Eur. J . Biochem. 148,29-34 (1985) 0 FEBS 1985

Structural studies on the linkage unit of ribitol teichoic acid of Lactobacillas plantarum Naoya KOJlMA, Yoshio ARAKI and Eiji IT0 Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo-shi

(Received October 22, 1984) - EJB 84 1122

Structural studies were carried out on the linkage unit which joins ribitol teichoic acid to peptidoglycan in the cell walls of Lactobacillusplantarum AHU 1413. The heating of the cell walls at pH 2.5 led to release of only 5% of ribitol teichoic acid components as water-soluble material. In contrast, the same treatment of the cell walls after N-acetylation led to release of about 80% of the teichoic acid moiety, giving a teichoic-acid-linked sugar preparation which contained about equimolar amounts of mannosamine, glucosamine and glycerol as minor components.

The teichoic-acid-linked sugar was hydrolyzed by mild alkaline treatment into a disaccharide, N-acetyl- mannosaminyl(~1+4)N-acetylglucosamine and ribitol teichoic acid linked to glycerol. The Smith degradation of the N-acetylated cell walls gave a characteristic fragment, 1,2-ethylenediol-phospho-glycerol-phospho-N- acetylmannosaminyl(P 1 -+4)N-acetylglucosamine. Furthermore, when the intact cell walls were subjected to the NaNOz treatment followed by NaBH4 reduction, the ribitol teichoic acid moiety was recovered for the most part in the water-soluble polymer fraction, from which a sugar, N-acetylmannosaminyl-2,5-anhydromannitol, was released by mild alkaline treatment.

These results lead to the conclusion that the ribitol teichoic acid chain in the intact cell walls of this organism is linked to peptidoglycan through a unique linkage unit, glycerol-phospho-N-acetylrnannosaminyl(P 1 +4)-glucos- amine. The anomalous stability of the linkage between the teichoic acid moiety and peptidoglycan against acid hydrolysis seems to be accounted for by the involvement of the N-unsubstituted glucosamine residue in the phosphodiester bridge that joins the two polymers.

A disaccharide, ManNAc(P 1 +4)GlcNAc, has been re- ported to be involved in the linkage regions between glycerol teichoic acids and peptidoglycan in the cell walls of several Bacillus subtifis strains and a strain each of Bacillus cereus and Bacillus licheniformis [l, 21. In addition, the same disaccharide has been found along with three glycerol-P units in the linkage region between ribitol teichoic acid and peptidoglycan in the cell walls of Staphylococcus aureus H [3]. Another linkage disaccharide, Glc(P 1 -+4)GlcNAc, has been detected in the linkage region between poly(galactosylglycero1 phosphate) and peptidoglycan in the cell walls of Bacillus coagulans AHU 1366 [4].

In order to obtain further information on the linkage units of acidic polymers in cell walls, we studied the structure of Lactobacillusplantarum cell walls which are known to contain ribitol teichoic acids. In preliminary experiments, the cell walls of L. plantarum AHU 1413 was shown to contain a small amount of mannosamine. However, the teichoic acid moiety was hardly released by mild acid hydrolysis under the conditions generally used for release of teichoic acid moieties from the cell walls. The present paper reports that the ribitol teichoic acid is joined to peptidoglycan through a new type of

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

Abbreviations. ManNAc, N-acetylmannosamine; TA-S, teichoic- acid-linked sugar; Gro-P, glycerol phosphate.

Enzymes. Alkaline phosphatase (EC 3.1.3.1); acid phosphatase (EC 3.1.3.2); lysozyme (EC 3.2.1.17).

the linkage unit, Gro-P-ManNAc(P1+4)GlcN, in the cell walls of this strain.

MATERIALS AND METHODS

Preparation of cell walls and N-acetylated cell walls

The methods used for the culture of Lactobacillus plantarum AHU 141 3 (kindly given by Dr S. Takao, Hokkaido University) and for the preparation of cell walls from the cells at late-log phase were the same as those described previously [5 ] . The cell walls (400 mg) were treated in 80 ml 25 mM glycine/HCl buffer (pH 2.5) at 100°C for 10 min, then cooled and centrifuged at 20000 x g for 45 min. The precipitate was again treated in the same way. The final precipitate was used as the acid-treated cell walls (260mg). In this process, a rhamnose-containing polysaccharide was for the most part released as a component of water-soluble material, whereas the ribitol teichoic acid moiety was retained in the insoluble fraction (acid-treated cell walls). The acid-treated cell wall preparation (120 mg) was then treated with 1 ml of acetic anhydride in 60 ml of a saturated NaHC03 solution for 1 day at 4°C as described previously [6]. The cell walls were washed several times with water and used as the acid-treated, N- acetylated cell wall preparation. Cell walls were also prepared from the cells of Staphylococcus aureus H, 209P and Copenhagen, Bacillus subtilis AHU 1035, AHU 1235, AHU 1392 and W23, Bacillus licheniformis AHU 1371, Bacillus coagulans AHU 1366 and L. plantarum AHU 1680 as de- scribed previously [I -51. B. subtitus W23 was furnished as

30

strain IAM 12021 by the Institute of Applied Microbiology of Tokyo University, and other microorganisms were the same as those in the previous papers.

Preparation of teich o ic- ac id- lin k ed sugar

The acid-treated, N-acetylated cell wall preparation (100 mg) was treated in 20 ml25 mM glycine/HCl buffer (pH 2.5) at 100°C for 10 min, then cooled and centrifuged. The precipitate was again treated in the same way. The supernatant fractions were pooled and dialyzed against water. The nondialyzable fraction was applied on a DEAE-cellulose column (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 NaCl in the same buffer. The fractions containing phosphorus and hexosamine, eluted at about 0.25 M NaCI, were pooled, dialyzed and then subjected to chromatography on a Sephacryl S-200 column (1 x 100 cm) in 50 mM (NH4)2C03. Fractions containing phosphorus and hexosamine were pooled, dialyzed and used as the teichoic-acid-linked sugar preparation (TA-S, 20.5 mg).

Deanzination of cell walls

The acid-treated cell walls (20 mg) before and after N- acetylation were treated with 0.5 M NaN02 in 8 ml 0.1 M acetic acid at 25°C for 3 h. The reaction mixtures were neutralized with 1 M NaOH, reduced with NaBH, and centrifuged at 20000 x g for 45 min. After dialysis, the supernatant fraction was subjected to successive chromatog- raphy on columns of DEAE-cellulose and Sephacryl S-200, as described for the isolation of TA-S. Phosphorus-containing material was used as the acidic polymer resulting from deamination.

Mild alkaline treatment of teichoic-acid-linked sugar

The TA-S preparation (before and after reduction with NaBH,) and the acidic polymer resulting from deamination were treated in 0.5 M NaOH at 37°C for 30 min. After passage through a column of Dowex 50 (H' form), the products were subjected to chromatography on a Sephadex G-25 column (1 x 147 cm, superfine) in 50 mM (NH&C03.

Smith degradation of cell walls

The acid-treated cell wall preparation (50 mg) before and after N-acetylation was oxidized with 0.1 M NaIO, in 0.1 M sodium acetate buffer (pH 5.0) in the dark at 4°C for 48 h. After centrifugation, the precipitate was reduced with NaBH,, washed with water, then treated with 25 mM glycine/HCl buffer (pH 2.5) at 100°C for 10min. The product was centrifuged, and the resulting supernatant was deionized by passage through a column of Dowex 50 (H' form) and sub- jected to chromatography on a Sephadex (3-25 column (1 x 147 cm) in 50 mM (NH&C03.

Other materials and analytical methods

Unless otherwise stated, materials and methods were the same as those described in previous papers [l, 31. The disac- charides, ManNAc(P 1 -4)GlcNAc and Glc(~1-+4)GlcNAc, were isolated as the linkage saccharides from teichoic-acid - glycopeptide complexes of B. cereus AHU 1030 [I] and B. coagulans AHU 1366 [4]. Total hexosamine was determined

by the method of Tsuji et al. [7] with glucosamine as a standard after N-deacetylation of samples (2 M HCI, IOOT, 2 h); N-unsubstituted glucosamine in polysaccharides, by the same method without hydrolysis [XI ; phosphorus, by the method of Lowry et al. [9]; N-acetylhexosamine, by the modified Morgan-Elson method [ 101 ; formaldehyde, with chromotropic acid [Il l . Amino acids, amino sugars and muramic acid 6-phosphate were determined by an amino acid analyzer after hydrolysis of samples (4 M HC1,IOO "C, 4 h) as described previously [ 121. Hexose, hexosamine, polyol, 2,5- anhydromannitol and erythritol were analyzed by gas-liquid chromatography after hydrolysis of samples (2 M HC1, 100' C, 1 h), followed by alkaline phosphatase treatment, N- acetylation and trimethylsilylation, as described previously [3]. Methyl sugars were also analyzed by gas-liquid chromatography. Reduction of saccharides was carried out with 0.1 M NaBH, in 50 mM borate buffer (pH 9.8) at 4°C for 16 h. 2,5-Anhydromannitol was prepared from glucos- amine by deamination and used as a standard.

RESULTS

Analysis of cell ~ v d l s before avid after treatment with mild acid

The cell wall preparation of L. plantarum AHU 1413 contained large amounts of ribitol, phosphorus, glucose, galactose and rhamnose, together with peptidoglycan components (Table 1). In addition, the preparation was found to contain small amounts of mannosamine, glycerol and inuramic acid 6-phosphate. The cell walls of this strain were shown to contain two different polymers, a rhamnose- containing neutral polysaccharide and a ribitol teichoic acid, by the separation of a ribitol teichoic-acid - glycopeptide complex and a neutral polysaccharide-glycopeptide complex from lysozyme digests of alkali-treated cell walls.

By heating the intact, non-N-acetylated cell walls in glycine/HCl buffer (pH 2.5) for 10 min, almost all of the rhamnose and galactose residues were released as components of water-soluble material along with small parts of the phosphorus and glucose residues. As analyzed by gel chromatography, the water-soluble material was shown to consist of large amounts of rhamnose-containing oligo- saccharides and only a very small amount of ribitol teichoic acid. More than 95% of the ribitol, glycerol and mannosamine residues remained in the residual, water-insoluble fraction (acid-treated cell walls, Table l) , which contained only negligible amounts of rhamnose and galactose and was therefore used in further studies on the teichoic acid moiety.

Isolation and analysis of teichoic-acid-linked sugar

When the acid-treated, rhamnose-free cell wall prepara- tion was first N-acetylated and then heated again at pH 2.5, about 80% of the ribitol, phosphorus, glucose, glycerol and mannosamine residues in the preparation were released as components of water-soluble material. In this process, the muramic acid 6-phosphate groups, which remained in the residual, water-insoluble fraction, were for the most part converted into an acid-phosphatase-sensitive form. Thus, the heating of the N-acetylated cell walls at pH 2.5 seems to cause cleavage of the linkage between the teichoic acid moiety and a phosphoryl group of the muramic acid 6-phosphate in the peptidoglycan moiety.

31

Table 1. Composition of' cell ~?al ls . acid-treuted cell walls, teichoic- acid- giycopeptide complex und teichoic-acid-linked sugar The values are expressed relative to the mass of dried materials. Each preparation was analyzed for composition after acid hydrolysis. Cell walls, acid-treated cell walls and teichoic-acid - glycopeptide also contained glutamic acid, alanine and diaminopimelic acid

Component Content in

cell acid- teichoic- TA-S walls treated acid-

cell glyco- walls peptide

Muramic acid 6-phosphate

Murdmic acid Glucosamine Mannosamine Glucose Galactose Rhamnose Ribitol Glycerol Phosphorus

31 345 590

17 829 292 641 480

34 950

39 41 5 70 1

28 867

38 0

73 1 32

841

42 256 41 3 47

1880 0 0

1460 56

1520

0 0

66 14

2720 0 0

2100 84

2090

Fractionation of the water-soluble material by successive chromatography on columns of DEAE-cellulose and Sephacryl S-200 gave a single peak of phosphorus-containing polymer, denoted as the teichoic-acid-linked sugar (TA-S). The TA-S preparation contained mannosamine, glucosamine, glycerol, ribitol, phosphorus and glucose in a molar ratio of 1 .O: 0.9: 1.1 : 28 : 28 : 37 (Table 1). This preparation contained a negligible amount of alanine, indicating that almost all of ester-linked alanine residues were removed from the teichoic acid chain by the repeated mild-acid treatment. Analysis of the TA-S preparation after reduction with NaBH4 indicated that the glucosamine residue was present at the reducing end of the polymer chain. These data suggest that the TA-S prepa- ration consisted of a mannosamine-containing unit, a glycerol phosphate unit and a ribitol teichoic acid chain with an approximate molelcular mass of 18 000.

Characterization of teichoic acid moiety

The H F treatment (47% HF, 25°C 16 h) of TA-S (2 mg) followed by chromatography on Sephadex G-25 gave three polyol-containing products (compounds A, B and C in order of decreasing molecular sizes). The yields of compounds A, B and C were 2.7 pmol, 0.6 pmol and 1.3 pmol as ribitol, respectively. Compound A was composed of ribitol and glucose in a molar ratio of 1 :2. The NaI04 oxidation gave 1 mol formaldehydelmol ribitol residue in this compound. Chromic anhydride oxidation caused no change in the glucose content, indicating an a-configuration for both glucose re- sidues. Permethylated compound A gave only a 2,3,4,6-tetra- O-methylglucose derivative as analyzed by gas-liquid chromatography after acid hydrolysis. Thus, the most prob- able structure for compound A is Glc(a I -+2/4)[Glc(a1+3)]- ribitol. Compounds B and C were also characterized to be Glc(a 1 +2/4)ribitol and free ribitol, respectively. Thus, the ribitol teichoic acid of this strain was presumed to consist of diglucosylribitol, monoglucosylribitol and unglucosylribitol

- I

2 6 \

L 0 L

\

- -

5 4 200 g v) 3

r

- B a, c

E 2 0 100 g f [L m 0

Y a,

0 o = 40 50 60 70 80

Fraction number

Fig. 1. Chromatography of teichoic-acid-linked sugar after treatment w t h mild alkali. The TA-S preparation (15 mg) was treated in 4 ml 0.5 M NaOH at 37°C for 30 min, and the product was subjected to chromatography on a Sephadex (3-25 column (1 x 147 cm) in 50 mM (NH&C03. 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-acetylglucosamine, respectively. Fractions indicated by bars were pooled

units, which were probably joined by phosphodiester bonds at C-I and C-5 of the ribitol residues.

Alkaline hydrolysis of teichoic-acid linked sugar

The mild alkaline treatment (0.5 M NaOH, 37"C, 30 min) of TA-S followed by chromatography on Sephadex (3-25 (Fig. 1) yielded two phosphorus-containing fractions (peaks 1 and 2). The glycerol residues as well as the teichoic acid components, ribitol, phosphorus and glucose residues, were recovered in the fraction excluded from the column (peak l), whereas the mannosamine and glucosainine residues were recovered in the second fraction (peak 2), which appeared at the position of standard chitobiose. The saccharide in the second fraction was identified as ManNAc(P 1 +4)GlcNAc on the basis of the following evidence. It gave equimolar amounts of glucosamine and mannosamine as analyzed after acid hydrolysis, whereas a reduced sample of this fraction gave glucosaminitol and mannosamine. The Smith degradation of the reduced sample yielded N-acetylxylosaminitol. The sugar in this fraction gave a much lower color yield than that of N-acetylglucosamine in the modified Morgan-Elson reaction (molar color yield relative to that of N-acetylglucosamine = 0.03). Furthermore, upon paper chromatography in I-butanol/pyridine/water (6: 4: 3 , v/v/v) on 15 mM borate- treated paper, the disaccharide was coincident with standard ManNAc(P1+4)GlcNAc (migration relative to that of chitobiose = 1.10) and distinguishable from standard Glc- (PI j4)GlcNAc (1 .00). By the chromic anhydride oxidation, the N-acetylmannosamine residue of the disaccharide dis- appeared completely within 20 min.

When the mild alkaline treatment was carried out after the TA-S preparation had been reduced with NaBH4, the product was eluted from the Sephadex G-25 column with a similar elution pattern. However, in this case, material in peak 2 was shown to be ManNAc-GlcNAc-01. Thus, the N-acetylglucosamine of the disaccharide ManNAc-GlcNAc represented the reducing terminus of TA-S. From the above result, it is suggested that the anomalous acid stability of the linkage between teichoic acid and peptidoglycan in the intact, non-N-acetylated cell walls may be explained by N-unsubsti-

32

tution at the glucosamine residue in the linkage disaccharide unit.

Deantinution qf acid-treated cell walls Further experiments were directed toward proving N-un-

substitution at the terminal glucosamine residue of the linkage disacharide in the non-N-acetylated cell walls. When the acid- treated cell wall preparation was reacted with 0.5 M NaNOz and then reduced with NaBH4, 72% of the phosphorus in the cell walls was released as a component of soluble acidic material. In contrast, when the same experiment was carried out after the cell wall sample had been N-acetylated, only a negligible amount of phosphorus (less than 0.90/,) was re- leased.

The water-soluble acidic material resulting from deamina- tion of the cell walls gave a single, phosphorus-containing polymer after successive chromatography through columns of DEAE-cellulose and Sephacryl S-200. This polymer contained mannosamine, glycerol, ribitol, phosphorus and glucose in a molar ratio of 1 .O: 1.2 : 29: 30 : 36, but contained only negligible amounts of glucosamine and glycopeptide components.

When this polymer was subjected to mild alkaline hydroly- sis followed by chromatography through Sephadex G-25 (Fig. 2), the ribitol, phosphorus, glucose and glycerol residues were for the most part recovered in the first fraction (peak I ) excluded from the column, whereas most of the mannosamine residues were recovered in the second fraction (peak 2). Mate- rial in the latter fraction gave equimolar amounts of N- acetylmannosamine and 2,5-anhydromannitol (aMan-01) as analyzed by gas-liquid chromatography after acid hydrolysis followed by N-acetylation and was characterized to be ManNAc-aMan-01. Thus, the acidic polymer released from the cell walls by the NaN02/NaBH4 treatment was shown to be the teichoic-acid-linked disaccharide deaminated at the reducing terminus. This result indicates the presence of an N-unsubstituted glucosamine residue as the reducing terminus of the linkage disaccharide.

Smith degradation of ucid-treated cell wialls

Attempts to obtain linkage-unit-containing fragments by means of Smith degradation of the teichoic-acid - glyco- peptide complex or of TA-S were unsuccessful because of incomplete destruction of the teichoic acid chain owing to glucosylation of ribitol residues. Therefore, we tried to obtain linkage-unit-containing fragments by the Smith degradation of cell walls. When the acid-treated cell walls were N - acetylated and then oxidized with NaI04, most of the phosphorus (goo/,) was released as a component of water- soluble material, whereas over 95% of the mannosamie re- sidues along with a small portion of the phosphorus (10%) were recovered in the residual, insoluble fraction. The in- soluble fraction was treated with NaBH4, and the reduction product, which contained equimolar amounts of man- nosamine and glycerol together with large amounts of peptidoglycan components, was analyzed as the linkage-unit- bound peptidoglycan. By heating the reduction product at pH 2.5 for 10 min at 10O"C, the mannosamine residue was for the most part released as a component of water-soluble material. Chromatography of the water-soluble material through Sephadex G-25 (Fig. 3) gave three phosphorus-containing fractions. The component of the first fraction (peak I), purified by chromatography on a DEAE-cellulose column, was shown to contain mannosamine, glucosamine, 1,2-

Fraction number

Fig. 2. Chromatography of acidic polymer resulting from deamination of cell walls after treatment with mild alkali. The acidic polymer fraction, obtained from intact, non-N-acetylated cell walls (20 mg) by deamination, was treated with 0.5 M NaOH at 37°C for 30 min, and the product was chromatographed under the same conditions as described in Fig. 3 . Fractions were analyied for phosphorus (0) and reducing groups (0 ) . The reducing groups were measured after hydrolysis of aliquots of each fraction in 4 M HCI at 100°C for 2 h and expressed as glucosamine. Fractions indicated by bars were pooled

0

E Fraction number

Fig. 3. Chromatography of Smith degradation products from N- acetylured ceN walls. The N-acetylated cell wall preparation (50 mg) was oxidized with NaI04 at 4 C for 2 days. After removal of reagents by centrifugation, the precipitate was reduced with NaBH4, then heated at pH 2.5 for 10 min. The resulting water-soluble products were chromatographed under the same conditions as described in Fig. 1. Symbols and arrows are the same as in Fig. 1. Fractions indicated by bars were pooled

ethylenediol, glycerol and phosphorus in a molar ratio of 1.0: 1.1 : 0.9:l .I : 2.0. Most of the mannosamine residues (71%) in the starting cell walls were recovered in this compound. The phosphoryl groups in this compound were unaffected by the treatment with alkaline phosphatase. By mild alka- line hydrolysis, this compound was hydrolyzed into the di- saccharide ManNAc(fl1+4)GlcNAc (peak 2) and a fragment that contained glycerol (Gro), 1 ,a-ethylenediol (EtOz) and phosphorus in a molar ratio of 1.0:0.9: 1.9 (peak 1, Fig. 4). Half of the phosphoryl (P) groups in this fragment were sensitive towards digestion with alkaline phosphatase, whereas the glycerol residues were unaffected by NaI04 oxidation. These data are consistent with the structure, Et0,- P-Gro-P, for the compound of peak 1 in Fig. 4. Thus, it is most probable that the component of peak 1 in Fig. 3 was Et02-P-Gro-P-ManNAc(P 1 -+4)GlcNAc.

Major components in peaks 2 and 3 (Fig. 3) were also characterized to be Et02-P-Gro-P and Et02-P-Gro, re-

33

I

L +- 1 E 1, 100 c .- E s :: Z 50 73 C

VI

2 s o 8 40 50 60 70 80 0 5 Fraction number

Fig . 4. Chroma tography qf linkage-un it-con tain ing fragmen t a f ter treatment with mild alkali. A linkage-unit-containing fragment (0.8 pmol phosphorus), obtained from peak 1 in Fig. 3 by chromatography on DEAE-cellulose, was treated with 0.5 M NaOH at 37' C for 30 min. The product was chromatographed under the conditions same as described in Fig. 1 . Symbols and arrows are the same as in Fig. 3 . Fractions indicated by bars were pooled

spectively, on the basis of their composition, molecular sizes and other analytical data. The former compound seems to be partly derived from the compound in peak 1 during the acid treatment.

When the acid-treated cell walls were directly subjected to the Smith degradation without prior N-acetylation, a frag- ment which contained mannosamine, erythritol (Ery-ol), glyc- erol, 1,2-ethylenediol and phosphorus in a molar ratio of 1.0:0.96: 1.0:0.87: 1.7 was obtained. By the mild alkaline treatment, this fragment was hydrolyzed into Et02-P-Gro- P and a hexosamine-containing compound characterized as ManNAc( 1 -+2)Ery-ol. The erythritol residue in this com- pound seems to arise from the N-unsubstituted glucosamine residue in the linkage disaccharide unit in the acid-treated cell walls.

The above results lead to a conclusion that Gro-P- (3/4)ManNAc(/3 1 -+4)GlcN represents the linkage unit for the ribitol teichoic acid chain in this organism. The 1,2-ethylenediol residues in the fragments resulting from the Smith degradation seem to arise from the terminal ribitol residues of the teichoic acid chains. The quantitative forma- tion of l ,2-ethylenediol-containing fragments in this proce- dure indicates that the terminal ribitol residue in each teichoic acid chain is unsubstituted at C-2 and C-3.

Occurrence of N-unsubstituted glucosamine residues in linkage sugars qf other niicroorganisms

It was uncertain whether the hexosamine residues are N-acetylated or not in the linkage regions of teichoic acids in various bacterial strains, because most of previous studies on the linkage regions were carried out with N-acetylated preparations of cell walls [l -41. In the cell walls of L. plantuvum AHU 141 3, N-unsubstitution at the glucosamine residue of the linkage disaccharide accounts for the acid stability of the junction between the teichoic acid moiety and peptidoglycan. Thus, it seems possible to estimate the extent of N-unsubstitution at this particular glucosamine residue by measuring acid-catalyzed teichoic acid liberation before and aftere N-acetylation of cell walls.

As summarized in Table 2, the N-acetylated cell wall prep- arations from different strains which are known to have ManNAc-GlcNAc or Glc-GlcNAc as the linkage disaccharide

Table 2. Liberation of teichoic acid moiety f rom cell walls of various bacterial strains by mild acid treatment The intact and N-acetylated cell wall preparations were treated twice in glycine/HCl buffer (pH 2.5) at 100°C for 10 min. The resulting water-soluble material was analyzed for phosphorus (or ribitol). Liberation of the teichoic acid moiety is shown as a percentage of ribitol (for L. plantarum) or phosphorus (all other microorganisms) recovered in the water-soluble material relative to that in the starting cell walls. The N-unsubstituted glucosamine residues in the intact, non-N-acetylated cell walls were also analyzed by the method of Tsuji et al. [8]. The extent of N-unsubstitution in glycan strands was tentatively calculated on the basis of the amount of N-unsubstituted glucosamine and the content of muramic acid in the cell walls and is shown as a percentage; the values have been corrected for the color developmcnt due to thc peptide moiety of peptidoglycan

Microorganism Liberation of Extent of teichoic acid moiety N-unsub- from cell walls stitution

at glucos- before after amine in N-acety- N-acety- intact lation lation cell walls

L. plantarum AHU 141 3 L. plantarum AHU 1680 S. aureus H S. aureus 209P S. uureus Copenhagen B. subfilis W23 B. subtilis AHU 1035 B. subtilis AHU 1235 B. subtilis AHU 1392 B. lichenijormis AHU 1371 B. coagulans AHU 1366

%

5 8

81 76 48 64 38 45 62 64 15

82 19 82 81 85 15 14 12 70 71 78 -

5 8 3 1 0

29 16 12 10 10 13

units, showed similar values (70-80%) of the extent of teichoic acid liberation by the treatment at pH 2.5, but the intact cell wall preparations showed various values. Thus, the cell wall preparations examined may be classified into three groups with respect to the extents of N-substitution at the glucosamine residues in the linkage units : the preparations almost completely N-unsubstituted (L. plantarum AHU 141 3 and AHU 3680), the preparations partially N-unsubstituted (S. aureus Copenhagen and B. subtilis AHU 1035 and AHU 1235) and the preparations almost fully N-substituted at these glucosamine residues (other strains). It is known that cell wall peptidoglycans of Bacillus are partially or completely N-unsubstituted at their glucosamine residues [5, 61. Therefore, the extent of N-unsubstitution of hexosamine re- sidues in each cell wall preparation was compared with that in the linkage region (Table 2). However, any direct correla- tion was not indicated in the extents of N-unsubstitution at the glucosamine residues between the linkage regions and the peptidoglycan moieties.

DISCUSSION The results described above led to a conclusion that in the

cell walls of L. plantarum AHU 141 3 the ribitol teichoic acid is linked to peptidoglycan through a linkage unit, Gro-P- ManNAc(fi 1 -+4)GlcN. This linkage unit is characteristic in that it contains a single glycerol-P unit. The Smith degrada- tion of the acid-treated, N-acetylated cell walls of this strain

34

gave a fragment, EtO,-P-Gro-P-ManNAc(Dl +4)GlcNAc (Fig. 3, peak 1). In the same procedures, ribitol teichoic- acid - glycopeptide complexes of B. subtilis W23 (unpublished data) and S. aureus H [3] gave homologous fragments, Et0,- P-(Gro-P),-ManNAc(P 1 +4)GlcNAc and EtOz-P-(Gro-P)3- ManNAc(0 1 +4)GlcNAc, respectively. These fragments were distinguishable from each other by chromatography through Sephadex G-25. In addition, 1,2-ethylenediol-P derivatives linked to different numbers of glycerol-P units resulting from mild alkaline treatment of these fragments were also dis- tinguishable from one another. The KD values in gel chromatography through Sephadex G-25 and the migration distances relative to sn-glycerol 3-phosphate in paper electrophoresis were respectively as follows : 1,2-ethylenediol- P-glycerol-P (from L. plantarum AHU 1413), 0.56 and 1.12; 1,2-ethylenediol-P-(glycerol-P)2 (from B. subtilis W23), 0.45 and 1.25; 1,2-ethylenediol-P-(gly~erol-P)~ (from S. aureus H), 0.32 and 1.35. Thus, each bacterial species may have a characteristic number of glycerol-P units in the linkage region between teichoic acid and peptidoglycan. The structure of the parts composed of glycerol-P units is expected to provide a valuable marker in phylogenetic classification of gram-posi- tive bacteria. However, the precise structure of these parts as well as their biological role remains to be clarified.

In the cell walls of B. cereus AHU 1030 [l] and S. aureus H [3], the reducing terminal glucosamine residues in the linkage regions between teichoic acids and peptidoglycan are fully N-acetylated. In contrast, a possibility of the presence of N-unsubstituted hexosamine residues in the linkage unit of the cell walls of L. plantarum AHU 1413 was suggested on the basis of the result of the following preliminary experiment. A radioactive TA-S was isolated from the acid-treated cell walls after N-acetylation with [I4C]acetic anhydride. Mild alkaline treatment of this material gave a radioactive disaccharide, which was coincident with standard ManNAc(P 1 +4)Glc- NAc on paper chromatography. In the present work, N- unsubstitution at the terminal glucosamine residue of the linkage disaccharide unit was confirmed either by the deamination or by the Smith degradation of the acid-treated, non-N-acetylated cell walls. In contrast, as shown in Table 2, the glucosamine residues in the peptidoglycan moiety are for the most part N-acetylated in the intact cell walls of this strain.

Unless the cell walls had been N-acetylated, the teichoic acid moiety could not be released from them by heating at pH 2.5. Although a small amount of ribitol-containing poly- mer (less than 5% of total ribitol) was released from the intact, non-N-acetylated cell walls in the same process, this polymer contained neither glucosamine nor mannosamine. Thus, the liberation of this polymer from the non-N-acetylated cell walls seems to be due to cleavage at the phosphodiester bonds in the ribitol teichoic acid chain, but not at the joint between the linkage saccharide and peptidoglycan. This anomalous acid

stability of the glucosamine-P linkage seems to be explained by the well known prohibitive effect of the free amino group on acid hydrolysis of a hexosaminide. Thus, when glu- cosamine I-phosphate prepared from N-acetylglucosamine 1 - phosphate [13] by alkaline hydrolysis was tested, less than 2% was dephosphorylated under the conditions for mild acid hydrolysis (10 mM HCl, 100°C, 10 min), and 50% hydrolysis of this compound required the treatment in 1 M HCI at 100°C for 10 min.

By analogy with the enzymatic deacetylation of the N-acetylglucosamine residues in the cell wall peptidoglycan of B. cereus [14] and in the chitosan of Mucor rouxii [15], it seems likely that the N-unsubstituted glucosamine residue in the linkage unit is derived from the N-acetylated one by the action of a specific deacetylase, which differs probably from the deacetylase specific for peptidoglycan. A preliminary study indicated that the membrane preparation of L. plantarum AHU 1413 catalyzes the formation of ManNAc- GlcNAc from UDP-ManNAc and UDP-GlcNAc in a lipid- bound form (unpublished data).

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

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