JOURNAL OF BACTERIOLOGY, JUlY 1976, p. 309-318 Copyright ©D 1976 American Society for Microbiology

Vol. 127, No. 1 Printed in U.S.A.

Peptidoglycan Biosynthesis in Micrococcus luteus (sodonensis): Transglycosidase and Phosphodiesterase Activities in Membrane Preparations SUSAN E. JENSEN'* AND J. N. CAMPBELL Department of Microbiology, University of Alberta, Edmonton, Alberta, Canada, T6G 2E1 Received for publication 13 February 1976

Two enzyme activities involved in the biosynthesis of peptidoglycan in Microluteus (sodonensis), a transglycosidase and a phosphodiesterase, have been demonstrated in isolated membrane preparations. The transglycosidase activity promotes the in vitro synthesis of an uncross-bridged peptidoglycan that is completely susceptible to lysozyme. This in vitro-synthesized peptidoglycan consists of 76%-"soluble" and 24% "insoluble" material. The soluble peptidoglycan is primarily a single low-molecular-weight species of approximately 20 disaccharide peptide units. "Insoluble" peptidoglycan, which likely represents newly synthesized material incorporated into an existing cell wall, was solubilized by butanol extraction, and the two were compared. The phosphodiesterase activity demonstrated in this system cleaves uridine diphosphate-N-acetylmuramyl-L-alanyl-D-isoglutamyl-L-lysyl-n-alanyl-D-alanine to yield N-acetylmuramyl-L-alanyl-D-isoglutamyl-L-lysyl-D-alanyl-D-alanine plus uridine 5'-monophosphate plus inorganic phosphate. This phosphodiesterase activity, not detected under normal transglycosidase assay conditions, is a recycling mechanism and acts indirectly through formation and subsequent cleavage of a lipidlinked intermediate.

coccus

The use of cell-free systems to study the multistep process of peptidoglycan biosynthesis in microorganisms has been reported for a number of different species. Most commonly, membrane preparations serve as a source of enzymes (1, 5, 12). With such preparations, however, the in vitro-synthesized peptidoglycan frequently differs significantly from native cell wall peptidoglycan in size, solubility, and degree of cross bridging. The relationship of in vitro- to in vivo-synthesized peptidoglycan must be established if the in vitro assay system is to be useful in understanding the in vivo synthesis of peptidoglycan. Transglycosidation involves a number of intermediate steps that are difficult to isolate and detect individually but which must occur for the final product to be formed. One such step is a phosphodiesterase-type activity needed to cleave the membrane-bound intermediates from the carrier lipid and to finally release the completed glycan strand from the membranes (26). This paper describes the transglycosidase enzyme system present in an in vitro system from Micrococcus luteus (sodonensis). A phos-

phodiesterase activity that has characteristics that indicate it to be functionally related to the transglycosidase system is also described. (Portions of this material were incorporated into a Ph.D. thesis submitted by the senior author to the University of Alberta. A preliminary report of this work was presented at the 74th Annual Meeting of the American Society for Microbiology, 12 to 17 May 1974, Chicago, 1l.)

MATERIALS AND METHODS Organism and culture conditions. The organism used was Micrococcus luteus (sodonensis) (ATCC 11880). Unless otherwise stated, cultures were grown in Trypticase soy broth (Baltimore Biological Laboratories, Cockeysville, Md.) at 30 C with vigorous aeration. Routinely a 0.1% (vol/vol) amount of stationary-phase (18 h) culture was used as inoculum. Cell walls. Purified M. luteus (sodonensis) cell walls were a gift from K. G. Johnson, National Research Council, Ottawa, Canada. Cell membranes. Stationary-phase cells were harvested and washed in 50 mM Tris-hydrochloride, pH 7.5, containing 1 mM 2-mercaptoethanol (TME). When indicated, MgCl2 was added in concentrations I Present address: Department of Microbiology, Univer- of up to 50 mM (TME-Mg) and used in all steps of sity of British Columbia, Vancouver, British Columbia, preparation. Cells were broken by one of three methods: (i) high-speed, short-term (2-min) disrupCanada V6T 1W5. 309

310

JENSEN AND CAMPBELL

tion with plastic beads (Bio-Beads S-X8, 200 to 400 mesh, Bio-Rad Laboratories, Richmond, Calif.) in an Omni-mixer (Ivan Sorvall, Inc., Norwalk, Conn.); (ii) grinding with alumina in a mortar and pestle; or (iii) lysozyme digestion (100 ,ug of enzyme per ml of cells for 1 h at 30 C) followed by disruption in the French pressure cell at 1.5 x 104 lb/in2. Membranes were purified as follows: the disrupted cell preparation was centrifuged for 5 min at 15,000 x g, the pellet was discarded, and the supernatant was recentrifuged for 45 min at 48,000 x g. This pellet was washed twice in TME buffer and resuspended to yield a final protein concentration of 7 mg/ml. Preparations were kept cold at all stages of the purification. Membranes were prepared from exponentialphase cells in like manner. Preparation of UDP-MurNAc-['4C]pentapeptide. '4C-labeled uridine diphosphate-N-acetylmuramylL-alanyl-D-isoglutamyl-L-lysyl-D-alanyl-D-alanine was prepared by modification of the method of Garrett (7). A 300-ml portion of Trypticase soy broth containing 1% sodium lactate was inoculated, and the cells were harvested at the midexponential phase of growth. The cell pellet was resuspended into 250 ml of 50 mM sodium phosphate buffer, pH 7.0, containing 1 mM ethylenediaminetetraacetic acid, 5 tig of chloramphenicol per ml, and 1% sodium lactate. Ten microcuries each of L-alanine-[U-'4C]uridine (157 mCi/mmol; New England Nuclear Corp., Boston, Mass.) and D-alanine-[U-'4C]uridine (36 mCi/mmol, Amersham/Searle Corp., Des Plaines, Ill.) were added, and the mixture was incubated for 5 min at 30 C. Unlabeled glutamic acid, lysine, and alanine were then added to a final concentration of 2 ,ug/ml, and incubation was continued for 1 h with shaking. The cells were harvested and extracted twice with 25 ml of 25% trichloroacetic acid for 30 min at 4 C. The extracts were pooled and back-extracted three times with diethyl ether to remove the trichloroacetic acid. The aqueous layer was adjusted to pH 7.0, applied to a Sephadex G-25 column (2.5 by 90 cm), and eluted with water. Fractions with radioactive, soluble hexosamine-containing, ultraviolet (UV)-absorbing material were pooled and lyophilized. The dried material was streaked on Whatman 3 MM paper strips at a rate of 0.5 ,umol/cm and developed in solvent A. The radioactive area of each strip was cut out, eluted with water, and then rechromatographed in solvent B. The radioactive spot was again eluted from the second chromatogram, desalted on the Sephadex G-25 column as before, and analyzed. The material was shown to contain N-acetylmuramic acid, glutamic acid, lysine, and alanine in a molar ratio of 1.0:0.99:1.04:3.2 and had a specific activity of 0.32 mCi/mmol (694 dpm/nmol). The final yield of the precursor was 7.4 tmol. Enzyme assays. Transglycosidase activity was routinely assayed in a reaction mixture consisting of: 0.875 M Tris-hydrochloride, pH 8.6, 20 ,ul; 0.1 M MgCl2, 10 ul; 2 mM uridine diphospho-N-acetylglucosamine (UDP-GlcNAc), 10 ,ul; 0.8 mM UDPMurNAc-['4C]pentapeptide, 15 ,l; and 0.5 mg of membrane protein in a final reaction volume of 140 ul. Incubation was 3 h at 30 C, and the reaction was stopped by boiling. Reaction mixtures were streaked

(UDP-MurNAc-['4C]pentapeptide)

a

J. BACTERIOL.

onto 4-cm wide strips of Whatman 3 MM paper and chromatographed 16 h in solvent A. The dried chromatograms were then cut into 1-cm strips, and radioactivity was measured in a Nuclear-Chicago (Nuclear-Chicago Corp., Des Plaines, Ill.) Mark I scintillation counter using Bray solution. Cephaloridine, if required, was added to the normal assay system to yield final concentrations of 10 or 100 ,ug/ml. Cephaloridine was used instead of penicillin because of the instability of penicillin to Tris buffers (3, 6). Phosphodiesterase activity was measured using an assay system containing: 0.875 M Tris-hydrochloride, pH 8.6, 20 ul; 0.6 M MgCl2, 10 ,1; 0.8 mM UDPMurNAc-['4C]pentapeptide, 15 u.l; and 0.5 mg of membrane protein in a final reaction volume of 140 ,ul. Incubation was for 3 h at 25 C. Reaction products were separated and quantitated as described for the transglycosidase assay. The effect of magnesium on transglycosidase and phosphodiesterase activities was tested by varying the concentration of MgCl2 up to 125 mM in the normal assay system while maintaining the final volumes constant at 140 ,u. Analytical methods. Protein content of the membrane suspensions was measured by the method of Lowry et al. (11). Samples were diluted sufficiently to eliminate interference by TME. Total hexosamines were determined by the modified MorganElson technique (8). Soluble N-acetylhexosamine was quantitated by the method of Strominger et al. (23). Total amino acids were determined as their dinitrophenyl derivatives (8). Derivatives were separated by thin-layer chromatography on MN Silica Gel G-HR (Machery, Nagel, & Co., Duren, W. Germany), developed sequentially in solvent C and then in solvent D. Dextran was measured by the anthrone reaction (22). Reducing sugars were measured by the modified Park-Johnson ferricyanide method (8). Developing solvents for paper chromatography. Solvent A consisted of isobutyric acid-0.5 N aqueous ammonia (5:3, vol/vol). Solvent B consisted of ethanol-1 M ammonium acetate (5:2, vol/vol). Solvent C consisted of 1% aqueous ammonia-saturated butanol. Solvent D consisted of chloroform-methanol-acetic acid (85:14:1, vol/vol). Molecular weight determinations. A calibrated, upward-flow Sephadex G-200 column (2.5 by 45 cm) equilibrated with 0.3% NaCl was used for molecular weight determinations. Two-milligram amounts (in a 1-ml volume) of each of a series of dextran standards of known molecular weights (Pharmacia Fine Chemicals, Inc., Piscataway, N.J.) were applied individually and eluted with 0.3% NaCl. Peaks were detected using anthrone reagent.

RESULTS Transglycosidase activity of membrane fragments. The multistep conversion summarized in Fig. 1 (left side) was monitored by following the incorporation of label from the UDP-MurNAc-1'4C]pentapeptide into a chromatographically immobile fraction.

~I

VOL. 127, 1976

The method of cell disruption used in preparing the membrane fragments was found to greatly affect the resulting activity of the membrane preparations. Membranes prepared by grinding cells with plastic beads for 2 min contained the most active transglycosidase system. Figure 2 shows the result of chromatographic separation of a reaction mixture. Peaks were UDP-MurNAc-pentapeptide

UMP

UDPGlcNAc

transglycosidase

linear peptidoglycan + carrier lipid-P-P carrier

li.pid-P

+ P1

(2) phosphodiesterase

MurNAc-pentapeptide + carrier lipid-P-P carrier lipid-P + Pi

FIG. 1. Transglycosidase- and phosphodiesterasemediated steps in peptidoglycan biosynthesis in M. luteus (sodonensis).

12

10 : l-o

311 identified as peak I (at origin), in vitro-synthesized peptidoglycan; peak II (retardation factor

PEPTIDOGLYCAN SYNTHESIS IN M. SODONENSIS

[Rf], 0.3) residual-labeled substrate; peak III (Rf = 0.55 to 0.65), a mixture of MurNAcL'4Clpentapeptide (due to phosphodiesterase activity) and ['4C]pentapeptide (due to amidase activity); and peak IV (Rf = 0.8 to 0.9), lipid precursors. The pH and temperature optima were 8.0 to 9.0 and 30 C, respectively. Under optimum conditions, using the standard transglycosidase assay system, there was an incorporation of 4.7 nmol of MurNAc-['4C]pentapeptide per mg of membrane protein per h into peptidoglycan. Membranes prepared by alumina grinding exhibited much lower activity (2.2 nmol of MurNAc-['4C]pentapeptide incorporated per mg of protein per h), whereas lysozyme-French pressure cell-prepared membranes contained no demonstrable peptidoglycan-synthesizing activity. Some elements of the overall system were present however, as small amounts of lipid-linked precursors (peak IV) could be detected. Role of magnesium. Mg2+ is essential for transglycosidase activity, with 7 mM giving maximal activity (Fig. 3). Some Mg2+ might remain associated with the membranes after purification, which would account for the small amount of activity that persists even in the absence of added Mg2+. Concentrations above 7 mM were inhibitory, and at 120 mM Mg2+ activity was essentially zero. Whereas the presence of Mg2+ was required in the assay system, the presence of Mg2+ during the preparation of the membranes was shown to be inhibitory. The presence of 50 mM Mg2+ in the washing buffer during preparation completely destroyed transglycosidase activity in the resulting mem-

IVI

I 71-~~~~~~~~~I

FIG. 2. Transglycosidase activity of stationaryphase membranes from M. luteus (sodonensis). A membrane suspension containing 7 mg ofprotein per ml was prepared from stationary-phase cells of M. luteus (sodonensis). A 70-pl sample was assayed for transglycosidase activity under standard conditions as described in Materials and Methods. Peak I, Newly synthesized pepti&6glycan; peak II, residual substrate

(UDP-MurNAc-["4Cjpentapeptide); peak III, phosphodiesterase and amidase products (MurNAc['4CJpentapeptide and ['4CJpentapeptide); and peak IV, lipid precursors.

IE cr

mg2# Concentration (mM) FIG. 3. Effect of Mg2" on transglycosidase activity. Standard transglycosidase assays were set up as described in Materials and Methods. Activity is expressed as the percentage of total radioactivity incorporated into peptidoglycan.

JENSEN AND CAMPBELL 312 branes. This lost activity could not be restored to the membranes either by dialysis of the membranes against Mg2+-free buffer or by addition of the concentration washings back to washed membranes. Furthermore, no transglycosidase activity could be found free in the washes of the inactivated membranes. Isolation and characterization of in vitrosynthesized peptidoglycan. The product of transglycosidase activity (peak I, Fig. 2) was tested for lysozyme susceptibility by adding 100 ,ug of lysozyme to a completed transglycosidase assay. After a 20-h digestion at 37 C, the mixture was chromatographed using solvent A, and the distribution of radioactivity was compared with that of a normal transglycosidase assay (Fig. 4). The complete disappearance of labeled material from the origin confirmed the presence of the lysozyme-sensitive ,B-1,4-N-acetylmuramyl-N-acetylglucosamine linkage. The two products of lysozyme activity (peaks III and IV) were identified as disaccharide-peptide and tetrasaccharide-peptide, respectively, on the basis of their chromatographic properties. The inclusion of cephaloridine in the transglycosidase assay system had no apparent effect on peptidoglycan biosynthesis. This fact, coupled with the inability to demonstrate release of free labeled alanine, was taken as evidence that neither transpeptidation (cross bridge for-

.t 0

v

Rf

FIG. 4. Lysozyme digestion of in vitro-synthesized peptidoglycan. Two standard transglycosidase assays were set up and after incubation the reactions were stopped by boiling for 1 min. One assay system then received 100 Mg of lysozyme. Incubation was for 20 h at 37 C in the presence of 0.02% NaNO:,. The reactions were again stopped by boiling, and the reaction mixtures were chromatographed in solvent A. Symbols: ( ) Peptidoglycan control; (- -) peptidoglycan plus lysozyme. Peak I, Newly synthesized peptidoglycan; peak II, residual substrate (UDPMurNAc- ['4C]pentapeptide); peak III, disaccharide peptide; peak IV, tetrasaccharide peptide; peak V, analogous to peak III, Fig. 2; and peak VI, analogous to peak IV, Fig. 2.

J. BACTERIOL.

mation) nor DD-carboxypeptidase activity was occurring under these in vitro conditions. To examine the physical characteristics of in vitro-synthesized peptidoglycan, a normal transglycosidase reaction mixture was scaled up 200-fold. The peptidoglycan formed represented an incorporation of 805 nmol of MurNAc-['4C]pentapeptide into peptidoglycan. The size and uniformity of this peptidoglycan were examined first by centrifuging the entire reaction mixture at 48,000 x g for 45 min. "Soluble peptidoglycan" was defined as that material remaining in the supernatant, whereas "insoluble peptidoglycan" was sedimented along with the membrane fragments. Soluble peptidoglycan comprised 76% of the total peptidoglycan formed. Insoluble peptidoglycan accounted for the 24%, and the proportion of soluble versus insoluble material could not be altered by the addition of cephaloridine. The soluble peptidoglycan-containing supernatant was lyophilized, resuspended in 2 ml of water, and chromatographed on a Sephadex G200 column (2.5 by 45 cm). Fractions were eluted with water and monitored for radioactivity. Results are summarized in Fig. 5. Essentially two main components were separated, a high-molecular-weight fraction (peak I, excluded with the void volume) and a low-molecular-weight fraction (peak II, K,,,. = 0.7; K,, = [Vf - VI][V, - VJ, where V, is the elution volume, V, is the void volume, and V, is the total volume) which contained the majority of the material. Peak III in this figure represents residual substrate. Peak II was further purified by rechromatographing on Sephadex G-200 (now eluted with K,,,. of 0.53), and then both high- and low-molecular-weight fractions were lyophilized separately. Insoluble peptidoglycan was demonstrated to owe its insolubility to the fact that it is associated with membrane fragments rather than to its intrinsic size. This peptidoglycan was solubilized by extraction with water-saturated butanol. Two successive butanol extractions released more than 90% of the insoluble peptidoglycan into the aqueous phase, and it was no longer sedimentable by centrifugation at 48,000 x g for 45 min. The chromatographic analysis on Sephadex G-200 of this solubilized insoluble peptidoglycan revealed a similar distribution as shown in the soluble peptidoglycan, with the exception that the amount of the low-molecular-weight material was much, much less (peak III, Fig. 5). As one would expect there was little, if any, residual substrate. The high- and low-molecular-weight fractions of insoluble peptidoglycan were lyophilized. The yield of each of the four species of in

VOL. 127, 1976

313

PEPTIDOGLYCAN SYNTHESIS IN M. SODONENSIS

with a Kav of 0.522, which corresponded to a molecular weight of 19,400. The separated peptidoglycan fragments (peaks I and II, Fig. 5) from soluble and insoluble peptidoglycan were resuspended in water to give 35,000 to 40,000 dpm/ml. This would correspond to a hexosamine content of 50 to 60 nmol/ ml, assuming that all peptidoglycan had been synthesized in vitro from radioactive substrate and that, for each mole of MurNAc['4C]pentapeptide incorporated, 1 mol of GlcNAc was incorporated. The samples were then assayed for actual hexosamine content (Table 2). Actual hexosamine values were higher than assumed, which indicated that the peptidoglycan fragments all contained unlabeled (i.e., preexisting) peptidoglycan material. The lower-molecular-weight species were the least diluted with unlabeled material. Although the membrane suspensions appeared

._

-o ._

I

TABLE 1. Distribution of fractions of in vitrosynthesized peptidoglycan of M. luteus (sodonensis) Total in vitro-

Kav FIG. 5. Fractionation of in vitro-synthesized peptidoglycan by Sephadex G-200 chromatography. The soluble and insoluble portions of in vitro-synthesized peptidoglycan were chromatographed on a Sephadex G-200 column (2.5 by 45 cm) as described in Materi) Soluble peptidoals and Methods. Symbols: ( glycan; (- -) insoluble peptidoglycan. Peak I, highmolecular-weight material; peak II, low-molecularweight material; and peak III, residual substrate

(UDP-MurNAc-['4C]pentapeptide).

vitro-synthesized peptidoglycan is shown in Table 1. The overall recovery of 788 nmol of an initial 805 nmol is high, because the insoluble high-molecular-weight peptidoglycan is about 20% contaminated with radioactive lipid intermediates (peak IV, Fig. 2) which do not separate out upon gel filtration. The molecular weights (size) of the high- and low-molecular-weight fractions of soluble peptidoglycan were determined by chromatography on a calibrated Sephadex G-200 column (9). The column was calibrated using a series of dextran standards wkch known average molecular weights (Fig. 6). Figure 6 shows the elution profiles of high- and low-molecular-weight soluble peptidoglycan fractions from the calibrated column. The high-molecular-weight material was essentially all excluded from the column, indicating a molecular weight in excess of 130,000 (peak I). The peak has a shoulder which ranges down to a Kay of 0.17, a molecular weight of 70,000. The low-molecular-weight material (peak ]E) eluted in a uniform peak

Am ounp

Peptidogl ycan specie s

8synthesized peptidoglycan (96)

16.1 127 Soluble, high molecular weight 41.0 402 Soluble, low molecular weight 17.5 138 Insoluble high molecular weight 15.4 121 Insoluble low molecular weight a Nanomoles of MurNAc-['4C]pentapeptide incorporated into peptidoglycan.

..

.,It

.3

K

.

.

FIG. 6. Chromatography of soluble in vitro-synthesized peptidoglycan fractions on a calibrated Seph-

adex G-200 column. Samples (1 ml) of the high- and low-molecular-weight species of soluble in vitro-synthesized peptidoglycan (each containing approximately 20,000 dpm/ml) were chromatographed on a calibrated Sephadex G-200 column. Symbols: ( Soluble high-molecular-weight material; (--) soluble low-molecular-weight material.

314

JENSEN AND CAMPBELL

TABLE 2. Hexosamine contentoffractions of in vitrosynthesized peptidoglycan of M. luteus (sodonensis) Peptidoglycan actually synMeathesized in Peptidoglycan species Estimated by sured by vitro (%) radioac- Morgan- [(A/B) x tivity (A) Elson (B) 1001 57.8 640 9.03 molecuSoluble, high lar weight 57.2 115 49.74 Soluble, low molecular weight 60.4 405 14.90 Insoluble, high molecular weight 55.4 255 21.72 nsoluble, low molecular weight a Nanomoles per milliliter. Hexosamine content"

free from cell wall contamination when examined in the electron microscope, the unlabeled peptidoglycan must have arisen from there, since all other components of the assay system are defined. This in vivo-synthesized peptidoglycan could be analogous to the soluble peptidoglycan that has been observed to be excreted from protoplasts of Streptococcus faecalis (21). The nature of the association between labeled and unlabeled material was not determined. They eluted simultaneously upon gel filtration, but this indicated only a similarity of size and not necessarily the presence of a covalent attachment. Phosphodiesterase activity of membrane fragments. Omission of UDP-GlcNAc from the transglycosidase assay system completely prevented peptidoglycan biosynthesis, as expected. The UDP-MurNAc-['4Clpentapeptide did not remain unchanged; however, it was converted into a new radioactive product (Fig. 7) with an Rf of 0.6 in solvent A, which corresponded with the minor peak at this Rf value seen in a normal transglycosidase assay. The product was demonstrated not to be D-alanine and hence had not resulted from either transpeptidase or D-carboxypeptidase activity. This phosphodiesterase activity was optimal at 25 C between a pH of 8.0 and 9.0. Like the transglycosidase system it required Mg2+ for activity, but unlike transglycosidase it was markedly less sensitive to excess Mg2+ in the reaction mixture or to the presence of Mg2+ during membrane preparation. Characterization of reaction products. The phosphodiesterase product was isolated from an assay system scaled up 100-fold. The radioactive, non-UV-absorbing product was first purified by gel filtration on Sephadex G-25 and then subsequently by paper chromatography in solvents A and B. After purification, approximately 400 nmol (based on radioactivity) of the

J. BACTERIOL.

material was available for assay. The material, resuspended in a 1-ml total volume and was assayed for total amino acids, soluble N-acetylhexosamine, and reducing groups as previously described. The results summarized in Table 3 showed a composition compatible with MurNAc-['4C]pentapeptide. The presence of a free reducing group and the absence of demonstrable UV absorbance confirmed that the nucleotide diphosphate portion had been removed from the Cl position. During the purification of MurNAc['4C]pentapeptide by gel filtration, some nonradioactive, UV-absorbing material was detected. These unlabeled, UV-absorbing products were isolated and identified chromatographically as uridine and uridine 5'-diphosphate (UMP). Envelope preparations of M. luteus (sodonensis) have been shown to contain at least one phosphomonoesterase active on UMP (4, 13). Uridine was thus apparently a secondary product resulting from this activity. Identification of the products as MurNAc-

L'4C]pentapeptide

and UMP (rather than

UDP), together with the absence of this activity

-

1

'

9

*'

A9 Rf

.0

..

*

M

FIG. 7. Phosphodiesterase activity of stationaryphase membranes from M. luteus (sodonensis). A standard transglycosidase assay was set up with UDP-GlcNAc omitted from the reaction mixture. After the addition of 70 pi ofstationary-phase membrane suspension (7 mg ofprotein per ml), the assay was incubated under standard conditions, chromatographed in solvent A, and counted. Peak I, Residual substrate (UDP-MurNAc-['4C]pentapeptide); peak II, phosphodiesterase product (MurNAc-['4CJpentapeptide). TABLE 3. Chemical composition of the radioactive product of phosphodiesterase activity in membrane preparations of M. luteus (sodonensis) Component nmol/ml Molar ratio Glutamic acid 397 1.0 Alanine 1330 3.3 Lysine 423 1.06 Soluble N-acetylhexosamine 370 0.93 Reducing groups 369 0.93

VOL. 127, 1976

in a normal transglycosidase assay system, indicated that UDP-MurNAc-['4Clpentapeptide was not the direct substrate for the enzyme. The possible sequence of reactions shown in Fig. 1 (right side) led to the description of the activity as a phosphodiesterase. The effect of adding equimolar. concentrations of various nucleotides to the phosphodiesterase assay was measured (Table 4). UMP, the proposed end product, was clearly the most inhibitory material, whereas uridine and UDP had almost no effect. UDP-glucose had a moderately inhibitory effect. The possibility that phosphodiesterase and transglycosidase activities are competing systems regulated by the availability of UDPGlcNAc was tested by measuring the effect of the addition of varying amounts of UDPGlcNAc to the phosphodiesterase assay system on the resultant distribution of the two activities. The maximum inhibition of phosphodiesterase activity occurred with equimolar amounts of UDP-GlcNAc and UDP-MurNAc[14C]pentapeptide, which supports the scheme presented in Fig. 1 (Fig. 8). Solubilization of transglycosidase and phosphodiesterase activities from membrane preparations. Three methods commonly used to solubilize membrane or wall-associated enzymes were used in an attempt to release the transglycosidase and phosphodiesterase in their active forms. (i) For butanol extractions, the membrane suspensions were mixed with equal amounts of water-saturated butanol for 15 min at 4 C. The emulsion was centrifuged at 48,000 x g for 45 min, and the aqueous layer and pellet were saved. This technique has been successfully used to solubilize several membrane-associated enzymes (3). (ii) For Triton X100 treatment, the membrane suspensions were made 1% with respect to Triton X-100. After brief mixing the suspension was centrifuged at 48,000 x g for 45 min to sediment residual membranes. Again this method was very effective for solubilizing DD-carboxypeptidase from Bacillus subtilis membranes (25). (iii) For LiCl extraction, the membranes were sedimented TABLE 4. Effect of added nucleotides on phosphodiesterase activity in membrane preparations of M. luteus (sodonensis) (2 nml) Addllon

Additions (25 nmol)

Radioactivity MurNAc-

as

s4cpentapeptide

Inhibition (96

and resuspended to their original volume in 6 M LiCl in 1 M Tris-hydrochloride, pH 8.0. The membranes were extracted for 60 min at 4 C and then centrifuged at 48,000 x g for 45 min. LiCl extraction has been used to solubilize several wall-associated autolysins (20). In each of these three methods, the residual membrane pellet was resuspended to its initial volume in TME buffer. Both the resuspended pellets and the supernatants from each procedure were dialyzed overnight against TME buffer. A 70-1.I portion of each was then assayed for transglycosidase and phosphodiesterase activities in the normal assay systems (Table 5). No completely satisfactory method of solubilization was found. Both butanol and LiCl extractions completely inactivated the enzymes. Triton X-100 treatment caused approximately 20% of both activities to be found free in solution, but the remaining 80% of both activities was destroyed. This suggested that the strict

UDP-GicNAc (nmoIes) FIG. 8. Effect of UDP-GlcNAc on the distribution of phosphodiesterase and transglycosidase activities. Normal phosphodiesterase assays (12 nmol of UDPMurNAc-/ 4C]pentapeptide in each) were set up containing varying amounts of UDP-GlcNAc, and phosphodiesterase and transglycosidase activities were measured. Results are expressed as the percentage of total radioactivity incorporated into MurNAc-peptide ) Transglycosiand peptidoglycan. Symbols: ( dase; (- -) phosphodiesterase.

TABLE 5. Solubilization of transglycosidase and phosphodiesterase activities from membrane preparations of M. luteus (sodonensis) Transglycosidase Phosphodiesterase Solubilization technique

Solubilized (%)

(%)

None Uridine UMP UDP

UDP-glucose

315

PEPTIDOGLYCAN SYNTHESIS IN M. SODONENSIS

37.2 39.8 11.4 34.2 23.1

0 0 69.4 8.1 37.9

Triton X-100 Butanol extraction LiCl extraction

21.7 3.7

O

RemainRemainSolubiing ing bound (%) lized bound

(%)

1.5 1.4

O

(%)

19.8 3.0

O

2.3 3.2

O

316

JENSEN AND CAMPBELL

orientation imposed by immobilization in the membrane is essential to the stability of both transglycosidase and phosphodiesterase activities. DISCUSSION The highly active transglycosidase system present in membranes from M. luteus (sodonensis) has made it possible to synthesize peptidoglycan in vitro in large enough amounts to examine its properties. Several similarities were noted between the present study and recent results obtained by Bordet and Perkins (5) and Pellon et al. (18, 19) in their examination of in vitro peptidoglycan biosynthesis by M. luteus (lysodeikticus). They found that newly synthesized peptidoglycan was mainly soluble (52 to 62%) and that a major part of the insoluble peptidoglycan could be solubilized by trypsin digestion, just as insoluble peptidoglycan from M. luteus (sodonensis) can be solubilized by butanol extraction. Insolubility was judged to be due to association with the membrane rather than to insolubility of the peptidoglycan itself. Unlike the situation in M. luteus (sodonensis), however, they found some evidence for cross bridging taking place, and they also found their soluble peptidoglycan to be polydisperse upon gel filtration. Their suggestion that soluble peptidoglycan may be a degradation product ofinsoluble peptidoglycan seems less likely in our system where the soluble peptidoglycan consists mainly of a single species. About 75% of the peptidoglycan synthesized in vitro by M. luteus (sodonensis) was soluble, which implied that most of it was not being attached to membrane-associated cell wall material but rather was being released free into the medium. This soluble peptidoglycan consisted of linear glycan strands, predominantly of one size. The molecular weight of 19,400 for the strands corresponded with an average length of 20 disaccharide-peptide units. Peptidoglycan biosynthesis in vivo results in the formation of a large, insoluble network of crossbridged glycan strands. If the soluble low-molecular-weight material, typical of in vitro-synthesized peptidoglycan, is also produced in vivo, then it must subsequently be incorporated into insoluble cell wall material. Evidence that such synthesis of soluble peptidoglycan does take place in vivo when transpeptidation is inhibited has been reported by Mirelman et al. (15). Other studies of cell wall growth indicate that peptidoglycan grows laterally by the addition of complete linear strands of peptidoglycan by transpeptidation (14, 16, 23). In such a system as this one from M. luteus (sodonensis), where transpeptidation is not taking place,

J. BACTERIOL.

transglycosidation proceeding in the absence of transpeptidation might be expected to result in formation of a population of soluble linear peptidoglycan strands of low molecular weight. Our results would tend to support such a theory. The high-molecular-weight peptidoglycan produced in vitro may be due to incorporation of some l-ow-molecular-weight material into preexisting peptidoglycan by transglycosidation. Mirelman and Sharon (16) showed that most peptidoglycan growth in M. luteus (lysodeikticus) occurred through transpeptidation, but that about 30% of growth occurred longitudinally through transglycosidation. The fact that in vitro-synthesized peptidoglycan from M. luteus (sodonensis) is 100%o degraded by lysozyme compared to only 50% susceptibility of native cell wall peptidoglycan again demonstrates the structural differences between the two (K. G. Johnson, Ph.D. thesis, Univ. of Alberta, Edmonton, Alberta, 1971). Lysozyme resistance in native cell wall peptidoglycan is related to the extent of cross bridging and also to O-acetyl substitution of muramic acid residues. In vitro-synthesized peptidoglycan is not cross bridged and, since 0 acetylation is an energy requiring process, is also not 0 acetylated. This would account for the increased susceptibility of this material to lysozyme. The phosphodiesterase activity detected in M. luteus (sodonensis) has not been reported from similar in vitro systems in other organisms. Heydanek et al. have described a pyrophosphatase activity in membrane preparations from Staphylococcus aureus which cleaves carrier lipid-P-P-MurNAc-peptide to yield carrier lipid-P plus P-MurNAc-peptide (10). The present study, however, found no evidence that P-MurNAc-peptide was being produced at any time during the 3-h phosphodiesterase incubation period. The phosphodiesterase has been postulated to use carrier lipid-P-P-MurNAc-pentapeptide rather than UDP-MurNAc-pentapeptide as a substrate because of the low levels of phosphodiesterase activity detected in a normal transglycosidase assay system. UDP-GlcNAc inhibits phosphodiesterase indirectly by permitting transglycosidation to occur. Omission of UDP-GlcNAc prevents peptidoglycan biosynthesis and allows carrier lipid-P-P-MurNAcpentapeptide to accumulate. The phosphodiesterase releases this unproductively bound MurNAc-pentapeptide and returns the carrier lipid to the membrane pool. UDP-glucose is also somewhat inhibitory to phosphodiesterase activity. This is perhaps the result of its ability to replace UDP-GlcNAc dur-

VOL. 127, 1976

PEPTIDOGLYCAN SYNTHESIS IN M. SODONENSIS

ing transglycosidation and therefore use up lipid intermediates in the formation of an incorrect "peptidoglycan" (17). Alternatively, UDPglucose may be a precursor of the glucose polymers found in cell walls of M. luteus (sodonensis). The synthesis of such compounds has frequently been found to share carrier lipids with the peptidoglycan-synthesizing system (2, 24, 28). Phosphodiesterase activity may be a normal component of the transglycosidase enzyme system, functioning as a scavenger enzyme only when peptidoglycan biosynthesis is prevented. The phosphodiester bonds linking disaccharidepeptide units to the carrier lipid must be broken during the course of glycan chain elongation. Similarly, phosphodiesterase activity is required to release the complete linear peptidoglycan strand from the carrier lipid (26). The ability of phosphodiesterase to degrade UDP-MurNAc-pentapeptide through formation and cleavage of lipid intermediates may also explain why whole cells of M. luteus (sodonensis) will not accumulate UDPMurNAc-pentapeptide when exposed to penicillin or cephaloridine. The ability to accumulate UDP-MurNAc-pentapeptide under conditions of cation deprivation is also consistent with this theory, since phosphodiesterase activity has an absolute requirement for Mg2+. ACKNOWLEDGMENTS We wish to thank Lucille M. Phillippe for her technical assistance. This work was supported by the National Research Council of Canada (operating grant A-1743). LITERATURE CITED 1. Anderson, J. S., M. Matsuhashi, M. A. Haskins, and J. L. Strominger. 1965. Biosynthesis of the peptidoglycan of bacterial cell walls. II. Phospholipid carriers in

the reaction sequence. J. Biol. Chem. 42:3180-3190. 2. Baddiley, J. 1972. Isoprenoid intermediate in the biosynthesis of bacterial cell walls, p. 337-352. In R. Piras and H. G. Pontis (ed.), Pan-American Association of Biochemical Societies Symposium, Argentina, 1971. Biochemistry of the glycosidic linkage: an integrated view, proceedings, vol. 2. Academic Press Inc., New York. 3. Barnett, H. J. 1973. D-alanine carboxypeptidases of

Bacillus stearothermophilus: solubilization of particulate enzymes and mechanism of action of penicillin. Biochim. Biophys. Acta 304:332-352. 4. Berry, S. A., and J. N. Campbell. 1970. The extracellular nuclease activity of Micrococcus sodonensis. III. Kinetic studies and control of production. Biochim. Biophys. Acta 220:256-268. 5. Bordet, C., and H. R. Perkins. 1970. Iodinated vancomycin and mucopeptide biosynthesis by cell free preparations from Micrococcus lysodeikticus. Biochem. J. 119:877-883. 6. Florey, H. W., N. J. Heatley, M. A. Jennings, A. G. Saunders, E. P. Abraham, and M. E. Florey. 1949. Antibiotics, vol. 2, p. 803. Oxford University Press, London.

317

7. Garrett, A. J. 1969. The effects of magnesium ion deprivation on the synthesis of mucopeptide and its precursors in Bacillus subtilis. Biochem. J. 115:419-430. 8. Ghuysen, J. M., D. J. Tipper, and J. L. Strominger. 1969. Enzymes that degrade bacterial cell walls, p. 685-699. In E. F. Neufeld and V. Ginsberg (ed.), Methods in enzymology, vol. 8. Academic Press Inc., New York. 9. Granath, K. A., and B. E.Kvist. 1967. Molecular weight

distribution analysis by gel chromatography on Sephadex. J. Chromatography 28:68-81. 10. Heydanek, M. G., W. G. Struve, and F. C. Neuhaus. 1969. On the initial stage in peptidoglycan synthesis. III. Kinetics and uncoupling of phospho-N-acetylmuramyl-pentapeptide translocase (uridine 5'-phosphate). Biochemistry 8:1214-1221. 11. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurements with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 12. Lugtenberg, E. J. J., A. van Schijndel-Van Dam, and T. H. M. van Bellegem. 1971. In vivo and in vitro action of new antibiotics interfering with the utilization of

N-acetyl-glucosamine-N-acetyl-muramyl-pentapep-

tide. J. Bacteriol. 108:20-29. 13. Mills, C., and J. N. Campbell. 1974. Production and control of extracellular enzymes in Micrococcus sodonensis. Can. J. Microbiol. 20:81-90. 14. Mirelman, D., R. Bracha, and N. Sharon. 1972. Role of the penicillin sensitive transpeptidation reaction in attachment of newly synthesized peptidoglycan to cell walls of Micrococcus luteus. Proc. Natl. Acad. Sci. U.S.A. 69:3355-3359. 15. Mirelman, D., R. Bracha, and N. Sharon. 1974. Penicillin-induced secretion of a soluble, uncross-linked peptidoglycan by Micrococcus luteus cells. Biochemistry 13:5045-5053. 16. Mirelman, D., and N. Sharon. 1972. Biosynthesis of peptidoglycan by a cell wall preparation of Staphylococcus aureus and its inhibition by penicillin. Biochem. Biophys. Res. Commun. 46:1909-1917. 17. Park, J. T., and A. N. Chatterjee. 1966. Membrane associated reactions involved in bacterial cell wall mucopeptide synthesis, p. 466472. In E. F. Neufeld and V. Ginsberg (ed.), Methods in enzymology, vol. 8. Academic Press Inc., New York. 18. Pellon, G., C. Bordet, and G. Michel. 1974. Membranepeptidoglycan association in the in vitro biosynthesis of the peptidoglycan of Micrococcus luteus. Ann. Mi-

crobiol. (Inst. Pasteur) 125(B):149-158.

19. Pellon, G., C. Bordet, and G. Michel. 1976.

Peptidogly-

cans synthesized by a membrane preparation of Micrococcus luteus. J. Bacteriol. 125:509-517.

20. Pooley, H. M., J. M. Porres-Juan, and G. D. Shockman. 1970. Dissociation of an autolytic enzyme-cell wall complex by treatment with unusually high concentrations of salt. Biochem. Biophys. Res. Commun. 38:1134-1140. 21. Rosenthal, R. S., and G. D. Shockman. 1975. Synthesis of peptidoglycan in the form of soluble glycan chains by growing protoplasts (autoplasts) of Streptococcus faecalis. J. Bacteriol. 124:419-423. 22. Scott, T. A., and E. H. Melvin. 1953. Determination of dextran with anthrone. Anal. Chem. 25:1656-1661. 23. Strominger, J. L. 1957. Microbial uridine-5'-pyrophosphate N-acetylamino sugar compounds. I. Biology of the penicillin-induced accumulation. J. Biol. Chem. 224:509-523. 24. Strominger, J. L., Y. Higashi, H. Sandermann, K. J. Stone, and E. Willoughby. 1972. The role of polyisoprenyl alcohols in the biosynthesis of peptidoglycan and other complex polysaccharides, p. 135-154. In R. Piras and H. G. Pontis (ed.), Pan-American Association of Biochemical Societies Symposium, Argentina, 1971. Biochemistry of the glycosidic linkage: an inte-

318

JENSEN AND CAMPBELL

grated view, proceedings, vol. 2. Academic Press Inc., New York. 25. Umbreit, J. N., and J. L. Strominger. 1973. Relation of detergent HLB number to solubilization and stabilization of D-alanine carboxypeptidase from Bacillus subtilis membranes. Proc. Natl. Acad. Sci. U.S.A. 70:2997-3001. 26. Ward, J. B., and H. R. Perkins. 1973. The direction of glycan synthesis in bacterial peptidoglycan. Biochem. J. 135:721-728.

J. BACTERIOL. 27. Ward, J. B., and H. R. Perkins. 1974. Peptidoglycan biosynthesis by preparations from Bacillus licheniformis: cross-linking of newly synthesized chains to preformed cell wall. Biochem. J. 139:781-784. 28. Wright, A., M. Dankert, P. Finnessey, and P. W. Robbins. 1967. Characterization of a polyisoprenoid compound functional in 0-antigen biosynthesis. Proc. Natl. Acad. Sci. U.S.A. 57:1798-1803.