasaccharide is linked to a polypre- nol by a diphosphate bridge. This alkaline solvent A has a. been used throughout this work because in a very simple u manner it allows the fractionation of the 1203 extracts almost 0r- 500o as efficiently as by DEAE-cellulose column chromatogra- (U phy, a more cumbersome procedure. The presence of poly- prenol-linked pentasaccharide (peak II) in the 1203 extracts ABC was, then, regularly determined by paper chromatography with buffer A, and the compound ofRf = 0.25 was submitted to paper electrophoresis with buffer C. The compound of RUMP = 1.0 in this system was counted for radioactivity: this U 40 60 80 100 120 datum was considered the amount of polyprenol-linked Fraction Number pentasaccharide originally present in the 1203 extract. Peak FIG. 4. Size of the oligosaccharides under study. A mixture of III, that is, polyprenol-linked pyruvylated pentasaccharide, unlabeled standards and ["4C-GlcA]pentasaccharide (A), [14C- submitted to the same procedure produced a substance of GlcA]tetrasaccharide (B), and ['4C-Man]trisaccharide (C) (15,000 RUMP = 1.4 upon paper electrophoresis with buffer C (15). cpm each) were sampled in a Bio-Gel P2 (200-400-mesh) column An even more reliable piece of information about the size (107 by 0.9 cm) in 0.1 M pyridinium acetate buffer, pH 5.0. Although of the from II was obtained Bio-Gel the 14C-labeled tri-, tetra-, and pentasaccharides were run individu- oligosaccharide peak by ally on several occasions with consistent elution volumes, in this P-2 gel filtration: once again, it behaved as a pentasaccharide particular experiment they were filtered together to point out their (Fig. 4, peak A). different elution volumes. Fractions of 0.5 ml were collected at a The pattern of pentasaccharide accumulation was identi- rate of 0.13 ml min-, and aliquots of 0.15 ml were counted for cal for incubations performed from 12 to 200C, and a plateau radioactivity. Glucose, (Suc), (Raf), and stachiose was reached in 20 to 30 min. On the contrary, polysaccharide (Sta) were added as standards of mono-, di-, tri-, and tetrasaccha- synthesis was maximal at 20'C and decreased to 40 to 60% at rides and located by the phenol- method (8). Blue 12'C, although it remained linear for at least 1 h. At 30'C, (BD) and CoCl2 were added as total exclusion and total incorporation of radioactivity into both fractions, 1203 ex- inclusion indicators, respectively. The elution volumes of glucu- tract and was diminished. Pentasac- ronic acid and of [14C]GlcA-labeled X6 oligosaccharide, indicated by polysaccharide, highly the open arrows, were determined in different runs under the same charide formation has an absolute requirement for Mg2+ conditions. ions, a plateau being reached at 6 mM MgCl2 under the incubation conditions used (12°C for 30 min). In addition, pentasaccharide formation reached a plateau at 285 ,uM UDP-Glc. Different concentrations of UDP-GlcA (from 14 to This result confirms the observation of Jansson et al. (17), 285 ,uM) or GDP-Man (from 14 to 540 pM) did not change whose structural studies indicated that no glucuronic acid this maximum of pentasaccharide accumulation. was linked to glucose, as suggested previously (25). As Sequential synthesis of polyprenol-linked pentasaccharide. expected, the pentasaccharide derivative was formed only (i) Glucose-(0-1,4)-glucose diphosphate polyprenol as a pre- when unlabeled UDP-GlcA and GDP-Man were present in cursor of trisaccharide and pentasaccharide diphosphate poly- the second incubation (Fig. SE). On the other hand, when prenol. Incubations carried out in the presence of UDP- GDP-Man alone was added, a compound with the properties [14C]Glc as the only sugar donor accumulate glucose-(p-1,4)- of a trisaccharide-polyprenol (see below), as well as a small glucose-diphosphate polyprenol in the 1203 extract. A amount of pentasaccharide-polyprenol, was formed (Fig. detailed analysis of this compound has been described in a SC). The formation of pentasaccharide-polyprenol observed previous communication (14) and will not be discussed here. in Fig. SC can be explained assuming that endogenous But, supporting the role of intermediate assigned to this glucuronic acid donor was present in the enzyme prepara- prenyl-phosphosugar, it has been used in the present work as tions, since it was highly decreased when dialyzed EDTA the starting point for the stepwise addition of the remaining cells were used. From these results, it was concluded that three components of the repeating unit. the polyprenol-bound was a precursor of the The incubations were performed in two steps and at 12°C trisaccharide- and the pentasaccharide-polyprenol interme- to minimize polysaccharide formation, and the results are diates. presented in Fig. 5. In the first incubation, carried out in the (ii) Analysis of the trisaccharide-polyprenol derivative. The presence of UDP-[14C]Glc alone, the only compound formed trisaccharide-polyprenol compound could be obtained either was the polyprenol-bound cellobiose (Fig. 5A). The excess [14C]Glc or [1 C]Man labeled, but not I'4C]GlcA labeled, and of labeled nucleotide was removed by centrifugation, and the had all of the properties expected for a prenyl-diphospho- EDTA cells were reincubated under different conditions. No sugar: it was totally extracted in 1203 solvent, and mild acid changes in the pattern of incorporation were observed when hydrolysis released the labeled sugar moiety, which mi- the second incubation was performed with no additions (Fig. grated as a neutral compound in electrophoresis with buffer SB) or in the presence of unlabeled UDP-GlcA (Fig. SD): the C and coeluted with the trisaccharide raffinose in a Bio-Gel cellobiose-P-P-polyprenol accumulated remained unaltered. P2 gel filtration column (Fig. 4, peak C). Because of the way 2494 IELPI ET AL. J. BACTERIOL.

I then oxidized with periodate, treated with mild acid to A,C2Pc -lcPc F remove oxidized glycosidic residues and to open the phos- 1000 phate cycle, and incubated with alkaline phosphatase to free the undegraded . ['4Clcellobiose was recovered in 0- more than 90% yield, indicating that the mannose was linked to C-3 of the nonreducing glucose and that the trisaccharide 1000 was linear. A branched trisaccharide would have produced IJ I_ 50%, or less, of the radioactivity as free [14C]Glc. The anomeric configuration was confirmed to be ca by - treatment with a-mannosidase. The trisaccharide was, then, 100psCPSc X3Pc mannose-(a-1,3)-glucose-(I3-1,4)-glucose (Fig. 1B, insert II) and identical to trisaccharide X3 from A. xylinum. (iii) Sequential synthesis of tetrasaccharide- and pentasac- charide-P-P-polyprenol from mannose-(ct-1,3)-glucose-(I3-1,4)- glucose-P-P-polyprenol. In the experiments described above, the cellobiose-P-P-polyprenol was shown to be the precursor of the trisaccharide and pentasaccharide derivatives. To obtain the tetrasaccharide-P-P-polyprenol that should be in 0 i between the latter two compounds, trisaccharide-P-P-poly- prenol was used as the starting point. Again, two-step 1000 incubation experiments were used. The first incubation was carried out in the presence of unlabeled UDP-Glc and

0 GDP-Man, to accumulate polyprenol-bound trisaccharide. 0 20 30 Only UDP-[14C]GlcA was present in the second incubation, and a new compound of Rf = 0.33 was detected upon paper Distance from origin (cm) chromatography with solvent A (Fig. 6A). As pointed out FIG. 5. Conversion of cellobiose-P-P-polyprenol to pentasaccha- above, this alkaline solvent breaks the diphosphate bridge of ride-P-P-polyprenol. Two-step incubations were performed as de- prenyl-phosphosugars, releasing the respective oligosaccha- scribed in Materials and Methods. The 1203 extracts from each ride cyclic phosphate ester. For this reason the compound of experiment were chromatographed with solvent A and scanned for Rf = 0.33 was submitted to paper electrophoresis with radioactivity. The cyclic phosphate esters that were observed with this solvent were referred to as the respective sugar diphosphate solvent C: it moved the same as the cyclic phosphate ester of polyprenol (see text). The first incubation, a fivefold standard A. xylinum X4 tetrasaccharide (X4Pc; RUMP = 1.15) (Fig. mixture, was incubated at 12'C in the presence of UDP-[14C]Glc to 6B). Furthermore, the only radioactive compound present in allow formation of [14C-Glc]cellobiose-P-P-polyprenol (A). The sec- the 1203 extracts behaved as a prenyl-phosphotetrasaccha- ond incubation was also at 12'C, with the following additions: (B) no ride: upon paper electrophoresis with solvent C it did not additions; (C) GDP-Man; (D) UDP-GlcA; (E) GDP-Man and UDP- migrate, but after mild acid hydrolysis a single compound GlcA. The mobilities of external standards of the cyclic phosphate with the mobility (RUMP = 0.7) of the A. xylinum X4 esters of glucose, cellobiose, and trisaccharide X3 from A. xylinum tetrasaccharide was released (Fig. 6C). This compound, (GlcPc, C2Pc, and X3Pc, respectively) are indicated. The cyclic phosphate ester of the pentasaccharide from X. campestris (psPc), upon paper chromatography with solvent D, also migrated as obtained as described in the legend to Fig. 3C, was run as a the tetrasaccharide X4 (Fig. 6D) and coeluted with the reference. F, solvent front. tetrasaccharide stachiose by Bio-Gel P2 gel filtration (Fig. 4, peak B). When the second incubation was carried out in the presence of UDP-[14C]GlcA and GDP-Man, a compound with the properties of the pentasaccharide-P-P-polyprenol in which it was obtained, it had to contain cellobiose at its described above was the main product detected (not shown). reducing end (Fig. 5C). The structure of the trisaccharide The quantitative aspects of the sequential synthesis of the moiety was mainly established by comparison with the polyprenol-linked pentasaccharide described above are sum- known linear trisaccharide X3 from A. xylinum, to which it marized in Table 2. In experiment A, the starting compound turned out to be identical. Its characterization as mannosyl was the [14C-Glc]disaccharide-P-P-polyprenol, accumulated cellobiose has already been described (14), but one point to in the first incubation (A1). Its amount remained constant clarify was whether the incorporated mannose generated a when it was reincubated alone (A2) or in the presence of branched or a linear trisaccharide (Fig. 1, dotted inserts I UDP-GlcA (A4). On the contrary, when GDP-Man was and II, respectively). The criteria used were the same present in the second incubation, about half of the radioac- applied to demonstrate that trisaccharide X3 fromA. xylinum tivity was converted into the trisaccharide and a measurable was linear (7): periodate oxidation of the 1,2-cyclic phos- amount of pentasaccharide was also detected (A3). Finally, phate ester of [f4C-Glc]trisaccharide. In this compound the when GDP-Man and UDP-GlcA were present in the second glucose at the reducing end is insensitive to periodate incubation, about 70% of the initial radioactivity was asso- oxidation because it is protected at C-1 and C-2 by the ciated with the only compound detected, the polyprenol- phosphate residue and at position 4 by the P-1,4 linkage of bound pentasaccharide (A5). In the last two experiments, the the nonreducing glucose. Since C-3 and C-6 are free, they slightly low radioactivity recovered has been justified by could bear the mannose as a branch (Fig. 1A, insert I). The taking into consideration that the pentasaccharide formed nonreducing glucose, on the other hand, is totally unpro- was partially derived into polymer, even in incubations tected, unless it carries the mannose at C-3 (Fig. 1B, insert carried out at 12°C. At 20°C, this drain was much greater. In II). In the latter case, it should be insensitive to periodate the second series of experiments (B to D), the starting oxidation. The unlabeled mannose is degraded in both cases. compound was the trisaccharide-P-P-polyprenol. This com- The 1,2-cyclic phosphate ester of [14C-Glcltrisaccharide was pound, either [14C]Glc (experiment B) or [14C]Man (experi- VOL. 175, 1993 XANTHAN GUM BIOSYNTHESIS 2495

X4Pc A GDP-Man in the second incubation, the tetrasaccharide- polyprenol was the only derivative observed (not shown), ruling out the possibility of a glucose-glucuronic acid linkage Rt=0.33 I (17). 0l (iv) Structure of the pentasaccharide. The sequential syn- X Ic X4PC thesis of the polyprenol-linked pentasaccharide starting out 300 B. from cellobiose-P-P-polyprenol or trisaccharide-P-P-poly- RUMP=l.15lS prenol demonstrated the presence of a mannose-(a-1,3)- E glucose-(0-1,4)-glucose fragment at the reducing end of the 0 iWP IIcA pentasaccharide. To find out to which sugar of this fragment the glucuronic acid was linked, [14C-Man]- or [14C-GlcA]- 300 + labeled pentasaccharide was partially hydrolyzed, since the 0 RUMp=0.7 aldobiuronic linkage is fairly acid resistant. The reaction v m products were submitted to paper electrophoresis ix with buffer C, and with either label a major radioactive, nega- UMP GIcA tively charged compound was observed (not shown). Its X4 X3 iJps D mobility (RUMP = 0.9) was identical to that of the disaccha- 300 ride glucuronyl-P-1,2-mannose, isolated from partial hy- drolysates of A. xylinum oligosaccharides (6). Treated with -glucuronidase, this compound liberated either [14C]GlcA or [14C]Man, according to the label, proving that it was a 0 10 20 30 40 P-glucuronyl-mannose disaccharide. Distance from origin (cm) Permethylation of the ["4C-Man]pentasaccharide followed FIG. 6. Analysis of the ["4C-GlcA]tetrasaccharide-P-P-polypre- by acid hydrolysis yielded 2,3,4,6-tetra-O-methyl-Man and nol. Two-step incubations were performed as described in Materials 3,4,6-tri-O-methyl-Man in almost equimolar amounts (not and Methods. The first reaction, performed at 20'C, was carried out shown). These results are in agreement with an internal in the presence of UDP-Glc and GDP-Man to allow formation of mannose substituted in position 2 by a glucuronic residue trisaccharide-P-P-polyprenol. The second incubation was also at and a terminal mannose at the nonreducing end of the 20'C, carried out in the presence of UDP-["'C]GIcA. The 1203 pentasaccharide, as proposed for the repeating unit of xan- extract was treated in the following way: (A) paper chromatography than gum (17, 21). with the alkaline solvent A, which breaks prenyl-P-P sugars produc- In the proposed structure of xanthan gum, the glucuronic ing the cyclic phosphate ester of the respective sugar; (B) the acid residue is substituted in C-4 by the terminal mannose. compound with Rf = 0.33 from panel A submitted to paper electro- was phoresis with solvent C; (C) mild acid hydrolysis (pH 2, 1000C, 10 This feature confirmed in the following way. The min) followed by paper electrophoresis with buffer C; (D) the carboxyl group of [14C-GlcA]pentasaccharide was reduced compound with RUMP = 0.7 from panel C chromatographed with to the respective primary alcohol, yielding a [14C]Glc-labeled solvent B. The arrows indicate the mobilities of the trisaccharide neutral oligosaccharide, which when submitted to Smith ["'C-Man]X3 and the tetrasaccharide ['4C-GlcA]X4 and its cyclic degradation produced [14C]erythritol (data not shown), thus phosphate ester derivative, [14C-GlcA]X4Pc, from A. xylinum, proving that in the original pentasaccharide the glucuronic added as external standards. Pentasaccharide (ps), obtained by mild acid was substituted at position 4. acid hydrolysis of peak (cf. Fig. 3B), was run as a reference. F, Polymer formation. The results shown in solvent front. Table 1 indicate that, with either label, a radioactive polysaccharide was formed in addition to the 1203-extractable material. The question arose whether the pentasaccharide diphosphate ment C) labeled or unlabeled (experiment D), could be polyprenol was the precursor of the building blocks of that converted into the tetrasaccharide derivative when UDP- polysaccharide. This precursor-product relationship was GlcA (either labeled [D1] or not labeled [B2 and C2]) was studied in two-step incubation experiments (Table 3, exper- present in the second incubation. In the last cases, about iment A). In the first incubation, [14 C-GlcA]pentasaccharide 50% of the labeled trisaccharide-bound lipid was converted diphosphate polyprenol was prepared at 12°C. After washing to the tetrasaccharide-P-P-polyprenol. Finally, no[("C]GlcA- off the excess sugar nucleotide donors and the polymer labeled oligosaccharide derivatives were detected in the formed, the incubation was continued without additions for absence of the sugar donors during the first incubation 30 min at 20°C, since at this temperature polysaccharide (experiment E), indicating the absence of endogenous trisac- formation is maximal. Under these conditions about 30% of charide acceptor. the initial radioactivity present in the lipid-linked pentasac- Polyprenol-bound tetrasaccharide, in turn, was able to charide was found in the polymer fraction (Table 3, aliquot accept a second mannose residue to form the polyprenol A2), while <2% was detected in a control not reincubated derivative of the pentasaccharide repeating unit. This was (Table 3, aliquot Al). It should be mentioned that the missing demonstrated in an experiment identical to the experiments radioactivity, about 30% of that initially associated with just discussed, except for the presence of unlabeled GDP- pentasaccharide-P-P-polyprenol, was detected in the inclu- Man, in addition to UDP-[14C]GlcA, during the second sion volume of the Bio-Gel filtration step. Although not incubation (experiment D2). In this case the major com- totally characterized, this compound very likely was free pound was the pentasaccharide-polyprenol derivative. The pentasaccharide. low recovery of radioactive pentasaccharide-polyprenol It was of interest to find out whether some of the precur- (30%) in experiment D2 was attributed to xanthan gum sors of the polyprenol-linked pentasaccharide were also able polymerization, because at the temperature of the experi- to be polymerized into some kind of polysaccharide. Previ- ment (12°C) some polymerization occurs. On the other hand, ous studies had shown that very small amounts of polymer if in a similar experiment UDP-Glc was substituted for were produced when cellobiose diphosphate polyprenol syn- 2496 IELPI ET AL. J. BACT1ERIOL.

TABLE 2. Sequential synthesis of the pentasaccharide-P-P-polyprenol Addition(s)a at time of incubation [14C] (pmol mg of protein-1)' in lipid-bound: Expt Second incubation First incubation Disaccharide Trisaccharide Tetrasaccharide Pentasaccharide Aliquot Addition A UDP-[14C]Glc A1 Not reincubated 60 A2 None 57 A3 GDP-Man 28 9 A4 UDP-GlcA 60 A5 UDP-GlcA + GDP-Man 43 B UDP-[14C]Glc + GDP-Man B1 None 110 B2 UDP-GlcA 60 43 C UDP-Glc + GDP-[14C]Man C1 None 154 C2 UDP-GlcA 77 70 D UDP-Glc + GDP-Man D1 UDP-[14C]GlcA 55 D2 UDP-[14C]GlcA + 1.4 18 GDP-Man E None UDP-['4C]GlcA a Two-step incubations were performed at 12'C as described in Materials and Methods with the components indicated. The first incubation was scaled up fivefold for experiment A and twofold for experiments B, C, and D. In each case, cells were washed, resuspended, and reincubated with the additions indicated. Aliquot A1 was a control not reincubated. Experiment E was a control for the presence of endogenous donors and/or acceptors with no added nucleotides in the first incubation. I The 1203 extracts of each experiment were analyzed by paper chromatography with solvent A and scanned for radioactivity. The peak areas were cut out and counted in a scintillator. Blank spaces indicate no detection (<1 pmol mg of protein-1).

thesized in the first incubation was reincubated in a similar a Bio-Gel A-5m column, coeluted with a sample of authentic way to the above-described experiment (14). Also, a small xanthan gum obtained in vivo, indicating a similar degree of incorporation of radioactivity into the polymer fraction was polymerization since both products had the same apparent observed when trisaccharide diphosphate polyprenol was molecular weight (about 4 x 106) (14). Similar results were reincubated with no additions (Table 3, aliquot B2) or in the obtained with polysaccharide synthesized in vitro, using presence of UDP-glucuronic acid to allow the formation of either UDP-['4C]GlcA or GDP-[14C]Man as the labeled tetrasaccharide-P-P-polyprenol (Table 3, aliquot B3). The precursor (Fig. 7). One point to make clear is the way in synthesis of polymer was evident only when the appropriate which the linear pentasaccharide generated a branched poly- sugar nucleotides were added during the reincubation to mer. The repeating units should be polymerized not in an produce polyprenol-pentasaccharide (Table 3, aliquot B4), as intermolecular head-to-tail manner but through a ,-1,4- formerly shown for the [14C-Glc]pentasaccharide precursor glucose-glucose linkage to form the trisaccharide branch. (14). These data suggest that under the conditions of the Thus, the polymeric product should contain -4- and -3,4- assay polyprenol-diphosphate pentasaccharide is the best substituted glucoses in a 1:1 ratio, as xanthan gum does (Fig. substrate for the polymerizing enzyme(s). 1). The presence of these structures was investigated by Characterization of the in vitro polymeric product. Previ- permethylation of the polymer isolated from an in vitro ous studies have shown that the [14C]Glc-labeled polymer standard incubation carried out in the presence of UDP- obtained by in vitro incubations, upon gel filtration through [14C]Glc. [14C-Glc]pentasaccharide, obtained from the 1203

TABLE 3. Ability of tri-, tetra-, and pentasaccharide diphosphate polyprenols to function as substrates of the polymerization reaction' Incorporation of radioactivity Addition(s) (pmol mg of protein-')' Lipid-bound Expt Aliquot in the second step oligosaccharide 1203 extract Polysaccharide A A1 Not reincubated 65 1.2 Pentasaccharide A2 None 24 21 Pentasaccharide B B1 Not reincubated 37 1.2 Trisaccharide B2 None 32 1.9 Trisaccharide B3 UDP-GlcA 28 1.2 Tetrasaccharide B4 UDP-GlcA + GDP-Man 14 20 Pentasaccharide a Two-step incubations, the first one at 12'C and the second one at 20°C, were performed as described in Materials and Methods. For the first step, the standard incubation mixture was scaled up either twofold (experiment A), containing UDP-Glc, GDP-Man, and UDP['4C]GlcA, or fourfold (experiment B), containing UDP-Glc and GDP-[14CJMan. The EDTA cells were then washed, resuspended, aliquoted, and reincubated as indicated, except for controls A1 and B2 which were not reincubated. b The 1203 extracts were analyzed as described in footnote" of Table 2. Polysaccharide was determined from supernatant fractions of the second step, as described in Materials and Methods. VOL. 175, 1993 XANTHAN GUM BIOSYNTHESIS 2497

EI I E .5 . 0 100 _ J 30 U '5 45° 20 30 4 15 -I Z'

200 6=aQQIwJ aq.,.6%0.976 18..o1 o. .1_1 1 1 ' C, 10 20 30 40 Fraction Number 100 1A. FIG. 7. Gel filtration of in vitro synthesized polymer. The incu- bations were performed and processed as in the footnote to Table 1, using either GDP-['4C]Man (@) or UDP-[14C]GlcA (0) as the labeled 01 donor. The respective aqueous incubation supernatants were fil- 0 5 10 15 tered through a Bio-Gel A-5m column (110 by 1.2 cm) in pyridinium Distance from origin (cm) acetate buffer, pH 5.0, at a rate of 0.25 ml min-1. Fractions of 2.0 ml of the methylated were collected, and radioactivity was determined with 0.5-ml ali- FIG. 8. Thin-layer chromatographic analysis to point out products obtained from in vitro synthesized ['4C]Glc-labeled poly- quots. The profiles obtained are shown superimposed saccharide and pentasaccharide. [14C]Glc-labeled polysaccharide their likenesses. The solid arrow indicates the elution position of cpm each) were perme- ['4C]Glc-labeled and unlabeled xanthan (14). The open arrow shows (A) and pentasaccharide (B) (about 15,000 added as the inclusion volume thylated as described in Materials and Methods. The acid hydrolysis the elution position of CoC12, products were analyzed by thin-layer chromatography with solvent indicator. F on silica gel 60 plates. Bands (0.5 cm wide) were scratched off the plates, and the powder was counted for radioactivity. Mobilities of standards are indicated by arrows: (1) 2,3,4,6-tetra-O-Me-Glc; (2) extract, was submitted to the same procedure, as a control. 2,3,6-tri-O-Me-Glc; (3) 2,4,6-tri-O-Me-Glc; (4) 2,3,4-tri-O-Me-Glc; Analysis of the hydrolysis products from the permethylated (5) 3,4,6-tri-O-Me-Glc; (6) 2,4-di-O-Me-Glc; (7) 3,4-di-O-Me-Glc; (8) polymer demonstrated the presence of similar amounts of 2,6-di-O-Me-Glc; (9) 4,6-di-O-Me-Glc. 2,3,6-tri-0-methyl-Glc and 2,6-di-0-methyl-Glc, as expected for the branched structure proposed (Fig. 8A). The perme- thylated pentasaccharide, instead, produced two different continued to increase as a function of time, indicating the tri-O-methyl-Glc derivatives, 2,3,6- and 2,4,6-tri-0-methyl- formation of new reducing ends during the chase. [14C]sor- Glc, showing once again that the pentasaccharide repeating bitol, on the contrary, remained constant at the level ob- unit was linear (Fig. 8B). tained at the end of the pulse, showing that the reducing ends All of these results taken together strongly support that of the exopolysaccharide formed during the pulse remained the polysaccharide obtained in vitro is xanthan gum and is as such and that the newly formed exopolysaccharide the result of the polymerization of the pentasaccharide chains, although 14C labeled, did not carry the label at the repeating units, originally assembled as prenyl diphosphate glucose reducing ends that were replaced by unlabeled derivatives. glucose during the chase. Direction of chain growth. To examine the direction of the From these results it was concluded that new repeating in vivo xanthan chain growth during the polymerization units were added at the reducing end of a growing chain. process, a pulse-chase experiment was performed. The Growth at the nonreducing end should also have shown growing polysaccharide being released into the culture me- increasing amounts of [14C]sorbitol during the chase period. dium was labeled by a [14C]Glc pulse followed by a chase with unlabeled glucose. The polymer liberated at the end of DISCUSSION the pulse and during the chase was isolated by gel filtration on Bio-Gel A-5m, and the reducing ends were labeled by In the present study on the biosynthesis of xanthan gum, reduction with Na[3H]BH4. The polysaccharide was then an enzymatic preparation consisting of EDTAX. campestris hydrolyzed with acid, and the neutral sugars were isolated cells as a source of lipid acceptor and glycosyl transferases by paper electrophoresis with solvent C. Labeled mannose, was used. Incubations performed in the presence of UDP- glucose, and sorbitol were fractionated by paper chromatog- Glc, GDP-Man, and UDP-GlcA, one of which was 14C raphy with solvent D. The sorbitol area was eluted and labeled, led to the formation of radioactive compounds purified by paper electrophoresis with solvent E. Finally, the soluble in organic solvents. The main compound formed was band of sorbitol, which was clearly separated from the other identified as the pentasaccharide that constitutes the repeat- labeled components, was eluted and differentially counted ing unit of xanthan gum, associated with a lipid component. for 3H and 14C. Previous results obtained with this system, but using only It could be observed that after the chase the [14C]Glc UDP-[14CIGlc as the sugar donor, indicated that the lipid incorporated into the polymer fraction (Fig. 9) continued to moiety very likely was an allylic polyprenol. This conclusion increase as a function of time, indicating the secretion of was supported on the basis of the properties and polysaccharide during the chase period. In addition, the the behavior of the lipid-bound derivatives after mild acid, [3H]sorbitol isolated after reduction and acid hydrolysis also catalytic reduction, and phenol treatments (14). Similar 2498 IELPI ET AL. J. BACTERIOL.

Gtc 1.0 UDP Gtc GDP Man Gtc UDP P GDP cr- 0.8 Gtc P P 2 Lipid 3 (n1- P Lip d-P-P-Gtc-GIC-Man 0. a UDP Gtc Lipid UDP GIcA SLI 0 3 0.4 n UMP UDP :5 vr 0 I P-Lipid Lipid -P-P-Gte -Gtc -Man - GIcA 0 4 023 xanthan | k 5 GDP Man JO U 5 15 45 Lipid-P-P-GGc-Gtc -Gte GOP Man Man Lipid-P-P-Gtc Time of chase tmin) GIcA GIcA 6 Gte 9. Direction of chain Cells were grown as FIG. growth. reported Man Man before (4) (up to an optical density of 1.0) and centrifuged, and the Manj nI cell pellet was washed three times with 3 mM phosphate buffer (pH GtcA 7.4) containing 0.13 M NaCl. To the cells (3 x 1011), resuspended in Man Lipid-P-P -Gtc the same buffer (1.0 ml), [14C]glucose (77 nmol; 285 Ci was mold) Gtc added, and the resuspension was incubated at 280C for 5 min. Three Man- aliquots were then taken: one was immediately inactivated by EDTA-Tris time of and unlabelled GIcA adding (zero chase), glucose (64 Man nmol) was added to the other two aliquots, which were reincubated, - n one for 15 min and the other for 45 min (15 and 45 min of chase, FIG. 10. Scheme of the proposed sequence for biosynthesis of respectively). For each time of chase xanthan was isolated from the the exopolysaccharide xanthan. culture supernatants by filtration through a Bio-Gel A-Sm column. The 14C-labeled polymer fractions were pooled, and aliquots were taken to measure radioactivity (A). The remaining 14C-labeled polysaccharide was treated with Na[ H]BH4 (18 mCi, 3.8 Ci mol', bound to C-3 of the nonreducing glucose of cellobiose and, in 10 mM NaOH) at 20'C overnight and freed from the excess Na[3H]BH4 by extensive dialysis and filtration through a Bio-Gel therefore, the trisaccharide was linear. The a-anomeric A-Sm column as described above. The doubly labelled xanthan was configuration of the mannose was established by treatment then hydrolyzed with 1 M HCl at 100'C for 16 h. The products were with a-mannosidase. A similar trisaccharide-bound polypre- fractionated by paper electrophoresis with solvent C. Sorbitol was nol has been described for an A. xylinum system in this isolated from the other neutral components by subsequent paper laboratory (7). chromatography with solvent D and purified by electrophoresis with The next step in the pathway is the transfer of glucuronic solvent E. The sorbitol area was eluted and differentially counted for acid from UDP-GlcA to trisaccharide-P-P-polyprenol to 14C (0) and 3H (@). form the tetrasaccharide-bound polyprenol (Fig. 10, reaction 4). Incubations carried out in the presence of UDP-Glc, GDP-Man, and UDP-GlcA produced essentially pentasac- treatments, as well as sensitivity to mild alkali, indicated that charide diphosphate polyprenol, and no tetrasaccharide a diphosphate was involved in the sugar-polyprenol linkage diphosphate polyprenol could be detected. This compound (14). could only be obtained in a two-step reaction (Table 2). Figure 10 summarizes the proposed pathway of xantham Finally, a second mannose residue from GDP-Man is gum biosynthesis. Reactions 1 to 5 represent the assembly of incorporated into the tetrasaccharide diphosphate polypre- the pentasaccharide repeating unit on a polyprenyl lipid nol to form the polyprenol-bound pentasaccharide (Fig. 10, acceptor by sequential addition of individual sugar residues reaction 5). from the corresponding sugar nucleotide precursors. All of Several lines of evidence support the structure proposed these intermediates were soluble in 1203 solvent. Recent by Jansson et al. (17) and Melton et al. (21) for the pentasac- studies have established that UDP-Glc, UDP-GlcA, and charide moiety. (i) The size was determined by gel filtration. GDP-Man are synthesized by X. campestris and are inter- (ii) The presence of the trisaccharide fragment a-mannose- mediates in the biosynthesis of xanthan gum (11). cellobiose at the reducing end of the pentasaccharide was The transfer of glucose-1-P from UDP-Glc to an endoge- shown by the sequential synthesis of the pentasaccharide-P- nous polyprenol phosphate acceptor was confirmed after P-polyprenol. (iii) Partial acid hydrolysis led to the isolation isolation of cellobiose-[1-32P]diphosphate polyprenol in in- and characterization of the disaccharide glucuronyl-0-1,2- cubations carried out in the presence of (13- 2P)UDP-Glc mannose residue. (iv) Results from permethylation of [14C- (data not shown). The synthesis of cellobiose-P-P-polypre- Man]pentasaccharide indicated the presence of an internal nol involves the transfer of a glucose moiety from UDP-Glc mannose substituted in position C-2 and a terminal mannose to a glucose diphosphate polyprenol (Fig. 10, reaction 2). residue at the nonreducing end of the pentasaccharide. (v) This precursor of xanthan gum was characterized in a Finally, Smith degradation of the [14C-GlcA]carboxyl-re- previous work (14). The transfer of mannose from GDP-Man duced pentasaccharide demonstrated that in the original to cellobiose diphosphate polyprenol resulted in the forma- pentasaccharide the glucuronic acid was substituted at posi- tion of the trisaccharide diphosphate polyprenol (Fig. 10, tion 4. reaction 3). The precursor-product relationship between The in vitro synthesis of pentasaccharide-P-P-polyprenol cellobiose-P-P-polyprenol and trisaccharide-P-P-polyprenol was also accompanied by the synthesis of polysaccharide was established from incubations carried out in two steps. chains. Incubation of EDTA cells with UDP-Glc, GDP-Man, Periodate oxidation of the cyclic phosphoric ester of the and UDP-GlcA, one of which was labeled, resulted in the [14C-Glc]trisaccharide indicated that the mannose was incorporation of radioactivity into a polymeric product. The VOL. 175, 1993 XANTHAN GUM BIOSYNTHESIS 2499 incorporation of a labeled sugar into this polymer was 4. Cadmus, M. C., S. P. Rojovin, K. A. Burton, J. E. Pittsley, C. A. dependent on the presence of the three sugar nucleotide Knutson, and A. Jeanes. 1976. Colonial variation in Xanthomo- donors in the reaction mixture. Results from two-step incu- nas campestris NRRL B-1459 and characterization of the poly- bation experiments showed that the pentasaccharide-P-P- saccharide of a variant strain. Can. J. Microbiol. 22:942-948. is far 5. Chapman, A., E. Li, and S. Kornfeld. 1976. The biosynthesis of polyprenol by the best polymerization substrate. the major lipid-linked oligosaccharide of Chinese hamster ovary Although X. campestnis mutants producing xanthan-like cells occurs by the ordered addition of mannose residues. J. polysaccharides with truncated side chains have been de- Biol. Chem. 254:10243-10249. scribed (3, 27, 28), using the wild-type in vitro system 6. Couso, R. O., L. Ielpi, and M. A. Dankert. 1987. A xanthan described here, only small quantities of radioactive polysac- gum-like polysaccharide from Acetobacter xylinum. J. Gen. charide were detected following the incubation of cellobio- Microbiol. 133:2123-2135. se-, trisaccharide-, or tetrasaccharide-P-P-polyprenol. 7. Couso, R. O., L. Ielpi, R. C. Garcia, and M. A. Dankert. 1980. The radiolabeled polysaccharide that was synthesized in Synthesis of mannosyl cellobiose diphosphate prenol in Aceto- vitro appeared to be identical in size to xanthan gum, as bacterxylinum. Arch. Biochem. Biophys. 204:434 443. 8. Couso, R. O., L. Ielpi, R. C. Garcia, and M. A. Dankert. 1982. determined by gel filtration (14; this paper). The apparent Biosynthesis of polysaccharides in Acetobacter xylinum. Se- degree of polymerization of the polymer produced in vitro quential synthesis of a heptasaccharide diphosphate prenol. was equivalent to that of the xanthan gum produced in vivo. Eur. J. Biochem. 123:617-627. The factors that govern the degree of polymerization of 9. Garcia, R. C., E. Recondo, and M. A. Dankert. 1974. Polysac- xanthan gum are unknown. charide biosynthesis in Acetobacter xylinum. Enzymatic syn- Acetyl and pyruvyl residues are added at the pentasaccha- thesis of lipid diphosphate and monophosphate sugars. Eur. J. ride-P-P-polyprenol level donated by acetyl-coenzyme A Biochem. 43:93-105. and phosphoenolpyruvate, respectively (15, 16). X. campes- 10. Harding, N. E., J. M. Cleary, D. K. Cabanas, I. G. Rosen, and tris mutants producing nonacetylated and/or nonpyruvylated K. S. Kang. 1987. Genetic and physical analysis of genes essential for xanthan gum biosynthesis in Xanthomonas xanthan gum have been described before (12, 20, 29). campestris. J. Bacteriol. 169:2854-2861. Although no quantitative studies have been reported, these 11. Harding, N. E., S. Raffo, A. Raimondi, J. M. Cleary, and L. mutants have the ability to produce the polysaccharide Ielpi. Identification, genetic and biochemical analysis of genes unaltered. Preliminary results suggest that addition of acetyl- involved in synthesis of sugar nucleotide precursors of xanthan coenzyme A and phosphoenolpyruvate to the reaction mix- gum. J. Gen. Microbiol., in press. ture does not alter significantly the amount of radioactivity 12. Hassler, R. A., and D. Doherty. 1990. Genetic engineering of incorporated into xanthan gum (data not shown). Further polysaccharide structure: production of variant gum in Xanth- studies are necessary to define the role, if any, of the acetyl omonas campestris. Biotechnol. Prog. 6:182-187. and pyruvyl substituents in the polymerization process. 13. Hotte, B., I. Rath-Arnold, A. Pdihler, and R. Simon. 1990. In this a Cloning and analysis of a 35.3-kilobase DNA region involved in work, mechanism for the assembly of xanthan exopolysaccharide production by Xanthomonas campestris pv. gum has been proposed. The synthesis of xanthan gum campestris. J. Bacteriol. 172:2804-2807. involves the polymerization of polyprenol-linked pentasac- 14. Ielpi, L., R. 0. Couso, and M. A. Dankert. 1981. Lipid-linked charide repeating units by transfer of the growing polysac- intermediates in the biosynthesis of xanthan gum. FEBS Lett. charide chain from its lipid carrier to a new pentasaccharide- 130:253-256. P-P-polyprenol, in a way similar to that described for 15. Ielpi, L., R. 0. Couso, and M. A. Dankert. 1981. Pyruvic acid 0-antigen synthesis in Salmonella newington (22). As shown acetal residues are transferred from phosphoenolpyruvate to the in Fig. 10, C-1 of glucose at the reducing end of a preassem- pentasaccharide-P-P-lipid. Biochem. Biophys. Res. Commun. bled nascent polymer is transferred to C-4 of glucose at the 102:1400-1408. of the 16. Ielpi, L., R. 0. Couso, and M. A. Dankert. 1983. Xanthan gum nonreducing position pentasaccharide-P-P-polyprenol biosynthesis: acetylation accurs at the prenyl-phospho-sugar newly formed, simultaneously producing the trisaccharide stage. Biochem. Int. 6:323-333. side chain. The final events involving the regeneration of the 17. Jansson, P. E., L. Kenne, and B. Lindberg. 1975. Structure of polyprenol phosphate, as well as the secretion and release of the extracellular polysaccharide from Xanthomonas campes- the polymer into the culture medium, deserve more study. tris. Carbohydr. Res. 45:274-282. 18. Koplin, R., W. Arnold, B. Hotte, R. Simon, G. Wang, and A. ACKNOWLEDGMENTS Puihler. 1992. Genetics of xanthan production in Xanthomonas campestris: the xanA and xanB genes are involved in UDP-Glc The skillful technical assistance of Susana Raffo is gratefully and GDP-mannose biosynthesis. J. Bacteriol. 174:191-199. acknowledged. 19. Li, E., I. Tabas, and S. Kormfeld. 1978. The synthesis of This work was supported in part by grants from CONICET to L.I. complex-type oligosaccharides. Structure of the lipid-linked and to M.A.D., who are members of Carrera del Investigador oligosaccharide precursor of the complex-type oligosaccharides (CONICET, Argentina). of the vesicular stomatitis virus G protein. J. Biol. Chem. 253:7762-7770. REFERENCES 20. Marzocca, M. P., N. E. Harding, E. A. Petroni, J. M. Cleary, 1. Baldessari, A., L. lelpi, and M. A. Dankert. 1990. A novel and L. Ielpi. 1991. Location and cloning of the ketal pyruvate galacturonide from Xanthomonas campestris. J. Gen. Micro- transferase gene of Xanthomonas campestris. J. Bacteriol. biol. 136:1501-1507. 173:7519-7524. 2. Barrere, G. C., C. E. Barber, and M. J. Daniels. 1986. Molecular 21. Melton, L. D., L. Mindt, D. A. Rees, and G. R. Sanderson. 1976. cloning of genes involved in the production of the extracellular Covalent structure of the extracellular polysaccharide from polysaccharide xanthan by Xanthomonas campestris pv. Xanthomonas campestris: evidence from partial hydrolysis campestris. Int. J. Biol. Macromol. 8:372-374. studies. Carbohydr. Res. 46:245-257. 3. Betlach, M. R., M. A. Capage, D. H. Doherty, R. A. Hassler, 22. Robbins, P. W., D. Bray, M. A. Dankert, and A. Wright. 1967. N. M. Henderson, R. W. Vanderslice, J. D. Marrelli, and M. B. Direction of chain growth in polysaccharide synthesis. Science Ward. 1987. Genetically engineered polymers: manipulation of 158:1536-1542. xanthan biosynthesis, p. 35-50. In M. Yalpani (ed.), Progress in 23. Sanderson, G. R. 1981. Applications of xanthan gum. Br. biotechnology, vol. 3. Industrial polysaccharides. Elsevier, Am- Polym. J. 13:71-75. sterdam. 24. Sandford, P. A., J. E. Pittsley, C. A. Knutson, P. R. Watson, 2500 IELPI ET AL. J. BACTERIOL.

M. C. Cadmus, and A. Jeanes. 1977. Extracellular microbial M. Tecklenburg. 1990. Genetic engineering of polysaccharide polysaccharides. Am. Chem. Soc. Symp. Ser. 45:192-210. structure in Xanthomonas campestris, p. 145-156. In V. 25. Siddiqui, I. R. 1967. An extracellular polysaccharide from Crescenzi, I. C. M. Dea, S. Paoletti, S. S. Stivala, and I. W. Xanthomonas campestris. Carbohydr. Res. 4:284-291. Sutherland (ed.), Biomedical and biotechnological advances in 26. Thorne, L., L. Tansey, and T. J. Pollock. 1987. Clustering of industrial polysaccharides. Gordon and Breach Science Publish- mutations blocking synthesis of xanthan gum by Xanthomonas campestris. J. Bacteriol. 169:3593-3600. ers, New York. 27. Vanderslice, R. W., D. H. Doherty, M. A. Capage, M. R. 28. Vojnov, A., and M. A. Dankert. Unpublished data. Betlach, R. A. Hassler, N. M. Henderson, J. Ryan-Graniero, and 29. Wernan, W. C. October 1981. U.S. patent 4,296,203.