Eur. J. Biochem. 1.55, 521 -526 (1986) cj FEBS 1986

Structural studies on the minor teichoic acid of BaciZZus coagulans AHU 1631 Naoya KOJIMA, Ken-ichi UCHIKAWA, Yoshio ARAKI and Eiji IT0 Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo

(Received September 30/December 13, 1985) - EJB 851079

The minor teichoic acid linked to glycopeptide was isolated from lysozyme digests of Bacillus coagulans AHU 1631 cell walls, and the structure of the teichoic acid moiety and its junction with the peptidoglycan were studied. Hydrolysis of the teichoic-acid - glycopeptide complex with hydrogen fluoride gave a nonreducing oligosaccharide composed of glucose, galactose and glycerol in a molar ratio of 3: 1 : 1 which was presumed to be dephosphorylaled repeating units of the polymer chain. From the results of structural analysis involving NaI04 oxidation, methyla- tion and acetolysis, the above fragment was characterized as glucosyl(j? 1 + 3)glucosyl([j 1 +6)gdlactosyi- (j? 1 +6)glucosyl(al+1/3)glycerol. In addition, the Smith degradation of the complex yielded a phosphorus- containing fragment identified as glycerol-P-6-glucosyl(~ 1 + 1/3)glycerol. These results led to the most likely structure for the repeating units of the teichoic acid, -6[glucosyl(/3 1 +3)]glucosyl(,h’1+6)galactosyl(,h’ 1 -6)glu- cosyl(a1 -+ 1/3)glycerol-P-.

The minor teichoic acid, just like the major teichoic acid bound to the linkage unit, was released by heating the cell walls at pH 2.5. The mild alkaline hydrolysis of the minor teichoic acid after reduction with NaB3H4 gave labeled saccharides characterized as glucosyl(j? 1 +6)galactitol and glucosyl(j? 1 +3)glucosyl(B 1 +6)galactitol, together with a large amount of the unlabeled repeating units of the teichoic acid chain. Thus, the minor teichoic acid chain is believed to be directly linked to peptidoglycan at the galactose residue of the terminal repeating unit without a special linkage sugar unit.

The cell wall teichoic acids seem to be constructed of two chemically different parts, main chains and linkage units which join the polymer chains to the peptidoglycan [I - 111. In a previous paper [12] we reported that the cell walls of Bacillus cougulans AHU 1631 possess two kinds of teichoic acids. The major teichoic acid made up of repeating -6Gal(al+2)[Glc(~1+1/3)]Cro-P- units was shown to be bound to peptidoglycan through a linkage unit, (Gro-&- Glc(,h’1+4)GlcNAc. On the basis of preliminary analytical data, the other minor teichoic acid seemed to have more complicated repeating units composed of glucose, galactose and glycerol in a molar ratio of 3 : 1 : 1, This situation provided us with an opportunity to compare modes of junction to peptidoglycan between different kinds of teichoic acids in the cell walls of a single strain. Furthermore, the results will provide us with useful information on the biosynthetic pathways for different types of teichoic acids.

The present paper describes the results of structural studies on the minor wail teichoic acid and its junction to peptidoglycan.

MATERIALS AND METHODS

Preparation of teichoic-acid -glycopeptide complexes and teichoic-acid-linked sugars

A minor teichoic-acid - glycopeptide complex (denoted as TA-GP-0 in a previous paper [12]) and major ones (TA-GP-I

Correspondence 20 E. Ito, Department of Chemistry, Faculty of Science, Hokkaido University, Kita-10-jyo, Nishi-8-chome, Kita-ku, Sapporo, Japan 060

Abbreviations. ManNAc, N-acetyl-D-mannosamine; GlcNAc, N- acetyl-D-glucosamine ; TA-GP, teichoic-acid - glycopeptide complex ; TA-S, teichoic-acid-linked sugar; P, phosphate; Gro, glycerol

and TA-GP-11) were isolated from lysozyme digests of the N- acetylated cell walls (500 mg) of Bacillus coagulans AHU 1631 by successive chromatography on columns of DEAE- cellulose, Sephacryl S-200 and DEAE-cellulose, as described previously [12]. The yield of TA-GP-0 was 26.5 mg. For the isolation of teichoic-acid-linked sugars, the cell walls (200 mg) were heated in 40 ml 25 mM glycine/HCl buffer (pH 2.5) at 100°C for 10 min, cooled and centrifuged at 20000 x g . The precipitate was treated twice more in the same way. After dialysis, the pooled supernatant was applied on a column of DEAE-cellulose (1.5 x 3 cm) equilibrated with 5 mM Tris/ HCl buffer (pH 7.2), and the column was eluted with a linear gradient of 0-0.4 M NaCl in the same buffer, as described previously [12]. Two acidic polymer fractions, eluted at NaCl concentrations of 0.15 M and 0.25 M, were respectively purified by chromatography of a Sephacryl S-300 column (1 x 100 cm) and used as teichoic-acid-linked sugars A (TA-S- A, 18 mg) and B (TA-S-B, 56 mg).

Preparation of dephosphorylated repeating units The TA-GP-0 preparation (2 mg) was treated in 0.2 ml

47% hydrogen fluoride (HF) at 25 “C for 16 h. After removal of H F by evaporation in an air flash and of anionic material by passage through Dowex 2 (acetate form), the product was subjected to chromatography on a Cellulofine GCL-25 m column (1 x 147 cm) in 50 mM (NH&C03. Fractions which contained hexose and were eluted near the position of stan- dard maltopentaose, were pooled and used as the dephos- phorylated repeating unit preparation (denoted as com- pound 1) after purification by rechromatography on the same column.

Mild alkaline hydrolysis of reduced teichoic-acid-linked sugar The TA-S-A preparation (5 mg) was treated first with

0.4 mg NaB3H4 (13.1 mCi/mg) in borate buffer (pH 9.5) at

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4°C for 16 h then with unlabeled NaBH,. After desalting by dialysis, the reduced TA-S-A preparation was hydrolyzed in 1 ml 0.5 M NaOH at 37 'C for 24 h. The resulting product was passed through a Dowex 50 (H' form) column and subjected to chromatography on a column of Cellulofine GCL-25m (1 x 147 cm) in 50 mM (NH4)2C03. Fractions (1 ml) were collected and analyzed for hexose, phosphorus and radioactivity.

Acetolwis and other analytical methods

The acetolysis of the dephosphorylated repeating units was performed by the method of Dickey and Wolfrom [13] and the product was fractionated by chromatography on Cellulofine GCL-25 m. Descending paper chromatography was carried out on Toyo no.50 filter paper in 1-butanol/ pyridine/water (6 : 4: 3, by vol.). Paper electrophoresis was performed in 50 mM borate buffer (pH 9.9). Unless otherwise indicated, other analytical methods and materials were the same as those described in the previous paper [12].

Treatment of cells with antibiotic 24010

B. coagulans AHU 1631 cells were grown in 2.5 1 of a broth medium, and antibiotic 24010 (0.5 pg/ml or 1.0 pg/mg) was added at the early log phase. After incubation for 2 h, the cells were collected, and cell walls were prepared as described previously [ll]. The yields of cell walls were 47.6 mg and 44.6 mg for the cells treated with the antibiotic at concentra- tions of 0.5 pg/ml and 1 .O pg/ml, respectively, as compared to 54.1 mg for the untreated cells.

RESULTS

Coniposition of minor teiclzoic-acid- gl-vcopeptide complex

As described previously [12], three teichoic-acid - glyco- peptide complexes (TA-GP-0, TA-GP-I and TA-GP-11) were separated from lysozyme digests of the cell walls of Bacillus coagulans AHU 1631 by successive chromatography on columns of DEAE-cellulose and Sephacryl S-300. The major components, TA-GP-I and TA-GP-11, have been shown to have the same teichoic acid chains composed of repeating -6Gal(al--+2)Gro-P- units with p-glucosyl branches linked to C-I or C-3 of the glycerol residues. The minor component, TA-GP-0, contained glucose, galactose, glycerol and phosphorus in amounts of 3.20, 1.29, 1.35 and 1.44 lmol/ mg dry weight, respectively, together with small amounts of muramic acid 6-phosphate (21 nmol/mg) and other glycopeptide components. On the basis of its high glucose content, the minor complex (TA-GP-0) seemed to have teichoic acid chains different from those of TA-GP-I and TA- GP-11. The apparent molecular mass of TA-GP-0, 65 kDa, was calculated from the molar ratio of the teichoic acid components to muramic acid 6-phosphate on the assumption that each polymer chain was bound to C-6 of muramic acid in the glycopeptide moiety through a phosphodiester bond.

Structure of teichoic acid moiety of TA-GP-0

The hydrolysis of TA-GP-0 with 47% HF followed by chromatography on Cellulofine GCL-25m gave a single peak of hexose-containing material, which was eluted near the posi- tion of standard maltopentaose and used as the dephospho- rylated repeating unit preparation (denoted as compound 1)

Table 1 . Composition of the dephosphorylated reputing unit und its ucetolysis products The dephosphorylated repeating unit (compound 1 ) was isolated from HF hydrolysates of TA-GP-0 by chromatography on Cellulofine GCL-25m and analyzed for components after acid hydrolysis (1 M HCI, 100"C, 2 h). The acetolysis products of compound 1 were fractionated by chromatography on the same column as shown in Fig.l and the materials in peaks 1 and 2 was purified by paper chromatography followed by gel chromatography. Resulting frag- ments 1 and 2 or their reduction products were analyzed after acid hydrolysis. Data are expressed in molar ratios to glucose

Component Molar ratio in

com- frag- fragment 1 frag- pound 1 ment 1 after re- ment 2

duction with NaBH4

Glucose 1.00 1 .oo 1 .00 1 .00 Glucitol 0 0 0.95 0 Galactose 0.32 0 0 0 Glycerol 0.34 0 0 0.97 HCHO

formation in NaIO, oxidation 0.29 0 1.87 1 .OO

of the teichoic acid chain. The yield of compound 1 was 4.5 pmol (as hexose)/mg of TA-GP-0. In this process, only a negligible amount of free N-acetylglucosamine or hexos- amine-containing material was given, suggesting the absence of a typical linkage unit.

Compound 1 contained glucose, galactose and glycerol in a molar ratio of 3: 1 : 1 (Table 1). The NaI04 oxidation of compound 1 gave 1 mol formaldehyde/mol glycerol residue, indicating that the terminal glycerol residue of compound 1 was substituted at C-1 or C-3. The acid hydrolysate of permethylated compound 1 gave 2,3,4,6-tet ra-0-methylgluci- to1 acetate, 2,4,6-tri-O-methylglucitol acetate, 2,3,4-tri-0- methylglucitol acetate and 2,3,4-tri-O-methylgalactitol ace- tate in a molar ratio of 1 .O : 1.03 : 0.85 : 0.93, as analyzed by gas-liquid chromatography after conversion to alditol acetates. This result indicates that in compound 1 a linear tetrasaccharide unit, comprising one residue each of non- reducing terminal glucose, 3-substituted glucose, 6-substi- tuted glucose and 6-substituted galactose residues, was linked to a glycerol residue at its reducing end.

On the other hand, the NMR spectrum given by TA-GP-0 exhibited signals corresponding to four anomeric protons (6 = 5.53 ppm, J = 3.71 Hz; 6 = 4.60 ppm; J = 6.85 Hz; 6 = 4.51 ppm, J = 7.73 Hz; 6 = 4.43 ppm, J = 7.53 Hz), indicating the presence of three a-glycosidically linked hexosyl residues and one a-glycosidically linked hexosyl residue. Moreover, the chromic anhydride oxidation of compound 1 led to degradation of the galactose residue and two-thirds of the glucose residues. The above results suggest that two p- glucosyl residues and one each of a-glucosyl and p-galactosyl residues were present in the repeating unit of the teichoic acid chain.

The acetolysis of compound 1 followed by chroma- tography on Cellulofine GCL-25 m gave three hexose- containing fragments (peaks 1, 2 and 3) and free glycerol (peak 4) (Fig. 1). Galactose residues were for the most part recovered in peak 3 as free galactose together with a small amount of free glucose. Materials in peaks 1 and 2 which

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g '1 5 1 01

Fraction number

Fig. 1. Separation qjucetolysis products qf compound I by gel chroma- tography. The dephosphorylated repeating unit preparation (com- pound 1, 4.1 pmol) was subjected to acetolysis and applied on a Cellulofine GCL-25m column. Fractions (1 ml) were collected and assayed for hexose (0), reducing groups (0 ) and glycerol (A). Glyc- erol was analyzed after acid hydrolysis (1 M HCI, lOO"C, 1 h) of samples. Fractions indicated by bars were pooled. Materials in peaks 1 and 2 were purified by paper chromatography and used as the fragments 1 and 2, respectively. Arrows 1,2, 3 and 4 indicate the elution positions of glycerol and the monomer, dimer and trimer of glucose, respectively

contained the majority of the glucose residues were purified by paper chromatography followed by chromatography on Cellulofine GCL-25 m, giving fragments 1 and 2, respectively.

Fragment 1 was a reducing disaccharide composed of glucose alone (Table 1) and identified to be Glc(p1+3)Glc. The NaI04 oxidation of this fragment after reduction with NaBH4 gave 2 mol of formaldehyde/mol, indicating nonsub- stitution of the reducing terminal glucose residue at C-2 and C-6. The methylation product of this saccharide gave 2,4,6- tri-0-methylglucitol acetate and 2,3,4,6-tetra-O-methylgluci- to1 acetate in a molar ratio of 0.65: 1.0. The low value for the former derivative may be explained by /?-elimination under the methylation conditions. In addition, the treatment with B- glucosidase led to hydrolysis of the fragment into free glucose. Fragment 2 was a non-reducing saccharide composed of glucose and glycerol in equimolar amounts (Table 1) and was characterized as Glc(cr1 + 1p)Gro by a procedure similar to that described above. Taking account of selective cleavage of glycosidic bonds at C-6 in acetolysis, these results led to the most likely structure for compound 1, Glc(j31+3)Glc(j31+6)- Gal(~1+6)Glc(~l+1/3)Gro.

Smith degradation of TA-GP-O

The NaI04 oxidation of TA-GP-0 resulted in degradation of two-thirds of the glucose residues and most of the galactose residues. After the Smith degradation, TA-GP-0 gave a single peak of phosphorus-containing material (denoted as com- pound 2) on chromatography through Sephadex G-25. Com- pound 2 was a nonreducing sugar composed of glucose, glyc- erol and phosphorus in a molar ratio of about 1 : 2: 1 (Table 2). The HF treatment of this compound yielded free glycerol, inorganic phosphate and Glc(fl1-+1/3)Gro. It gave 2 mol formaldehyde/mol phosphorus in NaI04 oxidation, whereas it was unaffected by alkaline-phosphatase treatment. The

Table 2. Composition of the Smith degradation product, compound 2 A phosphorus-containing product of the Smith degradation of TA- GP-0 was separated by chromatography on Sephadex G-25 and analyzed as compound 2 after acid hydrolysis. Compound 2 was sub- jected to Smith degradation, and the resulting phosphorus-containing product was separated by the same procedure as above and also analyzed. Data are expressed in molar ratios to glycerol taken as 2 (for compound 2) and 1 (for its Smith degradation product)

Component Molar ratio in

compound 2 Smith degradation product from compound 2

Glucose 1.21 0 Glycerol 2.00 1 .oo Phosphorus 1.07 1.20 1,2-Ethylenediol 0 0.96 HCHO formation in NaI04

oxidation 1.97 0.93 Alkaline-phosphatase

sensitive phosphoryl group 0 0

partial acid hydrolysis (1 M HC1, 100°C, 50 min) of com- pound 2 gave glucose 6-phosphate and glycerol phosphates, which were characterized by the same methods as described previously [14]. In addition, the Smith degradation of compound 2 yielded a phosphorus-containing fragment, which was characterized as 1,2-ethylenediol-P-glycerol from the analytical data data (Table 2). These data lead to the most probable structure for compound 2, Gro-P-6-Glc(pl-+ 1/ 3)Gro.

Taking account of the structure proposed for compound 1, the glucose residue in compound 2 seems to arise from the 3-substituted glucose residue of the repeating unit, and the glycerol residue linked to the reducing end seems to be derived from the 6-substituted galactose residue of the same unit. Therefore, the teichoic acid chain of the minor complex was probably composed of the repeating units, -6[Glc(/?1+3)]- Glc(/?l +6)Gal(fil+6)Glc(aI + 1/3)Gro-P-.

Isolation of teichoic-acid-linked sugurs

As described above, the HF hydrolysis of TA-GP-0 gave only a negligible amount of hexosamine-containing frag- ments, suggesting the possibility that in this complex the teichoic acid chain may be directly linked to a muramic acid 6-phosphate residue of the glycan chain without participation of a special structural part such as the glucosamine-containing saccharides reported previously [l - 121. To confirm this possibility, the cell wall preparation was heated at pH 2.5, and the released polymers were isolated and analyzed. About 8O0/" of the phosphorus and hexose residues of the cell walls were recovered in the water-soluble polymers. As shown in Fig. 2, chromatography of the resulting polymer fraction on DEAE- cellulose gave two distinct peaks (peaksA and B) of phosphorus-containing material. The components of these peaks were respectively eluted as single peaks of polymers containing both phosphorus and hexose on chromatography through Sephacryl S-300, giving teichoic-acid-linked sugars A (TA-S-A) and B (TA-S-B). As estimated by chromatography on Sephacryl S-200 and S-300 with dextrans T-20, T-40 and T-70 as reference standards, the apparent molecular masses of TA-S-A an TA-S-B were about 60 and 18 kDa, respectively.

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- L - \ - g 15 =I. - Lo

2 10 a r 0 0.4 a

6 5 - 0.2 - 3 s

v)

- I U

z W I

0 0.0 20 40 60 80

Fraction number

Fig. 2. Separation of teichoic-acid-linked sugars. The cell wall prepara- tion (200 mg) was treated with mild acid as described under Materials and Methods. After dialysis, the resulting water-soluble polymer was applied on a DEAE-cellulose column (1.5 x 6 cm) in 5 mM Tris/HCl buffer (pH 7.2), and the column was eluted with the same buffer and then .with a linear gradient of 0-0.4 M NaCl in the same buffer. Fractions (1 ml) were collected and analyzed for phosphorus (0) and hexose (0). Fractions indicated by bars were pooled and used as TA- S-A and TA-S-B after purification by chromatography on Sephacryl S-300

Table 3. Composition of teichoic-acid-linked sugar Each preparation was analyzed for components after acid hydrolysis. Data for TA-GP-0 and TA-GP-I1 were also as found previously [12]. Muramic acid is shown as a representative component of the glycopcptide moiety

Component Content

Ta-S-A TA-GP-0 TA-S-B TA-GP-I1

nmol/mg dry weight

Phosphorus 1540 1440 2220 2020 Glycerol 1580 1350 2050 1890 Glucose 4370 3200 1750 1660 Galactose 1500 1290 2000 1890 Glucosamine 0 92 102 232 Muramic acid 0 72 0 165 Muramic acid 6-

phosphate 0 21 0 67

The value for TA-S-A was coincident with that of TA-GP-0. The composition of TA-S-A was also in agreement with that of TA-GP-0 except for the absence of the glycopeptide components (Table 3). A similar relationship was shown be- tween TA-S-B and TA-GP-11. Thus, teichoic-acid-linked sug- ars A and B seem t o represent, respectively, the minor and major teichoic acid components released from the cell walls by the cleavage of acid-labile terminal linkages, probably sugar 1 -phosphate bonds.

The TA-S-B preparation contained a small amount of glucosamine residues, which have proved to be a component of the linkage saccharide unit, Glc(/l1+4)GlcNAc [12]. In contrast, the TA-S-A preparation contained no significant amount of glucosamine or other hexosamine. This result sup- ports the possibility that there is no glucosamine-containing linkage saccharide unit between the minor teichoic acid and peptidoglycan. However, there may be another possibility that

I 0.4

0 0.0 20 30 40 50

Fraction number Fig. 3. Elution profire of water-soluble material liberated by mild acid .from cell walls of antibiotic-treated cetls. Cells were incubated in the absence or in the presence of antibiotic 24010 (0.5 pg/ml and 1.0 pg/ ml) for 2 h. Each cell wall preparation (10 mg) was heated at pH 2.5 for 10 min, and resulting water-soluble material was chroma- tographed on DEAE-cellulose as described in Fig.2. (A) Control cell walls; (B) cell walls from cells treated with the antibiotic (0.5 pg/ ml); (C) cell walls from cells treated with the antibiotic (1 .O pg/ml). Symbols are the same as in Fig.2

I 1 - 8 1 L

E 3 3

vo

1

- 40

4 3 2 I

peak1 peak2

50 60 70

I *- 13 ‘s

JO

Fraction number Fig. 4. Separation oxfragments from mild alkaline treatment ojreduced teichoic-acid-linked sugar. The TA-S-A preparation was first reduced with NaB3H4 and then treated with mild alkali as described under Materials and Methods. After desalting, the resulting hydrolysate was applied on a Cellulofine GCL-25m column. Fractions (1 ml) were collected and analyzed for hexose (0), phosphorus (A) and radioac- tivity (0). Fractions indicated by bars were pooled. Arrows 1, 2, 3 and 4 indicate the elution positions of the monomer, dimer, trimer and tetramer of glucose, respectively

a unique component other than hexosamine is present in the linkage unit for this polymer.

Influence of antibiotic 24010 on the in vivo synthesis of’ teichoic acids

To obtain further evidence for the absence of any glucosamine-containing unit from the linkage region, the in- fluence of antibiotic 24010 on the in vivo synthesis of teichoic

525

H+

HO - 70

I I

Actual repeating unit

lactyl peptide

Fig. 5. Most probable structure of the minor teichoic-ucid-glycopeptide complex (TA-GP-0) in cell wulls of B. coagulans A HU 1631. The phosphate bonds labile towards mild acid (H') and mild alkali (OH-) are indicated by vertical lines. About 33% of the proximal terminal repeating units of teichoic acid chains lack the lateral branch glucose residues

acids was examined. This antibiotic belongs to the group of tunicamycin and is known to inhibit the formation of N- acetylglucosaminyl diphosphorylpolyprenol, a key lipid inter- mediate in the synthesis of the linkage units and also in the de novo synthesis of tcichoic acids [15, 161 and a teichuronic acid [17]. The cell wall preparations obtained from the cells treated with the antibiotic at concentrations of 0.5 pg/ml and 1.0 pg/ml showed a small decrease in the contents of total hexose and phosphorus (5 - 30%). On the other hand, the molar ratio of glucose/galactose increased from 1.29 (for the control cells) to 2.23 (for the antibiotic-treated ones). Further- more, when each cell wall preparation was heated at pH 2.5 and the resulting water-soluble polymer fraction was subjected to chromatography on a DEAE-cellulose column (Fig. 3), it was shown that whereas the amounts of hexose and phosphorus recovered in peak B (TA-S-B) were markedly decreased by the treatment with the antibiotic, those recovered in peak A (TA-S-A) were rather increased. Thus, it seems that the minor teichoic acid is synthesized without participation of N-acetylglucosaminyl diphosphorylpolyprenol and that this polymer possesses no glucosamine-containing linkage unit.

Mild alkaline treatment of reduced teichoic-acid-linked sugar

To determine the reducing terminus, the TA-S-A prepara- tion was first reduced with NaB3H4 and then hydrolyzed in 0.5 M NaOH at 37°C for 24 h. The hydrolysate gave a large amount of phosphorus-containing material (peak 1) and very small amounts of hexose-containing materials (peaks 2 and 3) on chromatography through Cellulofine GCL-25m (Fig. 4). The latter fragments (peaks 2 and 3) possessed radioactivity, indicating that both fragments arose from the reducing termini of the teichoic acid chains. Material in peak 1 contained glucose, galactose glycerol and phosphorus in an approximate molar ratio of 3 : 1 : 1 : 1. The phosphoryl group of this fragment was for the most part sensitive to alkaline- phosphatase treatment. The dephosphorylation product was characterized to be Glc(,61-+3)Glc(~1+6)Gal(p1+6)- Glc(ctl-+1/3)Gro by a procedure similar to that described for compound 1. The NaI04 oxidation of material in peak 1 gave a negligible amount of formaldehyde, whereas the oxidation of its dephosphorylation product gave 1 mol of formalde- hyde/mol. Thus, the phosphorus-containing material in peak 1 was Glc(~1-+3)Glc(~1+6)Gal(~1-+6)Glc(crl-+l/ 3)Gro-P, the repeating unit of the teichoic acid chain.

The 3H-labeled fragments in peaks 2 and 3 were respective- ly purified by paper chromatography and subsequent chromatography on Cellulofine GCL-25 m. The smaller radioactive fragment (peak 3) gave glucose and galactitol in equimolar amounts, as analyzed by gas-liquid chromatog- raphy after acid hydrolysis. The hydrolysis of this fragment yielded [3H]galactitol but no other labeled hexitol or hexosaminitol. In addition, this 3H-labeled fragment was shown to be sensitive to B-glucosidase treatment, whereas it was shown to be insensitive to the treatment with other exo- glycosidases, such as a-glucosidase, a- and 1-galactosidases and a- and p-N-acetylglucosaminidases. Thus, the labeled fragment in peak 3 was probably 8-glucosyl-galactitol. The larger radioactive fragment in peak 2 contained glucose and galactitol in an approximate molar ratio of 2: 1. The acid hydrolysis of this fragment gave [3H]galactitol as a labeled product. The Smith degradation of this fragment gave glucose-containing material characterized as glucosyl-l,2- ethylenediol, indicating the presence of 3-substituted glucose and 6-substituted galactitol residues at the inner and the re- ducing terminal positions, respectively. In addition, when the 'H-labeled fragment in peak 2 was digested with ,6- glucosidase for a short period, a radioactive disaccharide iden- tical with the 'H-labeled fragment in peak 3, p-glucosyl- galactitol, was mainly yielded, while prolonged b-glucosidase treatment gave free [3H]galactitol as a labeled product, suggesting that this fragment had the sequence p-glucosyl-,6- glucosyl-galactitol. Methylation analysis was not carried out because of its limited amount. The most likely structure for this fragment (peak 2) is glucosyl(al+ 3)glucosyl- (pl-+6)galactitol, and that for the small galactitol-containing fragment (peak 3 ) is glucosyl(pl+(i)galactitol. This result shows that Glc(/?1+3)Glc(,61+6)Gal were present at the re- ducing termini of the teichoic acid chains released from the cell walls by the mild acid treatment.

DISCUSSION

The results described above show that the minor teichoic acid component in the cell walls of Bacillus coagulans AHU 163 1 possesses apparent repeating saccharide units, Glc- (B1-+3)Glc(BI +6)Gal(~1+6)Glc(crl+1/3)Gro, which are joined to each other through the phosphodiester bonds be- tween C-I or C-3 of the glycerol residues and C-6 of the 3- substituted glucose residues, as shown in Fig. 5. The apparent

526

molecular mass of the minor teichoic-acid-linked glycopeptide (TA-GP-0) estimated by chromatography on Sephacryl S-300 was about 65 kDa, whereas that of the major one (TA-GP-11) was about 18 kDa [12]. In contrast with the major teichoic acid chain, which is linked to peptidoglycan through a linkage saccharide unit, Glc(P1+4)GlcNAc [12], each polymer chain of the minor teichoic acid is believed to be directly linked to peptidoglycan at the galactose residue of the terminal re- peating unit through a phosphodiester bond, on the basis of the following evidence. The minor teichoic acid fraction obtained from the cell walls after heating at pH 2.5 was shown to contain only a negligible amount of glucosamine (Table 3). The NaB3H4 treatment of this fraction followed by mild alkaline hydrolysis gave two 3H-labeled fragments, gluco- syl(~1+3)glucosyl(~l+6)galactitol and glucosyl(pl+6)ga- lactitol, in an approximate molar ratio of 2: 1 (Fig.4). These results are consistent with the most likely structure of the actual repeating units shown in Fig.5. About 33% of the proximal terminal repeating units of teichoic acid chains seem to lack the lateral branch glucose residues, as judged from the production of the 3H-labeled fragment, glucosyl(Pl-*6)galac- titol.

As far as we are aware, teichoic acid chains with rather simple backbone repeating units, such as glycerophosphate, monoglycosyl-glycerol phosphate, ribitol phosphate and N- acetylglucosamine 1-phosphate, seem to be linked to peptidoglycan through glucosamine-containing saccharide units, N-acetylglucosamine [l - 31, ManNAc(P1+4)GlcNAc [4- 101 and Glc(P1-+4)GlcNAc [ l l , 121, irrespective of their structural diversity in the backbone chains and glycosidic branches. In addition, the teichuronic acid of Micrococcus luteus, comprising glucose and N-acetylmannosaminuronic acid, is known to be linked to peptidoglycan through a special disaccharide unit, N-acetylmannosaminuronosyl-N-acetyl- glucosamine [17, 181. However, in the cell walls of Bacillus /icheniformis 6346 [19] and Bacillus subtilis AHU 1031 [14], the teichuronic acid chains are believed to be directly linked to peptidoglycan. In the cell walls of B. coagulans AHU 1631, the junction between the minor teichoic acid and peptido- glycan seems to be of this type. Thus, the mode of junction between a cell wall polymer and peptidoglycan may be corre- lated with the structure of the backbone chain of the polymer. The teichoic acids which have three or more sugar residues in each backbone repeating unit may be directly linked to peptidoglycan, making a group of anomalous teichoic acids.

The types of junction of wall polymers to peptidoglycan seem to be essentially related to the modes of biosynthesis of the polymer chains. Results of biosynthetic studies [16, 201 suggest that the ordinary teichoic acids are synthesized through the prior formation of lipid-bound linkage sugars. In contrast, the teichoic acids of the other group may be synthesized through the formation of lipid-bound repeating units without participation of lipid-bound linkage sugars, as in the synthesis of 0-polysaccharide chains [21], wall neutral

polysaccharides [22] and some teichuronic acids [19, 23, 241. In a preliminary experiment, the membrane preparation from B. couguluns AHU 1631 was shown to catalyze the formation of galactose-linked lipid(s), which may serve as a key inter- mediate in the formation of a lipid-bound repeating unit, and studies on this line are in progress.

This study was supported in part by a grant from the Ministry of Education, Science and Culture of Japan.

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