membrane intermediates peptidoglycan metabolism of … · 3550 van heijenoort et al. table 1. e....

Vol. 174, No. 11

Membrane Intermediates in the Peptidoglycan Metabolism ofEscherichia coli: Possible Roles of PBP lb and PBP 3YVELINE VAN HEIJENOORT,' MANOLO GOMEZ,2 MARCEL DERRIEN,'t JUAN AYALA,2

AND JEAN VAN HEIJENOORT1*

Centre National de la Recherche Scientifique, Unite de Recherche Associee 1131, Biochimie Moleculaire et Cellulaire,Universite Paris-Sud, 91405 Orsay, France, 1 and Instituto de Biologia Molecular, Centro de Biologia Molecular, Consejo

Superior de Investigationes Cientificas, Universidad Aut6noma de Madnid, Canto Blanco, 28049 Madrid, Spain2

Received 10 January 1992/Accepted 19 March 1992

The two membrane precursors (pentapeptide lipids I and II) of peptidoglycan are present in Escherichia coliat cell copy numbers no higher than 700 and 2,000 respectively. Conditions were determined for an optimalaccumulation of pentapeptide lipid II from UDP-MurNAc-pentapeptide in a cell-free system and for itsisolation and purification. When UDP-MurNAc-tripeptide was used in the accumulation reaction, tripeptidelipid II was formed, and it was isolated and purified. Both lipids II were compared as substrates in the in vitropolymerization by transglycosylation assayed with PBP lb or PBP 3. With PBP lb, tripeptide lipid II was usedas efficiently as pentapeptide lipid II. It should be stressed that the in vitro PBP lb activity accounts for at bestto 2 to 3% of the in vivo synthesis. With PBP 3, no polymerization was observed with either substrate.Furthermore, tripeptide lipid II was detected in D-cycloserine-treated cells, and its possible in vivo use inpeptidoglycan formation is discussed. In particular, it is speculated that the transglycosylase activity of PBP lbcould be coupled with the transpeptidase activity of PBP 3, using mainly tripeptide lipid II as precursor.

It is now established that in Escherichia coli there are atleast two distinct modes for the insertion of newly polymer-ized peptidoglycan material, one for elongation and the otherfor septation (30). Several proteins have been described asdirectly involved in the polymerization reactions (34, 47). Inparticular, certain penicillin-binding proteins (PBPs) werefound to catalyze in vitro polymerization reactions with N-acetylglucosaminyl -N- acetylmuramyl(pentapeptide) - pyro-phosphate-undecaprenol as a substrate (17, 18, 22, 29, 39,41).In vivo, this membrane intermediate (lipid II) is formedfrom UDP-N-acetylmuramoyl-pentapeptide by a two stepprocess (34). First, a translocase catalyzes the transfer of thephospho-MurNAc-pentapeptide moiety of UDP-MurNAc-pentapeptide to the membrane acceptor undecaprenol phos-phate, yielding MurNAc(pentapeptide)-pyrophosphate-un-decaprenol-(lipid I). Thereafter, a transferase catalyzes theaddition of N-acetylglucosamine, yielding lipid II. The trans-locase also catalyzes the reverse reaction, and the equilib-rium is in favor of the formation of UDP-MurNAc-pentapep-tide (10, 26). The irreversibility of the transferase reactionallows for the in vivo formation of lipid II. Recently, thetranslocase and the transferase have been identified as theproducts of gene mraY and gene murG, respectively (15, 26).From the data of Ramey and Ishiguro (33), the ratio of

lipid I to lipid II in growing cells can be estimated at 1:1.5.More recently, the UDP-MurNAc-pentapeptide/lipid I/lipidII ratio was estimated at ca. 300:1:3 (20) or 140:1:2.7 (26).Considering that there are ca. 105 molecules of UDP-Mur-NAc-pentapeptide per cell (23), there would thus be only1,000 to 2,000 molecules of lipids II and less than 700 of lipidI. Such extremely low pool levels explain why the isolationand purification of lipids I and II directly from cells is not aneasy matter. However, lipid II has been isolated from a

* Corresponding author.t Present address: Sanofi Recherche, 94256 Gentilly, France.

cell-free system in which it was allowed to accumulate to acertain extent by incubation of membranes with UDP-GlcNAc and radiolabelled UDP-MurNAc-pentapeptide (44).

In this study, a set of optimal conditions for a maximal invitro formation of lipid II with particulate fraction from E.coli was sought, as well as substantial improvements in itspurification. A lipid II containing tripeptide Ala-y-D-Glu-meso-DAP instead of the pentapeptide was prepared in asimilar way. Its possible in vivo presence was investigated.Both types of lipid II were compared as substrates in the invitro polymerization reactions assayed with PBP lb or PBP3. The possible physiological significance of such reactionsare discussed.

MATERUILS AND METHODS

Bacterial strains, plasmids, and growth conditions. The E.coli strains used in this study are listed in Table 1. Cells weregrown as previously described in Penassay broth (AM3;Difco Laboratories, Detroit, Mich.), LB medium (28), orminimal medium M63 (28) supplemented with glucose (0.2%)and thiamine (0.5 mg x liter-'). Cultures (40 or 400 ml) in200- or 2,000-ml flasks were inoculated with 1% of overnightprecultures, and growth was monitored by measuring theoptical density (OD) at 600 nm in a spectrophotometer model240 (Gilford Instruments, Oberlin, Ohio).

Plasmid pJP13 was constructed by insertion of a 2.4-kbpXhoII-XhoII DNA fragment containing the Pr promoter intothe BamHI site of plasmid pFR5A (35). Plasmid pFR5A wasconstructed by insertion of an 18-kbp BamHI-EcoRI DNAfragment from pLC19-19 containing the ponB gene into thecloning site of plasmid pUC9. Plasmid pJP14 contained thesame insert as pJP13 but in the orientation opposite that ofthe lac promoter. Plasmid pJP15 was derived from pJP13 bydeletion of a 17-bp BglII-BglII fragment, and the readingframe of PBP lb was disrupted at amino acid 432. PlasmidpXEPR was constructed by insertion of a 2.4-kbp XhoII-XhoII DNA fragment containing the Pr promoter into the

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3550 VAN HEIJENOORT ET AL.

TABLE 1. E. coli K-12 strains

Strain Genotype or Source orcharacteristics reference

HfrH thi-I rel 28FB81ysA FB8 lysA::KMr 25JM83 ara A(lac-proAB) rpsL thi strA 48

4(80 (lacZ M1S)JA200 F+ trpE5 recAI thr leuB6 lacY 4QCB1 MC6RP1 ponB::Spcr 8

BamHI site plasmid pXE15 (13). Plasmid pXE15 was con-structed by insertion of a 6.4-kbp XmaI-EcoRI DNA frag-ment from pLC26-6 into the cloning site of pUC9.

Chemicals. D-[14C]Ala-D-[14C]Ala (4.5 GBq x mmol-1),UDP-MurNAc-L-Ala--y-D-Glu-meso-diaminopimelic acid(A2pm), UDP-MurNAc-L-Ala-y-D-Glu-meso-[ C]A2pm (12GBq x mmol-1), UDP-MurNAc-L-Ala-y-D-Glu-meso-A2pm-D-Ala-D-Ala, and UDP-MurNAc-L-Ala-y-D-Glu-meso-A2pm-D-[14C]Ala-D-['4C]Ala (4.5 Bq x mmol-1) were pre-pared as previously described (6, 7, 14). [14C]A2pm (~l2 GBqx mmol-1) and [3H]A2pm (850 GBq x mmol- ) werepurchased from CEA (Saclay, France). Owing to radiolysis,they were regularly purified by paper rheophoresis (16 V/cm)on Whatman 3MM filter paper in 0.1 N formic acid (pH 2.3),detection by autoradiography, and elution with water. ATP,penicillin G, DNase, and RNase were purchased from Serva(Heidelberg, Germany), DEAE-cellulose DE32 was pur-chased from Whatman (Maidstone, England), and activatedsilicic acid Unisil was purchased from Clarkson ChemicalCo. (Williamsport, Pa.). Moenomycin A was a gift from G.Huber (Hoechst, Mainz, Germany). D-Cycloserine andN-octyl-o-D-glucopyranoside were bought from Sigma (LaVerpilliere, France). Clavulanic acid was purchased fromBeechman (Philadelphia, Pa.).

Analytical procedures. Protein, amino acid, and hexosa-mine contents were determined as previously described (23).Membrane preparations for the in vitro formation of lipid

II. Membrane preparations for the in vitro formation of lipidII were secured from spheroplasts of E. coli K-12 HfrH.Cultures were carried out at 37°C in LB medium in a 200-literfermentor. Cells were harvested at OD 0.9 and stored at-20°C. An aliquot (100 g) of frozen cells was thawedovernight at 4°C and suspended in 750 ml of 0.02 M Tris-HClbuffer (pH 8) containing 20% sucrose. The suspension wasgently stirred 10 min in the cold. Thereafter, a solution of eggwhite lysozyme (20 mg x ml-' in 0.02 M Tris-HCl [pH 8])was added to a final concentration of 0.2 mg x ml-1. After 10min of gentle stirring at 0°C, a solution of 0.2 M EDTA in0.02 M Tris-HCl buffer (pH 8) was slowly added over a i-hperiod to a final concentration of 0.02 M. Spheroplasts wererecovered by centrifugation at 12,000 x g for 20 min, andpellets were suspended with DNase and RNase, each at 20,ug x ml-l, in 750 ml of 0.05 M Tris-HCI buffer (pH 7.5)containing 10-' M MgCl2 and 10-3 M 2-mercaptoethanol(buffer A). After being stirred for 1 h at room temperature,the suspension was centrifuged for 1 h at 100,000 x g. Thepellet was suspended in the same buffer and treated againwith DNase and RNase. The final pellet was suspended in 55ml of buffer A at a protein concentration of 31 mg x ml-1'.

In vitro formation of lipid II and its extraction. An aliquot(45 ml, 1.4 g of protein) of the membrane preparation wasadded to 30 ml of a solution containing 0.5 M Tris-HCl buffer(pH 7.5), 40 mM MgCl2, 16 mM ATP (pH 7), penicillin G(100 ,ug x ml-'), 0.4 mM UDP-GlcNAc, 4 ,uM UDP-

MurNAc-pentapeptide, and an appropriate amount of UDP-MurNAc-L-Ala-y-D-Glu-meso-DAP-D-[14C]Ala-D-["4C]Ala.The suspension was incubated under stirring for 1 h at 35°C.To the reaction mixture, a solution (75 ml) of 6 M pyridiniumacetate in 1-butanol (1:2, vol/vol) was added (6 M pyridiniumacetate was prepared by mixing 51.5 ml of glacial acetic acidwith 48.5 ml of pyridine). After incubation for 2 h at 35°C,the mixture was centrifuged for 1 h at 1,500 x g. The upperorganic phase was recovered, and the remaining aqueousphase was submitted twice again to the same extractionprocedure. The three organic extracts were pooled and keptat 4°C overnight. The small turbid lower phase which hadappeared was recovered and centrifuged. The upper phase ofthe supernatant was added to the main organic solution,which was then back-washed three times with water satu-rated with 1-butanol. The final organic phase was evaporatedto dryness under vacuum at 5°C. The residue was dissolvedin 20 ml of chloroform-methanol (1:1, vol/vol).

Purification of lipid II. The chloroform-methanol extractwas applied to a column (1 by 40 cm) filled with DEAE-cellulose (Whatman DE32 in the acetate form) treated aspreviously described (5) and equilibrated with methanol(Fig. 1A). The elution was run first with 250 ml of methanol,with 40 ml of 0.4 M ammonium acetate in methanol, andthereafter with a linear gradient from 0.4 to 4 M ammoniumacetate in methanol (250 ml of 4 M ammonium acetate inmethanol connected with 250 ml of a solution of 0.4 ammo-nium acetate in methanol running onto the column). The flowrate was ca. 1 ml x min-'. The fractions containing radio-active lipid II were pooled and strongly mixed with 2volumes of water and 2 volumes of 1-butanol saturated withwater. After one night at 4°C, the upper organic phase wasrecovered. The remaining lower aqueous phase was submit-ted twice again to the same extraction procedure. The threebutanol extracts were pooled and evaporated to drynessunder vacuum at 5°C. The residue was dissolved in 10 ml ofchloroform-methanol (3:1, vol/vol). No loss of radioactivitywas observed during the extraction and evaporation proce-dures. The chloroform-methanol solution was applied to acolumn (1 by 35 cm) filled with Unisil silicic acid andpreviously equilibrated with chloroform-methanol (3:1, vol/vol) (Fig. 1B). The elution was first run with 150 ml of thissolvent and thereafter with a linear gradient from this solventto pure methanol (250 g of methanol connected with 250 g ofchloroform-methanol [3:1, vol/vol] running onto the col-umn). The fractions containing radioactive lipid II werepooled and kept at -25°C. The amino acid and hexosaminecontents of the product was determined: glutamic acid, 1;alanine, 2.6; A2pm, 1.06; glucosamine, 0.92; and muramicacid, 0.94. Traces of glycine, serine, leucine and isoleucinewere present.

Incorporation of I3HJA2pm and analysis of lipid I and II cellcontents. Cultures (40 ml) of strain FB8rel+lysA were grownat 37°C in 200 ml flasks under strong aeration in M63 mediumsupplemented with 0.2% glucose, L-lysine, L-methionine,and L-threonine, each at 100 ,ug x ml-', as previouslydescribed (25). When A6. reached 0.3 (2 x 108 cells xml-'), 5-ml aliquots were rapidly transferred to 25-ml flasksstirred at 37°C containing 1.5 MBq (850 GBq x mmol-1) oflyophilized [3HJA2pm. For the analysis of the incorporationof [HJA2pm into peptidoglycan and its precursors at varioustimes thereafter, aliquots (1 ml) were rapidly harvested bycentrifugation in the cold and pellets were suspended in 30 IlIof a mixture of isobutyric acid and 1 M ammonia (5:3). Themixture was applied to Whatman 3MM filter paper, andchromatography was run overnight in isobutyric acid-1 M

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LIPIDS I AND II IN PEPTIDOGLYCAN METABOLISM 3551

E 2.0 3

I0

1.5

0.5

10 20 30 40

Fraction number

FIG. 1. Purification of pentapeptide lipid II by column chroma-tography. As described in Materials and Methods, the crude chlo-roform-methanol extract (11 x 106 cpm) containing lipid II wasapplied to a DEAE-cellulose column eluted successively with meth-anol, 0.4 M ammonium acetate in methanol (starting at arrow 1), anda linear gradient from 0.4 to 4 M ammonium acetate in methanol(starting at arrow 2). Fractions (9 to 10 ml each) were tested forradioactivity. The second radioactive peak (fractions 20 to 40)corresponded to lipid II. After pooling, extraction, evaporation, anddissolution, the product was applied to a silicic acid column (B)eluted first with chloroform-methanol (3:1, vol/vol) and thereafterwith a linear gradient (starting at the arrow) from this solvent to puremethanol. Fractions (10 ml each) were tested for radioactivity.

ammonia (5:3). Radioactive compounds were detected witha Berthold scanner model LB283 (Berthold, Elancourt,France).The lipid I and II cell contents of the 5 ml of [3H]A2pm-

labelled cultures were analyzed after incubation for 25 min at37°C. Cultures were boiled for 4 min. Insoluble material wasrecovered by centrifugation at 12,000 x g, suspended in 200PI1 of water, and boiled for 4 min to remove residual[3H]A2pm and radioactive UDP-MurNAc-tri- and pentapep-tide nucleotides. After centrifugation, pellets were sus-pended in 10% acetic acid and material was transferred toglass vials, which were sealed and heated at 105°C for 1 h.After evaporation to dryness, residues were suspended inwater (80 p,l) and the soluble N-acetylmuramoyl-peptideswere reduced to their muramicitol derivatives as previouslydescribed (20). Accordingly, samples were diluted with 80 PI1of 0.5 M sodium borate buffer (pH 9) and incubated in the

presence of sodium borohydride (1 mg x ml-') for 30 min atroom temperature. The reaction was stopped by adjustingpH to 4 to 5 with acetic acid. The reduced muropeptideswere analyzed by high-pressure liquid chromatography(HPLC). When the lipid I and II contents of D-cycloserine-treated cells were examined, the drug was added at a finalconcentration of 0.4 ,ug x ml-' 10 min after addition of[3H]A2pm acid, and cells were further incubated for 15 minbefore the above-described analytical procedure was carriedout.Membrane preparation for polymerization reactions. Cul-

tures of strain JA200(pLC19-19) were prepared under strongstirring at 37°C in 2-liter flasks containing 500 ml of Penassaybroth (AM3; Difco) and inoculated with 5 ml of overnightprecultures. Cells from 2.5 liters of culture were harvested atOD 0.8 by centrifugation in the cold for 10 min at 10,000 xg (yield, 3 g [wet weight]), washed once with 0.02 MTris-HCl buffer (pH 8), and disrupted by grinding at 4°C in alarge mortar with levigated alumina (4 g/g [wet weight] ofcells) as previously described (45). After suspension of themixture in 45 ml of buffer A and centrifugation for 5 min at7,500 x g, the supernatant was centrifuged for 1 h at 100,000x g. The pellet was suspended in 0.5 ml of buffer A at ca. 30mg of protein x ml-1, and the suspension was stored at-20°C. Cultures of strain JM83(pJP13) or JM83(pXEPR)were performed under strong aeration in 2-liter flasks con-taining 500 ml of LB medium and ampicillin (100 Fg x ml-'),inoculated with 5 ml of overnight precultures. Cells grownfirst at 30°C up to OD 0.2 and for 1.5 to 4 h at 42°C werethereafter disrupted as described above.

Partial purification of PBP lb and PBP 3. Cultures of strainJA200(pLC19-19) were prepared overnight under strongaeration in seven 2-liter flasks containing 250 ml of Lmedium (28). After dilution with 250 ml of fresh medium,growth was allowed to proceed for 90 min before harvestingof cells in the cold by centrifugation for 15 min at 13,000 xg. Cells were washed with buffer A (yield, 15.5 g [wetweight]) and disrupted by sonication (Sonicator 150 TS;Ultrason, Annemasse, France) in the same buffer. Afterremoval of cell debris by centrifugation for 10 min at 3,000 xg, the supernatant was centrifuged for 1 h at 100,000 x g.The pellet was suspended in 11 ml of buffer A at 50 mg ofprotein x ml-'. The suspension was heated for 10 min at60°C to inactivate cell PBPs except PBP lb according toNakagawa et al. (29). After cooling in ice and addition ofn-octylglucopyranoside up to a final concentration of 1.5%(165 mg in 11 ml), the mixture was vigorously shaken for 30min at room temperature, allowed to stand for 15 min, andcentrifuged for 30 min at 27,000 x g. The pellet wassuspended in buffer A and treated again in the same way withthe detergent. The two resulting supernatants were pooledand fractionated with ammonium sulfate between 0 and 41%saturation. Precipitated material was recovered by centrifu-gation for 30 min at 27,000 x g, suspended in buffer A,dialyzed against the same buffer, and stored at -20°C. PBPlb was the only PBP detected when this preparation wasanalyzed by gel electrophoresis according to Spratt (37).

Partial purification of PBP 3 was carried out according toIshino and Matsuhashi (17), with minor modifications. Aculture of QCB1(pXEPR) was grown at 30°C in LB mediumto OD 0.6 and then transferred to 42°C for 1.5 h (conditionsshown to maximize the yield of PBP 3). Cells were harvestedby centrifugation, and a particulate fraction was obtained bysonication and differential centrifugations. An aliquot (350mg of protein) of this particulate fraction was extracted firstat 20°C with 50 mM Tris-HCl (pH 7.6) buffer containing 1%

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TABLE 2. Extent of accumulation of lipid II with variousmembrane preparations"

Membrane Protein yield (mg/g Activity"prepn [wet wtl of bacteria)

Alumina 4 6.5Sonication 8.9 6French press 6.8 8.7Spheroplasts 7 40-60

" Experiments were carried out with mid-exponential-phase cells of E. coliK-12 HfrH.

' The assay for the accumulation of lipid 11 was performed as described inMaterials and Methods. Activities are expressed as the percentage of conver-sion of UDP-MurNAc-pentapeptide into lipid 11.

Triton X-100 and 0.1 mM MgCl2 (buffer B). The pellet wasextracted again with buffer B containing 1 M NaCl. Theextract containing PBP 3 was desalted on a Sephadex G-50column eluted with buffer B. After cephalexin-Sepharoseaffinity chromatography and dialysis against 50 mM Tris-HCI-0.1% Triton X-100, the pooled fractions containing 80%purified PBP 3 on a protein basis were used directly in thepolymerization assay.Assay for the polymerization by transglycosylation. The in

vitro polymerization by transglycosylation catalyzed by PBPlb was assayed as described previously (45). The assay wascarried out with lipid II as a substrate at concentrationsranging from 10-5 to 10-4 M, depending on the lipid IIpreparation. The assay was very reproducible with 10- Mlipid II. However, at 10-5 M, a variability in the results wasobserved, owing presumably to an insufficient redissolutionof lipid II up to a saturating concentration. The apparent Kmof purified PBP lb for pentapeptide lipid II from E. coli hasbeen estimated at 3 ,uM (29). As previously recommended(29), the addition of methanol (15% final concentration) tothe assay will ensure proper solubility of lipid II and repro-ducible results. Penicillin is added to the assay to block alltranspeptidase and DD-carboxypeptidase activities. Withsome particulate fractions, D-alanine was clearly detectableon chromatograms, presumably because of the presence ofendogenous 1-lactamase activity. In such cases, clavulanicacid at a final concentration of 100 ,ug x ml-' was added tothe assay.

RESULTSConditions for the in vitro formation of lipid II. The

formation of lipid II by incubation of UDP-MurNAc-pen-tapeptide and UDP-GlcNAc with particulate fractions fromE. coli is a complex process, since they contain not only thetranslocase and transferase activities but also the membraneacceptor undecaprenyl-phosphate. The extent of in vitroformation of lipid II is greatly dependent on how the partic-ulate fraction was prepared and on the concentrations ofUDP-MurNAc-pentapeptide, UDP-GlcNAc, and protein.Preparations from spheroplasts were initially used (44).When various methods were compared (Table 2), sphero-plasts indeed led by far to the highest yields. A set of optimalconditions for a maximal synthesis was sought by fixing theUDP-MurNAc-pentapeptide and UDP-GlcNAc concentra-tions and by varying the protein concentration. Not being acommercially available product, UDP-MurNAc-pentapep-tide was fixed at 5 ,uM, whereas UDP-GIcNAc was fixed at5.10-4 M, a 100-fold-higher concentration, to ensure a highlipid II/lipid I ratio. The temperature optimum was found tobe 35°C, and the pH optimum was between 7 and 8 (in

A B C D E F a H I

START -I~~~'91 '.1~I#P '

i..I'. 1h|i

FRONT

FIG. 2. Analysis by paper chromatography of the formation oftripeptide and pentapeptide lipids II and their use in polymerizationcatalyzed by PBP lb. Chromatographies were run overnight onWhatman 3MM filter paper in isobutyric acid-1 M ammonia (5:3).Radioactive compounds were detected with a Berthold scannermodel LB283. (A) UDP-MurNAc-tripeptide (with [14C]A2pm); (B)UDP-MurNAc-pentapeptide (with [14C]Ala-D-[`4C]Ala); (C) in vitroformation of tripeptide lipid II; (D) purified tripeptide lipid II; (E) invitro formation of pentapeptide lipid II; (F) purified pentapeptidelipid II; (G) in vivo [3HA2pm-labelled peptidoglycan and precursors(peptidoglycan, Rf 0.0; UDP-MurNAc-tripeptide and UDP-Mur-NAc-pentapeptide, Rf0.1; [3H]A2pm, Rf 0.5; lipids intermediates, Rf0.8 to 0.9); (H) in vitro polymerization catalyzed by PBP lb withpentapeptide lipid II as a substrate (100 pmol in the assay); (I) invitro polymerization catalyzed by PBP lb with tripeptide lipid II asa substrate (100 pmol in the assay). The in vitro formation of lipid IIfrom UDP-MurNAc-tri- or pentapeptide (C and E) was carried outat an analytical level by scaling down by 1/10,000 the preparativeassay described in Materials and Methods.

Tris-HCl or potassium phosphate buffer). The incubationtime was limited to 60 min to avoid the degradative effects ofside reactions. Under these conditions, the yield of lipid IIincreased with the amount of particulate fraction in the assayand reached a plateau value at 8 mg of protein x ml-'. ATPwas found to stimulate the formation of lipid II by 35% whenadded to the assay at ca. 10 mM. This result was inagreement with a previous observation made by Araki et al.(1) for the in vitro synthesis of peptidoglycan in a cell-freesystem from E. coli. Presumably ATP is necessary for the invitro conversion of undecaprenol into undecaprenyl phos-phate catalyzed by a membrane phosphokinase. Such akinase has been isolated from Staphylococcus aureus (36),and the ratio of undecaprenol to undecaprenol phosphate hasbeen estimated at 1:1 in Streptococcus faecalis (43). Takinginto account the various results, an assay leading to 40 to60% conversion of UDP-MurNAc-pentapeptide into lipid IIwas developed (Fig. 2E). It was noteworthy that no pepti-doglycan material was formed under these conditions.

Purification of lipid II. The isolation and purification oflipid II from the in vitro assay were carried out by adaptingthe procedure of Umbreit and Strominger (44). A number ofmodifications (see Materials and Methods) were introducedto increase the final yield of lipid II from UDP-MurNAc-pentapeptide, which was only 2% in the study by theseauthors. The overall yield of five preparations that we maderanged from 12 to 18%. The specific radioactivity of theUDP-MurNAc-pentapeptide used varied from 0.2 to 3 GBq

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TABLE 3. Purification of lipid II

Pentapeptide Tripeptidelipid II lipid II

Step Yield of Yield of(1p6) each step cpm each step(106)(106) (%

Initial UDP-MurNAc-peptidea 75 31Butanol extract after the ac- 40.1 54 5 16

cumulation reactionDEAE-cellulose column 26.5 66 3.2 64Silicic acid column 11.6 44 1.51 47Overall yield' 15.5 4.8

a The specific radioactivity of UDP-MurNAc-pentapeptide was 3 GBq xmmol-', and that of UDP-MurNAc-tripeptide was 0.67 GBq x mmol-'.

b Overall yields were estimated as the ratio of purified lipid II obtained fromthe silicic acid column to the initial UDP-MurNAc-peptide.

x mmol-'. Yields of some steps of the preparations werefound to be greatly dependent on temperature. In particular,the removal in vacuo of butanol from extracts was per-formed at 5°C, and chromatographies on silicic acid columnswere run in the cold at 4°C. In Table 3, the yields of thevarious steps of a preparation from UDP-MurNAc-pen-tapeptide at 3 GBq x mmol-' are reported. In this case, 0.56,umol of lipid II was formed with 1.4 g of membrane proteinand 0.16 ,umol of purified lipid II was secured. Analysis ofthe amino acid and hexosamine contents of the purifiedproduct indicated a stoichiometry close to that of the

-DCS at pH 3.651500

1000 b c d

500-

o 1500 _ +DCS St pH 3.65

GlcNAc-MurNAc-pentapeptide moiety. The slightly lowerglucosamine content (see Materials and Methods) suggestedthe possible presence of some lipid I. This was confirmed byHPLC analysis (data not shown) after partial acid hydrolysisand reduction according to the method described for deter-mining lipid I and lipid II contents (see below). The ratio oflipid I to lipid II was estimated at 1:6. The mass spectrumanalysis of the material recovered in the ether phase aftermild acid hydrolysis was characteristic of undecaprenol asthe major constituent (data not shown).

In vitro formation of tripeptide lipid II. When UDP-MurNAc-pentapeptide was replaced by UDP-MurNAc-tripeptide as the substrate in the assay for the in vitrosynthesis of lipid II, a radioactive product, presumablytripeptide lipid II, was detected by paper chromatography, inwhich it migrated as pentapeptide lipid II (Fig. 2). However,it accumulated about three to four times less than didpentapeptide lipid II (Table 3). Its purification was carriedout under the conditions used for pentapeptide lipid II (Table3), and the chromatography elution patterns (data notshown) were very similar to those of pentapeptide lipid II.HPLC analysis (data not shown) carried as described belowafter partial acid hydrolysis and reduction revealed that 72%of the radioactivity of the product was recovered in a majorpeak eluting as the reduced dissaccharide tripeptide (com-pound b in Fig. 3).

Analysis of cell lipids I and II. The analysis of cell lipids Iand II was undertaken for two reasons. First, the in vitroformation of tripeptide lipid II raises the question of the

Rqtention time (min)

FIG. 3. Analysis of cell lipids I and II by HPLC of their reduced muropeptides. Experiments were carried out as described in Materialsand Methods with 5-ml cultures of strain FB8rel+lysA specifically labelled with [3H]A2pm. The reduced muropeptides originating from lipidsI and II were separated by HPLC on a ,u-Bondapak C18 column (3.9 by 30 cm) eluted with 0.05 M ammonium formate at a flow rate of 0.5ml x min-1. Fractions (0.5 ml) were collected, diluted with 4.5 ml of scintillation liquid, and counted for radioactivity. (A) Fifteen microlitersfrom 200 IL1 of the extract from untreated cells with an eluent at pH 3.65; (B) the same with an eluent at pH 3.20 (the upper curve correspondsto a threefold increase in the amount of injected material); (C) 15 ,ul from 200 p.l of the extract from D-cycloserine-treated cells with an eluentat pH 3.65; (D) the same with an eluent at pH 3.20. The excluded radioactive peak corresponds to residual [3H]A2pm. Arrows indicate thepositions of elution of the reduced muropeptides which correspond to MurNAcOH-tripeptide (a), GlcNAc-MurNAcOH-tripeptide (b),MurNAcOH pentapeptide (c) and GlcNAc-MurNAcOH-pentapeptide (d). Compounds a and c were secured by mild acid hydrolysis (5 minat 100°C in 0.05 M HCI of UDP-MurNAc-tripeptide and UDP-MurNAc-pentapeptide. Compounds b and d were secured according to Glauner(11). DCS, D-cycloserine.

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possible in vivo presence of tripeptide lipids I and II. Apriori, the 10- to 40-fold-lower pool of UDP-MurNAc-tripep-tide than of UDP-MurNAc-pentapeptide in normally grow-ing cells (27), and the 3- to 4-fold less efficient in vitroformation of tripeptide lipid II, would suggest very lowpool values for tripeptide lipids (at most ca. 100 copies percell). However, it has recently (3, 32) been proposed thatMurNAc-tripeptide precursors could be involved in pepti-doglycan polymerization during septation. It was thereforeessential to determine whether tripeptide lipids could bedetected in cell extracts. Second, it was also of interest tocompare purified lipid II after accumulation in cell-freesystems with the in vivo material.For these purposes, strain FB8rel+lysA was used for the

convenient specific pulse-labelling of peptidoglycan precur-sor by [3H]A2pm (25). The distribution of [3H]A2pm betweenpeptidoglycan and its A2pm-containing nucleotide and lipidprecursors was analyzed by paper chromatography (Fig.2G), and the separated products were quantified at varioustime intervals (data not shown). Between 10 and 30 min afterthe addition of [3H]A2pm, the radioactive pool of lipids I andII remained constant, whereas peptidoglycan steadily in-creased and [3H]A2pm steadily decreased. No clear separa-tion between lipids I and II could be observed. Purified tri-and pentapeptides lipids II migrated with the same Rf as thein vivo-labelled material (Fig. 2). Furthermore, D-cycloser-ine was used to promote the increase of the UDP-MurNAc-tripeptide pool and the depletion of the UDP-MurNAc-pentapeptide pool (16, 21, 31) so as to increase the pools ofthe putative tripeptide lipids. The tripeptide and pentapep-tide lipid cell contents were analyzed before and afterD-cycloserine treatment by adapting two previously de-scribed methods (20, 33). After boiling, harvesting, andthorough washing, cells were submitted to mild acid hydro-lysis to cleave the muropeptides from undecaprenol pyro-phosphate. After reduction, muropeptides were analyzed byHPLC according to two sets of conditions. For reducedmuropentapeptides (compounds c and d in Fig. 3), theelution was carried out at pH 3.65, whereas for reducedmurotripeptides (compounds a and b in Fig. 3), the elutionwas performed at pH 3.20 to ensure a proper separation fromresidual [3H]A2pm.

In untreated cells (Fig. 3A and B), reduced muropentapep-tides c and b were present in a 1:3 ratio as previouslydescribed (20, 26), and reduced murotripeptides were notclearly detectable. However, a very small radioactive peakwas observed at the position of reduced murotripeptide b(Fig. 3B). It was better visualized when larger amounts ofthe extract were injected and accounted for ca. 10% of thatof reduced muropentapeptide c (upper curve in Fig. 3B). InD-cycloserine-treated cells (Fig. 3C and D), reduced muro-pentapeptides c and d decreased, whereas the peak corre-sponding to reduced murotripeptide b increased signifi-cantly. When the material recovered under this latter peakwas analyzed by HPLC according to Glauner (11), it comi-grated with reduced murotripeptide b. It was necessary toverify that the observed reduced murotripeptide b did notoriginate from the labelled peptidoglycan material presentwith lipids I and II in the washed boiled cells in the course ofthe mild acid treatment. For this purpose, a sample of[3H]A2pm-labelled and D-cycloserine-treated cells wasboiled with 4% sodium dodecyl sulfate for 10 min. Afterrepeated washings with water, the resulting insoluble mate-rial was submitted to the same analytical procedure as forlipids I and II. No reduced muropentapeptide c or d wasdetected in the three different experiments carried out, and

TABLE 4. Transglycosylation activity of PBP lb and PBP 3present in extracts from various E. coli K-12 strainsa

Strain Activity" PBP overproduction (fold)

JM83 1.0 1QCB1 0.0 0JM83(pJP14) 1.0 1JM83(pJP15) 0.95 1JM83(pJP13) 28.6 1,000JA200(pLC19-19)Crude extract 4.0Purified fraction 23.8

QCB1(pXEPR)Crude extract 0.0 26Purified fraction 0.0a Extracts from the various strains were prepared and the assay for the in

vitro transglycosylation activity of polymerase PBP lb was carried out asdescribed in Materials and Methods. The putative in vitro transglycosylationactivity of PBP 3 (crude extract or purified fraction) was tested by the sameassay and by the assay of Ishino and Matsuhashi (17).

' Expressed as the percentage of conversion of lipid II into polymerizedmaterial for 10 ,ug of protein introduced in the assay.

no reduced murotripeptide b was detected in two of theexperiments. However, in one experiment a small peakeluting as reduced murotripeptide b accounted for less than20% of the level found in non-sodium dodecyl sulfate-treatedcells.

In vitro polymerization by transglycosylation catalyzed byPBP lb. It was previously established that purified PBP lb orparticulate fractions containing PBP lb can catalyze the invitro formation of cross-linked peptidoglycan material withpentapeptide lipid II as a substrate (29, 39). PBP lb is abifunctional enzyme which catalyzes transglycosylation andtranspeptidation reaction involved in peptidoglycan forma-tion. In the in vitro assay, transglycosylation can be uncou-pled from transpeptidation by the addition of penicillin (45).Moreover, it was previously established that such an uncou-pling leads to an important stimulation of the transglycosyl-ation reaction (39). In the present study, only the polymer-ization by transglycosylation was considered.

Particulate fractions from various strains and partiallypurified PBP lb from strain JA200(pLC19-19) were exam-ined as sources of transglycosylase activity (Table 4). Nopolymerase activity was detected in extracts from strainQCB1 devoid of PBP lb. Plasmid pLC19-19 carries theponBgene coding for PBP lb (40), and in strain JA200(pLC19-19),it led to a fourfold increase of activity. In strainJM83(pJP13), the plasmid ponB gene was under the controlof the Pr promoter. After full expression at 42°C, immuno-logical estimations indicated a ca. 1,000-fold increase in theamounts of PBP lb compared with that of the control strainharboring the plasmid without ponB. However, when thePBP lb transglycosylation activity was considered, only a28- to 29-fold increase was found (Table 4). When tripeptidelipid II instead of pentapeptide lipid II was used as thesubstrate in the assay, a similar extent of peptidoglycanformation was observed (Fig. 3). Moenomycin was previ-ously shown to specifically inhibit the transglycosidase ac-tivity of PBP lb at concentrations of 0.01 to 0.1 ,ug x ml-'(39, 45, 46). The same effect was observed in the in vitropolymerization reaction carried out with tripeptide lipid II asa substrate (data not shown).

In vitro polymerization catalyzed by PBP 3. PBP 3 has beenshown to be essentially involved in peptidoglycan biosyn-thesis at the time of septum formation during cell division in

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E. coli (30). Peptidoglycan synthesized in a defilamentationsystem which elicited the activity of PBP 3 in vivo containedincreased amounts of cross-linkage as well as a higher ratioof tripeptide-containing cross-linked subunits (32). The pos-sible use of tripeptide lipid II as a substrate for this enzymehas been postulated to be a signal that triggers the changefrom lateral to septal peptidoglycan biosynthesis (3).

Therefore, we have tried to determine whether the tripep-tide lipid II could serve as substrate for PBP 3 in an in vitrosystem. Particulate fractions from various strains (not pro-ducing PBP lb) and partially purified PBP 3 (-80% purifi-cation) from strain QCB1(pXEPR) were examined assources for transglycosylase activity (Table 4). PlasmidpXEPR carries the pbpB gene, coding for PBP 3, under thecontrol of Pr promoter. The amount of PBP 3, detected byantibodies raised against the protein, increased 26-fold whenstrain QCBl(pXEPR) was induced for 2.5 h at 42°C com-pared with wild-type expression of the protein. In the sameexperiment, P-lactam-binding activity showed only a nine-fold increase. However, when the transglycosylase activityof PBP 3 was analyzed in vitro, no polymerization occurredat 30°C, and no increase was seen in the induced fractioncompared with the level at 30°C, independently of thesubstrate (tripeptide or pentapeptide lipid II) used in theassay. This finding contradicted the previously described(17) polymerase activity of purified PBP 3 determined withpentapeptide lipid II used as the substrate. We have noexplanation for this discrepancy.

DISCUSSION

A set of optimal conditions was determined for the in vitroformation of radioactive pentapeptide lipid II in a cell-freesystem from E. coli and for its isolation and purification.These conditions led to the formation of ca. 0.68 ,Lmol oflipid II with membranes from 100 g (wet weight) of cells.Since the normal endogenous pool of lipid II for this amountof cells can be estimated at 0.1 to 0.2 ,umol, this means athree- to sixfold accumulation. Presumably the undecapre-nyl pyrophosphate pool could be the limiting factor for the invitro formation of lipid II under the conditions considered.Assuming a more or less complete depletion during the invitro assay, its in vivo pool would therefore be a few foldhigher than the lipid II pool.The polymerase activity of PBP lb was conveniently

determined by the in vitro assay using pentapeptide lipid IIas a substrate at a concentration near the estimated Km value(29). As previously observed (39), membrane preparationsfrom a strain lacking PBP lb were devoid of in vitropolymerase activity, whereas those from overproducingstrains possessed much higher levels. However, in the caseof the PBP lb overproducer harboring a plasmid with theponB gene under the control of the Pr promoter, the increasein polymerase activity was far from paralleling the consider-able increase in PBP lb copies. This result seemed to implythat a great part of the overproduced protein was in aninactive form, because of either an improper processing orthe lack of a nonoverproduced effector. When the in vitroPBP lb activity of particulate fractions was compared withthe amounts of peptidoglycan formed in vivo in the corre-sponding cells (27), the in vitro system appeared extremelyinefficient and accounted for at best 2 to 3% of the in vivopolymerization. This discrepancy was presumably due to thefact that the in vitro conditions, although optimized, are stillfar from the in vivo ones. In particular, in the in vitro assay,both lipid II and polymerase PBP lb are randomly dispersed

in detergent micelles, whereas it can be speculated that invivo there is some kind of integrated organization wherebythe formation of lipid II and its use are coupled.

In this study, the formation of tripeptide lipid II fromUDP-MurNAc tripeptide in a cell-free system was estab-lished and its purification was carried out. Since four timesless tripeptide lipid II than pentapeptide lipid II was formed,the efficiency of the translocase and transferase activitieswas thus partly dependent on the structure of the peptidemoiety. Moreover, tripeptide lipid II was used as a substratein the in vitro assay for PBP lb polymerase activity appar-ently as efficiently as was pentapeptide lipid II. A number ofobservations indicate that in vivo the E. coli peptidoglycanbiosynthesis system will accept modifications in its peptidemoiety (24).

It was previously reported (32) that when ether-treatedcells were incubated with exogenous [14C]UDP-GIcNAc andUDP-MurNAc-tri- or pentapeptide, incorporation of radio-activity into insoluble peptidoglycan material was observed.These results are very similar with those described here. It isnoteworthy that the efficiency of this system is also fourtimes lower with the tripeptide precursor than with thepentapeptide precursor. The use of the tripeptide precursorin our cell-free system or in ether-treated cells raised thepossibility of its use in the in vivo formation of peptidoglycanvia tripeptide lipids I and II. It was recently proposed that inthe cell cycle of E. coli, the alternating phases of cellelongation and septation depend on a regularly shiftingbalance between activities of two competing morphogeneticsystems (3). In growth by elongation of the cylindrical cell,transpeptidation would be carried out preferentially withpentapeptide side chains, whereas tripeptide subunits wouldbe the preferred acceptors for transpeptidation carried outby PBP 3 during septum formation. Variations in the tripep-tide subunit content would determine the shift from cellelongation to septation (3).

In peptidoglycan of normally growing cells, mainly tet-rapeptide subunits are present (12). The tripeptide subunitcontent is fairly low (12), and it does not seem to varysignificantly with elongation or septation (30). However,under a number of circumstances, such as the stationaryphase (12) or amino acid starvation (42), the tripeptidecontent of peptidoglycan will increase considerably. What-ever the case, the presence of tripeptide subunits in pepti-doglycan can a priori be explained in two ways. Either theyarise by polymerization of tripeptide lipid II after formationof tripeptide lipids from UDP-MurNAc-tripeptide or they areformed by action of a DL-carboxypeptidase on peptidoglycantetrapeptide subunits (2, 19). Finally, the possibility thatpentapeptide lipids I and II are to some extent converted tothe corresponding tripeptide lipids by specific hydrolyticremoval of the two C-terminal D-alanine residues cannot beentirely excluded. The first possibility was investigated here.The detection of tripeptide lipid II in D-cycloserine-treatedcells confirms that its formation from the tripeptide nucleo-tide can function in vivo and not only in cell-free systems orin ether-treated cells. The substantial increase of peptidogly-can tripeptide observed after treatment with sublytic con-centrations of D-cycloserine (32) could suggest that thetripeptide lipid II is used in vivo for polymerization. How-ever, to truly distinguish between the use of tripeptide lipidII and modifications after polymerization, control experi-ments with a DL-carboxypeptidase-deficient strain will haveto be carried out.

It was initially proposed that in vivo PBP lb was respon-sible for peptidoglycan synthesis during cell elongation,

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whereas PBP 3 was required for peptidoglycan synthesisduring septation (see reference 47 for references). Morerecent results suggest that PBP lb is also involved inseptation and that there is some connection between theactivation of PBP lb and PBP 3. For instance, we haveshown (9) that PBP lb is involved in some step at themoment of initiation of septation; also, we have found a newmutation that suppresses a pbpB(Ts) phenotype only in thepresence of PBP lb. On the basis of a study of the effects ofspecific P-lactams on the rate of peptidoglycan synthesis andthe timing of cell lysis, Wientjes and Nanninga (47) haveproposed that in vivo PBP lb catalyzes the synthesis of newcross-linked peptidoglycan chains which are used as primersby PBP 2 and PBP 3 to construct peptidoglycan for eitherelongation or septation. Furthermore, from the analysis ofthe mutagenic positions found in a number of PBP 3 mutantstrains, it was suggested (38) that the transglycosylase do-main assigned at the N-terminal end of PBP 3 does not exist.If we take into account these previous results and thefindings that (i) tripeptide lipid II can be used by PBP 1Bboth in vivo and in vitro for the polymerization reaction, (ii)no polymerization activity was found for PBP 3 in any of theassayed conditions, and (iii) increased amounts of cross-linked subunits containing tripeptide moieties that elicitseptum formation activities are found in a defilamentationsystem (32), we can speculate that the transglycosylaseactivity of PBP 1B is coupled with the transpeptidase activ-ity of PBP 3, using mainly tripeptide lipid II as precursor,and possibly mediated by other components of the septumformation structure (septosome).

ACKNOWLEDGMENTSThis work was supported by grants from the Centre National de la

Recherche Scientifique (URA 1131), the Institut National de laSante et de la Recherche Medicale (90 0314), the Actions Integreesfranco-espagnoles (1989-1991), and the Fundaci6n Ram6n Areces.

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