Eur. J. Biochem. 149, 331 -336 (1985) 0 FEBS 1985

Structural studies on the linkage unit between poly(N-acetylglucosamine 1-phosphate) and peptidoglycan in cell walls of Bacilluspumilus AHU 1650 Naoya KOJIMA, Johji IIDA, Yoshio ARAKI and Eiji IT0 Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo

(Received January 3/March 11, 1985) - EJB 85 0001

Structural studies were carried out on the polymer chains and their linkage regions in two kinds of teichoic acids, poly(N-acetylglucosamine 1 -phosphate) [poly(GlcNAc-1-P)] and glycerol teichoic acid, bound to peptidoglycan in the cell walls of Bacillus pumilus AHU 1650. The poly(G1cNAc-1-P)-glycan complex isolated from lysozyme digests of the cell walls contained mannosamine and glycerol as minor components. On the basis of proton NMR spectroscopic data and isolation of N-acetylglucosamine 4-phosphate from acid hydrolysates, the poly(G1cNAc-1-P) was shown to be a polymer in which N-acetylglucosamine 1-phosphate units are joined at C-4 of the glucosamine residues. Mild alkaline hydrolysis of the poly(G1cNAc-1-P)-glycan complex gave a mannosamine-linked glycan fragment and the acidic polymer fraction that contained glycerol residues. Mild acid treatment of the mannosamine-linked glycan fragment gave the linkage disaccharide, ManNAc(p1 + 4)GlcNAc, whereas the acidic polymer fraction was degraded by this treatment into N-acetylglucosamine 4-phosphate and a glycerol-containing fragment characterized as P-(Gro-P), (Gro = glycerol). On the other hand, direct mild acid hydrolysis of the complex gave a fragment characterized as P-(Gro-P),-ManNAc(p1 -+ 4)GlcNAc. These results lead to a conclusion that in the cell walls the poly(G1cNAc-1-P) chain is attached to peptidoglycan through a linkage unit, (Gro-P),-ManNAc(p1 --f 4)GlcNAc. By means of similar procedures, it was shown that the other cell wall polymer, glycerol teichoic acid, is also attached to peptidoglycan through the same disaccharide, ManNAc(pl+ 4)GlcNAc.

The majority of teichoic acids are phosphorylated polymers having glycerol phosphate, glycosylglycerol phosphate, ribitol phosphate, or glycosylribitol phosphate as the repeating units of their backbone chains. In addition, polymers of -6-GlcNAc-1-P-, -6-Glc(crl+ 3)GlcNAc-1 -P-, -6- Glc(cr1 --f 3)GalNAc-1-P- and Gro-P-4-GlcNAc-1-P- have also been reported [l -41 and are classified into a group of teichoic acid. These phosphorylated polymers are believed to be bound to muramic acid residues of peptidoglycan through phosphodiester bonds in cell walls. Baddiley and his coworkers reported that the 1,6-linked poly(G1cNAc-P) of Micrococcus varians [5] as well as the ribitol teichoic acids of Staphylococcus aureus H [6] and Bacillus subtilis W23 [7] is attached to peptidoglycan through a linkage unit, (Gro-P)3- GlcNAc.

However, the glycerol teichoic acids of Bacillus cereus AHU 1030 [8] and a number of other Bacillus strains [9] were shown to be attached to peptidoglycan through a common disaccharide unit, ManNAc@l+ 4)GlcNAc. In addition, the

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

Abbreviations. TA-G, teichoic-acid - glycan complex; ManNAc, N-acetylmannosamine; Gro, glycerol.

Enzymes. Glycerol 3-phosphate dehydrogenase (EC 1.1.1 .8); alkaline phosphatase (EC 3.1.3.1); acid phosphatase (EC 3.1.3.2); lysozyme (EC 3.2.1.1 7); N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28)

ribitol teichoic acids of S. aureus H and 209P [lo, 111, B. subtilis W23 and AHU 1390 [ l l ] and Listeria monocyto- genes EGD [12] were revealed to be attached to peptidoglycan through linkage units comprising ManNAc(p1+4)GlcNAc and a few glycerol phosphate residues. Thus, the disaccharide ManNAc-GlcNAc seems to occur widely as a common linkage saccharide unit for ribitol teichoic acids and glycerol teichoic acids in cell walls of various gram-positive bacteria. On the other hand, a linkage saccharide of another type, Glc(p1 -+ 4)GlcNAc, is present in the poly(galactosylglycero1 phosphate)-glycopeptide complexes obtained from the cell walls of Bacillus coagulans [13, 141.

It therefore seemed likely that similar linkage disaccharide units are also involved in the linkage regions for the hexosamine 1 -phosphate polymers.

MATERIALS AND METHODS

Materials

The methods used for the culture of Bacillus pumilus AHU 1650 (kindly supplied by Dr S. Takao, Hokkaido Uni- versity) and for the isolation and N-acetylation of the cell walls of this strain were the same as those described previously [15]. N-Acetylmuramoyl-L-alanine amidase was prepared from Flavobacterium L-11 enzyme given by Dr S. Kotani (Osaka University) [16]. Sephacryl S-300 and sn-glycerol-3- phosphate dehydrogenase were purchased from Pharmacia

332

Fine Chemicals and Sigma Chemical Co., respectively, and other materials were the same as in previous papers [8, 91.

Preparation of teichoic-acid - glycan complexes

The N-acetylated cell walls of B. pumilus AHU 1650 (450 mg) were treated with N-acetylmuramoyl-L-alanine amidase (500 units) at 37°C for 24 h in 500 ml 10 mM Tris/ HCl buffer (pH 8.2) and further digested for 48 h with lysozyme (5 mg). The polymer fraction, obtained from the digest by dialysis followed by chromatography on Sephadex G-50, was applied on a column of DEAE-cellulose (1.5 x 8 cm) equilibrated with 5 mM Tris/HCl buffer (pH 7.2) containing 0.2 M NaCl. The column was eluted with the same buffer followed by a linear gradient of NaCl from 0.2 - 0.6 M in the same buffer. The phosphorylated polymer fraction (eluted at 0.30-0.38 M NaC1) which contained a relatively large amount of N-acetylhexosmine residues bound through mild-acid-labile linkages was separated from the fraction (0.40 - 0.46 M NaCl) containing a smaller amount of these residues. The former fraction was further chromatographed twice on DEAE-cellulose to remove the component of the latter fraction. The resulting polymer was then subjected to chromatography on a Sephacryl S-300 column (1 x 100 cm). Material containing both hexosamine and phosphorus emerged as a single symmetrical peak and was used as the teichoic-acid - glycan complex I (TA-G-I, 20 mg) after re- chromatography on the same column. From the latter phosphorylated polymer fraction, another teichoic-acid - glycan complex (TA-G-11) was purified by similar procedures. The yield of TA-G-I1 was 120 mg.

Mild alkaline treatment

Teichoic-acid - glycan complexes (TA-G-I and TA-G-11) were first treated with NaBH,, and each of the reduced prod- ucts (12.5 pmol as phosphorus) was treated in 5 ml 0.5 M NaOH at 37°C for 25 min. After passage through a column of Dowex 50 (H'), the products were applied on a Sephacryl S-200 column (1 x 100 cm). Fractions (1 ml) were collected and analyzed for phosphorus and total hexosamine.

Preparation of linkage unit

TA-G-I (24 pmol as phosphorus) was hydrolyzed in 1 ml 10 mM HCl at 100°C for 10 min, and the product was applied on a Sephadex G-25 column (1 x 147 cm, superfine). The fragments excluded from the column were further fractionated by successive chromatography on columns of Sephadex G-50 (1 x 140 cm) and DEAE-cellulose (1 x 2 cm). Material, con- taining phosphorus, glycerol and hexosamine and eluted at 0.2 M NaCl from the DEAE-cellulose column, was pooled, dialyzed and used as the linkage unit preparation.

Analytical methods

Unless otherwise indicated, analytical methods were the same as those described in the previous papers [8, 91. Total hexosamine was determined by the method of Tsuji et al. [17] after N-deacetylation of samples by hydrolysis (2 M HCl, 100°C, 2 h) with glucosamine as a standard; phosphorus by the method of Lowry et al. [18]; N-acetylhexosamine by the modified Morgan-Elson method [ 191; formaldehyde with chromotropic acid [20]. Glycerol, hexosamine and hexosaminitol were analyzed by gas-liquid chromatography

Table 1 . Composition of cell walls and teichoic-acid-glycan complexes Contents are expressed relative to dry weight. Each preparation was analyzed for components after hydrolysis in 4 M HCI at 100°C for 4 h. Excess glucosamine is the difference between total amount of glucosamine derivatives and total amount of muramic acid derivatives. Glutamic acid is shown as a representative component of peptide moiety

Component Amount in

Cell walls TA-G-I TA-G-I1

nmoi . mg-'

Muramic acid 300 Muramic acid 6-phosphate 39 Glucosamine 440 Glucosamine phosphate 290 Excess glucosamine 390 Mannosamine 39 Glycerol 1350 Phosphorus 1880 Glutamic acid 290

140 48

890 2300 3000 48

260 3200

50

130 73

380 60

240 82

5060 5200

55

after hydrolysis of samples in 2 M HCl at 100°C for 2 h followed by alkaline-phosphatase treatment, N-acetylation and trimethylsilylation [lo]. Amino sugar and its phosphorylated derivative were analyzed on an amino acid autoanalyzer as described previously [21]. NaIO, oxidation and Smith degradation were performed under conditions similar to those described previously [lo]. 'H-NMR analysis of TA-G-I and 13C-NMR analysis of TA-G-11, respectively, were carried out on Jeol FX-500 and FX-125 spectrometers at 25 "C as described previously [9]. Chemical shifts were given relative to internal standards, 3-methylsilylpropane sulfonate (for 'H-NMR) and methanol (for 13C-NMR). Gel chromatography on columns of Sephadex G-25 and G-50 and Sehacryl S-200 and S-300 was carried out in 50mM (NH412C03.

RESULTS

Isolation of teichoic-acid- glycan complexes

The N-acetylated cell wall preparation of Bacillus pumilus AHU 1650 contained large amounts of phosphorus, glycerol and glucosamine, together with peptidoglycan components (Table 1). The polymer fraction, obtained from the N-acet- ylated cell walls by digestion with N-acetylmuramoyl-L- alanine amidase and lysozyme followed by chromatography on Sephadex G-50, was roughly separated into two major phosphorylated polymer fractions (Fig. 1, fractions I and 11) by chromatography on a DEAE-cellulose column. The polymers of fractions I and I1 were purified by successive chromatography on columns of DEAE-cellulose and Sephacryl S-300 respectively, giving final preparations of teichoic-acid - glycan complexes I (TA-G-I) and I1 (TA-G-11). The molecular masses of TA-G-I and TA-G-I1 were about 30 kDa and 25 kDa, respectively, as estimated by chromatography on a Sephacryl S-300 column with dextrans as references.

The acid hydrolysate of TA-G-I gave large amounts of glucosamine and glucosamine phosphate together with small amounts of mannosamine, glycerol, muramic acid 6-phos- phate and muramic acid, as analyzed mainly by an amino acid

333

I I II -- I 10

0.6

0.4 2 0.2

5

0 0.0 = 0 20 40 60 80

Fraction number

Fig. 1. Separation of teichoic-acid-glycan complexes by DEAE- cellulose column chromatography. The polymer fraction, obtained after digestion of N-acetylated cell walls (450 mg) with N-acetyl- muramoyl-L-alanine amidase and lysozyme, was applied on a DEAE- cellulose column (1.5 x 8 cm). Fractions ( 5 ml) were collected and analyzed for phosphorus (0). The elution of poly(G1cNAc-1-P) was monitored by measuring N-acetylhexosamine by the modified Morgan-Elson reaction [I91 after mild acid hydrolysis (50 mM HCl, I O O T , 10 min) of samples (0). Fractions indicated by bars I and I1 were separately pooled, rechromatographed on DEAE-cellulose, and used as TA-G-I and TA-G-11, respectively, after further purifcation by gel chromatography

analyzer and gas-liquid chromatography (Table 1). From the analytical data, the teichoic acid moiety of TA-G-I seems to be a polymer composed of glucosamine and phosphorus in equimolar amounts. On the other hand, the TA-G-I1 prepara- tion contained nearly equimolar amounts of phosphorus and glycerol as the major components, together with small amounts of mannosamine, muramic acid 6-phosphate, muramic acid and glucosamine. Thus, the cell walls of this strain seem to have glycerol teichoic acid and poly(G1cNAc-P) attached to peptidoglycan. The acid hydrolysate of either complex gave mannosamine in an amount nearly equimolar to muramic acid 6-phosphate, indicating that the mannosamine residues are probably involved in the linkage region of each of these phosphorylated polymers.

Characterization of teichoic acid moieties

The poly(G1cNAc-P) obtained from B. pumilus AHU 1650 was shown to differ from 1,6-1inked poly(G1cNAc-P) of Micrococcus varians on the basis of the following evidence. The NaIO, oxidation of TA-G-I led to only a negligible loss of the N-acetylglucosamine residues, suggesting substitution of these residues at either C-3 or C-4. Proton NMR spectrum given by TA-G-I exhibited peaks at 5.493 (1 H), 4.170 (1 H), 3.960 (2 H), 3.887 (1 H), 3.808 (1 H), 3.643 (1 H) and 2.105 ppm (3 H). The signal at 5.493 ppm ( J = 2.8 Hz) is as- signed to anomeric protons of a-glycosidically linked N-acetylglucosamine, though it was found in a lower-field region as compared with that of a-N-acetylglucosaminide re- ported previously [22]. This downfield-shift may be explained by a deshielding effect of phosphoryl substitution at C-1 of the saccharide residues. In addition, the signal of H-4 (6 = 4.170 ppm) was also found in a lower-field region, prob- ably by deshielding effect of phosphoryl substitution at C-4, whereas differences in chemical shifts of other protons were small or negligible. Thus, the N-acetylglucosamine residues in the teichoic acid moiety of TA-G-I seem to be linked to phosphoryl groups at C-4 and a-glycosidically at C-1.

When the TA-G-I preparation was treated in 10 mM HCl at 100°C for 20 min, the acidic polymer chain was completely

Vi yo Fraction I

1

- 60

Fraction number

Fig. 2. Chromatography of teichoic-acid-glycan complex after treat- ment with mild alkali. TA-G-I was treated with 0.5 M NaOH at 37°C for 25 min, and the product was applied on a Sephacryl S-200 column. Fractions (1 ml) were collected and analyzed for phosphorus (0) and total hexosamine (0). Pooled fraction are indicated by bars

degraded into a phosphorylated reducing monosaccharide, which was shown to be N-acetylglucosamine 4-phosphate as follows. The strong acid hydrolysis of this saccharide gave glucosamine and glucosamine phosphate, whereas alkaline- phosphatase treatment gave equimolar amounts of N-acetyl- glucosamine and inorganic phosphate. The NaIO, oxidation of a reduced sample of this saccharide yielded formaldehyde in an amount equimolar to phosphorus, indicating unsubstitution at C-6. The Smith degradation of the same reduced sample, followed by enzymatic dephosphorylation of the product, gave N-acetylxylosaminitol, but not N-acetyl- threosaminitol.

The above results together with the data on proton NMR spectroscopic analysis lead to a conclusion that the teichoic acid moiety of TA-G-I consisted of a-N-acetylglucosamine 1-phosphate residues which were joined by phosphodiester bonds at C-4 of the saccharide residues.

On the other hand, the teichoic acid moiety of TA-G-I1 seems to be a 1,3-1inked poly(glycero1 phosphate) on the basis of the analytical data, 13C-NMR spectrum [67.46 pppm, 2 C (d); 70.79 ppm, 1 C (t)] and isolation of glycerol phosphates and glycerol diphosphates from partial acid hydrolysates of TA-G-I1 [23].

Preparation and characterization of linkage-saccharide -glycan fragment

To study the structure of the linkage region between the polymer and the glycan moiety, the TA-G-I preparation was first reduced with NaBH, and then subjected to mild alkaline hydrolysis (0.5 M NaOH, 37"C, 25 min). As shown in Fig. 2, the product was separated by chromatography on a column of Sephacryl S-200 into a major, acidic polymer fraction (frac- tion 1) and a minor fraction (fraction 2) which contained almost all of the mannosamine residues and glycan components recoverd. The mannosamine-containing frag- ment in fraction 2 was used as the disaccharide-linked glycan fragment after purification by chromatography on DEAE- cellulose. The acid hydrolysate of this material gave muramic acid 6-phosphate, mannosamine and excess glucosamine (ex- cess amount of glucosamine over the total amount of muramic acid derivatives) in a molar ratio of 1 .O : 0.95 : 1.3.

When the disaccharide-linked glycan fragment was sub- jected to the treatment with mild acid (10 mM HCl, 100°C, 30 min) followed by chromatography on a Sephadex G-25

334

I

Fraction number

Fig. 3. Chromatography of a disaccharide linked to a glycan fragment after treatment with mild acid. The disaccharide-linked glycan frag- ment (Fig. 2, peak 2; 0.2 pmol as phosphorus) was hydrolyzed in 1 ml 10 mM HCI at 100°C for 30 min and applied on a Sephadex G-25 column. Fractions (1 ml) were collected and analyzed for phosphorus (0) and total hexosamine (0). Arrows 1 , 2, 3 and 4 indicate the elution positions of monomer, dimer, trimer and tetramer of N-acetyl- glucosamine, respectively. Fractions indicated by bars were pooled

column (Fig. 3), most mannosamine residues were recovered as a component of a disaccharide (peak 2), which was separated from the glycan fragment (peak 1). However, no appreciable amount of free N-acetylglucosamine was detected in this procedure, suggesting that the linkage unit of the N-ace- tylglucosamine type proposed by Heptinstall et al. [5] and Coley et al. [7, 81 was absent from the teichoic-acid-glycan complex of B. pumilus AHU 1650. In addition, acid- phosphatase treatment of the glycan fragment in peak 1 re- sulted in liberation of the phosphoryl groups with loss of muramic acid 6-phosphate, whereas the same treatment had no influence on the original disaccharide-linked glycan prepa- ration. Thus, it seems that the disaccharide moiety of the disaccharide-linked glycan fragment was linked through a phosphodiester bridge to C-6 of a muramic acid residue of the glycan fragment.

The mannosamine-containing disaccharide (peak 2) was shown to be identical with the previously reported linkage disaccharide, ManNAc(B1 --f 4)GlcNAc [8 - 121, on the basis of the data of analysis involving the measurement of composi- tion (before and after reduction with NaBH,), the chromic anhydride oxidation, the Smith degradation, the Morgan- Elson reaction and paper chromatographic comparison with the standard samples.

A disaccharide-linked glycan fragment was also isolated from the alkaline hydrolysate of TA-G-I1 in an identical proce- dure as the TA-G-I hydrolysate. The fragment gave the same disaccharide, ManNAc(fll-+ 4)GlcNAc, after mild acid hy- drolysis. Therefore, either poly(G1cNAc-1-P) in TA-G-I or poly(glycero1 phosphate) in TA-G-I1 seems to be linked to the glycan moiety through the disaccharide, ManNAc- (P l+ 4)GlcNAc.

Characterization of oligo(glycero1 phosphate) isolated,from the poly(G1cNAc-I-P) fraction

The major portion (60%) of the glycerol residues of TA- G-I was recovered in the acidic polymer fraction (Fig. 2, fraction 1) obtained from the mild alkaline hydrolysis of TA- G-I. When the polymer in this fraction was subjected to hy- drolysis in 10 mM HCl at 100°C for 10 min followed by chromatography on a column of Sephadex G-50 (Fig. 4), most phosphorus was recovered as N-acetylglucosamine 4-phos-

60 70 80 90 100 2 LL

Fraction number

Fig. 4. Separation of oligo (glycerol phosphate) ,from mild acid hydrolysates of acidic polymer fraction. The polymer fraction (Fig. 2, peak l) , obtained from mild alkaline hydrolysis of TA-G-I, was fur- ther treated in 10 mM HCI at 100°C for 10 min and applied on a Sephadex G-50 column. Fractions (1 ml) were collected and analyzed for phosphorus (0) and total hexosamine (0) . Arrows 1, 2 and 3 indicate the elution positions of standards, glucose, a decamer of glucose and dextran T-5, respectively. Fractions indicated by bars were pooled

phate (peak 3) and components of peak 2 tentatively characterized as oligo(GlcNAc-1 -P). However, the majority (79%) of the glycerol residues together with about 5% of phosphorus were recovered in peak 1 which appeared near the position of standard dextran T-5. The phosphorus-containing material in peak 1 had an obviously greater molecular mass than that of (Gro-P)3 obtained from the Smith degradation of the ribitol - teichoic-acid - glycopeptide complex of Bacillus subtilis W23 [ l l ] and gave a single peak of phosphorus in chromatography either on a DEAE-cellulose column or on a Sephadex G-50 column. The purified material was composed of glycerol and phosphorus in an approximate molar ratio of 1 : 1.14. The NaI04 oxidation had no appreciable effect on this compound. The alkaline-phosphatase treatment of this material led to liberation of a quarter of the phosphorus residues, suggesting the presence of two terminal phosphoryl groups. The NaIO, oxidation of the resulting dephosphoryla- tion product gave formaldehyde in an amount equimolar to that of the phosphoryl groups liberated. These results lead to the most likely structure for this compound, P-(Gro-P)7. However, the number of the repeating glycerol phosphate units in this compound is ambiguious and probably in the range between 6 and 8.

The above results suggest that the oligomer of glycerol phosphate (Fig. 4, peak 1) together with the disaccharide ManNAc(pl+ 4)GlcNAc (Fig. 3, peak 2) constitutes the linkage unit between poly(G1cNAc-1-P) and peptidoglycan in the cell walls of this strain.

Isolation and characterization of oligo (glycerol phosphate) -linked disaccharide

An attempt to obtain a teichoic-acid-bound linkage saccharide by heating the cell walls at pH 2.5 as reported previously [9 - 121 was unsuccessful because of acid-lability of the poly(G1cNAc-1-P) chains. Thus, we tried to obtain the presumptive linkage unit by the hydrolysis of TA-G-I in 10 mM HCl at 100°C for 10 min, and fragments containing most of the glycerol, mannosamine and glycan components were separated from the major fragment, N-acetyl- glucosamine 4-phosphate, by passage through a Sephadex G- 25 column and then fractionated by chromatography on a

335

Table 2. Analysis of linkage unit and phosphorus-containing fragment released from linkage unit by mild alkaline hydrolysis The linkage unit was purified from phosphorus-containing material (Fig. 5B, peak 2) resulting from the mild acid hydrolysis of TA-G-I and analyzed after strong acid hydrolysis. A phosphorus-containing fragment (Fig. 6, peak I), released from the linkage unit by mild alkaline hydrolysis, was also analyzed. Glycerol was assayed by the enzymatic method [24]. Analytical data for enzymatic dephosphorylation products of both fragments were also shown. Data are expressed in molar ratios to glycerol taken as 7

Component Relative amount in

linkage unit phosphorus-containing fragment

before after before after dephosphorylation dephosphorylation dephosphorylation dephosphorylation

Glucosamine 1.03 Mannosdmine 1.04 Phosphorus 8.21 Glycerol 7.00 Phosphatase-sensitive phosphoryl group 1.14 HCHO formed in NaIO, oxidation 0

0.96 0.98 7.18 7.00

1.05 -

0 0 7.89 7.00 1.91 0

0 0 6.03 7.00

1.93 -

200 I p l A

Fraction number

Fig. 5. Separation of linkage unit from mild acid hydrolysates of TA- G-I. (A) TA-G-I (7.5 mg) was hydrolyzed in 1 ml HC1 at 100°C for 10 min. Material excluded from Sephadex G-25 was then applied on a Sephadex G-50 column. Symbols are the same as in Fig. 4. Arrows 1, 2, 3 and 4 indicate the elution positions of standards, glucose, a decamer of glucose, (Gr0-P)~-ManNAc(~l+4)GlcNAc [ l l ] and dextran T-5. Fractions indicated by bars were pooled. (B) Material in the pooled fractions (fractions 62 - 70 in Fig. 5A) was applied on a DEAE-cellulose column (1 x 2 cm) equilibrated with 5 mM (NH&CO3. The column was eluted with a linear gradient of NaCl from 0-0.3 M. Fractions under bars were pooled and material in peak 2 was used as the linkage unit preparation after purification by rechromatography on a DEAE-cellulose column

Sephadex G-50 column, giving three peaks of phosphorus- containing material (Fig. 5A). The majority of the mannosamine and glycerol residues were recovered in the fraction eluted near the position of standard dextran T-5, and this fraction was further separated into three peaks of phosphorus-containing material by chromatography on a DEAE-cellulose column (Fig. 5B). The material of peak 2, in which about 60% of the mannosamine and glycerol residues of the starting TA-G-I preparation were recovered, was fur- ther purified by rechromatography on a DEAE-cellulose column and used as the linkage unit preparation. This prepa-

- c 2 E ._ e 40 5 B 6 20

-

.c 'D

VI 2 n r

40 50 60 70 00 90 E o

Fraction number

Fig. 6. Chromatography of linkage unit after mild alkaline treatment. The linkage unit preparation (2 pmol as phosphorus) was treated with 0.5 M NaOH at 37°C for 25 min, and the product was applied on a Sephadex G-50 column. Symbols and arrows are the same as in Fig. 4. Fractions indicated by bars were pooled

ration contained mannosamine, glucosamine, glycerol and phosphorus in an approximate molar ratio of 1 : 1 : 7 : 8 (Table 2). The result of analysis of this compound after reduc- tion with NaBH4 indicated that the glucosamine residue was at the reducing end. The alkaline-phosphatase treatment re- sulted in liberation of one mole of phosphorus per mole of mannosamine residue. In addition, the glucosamine residue was converted into N-acetylxylosaminitol by Smith degrada- tion of the reduced sample of this preparation, whereas the mannosamine residue was unaffected by this procedure. The linkage unit preparation gave ManNAc(fl1 --f 4)GlcNAc (peak 2) and phosphorus-containing material (peak 1) by mild alkaline hydrolysis followed by chromatography on a Sephadex G-50 column (Fig. 6). The analytical data in Table 2 show that the phosphorus-containing material (peak 1) was probably P-(Gro-P)7 and identical with the oligo(glycero1 phosphate) resulting from mild acid treatment of the acidic polymer fraction (Fig. 4, peak 1). The above data are consis- tent with P-(Gro-P),-ManNAc(pl+ 4)GlcNAc for the structure of the linkage unit preparation.

DISCUSSION The results described above lead to a conclusion that in

the cell walls of Bacillus pumilus AHU 1650, the 1,Clinked N-acetylglucosamine phosphate polymer [poly(GlcNAc- 1 -P)] chain is linked to peptidoglycan through a linkage unit,

336

(Gro-P),-ManNAc(pl + 4)GlcNAc. The oligo(glycero1 phos- phate) moiety of this linkage unit is probably bound at its one end to C-3 or C-4 of the N-acetylmannosamine residue through an alkali-labile phosphodiester bond. The binding of this linkage unit to peptidoglycan at the disaccharide moiety was demonstrated by the separation of the disaccharide-linked glycan fragment from the mild alkaline hydrolysates of TA- G-I (Fig. 2, fraction 2) , and its binding to the poly(G1cNAc- 1-P) chain at the oligo(glycero1 phosphate) moiety was in- dicated by the separation of the oligo(glycero1 phosphate)- linked acidic polymer (Fig. 2, fraction 1).

The linkage unit for poly(G1cNAc-1-P) of B. pumilus is characteristic in having an anomalously great number of glyc- erol phosphate residues. The average number of glycerol phosphate residues in this unit, about 7, was calculated from the analytical data (Table 2). On the other hand, the cell walls of this strain were shown to have teichoic acid of another type, 1,3-1inked poly(glycero1 phosphate), which is also attached to peptidoglycan through the same linkage disaccharide, ManNAc(p1 + 4)GlcNAc. As judged the contents of glycerol and excess glucosamine in the cell walls (Table l), the glycerol teichoic acid seems to be the predominant acidic polymer component of the cell walls, and the isolation of the poly(G1cNAc-1-P)-glycan complex from cell wall digests re- quired chromatography repeated on columns of DEAE- cellulose (three times) and Sephacryl S-300 (twice). In view of the similarity of the two acidic polymer preparations in the molecular masses (TA-G-I, 30 kDa; TA-G-11, 25 kDa) and ionic properties, the TA-G-I preparation might be contaminated by a small amount of material linked to glyc- erol - teichoic-acid. Therefore, it is possible that the great value of the molar ratio of glycerol to mannosamine in the oligo(glycero1 phosphate)-disaccharide preparation (Table 2) is at least partly due to contaminating material linked to glycerol-containing fragments derived from the glycerol - teichoic-acid. However, this possibility seems to be excluded on the basis of the following evidence. The majority of the glycerol and mannosamine residues in TA-G-I were recovered in the oligo(glycero1 phosphate)-disaccharide fraction, which appeared in gel chromatography as a distinct peak at the position corresponding to a much greater molecule than stan- dard (Gro-P)3-ManNAc(pl -P 4)GlcNAc obtained from Bacillus subtilis W23 [l 11 (Fig. 5A). The oligo(glycero1 phosphate)-disaccharide preparation did not contain fragments with periodate-sensitive terminal glycerol residues, which are expected to be formed in the acid hydrolysis of 1,3- linked poly(glycero1 phosphate) chains (Table 2), but it had phosphatase-sensitive terminal phosphoryl groups, which seem to have been derived from the proximal terminal N-ace- tylglucosamine 1-phosphate units of the poly(G1cNAc-1-P) chains.

The linkage units for teichoic acids so far studied contain only a few glycerol phosphate residues. For example, the ribitol teichoic acids of Lactobacillus plantarum AHU 1413 (unpublished data), B. subtilis W23 [I I] and Staphylococcus aureus [lo, 111 contain one, two and three glycerol phosphate residues, respectively, in their linkage units. Recently, a unique saccharide, glucosyl(p1 + 3)glucosyl(p1 + 1/3)glycerophos- phate was shown to be present between ribitol teichoic acid and the linkage saccharide ManNAc(pl-+ 4)GlcNAc in the

cell walls of Listeria monocytogenes EGD [12]. The result of the present study together with the previous data described above suggests that the molecular sizes and constitution of the parts containing glycerol phosphate units may differ diversely in various bacterial species, whereas the linkage dis- accharide parts seem to be rather uniform.

This work was partly supported by a Scientific Research grant from the Ministry of Education, Science and Culture of Japan.

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