Biosynthesis of the peptidoglycan of bacterial cell walls

ARCHIVES

OF

BIOCHEMISTRY

Biosynthesis I. Utilization Diphosphate Enzymes

AND

BIOPHYSICS

of the

of Uridine

487-515

(1966)

Peptidoglycan

Diphosphate

Acetylglucosamine from

116,

of Bacterial

Acetylmuramyl for

Staphy/ococcos

Pentapeptide

Peptidoglycan

aureus

and

Synthesis Micrococcus

JOHN S. ANDERSONS, PAULINE M. MEADOW: AZSD JACK L. STROMINGER5 Department

of Pharmacology,

University

of Wisconsin

Received

June

Cell

Medical

and

Uridine

by Particulate lysodeikticus*

MARY

School,

Walls’

A. HASKIN,

Afadison,

Wisconsin

13, 1966

Particulate enzymes have been obtained from Staphylococcus aureus and Micrococcus Zysodeikticus which utilize UDP-acetylmuramyl-pentapeptide and UDPacetylglucosamine to form a linear peptidoglycan consisting of alternating residues of acet,ylglucosamine and acetylmuramyl-pentapeptide. This product retains both of the n-alanine residues present in the uridine nucleotide precursor. The other reaction products have been identified as UMP and inorganic phosphate formed from UDPacetylmuramyl-pentapeptide and UDP formed from UDP-acetylglucosamine. The synthesis of peptidoglycan is specifically inhibited by ristocetin and vancomycin at concentrations that are equivalent to the growth inhibitory concentrations of these antibiotics for the two organisms studied. Bacitracin aIso inhibits this synthesis at low concentrations, but the effects of this substance require further study. Penicillin G and novobiocin do not inhibit, except at concentrations which are far ab’ove the growth inhibitory concentrations. Some of the properties of the enzyme systems in the two organisms have been presented. The particulate enzyme prepared from cells of S. aureus after sonic disintegration is unusual in that it is inactive unless incubation mixtures containing it, are spread on filter paper. Enzyme preparations obtained from S. aureus after alumina grinding or from Al. lysodeikticus by either method are active in the usual assays.

Soon after the discovery of uridine diphosphate glucose by Leloir and his collabo1 Dedicated to Luis F. Leloir on the occasion of his sixtieth birthday. * Supported by research grants from the U.S. Public Health Service (AI-06247) and National Science Foundation (GB-4552). This work was initiated under earlier grants at Washingt,on University School of Medicine, St. Louis, MO. 3 Supported by a postdoelorai feiiowship of the National Science Foundation. Present address: M.R.C. Laboratory of Molecular Biology, Hills Road, Cambridge, England. 4 Present address: Department, of Biochemistry, University College, London W.C. 1, England. 5 I secured my first independent posit,ion in 1951, soon after the discovery of uridine diphosphate glucose by Leloir and his collaborat,ors.

rators (I), a uridine diphosphate derivat’ive containing an unknown acetamido sugar, m-alanine, L-lysine, and n-glutamic acid was isolated from cells of Staphylococcus aureus in which it accumulated under the influence of penicillin (a, 3). The compound was also found in much smaller amounts in untreated cultures (4). Subsequent studies, establishing the structure of the sugar (5-S) and the sequenceof the peptide (g-12), have allowed representation of t’he structure of the nucleotide as IJDP-Mm-X-AC .L-ala .n-r-glu . My scientific career has been spent in exploring in one way or another the field of investigation which he opened up. It, is therefore a privilege for me to be able to contribute this paper in honor of his sixtieth birthday.

487

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ANDERSON,

L-lysen-alaen-ala. The sugar is N-acetylmuramic acid, a 3-O-n-lactic acid ether of N-acetyl-D-glucosamine The main features of the biosynthetic pathway t’o the nucleotide have been established (13, 14). At about the same time as this nucleotide was discovered, bacterial cell walls were isolated for the first time. It became apparent that all bacterial cell walls contain a peptidoglycan6 whose components are similar to those of the nucleotide. It was therefore proposed that t’he nucleotide is a precursor of the bacterial cell wall and that its accumulation in S. aureus is the consequence of inhibition of cell wall synthesis by penicillin (7, 8). This hypothesis was arrived at independently from morphological studies of the effects of penicillin on bacteria (15, 16) and has been amply supported by direct isotopic measurements of wall synthesis in the presence and absence of the antibiotic. The utilization of UDP-MurNAc-pentapeptide and UDP-GlcNAc for the synthesis of a chromatographically immobile (17) or acid precipitable (IS) product was reported in 1964, and confirmed in a third laboratory (19). Only in one of these systems could the product of the reaction be identified as a peptidoglycan (17). Subsequent, studies (20-23) have revealed that the reaction mechanism is complex. The initial acceptor (19, 20) is a membrane-bound phospholipid (20, 24, 25). A phosphodisaccharide-pentapeptide derived from the two uridine nucleotides is covalently linked to the phospholipid and may be further modified while attached to the phospholipid prior to utilization for peptidoglycan synthesis. With some types of enzyme preparations (18), for the most part only the initial stages of the 6A variety of nameshas been applied to the polymer found in all bacterial cell walls, including mucopeptide, glycopeptide, murein, and peptidoglycan. The latter term seems most, descriptive of the chemical nature of the material to several workers in this field and will be employed in this and future publications from our laboratory. Linear peptidoglycan strands do not have interpeptide cross links, in contrast to the cross-linked peptidoglycan. MurNAc and GlcNAc are abbreviations for N-acetylmuramic acid and N-acetylglucosamine, respectively, the sugars in the peptidoglycan.

ET

AL.

reaction, i.e., attachment of the sugars to the phospholipid, occur (21, 23). A detailed account of the experiments which have been carried out in our laboratory is reported in this and following papers. MATERIALS

AND

METHODS

General procedures and materials. Staphylococcus al(Teus, strains H and Copenhagen, were laboratory cultures maintained on agar slants with monthly transfers. Their identification by phage typing and immunological reactions have been described (26). Broth cultures of S. a?,relLs were grown with vigorous shaking at 37” in a medium consisting of 0.5$& pept,one (Difco), 0.5y0 yeast extract (Difco), 0.2% glucose, and 0.1% KzHPOd at pH 7. Cultures of Micrococcus lysodeikticus, ATCC 4698, were grown under similar conditions in a broth containing 3.7yG brain heart infusion (Difco). ‘4C-L-Lysine (uniformly labeled, 246 mC per millimole), 3H-nL-lysine (4,5-3H, 3.9 C per millimole), 3H-nL-alanine (3-3H, 236 mC per millimole), and sodium acetate (l-l%, 26.6 mC per millimole) were purchased from New England Nuclear Corp., Boston, Massachusetts. 14C-n-Alanine (l-14C, 7.5 mC per millimole) was obtained from U.S. Nuclear Corp., Burbank, California. Carrierfree 3’P-disodium phosphate was purchased from Volk Radiochemical Co., Skokie, Illinois. IXPacetylglucosamine, lysozyme, and Escherichia coli alkaline phosphate were products of Sigma Chemical Co., St. Louis, Missouri. P-Acetylglucosaminidase was prepared from pig epididymis (27). Phosphodiesterase from Russell’s viper venom was obtained from Worthington Biochemical Corp., Freehold, New Jersey. Penicillin G, vancomycin, and bacitracin were a gift of Eli Lilly and Co., Indianapolis, Indiana. Ristocetin was a gift of Abbott Labs., North Chicago, Illinois, and novobiocin was a gift of the Upjohn Company, Kalamazoo, Michigan. The following solvents were used for descending paper chromatography on Whatman No. 1 or No. 3 MM filter paper: A, isobutyric acid-l M NH,OH (5:3); B, 95% ethanol-l M ammonium acetate, pH 7 (15:6); C, n-butanol-ethanol-water (13:8:4); I), n-butanol-pyridine-water (6:4:3); and E, n-butanol-acetic acid-water (4:1:5). After chromatography in solvent A, chromatograms were routinely washed three times in acetone while wet before drying. Radioactivity on paper chromatograms was detected with a Vanguard model 880 paper strip scanner equipped with automatic data system, by radioautography, and by liquid scintillation counting on a Packard Tri-Carb liquid scintilla-

BIOSYNTHESIS

OF

tion spectrometer. Radioactive spots were cut from chromatograms and immersed directly in scintillation solution consisting of 4.0 gm 2,5diphenyloxazole and 0.3 gm l,l-bis-2-(4-methyl-5phenyl-oxazolyl)-benzene per liter of toluene. Protein was determined by the method of Lowry et al. (28). Uridine nucleotide concentrations were determined from nbsorbancy at 262 rnp and a molar extinction coefficient of lo*. Preparaiion of enzymes for synthesis of labeled uricline nucleotides. Four different preparations of UDP-MurNAc-pentapeptide labeled with ‘“C- and %-amino acids of high specific activity were prepared by enzymic addit)ion of amino acids to UDP-MurNAc’n-a1a.nglu or UDP-MurNAc.La1a.nglu.r,-lys. Crude cell extract from cells of S. aureus, strain Copenhagen, harvested at halfmaximal growth, was prepared as previously described (14). After dilution of 25 ml of the crude cell extract. with an equal volume of 0.02 M TrisHCl, pH 7.8, the n-lysine-adding enzyme and the n-ala.])-ala-adding enzyme were precipitated between 0.32 and 0.557, protamine sulfate by addition of 27; protamine sulfate. The protamine sulfate precipitate was eluted successively with 5 ml of pota.ssium phosphate, pH 7, of the following concentrations: 0.025, 0.05, 0.075, 0.10, 0.10, and 0.20 M. All buffers contained 0.001 M EDTA. Each eluate of the protamine sulfate precipit,ate was treated with 2.5 volumes of saturated ammonium sulfat,e. The resulting precipitates were collected by centrifu.gation and each dissolved in 1 ml of 0.02 M TrisHCl, pH 7.8. The major portion of the n-lysine-adeding enzyme was recovered in the 0.05 and 0.075 nr phosphate eluates. The former fraction was used for addition of labeled L-lysine to UDP-MurNAc.n-a1a.nglu. The n-a1a.nalaadding enzyme was eluted from the same protamine sulfate precipitate primarily in the 0.1 M phosphate eluates. n-A1a.nala synthetase (kindly provided by Dr. F. C. Neuhaus) was a t&-SOY0 ammonium sulfate fraction obtained from Streplococcus faecalis R (29). UDP-MurNAc.n-ala.n-glu was isolated from extracts of cells of S. aureus grown in lysine-deficient medium as previously described (30), and IJDP-MurNAc.n-a1a.nglu. ~-1~s was isolated from cells of S. auretLs inhibited with u-cycloserine (31). UDP-MurXL4c . L-ala . n-glu . 14c-~-lys ’ Dala.u-ala. IFor addition of l*C-L-lysine the reaction mixture contained 4.3 pmoles of UDP-MurNAc.n-a1a.nglu, 2pmoles of “C-n-lysine (5OOrC), 118 rmoles of ATP, 1.56 mmoles of Tris-HCl, pH 8.8, 126 rmoles of MgCh, 156 Mmoles of KF, and 600~1 of n-lysine-adding enzyme (912pg of protein) in a total volume of 5 ml. After incubation of the reaction mixture for 8 hours at 37”, paper chromatography of an aliquot in solvent A indicated that

PEPTIDOGLYCAN

‘Is9

more than 907n of the rlC-n-lysine had been incorporated into UDP-MurNAc~n-ala~n-glu~~4C~-1~s. The total reaction mixture was applied to t.wo sheets of Whatman No. 3 MM filter paper and developed for 24 hours in solvent A. The UDPMurNAc~n-ala~n-glu~‘4C-~-lys was located by radioautography and eluted wit,h water. The eluate was concent,rated in ZJUCUO to dryness. For addition of n-a1a.n.ala, the reaction mixture contained three-fourths of the preparation of UDP-MurNAc~L-ala~n-glu~14C-~-lys described above, 8.2pmoles of n-ala.n-ala, 5Opmoles of ATP, 800 rmoles of Tris-HCl, pH 8.8, 40 /*moles of MnCl?, and 2.3 ml of u-a1a.n.ala-adding enzyme (6.7 mg of protein) in a total volume of 4 ml. After incubation for 2 hours at 37”, paper chromatography of an aliquot in solvent A indicated that, more than 95y0 of the IJDP-MurNAc~n-ala~u-glu~l~C~-1~s had been converted to UDP-MurNAc’n-ala. n-glu.l”C-L-lys.n-ala.n-ala. The total reaction mixture was applied to five sheets of Whatman No. 3 MM filter paper and chromatographed for 48 hours in solvent A. In this solvent, ITDPMurNAc-pentapeptide has an RF of O.li whereas the UDP-MurNAc-tripeptide has an KF of 0.14. The labeled UDP-MurNAc-pentapeptide was located by radioautography and eluted from the paper with a minimum volume of water. The recovered material had the absorption spectrum of a uridine nucleotide and had a specific activity of 100 PC per micromole. Approximately 3 pmoles of labeled UDP-MurNAc-pentapeptide was recovered. The apparent, yield of more than 1OO7o (based on ‘“C-lysine added) probably resulted from the presence of lysine in one or both of the enzyme preparations employed as indicat,ed by the threefold dilution of t,he specific activity relative to the ‘C-n-lysine used. The isolated labeled UDP-MurNAc-pentapeptide was found to be essentially free of other radioactive compounds and at least 9OPr, pure with respect to ultravioletabsorbing compounds as revealed by paper chromatography in solvents A and B and by paper electrophoresis. UDP - MurNAc L-ala . D-glu . 3H-~-1~s . o-aZa.n-ala. For addition of 3H-n-lysine, the reaction mixture contained 1.4 #moles of UDPMurNAc.n-ala.n-glu, 1.3 pmoles of 3H-nn-lysine (2.5 mC), 39 pmoles of ATP, 520 rmoles of TrisHCl, pH 8.8, 42 pmoles of MgC12, 52 pmoles of KF, and 250 ~1 of n-lysine-adding enzyme (380 pg of protein) in a total volume of 1.7 ml. Strip scanning of a paper chromatogram on which an aliquot had been developed in solvent A revealed that about 25% of the 3H-lysine had been added to the nucleotide, i.e., about 5O7o of the 3H-n-lysine present initially. An additional 80 ~1 of L-lysineadding enzyme was added and the incubation was

490

ANDERSON,

continued for an additiotlal 4 hours, at which time the entire reaction mixture was applied to Whatman No. 3 MM filter paper and developed for 24 hours iu solvent A. Kodak Royal Blue X-ray film was exposed to the paper chromatogram for 4 days, at which time an image of the band of UI>P-MurNAc~~-ala~t~-glu~311-~-lys was clearly discernible. After elution with water and concentrat,iou in v(ccuo, a react,ion mixture containing the UDP-MurNAc.L-ala.D-glu.3H-~-lys, 7.2 pmolcs of u-a1a.D.ala, 7.6 pmoles of ATP, 38 pmoles of TrisHCl, pH 8.8, 1.9 pmoles of MnC12, and 100 ~1 of D-ala.o-ala-adding enzyme (290 pg of protein) in a total volume of 420 ~1 was incubated at, 37” for 8 hours. The reaction mixture was applied to What,man No. 3 MM filter paper and developed for 48 hours in solvent A. Radioactive materials were located by radioautography. The component with greater mobility, UDP-MurNAc.L-ala’nglu’3H-L-1~~s.o.ala’n-ala, R-as eluted with water and shown to co-chromatograph with authentic UDP-MurNAc-pentapeptide in solvents A and B. Approximately 0.2 rmole of UDP-MurNAc. L-ala. o-glu.3H-~-lys.D-ala.n-ala was recovered with a specific activity of 950 NC per micromole. UDP-MurNAc ’ L-ala D-gh . L-lys . ‘%-Dala.‘%‘-o-ala. For conversion of 1%.o-alanine to the dipeptide, the reaction mixture cont.ained 27 pmoles of ‘%-D-alanine (200 PC), 100 rmoles of ATP, 2000 pmoles of Tris-HCl, pH 8.8, 50 pmoles of MnCl?, and 4 ml of n-ala.D-ala synthetase (160 mg of protein) in a total volume of 6 ml. After incubat,ion of the mixture for 6 hours at 37”, the entire reaction mixture was applied to 4 sheets of Whatman No. 3 MM filter paper and developed for 24 hours in solvent A. A radioautogram indicated that 75P;, of the 1°CD-alanine (R,v = 0.65) had been converted t,o 1Gr-ala. XLD-ala (RF = 0.75). The dipeptide was eluted with water and concentrated to dryness in oacuo. For addition of radioactive dipeptide to UDPMurNAc-tripeptide, the reaction mixture contained t,he 14C-o-ala.14C-r-ala, 15 pmoles of UDPMurNAc.L-ala.D-glu.~-lys, 88 bmoles of ATP, 1500 pmoles of Tris-HCl, pH 8.8, 70 rmoles of MnC12, and 600 ~1 of o-a1a.D.ala-adding enzyme (1.7 mg of protein) in a total volume of 6.1 ml. The mixture was incubated for 6 hours at 37”, applied to 4 sheets of What,man No. 3 MM filter paper, and developed for 24 hours in solvent A. Iladioautograms located the UDP-MurNAc. L-ala.D-glU.~-ly~~l~C-o-ala~~%-o-ala and indicated that about 55% of the radioactive dipeptide had been incorporated into UDP-MurNAc-pentapeptide. Since the radioactive product was poorly separated from ATP, it was eluted with water and

ET

AI,

rechromatographed in solvent A. The IJDPMurNA.L-ala.D-glu.L-1ys.o.a1a.D.ala was again located by radioautography, eluted with water, and shown to be essentially free of BTP. It chromatographed with aut,hentic Ul>P-MurNAcpentapept,ide on two-dimensional paper chromatograms developed with solvents A and B. About 8 bmoles of the purified GDP-MurNAc.L-ala.uglu.~-lys.‘G-n-ala.‘4C-o-ala was obtained with a specific activity of 12 PC per micromole. UDP - XurNBc L-ala . D-glrc . ~~lys . 3HD-ala.3H-D-a/u. For conversion of 311-o-alanine to 3H-r-ala.3H-u-ala the reaction mixture contained 4.2 pmoles of 3H-DL-alanine (1 mC), 21 pmoles of ATP, 400 pmoles of Tris-HCl, pH 8.8, 10 pmoles of MnC12, and 1 ml of D-ala.o-ala synthetase (40 mg of protein) in a total volume of 2.4 ml. ilfter incubation for 6 hours at 37”, the reaction mixture was applied to 2 sheets of Whatman No. 3 MM filter paper and developed 21 hours in solvent A. Radioautograms exposed for 4 days clearly indicated the position of the 3H-D-ala.3H-o-ala and residual 3H-Dr,-alanine. Approximately 70% of the istope was recovered as the dipeptide. Since the D-a1a.D.ala synthetase has been shown to be highly specific for o-alanine (29), the relatively crude preparation employed must have contained a significant amount of alanine racemase. The 3H-o-ala.3H-o-ala was eluted with water and concentrated to dryness in uaczco. For addition of the a&labeled dipeptide to UDP-MurNAc-tripeptide, the reaction mixture contained the 3H-n-ala.3H-D-ala, 2 pmoles of UDPMurNAc.L-a1a.u.glu.~-lys, 12 Hmoles of ATP, 310 pmoles of Tris-HCl, pH 8.8, 10 rmoles of MnClp, and 500 ~1 of u-ala.D-ala-adding enzyme (810 pg of protein) in a tot,al volume of 2.7 ml. After 5 hours incubation at 37”, paper chromatography of an aliquot in solvent A indicated that 3570 of the dipeptide had been added to the nucleotide. ilfter addition of 150 ~1 additional n-ala’o-ala-adding enzyme and incubation for 5 more hours at 37”, the mixture was applied to 2 sheets of Whatman No. 3 MM filter paper and developed in solvent A for 26 hours. After drying, the chromatograms were also developed in solvent C for 16 hours. ltadioautograms were exposed for 4 days to locate t,he radioactive UDP-MurNAcpentapeptide. The UDP-MurNAc’L-ala.o-glu. L-lys.aH-D-ala.%-ala was eluted with a minimum volume of water and shown to co-chromatograph with authentic UDP-MurNAc-pentapeptide. About 0.8 pmole of the compound was recovered with a specific activity of 264 pC per micromole. 32P-UDP-MurNAc-pentapeptirle. =P-UDPMurNAc-pentapeptide was isolated and purified from cells of S. ourelLs inhibited with penicillin.

BIOSYNTHESIS

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Cells from a 100 ml culture of S. aureue, strain Copenhagen, at half-maximal growth, were collected by centrifugation at O”, washed once with water, and resuspended in an equal volume of fresh medium containing 0.5rc peptone (Difco), 0.5yc yeast extract, (Difco), and 0.2c< glucose. Fifteen mC of carrier-free 32P-sodium phosphate solution was added to the resuspended culture at 37”, and the pH was quickly adjusted to 7-7.5 with 4 x KOH. After 7 minutes incubation with shaking, 25 mg of penicillin G was added and the incubation was continued for 90 minutes with periodic addition of KOH to maintain the pH at 7-7.5. The labeled cells were harvested by centrifugation at 0”, washed once with 100 ml of 0.01 M potassium phosphate, pH 7, washed t,wice more with 100 ml volumes of water, and finally suspended in 7 ml of water. The suspension was heated in ,a boiling water bath for 8 minutes to extract cytoplasmic constituents. After cooling, 1 ml of 4O’z trichloroacetic acid was added, and 30 minutes later cellular debris and denatured protein were removed by centrifugation. The pellet was washed once with 1 ml of water, and t.he supernatant solution and washing were combined. On the basis of the absorbancy of the solution at) 262 rnp and a molar extinction coefficient of 104, 20 mg of charcoal (DarcoKB) was added for each micromole of ultraviolet-absorbing material. After thorough mixing of the extract and charcoal for 30-40 minutes at O”, the charcoal was collected by centrifugation at 35,000 g. The charcoal was washed twice with IO-ml volumes of lop3 M pot.assium phosphate adjusted to pH 4 with trichloroacetic acid and once with 10 ml of water. After each wash i,he charcoal was recovered by centrifugation at 35,000 g for 5 minutes. Finally, the charcoal was eluted at room temperature by mixing for 20 minutes with 30 ml of solution containing equal volumes of 95yc ethanol and 0.03 M NH,OH. The charcoal was removed by centrifugation at 35,000 g for 5 minutes. The elution step was repeated twice. The combined supernat,ant solutions were adjusted to pH 5 with isobutyric acid, concentrated in VQCUO to about 2 ml, and applied to Whatman No. 3 MM filter paper. The paper chromatogram was developed for 36 hours in solvent A. A radioautogram indicated the presence of several labeled compounds, only four of which received additional attent,ion. UDPMurNAc-pentapeptide: UDP-MurNAc.L-ala, and UDP-MurNAc were present as a broad band having a mobility of 0.6-0.7 relative to i17’P. CDP-ribitol (R~rr = 0.8) was also identified. The band containing UDP-MurNAc-pentapeptide was eluted, applied to another sheet of Whatman No.

PEPTIDOGLYCAN

491

3 MM filter paper, and developed in solvent B for 22 hours. Before drying, the chromatogram was washed twice in 80% ethanol and once in acetone in order to remove salts from the paper. Further removal of salts was accomplished by development for 24 hours in solvent C in which nucleotides are immobile. A radioautogram showed that the 32P-UDP-MurNAc-pentapeptide (RCYP = 1.02) was clearly separated from the mixture of 3?PUDP - MurNAc and a2P - UDP - MurNAc.L-ala (RL-MP = 1.35). The 32P-UDP-MurNAc-pentapeptide was eluted and shown to co-chromatograph with autheutic UDP-MurNSc-pentapeptide in solvents A and B. The ultraviolet absorption spectrum of the material was that of a uridine nucleotide. Several preparations were made according to this procedure and in most instances the isolated material had an initial specific activity of 50-100 & per micromole. 32P-I;DP-GlclV~lc. 3?P-UDP-GlcNAc was isolated and purified from cells of S. aureus inhibited with gentian violet. The procedure followed was very similar to that described above except that 10 mg of gentian violet (G. T. Gurr, lot G35) was substituted for penicillin. After chromatography in solvent A, many radioactive compounds were observed including UDP-GlcNAc (R.irr = 0.55), CDP-ribitol (RATP = 0.8), and a compound presumed to be UDP-GlcNAc-pyruvate enol ether (R~TP = 0.4). After chromatography in solvents B and C, the material containing UDP-GlcNAc was subjected to further purification by elect.rophoresis on Whatman No. 3 MM filter paper in 0.05 M ammonium acetate, pH 4.8, at 40 V per centimeter for 4 hours at 2”. The resultant electropherogram was washed three times in SOg;b ethanol, once in acetone and dried. A radioautogram showed two distinctly separate bands, one of which migrated toward the anode with the same mobility as authentic UDP-GlcNAc (10.5 cm). The other band (UDP-MurNAc) had a slightly faster mobility (12.3 cm). The 32P-UDP-GlcNAc had the ultraviolet absorption spectrum of a uridine nucleotide. The materials isolated in several such preparations all had initial specific activities of 50-100 PC per micromole. UDP-W-GZeNAc. UDP-I%-GlcNAc, labeled in the acetate moiety, was prepared from yeast enzymes (32) with minor modifications (33). Preparation of particulate enzyme. A 1% inoculum of an overnight culture of 8. auretts, strain H, was added to fresh culture medium at 37”. The growth of the culture was followed turbidimetritally at 700 rnp in 1:5 or 1: 10 dilutions. At quart,ermaximal growth, usually attained 120-140 minutes after inoculation, the cells were quickly harvested by centrifugation at 12,000 g for 5 minutes at, 0”.

492

ANDERSON,

The cells from each liter of culture were suspended in 25 ml of Tris-HCI, pH 7.8, and treated in the IO-kc Rayt,heon sonic oscillat,or for 10 minut,es. After centrifugation at 35,000 g for 1 minute to sediment unbroken cells and cell debris, the resulting supernatant solut,ion was centrifuged at 105,000 g for 1 hour. The pellet was resuspended in 25 ml of 0.02 M Tris-HCI, pH 7.8, and again sedimented by centrifugation at 105,000 9 for 1 hour. After discarding the supernatant solution, the particulate enzyme material was suspended in 0.5 ml of 0.02 M Tris-HCl, pH 7.8, for use as the particulate enzyme fraction. Particulate enzyme not used immediately was stored at -20” until needed. Such preparations of the particulate enzyme usually contained 12-18 mg of protein per milliliter. Particulate enzyme from Jf. Zysodeikticus was prepared in essentially the same manner. A al-hour cult,ure of M. lysodeikticus was used for inoculum. Cultures were usually ready to harvest about 12 hours after inoculation with a 2% inoculum. Washing of the M. Zysodeikticus particulate enzyme was essential if an absolute dependence on UDP-GlcNAc for incorporation of UDPMurNAc-%-pentapeptide into peptidoglycan was to be observed. Particulate enzyme was also prepared after disruption of the cells by grinding with alumina (20, 21, 34) ? Assay of peptidoglycan synthetase activity. A typical reaction mixture for assay of peptidoglycan synthesis by t,he particulate enzyme of Jf. Zysodeikticus contained 2 mpmoles of 14C- or VI-labeled UDP-MurNAc-pentapeptide (2-10 X lo4 cpm), 2 mpmoles of UDP-GlcNAc, 0.8 pmole of MgCI,, 2.5 pmoles of Tris-HCl, pH 8.6, and 60 rg of enzyme protein in a total volume of 25 ~1. Incubation was at 37”, usually for 60 minutes. After inactivation, reaction mixtures were subjected to paper chromatography on Whatman No. 3 MM filter paper in isobutyric acid-l N NH,OH (5:3). The peptidoglycan remained at the origin of the chromatogram and was counted in a liquid scintillation spectrometer. Peptidoglycan synthetase activity in the particu1at.e enzyme of 8. aUreU.s was assayed by the above procedure with the following modifications: the amount of MgC12 added was reduced from 0.8 to 0.08 pmole, 25 mwmoles of ATP was added, and the incubation was at 20”. ’ The advantageous properties of enzyme prepared after alumina grinding and the advantages of incubation at 20” were discovered by Dr. Michio Matsuhashi during studies of the system in which glycine is incorporated into the peptidoglycan (21). Details will be presented in a forthcoming publication on that system.

ET AL. Activity was 50% greater than in its absence. At siderably greater than at Incubations were carried or with incubat,ions spread The latter will be described

in the presence of ATP 20” activity was con37O.1 out either in test tubes as a film on filter paper. further.

RESULTS

Detection of the Reaction with Particles Prepared after Sonic Disintegration of S. aureus Initially, utilization of 14C-lysine-labeled UDP-MurNAc-pentapeptide and UDPGlcnTAc for synthesis of peptidoglycan, catalyzed by a particulate enzyme prepared after sonic disintegrat,ion of S. aureus, was observed when the reaction mixtures were allowed to incubate on filter paper. Conventional methods of assaying enzyme-catalyzed reactions with t’his enzyme preparat’ion had failed to show product formation. By chance, reaction mixtures were spotted on filter paper for assay without prior inactivation of the enzyme. In these instances a portion of the radioactivity was found to be chromatographically immobile. For example, with 10 ~1 of enzyme, 110 rpmoles of product were formed in 1 hour if the mixture was incubated on the paper at 37”, while an identical reaction mixture incubated in a tube at 37” and inactivat’ed prior to application to filter paper yielded only 2 ppmoles of product. This immobile radioactive material was presumed t’o be a peptidoglycan since its formation from UDP-R’IurNAc-14Cpentapeptide was dependent on the presence of UDP-GlcNAc. To demonstrate that the reaction was indeed t,aking place after application of the mixtures to the filter paper, reaction mixtures containing 14C-lysinelabeled UDP-YIurNAc-pentapeptide, UDPGlcNAc, and particulate enzyme from S. auyeus were applied to a series of small squares of filter paper and allowed to dry for various periods of time. At s-minute intervals the filter paper squares bearing the reaction mixtures were dropped into a beaker containing 100 ml of chromatography solvent A. Addition of solvent A is a convenient method for stopping enzymecatalyzed reactions. Three extractions of the paper squares with solvent A approximates development of a paper chromatogram in

BIOSYNTHESIS

OF

t,hat all materials which are mobile in this solvent system are extracted from the paper and only immobile materials remain. After extra&ion of the squares by stirring them in the beaker (with a magnetic stirrer underneath a wire mesh screen) and washing them several times with acetone, the amount of radioactivity remaining on the filter paper was determined. During Dhe first 10 minutes after application to the paper no reaction occurred, but during the interval between 10 and 20 minutes considerable radioactive product was formed. After 30 minutes no further reaction was observed. These results suggested that part,ial drying of the paper discs was necessary and that the reaction took place only at a certain degree of hydrat,ion.. In order to control the variables of temperature and humidity and consequently the state of hydration of the reaction mixtures on the filter paper, subsequent reactions on paper were carried out in a 37” room in a closed chamber containing an atmosphere saturated with water vapor. This humid chamber was a covered glass chromatography tank (12 X 12 X 24 inches) filled to a depth of one-half inch with water. Strips of Whatman h’o. 3 A’IR’I filter paper (15,; X 22 inches) bearing the reaction mixture at one end of thl? strip were suspendedfrom a supporting rack within the chamber. After incubation, the strips were subjected to descending paper chromatography in solvent A for separation of residual radioactive substrate from the immobile product. Radioautograms were prepared to provide unequivocal evidence for the completeness of the chromatographic separation. Optimal conditions for incorporation of radioactivity from UDP - ;IlurNAc - 14Cpentapep tide into product were investigated. Reaction mixtures containing ‘“Clysine labeled UDP-?tlurNAc-pentapeptide, UDP-GlcNAc, and enzyme were spotted on filter paper strips, allowed to dry for various lengths of time at 37”, and then placed in the humid chamber where they were incubated for 242 hours. Although earlier experiments had indicated that optimal reaction was obtained with 10 minutes of drying if the mixtures were spotted at, room t’emperature, in

493

PEPTIDOGLYCAN

I 0

I 20 OF DRYING

I IO MINUTES

I 30

FIG. 1. Effect of drying of reaction mixtures with 8. aUreU.s enzyme on filter paper strips at 37” before incubation total reaction mixture

in

a humid contained

chamber. The 14 mpmoles of

UDP-MurNAc-pentapeptide (labeled with 14Clysine), 14 mpmoles of UDP-GlcNAc, 64 @moles of Tris-HCl, pH 7.8, 1.9 pmoles of MgC12, and 255 ~1 of S. aUreU.s particulate enzyme, prepared after sonic disintegration, in a total volume of 510 ~1. Aliquots of 20 ~1 were applied to strips of Whatman No. 3 MM filter paper. After drying at 37” for the intervals indicated, the strips were incubated for 2.5 hours in a humid chamber at 37”. At the end of the incubation period, the strips were removed, dried quickly, and subjected to chromatography in solvent A. Material remaining at the origin was counted.

the 37” room the optimum time was the minimum time for application of the mixture to the paper and for placing it in the humid chamber (30-60 seconds) (Fig. 1). Only a small amount of product was formed during application of the reaction mixture to the paper. In the humid chamber nearly linear formation of product occurred during the first 40 minutes (Fig. 2). Several kinds and weights of filter paper and other materials were tested as possible supports for incorporation of radioactivity into peptidoglycan. Whatman No. 1, No. 3 MM, and No. 40 papers were about equally effective as support’sfor the reaction. Schaar filter paper (hexagon brand) was only about 50% as effective. Practically no product was formed when the reaction mixtures were applied to strips of glass fiber filter paper or to cellulose acetate. Whatman No. 3 34RI was the filter paper of choice because

494

AXDERSOS.

ET

of its skength and durability during manipulation. As a consequence of the results described above, Ohc procedure routinely used to assay peptidoglycan formation with particulate enzyme prepared aft’er sonic disintegration of S. aweus involved spotting of the reaction mixture on Whatman Ko. 3 MM filter paper in the 37” room and then incubating in the humid chamber at’ 37”

I 0.5 TIME

I I.0 IN HOURS

I 1.5

i 2.0

FIG. 2. Time course of peptidoglycan synthesis with S. aureus enzyme on filter paper strips in a humid chamber. The reaction mixture was the same as described in Fig. 1. Aliquots of 20 ~1 were applied to strips of Whatman No. 3 MM filter paper at 37” and after 2 minutes drying, were placed in a humid chamber at 37”. At the end of the indicated incubation periods, the strips were removed, dried quickly, and subjected to chromatography in solvent A. The slight lag observed during the first 10 minutes is probably due to the time required for formation of the phospholipid intermediates in the reaction (20).

TABLE REQUIREMENTS

FOR

FORMATION

AL.

for 1 hour. The renct,ion was stopped by removing the strips from the humid chamber rind drying them at :37”. Under t#hcse conditions, the extent of reaction was proportional t,o amount of cuzymc. Properties of the Reaction Catalyxetl by the Particulate Enzyme Prepared from S. aureus after Sonic Disintegrntion Incorporation of radioactivit8y from UDP-\IurNAc-pentapeptide into immobile product in the filter paper assay was dependent on the presence of UDP-GlcNAc (Table I). Of other nucleotides tested (see Fig. 1 in reference 17), only UDP-Glc was able to substitute for UDP-GlcKAc. However, the slight, incorporation of radioact,ivity reported earlier with UDP-Glc is now thought to be due to slight contaminat,ion of the preparation of UDP-GlcKAc. More recent preparations of UDP-Glc have not been able to substitute for UDP-GlcKAc, even to a slight extent. GlcKAc-l-P, obtained by treatment of UDP-GlcKAc with venom phosphodiesterase, could not support’ peptidoglycan synthesis. UDP-T\lurKAc~L-ala~n-glu~14C-~-lys did not substitute for UDP-?tIurKAc-pentapeptide, nor did the addition of unlabeled UDPi\IurEAc-pentapeptide to a system cont#aining UDP-MurKBe .L-ala .D-&I . 14C-~-lys and UDP-GlcNAc lead to coupled or simult,aneous incorporation of MurNAc-tripepticle and MurNAc-pentapeptide (Table I). Prior treatment of UDP-PIIurNAc-14C-pentapcpI

OF PRODUCT

WITH

S.

AL~REOS

ENZYME

Reaction mixtures contained 0.9 mpmole of either 1%-1ysine labeled UDP-MurNAc-pentapeptide or UDP-MurNAc-tripeptide or 14C-lysine itself or 0.6 mpmole of unlabeled UDP-MurNAc-pentapeptide or 0.8 mpmole of UDP-GlcNAc, 3.8 pmoles of Tris-HCl, pH 7.8, 0.11 pmole of MgC12, and 15 Fit volume of 29 ~1. Reaction of S. uureus particulate enzyme (220 pg of protein) in a total mixt)ures were spotted on strips of Whatman No. 3 MM filter paper at 37” and immediately transferred to the 37” humid chamber to incubate for 1 hour, and were then assayed. Substrates

present

Radioactivity incorporated (Wd~S)

UDP-MurNAc-14C-pentapeptide UDP-MurNAc-14C-pentapeptide UDP-MurNAc-14C-t.ripeptide UDP-MurNAc-14C-tripeptide 14C-Lysine

+ UDP-GlcNAc + +

UDP-GlcNAc UDP-GlcNAc

+ UDP-MurNAc-pentapeptide

99 4 2 1 2

BIOSYNTHESIS

OF

tide witlh venom phosphodiestersse or hydrolysi:3 of the nuclcotide in 0.01 N HCl also prevented incorporation of radioactivity into the immobile product. 14C-Lysine itself was not incorporated. Incorporation of radioactivity from UDPMurSAc--laC-pentapeptide was stimulated by Mg++ and AIn++ t,o a maximum of Sfold. Magnesium ions gave maximal stimulation of the reaction at a concentrat,ion of 3 nq but showed slightly great’er stimulation at a concentra8tion of 1.5 mill (Fig. 3). Calcium and cobalt ions did not stimulate and were inhibitory at concentrations greater than 5 nmr. The pH optimum was 7.5-S in 0.1 ill Tris buffer. The apparent K, for UDP-IIur-NAc-pentapeptide in the presence of excess UDP-GlcNAc was G X lop5 111(Fig. 4). The similar constant for UDP-GlcSAc in the presence of excess UDP-i\IurNAc-pentapeptide was 7 X 10w5fi1. In bot’h cases the concentrations of substrates indicated are the concentrations in the reaction solutions prior to application to the filter paper. The actual concentrations of the substrates at t’he time the reaction proceeded is not known.

I

0' -LOG

4

2 METAL

ION COkENTRATION

FIG. 3. Metal ion dependence of peptidoglycan synthesis by S. UUT~US enzyme in the filter paper reaction. R.eaction mixtures contained 1.8 mpmoles of ‘4C-lysine labeled UDP-MurNAc-pentapeptide, 2.1 mrmoles of UDP-GlcNAc, 2.5 pmoles of TrisHCl, pH 8.6, metal ions as indicated, and 10 ~1 of 8. aure~s particulate enzyme, prepared after sonic disintegration (160 pg of protein) in a total volume of 22 ~1. All reaction mixtures were spotted on Whatman No. 3 MM filter paper, dried for 2 minutes, and then incubated for 1 hour in the 37” humid chamber.

495

PEPTIDOGLYCAN

S (UDP-

MurNAc

-PENTAPEPTIDE)

Km-7xlO=‘~ 16-

I

0

-10

0

0.1 0.2 S (UDPGlcNAc

gx

0.3 )X103

,&*;-I

30 -

0.4

0.5

M

FIG. 4. Michaelis constants for UDP-MurNAcpentapeptide and UDP-GlcNAc with 8. aweus particulate enzyme in the filter paper assay. Reaction mixtures each contained &lo mpmoles of UDP-MurNAc-pentapeptide (labeled with i4Clysine), O-10 mpmoles of unlabeled UDP-GlcNAc, 2.6 pmoles of Tris-HCl, pH 7.8,0.08 pmole of MgCl*, and10 ~1 of S. aurerLs particulate enzyme, prepared after sonic disintegration (155 pg of protein) in a total volume of 20 ~1. When one subst,rate was varied, the amount of the other was 10 mfimoles. The reaction mixtures were spotted on Whatman No. 3 MM filter paper strips, dried 2 minutes at 37”, and then incubated 1 hour in a humid chamber at 37”.

Properties of the Reaction Catalyzed by the Particulate Enzyme Prepared from M. lysodeikticus ajtey Sonic Disintegration Because of the unusual features of the S. aureus enzyme and difficulties in handling it, it was decided to investigate the properties of similar preparations from M. lysodeikticus. Particulate enzyme prepared after sonic disintegration of M. lysodeiktkus also catalyzed utilization of UDP-MurNAcJ4Cpentapeptide in a UDP-GlcNAc-dependent reaction wit,h formation of a radioactive,

496

ANDERSON,

0

2

I -LOG

METAL

3

4

5

ION CONCENTRATION

ET

AL.

PH

ion dependence of peptidoglycan synthesis with enzyme from M. Zysodeikticus. Reaction mixtures contained 1.9 mpmoles of UDP. L-ala * D-glU . ~-1~s * %-n-ala . 1% MurNAc D-ala, 2.1 mfimoles of UDP-GlcNAc, 2.5 Fmoles of Tris-HCl, pH 8.6, and 10 bl of M. Zysodeiklicus particulate enzyme, prepared after sonic disintegration (145 pg of protein) in a total volume of 22 ~1. MgCl, and MnClz were present as indicated. The reaction mixtures were incubated 1 hour at 37” in test tubes and then assayed.

FIG. G. Optimum pH for peptidoglycan synthesis with enzyme from 1lf. Zysodeikticvs. Each reaction mixture contained 2.1 mpmoles of UDPMurNAc + L-ala n-glu . ~-1~s . W-o-ala . IdC n-ala; 2.4 mpmoles of UDP-GlcNAc; a mixed buffer containing 1.3 kmoles each of Tris, K,HP04, and 2-amino-2-methyl-1,3-propanediol adjusted to the indicated pH; 1 pmole of MgCls; and 11 ~1 of AI. lysodeikticus particulate enzyme, prepared after sonic disintegration (165 pg of protein) in a total volume of 21 ~1. All reactions were incubated 1 hour at 37”.

chromatographically immobile product. The M. lysodeikticus enzyme, however, was able to catalyze formation of the product when incubations were carried out in a test tube at 37” as well as in the filter paper assay. Although under the conditions used initially at relatively low concentration the paper assay yielded about twice as much product as the tube assay, the velocity of the reaction in the tube was markedly accelerated at high Mg++ concentration (Fig. 5). The maximum velocity, observed in the tube reaction at 30 mM WP, was greater than the velocity of the paper reaction at any cation concentration. The reaction was activated by Mn++ as well as by Mg++ (Fig. 5). However, no unusual stimulation of the test tube reaction at high Mn++ concentration was observed, and at all Mn++ concentrations the reaction in the tube was slower than that on the paper. The optimal Mn++ concentration between 1 and 10 mM, whether in the tube or the paper, was lower than the optimal Mg++ concentration. Co*, Cu++, Aland Ca* were inhibitory at 15 mu. The pH optimum was 8.6, measured in the tube reaction (Fig. 6).

The reaction in the tube assay with t,he .iif. lysodeikticus particulate enzyme was absolutely dependent upon the addition of UDP-GlcKAc (Table II). UDP-Glc, UDPGaliL’Ac, UDP-Gal, TDP-Glc, GDP-Glc, GCP-Gal, and GlcNAr-1-P could not substitute for UDP-GlcNAc. The specificity of the reaction catalyzed by the M. lysodeikticus particulate enzyme for UDP-MurNAc-pentapeptide was examined. UDP-?(IurNAc-pentapeptide was the only unlabeled nucleotide which, when added t’o UDP-MurNAc-14C-pentapeptide, diluted incorporation to an extent commensurate with the amount of diluent added (Table III). The slight reduction of glycopeptide formation on addition of excess unlabeled UDP - MurSAc - tripeptide or UDP-MurNAc-dipeptide is, however, probably due to some competition for utilization of the natural substrate by these nucleotides. P-MurNAcJ4C-pentapeptide, formed by treatment of UDP-MurNAc-14C-pentapeptide with phosphodiesterase, was not a substrate. UDP-MurNAc-14C-tripeptide was utilized to a slight extent, but addit,ion of the preferred substrate, unlabeled UDPMurNAc-pentapeptide, reduced this slight

FIG. 5. Metal

n!fg++

BIOSYNTHESIS TABLE SPECIFICITY OF FOR UDP-GLCNAC

TABLE SYNTHESIS

I%{. LYSODEIKTICUS

REQUIREMENT FOR TIDE IN PEPTIDOGLYCAN

ENZYME

LYSODEIKTICUS

Each reaction mixture contained 2 mrmoles of UDP -MurNAc * L-ala . D-glU . ~-1~s . 14C-D-ala . W-n-ala, 2.5 pmoles of Tris-HCl, pH 8.6, 0.8 rmole of MgClp, 10 ~1 of M. lysodeikticus particulate enzyme (150 pg of protein), and the compormds indicated, in a total volume of 26 ~1. All reactions were incubat,ed 1 hour at 37”. Additions Compound

None UDP-GlaNAc UDP-GlcNAc UDP-010 UDP-GalNAc UDP-Gal TDP-Glc GDP-Glt CDP-Gal GlcNAc-l-Pa

Amount (mpmoles) -

0.38 1.38 2.3 3.3 5.5 1.1 1.1 5.0 7.6

497

PEPTIDOGLYCAN

II

PEPTIDOGLYCAN \VITH

OF

Peptidoglycan CJl/Lmoles)

4.9

217 374 5.4 7.4 5.7 6.3 5.7 5.7 6.1

u GlcNAc-1-P was prepared from UDP-GlcNAc by preiucubatiou with venom phosphodiesterase for 30 minlltes at 37”. The phosphodiesterase was inactivated by boiling for 2 minutes before addit ion of the UDP-MurNAc-pentapeptide and particulate enzyme. Inactivation of phosphodiesterasp by heating was demonstrat,ed by the fact that none of the ‘“C-labeled UDP-MllrNAc-pentapept itlc was degraded during the subsequent incubation.

incorporation to the level of the controls. Exchange experiments to be reported in a following paper support the fact that this system can utilize UDP-MurNAc-‘*C-tripeptide to this slight extent. The alpparent K, for UDP-RIurNAcpentapeptide was 2.4 X low4 M and for UDP-GlcNAc, 2.0 X 10h4 M (Fig. 7). Properties of the Particulate Enzyme Psrepared from S. aureus after Grinding wit,h Alumina This patrticulate fraction catalyzed formation of peptidoglycan if the reaction mixture was incubated in test tubes at 20’. If incubatio:n was carried out at 37” the system was rapidly inactivated and litt’le product

III

UDP-MCIRNAC-PENTAE>EF SYNTHESIS ENZYME

WITH

1%'.

The complete reaction mixture contained 2.4 mpmoles of UDP-MurNAc.L-a1a.n.g1u.L.lys. 14C-n-ala.14C-n-ala or TJI~P-nfurNAc.L-ala.nglu.W-L-lys, 1.9 mrmoles of UI)P-GlcNAc, 2.5 rmoles of Tris-HCl, pH 8.6, 0.8 Fmole of MgClz, and 10 bl of M. lysodeikticus particulate enzyme (150rg of protein) in a total volume of 1’2~1. Other nonradioactive components added as indicated were 2.4 mpmoles of UDP-MurN~~~-prntnpeptide, 15 mpmoles of UDP-MurNAc.~~ala.r,-gllr, 13 mrmoles of UDP-MurNAc.1,.ala.l)-glll. L-lys, or 80 mpmoles of D-ala.o-ala. All rcact,iuu rllixtures were incubated for 1 hour at 37". Pegtidoglycan formed Additions

and deletions

Complete Minus UDP-GlcNAc Boiled enzyme UDP-MllrNAc-pentapeptide UDP-MllrNAc-tripeptide TJDP-MurNAc-dipeptide u-afa.D-ala P-MurNAc-‘“C-pentapeptide”

UDP-Mur NAcm~aCpentapeptide

505 4 3 “22

C~~~moles) f ram -~ UDP-Mar.XAc-1% trlpeptide

18 4 6 5

438 J-15 20 li

0 Prepared from ITDP-MrlrNAc-‘~<‘-l,elltapeptide by preincubation with venom phosphodiesterase. The phosphodiesterase was inactivated b> boiling prior to addition of the other srtbst,rate aud particulate enzyme.

was formed.’ The effects of temperature on the reactions will be more fully documented in a following paper. This system was also completely dependent upon t,he presence of UDP-GlcKAc and was stimulated by l\Ig++ (Table IV). The optimal nIg++ concentration was 3 mM for this X. aureus enzyme (Fig. S), in marked contrast to the M. lysodeikticus enzyme described earlier which required 30 m&I Mg++ in the tube assay. The pH optimum for peptidoglycan synthesis by the enzyme prepared by alumina grinding was 9 (Fig. 9). Under optimal conditions this test tube react’ion was proportional to time (as in Fig. 2) and to amount of enzyme (Fig. 10). hdenosine

498

ANDERSON,

ET

AL. TABLE

IV

REQUIREMENTS FOR PEPTIDOGLYCAN SYNTHESIS BY S. Km=

2.4x10-4

6r

i

.

AUREUS

Additions

S
y

FIG. 7. Michaelis constants for UDP-MurNAcpentapeptide and UDP-GlcNAc with enzyme from $1. ly.sodeik&~s. Reaction mixtures each contained O-10 mpmoles of MurNAc-pentapept,ide (labeled with V-lysine), O-10 mpmoles of UDPGlcNAc, 3 pmoles of Tris-HCl, pH 7.8,QO mpmoles of MgCl,, and 9.5 ~1 of kf. Zysodaikticus particulate enzyme, prepared after sonic disintegration (96 pg of protein) in a total volume of 20 ~1. When one substrate was varied, the amount of the other was 10 mpmoles. The reaction mixtures were incubated in tubes for 1 hour at) 37”.

triphosphate stimulated the reaction for reasons which are not known* (Table IV). This preparation also catalyzed incorporation of glycine residues into the peptidoglycan (21). Incorporation of glycine has been shown to be dependent on the presence of UDP-MurNAc-pentapeptide and is sensi8 More recently, it has been found that stimulat.ion by ATP is at least partly due to the utilization of ATP in a reaction in which ammonia is utilized in the amidation of the ol-carboxyl group of glutamic acid in the pentapeptide. This amidation occurs with one of the lipid intermediates as substrate. The reaction cycle proceeds more rapidly with the amidated intermediate (G. Siewert. unpublished results; see reference 35).

PARTICULATE

ENZYME

The complete system contained 2.4 mMmolcs of UDP-MurNAc L-ala. n-glu ~-1~s. 14C-o-ala . 14C-n-ala, 5.0 mkmoles of UDP-GlcNAc, 26 mpmoles of ATP, 2.6 pmoles of Tris-HCl, pH 8.6, 120 mpmoles of MgC12, and 3.8 ~1 of S. aureus particulate enzyme, prepared after alumina grinding (52 pg of protein), in a tot.al volume of 25~11. Additions included 5.3 mpmoles of glycine, 1.3 pmoles of KCI, 2.6 rg of chloramphenicol, 65 mpmoles of 2-mercaptoethanol, 1 pg of RNase, or 5 rg of lysozyme as indicated. All reactions were incubated 1 hour at 20” and were inactivated prior to being spot,ted on filter paper for assay. OI deletions

Complete Boiled enzyme Minus UDP-GlcNAc Minus ATP Minus MgCk + Glycine + Glycine, KCl, chloramphenicol, and 2-mercaptoethanol + RNase + Lysozyme

Glycopeptide formed &moles)

188 5.5 5.1 76 7fi

175 184 187 8.8

Q Although MgClz was deleted from the react ion mixture, the enzyme preparation employed contained about 38 mpmoles of Mg++. to RNase (18, 21). However, the components of t.he glycine incorporating system, added either singly or collectively, did not stimulate incorporation from UDPMurKAc - 14C - pentapeptide (Table IV). Furthermore, incorporation from UDPMuriYAcJ4C-pentapeptide was unaffected by a level of RNase which completely inhibited glycine incorporation, Although incorporation of glycine can occur in this system, it is not a prerequisite for formation of the linear peptidoglycan. Peptidoglycan formation was sensitive to the action of lysozyme. If lysozyme was tive

present

from

the

beginning

of incubation,

or

if it was added after incubation, little net peptidoglycan formation could be demonstrated. Radioautograms prepared after paper chromatographic development of these reaction mixtures in solvent A showed that 14C-disaccharide-pentapeptide and 14C-

BIOSYNTHESIS

t

OL

PEPTIDOGLYCAN

I 2

; -LOG

OF

MAGNESIUM

i 10.0

CONCENTRATION

FIG. 8. Dependence of peptidoglycan synthesis on metal ion concentration with enzyme from S. aureu~ in the test tube reaction. Each reaction mixture contained 2.4 mrmoles of UDPMurNAc . L-ala . n-glu . ~-1~s . 14C-n-ala . 14C n-ala, 5.6 mpmoles of UDP-GlcNAc, 26 mpmoles of ATP, 2.6 rmoles of Tris-HCl, pH 8.6, 3.8 ~1 of S. aureus particulate enzyme, prepared after alumina grinding (52 pg of protein), and the indicated amount of MgCl*, in a total volume of 25 ~1. All reactions were incubated 1 hour at 20”.

t~trasaccharide - bis(pentapeptide) were formed under these conditions (see below).

FIG. 9. Opt,imum pH for peptidoglycan synthesis by S. CLZLWUS in the test tube reaction. Each reaction mixture contained 2 mfimoles of UDP-MurNAc-pent,apeptide labeled with W-Dala-W-n-ala, 9.9 mpmoles of UDP-GlcNAc, 0.11 pmole of ATP, 0.13 pmole of MgCl,, a mixed buffer containing 1.3 pmoles each of Tris, K2HPOI, and 2-amino%methyl-1,3-propanediol of the indicated pH values, and 5.0 ~1 of S. aureus particulate enzyme, prepared after alumina grinding (79 rg of protein) in a total volume of 25 ~1. All reactions were incubated 1 hour in tubes at 20”.

Properties of the reaction catalyzed by particulate enzyme prepared from M. lysodeikticus after grinding with alumina. Although the properties of the S. aureus particulate enzyme were rather markedly dependent on the method of disruption of the cells, t,he particulate enzyme obtained from &‘. lysodeikticus after grinding with alumina did not differ significantly in properties from that obtained after sonic disintegration. A 2-3 fold increase in specific activity was noted when the enzyme was prepared by grinding with alumina. This enzyme was capable of catalyzing peptidoglycan formation in either the conventional tube assa,y procedure or by the filter paper assay method. Speci,fic activities of enzymes prepared by various procedures The specific activities in mpmoles of product formed per mg per hour of the various enzymes, measured under optimum conditions in each case,were as follows: S. aureus enzyme prepared after sonic disintegration, about 0.04 in the tube assay and 0.6 in the

0 /

,

4

8

I

JJI OF ENZYME

FIG. 10. Proportionality of assay to amount of S. aureus enzyme in the test tube reaction. Each reaction mixture contained 2.4 mpmoles of UDPMurNAc . L-ala n-glu . ~-1~s . 14C-n-ala . 1% n-ala, 5.6 mpmoles of UDP-GlcNAc, 26 mrmoles of ATP, 2.6 rmoles of Tris-HCl, pII 8.6, 120 mpmoles of MgCL, and the indicated amount of S. aureus particulate enzyme, prepared after alumina grinding (13.7 rg of protein/pi) in a total volume of 25 ~1. All reactions were incubated 1 hour at 20”.

500

ANDERSON,

filter paper assay; 8. aweus enzyme prepared nft,cr alumina grinding, 3.5 in the tube assay; 31. I~sorleikticus enzyme prepared after sonic disintegrat,ion, 3.0 in the tube nss:~y; and .U. lysodeikticus enzyme prepared aft,er alumina grinding, 15 in the tube assay. Since these represent activit’ies of 0.5 ml of enzyme preparation with a protein caontent. of about 15 mg/ml obtained from the cells from 1 lit,er of culture at, onequarter maximum growth, the maximum a,ctivity obtained in X. aweus was about 0.026 pmole/hr/liter or in JL. lysodeikticus 0.11 ~mole/hr/liter. These values correspond to approximate values for the rate of synthesis of peptidoglycan in whole cells at one-quarter maximum growth of 100 pmoles/ hr/litcr for 8. au?.eus and 33 pmoles/hr/liter for 31. lysodeikticus. Thus, under the present conditions of enzyme preparation and assay, only a small frackion of the total activity which must be present, has been expressed. These values may be compared to values previously obt,ained for two enzymes involved in the biosynthesis of the teichoic acid in tbe cell wall of S. aureus, viz, 75 pmoles/ hr/litcr for the UDP-GlcKAc-polyribitol phosphate GIcKAc kansferase and 7.5 pmoles/hr/liter for t,he CDP-ribit,ol-phosphoribitol transferase. Xtoichiometry of MurNAc-Pentapeptide and GkVAc Incorporation An incubation was carried out utilizing UDP-14C-GlcNAc, UDP-MurSAc-3H-pentapeptide, and particulate enzyme from M. lysodeikticus. Aliquots of the reaction mixture were inactivated after various times of incubation. The amounts of 3H and 14C incorporated into peptidoglycan were then determined. Both isotopes were incorporated linearly as a function of incubation time (Table V). At all times the amounts of GlcNAc and MurNAc-pentapeptide incorporated were approximately equivalent.g 9 The ratio of GlcNAe to MurNAc-pentapeptide actually observed, 0.9, is not believed to be significantly different from 1. It was necessary to apply a correction factor to observed 3H-counts in peptidoglycan which introduced some inaccuracy into the measurement. Every radioactive spot observed in each reaction mixture was count.ed. When the total 3H a.nd 1% counts ob-

AL.

ET

TABLE SIMULTANE~C~ PENTAPEPTIJ~E

1-

JNC~RPORATWN

OF

W-GLCNAC LYSGDEIXTICLTS ENZYME .JND

MunNAc-3Hw JTH .%f.

A reaction mixt,ure containing 29 m/*moles of UI)P-MurNAc-3H-pentapeptide, 28 mpmoles of T;DP-l~C-C~lcNAc, 36 pmoles of Tris-HCl, pH 8.6, 11 pmoles of MgCl?, and 140 ~1 of Af. 2ysocleikticcb.s enzyme (2 mg of prokirl) in a t.otal volume of 285 ~1 was incubated at 37”. Aliquots of I9 ,IJ were rcmoved at intervals, inactivated, spotted on Whatman No. I. filter paper, and chromatogrnphed in solvent A. Relative amounts of 3H and ‘T incorporated into peptidoglycan were determined by counting the chromatogram origins in the licluid scintillation spectrometer. See footnote 9. Peptidoglycan IncubTzat&

MuNAc-~Hpentapeptide

0 10 20 30 GO 90 120

formed

&u~~oles)

tune

4 82 197 311 G12 868 1036

‘“C-GlcXAc

3 88 179 278 537 760 907

Ratio of “C-GlcKAc to XIurNAc-3Hpentapeptide

1.07 .!I1 .8!) .88 .88 .87

Ineoq,poration of the irzta.ct pentapeptide moiety. The nucleotide, UDP-MurNAcpentapeptide, contains five amino acid residues including 2 n-alanine and one L-alanine residue. However, enzymic dcgradation of t,he cell wall from S. aweus served in each reaction mixture were compared as a function of incubation time, the total laC counts were essentially constant, but the total 3H counts increased. The increase was correlated with peptidoglycan formation. Because of the size of the peptidoglycan polymer formed and possibly because of attachment to the particulate enzyme, the radioactive product did not fully penetrate the filter paper used for chromatographic separation and on which counting was done. Because of the differences of average energy of t’he beta particles emitted, I% was counted with essentially equal efficiency throughout the paper, hut VII was counted more efficiently when the materials containing it did not fully penetrate the paper. When all 3H counts observed at the origin were multiplied by a factor of 0.7, the total 3H counts observed in each reaction mixture were then essentially constant, regardless of the amount of peptidoglycan formed. The correction factor was therefore employed in all caleuialions.

BIOSYNTHESIS

OF TABLE

I~ETEKTION

OF BOTH

I~FC*ORPORATION

D-ALANINE OF

rio1

PEPTIDOGLYCAN VI

I!ERIDTXS

OF

MuRNAc-PENTAPEPTIDE

THE

PENTAPEPTIUE

INTO

DCRIN~

PEPTIDOGLYCAK

Reaction mixtnre A contained 16.2 mpmoles of Ul)P-MurNAc’L-ala~u-glu~3H-~-lys~1~C-~-ala~~~CD-ala with an observed ratio of cpm of 3H to ‘4C of 2.75. React,ion B contained 15.8 mpmoles of UDPMurNAc~~-ala~~-gl~~~~~C-~~-l~~~~~H-~-ala~”H-~-ala with an observed rat,io of cpm of “H to 1% of 0.96. Both reaction mixtures also contained lG.2 mpmoles of Ul>P-GlcNAc, 18.9 pmoles of Tris-HCI, pH 7.8, 0.57 pmole of MgCl+, and 72~1 of M. lysotleikticus particlllate enzyme (1.3 mg of protein) in a total volume of 148 ~1. Aliqllots of 18.8 ~1 were added to an eqllal volLmle of solvent, A at the indicated illtervals. Further manipldation was the same as that described in Table V. Carrying out the experiment with 3H in L-lysine and 1% in o-a1a.o.ala in one case and with llC in L-lysine and 3H in D-ala.o-ala in the other served as a Imeans of checking the met)hods, cotmting procedlues, and calculations. Reaction

time

Incub;;%

Reaction

B

~~~ Peptidoglycan ‘H-~-1~s

0 10 25 40 Ii0 90 120

A

2.7 G8.1 169 288 455 Mi4 741

formed ‘CD-ala

(~~moles)

Peptidoglycan

formed

(~,mmles~

~‘ct-ala

4.0 64.7 lG2 273 Gli 630 725

~

-~~ 1.05 1.04 1.05

I

1.04

i

1.05 1.0’2

’

has show1 that about 90% of the peptide units in the wall are tet’rapept’ides containing one walanine and one L-alanine residue (36). Only 10% of pentapeptide was present. In order to det#ermine whether one or 2 u-alanine residues were present in t’he enzymically synt’hesized linear peptidoglycan, incubation mixtures were prepared containing UDl’ - XurNAc - pentapeptide, doubly labeled in L-lysine, and n-alanyl-nalanine. In one case the subskate was UDP-:\Iu~rSAc~.~-al:l.o-glu.l~C-~-lys.~H-~)ala.“-H-n-ala, and in t’he other, UDP-NurKAc +L-alla .u-glu .“H-~-1~s .14C-u-ala.*4C-u ala.14C-n-ala. Incorporation of 14C and 3H into peptjidoglyc*an was observed as a function of time with 111.lysorleikticzcs particulate enzyme. Again a linear rate of iworporation of 3H and 14Cwas observed. After correction of tritiurn count,s for the observed greater caounting efficiency for tritium at chromatogram origins9 t,he ratio of “H-1-1~s to ‘Y-D-ala .14C-~-ala incorporat,ed was about 1.04, mtl the ratio of 14C-~-lysto 3H-u-ala. WI)-& was about, 0.92 (Table VI). These results indicate that t’he pcntapeptide moiety ~1s incorporated without) loss of u-alanine.

5.4 70.5 192 304 478 fi(i‘4 835

2.3 75.3 210 333 52li 727 924

I

I

.9-I .91 .R2 .91 .91 I)0

l\Loreover, when UDP-i\rur,l-Ac-pcntapeptide labeled wit’h 14C-n-:da.14C-~-:tl:~ was used as substrat#ein rea&ion mixt,ures, there was never any indication OII raclioautograms of release of any “C-w:tlanine cit#hcr as the amino acid or as any radioactive breakdown product. The terminal u-alanine of the perkapcptide must he lost, in some subsequent reaction in cLel1 wall synthesis. Such a reaction has not so far been obtained with enzyme preparat#ions from S. aureus or X. l~sdeiliticus, but wit’h enzyme preparations from R. co/i the reaction in which the t,crminnl walanine residue is eliminated occurs. It, is a transpeptidation by means of which linear pcptidoglycan strands become woss linked (37).

IniMly, product analysis was carried out peptidoglycan formed by enzyme from ,S. aweus in t,hc filter paper assay (17). Chromatogram origins bearing W-lysinelabeled pept,idoglycan mere extracted several times wit,h 0.02.5 N ammonium awtnt8e, pH 5.2. After lyophiliznt’ion of t11c poolY1 011

502

ANDERSON, UDP-

ET AL.

Acetylmuromyl-peptlde Acetylmuramyl-peptlde

A C-Product

C J

03 0.00t

0.20

B Further with p-

t 0.40

FIG. II. Effect of various treatments on the peptidoglycan UZLT~USparticulate enzyme. For details, see text.

extracts, aliquots mere subjected to digestion by lysozyme, by P-acetylglucosaminidase, or by bot)h enzymes and then rechromatographed in solvent A (Fig. 11). Lysozyme digestion yielded a radioactive compound, now known to be GlcSAcMuSAc-pentapeptide (seebelow), having a mobility slightly less than that of authentic MurNAc-pentapeptide. A11 untreated aliquot contained only the undegraded pept’idoglycan product. Coupled action of lysozyme and ,&acetylglucosaminidase yielded a radioactive product indistinguishable from authent’ic XurNAc-pentapeptide. Treatment of the peptidoglycan with the exo-Pacetylglucosaminidase prior to treatment with lysozyme had no effect. After rechromatography only undigested peptidoglycan could be detected, indicating that the exo-enzyme was unable to hydrolyze the polymeric peptidoglycan. These results provided the basis for a more extensive analysis of the peptidoglycan formed in a tube reaction with M. lysodeikticus particulate enzyme. Two similar reaction mixtures were employed which differed only in the manner in which the substrates were labeled. For reaction A, 3H-D-ala . 3H-n-ala-labeled UDP -

Treated Acetylglucosamlnldase

0.60 product

Rf

synthesized

with S.

MurNAc - pentapeptide and UDP - 14CGlcNAc were the substrates, while for reaction B, 14C-n-ala.14C-n-ala-labeledUDPMurNAc-pentapeptide and unlabeled UDPGlcNAc were employed. In preliminary experiments, it had been established t’hat at least half of the product’ could be sedimented by centrifugation at 100,000 g for 30 minutes. It was presumably attached to some insoluble component of the particulate enzyme. The remainder of the product remained in solution. The soluble portion was immobile on paper chromatography and susceptibie t,o hydrolysis by lysozyme. It has not been further characterized. The peptidoglycan associated with the part.iculate enzyme in a large scale incubation was collect’ed by centrifugation and digested with lysozyme. Paper chromatography of the lysozyme digest yielded two major radioactive bands as well as a number of minor components (Fig. 12). The major components were identified as disaccharide pentapeptide and tetrasaccharide-bis(pentapeptide) as described below. The minor components of the digest were not investigat,ed but were assumedlo be higher homologs of the two major component’s. After treatment of the compound pre-

BIOSYNTHESIS

OF

PEPTIDOGLYCAN

Gk;NAc .tvitiAcpentapept~ FIG. 12. Hydrolysis by lysozyme of peptidoglycan formed by M. Zysodeikticus particulate enzyme. Reaction A contained 60 mmoles of UDP-MurNAc~L-ala~D-gIU~n-lys~3H-n-aIa~3Hn-ala and 60 mpmoles of UDP-“C-GlcNAc. Reaction B contained 60 mpmoles of UDPMurNAc.n-ala.n-glu.~-Iy~~~~C-n-ala~ K-n-ala and 64 mpmoles of unlabeled UDP-GlcNAc. Both reaction mixtures also contained 82 pmoles of Tris-HCI, pH 8.6, 16 Fmoles of MgCl2, and M. Zysodeikticus particulate enzyme (0.59 mg of protein) in a total volume of 0.75 ml. Both were incubat’ed 3 hours at 37”. Peptidoglycan associated with the particulate enzyme was separated from residual radioactive substrate by centrifugation at 100,006 g and washed once. Treatment wit,h lysozyme (222rg) was carried out for 3 hours at 37” in 0.30 ml of 0.02 M Tris-HCI, pH 8.2,0.1 mM MgCls. After centrifugation to remove the particulate enzyme and the particle-bound intermediates, the supernatant solution was chromatographed on Whatman No. 1 filter paper in solvent A. A radioautogram is shown.

sumed toI be disaccharide-pentapeptide with lysozyme, /%acetylglucosaminidase, and acetylmmamyl-L-alanine amidase (38), aliquots of the respective hydrolyzates were chromatographed on paper in solvents A, D, and E (Table VII) and subjected to paper electrophoresis at’ pH 5.2 (Fig. 13). The results obtained were compatible with the structure, GlcNAc-MurNAc-pentapeptide, for the disaccharide-pentapeptide. Exhaustke treatment of disaccharide-pentapeptide with lysozyme had no effect, but P-acetylglucosaminidase released 14c _ GlcNAc and ;\IurNAc-3H-pentapeptide in the case of the material originating from reaction A, and MurNAc-14C-pentapeptide in the case of material from reaction B. The acetylmuramyl-L-alanine amidase acted

on the disaccharide-pentapeptide from reaction A to yield 14C-disaccharideand 3H-pentapeptide, whereas in the caseof reaction B, the product was 14C-pentapeptide. The 14C-disaccharide cochromatographed with authentic 4-O+N-acetylglucosaminyl-N-acetylmuramic acid (39) in solvents A, D, and E and had the same mobility on paper electrophoresis. Similar treatment of the tetrasaccharidebis(pentapeptide) with lysozyme, p-acetylglucosaminidase, and acetylmuramyl-nalanine amidase was carried out (Table VII, Fig. 14). The results are compatible with the st’ructure, GlcNAc-i\ 1urNAc (-pentapeptide)-GlcNAc - MurNAc -pentapeptide. Treatment with lysozyme converted tetrasaccharide-bis (pentapeptide) to disaccha-

CIIILO~~A’L’O(:KA~HI(’ MOBILITIER .4iw

Plt(>urjc"rs

OF

TABLE

\-II ENZYMIC

TETRAsA~'~.HAI~IDE-BIS(~E.U.I.AI'EPTII)E)

OF

HYDIU)I,~SIS OF

~)IS~~~.~:HAKIDE-~'EN'I.AFEF.I.II)E

and

Amidase

Reference cornpo~ulds GlcNAc MurNAc-pentapeptidc Pentapeptide (;lcNAc-MurNAc

+

:

idr

treatment

Tetrasaccllitridc-~)is(~-‘el~tapcptidcJ + lysozyme + fi-GlcNAc-sse

T_isaccharidc-pelltapept + lysozyme + p-GlcNAc-ase + Amidase

Samples products

Disacrharidc-pentapcptide GlcNAc + trisaccharidc-bis (pentnpcpt ide) Tet rnsaccharide + pentapept

No hydrolysis C+lcNAc + MIlrNAr-pentapept GlcNAr-MurNAc + pentapeptide

Presumed

idc

-.~

ide

0.01 0.10

0.41; 0.62 0.35

0.11

0.38

0.43 0.02

P'

(0.01)

(0 .ooy

(0.01)

(0.01)

0.01

(0.00)

0.11

(0.01

u

in solvent:

0.40

0.01

0.4i

(O.(i-L)

(0.2.5

0.43

0.32

(0.16) (0.38)

(0.38) (0.48) (0.04)

(0.30)

0. lfi 0.38

0.38 0.48 0.32 ____

0.39

RF

i 0.27

0.24

0.20 0.0s 0.01

0.21

(0.03)

(0.07)

-

0.01

0.00 0.01

0.01

D

-

c

0 o:j

0.01)

-

0.07 0.03

-~

The materials l~sed were the major radioactive bands shown ill Fig. 12. Aliyllnta WCI’P digested with: (1) l~soa~rne cl.4 pg/r1,) it1 0.1 51 ‘rris.HCl, IjH 8.6, 5m~ MgCl!. (~)B-acetylglu~osmnillitlase (6.8 pg/plj ill 25 mxf strdillnl citrate, pH 4.2, 0.1 MKC~; or (3) :tcet~lllr~lr:tln~l-L-;ll:tlriI1c ;~rl~idase (‘2 Pg/P~) in 25 mh~ :unmonium acetate, pH 5.2. All iucltbations were for 3 hcj~~rs at 37”. After paper rhromatogrnph~~, radioactivr materials were located \vith tllr \.angrlard paper strip scarer. Radioactive compo~ulds so located were the11 counted ill the liquid scintill:>t io11 spcct rorneter (0 tlrterrllille the :Lrnor,,,t ‘and nat Ilre of the isotope(s) pIesent. Vdues recorded in the 1 able are the observed T
I'APEK

BIOSYNTHESIS

OF

PEPTIDOGLYCAN

505

FIG. 13. Paper electrophoresis of disaccharide-pentapeptide from 112. Z~sotleikticzls and it,s enzymic hydrolysis products. The compounds and enzymic hydrolyses were those described in Fig. 12 and Table VII. Eleckophoresis was on Whatman No. 3 MM filter paper in 0.05 M pyridinium acetate, pH 5.2, at a potential of 20 V/cm for G hours. Only the XXabeled compounds were detected by the radioautograms shown. Authentic ClcNAc-MurNilc was detected by fluorescence in ultraviolet lighi after heat treatment with alkali.

ride-penbapeptide. Although P-acetylglucosaminidase released 14C-GlcKAc from the tetrasaccharide-bis(pentapeptide) obtained from react,ion A, half of t#he 14C was inscparnble from the “H in the compound. Since fl-acetylglwosnminidase is an esoenzyme, it, should have no effect on the trisacchnride, lYIurKAc (-pentapeptide)-GlcXAc-JIurSAcpentapeplidc. Treatment’ of ktrasarcharidebis(penta:peptide) obt8ained from react,ion B with &acetylglucosaminidase yielded only one raclioact,ive component which had the smx chromatjographic and electrophorct’ic mobilities as the material presumed to be trisaccharide-bis(pentapeptide). Treatment with wetylmuramyl-L-alanine amidase released 3H-pcntapeptide from the tetrasaccharide-bis(pentapeptide) of renct’ion A. It, should have also yielded W-ktrasaccharide; however, the 14C product observed was 14C-tliaaccharicle which resulted from the a&ion of a contaminating acetylmuramidase activity in t’he amidasc. Treatment of the

tetrasawharide-bis(pentapeptide) from reaction B with amidase yielded 14C-pentapepticle as expected. Productj identification has thus been based on the ratios of incorporated isotopes and on the degradation of the radioactive product, by enzymes of known specificities. Insufficient produck has been obtained at the present time to permit characterization by chemical analyses, but its susceptibility to enzymes of known specificities appears to provide adequate support, for the supposition that the product, contains equimolar amounts of alternat’ing Glcn’Ac and RIurNAc-pentapeptide residues. From the mechanism of synthesis, GlcNAc must be the nonreducing end of the enzymically synthesized product. The failure to detect the release of “C-GlcNAc on treatment of product with the exo-fl-acetylglucosaminidase suggests that the product contains a chain of at least 10 sugar residues. The formation of tetrasnccharicle-bis(pentapep-

FIG. 14. Paper and

its enzymic

electrophoresis of tetrasaccharide-biscpentapeptide) hydrolysis products. See legend to Fig. 13.

tide) indicates its minimum size. No other information is available concerning the chain length of the enzymically synthesized product.

fromM.

lysockikticus

The basic experiment consisted of four reaction mixtures, two of which contained 32P- and ‘“C-labeled UDP-MurKAc-pentapeptide and two of which contained 3sPand 14C-labeled UDP-GlcNAc. Unlabeled Identification of UDP, UMP, and UDP-GlcNAc was added to only one tube Pi as Reaction Pmducts of the first pair and unlabeled UDPwas added to only 32P-Labelednucleotides were employed to MurNAc-pentapeptide determine the fate of the nucleotide moiety one tube of the second pair. Appropriate of each of the substrates. In all reactions amounts of buffer and metal ions were previously described in which uridine present in all tubes and the reactions were nucleotides are donors of monosaccharide initiated by addition of the particulate units in the biosynthesis of polysaccharides, enzyme. After incubation at 20” for S. UDP has either been identified as the aweus or at 37” for ill. lysodeikticus, the enzymes were inactivated, and about 0.1 product or presumed to be the product. However, it soon became apparent that in pmole each of carrier UDP and UMP was the peptidoglycan-forming system described added to each reaction mixture. After twohere, -only 3*P-UDP-GlcNAc gave rise to dimensional paper chromatography in solUDP. 3*P-UDP-MurNAc-pentapeptide al- vents A and B, radioautograms were prepared and the UDP and UMP were ways yielded UMP and Pi, and no UDP was detected in reaction mixtures containing located by the absorption of ultraviolet this labeled substrate. Experiments were light by the carriers added. Figure 15 shows designed to permit the determination of the the qualitative result obtained with enzyme stoichiometric relationship between the from S. aureus when 32P-UDP-MurNAcamount of MurNAc-pent’apeptide incor- 14C-pentapeptide was employed, with and porated and the amount of URIP and Pi without addition of UDP-GlcNAc. Peptidoformed as well as between the amount of glycan (at the origin) was formed only when GlcKAc incorporated and the amount of UDP-GlcNAc was present. Similarly, Pi UDP formed. was formed in significant amount only in

BIOSYNTHESIS

OF PEPTIDOGLYCAN

507

FIG. 15. Formation of UMP, Pi, and peptidoglycan from UDP-MurNAc-pentapeptide in the presence of UDP-GlcNAc. Reaction mixture A (right) contained 2.1 mpmoles of JZP-PDP-MurNAc-W-pentapeptide, 3.3 mpmoles of UDP-GlcNAc, 24 mpmoles of ATP, 2.5 pmoles of Tris-HCl, pH 8.6, 0.12 pmole of MgC12, and 4.8 ~1 of S. aureus particulate enzyme, prepared after alumina grinding (70 fig of protein), in a total volume of 25 ~1. Reaction mixture B (left) was identical except that the UDP-GlcNAc was omitted. Both were incubated for 2 hours at 20”. After inactivation, 0.1 Nmole each of carrier UDP and UMP was added to each reaction mixture. The radioautograms shown are of portions of twodimensional paper chromatograms developed in solvenk A and B. UDP and UMP were located by absorption of ultraviolet light by the added carriers.

activity found in immobile product and UDP were greatly increased. Enzyme preparat’ions from M. lysodeikticus were investigated to confirm these results with a second organism and to carry out experiments where the blank due to glycosylation of teichoic acid would be eliminated (since this organism does not contain a teichoic acid). This expectation was realized in that essentially no ut#ilization of 33P-UDP-14C-GlcNAc occurred in the absence of UDP-MurNAc-pentapeptide. 32P-UDP-14C-GlcNAc was incubated with and wit,hout UDP-MurNAc-pentapept’ide is However, as in the case of enzyme from S. shown in Icig. 16. In this instance a small aweus, considerable formation of 32P-UMP 32P-UDP-MurNAc-14C-pentapeptide amount of 14C-GlcKAc was incorporated from into immobile producO from 32P-UDP-14C- occurred in the absence of added UDPGlcSAc. GlcNAc, even in the absence of UDPCalculation of the stoichiometric relationBIurNAc-pentapeptide. A small amount of 32P-UDP was formed simultaneously. This ships between the amounts of peptidoglycan formed and the amounts of UDP, UMP, reaction in the absence of added UDPi\IqrNAc-pentapeptide was due to transfer and Pi released were carried out for the S. of 14C-GlcNAc to some endogeneous accep- aureus and M. Zysodeikticus syst,ems (Table tor, probably transfer into teichoic acid by VIII). When correction was made for the the UDP-GlcNAc-polyribitol phosphate- exchange reaction in both cases and for GlcNAc isansferasc known to be present in teichoic acid glycosylation in S. aureus, the the particulate enzyme fraction of S. aureus stoichiometry indicated that 1 UDP residue was formed for each GlcNAc incorporated ~(33). In the presence of unlabeled UDPRIurNAc-pentapept#ide the amounts of radio- into pept’idoglycan, and 1 residue each of the presence formation of instances; its greater in the

of UDP-GlcNAc.

However,

UhlP was observed in both amount, however, was much reaction mixture containing UDP-GlcXAc. This formation of U;\IP from UDP-hiIurnTAc-pentapeptide in the .absence of added UDP-GlcKAc is due to an exchange reaction (19) and is discussed below. Essentially no radioactive UDP could be det’ect,ed. The companion experiment in which

50s

AiWEICSOS,

ET

AL

FIG. 1G. Formation of UI>P and pept,idoglycall from UI)P-(+lc?;Ac itt the prwe~~cr of UDP-MurNAc-pentapept,ide. Reaction mixt,ure A (right) corltailled 21 m~moles of “*l’-L7)P“C-GlcNAc and 2.2 m,.moles of unlabeled UDP-MurSAc-pent aprptide. Reaction nkture B (left) was identical expect that 1JL)P-MurNAc-pentape~)title was nmittrd. 911 other procedures were the same as described in Fig. 15. The small amount, of radioactive compound just below IJDP is UTP, which was sometimes formed from TrI)P by the ellzymr prepamtion,

UMP and Pi mereformed for each ;\IuriYAcpentapeptide incorporated into product’. Moreover, these experiments illustrate that in both organisms GlcSAc and L\IurNAcpentapeptidc were incorporated int#o the product in equivalent’ amounts. In other experiments with both organisms, incubations were carried out with both substrates labeled simultaneously, i.e., 3aPUDP-14C-Gl~i\‘A(~ and 32P-UDP-RlurKAc3H-pentapeptide. Under these conditions 1 residue each of UDP, UMP, and Pi were formed for each GlcNAc and MurNAcpentapeptide incorporat8ed. These experiments conclusively established that UT\IP and Pi formed from UDP-3lurKAc-pentapeptide could not have been formed from degradation of UDP, since UDP formed at the sametime from UDP-GlcSAc was stable under the incubat’ion conditions. It should be not)ed that in these experiments with 32P-labeled nucleotides essentially no 33Pwas detected in t,he peptidoglycan product at the origin of t,hc rhromatograms. The large amount of UMP formed in reaction mixtures containing 33P-UDP-

MurNAc-penk~peptide in the absence of UDP-GlcKAc resulted from an exchange ““P-UDPreaction (19, 2”) involving nrur~Acprrlt,apeptide, UAIP, and the first lipicl intermediate in the reaction cy(‘le, lipid - phosphoacetylmuramyl - pentapeptide (20). Because of the extensive amount of 331’~umformed and trapped by the exchange reaction in the presence of :I pool of unlabeled UhIP, good agreement between the amount of 33P-UMP released as a reaction product and the amount of pepkloglycan formed was often difficult to achieve. The UMI’ for this exchange is presumably derived from degradation of endogenous nwleot~idesor nucleic acid, and cspcriments giving the best, stoichiometry were those in which t,hc exchange in the absenceof added Un II’ was minimum. Bflects

of Antibiotics Peptidoglycan

on Growth Synfh.esis

am1

Five arltibiotics, perlicilliI1 (-+ (3, 4), bacit,racin (-1-O),ristocdin (II), vawomycin (42, 43), and norobiocin (M), cause accumulation of UDI’-~lur-\;Ac-pentupeptide in S. uwez6.s.Penicillin, bacitracin, ri+kocetin,

BIOSYNTHESIS

OF TABLE

PEPTII

509

)OGLYCAN

VIII

STOICHIOMETRY OF REACTION PRODUCTS IX PEPTIDOGLYCAN SYNTHESIS Reactions were carried out essentially as described in thelegends toFigs. 15andl6.After inactivation of enzymes, 80 mpmoles each of carrier UDP and UMP were added to each reaction mixture. Residual substrates and reaction products were separated by two-dimensional paper chromatography in solvents A and B. Carrier UDP and UMP were located by adsorption of ultraviolet light. Peptidoglycan remained at the origin. Radioautograms were prepared to locate all of the radioact,ive materials which were then counted on 1 he paper in a liqllid scint,illation spectrometer. I

Substrates UDP-~lur~Ac.pentapeptide

Lv a?,)‘C?lS :+?p,“H-I,-[~s =P,

Labeled

UDP-GlcNAc

No

Origin

UDP

1185 5

products

&moles)

UMP

Pi

16 16

1500 30fY

1022 50

A 1180 1232 112c

0 1289 247c --.-

1194 46 49

972 226 51

A 1120

1042

0

172

796 5

19 17

1442 (i44*

788 l-46

A

791 804 40

2 776 103

798 21 150

642 38 28

A

764

674

0

10

label -u

“H-~-1~s

reaction

-No

M.

label

=P, =I’,

lysodeikticus :‘:p, 14C-L-lYS ::?p , 'CL-lys

No

label -

No

1%.GlcNAc 1%.GlcNAc

label -

a2P, ‘*C-GlcNAc azP, W-GlcNAc

a Substrate omitted. b This value is the important, correction of UMP formed due to the exchange reaction. c These values are the correcl.ions due to transfer of GlcNAc t,o an endogenous acceptor, teichoic acid.

and vancomycin specifically inhibit cell wall synthesis, whereas novobiocin also inhibits other cellular processes (44). In order to ascertain whether or not the specific locus of action of these antibiotics was the inhibition of the reaction in which UDP-MurNAc-pentapeptide and UDPGlcNAc were utilized for peptidoglycan synthesis,, the concentration of antibiotic which caused 50% inhibition of growth of the organism in liquid cultures was compared with the concentration of antibiotic which caused 50 % inhibition of in vitro peptidoglycan synthesis and which caused a 50% reduct’ion of the activity of particulate enzyme prepared from cultures pretreated with antibiotic. For growth inhibition studies, a 1% inoculum of an overnight culture of X. uuwus

probably

a

or a 24-hour culture of M. lysodeikticus was added to fresh medium. Growth of t,he culture was measured turbidimetrically in lo-fold dilutions of aliquobs taken during the course of the incubation. At quartermaximal growth, the culture was divided into several subcultures, all but one of which received ant.ibiotic. Each successive flask contained antibiotic at a concentration two to three times that of the previous flask. Incubation of cultures was continued until a definite pattern of antibiotic effects was apparent (Fig. 17). From these data, concentrations required for 50 % inhibition of growth were estimated. The concentrations of antibiotics required for inhibition of peptidoglycan synthesis in the usual assay with particulate enzyme

I 8

I 4

I 16

I 6

2L L

I

>

7

4

i

2

81HO”R:8

4

I

6 0.04

24

T /

/ 0

8

1

4

,

2

16

t

6

I - 24

Fra. 17. Inhibition of bacterial growth by antibiotics. Cultures of S. aure-c~s (upper series) xnd .V. lysotkiklir~s (lower series) were grown at 37’ from 1% incoula’ At quarter-maximal growth the cultures were divided and lhrt :I((tibiotics were added. (irowth was followed turbidimet.rically at 700 n,h in IO-fold diluted aliquots. Growth CUI’WS rlf control cultures are shown by heavy lines. The numbers associated with the curves are the rcsprc~ ivc: :intihiotic ULIIcentrations in fig/ml. Concentrations higher and lower than those which produced nxtximum and rnic~inrurn cil’c~cls are not jllustratetl. Note the difference in the velocity of growth of the two orgnliisms. In n fast-growing org:lriisnl such as S. uureus, some lag is evident before the antibiotic inhibits growth.

oO++--+-

2

5$ 3 L

1,

L8

--

BIOSYNTHESIS TABLE INHIBITION BY

OF

IX

OF PEPTIDOGLYCAN ANTIBIOTICS ADDED

TABLE IN

INHIBITION

SYNTHESIS VITRO

All reaction mixtures contained 1.9 mpmoles of IJDP-MurNAc-pentapeptide, labeled with Wn-ala.14C-n-ala; 2 mrmoles of UDP-GlcNAc; 2.5 pmoles of Tris-HCl, pH 8.6; and antibiotics as indicated in a total volume of 25 ~1. Reaction mixtures with 3.8 ~1 of S. aureus enzyme, prepared after alumina grinding (52 pg of protein), also cont,ained 25 mpmoles of ATP and 0.12 pmole of MgClz and were incubated at 20” for 1 hour in test t,ubes. Reaction mixtures with 3.8 ~1 of M. Zysodeikticus enzyme, prepared after alumina grinding (25 pg of protein), also contained 0.84 Fmole of MgCls and were incubated for 1 hour at 37” in test tubes.

pe tide P f”ZEl brmoles)

Antlbzg:,;;l;dcled

None

Inhibition (%)

Peptidoglycan formed &moles)

Illtl,h’,t’o” O

367

-

122 85 33 10

7 35 75 92

380 337 220 25

0 8 40 93

\-ancomycin 2 6 17 51

105 86 38 14

20 34 71 89

352 299 134 23

4 19 63 94

16 14

323 229

12 38

44 5G A2 G2

133 106 97 45

64 71 74 88

8 10 24 66

310 262 89 13

16 29 76 96

G

-

-

Bacitracin 49 150 440 1300 Novobiocin 42 130 380 1150

120 118 100 45

from both organisms were determined (Table IX). Similarly, growing cultures of X. aureus and M. lysodeikticus at one-quarter maxi-

preparations

OF

X

PEPTIDOGLYCAN

PARTICULATE ENZYME TURES PRETREATED

SYNTHESIS

PREPARED FROM WITH ANTIBIOTICS

IN CUL-

Triplicate or quadruplicate cultures of either M. lysodeikticus or S. aureus were grown to quarter-maximal growth. One culture was reserved as a control. Varying amount,s of antibiotic were added to the other cult’ures. After 15 minutes additional incubation, the cells were harvested and particulat,e enzyme was prepared from each batch of cells. The cells were disrupted by treatment with the sonic oscillat,or. The enzyme from S. auTeu.s was assayed by the filter paper assay and enzyme from M. lysodeikticus was assayed by the filter paper and test tube procedures. Results are expressed as the percent,age inhibition of peptidoglycan synthesis catalyzed by the enzyme from antibiotic-treated cells relat,ive to the peptidoglycan synthesis catalyzed by t,he enzyme from the control cells.

-

131

Kistocetin 1 4 13 40

Penicillin 1300 3800

511

PEPTIDOGLYCAN

Inhibition Antibiotic

Ristocetin

Vancomycin

Penicillin Bacitrarin

G

Inhibition

Antibiotic cont. CM/ml)

Filter paper assay

3 10 25 250 5 7 10 25 50 250 15 50 250 5 10

0 lx 08 9G 0 59 92 93 37 29 30 64 74 73 81

(‘7,)

Antibiotic cont. Gdml)

Filter PaPer aSSay v% I

assay (“,,I

5 10 20 50 5 10 20 30 50 250 15 50 150 1 10 30 100

17 31 78 99 17 28 87 79 32 18 59 69 81 0 12 0 0

24 33 71 99 4 "5 81 74 16 10 30 45 69 7 G 0 8

%

mum growth were treated with various concentrations of antibiotics for 15 minutes. After harvesting, particulate enzyme was prepared and assayed for enzymic activity (without further addition of antibiotics to the assay mixtures) (Table X). The data obt,ained are summarized in Table XI. The concentrations of ristocetin and vancomycin required to inhibit the particulate enzyme and those required t,o

51’2

ASUERSOPU’,

ET

TABLE ANTIBIOTIC

SENSITIVITY

OF CELL 8.

I)atn are expressed to 5K. of the control and S.

ACREUS

Xl

GROWTH AKD

AL.

‘$1.

ASI)

PEPTIDOGLYCAN

SYSTHICSIS

IS

LI'SODEIPTICUS

as the concentratiort of antibiotic which reduced growth value and were obtained from the experiments recorded

rate or ct1zyttte act ivit.y in Fig. 17 :IILCI Tnhles IS

u. lysedeikliclrs

s. nll”ClbS _____. Peptidoglycan

Synthesis

1

Peptldoglycan

synthesis

Antibmtic Growth

ltistocetin Vancomycin Bacitracin Novobiocin Penicillin

G

&g/ml)

12 0 35 0.03 0.04

I

I I

~Antibiotic -

~~~ added

(pg/ml)

in Tim

in aitro

12 (i 25 5 > 250

8n 1oa 50 500 >4000

mpl Growth

(fig/ml)

1.

Antibiotic Ida aim

(

7 7 3 0.3 0.2

I

:

15 15 50 >I00 >750

added

-~ (pg,‘mlj ~~~

-

in vitro

15 10 30 150 4000

a These values for 50CjA inhibition by vsncomycin and ristocetin of the S. aureus enzyme prepared after alumina grinding and assayed by the test tube method are considerably lower t,han values previously obtained with the enzyme prepared after sonic disintegration and assayed by the filter paper method (200 pg/ml for each (17)). These differences could be due to differences in the nature of the particulate enzyme or t,o adsorption of the antibiotics on the filter paper.

inhibit growth were virtually identical in both organisms. It is also noteworthy that the concentrations required to inhibit, the particulate enzyme CA vitro wert virtually identical to those required if the cells were treated with these antibiotics prior to preparation of enzymes. These t,wo amibiot’ics appear t,o be highly selectjive inhibitors of pepticloglycan synthesis. The inhibition of the system in imart cells is not reversed during preparation and washing of the particulate enzyme. Bacitracin also inhibited the enzymic reaction in X. aul’eus at a concentration virtually identical to that required to inhibit growth of this organism. However, with enzyme from AI. lgsodeikticus, the inhibitory concent)ration was Den times greater than that required to inhibit growth, whether the antibiotic was added in z!it~o to the particulate enzyme or in viva to the cells prior to preparation of enzyme. Another peculiarity of the inhibition by bacitracin is that total inhibition was not obtained even at very high concentrations (Tables IX and X); rather, activity appeared to plateau at a value between 50 and 75 70. In both organisms, the inhibition of activity by bacitracin

was partial over a broad range of conczentrstions, in contrast to the sharply defined range of concentrations of ristocctin and vancomycin which include barely detectable inhibition and almost complete inhibition. Total inhibition (> 95 %) could be obtained mit,h bot,h of these latter substances at low concentrations. Thus, although bacitracin has some inhibitory effects on the enzymic synthesis of peptidoglycan, it is not yet cert’ain whether these are directly related to inhibition of cell growth by low concentrations of this substance. This question requires further investigation. Penicillin G and novobiocin inhibited pept,idoglycan synt,hesis only at- concentrations which were much greater than those required to inhibit growt,h. These antibiotics thus have no specific effect on this reaction sequence. The effects of penicillin G were inventigated more extensively since, in contrast, to data reported in a preliminary account of the present studies (17), it had also been reported (18) that’ penicillin G does inhibit this reaction if it is added to the cells prior to preparat’ion of the enzyme. In the present experiments particles prepared from cells

BIOSYNTHESIS

OF PEPTIDOGLYCAN

513

bility to enzymes of known specificities. The absenceof cross liiing in this product has been demonstrated by the fact that both of the n-alanine residuespresent in the uridine nucleotide precursor are retained in the peptidoglycan. Moreover, no trace of free ‘*C-n-alanine has been detected when reactions were carried out with UDPMurNAc-pentapeptide labeled with ‘*C-Dala+l*C-n-ala. It is now known that the terminal n-alanine residue is removed in the cross linking reaction (37, 45-47) and that the interpeptide bridge in many bacterial species is linked to the remaining n-alanine residue of bhe resulting tetrapeptide (48). The ident’ification of the uridine nucleotide reaction products provided the most unexpected findings in the study of this reaction. In addition to discovering UDPglucose, Leloir and his collaborators also discovered transglycosylation reactions involving nucleoside diphosphate sugar compounds. The reaction which they studied was the biosynthesis of sucrose from UDPglucose and fructose (49). They identified UDP as the nucleotide product of t.his reaction. Subsequently, many other transglyeosylations involving nucleoside diphosDISCUSSION phat,e sugar compoundshave been discovered These experiments have thus demon- (seerevicm by Leloir et al. (50)), and in every strated t,hat the two uridine nucleotides, case where it has been studied, a nucleoside UDP - J,IurNAc - pentapeptide and UDPdiphosphate has been identified as the GlcXAc, :arc utilized by particulate enzyme nucleotide reaction product. In the present preparations from X. aweus and M. tyy.so- study, UDP was identified as the nucleotdde deikticus, presumably fragments of the cell product arising from the glycosylat8ion membrane, to form a linear peptidoglycan involving UDP-GlcNAc. However, in the reaction involving UDP-MurNAc-pentaconsisting of alternating residuesof GlcNAc and MurYAc-pentapeptide. That the resi- peptide UMP and inorganic phosphate mere dues alternate is indicated by the obligatory formed instead. Extensive study has revealed requirement for both substrates before that’ t’hey are the primary reaction products peptidogl,ycan synthesis can be observed, and t,hat they do not arise by degradation of by t,he 1: 1 stoichiometric ratio of incorpora- UDP formed in the reaction. This was the first example of format’ion of this t,ype of tion of GlcN,4c and MurNAc-pentapeptide, and by the isolation of GlcNAc-XlurNAcproduct and it led to t(he discovery of the pentapeptidc from lysozyme digests as well phospholipid intermediates in the reaction as the dimer of this compound, tetrasac- mechanism. The initial accept,or is a memcharide-bis(pentapeptide). In addition, the brane-bound phospholipid to which phosmechanism for synthesis of the peptido- phoacetylmuramyl pentapeptide is t’ransglycan insures strict alternation of the sugars ferred wit’h formation of UMP. Then Glc(20). The peptidoglycan product of this NAc is transferred from UDP-GlcSAc to reaction has been identified by its suscepti- form lipid-phosphodisaccharide-pentapep-

treated with 50 pg per microliter of penicillin for 15, 30, or 60 minutes prior to harvesting and washed in normal buffers or in buffers containing 50 pg per microliter of penicillin G had normal enzymic activity. In contrast,, little or no apparent activity was found in unwashed particles from S. uureua cells pretreated with penicillin G. Unwashed particles from M. lysodeikticus, however, had normal enzymic activity. These experiments are best explained by the presenceof unlabeled UDP-MurNAc-pentapeptide in lthe particles prepared in this way from cells of S. uureus. This nucleotide accumulates in the cells and could contaminat,e the particulate enzyme. It would therefore dilute the labeled substrate employed in the assay (UDP-MurNAc-pentapeptide-‘“C) and thereby reduce apparent activity, unless removed by washing. Cells of M. lysodeikticus do not accumulate uridine nucleot,ides under the influence of penicillin G and hence unwashed particles prepared in the presence of penicillin H had normal cnzymic act,ivity. It is concluded t,hat penicillin G has no effect on the synthesis of t’he linear peptidoglycan strands st#udiedhere.

514

ANI)ERSON.

tide and UDP. Finally, the lipid-phosphodisaccharide-pentapeptide is utilized to form the linear peptidoglycan with release of a phosphorylated phospholipid. Nothing is known about the acceptor of disaccharidepentapeptide units for this transfer reaction. This acceptor is contained in the part’iculate enzyme preparation and is presumed to be an incomplete peptidoglycan. After t’he transfer reaction, inorganic phosphate (derived originally from UDP-MurKAc-pentapeptide) is released from the phosphorylated phospholipid. The phospholipid can then recycle and catalyze further addition of a disaccharide unit to the peptidoglycan. A similar type of reaction mechanism has been found to operat#e in the biosynthesis of the bacterial lipopolysaccharides (51, 52). However, formation of URIP in the first step in these reaction cycles has not yet been demonstrable because of the presence of phosphatases in the enzyme preparations. The particulate enzyme prepared from X. auwus after disrupt’ion of the cells by sonic disintegration catalyzed pept’idoglycan synthesis only when incubation mixtures were spread on filter paper. In contrast, enzyme prepared after alumina grinding of cells of S. aureus was active when assayed in the usual way, and particulate enzymes prepared from M. lysodeikticus after disruption either with alumina or by sonic oscillation were similarly active. Information presently available suggests that the filter paper is serving as an inert support for the rcact’ion. A great deal of the product can be extracted from the paper with water, and thus it. is not attached to cellulose fibers in Dhe filter paper. It has also not been possible to obtain activity with this enzyme by adding various acceptors to it in conventional assays. Small amounts of filter paper added to assay mixtures in test tubes do not initiate peptidoglycan synthesis. It may be that the reaction which occurs on filter paper results from stabilization of the particulate enzyme or from some kind of reaggregation of particles which had been severely disrupted by sonic oscillation. It should be mentioned that peptidoglycan synthesis occurs in three stages: 1, synthesis of the uridine nucleotide presurors; 9,

ET

AL.

utilization of t,hose precursors to form linear peptidoglycan st,rands; and 3, cross linking of the strands. Antibiotics are known which inhibit each of these stages. The second stage, studied here, appears to be specifically inhibited by ristocetin and vancomycin and possibly by bacitracin.lO REFERENCES 1. CaPrWO, Ii., LELoIR, L. F., C.\RI)INI, C. E., ~NI) PAL.IDINI, A. C., J. Riol. C’hem. 184, 333 (1950). 2. Pam, J. T., .&ND JOHNSON, M., J. Hiol. Chem. 179, 585 (1949). 3. PARK, J. T., J. Biol. Chem. 194, 877 (1952). 4. STRDMINGER, J. L., J. Biol. Chem. 224, 509 (1957). 5. STILBNGE, R. E., AND DARK, F. A.,:l’ntlrre 177, 186 (1956). 6. STRANGE, R. E., .~ND KENT, 1,. II., Biocheva. J. 71, 333 (1959). 7. PARK, J. T., .~ND STROMINGER, J. L., Science 126, 99 (1957). 8. STROMINGER, 5. I,., Pam, J. T., .\w THOMPSON, K. IS., J. BioZ. Chem. 234, 3263 (1959). 9. STROMINGER, J. L., Compt. Ifend. Il’rno. Lab. Curlsberg 31, 181 (1959). 10. STROMINGER, J. I,., .\su THRENS. IL. IT., Biochim. Hiophys. Scta 33, 280 (1959). 11. ITO, E., END STROMINGER, J. L., .J. Biol. Chem. 239, 210 (1964). 12. LANZILLOTI. A. E., BENZ, E., .INII GOLDMAN, I,., J. Am. Chem. Sot. 86, 1880 (1964). 13. ITO, R., A4x~ STROMINGER, J. L., .I. Biol. Chem. 237, 2689 (1962). 14. STROMINGER, J. L., in “The Bacteria” (1. C. Gunsalus and R. Stanier, eds.), 1’01. III, p. 413. Academic Press, New York (1962). 15. DUGUID, J., Edinburgh Med. J. 53, -401 (1916). 16. LEDERBERG, J., Proc. Matl. Acad. Sci. ITS. 42, 574 (1956). 17. MENIOW, P. M., ANDERSON, J. S., .~ND SmomNGEI~, J. L., Biochem. Biophys. Res. Commun. 14, 382 (1964). 18. CH.\TTERJEE, A. N., AND P.~HK, J. T., Proc. h;atl. dcad. Sci. U.S. 61, 9 (1964). lo Peptidoglycan synthetase, the enzyme which catalyzes the last step in the reaction, transfer of the disaccharide-pentapeptide units from the lipid carrier to the acceptor, is the component most sensitive to ristocetin and vancomycin (20). The failure of other workers to detect inhibition of the system by vancomycin in vitro (18) is probably due to the fact that these investigators were studying only the initial stages of the reaction.

BIOSYNTHESIS 19.

STRUVE,

Biophys. 20.

21 .

22.

23.

24.

25.

26.

27. 28.

29. 30. 31.

32. 33. 34.

35.

ANDERSON,

PI. G.,

AND

Res. J.

NEUHAUS,

Commun. S.,

MATSUHBSHI,

OF

F. C., Biochem. 18, 6 (1965). M.,

HASKIN,

M. A., END STROMINGER, J. L., Proc. Xatl. Acad. Ski. U.S. 63, 881 (1965). M;\TSUHABHI, A M., DIETRICH, C. P., AND STROMII~GER, J. L., Proc. Natl. dcad. Sci. U.S. 64, 587 (1965). STRUVE, W. G., SINHA, R. K., AND NEUHAUR, F. C., Biochemistry 6, 82 (1966). PARK, J. T., “16th Symposium of the Society for General Microbiology, Cambridge Univ. Press, Cambridge. p. 70 (1966). ANDERSON, J. S., ,~ND STROMINGER, J. L., Biochem. Biophys. Res. Cornmzcn. 21, 516 (1966). I)IETRICH[, C. P., MATSUHASHI, M., AND STROMINGER, J. L., Biochenc. Biophys. Res. Conmm. 21, 619 (1965). NATHEN~ON, S. G., ISHIMOTO, N., ANDERSON, J. S., AND STROMINGER, J. L., J. Biol. Chem. 241, 651 (1966). FINDI,AY, J., AND LEVVY, S. A., Biochem. J. ‘77, 170 (1960). Lower-, 0. H., KOSENBROUGH, N. J., FARR, A. L., AND R.\ND.\LL, H. J., J. Biol. Chern. 193, 265 (1951). NEUHAUR, F. C., J. Biol. Chem. 237,778 (1962). STROMINGER, J. L., AND THRENN, R. H., Biochim. Biophys. Acta 36, 83 (1959). STROMINGER, J. L., THRENN, R. H., AND SCOTT, S. S., J. Sm. Chem. Sot. 81, 3803 (1959). D. H., Proc. Null. GLASER, L., AND BROWN, dcad. Sci. c:.S. 41, 253 (1955). NATHEE~SON, S. G., AND STROMINGER, J. L., J. Biol. Chem. 238, 3161 (1963). STROMINGER, J. L., M~TSUHASHI, M., ANDERSON, J. S., MEADOW, P. M., DIETRICH, C. P., KATZ, W., SIEWERT, G., AND GILBERT, J. M., in “Methods in Enzymology” Vol. VIII, p. 473. Academic Press, New York (1966) . DIETRI~H, C. P., MATSUHASHI, M., KATZ, W., SIE~ERT, G., THOMAS, P., AND ANDERSON, J. S., Federation Proc. 26, 588 (1966).

a-1 3r

PEPTIDOGLYCAN 36. GHUYSEN, AND

J. M., STROMINGER,

D. J., BIRGE, C., J. L., Biochemistry 4,2245

TIPPER,

(1965). 37. IZAKI, K., MATSUHASHI, M., AND STROMINGER, J. L., Proc. Katl. Acad. Sci. Ir.S. 66, 656 (1966). 38. GHUYSEN, J., LEYH-BOUILLE, M., AND DIERICKX, L., Biochim. Hiophys. Acta 63, 286 (1962). 39. TIPPER, D. J., GHUYSEN, J. RI., AND STROMINGER, J. L., Biochemistry 4, 468 (1965). 40. ABRAHAM, F,. P., AND NEWTON, G., Ciba Found. Sywlp. Amino Acids and Peptides with Antimetabolic Activity, p. 205 (1958). 41. WALLAS, C., AND STROMINGER, J. I,., J. Biol. Chem. 238, 2264 (1963). 42. REYNOLDS, P. R., Biochim. Biophys. Llcta 62, 403 (1961). 43. JORDAN, Il. C., Biochem. Biophys. Res. Comwmn. 6, 167 (1961). 44. WISHNOW, R. M., STROMINGER, J. I,., BIRGE, C. H., AND THRENN, I:. H., J. Harferiol. 89, 1117 (1965). 45. WISE, E. M., AND PARK, J., I’roc. Sr11l. Acad. Sci. U.S. 64, 75 (1965). 46. TIPPER, 1). J., AND STRO~LIINGER, J. I,., Proc. Natl. Bead. Sci. U.S. 64, 1133 (1965). 47. ARAKI, Y., SHIMAI, R., RHIBIODA, A., ISHIMOTO, N., .YND ITO, E., Hiochem. ISiophys. Res. Commun. 23, 466 (1966). 48. GHUYSEN, J. M., PETIT, J. F., MUNOZ, E., AND KATO, K.. Federation I’TOC. 26, 410 (1966). 49. LELOIR, L. F., .\ND CAKDISI, C. II:., J. -4m. Chem. Sot. 76, 6084 (1953). 50. LELOIR, L. F., CARDINI, C. E:., AND CABIB, E., in “Comparative Biochemistry” (M. Florkin and H. S. Mason, eds.), Vol. 2, p. 97. Academic Press, New York (1960). 51. WEINER, I. M., HIGUCHI, T., ROTHFIELD, L., SALTMARSH-ANDREW, M., OSBORN, M. J., AND HORECKER, B. L., Proc. Nail. /{cad. Sci. U.S. 64, 228 (1965). 52. WRIGHT, A., D~\NKERT, M., AND I?OBBINS, P. W., Proc. Natl. Acad. Sci. r:.S. 64, 235 (1965).