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 Microbiology Sequential Assembly and Polymerization of the Polyprenol- Linked Pentasaccharide Repeating Unit of the Xanthan Polysaccharide 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-glucuronic acid were used as sugar donors. A linear pentasaccharide unit is assembled on a polyprenol-P lipid carrier by the sequential addition of glucose-1-P, glucose, mannose, 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 trisaccharide side chain. Microbial exopolysaccharides are produced during the MATERIALS AND METHODS growth of various genera of bacteria 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 (cellobiose, 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 xanthan gum, an exopolysaccharide liberated into the (13-1,2)-Man-(oa-1,3)-Glc-(1-1,4)-Glc (tetrasaccharide 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 cellulose cyclic phosphate esters in position 1,2 of the reducing (Fig. 1): the main chain is a 3-1,4-glucan 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 glucoses. 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 hydrolysis 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 disaccharides. 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 ethanol (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 chloroform-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]oligosaccharides, 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 polysaccharides 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.