Structures of Two Monomeric Units of Teichoic Acid Preparedfrom the Cell Wall of Lactobacillus plantarum NRIC 1068

Satoru TOMITA,1;y Kazuo FURIHATA,2 Tomoo NUKADA,3 Eiichi SATOH,1

Tai UCHIMURA,1 and Sanae OKADA1

1Department of Applied Biology and Chemistry, Faculty of Applied Bio-Science, Tokyo University of Agriculture,1-1-1 Sakuragaoka, Setagaya, Tokyo 156-8502, Japan2Division of Agriculture and Agricultural Life Science, The University of Tokyo,1-1-1 Yayoi, Bunkyo, Tokyo 113-8657, Japan3Department of Fermentation Science, Faculty of Applied Bio-Science, Tokyo University of Agriculture,1-1-1 Sakuragaoka, Setagaya, Tokyo 156-8502, Japan

Received August 22, 2008; Accepted November 14, 2008; Online Publication, March 7, 2009

[doi:10.1271/bbb.80582]

The cell wall of Lactobacillus plantarum containslarge amounts of cell wall teichoic acid (WTA). WTAwas isolated from the cell wall of L. plantarum NRIC1068 (= ATCC 8014 and 17-5) by extraction withtrichloroacetic acid, and two monomeric units (F1 andF2) were prepared from the alkaline hydrolysate ofWTA. Componential analysis by HPLC showed thatthese monomers were composed of ribitol, glucose, andphosphoric acid. Structural analyses of the monomerswere performed by NMR spectroscopy with comparisonto chemically synthesized monomers. The structures ofF1 and F2 were determined to be 3,4-�-D-diglucosyl-2-phosphoryl ribitol and 3,4-�-D-diglucosyl-1-phosphorylribitol respectively. The unique structure of WTA inL. plantarum results from modification of the mainchain with multiple glucose residues.

Key words: teichoic acid; cell wall structure of lacticacid bacteria; Lactobacillus plantarum

The cell walls of Gram-positive bacteria consistof peptidoglycan, polysaccharide, and teichoic acid.These structures thickly cover the cell membrane andprovide cell shape, and they play important roles inresistance to external mechanical, chemical, thermal,and osmotic stresses.1,2) Furthermore, the cell walls oflactic acid bacteria not only are indispensable to thelife support of the bacteria, but also affect bacterialcolonization of the host intestinal epithelial cells andimmune stimulation of the host.3,4) It was recentlymade clear that the cell wall plays an important role inthe beneficial effects of intestinal lactic acid bacteriaon human health, for example, through intestinal andimmune system regulation. These effects are oftenstrain-dependent,5,6) but little is known about theirmechanisms. The compositions of the cell walls arebasically similar within a species, but sometimesdifferences in components are observed among strains.These differences are used to characterize the strains.Consequently, to understand the role of the cell wallsof lactic acid bacteria and the structure-functionrelationship at the strain level, elucidation of the

intraspecific diversity of the cell wall is absolutelynecessary.Lactobacillus plantarum is a lactic acid bacterium that

is widely isolated from the environment and fermentedfoods, and is expected to be applied in the production ofvarious fermented foods and useful materials. The cellwalls of L. plantarum are composed of peptidoglycancontaining meso-DAP; teichoic acid (glycerol or ribitoltype); and polysaccharide composed of glucose andrhamnose.7) Although the peptidoglycan is a majorcomponent of the cell wall, its analysis is used forspecies identification and is not appropriate for estimat-ing intraspecific diversity. Cell wall teichoic acid (WTA)is a second abundant component next to the peptidogly-can in the cell walls of lactic acid bacteria. It is known tohave various functions8,9) and to differ in componentsand configuration at the strain level within the samespecies.10–13) Hence, we anticipated that the structuraldiversity of the cell wall among strains of L. plantarumcould be defined by investigating WTA structure.The chemical composition of WTA in Lactobacillus

species has been examined widely for species character-ization. Lactobacillus plantarum has been found tobe unique, because either glycerol-type or ribitol-typeWTA is contained within the same species.14) Thecell wall of L. plantarum generally contains a consid-erable amount of WTA. The structures of glycerol- andof ribitol-type WTA were originally analyzed andestimated by chemical decomposition methods,15,16)

but in recent years, the structures of the WTA ofBacillus subtilis and Staphylococcus aureus have beendetermined by modern analytical methods. Hence, weused nuclear magnetic resonance (NMR) spectroscopyfor structural determination. NMR is useful for thenondestructive analysis and comparison of spectraamong samples. Additionally, we used several syntheticmonomers to determine the absolute stereostructure ofWTA. Here we report the structure of the monomericunits of WTA isolated from L. plantarum NRIC* 1068

y To whom correspondence should be addressed. Fax: +81-3-5477-2537; E-mail: ttagggtelomere@gmail.com

* NODAI Culture Collection Center, Tokyo University of Agricul-

ture, Tokyo, Japan

Biosci. Biotechnol. Biochem., 73 (3), 530–535, 2009

(= ATCC 8014 and 17-5) as determined by thesemethods.

Materials and Methods

Preparation of WTA. Lactobacillus plantarum NRIC 1068 was

grown at 30 �C in 20 liters of GYP medium composed of 20 g of D-

glucose, 10 g of yeast extract, 10 g of peptone, 10 g of sodium acetate

and minerals per liter (pH 6.8). The cells were collected by continuous

centrifugation at 10,000 rpm and washed twice with 20mM phosphate

buffer (pH 7.0). The harvested cells were suspended in the same buffer

and disrupted by ultrasonic disruptor (UD-201, Tomy Seiko, Tokyo)

while they were cooled with iced water. After centrifugation

(12,000 rpm, 4 �C, 30min), the whitish upper layer of the pellet was

collected as the disrupted fraction, and was washed with the phosphate

buffer. The crude cell wall fraction was treated with 200ml of 2%

sodium dodecyl sulfate at 100 �C for 1 h, and then was washed with

distilled water thoroughly. Subsequently, the fraction was treated with

200ml of each enzyme solution, Pronase (Roche Diagnostics,

Mannheim, Germany), DNase (Wako Pure Chemical Industries,

Osaka, Japan) and RNase (Sigma-Aldrich, St. Louis, MO), and Pepsin

(Sigma-Aldrich) in that order. The enzyme-treated cell wall was finally

washed with distilled water. WTA was then extracted from the cell

wall fraction with 100ml of 10% trichloroacetic acid (TCA) at 4 �C for

72 h and precipitated with ethanol (5 vol) at 4 �C for 48 h. The

precipitate was purified by repetition of dissolution with TCA and

precipitation with ethanol to remove low-molecular-weight substances.

After it was washed with ethanol several times, until the precipitate

became powdered, the WTA was dried in a centrifugal evaporator

(CVE-2000, Tokyo Rikakikai, Tokyo). It was then fractionated on a

DEAE-Toyopearl 650M (Tosoh, Tokyo) column (1:6� 15 cm) in a

gradient of water-0.5M sodium chloride to remove neutral polysac-

charides, and on a Sephadex G-75 (GE Healthcare, Buckinghamshire,

UK) column (2:6� 70 cm) in water to prepare homogeneous WTA.

Since the WTA in this study contained glucose residues, the fractions

containing WTA were detected by the phenol-H2SO4 method. The

WTA-containing fractions were collected and lyophilized.

Preparation of monomeric units of WTA. Monomeric units of WTA

were prepared as described by Archibald et al.15) The purified WTA

was dissolved in 1ml of 1N sodium hydroxide and hydrolyzed at

100 �C for 3 h. After neutralization with 0.5ml of 2N hydrochloric

acid, the hydrolysate was demineralized on a Sephadex G-25 (GE

Healthcare) column. Sugar phosphates in the hydrolysate were

analyzed by high-performance liquid chromatography (HPLC, LC-10

system, Shimadzu, Kyoto, Japan) on an Shodex Asahipac NH2P-50 4E

(Showa Denko, Tokyo) column with 50mM sodium-phosphate buffer

(pH 4.4). The peaks of sugar phosphates were detected with the

refractive index detector (RID-10A, Shimadzu), and were collected

with a fraction collector (FRC-10A, Shimadzu). The sugar phosphates

in the fraction were separated by charcoal chromatography on a

charcoal powder-Celite 535 (1:1, w/w, Wako) column and then eluted

with 50% ethanol. In this way WTA monomers were prepared from

purified WTA.

Additionally, dephosphorylated monomers were prepared from the

WTA monomers by enzyme treatment. The WTA monomers were

dissolved in 1ml of 50mM glycine-sodium hydroxide buffer (pH 10.4)

containing 0.5U/ml alkaline phosphatase (Sigma) and incubated

overnight at 37 �C. From the reaction mixture, dephosphorylated

monomers were separated by charcoal chromatography under the same

conditions, and the fraction absorbed to the column was collected and

lyophilized. In this way the dephosphorylated WTA monomers were

prepared.

Analytical methods. Constituent sugars of the dephosphorylated

monomers were analyzed by HPLC on an Asahipac NH2P-50 4E

column with water-acetonitrile (25:75, v/v). The monomers were

hydrolyzed with 1ml of 6N hydrochloric acid at 100 �C for 3 h in a

vacuum reaction tube. The hydrochloric acid in the hydrolysate was

removed in a rotary vacuum evaporator (Tokyo Rikakikai). The dried

residue was dissolved in a small amount of water and used in the

analysis by HPLC. In this analysis, most of the ribitol in the monomers

was changed to anhydroribitol by the acid hydrolysis, and the

chromatogram of HPLC showed the peak of anhydroribitol instead

of ribitol. Therefore, the ribitol content was determined by creating a

calibration curve of the anhydroribitol. Optical rotation was analyzed

on the digital polarimeter DIP-140 (Jasco, Tokyo) at 589 nm in

distilled water. High-resolution mass spectra of the monomers were

obtained by time-of-flight mass spectrometry (TOF-MS) using an

AQUITY UPLC System coupled with an LCT Premier XE Mass

spectrometer (Waters, Tokyo). Leucine (molecular weight, 131.1729)

was used as the reference.

NMR spectra were recorded on an INOVA-500 (Varian, Palo Alto,

CA) spectrometer in D2O at ambient temperature with proton and

carbon frequencies of 499.86 and 125.69MHz respectively. Dioxane

(�C 67.5 ppm) and sodium 2,2-dimethyl-2-silapentane-5-sulfonate (�C0 ppm) were used as the references. H–H correlation spectroscopy

(COSY) spectra were recorded with a spectral width of 1324�1324Hz, eight repetitions, and 2� 512 increments. H–C hetero-

nuclear single quantum coherence (HSQC) spectra were recorded with

a width of 6525� 2142Hz, a 2.0-s relaxation delay, eight repetitions,

and 2� 256 increments. Additionally, H–C constant time hetero-

nuclear multiple-bond connectivity (CT-HMBC) spectra were recorded

using pulse field gradient programs with a width of 6525� 2142Hz, a

2.0-s relaxation delay, 32 repetitions, and 256 increments. H–P HMBC

NMR analysis was performed on an ALPHA-500 (Jeol, Tokyo)

spectrometer with proton and phosphorus frequencies of 500.00 and

202.35MHz respectively. The spectrum of F1 was recorded with a

spectral width of 5070� 1272Hz, a 1.4-s pulse delay, and 32 times �256 total scans.

Results

Preparation of WTAThe crude WTA extracted from the cell wall of

L. plantarum NRIC 1068 was purified on the DEAE andSephadex columns. A neutral polysaccharide withglucose and rhamnose has been reported in the cellwall of L. plantarum,7) but we did not detect it duringthe WTA purification process.

Preparation of monomeric units of WTAHPLC profiles of an alkaline hydrolysate of WTA

revealed two peaks of sugar phosphates (Fig. 1). Thesepeaks were fractionated as fraction 1 (F1) and fraction 2(F2) of the WTA monomers, and were purified bycharcoal chromatography. After lyophilization, approx-imately 30mg of each monomer was obtained from200mg of the purified WTA. Additionally, we usedalkaline phosphatase to prepare dephosphorylatedmonomers from F1 and F2, and the digested monomerswere isolated by charcoal chromatography. Approxi-mately 10mg of each dephosphorylated monomer (DPf1or DPf2) was obtained from 20mg of F1 or F2.

Structural analysis of DPf1 and DPf2Component analysis of DPf1 and DPf2 by HPLC

showed the presence of ribitol and glucose in bothmonomers. Additionally, there were no differences inthe 1H and 13C NMR spectra of DPf1 and DPf2: thesestructures were entirely identical. Therefore, DPf1 andDPf2 were dealt with in the subsequent investigation asthe same dephosphorylated monomer.The content of ribitol and glucose in the hydrolysate

of the dephosphorylated monomer was measured byHPLC analysis, and the molar ratio of ribitol to glucosewas calculated to be approximately 1:2. Examination ofthe NMR spectra of the dephosphorylated monomer alsoshowed two anomeric signals of glucose, at 5.18 and

Structure of Teichoic Acid from Lactobacillus plantarum 531

5.08 ppm on 1H NMR and at 100.75 and 96.61 ppm on13C NMR. These results suggest that the dephosphory-lated monomer consisted of one molecule of ribitol andtwo glucose residues. The coupling constant of theanomeric signals was 3.90Hz on 1H NMR and170.37Hz on 13C nuclear overhauser effect (NOE) 1Hnon-decoupling NMR. These values indicated that theglucose residues had �-glycosidic linkages. Addition-ally, the high positive optical rotation of the dephos-phorylated monomer (½��20D +123.2) also indicated thepresence of �-D-glycosidic linkages. Hence, the dephos-phorylated monomer was assumed to be �-D-diglucosylribitol.

On the basis of this structural assumption, threesynthetic monomers, 2,3-, 2,4- and 3,4-�-D-diglucosylribitol, were prepared to determine the steric config-uration of the glucose residues of the dephosphorylatedmonomer (Nukada et al., in preparation). The structuresof these synthetics were clear from the exclusivesynthetic pathways, and we were able to distinguishthem by NMR spectroscopy. On the basis of compar-isons between the isolate and the chemical synthetics,the optical rotation and 1H and 13C NMR spectra ofthe isolate corresponded to that of 3,4-�-D-diglucosylribitol. Thus the structure of the dephosphorylatedmonomer of WTA from the L. plantarum NRIC 1068cell wall was identified as 3,4-�-D-diglucosyl ribitol.The molecular formula of the dephosphorylated mono-mer was identified as C17H32O15 by TOF-MS (m=z477.1808 ½Mþ H�þ, calculated for 477.1819).

To assign the signals in the 1H and 13C NMR spectraof the dephosphorylated monomer, we carried outvarious two-dimensional NMR studies: H–H COSY,

H–C HSQC, and H–C CT-HMBC. In the COSY andHSQC spectra, we assigned the signals of two glucoseresidues using anomeric resonance as a starting point forthe analysis. Among the other signals, signals at 62.38and 63.29 ppm were assigned to C-1 and C-5 of ribitolbecause they were methylene carbons, and signals at80.59 and 80.20 ppm were identified as C-3 and C-4 ofribitol by observation of the lower field shift caused bybinding of the glucose residue. Therefore, the signal at71.45 ppm was logically assigned to C-2 in the ribitol.Finally, the 1H signals of the ribitol residue were fullyassigned using the CT-HMBC spectrum. The chemicalshifts were listed in Table 1.

Structural analysis of F1 and F2The structures of F1 and F2 were analyzed on the

basis of assignment of the dephosphorylated monomer,and the glucose residues were assigned on the basis ofthe two-dimensional NMR spectra of the monomers(Fig. 2). Although F1 and F2 were isolated as differentpeaks on the HPLC chromatogram, their dephosphor-ylation products were both determined to be 3,4-�-D-diglucosyl ribitol. This indicated that the structuraldifference between F1 and F2 existed at the binding siteof the phosphoric acid group. The coupling that occurredbetween carbon and phosphorus was useful in identify-ing the position of that group. Signals with couplingwere extracted by observation of the 13C NMR spectraof F1 and F2 (Fig. 3B and C).In the 13C NMR spectrum of F1, three signals with

coupling appeared at 78.85 (JCP ¼ 6:5), 76.14 (JCP ¼5:5) and 61.77 (broad) ppm (Fig. 3B). These signalswere assigned to C-3, C-2, and C-1 respectively, in theribitol residue by two-dimensional NMR. Hence wesuggest that the phosphoric acid of F1 was linked to C-2of the ribitol residue. This suggestion was confirmed byexamination of the H–P CT-HMBC spectrum, whichrevealed a correlation between phosphorus and H-2 inthe ribitol residue (data not shown). Thus F1 wasdetermined to be 3,4-�-D-diglucosyl-2-phosphoryl ribi-tol (Fig. 4). The chemical shifts are listed in Table 2.The molecular formula of F1 was identified asC17H33O18P by TOF-MS (m=z 557.1442 ½Mþ H�þ,calculated for 557.1483).We then analyzed the structure of F2 by a similar

procedure. In the 13C NMR spectrum of F2, only onesignal with coupling appeared, at 66.57 (broad) ppm(Fig. 3C), and it was assigned to C-1 of the ribitolresidue. Thus, F2 was determined to be 3,4-�-D-diglucosyl-1-phosphoryl ribitol (Fig. 4). The chemicalshifts are listed in Table 3. Although C-2 of the ribitolresidue was invisible due to overlapping with the signalof C-4 of the glucose residues, the signal of C-2

Table 1. 1H and 13C Chemical Shifts of the Dephosphorylated Monomer

Unit Nucleus 1 (1a, 1b) 2 3 4 5 (5a, 5b) 6 (6a, 6b)

Rbo1H 3.78, 3.85 4.03 3.93 4.07 3.82, 3.8413C 63.29 71.45 80.59 80.20 62.38

Glc1 ! 31H 5.08 3.55 3.66 3.43 3.84 3.78, 3.8613C 100.75 72.25 73.44 70.19 73.36 61.19

Glc1 ! 41H 5.18 3.56 3.75 3.44 3.87 3.77, 3.8213C 98.61 72.34 73.65 70.22 72.66 61.08

F1

F2

0 5 10

RIU

Retention time (min)

Fig. 1. Chromatogram of NaOH Hydrolysate of WTA Isolated fromthe L. plantarum NRIC 1068 Cell Wall.HPLC analysis was carried out on an Asahipac NH2P-50 4E

column. Sugar phosphates were eluted with 50mM sodium phos-phoric acid buffer (pH 4.4, 1.0ml/min, 30 �C), and were detectedwith a refractive index detector.

532 S. TOMITA et al.

probably showed coupling between carbon and phos-phorus. The molecular formula of F2 was identified asC17H33O18P by TOF-MS (m=z 557.1484 ½Mþ H�þ,calculated for 557.1483).

Discussion

We used NMR spectroscopy to analyze two mono-meric units (F1 and F2) prepared from ribitol-type WTA

B1

C1

B3 B2,C2

B6,C6

C3

A1

B4,C4

B5

C5

A2A3

A4

A5

B1C1

B3 B2,C2

B6,C6

C3

A1

B4,C4

B5

C5A2

A3A4

A5

F1

F2

Fig. 2. HSQC Spectra of Monomeric Units of WTA Isolated from the L. plantarum NRIC 1068 Cell Wall.

∗ ∗ ∗

∗

A

B

C

100 90 80 70 60 ppm

100 90 80 70 60 ppm

100 90 80 70 60 ppm

Fig. 3. 13C NMR Spectra of WTA Monomers.A, Dephosphorylated monomer; B, F1; C, F2. At asterisked peaks, coupling between carbon and phosphorus was observed.

Structure of Teichoic Acid from Lactobacillus plantarum 533

of L. plantarum NRIC 1068 cell walls. F1 and F2 werenovel compounds, identified as 3,4-�-D-diglucosyl-2-phosphoryl ribitol and 3,4-�-D-diglucosyl-1-phosphorylribitol respectively, and the chemical shifts of thesemonomers were obtained as novel information on WTAstructure. In an earlier study, Archibald et al. analyzedthe structure of ribitol-type WTA in the cell walls ofL. plantarum and discovered the presence of 3,4-�-D-diglucosyl ribitol as a monomeric structure.15) Thepositions of the glucose residues were also confirmed byNMR spectroscopy in this study. In addition, wedetermined the positions of the phosphoric acid residues.The phosphoric acid residue of F1 was bound to C-2 ofribitol, and that of F2 was bound to C-1 of ribitol.Generally, the phosphoric acid residues of ribitol-typeWTA bind to the terminal of ribitol, and the WTAconsists of phosphodiester linkages between C-1 andC-5 of the ribitol.8) However, we obtained not only a1-phosphoryl ribitol unit (F2) but also a 2-phosphorylribitol unit (F1). Hence we suggest that the WTA

configuration of this strain consists of a complicatedrepeating unit. The existence of a WTA structure thatcontains two monomers differing in the position of thephosphoric acid residue is exceptional.F1 and F2 were also characterized by a structure

with two glucose residues. Ribitol-type WTA is presentin the cell walls of B. subtilis, S. aureus, and Listeriamonocytogenes as well as L. plantarum, and the WTAstructures of these organisms have been elucidated,17,18)

but multiple glucosylated monomers such as F1 and F2are unknown outside L. plantarum NRIC 1068. Sofar, several rare WTA structures have been found inother organisms, which contain arabitol, erythritol, ormannitol in the main chain and are modified byN-acetylglucosamine or 6-O-alanylglucose.19–23) Theseorganisms have unique WTA structures with variedstructural components. In contrast, L. plantarum appearsto use only glucose and ribitol, the most generalcomponents of WTA, and to have a unique structurecontaining two glucose residues. The unique structure ofthe WTA in L. plantarum is created by modificationwith multiple glucose residues, which makes it possible,in theory, to expand the structural diversity of WTA inL. plantarum cell walls. This characteristic modificationcontributes to the diversity of WTA at the strain level.The structural diversity provided by WTA probably

correlates with functions of the cell wall, but therelationships between the structural diversity, especiallythe glycosyl residues, and the functions of WTA arenot yet established. Neuhaus and Baddiley explainedthat many bacteriophages showed binding specificityfor accessible WTA on cell surface of Gram-positivebacteria.8) Additionally, several studies of bacterio-phages have reported a relationship between glycosylresidues in WTA and phage adsorption to the bacterialcell wall.24–26) Therefore, the WTA of L. plantarumare also clearly required for avoiding bacteriophageinfection. The multiple glucosylated monomers ofWTA of L. plantarum are suggested to be more usefulagainst the infection by bacteriophages due to thestructural diversity by modification with multiple glu-cose residues.

O

HO

HO

O

OH

HO

OR1

O OR2

HO

HOO

HOOH

OH R1 = HR2 = H

R1 = HR2 = PO3H2

R1 = PO3H2R2 = H

Dephosphorylatedmonomer

F1

F2

12

34

5

6"

1"2"

3"

4"5"

6'

1'2'

3'

4'

5'

Fig. 4. Structures of the Monomeric Units of WTA Isolated from theL. plantarum NRIC 1068 Cell Wall.

Table 2. 1H and 13C Chemical Shifts of F1

Unit Nucleus 1 (1a, 1b) 2 3 4 5 (5a, 5b) 6 (6a, 6b)

Rbo (A)1H 3.95, 3.96 4.47 4.17 4.10 3.84, 3.8613C 61.77 (broad) 76.14 (JCP ¼ 5:5) 78.85 (JCP ¼ 6:5) 79.98 62.32

Glc(1 ! 3) (B)1H 5.18 3.55 3.67 3.43 3.84 3.79, 3.8813C 100.53 72.22 73.42 70.13 73.36 61.17

Glc(1 ! 4) (C)1H 5.19 3.54 3.77 3.43 3.89 3.79, 3.8313C 98.70 72.48 73.64 70.20 72.63 61.05

Table 3. 1H and 13C Chemical Shifts of F2

Unit Nucleus 1 (1a, 1b) 2 3 4 5 (5a, 5b) 6 (6a, 6b)

Rbo (A)1H 4.08, 4.12 4.13 3.95 4.10 3.82, 3.8213C 66.57 (broad) 70.08 79.59 80.15 62.39

Glc(1 ! 3) (B)1H 5.10 3.55 3.64 3.41 3.84 3.76, 3.8513C 100.66 72.02 or 72.23 73.12 70.08 73.18 61.05

Glc(1 ! 4) (C)1H 5.20 3.55 3.75 3.42 3.86 3.75, 3.8013C 98.42 72.02 or 72.23 73.42 70.08 72.45 60.91

534 S. TOMITA et al.

It is conceivable that the structural diversity seen inWTA is also present in other lactic acid bacteria, butlittle is known about the structures of these otherspecies.27,28) Minimal knowledge of the WTAs ofLactobacillus buchneri and L. helveticus has beenobtained by chemical decomposition methods. Althoughthe WTA of L. plantarum has been the most frequentlystudied among the WTAs of lactic acid bacteria, themonomeric structures of glycerol-type WTA have notyet been determined. Therefore, it is not too much to saythat our knowledge of the WTA structure in lactic acidbacteria is still at a basic level. In this context, ourelucidation of the structure of WTA in L. plantarum bymodern NMR techniques is a step toward understandingthe importance of WTA in the cell wall of lactic acidbacteria.

Acknowledgments

We thank Professor H. Seto (Faculty of AppliedBioscience, Tokyo University of Agriculture) forvaluable discussions on NMR analysis. We are alsograteful to M. Koumoto for preparation of the syntheticWTA monomers.

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Structure of Teichoic Acid from Lactobacillus plantarum 535