Escherichia coli: Possible Roles of PBP lb and PBP 3

Transcription

1 JOURNAL OF BACTERIOLOGY, June 1992, p /92/ $02.00/0 Copyright C) 1992, American Society for Microbiology Vol. 174, No. 11 Membrane Intermediates in the Peptidoglycan Metabolism of Escherichia coli: Possible Roles of PBP lb and PBP 3 YVELINE 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, Orsay, France, 1 and Instituto de Biologia Molecular, Centro de Biologia Molecular, Consejo Superior de Investigationes Cientificas, Universidad Aut6noma de Madnid, Canto Blanco, 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 coli at cell copy numbers no higher than 700 and 2,000 respectively. Conditions were determined for an optimal accumulation of pentapeptide lipid II from UDP-MurNAc-pentapeptide in a cell-free system and for its isolation and purification. When UDP-MurNAc-tripeptide was used in the accumulation reaction, tripeptide lipid II was formed, and it was isolated and purified. Both lipids II were compared as substrates in the in vitro polymerization by transglycosylation assayed with PBP lb or PBP 3. With PBP lb, tripeptide lipid II was used as efficiently as pentapeptide lipid II. It should be stressed that the in vitro PBP lb activity accounts for at best to 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 in peptidoglycan formation is discussed. In particular, it is speculated that the transglycosylase activity of PBP lb could 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 at least two distinct modes for the insertion of newly polymerized peptidoglycan material, one for elongation and the other for septation (30). Several proteins have been described as directly involved in the polymerization reactions (34, 47). In particular, certain penicillin-binding proteins (PBPs) were found to catalyze in vitro polymerization reactions with N- acetylglucosaminyl -N- acetylmuramyl(pentapeptide) - pyrophosphate-undecaprenol as a substrate (17, 18, 22, 29, 39, 41). In vivo, this membrane intermediate (lipid II) is formed from UDP-N-acetylmuramoyl-pentapeptide by a two step process (34). First, a translocase catalyzes the transfer of the phospho-murnac-pentapeptide moiety of UDP-MurNAcpentapeptide to the membrane acceptor undecaprenol phosphate, yielding MurNAc(pentapeptide)-pyrophosphate-undecaprenol-(lipid I). Thereafter, a transferase catalyzes the addition of N-acetylglucosamine, yielding lipid II. The translocase also catalyzes the reverse reaction, and the equilibrium is in favor of the formation of UDP-MurNAc-pentapeptide (10, 26). The irreversibility of the transferase reaction allows for the in vivo formation of lipid II. Recently, the translocase and the transferase have been identified as the products 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/lipid II 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 only 1,000 to 2,000 molecules of lipids II and less than 700 of lipid I. Such extremely low pool levels explain why the isolation and purification of lipids I and II directly from cells is not an easy matter. However, lipid II has been isolated from a * Corresponding author. t Present address: Sanofi Recherche, Gentilly, France cell-free system in which it was allowed to accumulate to a certain 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 in vitro formation of lipid II with particulate fraction from E. coli was sought, as well as substantial improvements in its purification. A lipid II containing tripeptide Ala-y-D-Glumeso-DAP instead of the pentapeptide was prepared in a similar way. Its possible in vivo presence was investigated. Both types of lipid II were compared as substrates in the in vitro polymerization reactions assayed with PBP lb or PBP 3. The possible physiological significance of such reactions are discussed. MATERUILS AND METHODS Bacterial strains, plasmids, and growth conditions. The E. coli strains used in this study are listed in Table 1. Cells were grown as previously described in Penassay broth (AM3; Difco Laboratories, Detroit, Mich.), LB medium (28), or minimal medium M63 (28) supplemented with glucose (0.2%) and thiamine (0.5 mg x liter-'). Cultures (40 or 400 ml) in 200- or 2,000-ml flasks were inoculated with 1% of overnight precultures, and growth was monitored by measuring the optical density (OD) at 600 nm in a spectrophotometer model 240 (Gilford Instruments, Oberlin, Ohio). Plasmid pjp13 was constructed by insertion of a 2.4-kbp XhoII-XhoII DNA fragment containing the Pr promoter into the BamHI site of plasmid pfr5a (35). Plasmid pfr5a was constructed by insertion of an 18-kbp BamHI-EcoRI DNA fragment from plc19-19 containing the ponb gene into the cloning site of plasmid puc9. Plasmid pjp14 contained the same insert as pjp13 but in the orientation opposite that of the lac promoter. Plasmid pjp15 was derived from pjp13 by deletion of a 17-bp BglII-BglII fragment, and the reading frame of PBP lb was disrupted at amino acid 432. Plasmid pxepr was constructed by insertion of a 2.4-kbp XhoII- XhoII DNA fragment containing the Pr promoter into the

2 3550 VAN HEIJENOORT ET AL. TABLE 1. E. coli K-12 strains Strain characteristics Genotype or Source reference or HfrH thi-i rel 28 FB81ysA FB8 lysa::kmr 25 JM83 ara A(lac-proAB) rpsl thi stra 48 4(80 (lacz M1S) JA200 F+ trpe5 recai thr leub6 lacy 4 QCB1 MC6RP1 ponb::spcr 8 BamHI site plasmid pxe15 (13). Plasmid pxe15 was constructed by insertion of a 6.4-kbp XmaI-EcoRI DNA fragment 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 (12 GBq 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 prepared as previously described (6, 7, 14). [14C]A2pm (~l2 GBq x mmol-1) and [3H]A2pm (850 GBq x mmol- ) were purchased 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 purchased from Whatman (Maidstone, England), and activated silicic acid Unisil was purchased from Clarkson Chemical Co. (Williamsport, Pa.). Moenomycin A was a gift from G. Huber (Hoechst, Mainz, Germany). D-Cycloserine and N-octyl-o-D-glucopyranoside were bought from Sigma (La Verpilliere, France). Clavulanic acid was purchased from Beechman (Philadelphia, Pa.). Analytical procedures. Protein, amino acid, and hexosamine 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 lipid II were secured from spheroplasts of E. coli K-12 HfrH. Cultures were carried out at 37 C in LB medium in a 200-liter fermentor. Cells were harvested at OD 0.9 and stored at -20 C. An aliquot (100 g) of frozen cells was thawed overnight at 4 C and suspended in 750 ml of 0.02 M Tris-HCl buffer (ph 8) containing 20% sucrose. The suspension was gently stirred 10 min in the cold. Thereafter, a solution of egg white 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 10 min of gentle stirring at 0 C, a solution of 0.2 M EDTA in 0.02 M Tris-HCl buffer (ph 8) was slowly added over a i-h period to a final concentration of 0.02 M. Spheroplasts were recovered by centrifugation at 12,000 x g for 20 min, and pellets 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. The pellet was suspended in the same buffer and treated again with DNase and RNase. The final pellet was suspended in 55 ml 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 was added 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- J. BACTERIOL. 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 pyridinium acetate in 1-butanol (1:2, vol/vol) was added (6 M pyridinium acetate was prepared by mixing 51.5 ml of glacial acetic acid with 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 upper organic phase was recovered, and the remaining aqueous phase was submitted twice again to the same extraction procedure. The three organic extracts were pooled and kept at 4 C overnight. The small turbid lower phase which had appeared was recovered and centrifuged. The upper phase of the supernatant was added to the main organic solution, which was then back-washed three times with water saturated with 1-butanol. The final organic phase was evaporated to dryness under vacuum at 5 C. The residue was dissolved in 20 ml of chloroform-methanol (1:1, vol/vol). Purification of lipid II. The chloroform-methanol extract was applied to a column (1 by 40 cm) filled with DEAEcellulose (Whatman DE32 in the acetate form) treated as previously 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, and thereafter with a linear gradient from 0.4 to 4 M ammonium acetate in methanol (250 ml of 4 M ammonium acetate in methanol connected with 250 ml of a solution of 0.4 ammonium acetate in methanol running onto the column). The flow rate was ca. 1 ml x min-'. The fractions containing radioactive lipid II were pooled and strongly mixed with 2 volumes of water and 2 volumes of 1-butanol saturated with water. After one night at 4 C, the upper organic phase was recovered. The remaining lower aqueous phase was submitted twice again to the same extraction procedure. The three butanol extracts were pooled and evaporated to dryness under vacuum at 5 C. The residue was dissolved in 10 ml of chloroform-methanol (3:1, vol/vol). No loss of radioactivity was observed during the extraction and evaporation procedures. The chloroform-methanol solution was applied to a column (1 by 35 cm) filled with Unisil silicic acid and previously equilibrated with chloroform-methanol (3:1, vol/ vol) (Fig. 1B). The elution was first run with 150 ml of this solvent and thereafter with a linear gradient from this solvent to pure methanol (250 g of methanol connected with 250 g of chloroform-methanol [3:1, vol/vol] running onto the column). The fractions containing radioactive lipid II were pooled and kept at -25 C. The amino acid and hexosamine contents of the product was determined: glutamic acid, 1; alanine, 2.6; A2pm, 1.06; glucosamine, 0.92; and muramic acid, Traces of glycine, serine, leucine and isoleucine were present. Incorporation of I3HJA2pm and analysis of lipid I and II cell contents. Cultures (40 ml) of strain FB8rel+lysA were grown at 37 C in 200 ml flasks under strong aeration in M63 medium supplemented with 0.2% glucose, L-lysine, L-methionine, and L-threonine, each at 100,ug x ml-', as previously described (25). When A6. reached 0.3 (2 x 108 cells x ml-'), 5-ml aliquots were rapidly transferred to 25-ml flasks stirred at 37 C containing 1.5 MBq (850 GBq x mmol-1) of lyophilized [3HJA2pm. For the analysis of the incorporation of [HJA2pm into peptidoglycan and its precursors at various times thereafter, aliquots (1 ml) were rapidly harvested by centrifugation in the cold and pellets were suspended in 30 IlI of a mixture of isobutyric acid and 1 M ammonia (5:3). The mixture was applied to Whatman 3MM filter paper, and chromatography was run overnight in isobutyric acid-1 M

3 VOL. 174, 1992 LIPIDS I AND II IN PEPTIDOGLYCAN METABOLISM 3551 I0 E Fraction number FIG. 1. Purification of pentapeptide lipid II by column chromatography. As described in Materials and Methods, the crude chloroform-methanol extract (11 x 106 cpm) containing lipid II was applied to a DEAE-cellulose column eluted successively with methanol, 0.4 M ammonium acetate in methanol (starting at arrow 1), and a linear gradient from 0.4 to 4 M ammonium acetate in methanol (starting at arrow 2). Fractions (9 to 10 ml each) were tested for radioactivity. The second radioactive peak (fractions 20 to 40) corresponded to lipid II. After pooling, extraction, evaporation, and dissolution, the product was applied to a silicic acid column (B) eluted first with chloroform-methanol (3:1, vol/vol) and thereafter with a linear gradient (starting at the arrow) from this solvent to pure methanol. Fractions (10 ml each) were tested for radioactivity. ammonia (5:3). Radioactive compounds were detected with a Berthold scanner model LB283 (Berthold, Elancourt, France). The lipid I and II cell contents of the 5 ml of [3H]A2pmlabelled cultures were analyzed after incubation for 25 min at 37 C. Cultures were boiled for 4 min. Insoluble material was recovered by centrifugation at 12,000 x g, suspended in 200 PI1 of water, and boiled for 4 min to remove residual [3H]A2pm and radioactive UDP-MurNAc-tri- and pentapeptide nucleotides. After centrifugation, pellets were suspended in 10% acetic acid and material was transferred to glass vials, which were sealed and heated at 105 C for 1 h. After evaporation to dryness, residues were suspended in water (80 p,l) and the soluble N-acetylmuramoyl-peptides were reduced to their muramicitol derivatives as previously described (20). Accordingly, samples were diluted with 80 PI1 of 0.5 M sodium borate buffer (ph 9) and incubated in the presence of sodium borohydride (1 mg x ml-') for 30 min at room temperature. The reaction was stopped by adjusting ph to 4 to 5 with acetic acid. The reduced muropeptides were analyzed by high-pressure liquid chromatography (HPLC). When the lipid I and II contents of D-cycloserinetreated cells were examined, the drug was added at a final concentration of 0.4,ug x ml-' 10 min after addition of [3H]A2pm acid, and cells were further incubated for 15 min before the above-described analytical procedure was carried out Ṁembrane preparation for polymerization reactions. Cultures of strain JA200(pLC19-19) were prepared under strong stirring at 37 C in 2-liter flasks containing 500 ml of Penassay broth (AM3; Difco) and inoculated with 5 ml of overnight precultures. Cells from 2.5 liters of culture were harvested at OD 0.8 by centrifugation in the cold for 10 min at 10,000 x g (yield, 3 g [wet weight]), washed once with 0.02 M Tris-HCl buffer (ph 8), and disrupted by grinding at 4 C in a large mortar with levigated alumina (4 g/g [wet weight] of cells) as previously described (45). After suspension of the mixture in 45 ml of buffer A and centrifugation for 5 min at 7,500 x g, the supernatant was centrifuged for 1 h at 100,000 x g. The pellet was suspended in 0.5 ml of buffer A at ca. 30 mg 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 containing 500 ml of LB medium and ampicillin (100 Fg x ml-'), inoculated with 5 ml of overnight precultures. Cells grown first at 30 C up to OD 0.2 and for 1.5 to 4 h at 42 C were thereafter disrupted as described above. Partial purification of PBP lb and PBP 3. Cultures of strain JA200(pLC19-19) were prepared overnight under strong aeration in seven 2-liter flasks containing 250 ml of L medium (28). After dilution with 250 ml of fresh medium, growth was allowed to proceed for 90 min before harvesting of cells in the cold by centrifugation for 15 min at 13,000 x g. Cells were washed with buffer A (yield, 15.5 g [wet weight]) and disrupted by sonication (Sonicator 150 TS; Ultrason, Annemasse, France) in the same buffer. After removal of cell debris by centrifugation for 10 min at 3,000 x g, 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 of protein x ml-'. The suspension was heated for 10 min at 60 C to inactivate cell PBPs except PBP lb according to Nakagawa et al. (29). After cooling in ice and addition of n-octylglucopyranoside up to a final concentration of 1.5% (165 mg in 11 ml), the mixture was vigorously shaken for 30 min at room temperature, allowed to stand for 15 min, and centrifuged for 30 min at 27,000 x g. The pellet was suspended in buffer A and treated again in the same way with the detergent. The two resulting supernatants were pooled and fractionated with ammonium sulfate between 0 and 41% saturation. Precipitated material was recovered by centrifugation for 30 min at 27,000 x g, suspended in buffer A, dialyzed against the same buffer, and stored at -20 C. PBP lb was the only PBP detected when this preparation was analyzed by gel electrophoresis according to Spratt (37). Partial purification of PBP 3 was carried out according to Ishino and Matsuhashi (17), with minor modifications. A culture of QCB1(pXEPR) was grown at 30 C in LB medium to OD 0.6 and then transferred to 42 C for 1.5 h (conditions shown to maximize the yield of PBP 3). Cells were harvested by centrifugation, and a particulate fraction was obtained by sonication and differential centrifugations. An aliquot (350 mg of protein) of this particulate fraction was extracted first at 20 C with 50 mm Tris-HCl (ph 7.6) buffer containing 1%

4 3552 VAN HEIJENOORT ET AL. TABLE 2. Extent of accumulation of lipid II with various membrane preparations" Membrane Protein yield (mg/g Activity" prepn [wet wtl of bacteria) Alumina Sonication French press Spheroplasts " Experiments were carried out with mid-exponential-phase cells of E. coli K-12 HfrH. ' The assay for the accumulation of lipid 11 was performed as described in Materials and Methods. Activities are expressed as the percentage of conversion of UDP-MurNAc-pentapeptide into lipid 11. Triton X-100 and 0.1 mm MgCl2 (buffer B). The pellet was extracted again with buffer B containing 1 M NaCl. The extract containing PBP 3 was desalted on a Sephadex G-50 column eluted with buffer B. After cephalexin-sepharose affinity 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 the polymerization assay. Assay for the polymerization by transglycosylation. The in vitro polymerization by transglycosylation catalyzed by PBP lb was assayed as described previously (45). The assay was carried out with lipid II as a substrate at concentrations ranging from 10-5 to 10-4 M, depending on the lipid II preparation. The assay was very reproducible with 10- M lipid II. However, at 10-5 M, a variability in the results was observed, owing presumably to an insufficient redissolution of lipid II up to a saturating concentration. The apparent Km of purified PBP lb for pentapeptide lipid II from E. coli has been estimated at 3,uM (29). As previously recommended (29), the addition of methanol (15% final concentration) to the assay will ensure proper solubility of lipid II and reproducible results. Penicillin is added to the assay to block all transpeptidase and DD-carboxypeptidase activities. With some particulate fractions, D-alanine was clearly detectable on chromatograms, presumably because of the presence of endogenous 1-lactamase activity. In such cases, clavulanic acid at a final concentration of 100,ug x ml-' was added to the assay. RESULTS Conditions for the in vitro formation of lipid II. The formation of lipid II by incubation of UDP-MurNAc-pentapeptide and UDP-GlcNAc with particulate fractions from E. coli is a complex process, since they contain not only the translocase and transferase activities but also the membrane acceptor undecaprenyl-phosphate. The extent of in vitro formation of lipid II is greatly dependent on how the particulate fraction was prepared and on the concentrations of UDP-MurNAc-pentapeptide, UDP-GlcNAc, and protein. Preparations from spheroplasts were initially used (44). When various methods were compared (Table 2), spheroplasts indeed led by far to the highest yields. A set of optimal conditions for a maximal synthesis was sought by fixing the UDP-MurNAc-pentapeptide and UDP-GlcNAc concentrations and by varying the protein concentration. Not being a commercially available product, UDP-MurNAc-pentapeptide was fixed at 5,uM, whereas UDP-GIcNAc was fixed at M, a 100-fold-higher concentration, to ensure a high lipid II/lipid I ratio. The temperature optimum was found to be 35 C, and the ph optimum was between 7 and 8 (in A B I~~~ C D E F a H I START - FRONT '91 '.1~I# P ' J. BACTrERIOL. i.. I'. 1 h i FIG. 2. Analysis by paper chromatography of the formation of tripeptide and pentapeptide lipids II and their use in polymerization catalyzed by PBP lb. Chromatographies were run overnight on Whatman 3MM filter paper in isobutyric acid-1 M ammonia (5:3). Radioactive compounds were detected with a Berthold scanner model LB283. (A) UDP-MurNAc-tripeptide (with [14C]A2pm); (B) UDP-MurNAc-pentapeptide (with [14C]Ala-D-[`4C]Ala); (C) in vitro formation of tripeptide lipid II; (D) purified tripeptide lipid II; (E) in vitro formation of pentapeptide lipid II; (F) purified pentapeptide lipid 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, Rf 0.8 to 0.9); (H) in vitro polymerization catalyzed by PBP lb with pentapeptide lipid II as a substrate (100 pmol in the assay); (I) in vitro polymerization catalyzed by PBP lb with tripeptide lipid II as a substrate (100 pmol in the assay). The in vitro formation of lipid II from UDP-MurNAc-tri- or pentapeptide (C and E) was carried out at an analytical level by scaling down by 1/10,000 the preparative assay described in Materials and Methods. Tris-HCl or potassium phosphate buffer). The incubation time was limited to 60 min to avoid the degradative effects of side reactions. Under these conditions, the yield of lipid II increased with the amount of particulate fraction in the assay and reached a plateau value at 8 mg of protein x ml-'. ATP was found to stimulate the formation of lipid II by 35% when added to the assay at ca. 10 mm. This result was in agreement with a previous observation made by Araki et al. (1) for the in vitro synthesis of peptidoglycan in a cell-free system from E. coli. Presumably ATP is necessary for the in vitro conversion of undecaprenol into undecaprenyl phosphate catalyzed by a membrane phosphokinase. Such a kinase has been isolated from Staphylococcus aureus (36), and the ratio of undecaprenol to undecaprenol phosphate has been estimated at 1:1 in Streptococcus faecalis (43). Taking into account the various results, an assay leading to 40 to 60% conversion of UDP-MurNAc-pentapeptide into lipid II was developed (Fig. 2E). It was noteworthy that no peptidoglycan material was formed under these conditions. Purification of lipid II. The isolation and purification of lipid II from the in vitro assay were carried out by adapting the procedure of Umbreit and Strominger (44). A number of modifications (see Materials and Methods) were introduced to increase the final yield of lipid II from UDP-MurNAcpentapeptide, which was only 2% in the study by these authors. The overall yield of five preparations that we made ranged from 12 to 18%. The specific radioactivity of the UDP-MurNAc-pentapeptide used varied from 0.2 to 3 GBq

5 VOL. 174, 1992 LIPIDS I AND II IN PEPTIDOGLYCAN METABOLISM 3553 TABLE 3. Purification of lipid II Pentapeptide lipid II Tripeptide lipid II Step Yield of Yield of (1p6) each step cpm each step (106)(106) (% Initial UDP-MurNAc-peptidea Butanol extract after the ac cumulation reaction DEAE-cellulose column Silicic acid column Overall yield' a The specific radioactivity of UDP-MurNAc-pentapeptide was 3 GBq x mmol-', 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 from the silicic acid column to the initial UDP-MurNAc-peptide. x mmol-'. Yields of some steps of the preparations were found to be greatly dependent on temperature. In particular, the removal in vacuo of butanol from extracts was performed at 5 C, and chromatographies on silicic acid columns were run in the cold at 4 C. In Table 3, the yields of the various steps of a preparation from UDP-MurNAc-pentapeptide at 3 GBq x mmol-' are reported. In this case, 0.56,umol of lipid II was formed with 1.4 g of membrane protein and 0.16,umol of purified lipid II was secured. Analysis of the amino acid and hexosamine contents of the purified product indicated a stoichiometry close to that of the GlcNAc-MurNAc-pentapeptide moiety. The slightly lower glucosamine content (see Materials and Methods) suggested the possible presence of some lipid I. This was confirmed by HPLC analysis (data not shown) after partial acid hydrolysis and reduction according to the method described for determining lipid I and lipid II contents (see below). The ratio of lipid I to lipid II was estimated at 1:6. The mass spectrum analysis of the material recovered in the ether phase after mild acid hydrolysis was characteristic of undecaprenol as the major constituent (data not shown). In vitro formation of tripeptide lipid II. When UDP- MurNAc-pentapeptide was replaced by UDP-MurNActripeptide as the substrate in the assay for the in vitro synthesis of lipid II, a radioactive product, presumably tripeptide lipid II, was detected by paper chromatography, in which it migrated as pentapeptide lipid II (Fig. 2). However, it accumulated about three to four times less than did pentapeptide lipid II (Table 3). Its purification was carried out under the conditions used for pentapeptide lipid II (Table 3), and the chromatography elution patterns (data not shown) were very similar to those of pentapeptide lipid II. HPLC analysis (data not shown) carried as described below after partial acid hydrolysis and reduction revealed that 72% of the radioactivity of the product was recovered in a major peak eluting as the reduced dissaccharide tripeptide (compound b in Fig. 3). Analysis of cell lipids I and II. The analysis of cell lipids I and II was undertaken for two reasons. First, the in vitro formation of tripeptide lipid II raises the question of the DCS at ph b c d 500- o 1500 _ +DCS St ph 3.65 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 Materials and Methods with 5-ml cultures of strain FB8rel+lysA specifically labelled with [3H]A2pm. The reduced muropeptides originating from lipids I 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 0.5 ml x min-1. Fractions (0.5 ml) were collected, diluted with 4.5 ml of scintillation liquid, and counted for radioactivity. (A) Fifteen microliters from 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 corresponds to 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 eluent at ph 3.65; (D) the same with an eluent at ph The excluded radioactive peak corresponds to residual [3H]A2pm. Arrows indicate the positions 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 min at 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.

6 3554 VAN HEIJENOORT ET AL. possible in vivo presence of tripeptide lipids I and II. A priori, the 10- to 40-fold-lower pool of UDP-MurNAc-tripeptide than of UDP-MurNAc-pentapeptide in normally growing cells (27), and the 3- to 4-fold less efficient in vitro formation of tripeptide lipid II, would suggest very low pool values for tripeptide lipids (at most ca. 100 copies per cell). However, it has recently (3, 32) been proposed that MurNAc-tripeptide precursors could be involved in peptidoglycan polymerization during septation. It was therefore essential to determine whether tripeptide lipids could be detected in cell extracts. Second, it was also of interest to compare purified lipid II after accumulation in cell-free systems with the in vivo material. For these purposes, strain FB8rel+lysA was used for the convenient specific pulse-labelling of peptidoglycan precursor by [3H]A2pm (25). The distribution of [3H]A2pm between peptidoglycan and its A2pm-containing nucleotide and lipid precursors was analyzed by paper chromatography (Fig. 2G), and the separated products were quantified at various time intervals (data not shown). Between 10 and 30 min after the addition of [3H]A2pm, the radioactive pool of lipids I and II remained constant, whereas peptidoglycan steadily increased and [3H]A2pm steadily decreased. No clear separation between lipids I and II could be observed. Purified triand pentapeptides lipids II migrated with the same Rf as the in vivo-labelled material (Fig. 2). Furthermore, D-cycloserine was used to promote the increase of the UDP-MurNActripeptide pool and the depletion of the UDP-MurNAcpentapeptide pool (16, 21, 31) so as to increase the pools of the putative tripeptide lipids. The tripeptide and pentapeptide lipid cell contents were analyzed before and after D-cycloserine treatment by adapting two previously described methods (20, 33). After boiling, harvesting, and thorough washing, cells were submitted to mild acid hydrolysis to cleave the muropeptides from undecaprenol pyrophosphate. After reduction, muropeptides were analyzed by HPLC according to two sets of conditions. For reduced muropentapeptides (compounds c and d in Fig. 3), the elution was carried out at ph 3.65, whereas for reduced murotripeptides (compounds a and b in Fig. 3), the elution was performed at ph 3.20 to ensure a proper separation from residual [3H]A2pm. In untreated cells (Fig. 3A and B), reduced muropentapeptides c and b were present in a 1:3 ratio as previously described (20, 26), and reduced murotripeptides were not clearly detectable. However, a very small radioactive peak was observed at the position of reduced murotripeptide b (Fig. 3B). It was better visualized when larger amounts of the extract were injected and accounted for ca. 10% of that of reduced muropentapeptide c (upper curve in Fig. 3B). In D-cycloserine-treated cells (Fig. 3C and D), reduced muropentapeptides c and d decreased, whereas the peak corresponding to reduced murotripeptide b increased significantly. When the material recovered under this latter peak was analyzed by HPLC according to Glauner (11), it comigrated with reduced murotripeptide b. It was necessary to verify that the observed reduced murotripeptide b did not originate from the labelled peptidoglycan material present with lipids I and II in the washed boiled cells in the course of the mild acid treatment. For this purpose, a sample of [3H]A2pm-labelled and D-cycloserine-treated cells was boiled with 4% sodium dodecyl sulfate for 10 min. After repeated washings with water, the resulting insoluble material was submitted to the same analytical procedure as for lipids I and II. No reduced muropentapeptide c or d was detected in the three different experiments carried out, and TABLE 4. Transglycosylation activity of PBP lb and PBP 3 present in extracts from various E. coli K-12 strainsa Strain Activity" PBP overproduction (fold) JM QCB JM83(pJP14) JM83(pJP15) JM83(pJP13) ,000 JA200(pLC19-19) Crude extract 4.0 Purified fraction 23.8 QCB1(pXEPR) Crude extract Purified fraction 0.0 J. BACTERIOL. a Extracts from the various strains were prepared and the assay for the in vitro transglycosylation activity of polymerase PBP lb was carried out as described in Materials and Methods. The putative in vitro transglycosylation activity of PBP 3 (crude extract or purified fraction) was tested by the same assay and by the assay of Ishino and Matsuhashi (17). ' Expressed as the percentage of conversion of lipid II into polymerized material for 10,ug of protein introduced in the assay. no reduced murotripeptide b was detected in two of the experiments. However, in one experiment a small peak eluting as reduced murotripeptide b accounted for less than 20% of the level found in non-sodium dodecyl sulfate-treated cells. In vitro polymerization by transglycosylation catalyzed by PBP lb. It was previously established that purified PBP lb or particulate fractions containing PBP lb can catalyze the in vitro formation of cross-linked peptidoglycan material with pentapeptide lipid II as a substrate (29, 39). PBP lb is a bifunctional enzyme which catalyzes transglycosylation and transpeptidation reaction involved in peptidoglycan formation. In the in vitro assay, transglycosylation can be uncoupled from transpeptidation by the addition of penicillin (45). Moreover, it was previously established that such an uncoupling leads to an important stimulation of the transglycosylation reaction (39). In the present study, only the polymerization by transglycosylation was considered. Particulate fractions from various strains and partially purified PBP lb from strain JA200(pLC19-19) were examined as sources of transglycosylase activity (Table 4). No polymerase activity was detected in extracts from strain QCB1 devoid of PBP lb. Plasmid plc19-19 carries theponb gene coding for PBP lb (40), and in strain JA200(pLC19-19), it led to a fourfold increase of activity. In strain JM83(pJP13), the plasmid ponb gene was under the control of the Pr promoter. After full expression at 42 C, immunological estimations indicated a ca. 1,000-fold increase in the amounts of PBP lb compared with that of the control strain harboring the plasmid without ponb. However, when the PBP lb transglycosylation activity was considered, only a 28- to 29-fold increase was found (Table 4). When tripeptide lipid II instead of pentapeptide lipid II was used as the substrate in the assay, a similar extent of peptidoglycan formation was observed (Fig. 3). Moenomycin was previously shown to specifically inhibit the transglycosidase activity 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 vitro polymerization reaction carried out with tripeptide lipid II as a substrate (data not shown). In vitro polymerization catalyzed by PBP 3. PBP 3 has been shown to be essentially involved in peptidoglycan biosynthesis at the time of septum formation during cell division in

7 VOL. 174, 1992 LIPIDS I AND II IN PEPTIDOGLYCAN METABOLISM 3555 E. coli (30). Peptidoglycan synthesized in a defilamentation system which elicited the activity of PBP 3 in vivo contained increased amounts of cross-linkage as well as a higher ratio of tripeptide-containing cross-linked subunits (32). The possible use of tripeptide lipid II as a substrate for this enzyme has been postulated to be a signal that triggers the change from lateral to septal peptidoglycan biosynthesis (3). Therefore, we have tried to determine whether the tripeptide lipid II could serve as substrate for PBP 3 in an in vitro system. Particulate fractions from various strains (not producing PBP lb) and partially purified PBP 3 (-80% purification) from strain QCB1(pXEPR) were examined as sources for transglycosylase activity (Table 4). Plasmid pxepr carries the pbpb gene, coding for PBP 3, under the control of Pr promoter. The amount of PBP 3, detected by antibodies raised against the protein, increased 26-fold when strain QCBl(pXEPR) was induced for 2.5 h at 42 C compared with wild-type expression of the protein. In the same experiment, P-lactam-binding activity showed only a ninefold increase. However, when the transglycosylase activity of PBP 3 was analyzed in vitro, no polymerization occurred at 30 C, and no increase was seen in the induced fraction compared with the level at 30 C, independently of the substrate (tripeptide or pentapeptide lipid II) used in the assay. This finding contradicted the previously described (17) polymerase activity of purified PBP 3 determined with pentapeptide lipid II used as the substrate. We have no explanation for this discrepancy. DISCUSSION A set of optimal conditions was determined for the in vitro formation of radioactive pentapeptide lipid II in a cell-free system from E. coli and for its isolation and purification. These conditions led to the formation of ca. 0.68,Lmol of lipid II with membranes from 100 g (wet weight) of cells. Since the normal endogenous pool of lipid II for this amount of cells can be estimated at 0.1 to 0.2,umol, this means a three- to sixfold accumulation. Presumably the undecaprenyl pyrophosphate pool could be the limiting factor for the in vitro formation of lipid II under the conditions considered. Assuming a more or less complete depletion during the in vitro assay, its in vivo pool would therefore be a few fold higher than the lipid II pool. The polymerase activity of PBP lb was conveniently determined by the in vitro assay using pentapeptide lipid II as a substrate at a concentration near the estimated Km value (29). As previously observed (39), membrane preparations from a strain lacking PBP lb were devoid of in vitro polymerase activity, whereas those from overproducing strains possessed much higher levels. However, in the case of the PBP lb overproducer harboring a plasmid with the ponb gene under the control of the Pr promoter, the increase in polymerase activity was far from paralleling the considerable increase in PBP lb copies. This result seemed to imply that a great part of the overproduced protein was in an inactive form, because of either an improper processing or the lack of a nonoverproduced effector. When the in vitro PBP lb activity of particulate fractions was compared with the amounts of peptidoglycan formed in vivo in the corresponding cells (27), the in vitro system appeared extremely inefficient and accounted for at best 2 to 3% of the in vivo polymerization. This discrepancy was presumably due to the fact that the in vitro conditions, although optimized, are still far 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 in vivo there is some kind of integrated organization whereby the formation of lipid II and its use are coupled. In this study, the formation of tripeptide lipid II from UDP-MurNAc tripeptide in a cell-free system was established and its purification was carried out. Since four times less tripeptide lipid II than pentapeptide lipid II was formed, the efficiency of the translocase and transferase activities was thus partly dependent on the structure of the peptide moiety. Moreover, tripeptide lipid II was used as a substrate in the in vitro assay for PBP lb polymerase activity apparently as efficiently as was pentapeptide lipid II. A number of observations indicate that in vivo the E. coli peptidoglycan biosynthesis system will accept modifications in its peptide moiety (24). It was previously reported (32) that when ether-treated cells were incubated with exogenous [14C]UDP-GIcNAc and UDP-MurNAc-tri- or pentapeptide, incorporation of radioactivity into insoluble peptidoglycan material was observed. These results are very similar with those described here. It is noteworthy that the efficiency of this system is also four times lower with the tripeptide precursor than with the pentapeptide precursor. The use of the tripeptide precursor in our cell-free system or in ether-treated cells raised the possibility of its use in the in vivo formation of peptidoglycan via tripeptide lipids I and II. It was recently proposed that in the cell cycle of E. coli, the alternating phases of cell elongation and septation depend on a regularly shifting balance between activities of two competing morphogenetic systems (3). In growth by elongation of the cylindrical cell, transpeptidation would be carried out preferentially with pentapeptide side chains, whereas tripeptide subunits would be the preferred acceptors for transpeptidation carried out by PBP 3 during septum formation. Variations in the tripeptide subunit content would determine the shift from cell elongation to septation (3). In peptidoglycan of normally growing cells, mainly tetrapeptide subunits are present (12). The tripeptide subunit content is fairly low (12), and it does not seem to vary significantly with elongation or septation (30). However, under a number of circumstances, such as the stationary phase (12) or amino acid starvation (42), the tripeptide content of peptidoglycan will increase considerably. Whatever the case, the presence of tripeptide subunits in peptidoglycan can a priori be explained in two ways. Either they arise by polymerization of tripeptide lipid II after formation of tripeptide lipids from UDP-MurNAc-tripeptide or they are formed by action of a DL-carboxypeptidase on peptidoglycan tetrapeptide subunits (2, 19). Finally, the possibility that pentapeptide lipids I and II are to some extent converted to the corresponding tripeptide lipids by specific hydrolytic removal of the two C-terminal D-alanine residues cannot be entirely excluded. The first possibility was investigated here. The detection of tripeptide lipid II in D-cycloserine-treated cells confirms that its formation from the tripeptide nucleotide can function in vivo and not only in cell-free systems or in ether-treated cells. The substantial increase of peptidoglycan tripeptide observed after treatment with sublytic concentrations of D-cycloserine (32) could suggest that the tripeptide lipid II is used in vivo for polymerization. However, to truly distinguish between the use of tripeptide lipid II and modifications after polymerization, control experiments with a DL-carboxypeptidase-deficient strain will have to be carried out. It was initially proposed that in vivo PBP lb was responsible for peptidoglycan synthesis during cell elongation,

8 3556 VAN HEIJENOORT ET AL. whereas PBP 3 was required for peptidoglycan synthesis during septation (see reference 47 for references). More recent results suggest that PBP lb is also involved in septation and that there is some connection between the activation of PBP lb and PBP 3. For instance, we have shown (9) that PBP lb is involved in some step at the moment of initiation of septation; also, we have found a new mutation that suppresses a pbpb(ts) phenotype only in the presence of PBP lb. On the basis of a study of the effects of specific P-lactams on the rate of peptidoglycan synthesis and the timing of cell lysis, Wientjes and Nanninga (47) have proposed that in vivo PBP lb catalyzes the synthesis of new cross-linked peptidoglycan chains which are used as primers by PBP 2 and PBP 3 to construct peptidoglycan for either elongation or septation. Furthermore, from the analysis of the mutagenic positions found in a number of PBP 3 mutant strains, it was suggested (38) that the transglycosylase domain assigned at the N-terminal end of PBP 3 does not exist. If we take into account these previous results and the findings that (i) tripeptide lipid II can be used by PBP 1B both in vivo and in vitro for the polymerization reaction, (ii) no polymerization activity was found for PBP 3 in any of the assayed conditions, and (iii) increased amounts of crosslinked subunits containing tripeptide moieties that elicit septum formation activities are found in a defilamentation system (32), we can speculate that the transglycosylase activity of PBP 1B is coupled with the transpeptidase activity of PBP 3, using mainly tripeptide lipid II as precursor, and possibly mediated by other components of the septum formation structure (septosome). ACKNOWLEDGMENTS This work was supported by grants from the Centre National de la Recherche Scientifique (URA 1131), the Institut National de la Sante et de la Recherche Medicale ( ), the Actions Integrees franco-espagnoles ( ), and the Fundaci6n Ram6n Areces. REFERENCES 1. Araki, Y., R. Shirai, A. Shimada, N. Ishimoto, and E. Ito Enzymatic synthesis of cell wall mucopeptide in a particulate preparation of Escherichia coli. Biochem. Biophys. Res. Commun. 23: Beck, B. D., and J. T. Park Activity of three murein hydrolases during the cell division cycle of Escherichia coli K-12 as measured in toluene-treated cells. J. Bacteriol. 126: Begg, K. J., A. Takasuga, D. H. Edwards, S. J. Dewar, B. G. Spratt, H. Adachi, T. Ohta, H. Matsuzawa, and W. D. Donachie The balance between different peptidoglycan precursors determines whether Escherichia coli cells will elongate or divide. J. Bacteriol. 172: Clarke, L., and J. 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