Eur. J. Biochem. 155, 513-519 (1986) c,) FEBS 1986

Biosynthesis of the wall acidic polysaccharide in Bacillus cereus AHU 1356 Naoya KOJIMA, Yoshio ARAKI and Fiji IT0 Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo

(Received Octobcr 14/December 9, 1985) - EJB 85 1127

Biosynthetic studies on an acidic polysaccharide, comprising galactose, rhamnose, N-acetylglucosamine and sn-glycerol I-phosphate, were carried out with a membrane system obtained from Bacillus cereus AHU 1356. Incubation of the membranes with UDP-[14C]Gal, TDP-[14C]Rha and UDP-['4C]GlcNAc resulted in the forma- tion of four or more labeled-sugar-linked lipids and a labeled polysaccharide. Data on structural analysis of the sugar moieties released from the glycolipids, together with results of enzymatic conversion of [ ''C]galactose- linked lipid and [ 14C]Rha-Gal-linked lipid to higher-oligosaccharide-linked lipids and polysaccharide, led to the conclusion that the acidic polysaccharide is probably synthesized through the following pathway: Gal-PP- lipid- Rha- Gal- PP-lipid+GlcNAc- Rha- Gal - PP-lipid- Rha- GlcNAc- Rha -Gal- PP-lipid +glycerol- P - Rha- GlcNAc- Rha- Gal - PP-lipid+[ -(glycerol-P-)Rha- GlcNAc- Rha- Gal -1,-PP-lipid+polysaccharide. Theglycero- phosphate residues seem to be derived from phosphatidylglycerol.

The cell walls of Bacillus cereus AHU 1356 are made up of at least three different polysaccharides, namely, peptido- glycan, a neutral polysaccharide composed of N-acetylglucos- amine, N-acetylmannosamine (ManNAc), N-acetylgalactos- amine and glucose [l] and an acidic polysaccharide with the repeating units, +4)[,sn-glycerol-l-P-2]Rha(crl+4)GlcNAc- (a1 +3)Rha(crl+3)Gal(al+ [2]. Previous studies [3, 41 demonstrated that the neutral polysaccharide is formed from UDP-GlcNAc, UDP-ManNAc, UDP-GalNAc and UDP-Glc via intermediates bound to undecaprenyl pyrophosphate (PP- CS5). Recently the membrane preparation of the same strain was shown to catalyze the incorporation of galactose, rhamnose and N-acetylglucosamine residues from the cor- responding sugar-linked nucleotides into glycolipids and a polymer presumed to be the wall acidic polysaccharide. The present paper reports the results of studies on the structure of these glycolipids and their function as intermediates in the synthesis of the wall acidic polysaccharide of this strain.

MATERIALS AND METHODS

Ma ter ials

UDP-[U-'4C]Gal (123 Ci/mol), UDP-[GlcN-'4C]GlcNAc (306 Ci/mol), [U-'4C]glycerol (171 Cilmol) and [U-'4C]glu- cose 1-phosphate (328 Ci/mol) were purchased from Amer- sham International. Labeled and unlabeled TDP-Rha were

Correspondence 10 E. Ito, Department of Chemistry, Faculty of Science. Hokkaido University, Kita-10-jyo, Nishi-8-chome, Kita-ku, Sapporo, Japan 060

Abbreviations. Cs5, undecaprenol; ManNAc, N-acetylmannos- amine; GalNAc, N-acetylgalactosamine; SDS, sodium dodecyl

Enzymes. cl-Rhamnosidase (EC 3.2.1.40); a-N-acetylglucosamini- dase (EC 3.2.1 S O ) ; acid phosphatase (EC 3.1.3.2); phosphodiesterase I1 (EC 3.1.4.-); lysozyme (EC 3.2.1.17).

Sulfate.

enzymatically prepared from D-glucose 1-phosphate via TDP- Glc [5, 61. Other unlabeled nucleotide sugars, acid phospha- tase and naringinase were obtained from Sigma Chemical Co. ; Sephadex G-25 (superfine), Sephacryl S-300 and Sepharose CL-6B from Pharmacia Fine Chemicals; silica gel H from E. Merck AG. Exo-a-N-acetylglucosaminidase and cr-rhamnosidase were prepared from human urine [7] and naringinase [8] respectively. Phosphodiesterase 11 was pre- pared according to the method of Schneider and Kennedy [9]. Standard glycolipids, cr-['4C]GlcNAc-PP-C55 and cr and /- ['4C]GlcNAc-P-C55, were prepared by using membranes from B. cereus AHU 1356 cells, as described previously [3,4]. Antibiotic 24010 was kindly provided by Dr M . Mizuno, Asahi Chemical Industry Co. [lo].

Assay qf enzymatic formation of glycolipids and polymer

The procedures for the culture of B. cereus AHU 1356 and for the preparation of membranes from the cells were essentially the same as those described previously [3]. The standard reaction mixture contained 10 pM UDP-Gal, 10 pM TDP-Rha, 40 pM UDP-GlcNAc, 1 mM ATP, antibiotic 24010 (2 pg/ml), 30 mM MgClz and the membranes (1 - 2 mg protein) in 50 p150 mM Tris/HCl buffer, pH 8.2. Appropriate 14C-labeled nucleotide sugar(s) was used according to the experimental purposes. The reaction mixture was incubated at 25°C for suitable time periods and subjected to paper chromatography in solvent A. The chromatogram was cut into segments and measured in a liquid scintillation counter for radioactivity of glycolipids (RF = 0.52 -0.87) and poly- mer remaining at the origin. For further analysis of the sugar moieties, the glycolipids were extracted from the paper segments with CHC13/CH30H/H20 (10: 10: 3, by vol.) and hydrolyzed with 10 mM HCl in 25% 1-propanol at 100°C for 15 min. The resulting water-soluble material was fractionated by paper chromatography in solvent B. The polymer remain- ing at the origin of the first chromatogram was extracted from

514

the paper with 50% aqueous phenol at 68 - 70°C for 1 h and used for further analysis after dialysis.

Preparation of [ ' " C ] Gal-linked and [' "C] Rha-Gal-linked lipids

To prepare ['4C]Gal-linked lipid, UDP-['"CIGal (1 nmol, 0.2 pCi) was incubated with the membranes (1 mg protein) at 25 "C for 15 min in a mixture the same as the standard reaction mixture except for the omission of other nucleotide sugars. The glycolipid was extracted from the reaction mixture twice with 1 ml CHC13/CH30H (2: 1, v/v) and subjected to thin- layer chromatography. The portion of the plate corresponding to the spot of the labeled lipid, located by radioautography, was scraped and extracted with CHC13/CH30H/H20 (10: 10: 3, v/v/v). Only ['4C]Gal-linked lipid (SO000 cpm) was yielded as a labeled glycolipid.

To prepare ['"CIRha-Gal-linked lipid, TDP-['"CIRha (10 nmol, 2 yCi) was incubated with the membranes (10 mg protein) at 25°C for 15 min in a reaction mixture containing 10 pM UDP-Gal, 1 mM ATP and 30 mM MgClz in 1 ml 50 mM Tris/HCl buffer, pH 9.2. The extraction and isolation of glycolipids were carried out as described above. Only ['4C]Rha-Gal-linked lipid (280000 cpm) was yielded as a labeled product.

Pulse-chase experiment

B. cereus AHU 1356 cells were grown in 150 ml Difco antibiotic medium no. 3 to the early log phase, harvested and suspended in 60ml fresh medium. The cell suspension was again incubated with 25 yCi [U-'4C]glycerol (171 Ci/mol) at 37°C for 30 min. The cells, harvested by centrifugation, were resuspended in 60ml fresh medium and then chased with 10 mM unlabeled glycerol. At appropriate intervals 10-ml portions were withdrawn, cooled and centrifuged. The col- lected cells were washed twice with cold water and extracted with 4 ml CHC13/CH30H (2: 1, v/v). The pooled extract was analyzed for lipids by thin-layer chromatography. The unextractable residue was treated with 4% sodium dodecyl sulfate (SDS) at 100°C for 15 min, giving the SDS-soluble fraction and the SDS-insoluble cell wall fraction. The cell wall fraction was treated with excess lysozyme after N-acetylation with acetic anhydride, and the resulting soluble polymer was analyzed as the polysaccharide-linked glycopeptide after chromatography on columns of DEAE-cellulose and Sepharose CL-6B, as described previously [l , 21.

Chromatograph?>

Gel chromatography on columns of Sephadex (3-25 (1 x 80 cm), Sephacryl S-300 (1 x 100 cm) and Sepharose CL-6B (1 x 85 cm) was carried out in 50 mM (NH4)2C03. For ion-exchange chromatography, glycolipids were applied on a DEAE-cellulose column (1 x 3 cm, acetate form) in CHC13/CH30H/Hz0 (10: 10: 3, by vol.). The column was eluted with the same solvent then with a linear gradient of ammonium formate (0 - 12 mM) in the same solvent. For ion- exchange chromatography of polysaccharides and oligosac- charides, each sample was applied on a DEAE-cellulose column (1 x 3 cm) equilibrated with 5 mM Tris/HCl buffer, pH 7.2. The column was eluted with the same buffer then stepwise with 0.05 M, 0.1 M, 0.2 M and 0.3 M NaCl in the same buffer. Descending paper chromatography was carried out on Toyo no. 50 filter-paper in isobutyric acid/0.5 M

NH40H ( 5 : 3 , v/v) (solvent A) and in I-butanol/pyridine/ acetic acid/water (6:4:0.3:3, by vol.) (solvent B). Paper electrophoresis was performed using the same paper in pyridine/acetic acid/water (35: 5: 960, by vol., pH 5.8) (buffer A) and in 50mM borate, pH9.5 (buffer B). Paper chromatograms and electrophoretograms were measured for radioactivity by autoradiography or by cutting the strip into 1-cm segments and counting in a liquid scintillation counter. Thin-layer chromatography was carried out on silica gel H plates in CHC13/CH30H/H20 (65 : 25 : 4, by vol.).

RESULTS

Influence of ancibiotic 24010 on the in vivo synthesis of acidic polysaccharide

The biosynthetic pathways involving GlcNAc-PP-poly- prenols as precursors are known to be inhibited by antibiotics of the tunicamycin group. When antibiotic 24010 of this group was added at a concentration of 0.2 pg/ml or 1 .O pg/ml to an exponentially growing culture of B. cereus AHU 1356, the resulting cell wall preparation contained greater amounts of rhamnose and phosphorus, the representative constituents of the acidic polysaccharide, than did the control cell wall preparation. In contrast, the contents of galactosamine and mannosamine, the constituents of the neutral polysaccharide, in the cell walls of antibiotic-treated cells decreased by 25 - 30% relative to those in the control cell wall preparation. This result suggests that GlcNAc-PP-polyprenol is not involved in the biosynthesis of the acidic polysaccharide. Therefore, antibiotic 24010 was used in the present study to inhibit selec- tively the formation of the neutral polysaccharide.

Incorporation of ['4C]galactose into glycolipidy and polymer

The incubation of the membranes from B. cereus AHU 1356 with UDP-[14C]Gal, UDP-GlcNAc and TDP-Rha, followed by paper-chromatographic separation of the prod- ucts, gave three bands of radioactive material with RF values of 0.71 -0.87 (band l), 0.52-0.71 (band 2) and 0.17-0.25 (band 3), in addition to the bands of the substrate (RF = 0.15, band 4) and a polymer remaining at the origin (Fig. 1). Analysis of labeled material after acid hydrolysis revealed that bands 1 and 2 contained glycolipids labeled in galactose (see below), whereas material in band 3 was characterized as galactose cyclic 1,2-phosphate, a degradation product of the substrate.

The requirements of the polymer and glycolipid synthesis for sugar-linked nucleotides were investigated with UDP- ['"CIGal as the labeled substrate (Table 1). The incubation of UDP-['"CIGal alone with the membranes led to incorpora- tion of a considerable proportion (about 17%) of the radioac- tivity into the glycolipid fraction (band 1). However, the incor- poration into polymer was negligible under these conditions. The addition of TDP-Rha alone gave no apparent effect on the reaction with UDP-[14C]Gal, and the addition of UDP- GlcNAc alone resulted in a marked reduction of the glycolipid formation. The supplementation of both TDP-Rha and UDP- GlcNAc led to the maximal incorporation of radioactivity from UDP-[ '"CIGal into polymer, indicating the requirement of the acidic polymer formation for a set of these three nucleotide sugars. Antibiotic 24010 seemed to rather enhance the formation of glycolipids and polymer. UDP-Glc had no influence on the formation of glycolipids and polymer.

51 5

I t -

1

I I

10 20 30 Distance from origin ( c m )

Fig. 1. Separation qf reaction products by puper chromatography. The membranes (1 mg protein) wcre incubated with UDP-[14C]Gal (10 pM, 120000 cpm),TDP-Rha(l0 pM)and UDP-GlcNAc(40 pM) at 25°C for 2 h under the standard assay conditions. After the termination of reaction by addition of 5 p1 isobutyric acid, the reac- tion mixture was subjected to paper chromatography in solvent A. The chromatogram was cut into segmenls and counted for radioactiv- ity (k cpm = lo3 cpm)

Table 1. Requirement ,for substrates of galactose incorporution into glycolipids and polymer UDP-['4C]Gal (10 pM, 120000 cpm) was incubated with membranes (1 mg protein) at 25°C for 2 h in the presence of the supplements indicated at the following concentrations: UDP-GlcNAc, 40 pM; TDP-Rha, 10 pM; UDP-Glc, 20 pM; antibiotic 24010,2 pg/ml. Each reaction mixture was then subjected to paper chromatography in solvent A. The glycolipid fraction (RF = 0.52-0.87) and polymer remaining at the origin wcre counted for radioactivity

Supplement Radioactivity

glycolipids polymer

CPm

None TDP-Rha UDP-GIcNAc UDP-Glc TDP-Kha, UDP-GlcNAc TDP-Rha, UDP-GlcNAc,

TDP-Rha, UDP-GlcNAc, UDP-Glc TDP-Rha, UDP-GlcNAc

(boiled enzyme control)

antibiotic 24010

21 500 22 500

8 970 19 200 8 990

19 500 8 500

260

320 290

1430 320

4360

4800 4200

140

Characterization ojmonosaccharide-linked lipid

The glycolipid produced by incubation of the membranes with UDP-[14C]Gal alone was isolated by extraction with CHC1,/CH30H followed by thin-layer chromatography. Mild acid hydrolysis of this glycolipid gave a labeled mono- saccharide identified as galactose. The lipid gave a single radioactive spot at the same position (RF = 0.36) as standard cr-GlcNAc-PP-CSS upon thin-layer chromatography on a silica gel H plate. The concentration of ammonium formate, 9 mM, required for the elution from DEAE-cellulose (acetate form) of this glycolipid was close to the value, 8 mM, for the elution of standard glycolipid, cr-GlcNAc-PP-C55, and much

I

A C

Distance from origin ( c m )

Fig. 2. Paper chromatography of sugar moieties resulting from mild acid hydrolysis of glycolipid,fraction. The rnembrancs ( 1 mg protein) were incubated with UDP-['4C]Gal (24 pM, 120000 cpm), TDP- [I4C]Rha (20 pM, 120000 cpm) and UDP-[14C]GlcNAc (50 pM, 490000 cpm) at 25°C for 2 h under the slmdard assay conditions, and the reaction mixture was subjected to paper chromatography in solvent A. The glycolipid fraction (corresponding to bands 1 and 2 in Fig. 1) was extracted with CHCI3/CH30H/H20 (10:10:3) and hydrolyzed with mild acid, and the resulting water-soluble material was subjected to paper chromatography in solvent B. Material re- maining at the origin of the first chromatogram was solubilized by the treatment with hot phenol and further analyzed as the polymcr

higher than the value, 4mM, for the elution of a and P-GlcNAc-P-CS s. Furthermore, the addition of UDP- GlcNAc at 60 pM caused 50% inhibition of this lipid forma- tion, suggesting competition between the N-acetylglucos- amine-transferring system and the galactose-transferring system for the same endogeneous acceptor, presumed to be undecaprenyl phosphate. Thus, it is most probable that this glycolipid was Gal-f f-C5=,. Its formation was also inhibited by UMP and UDP. Triton X-100 and Nonidet NP-40 at a concentration of 0.1 % completely inhibited this glycolipid formation, whereas Targitol NP-40 at a concentration of 0.2% had no influence. Thus, Targitol NP-40 was used for dissolv- ing lipid intermediates.

Characterization of' saccharide moieties of other glycolipids

After the membranes were incubated with UDP-[ 14C]Gal, TDP-[ 14C]Rha and UDP-['4C]GlcNAc under the standard assay conditions, the reaction mixture was subjected to paper chromatography in solvent A, and glycolipids corresponding to bands 1 and 2 in Fig. 1 were extracted with CHC13/ CH30H/H20 and then hydrolyzed with 10 mM HC1 in 25% 1-propanol at 100°C for 15 min. The resulting water-soluble sugars were separated into four fractions (fractions A - D) by paper chromatography in solvent B (Fig. 2), then each frac- tion was subjected to chromatography on a Sephadex G-25 column. In this process, fraction A was separated into two sugars, A-I and A-2, and major components of fractions B, C and D were purified, giving sugars B, C and D respectively (Fig. 3).

The composition of these sugars is summarized in Table 2, together with that of polymeric material obtained from the oiigin of the first chromatogram by hot phenol treatment. The acid hydrolysate of a reduced sample of each sugar gave ['4C]galactitol, as analyzed by paper electrophoresis in buff-

516

I

vo 3 2 I 800 1 1 1 - 1

40 50 60 70 80 Fraction number

Fig. 3. Purification of sugar moieties. Material in each of bands A-D, shown in Fig. 2, was applied on a Sephadex G-25 column. Fractions indicated by bars were pooled and used as the purified sugars except for sugar B. Sugar B was further purified by rechromatography on the same column. Arrows 1, 2 and 3 indicate the respective elution positions of the monomer, dimer and pentamer of glucose

Table 2. Composition of sugars liberated from glycolipids by mild acid hydrolysis Sugars A-1, A-2, B, C, and D were separated from the mild acid hydrolysate of glycolipids as described in Figs 2 and 3. Each purified sugar was hydrolyzed in 1 M HCI at 100°C for 2 h, N-acetylated twice with acetic anhydride/pyridine/methanol ( 5 : 1 : 50, by vol.) and subjected to paper electrophoresis in buffer B. Areas corresponding to galactose, rhamnose and N-acetylglucosamine were cut out and counted for radioactivity. Data are expressed in molar ratios to galactose, calculated from the radioactivity found and the specific radioactivity of the starting substrates. The polymer product, obtained from the origin of the first chromatogram by the treatment with hot phenol, was also analyzed

Sample Galactose Rhamnose N-Acetyl- glucosamine

mol/mol

A- 1 1 .oo 0 0 A-2 1 .oo 0.89 0 B 1 .oo 2.04 1.19 C 1 .oo 0.87 0.78 D 1 .oo 1.92 0.97 Polymer product 1 .OO 1.81 1.14

er B, indicating the presence of galactose at the reducing terminus in each sugar. Sugar A-2 was a disaccharide, which was hydrolyzed into rhamnose and galactose by a-rham- nosidase. Thus, sugar A-2 was probably a-Rha-Gal. Sugar C was a linear trisaccharide composed of galactose, rhamnose and N-acetylglucosamine in equimolar amounts. The NaI04 oxidation of this sugar after reduction with NaBH4 led to degradation of the galactose and N-acetylglucosamine re- sidues, leaving the rhamnose residues undegraded. Further- more, the treatment of this sugar with a-N-acetyl- glucosaminidase gave free N-acetylglucosamine and a disac- charide identical with sugar A-2. Thus, the most probable

structure for sugar C was GlcNAc(a1-3)Rha-Gal. Sugar B was a linear tetrasaccharide composed of galactose, rhamnose and N-acetylglucosamine in a molar ratio of 1 :2: 1 . It was insensitive to a-N-acetylglucosaminidase. The NaI04 oxida- tion of this sugar after reduction with NaBH4 led to degrada- tion of 1 mol each of the galactose and rhamnose residues, leaving 1 mol each of the N-acetylglucosamine and rhamnose residues undegraded. The treatment with a-rhamnosidase led to hydrolysis of sugar B into free rhamnose and a trisaccharide identical with sugar C. Thus, the most probable structure for sugar B was Rha(al~3/4)GlcNAc(al+3)Rha-Gal.

Sugar D had the same sugar components as sugar B, but it was insensitive to a-rhamnosidase, suggesting the presence of an unlabeled substituent at the non-reducing terminal sugar residue. Sugar D gave a single spot with MGro-l-P value of 0.27 upon paper electrophoresis in buffer A, indicating the presence of a negatively charged substituent. The treatment of sugar D with acid phosphatase alone gave no appreciable change, while the treatment with a mixture of phosphodi- esterase I1 and acid phosphatase resulted in the formation of an uncharged sugar identical with sugar B. Taking account of the substrate specificity of phosphodiesterase I1 [9], the unlabeled substituent in sugar D seemed to be glycerophos- phate or its related residue. In addition, the mild acid hydro- lysis (0.1 M HCI, 100°C, 15 min) of sugar D gave two com- pounds tentatively characterized as glycerophosphorhamnose and phosphorhamnose. These results indicate that sugar D was probably glycerol-P-Rha(al+ 3/4)GlcNAc(al-t3)Rha- Gal, corresponding to the repeating unit of the wall acidic polysaccharide chain in the B. cereus AHU 1356 cell walls.

Time course of formation of glycolipids and polymer

The membranes were incubated with TDP-[ 14C]Rha in the presence of UDP-Gal and UDP-GlcNAc for various time periods, and the incorporation of radioactivity into glycolipids and polymer was measured after the separation of the sugar moieties by paper chromatography in solvent B. The data (Fig. 4) show that the rapid formation of [14C]Rha- Gal-linked and GlcNAc-[ ''C]Rha-Gal-linked lipids preceded the formation of ['4C]tetrasaccharide-linked lipid (sugar-B- linked lipid). Glycerophosphorylated tetrasaccharide-linked lipid (sugar-D-linked lipid) and higher-oligosaccharide-linked lipids were formed after a lag of a few minutes, whereas the polymer formation started after about 30 min.

To clarify the formation of oligosaccharides in a lipid- bound form, ['4C]Gal-PP-lipid or [14C]Rha-Gal-PP-lipid was again incubated with the membranes under the conditions similar to the standard assay conditions except for the pres- ence of O.l0/o Targitol NP-40. The results show that [I4C]Gal- PP-lipid could be quantitatively converted to Rha-[ 14C]Gal- linked lipid by incubation with the membranes in the presence of TDP-Rha alone. Similarly [ ''C]Rha-Gal-linked lipid was converted to GlcNAc-[ ''C]Rha-Gal-linked lipid by the in- cubation with the membranes in the presence of UDP- GlcNAc, whereas this lipid intermediate was unchanged by incubation with either TDP-Rha or UDP-Gal.

When ['4C]Rha-Gal-linked lipid was incubated with the membranes in the presence of both TDP-Rha and UDP- GlcNAc and the products were analyzed as above, a lipid linked to Rha-GlcNAc-Rha-Gal was formed first, then glycerophosphate-containing lipid (sugar-D-linked lipid) in- creased gradually for up to 30 min. After the formation of this glycolipid, the polymer formation started. However, when

51 7

I I >

1 2 3 4 I 2 3 4 1 1 1 1 1 1 1 1

1

Incubation time ( m i n ) Fig. 4. Time course of glycolipid and polymer formation. TDP- [14C]Rha (10 FM, 120000 cpm) was incubated with the membranes in the presence of UDP-Gal and UDP-GlcNAc under the standard assay conditions. At the indicated time periods the reaction was terminated, and each reaction mixture was subjected to paper chromatography in solvent A. Material remaining at the origin was measured for radioactivity as the polymer product (0). The glycolipids (RF = 0.52-0.87) were extracted from the paper, hydrolyzed with mild acid and then fractionated by paper chromatography in solvent B. Radioactivities in paper sections cor- responding to bands A, B, C and D, shown in Fig. 2, were counted in a liquid scintillation counter as the lipids linked to sugars A-2 ( W), B (0). C ( A ) and D (0 ) respectively. Material migrating slower than sugar D was also counted as higher-oligosaccharide-linked lipids (A)

either TDP-Rha or UDP-GlcNAc was omitted from the reac- tion mixture, only a negligible amount of polymer resulted. Thus, it is most likely that in B. cereus AHU 1356 the wall acidic polysaccharide was synthesized through the formation of the intermediate, glycerol-P-Rha-GlcNAc-Rha-Gal-PP- lipid (sugar-D-linked lipid), which was produced by the sequential addition of a rhamnose residue, an N-acetyl- glucosamine residue, another rhamnose residue and a putative glycerophosphate residue to Gal-PP-lipid.

Characater ization of polymer synthesized

Material remaining at the origin of the chromatograms in the above experiments could not be extracted with either CHCl3/CH30H/Hz0 (10: 10: 3) or water, but it was solubi- lized by the treatment with 50% phenol (68 - 70 "C, 1 h). As shown in Table 2, the solubilized polymer contained galactose, rhamnose and N-acetylglucosamine in a molar ratio of 1 : 2: 1 just as sugars B and D did. This polymer fraction was separated by DEAE-cellulose column chromatography into four fractions, fractions I-IV, which were eluted with 0, 0.05 M, 0.1 M and 0.2 M NaCI, respectively, indicating differences in their net negative charges (Fig. 5). The treat- ment of each of the latter two fractions, fractions 111 and IV, with a mixture of phosphodiesterase I1 and acid phosphatase resulted in loss of net negative charges from a major part of each fraction, whereas the treatment with acid phosphatase alone led to no change in their elution profiles (Fig. 6). From the substrate specificity of phosphodiesterase I1 it seems that the polymers synthesized had glycerophosphate residues like sugar D. Particularly the polymer in fraction IV, which was eluted with a high concentration of NaCl(0.2 M), is presumed to be highly substituted by glycerophosphate residues and

0 5 10 15 20 Fraction number

Fig. 5 . Ion-exchange chromatography of' polymer product. TDP- [14C]Rha (10 pM, 120000 cpm) was incubated with the membranes for 2 h under the same conditions as those in Fig. 4. Polymer remain- ing at the origin of chromatogram in solvent A was solubilized by the treatment with hot phenol. The resulting water-soluble material was applied on a DEAE-cellulose column after dialysis. At arrows 1, 2, 3 and 4, solvent was changed to buffers containing 0.05 M, 0.1 M, 0.2 M and 0.3 M NaCl respectively. Fractions indicated by bars were pooled and further analyzed

Fraction number

Fig. 6. Enzymatic digestion of acidic polymers. Each polymer product (Fig. 5, fraction 111 or IV, 800 cpm each), after digestion with the indicated enzyme@), was subjected to ion-exchange chromatography under the same conditions as in Fig. 5. (A) Fraction 111 digested with acid phosphatase alone; (B) fraction 111 digested with a mixture of phosphodiesterase I1 and acid phosphatase; (C) fraction IV digested with acid phosphatase alone; (D) fraction IV digested with a mixture of phosphodiesterase I1 and acid phosphatase

appears to correspond to the natural acidic polysaccharide in this strain.

Origin of glycerophosphate residues

As described above, some of glycolipid intermediates as well as the polymer products seemed to be substituted by glycerophosphate residues. However, when CDP-['4C]glyc- erol was incubated with the membranes and other nucleotide substrates under the standard reaction conditions, no appreci- able incorporation of ['4C]glycerol into polymers or lipid

518

UDP-Gal + P-C55- - - - 1

Go 1 -PP-C55 I

I I

I I I I I I I

I I I

t UMP

TDP-Rho + TDP

Rha-Gal -PP-c55

+UDP I

'-+TDP I

-1

UDP-GlCNAC

G 1 cNAc- Rha-GO 1 -PP-C55

TDP-Rha

Rho-G 1 cNAc-Rho-Go 1 -PP-C55 I I I

I I

Gro-P-Rha-G1 cNAc-Rha-Ga 1 -PP-C55 I

t [(Gro-P-Rho-GlcNAc-Rho-Gal ),-PP-C55] !

Phosphat i d v l s l y c e r o l ( G ro-P)

0 20 40 60 Chase time i min 1

Fig. I . Kinetics of incorporation of [14C]glycerol into cell wall and SDS-soluble fractions and phospholipids. B . cereus AHU 1356 cells were pulsed, chased and fractionated as described in Materials and Methods. Each fraction was measured for radioactivity in a liquid scintillation counter. The absorbance at 575 nm of the culture in- creased from 1.27 to 1.50 during chase (60 min). Cell wall fraction (0); SDS-soluble fraction (A) ; phosphatidylglycerol(0); phosphat- idylethanolamine (0); cardiolipin ( A )

intermediates was observed. To examine the possibility that some endogenous phospholipids may serve as donors of glycerophosphate residues, a pulse-chase experiment was carried out in vivo. The kinetics of incorporation of ['4C]glycerol into the lipid fractions (phosphatidylglycerol, phosphatidylethanolamine and cardiolipin), the SDS-soluble fraction (lipoteichoic acid fraction) and the SDS-insoluble fraction (cell wall fraction) are shown in Fig. 7. After a 30-min pulse period about 55% of the radioactivity was found in the total lipid fraction (CHC1&H30H extract). Thin-layer chromatography of the lipid fraction showed that the majority (86%) of the radioactivity was in phosphatidylglycerol. During the chase period a marked decrease of radioactivity in phosphatidylglycerol, accompanied by a corresponding in- crease of radioactivity in the cell wall fraction, was observed, whereas only a small change was shown in other lipid fractions.

The lysozyme digestion of the cell wall fraction, obtained from a 60-min-chased sample, gave quantitatively a radio- active polymer which was coincident with the natural, acidic- polysaccharide-linked glycopeptide [2] in its chromatographic behavior. Furthermore, either after acid hydrolysis or after treatment with a mixture of phosphodiesterase I1 and acid phosphatase, this preparation gave only ['4C]glycerol as a labeled product. Therefore, endogenous phosphatidylglycerol seems to serve as a main glycerophosphate donor in the syn- thesis of the cell wall acidic polysaccharide of B. cereus AHU 1356.

DISCUSSION

The wall acidic polysaccharide of B. cereus AHU 1356 is characteristic in having a repeating tetrasaccharide unit with an sn-glycerol 1-phosphate residue attached as a branch [2]. In a preliminary experiment it was shown that the membranes from this strain catalyze the formation of galactose-linked

PeDtidoglycon-- x--c p- - - - ; 1 55- (Gro-P-Rho-G1cNAc;Rha-Gal )n-P-PePtidoplYcon

Scheme 1. Proposed pathway for acidic polysaccharide synthesis in B. cereus A H U 1356. Reaction steps for the polymerization of the repeating units on a lipid intermediate, the transfer of the synthesized polymer chain to peptidoglycan and the conversion of pyrophospho- rylprenol to monophosphorylprenol were not established in this study and are expressed by dotted arrows

lipid from UDP-Gal as well as the formation of GlcNAc-PP- undecaprenol from UDP-GlcNAc [3]. Since both galactose and N-acetylglucosamine were contained in the repeating units of the acidic polysaccharide, either the galactose-linked lipid or N-acetylglucosamine-linked lipid was presumed to be involved in the initiation process of the acidic polysaccharide synthesis. However, the possibility of the involvement of GlcNAc-PP-undecaprenol was excluded, because antibiotic 24010 did not inhibit the in vivo and in vitro synthesis of this polysaccharide. On the contrary, the antibiotic stimulated the incorporation of galactose into glycolipids and polymer, particularly the incorporation observed in the presence of UDP-GlcNAc. This stimulatory effect of the antibiotic suggests that undecaprenyl phosphate serves as a glycosyl acceptor in the formation not only of the N-acetyl- glucosamine-linked lipid but also of the galactose-linked lipid.

The coincidence of the construction of the sugar moieties of the glycolipids, Rha-Gal (sugar A-2), GlcNAc-Rha-Gal (sugar C) and Rha-GlcNAc-Rha-Gal (sugar B), with the partial structures of the repeating unit of the acidic polysaccharide, in combination with the time course of the glycolipid formation and the demonstration of the conversion of lipid-linked ['4C]galactose or ['4C]Rha-Gal to lipid-linked higher oligosaccharides and polysaccharide, leads to the most likely biosynthetic pathway, Scheme 1, in which the repeating units of the acidic polysaccharide are synthesized through the stepwise addition of the sugar residues to Gal-PP-lipid and then joined together to form the polymer.

It is noteworthy that under the standard assay conditions a glycolipid with the sugar moiety, sugar D, presumed to be an sn-glycerol-1-phosphate-linked tetrasaccharide, was pro- duced without supply of any exogenous glycerophosphate donor. Although there was no direct evidence of the presence of the glycerophosphate residues, the possibility of the attach- ment of this residue at the non-reducing terminal rhamnose residue was strongly supported by the conversion of this

tetrasaccharide from a negatively charged, a-rhamnosidase- resistant form to a non-charged, a-rhamnosidase-sensitive form by the treatment with a mixture of phosphodiesterase I1 and acid phosphatase. In addition, the result of a pulse-chase experiment (Fig. 7) suggests that phosphatidylglycerol may serve as a main donor of the glycerophosphate residues. Pre- viously Kennedy and his coworkers have shown that phosphatidylglycerol is a donor of sn-glycerol 1-phosphate residues linked to sugar backbones in the membrane-derived ohgosaccharides of Escherichiu coli [ l l , 121. The same lipid has also been shown to serve as an sn-glycerol 1-phosphate donor in the elongation of polyglycerophosphate backbone chains of lipoteichoic acids [13, 141. The present result in- dicates that in the synthesis of the acidic pnlysaccharide the introduction of the sn-glycerol 1 -phosphate residues most probably takes place at the stage of the formation of the repeating units in the form bound to polyprenylpyrophos- phate. However, there is a possibility that the transfer of the glycerophosphate residues occurs also in the polymer level, as described below.

The polymers synthesized were roughly separated into four fractions by ion-exchange chromatography, showing di- versity in their content of negatively charged groups (Fig. 5). Taking account of chromatographic behavior of the polymers after the treatment with a mixture of phosphodiesterase I1 and acid phosphatase (Fig. 6), lateral glycerophosphate branches seemed to be present in the polymers of fractions 111 and IV. Even after the treatment with the phosphoesterase mixture, these fractions, particularly fraction IV, gave considerable amounts of a polymer which was retained on a DEAE- cellulose column and eluted from it at a low concentration (0.05 M) of NaC1. The above result suggests that fractions I11 and IV contained a component which had, in addition to the glycerophosphate residues, a negatively charged group resistant towards the phosphoesterase treatment. It seems likely that this component was pyrophosphate-linked polysac- charide, which was probably derived from polyprenylpyro- phosphate-linked polysaccharide by the phenol treatment [4]. Moreover, the polymer in fraction I1 (Fig. 5) seemed to have a similar phosphoesterase-resistant acidic group. The attach- ment of lateral glycerophosphate branches to the repeating units may not be absolutely required for the polymerization of these units, because a polymer fraction devoid of net negative charges was also obtained (Fig. 5 , fraction I). However, a possibility of enzymatic removal of the glycerophosphate re- sidues from the polysaccharide during the incubation can not be excluded.

The inability of antibiotic 24010 to inhibit the poly- saccharide synthesis suggests that the polymerization pro-

51 9

ceeds without participation of any linkage-saccharide-linked lipids, such as GlcNAc-PP-lipid [15], ManNAc-GlcNAc-PP- lipid [16], Glc-GlcNAc-PP-lipid [17] and N-acetylmannos- aminuronosyl-N-acetylglucosamine-PP-lipid [18]. Thus, the elongation of the acidic polysaccharide chain in this organism seems to progress either by the transfer of the repeating unit from the glycolipid to a growing polymer chain linked to lipid or by the transfer of a growing polymer chain from a lipid- linked intermediate to the lipid-linked repeating unit, as in the synthesis of 0 antigens of lipopolysaccharides [19].

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