JOURNAL OF BACTZIUOLOGY, June 1977, P. 1055-1063Copyright 0 1977 American Society for Microbiology

Vol. 130, No. 3Printed in U.S.A.

Biosynthesis of Wall Polymers in Bacillus subtilisANNE W. WYKE AND J. BARRIE WARD*

National Institute for Medical Research, Mill Hill, London, NW7 1AA, England

Received for publication 15 February 1977

Preparations of membrane plus wall derived from Bacillus subtilis W23 wereused to study the in vitro synthesis of peptidoglycan and teichoic acid and theirlinkage to the preexisting cell wall. The teichoic acid synthesis showed anordered requirement for the incorporation ofN-acetylglucosamine from uridine5'-diphosphate (UDP)-N-acetylglucosamine followed by addition of glycerolphosphate from cytidine 5'-diphosphate (CDP)-glycerol and finally by addition ofribitol phosphate from CDP-ribitol. UDP-N-acetylglucosamine was not onlyrequired for the synthesis of the teichoic acid, but N-acetylglucosamine residuesformed an integral part of the linkage unit attaching polyribitol phosphate tothe cell wall. Synthesis of the teichoic acid was exquisitely sensitive to theantibiotic tunicamycin, and this was shown to be due to the inhibition ofincorporation ofN-acetylglucosamine units from UDP-N-acetylglucosamine.

It is well established that in many gram-positive bacteria the two major polymers of thewall, peptidoglycan and teichoic acid, arelinked covalently. In Staphylococcus lactis 13,the teichoic acid, a polymer ofN-acetylglucosa-mine-i-phosphate and glycerol, is linkedthrough a phosphodiester bond to the 6-hy-droxyl group of a muramic acid residue in thepeptidoglycan (4). Chemical evidence also es-tablished that in S. aureus a trimer of glycerolphosphate acts as a linkage unit between thepolyribitol phosphate teichoic acid and the pep-tidoglycan (8), although the nature of the bondbetween the peptidoglycan and the linkage unitwas not determined.

In vivo experiments with Bacillus subtilis(19) and Diplococcus pneumoniae (25) haveshown that newly synthesized teichoic acid islinked only to peptidoglycan synthesized con-comitantly. These observations were largelyconfirmed in vitro with preparations of mem-brane plus wall from B. licheniformis (31),where the linkage of 80% of the newly synthe-sized teichoic acid, a 1,3 poly(glycerol phos-phate), required the concomitant synthesis ofcross-linked peptidoglycan. However, 20% ofthe teichoic acid was linked to the preexistingwall in the absence of peptidoglycan synthesis.More recently, we have isolated newly synthe-sized muramic acid phosphate from membrane+ wall preparations from B. licheniformis inwhich in vitro synthesis of both teichoic acidand peptidoglycan had occurred (32). Synthesisof the muramic acid phosphate required thesimultaneous synthesis of both polymers, andthe phosphate moiety was derived from uridine

5'-diphosphate-N-acetylglucosamine (UDP-GlcNAc).Bracha and Glaser (5) used similar enzyme

preparations to investigate the linkage of poly-ribitol phosphate teichoic acid to the peptido-glycan ofS. aureus. In this organism, synthesisof linked teichoic acid required adenosine 5'-triphosphate, cytidine 5'-diphosphate (CDP)-glycerol and UDP-GlcNAc in addition to CDP-ribitol. Moreover, increased concentrations ofUDP-GlcNAc stimulated the incorporation ofglycerol phosphate from CDP-glycerol intopreexisting wall. Simultaneously, Hancock andBaddiley (14) reported similar results showingthat UDP-GlcNAc stimulated the incorporationof glycerol phosphate from CDP-glycerol intopolymeric material synthesized by membranepreparations from a number ofother gram-posi-tive organisms.

In this communication, we describe a mem-brane + wall preparation from B. subtilis W23,which utilized UDP-GlcNAc, CDP-glycerol,and CDP-ribitol to synthesize polymeric mate-rial linked to the preexisting wall. The se-quence of addition from these nucleotide pre-cursors in the synthesis of this polymer andsome aspects of its chemical composition wereinvestigated. These membrane + wall prepara-tions can also synthesize peptidoglycan and awall-linked polymer derived from UDP-GlcNAc.

MATERIALS AND METHODSThe organism used, B. subtilis B7130, was a mu-

tant defective in the glucosylation of teichoic acid,derived from B. subtilis B71 met glyc glpD (a strain

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ofB. subtilis W23) (20): Cultures were grown underthe conditions described previously for B. lichenifor-mis (28) in mediumi supplemented with methionine(100 ,ug/ml) and glycerol (0.01%, vol/vol). Organismswere harvested in the mid-exponential phase ofgrowth, and the membrane + wall preparationswere isolated as described previously (28), exceptthat the buffer used for washing and final suspen-sion of the enzyme preparation was 50 mM tris(hy-droxymethyl)aminomethane-hydrochloride buffer(pH 7.8) containing 60 mM MgCl2 and 1 mM dithio-threitol (TMD-buffer). As used, the membrane +wall preparations contained 10 to 15 mg of proteinper ml and 40 to 60 mg (dry weight) of walls per ml.

Materials. UDP-N-acetyl[U-_4C]glucosamine (300mCi/mmol), [U-14C]glycerol (46 mCi/mmol), [y-32P]adenosine 5'-triphosphate (16.25 Ci/mmol), andNaB3H4 (870 mCi/mmol) were purchased from theRadiochemical Centre, Amersham, United King-dom. L-a-Glycerol 3-phosphate radioactively labeledin either the glycerol or phosphate moiety was pre-pared from [U-14C]glycerol or [y-32P]adenosine 5'-tri-phosphate, respectively, and was used for the enzy-matic synthesis of CDP-glycerol as previously de-scribed (22). The nucleotides were routinely used atspecific activities of 2.5 mCi/mmol for 14C and 5 mCi/mmol for 32p. Non-radioactive CDP-glycerol wasalso prepared by this method. Similarly, CDP-ribi-tol was synthesized from D-ribitol 5-phosphate andCTP by using a cytoplasmic extract obtained from B.subtilis B71 as a source of CDP-ribitol pyrophospho-rylase (EC 2.7.7.40). D-Ribitol 5-phosphate was pre-pared by reduction of D-ribose 5-phosphate withNaBH4. Reduction with NaB3H4 yielded D-[3H]ribitol 5-phosphate, which was used for synthe-sis of CDP-[3H]ribitol at specific activity of 6.28 mCi/mmol. UDP-N-acetylmuramyl-LAla-DisoGlu-mA2-pm-DAla-DAla (UDP-MurAc-pentapeptide) radioac-tively labeled with either diamino[3H]pimelic acid(17.4 mCi/mmol) or [14C]muramic acid (12.4 mCi/mmol) were prepared as previously described (28,29). Non-radioactive UDP-MurAc-pentapeptide wasisolated from B. licheniformis after accumulation ina medium lacking Mg2+ (11). [32P]UDP-GlcNAcwas obtained from cultures of Micrococcus luteusgrown in 1% peptone (Difco) containing glucose(0.2%), NaCl (0.5%), and 32P-labeled inorganic phos-phate and inhibited with vancomycin. The specificactivity of the nucleotide obtained ranged from 2.5to 5.6 mCi/mmol. Tunicamycin, supplied by G. Ta-mura, University of Tokyo, Tokyo, Japan, was thekind gift of A. H. Rose, University of Bath, Bath,United Kingdom. Other antibiotics were obtainedfrom various commercial sources as previously de-scribed.

Determination of polymer synthesis. Biosyn-thesis of polymeric material was carried out for 30min at 28°C. The complete reaction mixture con-tained in a final volume of 200 ,ul: 0.5 mMUDP-GlcNAc, 0.5 mM CDP-glycerol, 0.31 mM CDP-[3H]ribitol (6.28 mCi/mmol), and membrane + wallpreparation (150 ,ul). The reaction was terminatedby the addition of sodium dodecyl sulfate (SDS;10%, 200 ,u), and the SDS-insoluble fraction wasisolated as previously described (31). In certain

cases, the SDS-soluble material was examined bypaper chromatography after removal of the bulk ofthe SDS by precipitation at 0°C followed by centrifu-gation. The supernatant was then concentrated byfreeze-drying prior to chromatography. In experi-ments involving the preincubation of the enzymepreparation with certain substrates, excess nucleo-tides were removed after the first incubation bydiluting the incubation mixture (200 ,l) with ice-cold TMD buffer (3 ml) and centrifuging (2 min,17,000 x g). After being washed twice more withcold buffer (3 ml), the membrane + wall prepara-tions were resuspended in buffer (100 AD) prior to thesecond incubation.

Analytical methods. Protein was determined bythe method of Lowry et al. (18), with bovine serumalbumin as the standard. Amino sugars were deter-mined with a Beckman-Spinco automatic aminoacid analyzer after hydrolysis of the samples in 4 MHCI at 100°C for 4 h. Glycerol and phosphorus weredetermined by published methods (1, 30). Periodateoxidation and the formation of the dimedone com-plex of the released formaldehyde were carried outas described by Kennedy and Shaw (16). Radioactiv-ity in SDS-insoluble material and in liquid sampleswas determined in a Packard Tri-Carb 3385 scintil-lation counter, using toluene-based scintillationfluid [toluene containing 2,5-diphenyloxazole(0.4%), 1,4-bis-(4-methyl-5-phenyloxazol-2-yl)ben-zene (0.01%), and Biosolv (20%)]. Radioactivity onpaper was determined by counting the paper di-rectly in toluene-based scintillation fluid.Whatman no. 3 paper was used for descending

chromatography in the following solvent systems:A, ethanol-i M ammonium acetate (pH 3.8; 5:2, vol/vol); B, propan-l-ol-NH3-water (6:3:1, by volume);C, butan-1-ol-pyridine-water (6:4:3, by volume); D,butan-1-ol-acetic acid-water (3:1:1, by volume); E,isobutyric acid-1 M NH3 (5:3, vol/vol). Electrophore-sis on Whatman no. 3 paper was carried out at 70 V/cm in the following buffer systems: F, pyridine-acetic acid-water (pH 6.5; 50:2:948, by volume); G,pyridine-acetic acid-water (pH 3.5; 1:10:989, by vol-ume).

RESULTSPreliminary experiments with membrane +

wall preparations derived from B. subtilis B71synthesized small and variable amounts of bothpeptidoglycan and teichoic acid. Polymer syn-thesis was measured by the incorporation ofeither diamino[3H]pimelic acid or [3H]ribitolinto SDS-insoluble material after incubationwith the appropriate nucleotide precursors. Re-sistance of the radioactive labels to extractionby hot SDS has previously been taken as indi-cating that the newly synthesized material iscovalently bound to the preexisting wall (5, 31).Experiments with B. licheniformis had shownpreviously that mutants that are phosphogluco-mutase deficient and have reduced levels ofautolytic enzymes are generally more efficient

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in cell-free synthesis of peptidoglycan than thewild-type organism (28). We therefore exam-ined mutants of B. subtilis B71 that were un-able to synthesize glucosylated teichoic acid forenhanced activity of polymer synthesis. Thefollowing experiments were all carried out withone such mutant, B. subtilis B7130, which syn-thesized increased amounts of both peptidogly-can and teichoic acid and which did not showthe variability in yields observed with the par-ent organism.

Biosynthesis of peptidoglycan. Incubationof the membrane + wall preparations withUDP-GlcNAc and UDP-MurAc-pentapeptideresulted in the synthesis of peptidoglycan (Ta-ble 1). Since this synthesis was not inhibited byeither benzylpenicillin or cephaloridine (10 ,ug/ml), we conclude that incorporation of thenewly synthesized material occurs by a trans-glycosylation mechanism rather than by trans-peptidation. The incorporation of radioactivityfrom UDP-MurAc-pentapeptide was, however,dependent on the presence of UDP-GlcNAc. Incontrast, when UDP-['4C]GlcNAc was incu-bated with the membrane + wall preparations,radioactivity was incorporated into the SDS-insoluble material whether or not UDP-MurAc-pentapeptide was present (Table 1). Again thisincorporation was insensitive to the presence ofbeta-lactam antibiotics. Further studies on thenature of the material synthesized from UDP-[14C]GlcNAc are described below.Biosynthesis of teichoic acid. The incorpo-

TABLE 1. Biosynthesis ofpeptidoglycan bymembrane + wall preparations from B. subtilis

B7130a

SDS-in-solublepolymer

Radioactive precursor Other additions (pmol/mg ofproteinper 30min)

UDP-MurAc-[3H-A2 UDP-GlcNAc 231pm]pentapeptide

UDP-MurAc-[3H-A2 21pm]pentapeptide

UDP-MurAc-[3H-A2 UDP-GlcNac + 251pm]pentapeptide cephaloridine (10

j.g/ml)UDP-['4C]GlcNAc UDP-MurAc-penta- 769

peptideUDP-['4C]GlcNAc 842

a The incubation mixture contained in a total volume of200 g1: 0.5 mM UDP-MurAc-pentapeptide labeled whereindicated with diamino[3H]pimelic acid (17.4 mCi/mmol),0.1 mM UDP-GlcNAc (2.5 mCi/mmol), and membrane +wall preparation (150 Ml). After incubation at 28°C for 30min, the SDS-insoluble fraction was prepared as describedin the text.

ration of [3H]ribitol from CDP-[3H]ribitol intoSDS-insoluble material required the presenceof both UDP-GlcNAc and CDP-glycerol (Table2). Incorporation increased with time, reachinga plateau after 20 to 30 min of incubation. Atthis time, polymeric material in the SDS-solu-ble fraction (i.e., material remaining on theorigin after paper chromatography in solventE) was generally present in a four- to fivefoldexcess over the SDS-insoluble material. In-creasing the concentrations of UDP-GlcNAc inthe incubation mixture from 0.1 to 0.5 mM ledto a fourfold increase in the amount of[3H]ribitol incorporated into SDS-insoluble ma-terial, but the amount of SDS-soluble polymerformed was not investigated.

In contrast to previous findings with B. Ii-cheniformis (31), synthesis of the SDS-insolu-ble teichoic acid was independent of the pres-ence of UDP-MurAc-pentapeptide. Under theassay conditions as described in Table 2, theamount of [3H]ribitol incorporated in the pres-ence and absence of UDP-MurAc-pentapeptide(0.5 mM) was 1,491 and 1,467 pmol, respec-tively. Pretreatment of the enzyme prepara-tions with uridine-5'-monophosphate to removeendogenous membrane-bound intermediates ofpeptidoglycan biosynthesis by reversal of thephospho-MurAc-pentapeptide translocase (13)was without effect on the subsequent incorpora-tion of radioactivity. Thus, in these enzymepreparations, the concomitant synthesis of pep-tidoglycan does not appear to be a requirementfor the synthesis of SDS-insoluble teichoic acid.

Incubation of membrane + wall preparationswith CDP-[14C]glycerol (0.25 mM) alone led tothe incorporation of [14C]glycerol (181 pmol/mgof protein per 30 min) into the SDS-insolublefraction. When UDP-GlcNAc (0.5 mM) was alsoincluded in the incubation mixture, the amountof [14C]glycerol increased (345 pmol); the pres-ence or absence of CDP-ribitol had no effect onthis incorporation.

TABLE 2. Biosynthesis ofteichoic acid by membrane+ wall preparations from B. subtilis B7130a

System

Complete- UDP-GlcNAc- CDP-glycerol

SDS-insoluble teichoic acid(pmol of [3H]ribitol/mg of

protein per 30 min)

71833104

a The complete system for teichoic acid biosyn-thesis included in a total volume of 200 ,ul: 0.1 mMUDP-GlcNAc, 0.05 mM CDP-glycerol, 0.31 mMCDP-[3H]ribitol (6.28 mCi/mmol), and membrane +wall preparation (150 ul). Incubation conditions andisolation of the SDS-insoluble material were as de-scribed in the text.

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Preincubation of the enzyme preparation at28°C for 20 min with UDP-GlcNAc (0.5 mM)and CDP-glycerol (0.5 mM), followed by exten-sive washing to remove nucleotides (see Mate-rials and Methods), reduced the uptake of['4C]glycerol from CDP-[U4C]glycerol in a sec-ond incubation in the absence of UDP-GlcNActo 19 pmol. Moreover, similar preincubation(280C for 20 min) with UDP-GlcNAc and CDP-glycerol completely removed the requirementfor these nucleotides for the incorporation of[3H]ribitol such that, in the second incubation,uptake of [3H]ribitol (352 pmol) occurred whenCDP-ribitol was the only nucleotide present.This value should be compared with an incorpo-ration of 429 pmol obtained when all three nu-cleotide precursors were present in the secondincubation. Such preincubation experimentswere then used to establish the sequence ofincorporation of the various components of theteichoic acid. GlcNAc must be incorporated be-fore the glycerol phosphate (Table 3), and, sincea short preincubation with UDP-GlcNAc andCDP-glycerol allows addition of ribitol phos-phate from CDP-ribitol alone, addition of ribi-tol phosphate units must be the final stage ofthe synthesis.However, in contrast to these observations,

on increasing the time of the preincubationwith UDP-GlcNAc and CDP-glycerol from 20 to30 min, no subsequent addition of [3H]ribitolfrom CDP-[3H]ribitol occurred when UDP-

TABLE 3. Sequence of addition from nucleotideprecursors in teichoic acid biosynthesisa

SDS-insolu-ble teichoic

lst Incubationb 2nd Incubationb acid (Pmol Of[3Hlribitol/mg of pro-

tein)UDP-GlcNAc (0.5) CDP-glycerol (0.28)

CDP-[3H]ribitol 1,848(0.31)

CDP-glycerol (0.55) UDP-GlcNAc (0.5)CDP-[3H]ribitol 281

(0.31)

Water UDP-GlcNAc (0.5)CDP-glycerol (0.28) 1769CDP-[3H]ribitol

(0.31)The initial incubation mixture contained in a total

volume of 200 1l: membrane + wall preparation (150 pl)and the teichoic acid precursors as shown. After incubationfor 20 min at 28C, the precursors were removed by wash-ing, as described in the text. The washed membrane + wallpreparations were then reincubated, together with the pre-cursors shown, for a further 30 min, after which SDS-insoluble material was isolated.

b Numbers in parentheses indicate millimolar concen-trations.

GlcNAc and CDP-glycerol were omitted fromthe second incubation. This suggested that ribi-tol phosphate units may be added only toGlcNAc-glycerol units present in the enzymepreparation in some intermediate form and notto GlcNAc-glycerol units already bound topreexisting wall. To test this hypothesis, mem-brane + wall preparations were preincubatedfor 30 min with UDP-GlcNAc (0.5 mM) andCDP-[P4C]glycerol (0.5 mM), and the SDS-insol-uble fraction was prepared from one-half of thematerial at this stage (fraction A). The remain-der was washed extensively prior to a secondincubation with UDP-GlcNAc (0.5 mM), CDP-glycerol (0.5 mM), and CDP-[3H]ribitol (0.34mM) (fraction B). The SDS-insoluble materialfrom both was then subjected to periodate oxi-dation, and the amount ofradioactive fonnalde-hyde released was measured as the dimedonecomplex (16). One mole of [14C]formaldehydewould arise from oxidation of each terninal[140]glycerol residue, and 1 mol of[3H]formaldehyde would arise from each termi-nal ribitol residue. The ratio of radioactiveformaldehyde formed to the total radioactivityincorporated gives a measure of the chainlength. In fraction A, the total 14C incorporatedwas 9,725 dpm, ofwhich 1,698 14C dpm (17.5% ofthe total) was released as [140]formaldehyde; infraction B, 956 dpm of [14C]formaldehyde(16.9%) was obtained from the total 5,673 14Cdpm incorporated. Thus, the number of[14C]glycerol termini was the same in both frac-tions and corresponded to an average chainlength of 1.95 glycerol units in each case. Inaddition, 2,047 dpm of [3Hlformaldehyde wasobtained from a total of 36,507 dpm of[3H]ribitol incorporated into fraction B, corre-sponding to a chain of 18.8 ribitol units in thisexperiment. Addition of ribitol phosphate unitsto glycerol termini already present in the wallafter the preincubation would lead to a decreasein the number of [14C]glycerol molecules sus-ceptible to periodate oxidation. Since this wasnot the case, we conclude that ribitol phosphateunits must be added to GlcNAc-glycerol unitspresent in the enzyme preparation at the sametime as the CDP-ribitol. In experiments usingpreincubation with UDP-GlcNAc and CDP-glycerol for periods of up to 20 min, followed byomission of these nucleotides in the second in-cubation, the portion of the carrier containingthe GlcNAc and glycerol residues must remainin the membrane + wall preparation duringthe second incubation. Ribitol phosphate unitsare then added to this carrier before transfer ofthe completed polymer to the wall. In the ab-sence of added CDP-ribitol, GlcNAc and glyc-

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erol units can be transferred from the carrier tothe wall.

Effect of antibiotics on teichoic acid syn-thesis. Among the antibiotics known to inhibitvarious stages of peptidoglycan biosynthesis, anumber have also been reported to inhibit tei-choic acid synthesis. Vancomycin inhibitedpolyribitol phosphate synthesis by an enzymepreparation from Lactobacillus plantarum (12)and the incorporation of glycerol into a water-soluble polymer by membranes of B. subtilisW23 (14); however, bacitracin only inhibitedteichoic acid synthesis by membrane prepara-tions of B. licheniformis if peptidoglycan syn-thesis was occurring simultaneously (3). In thepresent investigation, these antibiotics had nosignificant effect on the incorporation of ribitolinto SDS-insoluble material, with vancomycin(1,250 ,tg/ml) and bacitracin (400 ,tg/ml) givingless than 10% inhibition. Teichoic acid synthe-sis had already been shown to be independentof concomitant peptidoglycan synthesis (seeabove) and, as expected, the beta-lactam anti-biotics benzylpenicillin and cephaloridine hadno inhibitory effect on the incorporation of[3H]ribitol. In contrast, the synthesis of teichoicacid was extremely sensitive to inhibition bytunicamycin, with greater than 90% inhibitionof [3H]ribitol incorporation occurring at an an-tibiotic concentration of 1 ,ug/ml (Fig. 1). Pre-incubation experiments, such as described inthe previous section, revealed that only theutilization ofUDP-GlcNAc was inhibited. Once

0 5 10 20Tunicomycin (ug/mni)

FIG. 1. Effect of tunicamycin on teichoic acid bio-synthesis in B. subtilis B7130. Assays containedUDP-[U-'4CJGlcNAc (0.48 mM), CDP-glycerol (028mM), CDP-[3H]ribitol (0.31 mM), and tunicamycinas indicated. Radioactivity in SDS-insoluble mate-rial was measured: *, [3H]ribitol; *, ['4C]GkcNAc.

the incorporation of GlcNAc had occurred, thesynthesis of the complete polymer from CDP-glycerol and CDP-[3H]ribitol was unaffected byconcentrations of tunicamycin that inhibitedsynthesis of the complete polymer by more than97% (Table 4). When UDP-[U-14C]GlcNAc wasincubated with the membrane + wall prepara-tion in the presence of CDP-glycerol and CDP-ribitol, the incorporation of GlcNAc into SDS-insoluble material was also inhibited by tunica-mycin but to a lesser extent than the ribitolincorporation (Fig. 1). This result could be ex-plained if GlcNAc is also incorporated into an-other SDS-insoluble polymer in addition to theteichoic acid, the synthesis of this other productbeing unaffected by tunicamycin. Tunicamy-cin-sensitive synthesis of lipid-linked interme-diates in teichoic acid synthesis has recentlybeen demonstrated in membranes ofB. subtilisW23 and S. aureus (6, 15). In addition, themembranes of S. aureus could synthesize poly-meric material from UDP-GlcNAc alone, whichwas not inhibited by tunicamycin (6).Participation of lipid intermediates in tei-

choic acid synthesis. Tunicamycin is known toinhibit the synthesis of polyisoprenyl N-acetyl-glucosaminyl pyrophosphate intermediates (17,23, 24) and, since teichoic acid synthesis wasexquisitely sensitive to tunicamycin, it seemslikely that a polyprenol-linked intermediatemay be involved. Attempts to isolate a lipidintermediate in teichoic synthesis were madeby incubating the membrane + wall prepara-tion with CDP-[3H]ribitol in the presence ofUDP-GlcNAc and CDP-glycerol with one orother of these latter nucleotides labeled with14C, followed by extraction with 2 volumes ofbutan-1-ol-6 M pyridinium acetate (pH 4.2; 1:1,

TABLE 4. Site of tunicamycin inhibition of teichoicacid biosynthesisa

UDP-G1cNAc

UDP-GIcNAcCDP-glycerol

UDP-GIcNAcCDP-glycerolCDP-ribitol

CDP-grYCerolCDP-ribitol

CDP-ribitOl1

Tunica-mycin (20ZgIml)

+

+

SDS-insolubleteichoic acid

(pmol of[3H]ribitol/mgof protein)

1,331

37

751729

638661

a Substrates and incubation times were as de-scribed in footnote a of Table 3. Tunicamycin waspresent during the second incubation where indi-cated.

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vol/vol). This solvent was chosen for the extrac-tion of possible teichoic acid-lipid intermediatessince it quantitatively extracts the undeca-prenyl lipid intermediates of peptidoglycansynthesis without extracting either the nucleo-tide precursors or the peptidoglycan (2). Bu-tanol-soluble material containing GlcNAc andglycerol was detected, but no [3H]ribitol wassolubilized. A carrier containing GlcNAc andglycerol residues attached to undecaprenolwould probably be soluble under these condi-tions, whereas the further addition of a longchain of ribitol phosphate units would increasethe hydrophilic nature of the material and thusrender it insoluble in the organic solvent. IfUDP-GlcNAc was omitted from the incubationmixture, incorporation of ['4C]glycerol was re-duced from 664 to 320 pmol/mg ofprotein per 30min. Small amounts of the glucosamine-con-taining acceptor preexisting in the membrane+ wall preparation could account for the incor-poration of [14C]glycerol under these conditions.Furthermore, formation of butan-1-ol-solubleGlcNAc increased from 646 to 1,617 pmol/mgper 30 min when CDP-glycerol and CDP-ribitolwere omitted from the incubation mixture.This could reflect either an increased synthesisof the GlcNAc lipid intermediate or, alterna-tively, the synthesis of polymeric GlcNAclinked to undecaprenol pyrophosphate as de-scribed for membranes ofB. subtilis 168 (G. E.Bettinger, A. N. Chatteijee, and F. E. Young,Fed. Proc. 34:668, 1973).Characterization of the newly synthesized

teichoic acid. Treatment with 60% HF (-2°Cfor 16 h) (26) of wall preparations containingteichoic acid synthesized from CDP-[3H]ribitolas the only radioactive precursor released morethan 90% of the radioactivity from the walls.Cleavage with HF would be expected to hydro-lyze all phosphoester linkages, but the releasedradioactivity remained at the origin upon chro-matography in solvent C. However, after alka-line hydrolysis (1 N KOH, 100°C for 3 h) fol-lowed by treatment with alkaline phosphatase,all of the radioactivity had the chromato-graphic mobility of ribitol.

Similar alkaline hydrolysis of SDS-insolublematerial prepared with CDP-[14C]glycerol asthe radioactive teichoic acid precursor gave amixture of glycerol, glycerol monophosphate,and glycerol diphosphate when chromato-graphed on a column of diethylaminoethyl-cel-lulose (HCO3i) (4). The identity of these var-ious components was further confirmed bychromatography in solvent B with the appro-priate marker compounds.

Cleavage with HF of material synthesized

from UDP-[14C]GlcNAc as the radioactive tei-choic acid precursor also released 90% of theincorporated radioactivity. Chromatography ofthe released material in solvent C showed that43.9% ofthe 14C present had a mobility identicalto that of authentic GlcNAc, a further 32.5%appeared as a discrete peak ofRG1CNAC = 0.5, andthe remaining 21.5% was located on the originof the chromatogram. Acid hydrolysis (4 NHCl, 100°C for 4 h) of these latter two fractionsgave glucosamine as the only radioactive com-ponent.

Periodate oxidation followed by isolation ofthe formaldehyde-dimedone complex gave anaverage chain length of 27.5 units for the newlysynthesized polyribitol phosphate. Similartreatment of material prepared from CDP-[14C]glycerol yielded no radioactive formalde-hyde. This would be expected if the glycerolphosphate units are part of a linkage unit be-tween the polyribitol phosphate and the preex-isting wall. On the assumption that newly syn-thesized polyribitol phosphate chains are onlylinked to newly synthesized glycerol phosphateunits, a calculation of the average chain lengthof glycerol phosphate units can be made froman average value of the polyribitol phosphatechain length compared with the total glycerolincorporation. On this basis, an average chainlength of 3.4 glycerol phosphate units was ob-tained.

Since the GlcNAc residues are also involvedin the linkage between the preexisting wall andthe glycerol phosphate oligomer, no free reduc-ing GlcNAc terminal is available for directmeasurement of the chain length. An indirectmethod of calculation of the chain length simi-lar to that used for the glycerol phosphate oligo-mer cannot be used for the GlcNAc portionsince UDP-[14C]GlcNAc is incorporated into an-other polymer (see below) in addition to theteichoic acid. SDS-soluble material was pre-pared by incubation of the enzyme preparationwith UDP- [14C]GlcNAc, CDP-glycerol, andCDP-[H]ribitol. Most of the SDS was removedby precipitation at 0C, and the water-solublepolymeric material was separated from theremaining detergent and the excess nucleo-tides by chromatography on a column (1 by70 cm) of Sephadex G75. Any polymeric ma-terial attached to a lipid carrier would havebeen expected to chromatograph in the deter-gent fraction. Periodate oxidation ofa sample ofthis material gave an average chain length of22.5 ribitol phosphate units, a value similar tothat found for SDS-insoluble material, suggest-ing that comparison of SDS-soluble and SDS-insoluble material is valid. An additional sam-

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ple of the SDS-soluble material was then re-duced with KBH4 (0.1 M in 0.01 M KOH) at 40Cfor 16 h and, after acid hydrolysis (4 N HCl,1000C for 4 h), the glucosamine and glucosami-nol were separated by chromatography on acolumn of Sephadex G10 equilibrated in boratebuffer (9). This gave a chain length of 1.4GlcNAc units.Release of the newly synthesized teichoic

acid from the preexisting wall. Since treat-ment with hot 5% SDS does not release thenewly synthesized teichoic acid from the preex-isting wall and further treatment with trypsinor with butan-1-ol-6 M pyridinium acetate (pH4.2; 1:1, vol/vol) is without effect, it is assumedthat the linkage involves a covalent bond. SDS-insoluble material containing [14C]GlcNAc and[3H]ribitol was synthesized in vitro, and thewall was solubilized completely by digestionwith Streptomyces muramidase in 0.05 M so-dium acetate buffer (pH 5.0; 350C for 48 h).Analysis showed that more than 90% of themuramic acid residues were then present asfree reducing groups. Chromatography of thesolubilized material on a column (0.5 by 17 cm)of diethylaminoethyl-cellulose (acetate form)gave two peaks, both ofwhich contained 3H and14C in a constant ratio. Extraction with 0.1 NNaOH under N2 at 00C for 24 h also completelysolubilized newly synthesized teichoic acid con-taining [14C]GlcNAc and [3H]ribitol. The alka-line extract was neutralized on a column (0.5 by5 cm) of Amberlite IR120 (H+) with 1Oo recov-ery of the radioactivity and then chromato-graphed on a column (0.5 by 17 cm) of Ecteola-cellulose (Cl-) eluted with a gradient of 0 to 0.5M LiCl. A single peak containing both radioac-tive labels was obtained (Fig. 2).Mild acid hydrolysis (0.1 N HCl, 1000C for 4

min) also solubilized the newly synthesized tei-choic acid, with all of the radioactivity beingreleased from the wall after four successive ex-tractions. This procedure also released morethan 97% of the phosphorus present in the cellwall of this organism. Material labeled in anytwo of the three teichoic acid components wassolubilized in this way and, upon electrophore-sis in buffers F or G or gel filtration on Sepha-dex G25, both labels always remained coinci-dent. We therefore conclude that the newlysynthesized teichoic acid contains GlcNAc,glycerol phosphate, and ribitol phosphate resi-dues linked together in a single polymer that iscovalently bound to the wall.Polymer synthesized from UDP-GIcNAc.

Membrane + wall preparations incorporatedradioactivity from UDP-[14C]GlcNAc into SDS-insoluble material in the absence of other nu-

ECL

0._

00

= .

xIn

2

0

00

Fraction

FIG. 2. Fractionation on Ecteola-cellulose of tei-choic acid newly synthesized from UDP-[U-'4C]GlcNAc, CDP-glycerol and CDP-[3H]ribitol andsolubilized by treatment with dilute alkali. 3H (A)and 14C (0) radioactivity was measured in 0.2-mlportions of the fractions.

cleotides (Table 1). This radioactivity couldsubsequently be solubilized by mild acid hy-drolysis and then remained at the origin ofchromatograms developed with solvents B andC. Further acid hydrolysis (4 N HCl, 1000C for 4h) of this solubilized material yielded only glu-cosamine when chromatographed in solvent C.Alkaline hydrolysis (1 N KOH, 1000C for 3 h) ofSDS-insoluble material yielded a major producthaving the electrophoretic behavior of glucosa-mine phosphate in buffer F. This also yieldedglucosamine after acid hydrolysis (4 N HCl,1000C for 4 h). This material has not been inves-tigated further but may be analogous to thepolymer synthesized from UDP-GlcNAc bymembranes from S. aureus (6).

DISCUSSIONMembrane + wall preparations from B. sub-

tilis B7130 were able to synthesize linear pepti-doglycan, teichoic acid, and a polymer ofGlcNAc, all of which were covalently bound tothe preexisting wall. Synthesis of these poly-mers can occur simultaneously, although eachalso occurs independently of the others. Tei-choic acid synthesis required an ordered incor-poration of residues from UDP-GlcNAc, CDP-glycerol, and CDP-ribitol, and all three compo-nents were linked together and attached to thepreexisting wall. Similar preparations from S.aureus (5) required these three nucleotides forteichoic acid synthesis, and toluenized cells ofMicrococcus sp. 2102 (14) similarly requiredUDP-GlcNAc for synthesis of the glycerol phos-phate linkage unit between teichoic acid andpeptidoglycan. However, in neither of thesecases was GlcNAc shown to be incorporatedinto the wall as part of the linkage region of thenewly synthesized teichoic acid.

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1062 WYKE AND WARD

The in vitro synthesis of teichoic acid by B.subtilis B7130 was.independent of peptidogly-can synthesis, unlike similar preparations fromB. licheniformis"in which teichoic acid synthe-sis required the concomitant synthesis of cross-linked peptidoglycan (31). In B. licheniformis,the two newly synthesized polymers are linkedtogether by a muramic acid phosphate bond,the phosphate arising from a GlcNAc residue inthe teichoic acid (32). However, no newly syn-thesized muramic acid phosphate was foundwhen enzyme preparations from B. subtilisB7130 synthesized teichoic acid alone or simul-taneously synthesized teichoic acid and linearpeptidoglycan. It has also been shown in B.licheniformis (27) and M. luteus (21) that linearpeptidoglycan synthesized in the presence ofbeta-lactam antibiotics has no associated phos-phorus. It therefore seems possible that mur-amic acid phosphate is only formed when newlysynthesized teichoic acid is linked to cross-linked peptidoglycan synthesized concomi-tantly.Recently two laboratories reported tunicamy-

cin-sensitive synthesis of lipid-linked interme-diates in the biosynthesis of teichoic acid bymembranes ofB. subtilis W23 andS. aureus (6,15). In both cases, UDP-GlcNAc was requiredfor synthesis of the intermediate. Bracha andGlaser (6) did not demonstrate that the inter-mediate synthesized by membranes from S. au-reus contained GlcNAc, although their pro-posed structure shows 1.5 to 2.0 GlcNAc resi-dues per molecule of intermediate, this figurehaving been calculated from measurements ofthe excess of glucosamine over muramic acid inthe teichoic acid-peptidoglycan complex ob-tained by digesting cell walls with lysostaphin.

CDfRglycerc

carrier-(Glc NAc)12

Tunicamycin

UD-01c NAc" ,

In the biosynthetic scheme proposed by Han-cock et al. (15), UDP-GlcNAc was required forthe addition of the glycerol phosphate trimer toa lipid acceptor, but GlcNAc was not incorpo-rated into the intermediate. The lipid portion ofthe intermediate was not identified in eithercase, although both used tunicamycin inhibi-tion to suggest that this was likely to be apolyprenol. Earlier, both Bracha and Glaser (5)and Hancock and Baddiley (14) had simultane-ously published similar schemes for the in vitrosynthesis of wall-linked teichoic acid in whichpolyribitol phosphate was transferred from alipoteichoic acid carrier either (i) to nascentpeptidoglycan that already contained glycerolphosphate and GlcNAc residues (5) or (ii) to aglycerol phosphate trimer that had previouslybeen attached to either a growing glycan chainor its polyprenyl-MurAc-pentapeptide-GlcNAcintermediate (14). Neither of these schemescould apply to the wall-linked teichoic acid syn-thesized in our present experiments since thisis clearly independent of any de novo peptido-glycan synthesis. The observations described inthis paper lead to the scheme for teichoic acidsynthesis shown in Fig. 3. GlcNAc residues arefirst added to a lipid carrier, which may be apolyprenol since this step is inhibited by tunica-mycin; secondly, a glycerol phosphate oligomeris added onto this carrier; and, finally, ribitolphosphate units are added onto the carrier mol-ecule. If ribitol phosphate units were addedindividually onto the carrier-GlcNAc-glycerolphosphate, the carrier molecules containingonly a few ribitol phosphate units would proba-bly have been solubilized by the butan-1-ol-6 Mpyridinium acetate; since this was not the case,we conclude that the polyribitol phosphate is

arrierlGlcNAc)-(glycerol-P)1.2 3

(ribitol-P)--CDP-ribitol28

arrier-(Glc NAc)-(glycerol-P)Hri bitol-P)/ 1.2 3 28

WA LL-PHGIcNAcHg1yceroI-PHribitot-P)13o 3 28

FIG. 3. Proposed scheme for the biosynthesis of teichoic acid linked to preexisting wall.

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BIOSYNTHESIS OF CELL WALL POLYMERS 1063

polymerized separately and then added to thecarrier-GlcNAc-glycerol phosphate. Such po-lymerization could occur on lipoteichoic acidcarrier as described by Fiedler and Glaser (10).Finally, the completed teichoic acid is cova-lently linked to the preexisting wall by a bondbetween a GlcNAc residue in the teichoic acidand an as yet unidentified component of thewall. This scheme is similar to those alreadypublished for the synthesis of teichoic acid in-termediates (6, 15) but also accounts for cova-lent linking of the new teichoic acid to preexist-ing wall in the absence of de novo peptidogly-can synthesis. Confirmation of this or any ofthe proposed biosynthetic schemes must awaitisolation of the intermediate at various stagesof its assembly and its subsequent utilizationfor synthesis of linked teichoic acid by cell-freeenzyme preparations.

ACKNOWLEDGMENTWe thank H. J. Rogers for his interest and encourage-

ment.

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mines in the neutralization ofbacteriophage deoxyri-bonucleic acid. J. Biol. Chem. 235:769-775.

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3. Anderson, R. G., H. Hussey, and J. Baddiley. 1972.Mechanism of wall synthesis in bacteria. The organi-sation of enzymes and isoprenoid phosphates in themembrane. Biochem. J. 127:11-25.

4. Archibald, A. R., J. Baddiley, and D. Button. 1968. Theglycerol teichoic acid of walls ofStaphylococcus lactis13. Biochem. J. 110.543-557.

5. Bracha, R., and L. Glaser. 1976. In vitro system for thesynthesis of teichoic acid linked to peptidoglycan. J.Bacteriol. 125:872-879.

6. Bracha, R., and L. Glaser. 1976. An intermediate inteichoic acid biosynthesis. Biochem. Biophys. Res.Commun. 73:1091-1098.

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tide translocase. The peptide subunit of uridine di-phosphate-N-acetylmuramyl-pentapeptide. J. Biol.Chem. 249:3140-3150.

14. Hancock, I., and J. Baddiley. 1976. In vitro synthesis ofthe unit that links teichoic acid to peptidoglycan. J.Bacteriol. 125:880-886.

15. Hancock, I. C., G. Wiseman, and J. Baddiley. 1976.Biosynthesis of the unit that links teichoic acid to thebacterial wall: inhibition by tunicamycin. FEBS Lett.69-.75-80.

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17. Lehle, L., and W. Tanner. 1976. The specific site oftunicamycin inhibition in the formation of dolichol-bound N-acetylglucosamine derivatives. FEBS Lett.71:167-170.

18. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J.Randall. 1951. Protein measurement with the Folinphenol reagent. J. Biol. Chem. 193:265-275.

19. Mauck, J., and L. Glaser. 1972. On the mode of in vivoassembly of the cell wall of Bacillus subtilis. J. Biol.Chem. 247:1180-1187.

20. Mindich, L. 1970. Membrane synthesis in Bacillus sub-tilis. I. Isolation and properties of strains bearingmutations in glycerol metabolism. J. Mol. Biol.49:415-432.

21. Mirelman, D., R. Bracha, and N. Sharon. 1974. Penicil-lin-induced secretion of a soluble, uncross-linked pep-tidoglycan by Micrococcus luteus cells. Biochemistry13:5045-5053.

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23. Takatauki, A., K. Kono, and G. Tamura. 1975. Inhibi-tion of biosynthesis of polyisoprenol sugars in chickembryo microsomes by tunicamycin. Agric. Biol.Chem. 39:2089-2091.

24. Tkacz, J. S., and J. 0. Lampen. 1975. Tunicamycininhibition of N-acetylglucosaminyl pyrophosphateformation in calf-liver microsomes. Biochem. Bio-phys. Res. Commun. 65:248-257.

25. Tomasz, A., M. McDonnell, M. Westphal, and E. Zan-ati. 1975. Coordinated incorporation of nascent pepti-doglycan and teichoic acid into pneumococcal cellwalls and conservation of peptidoglycan duringgrowth. J. Biol. Chem. 250:337-341.

26. Toon, P., P. E. Brown, and J. Baddiley. 1972. Thelipid-teichoic acid complex in the cytoplasmic mem-brane of Streptococcus faecalis N.C.I.B. 8191. Bio-chem. J. 127:399-409.

27. Tynecka, Z., and J. B. Ward. 1975. Peptidoglycan syn-thesis in Bacillus licheniformis. The inhibition ofcross-linking by benzylpenicillin and cephaloridine invivo accompanied by the formation of soluble peptido-glycan. Biochem. J. 146:253-267.

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