JOURNAL OF BACTERIOLOGY, Aug. 1991, p. 4618-4624 0021-9193/91/154618-07$02.00/0 Copyright C) 1991, American Society for Microbiology
Vol. 173, No. 15
In Vitro Synthesis and 0 Acetylation of Peptidoglycan by Permeabilized Cells of Proteus mirabilis CLAUDE DUPONTt AND ANTHONY J. CLARKE* Guelph- Waterloo Centre for Graduate Work in Chemistry, Department of Microbiology, University of Guelph, Guelph, Ontario NJG 2W1, Canada Received 16 April 1991/Accepted 30 May 1991
The synthesis and 0 acetylation in vitro of peptidoglycan by Proteus mirabilis was studied in microorganisms made permeable to specifically radiolabelled nucleotide precursors by treatment with either diethyl ether or toluene. Optimum synthesis occurred with cells permeabilized by 1% (vol/vol) toluene in 30 mM MgCl2 in in vitro experiments with 50 mM Tris-HCl buffer (pH 6.80). Acetate recovered by mild base hydrolysis from sodium dodecyl sulfate-insoluble peptidoglycan synthesized in the presence of UDP-[acetyl-j-_4CjN-acetyl-Dglucosamine was found to be radioactive. Radioactivity was not retained by peptidoglycan synthesized when UDP-[acetyl-1-_4C]N-acetyl-D-glucosamine was replaced with both unlabelled nucleotide and either [acetyl3H1N-acetyl-D-glucosamine or [glucosamine-1,6-3HJN-acetyl-D-glucosamine. In addition, no radioactive acetate was detected in the mild base hydrolysates of peptidoglycan synthesized in vitro with UDP-[glucosamine-63HJN-acetyl-D-glucosamine as the radiolabel. Chasing UDP-[acetyl-1-_4C]N-acetyl-D-glucosamine with unlabelled material served to increase the yield of 0-linked ['4C]acetate, whereas penicillin G blocked both peptidoglycan synthesis and ['4C]acetate transfer. These results support the hypothesis that the base-labile 0-linked acetate is derived directly from N-acetylglucosamine incorporated into insoluble peptidoglycan via N-*O transacetylation and not from the catabolism of the supplemented peptidoglycan precursors followed by subsequent reactivation of acetate.
The peptidoglycan (PG) of Proteus mirabilis is similar in composition and structure to those of other members of the family Enterobacteriaceae, except for one important distinction, the presence of 0-linked acetyl groups. This modification to PG occurs at the C-6 hydroxyl group of N-acetylmuramyl residues, producing the corresponding 2,6diacetylmuramyl derivative. O-Acetylated PG is resistant to the hydrolytic activity of many lysozymes, including human ones (11 and references therein). Thus, following infection, large fragments of 0-acetylated PG persist and circulate in a host organism, enhancing the induction of the many pathobiological effects of PG (for a review, see reference 30), including rheumatoid arthritis (14). That a number of bacteria, including many pathogenic species (both gram-positive ones, e.g., Staphylococcus aureus [16, 31, 35], and gram-negative ones e.g., Neisseria gonorrhoeae [34] and P. mirabilis [11, 13]) possess 0-acetylated PG makes this an important phenomenon that ironically has received little attention. The PG of some species of bacteria has been reported to be 0 acetylated to the extent of up to 70%, so that it can confer both intrinsic resistance and complete resistance to lysozyme hydrolysis (4, 11, 20, 25, 27). Although 0-acetylated PG was first observed more than 30 years ago (1, 7) and the biological significance of this modification was discerned soon after (6), very little is known about the biosynthetic process involved in PG 0 acetylation. It is known to be stimulated either when cells are in the stationary phase of growth or under conditions which mimic the stationary phase (e.g., under the influence of chloramphenicol [28]). Other studies with P. mirabilis, N. gonorrhoeae, and S. aureus have suggested that PG 0
acetylation occurs within the PG sacculus and outside the cytoplasm (9, 17, 18, 21-23, 33). Indeed, PG 0 acetylation has also been demonstrated to continue in an in vitro P. mirabilis PG biosynthetic system (25), albeit to a lesser extent than that observed in vivo. Furthermore, searches for lipid(bactoprenyl)-linkedN-acetylglucosaminyl-N,O-diacetylmuramyl-pentapeptide precursors in either the cytoplasm or the cytoplasmic membrane have proved futile (22, 33). These observations thus suggest that both an acetyltransferase and a source of transferable acetate must be present outside the cytoplasm. Such conditions would preclude the utilization of the typical activated acetate precursors, such as acetyl coenzyme A or acetyl phosphate, since they are not transported out of the cytoplasm. Through in vivo labelling experiments with P. mirabilis, we recently provided evidence to suggest that acetate is transferred from the N-2 position of PG-bound N-acetylglucosaminyl or N-acetylmuramyl residues to the C6 hydroxyl group of the latter (12). The activity of the putative enzyme responsible for PG N-*O transacetylation was therefore proposed to be analogous to that of aromatic-hydroxylamine acetyltransferase (EC 2.3.1.56), which transfers the N-acetyl group of some aromatic acetohydroxamates to the 0 position of some aromatic hydroxylamines (32). This mechanism of PG 0 acetylation would negate the requirement for readily available activated acetate precursors (e.g., acetyl coenzyme A or acetyl phosphate) in the milieu outside the cytoplasm, while utilizing the conserved bond energies stored within the PG sacculus in a manner analogous to transpeptidation. The fact that free amino sugars were not detected in the PG sacculus but were observed in the spent culture medium as turnover products indicated that the processes of PG turnover and PG N-*O transacetylation are associated (12). In the present study, using an in vitro PG biosynthesis system supplemented with specifically radiolabelled precur-
* Corresponding author. t Present address: Department of Chemistry, Carlsberg Laboratorium, Gamle Carlsbergvej 10, Valby, Copenhagen DK 2500,
Denmark. 4618
VOL. 173, 1991
IN VITRO PEPTIDOGLYCAN SYNTHESIS AND 0 ACETYLATION
sors, we provide further evidence to support the proposal of PG N-+O transacetylation. Sodium dodecyl sulfate (SDS)insoluble PG was found to contain ester-linked [14C]acetate only when synthesized by both ether- and toluene-permeabilized cells of P. mirabilis in the presence of UDP-[acetyl-l"4CIN-acetylglucosamine (UDP-[acetyl-1-"4C]GlcNAc). The transfer of radiolabelled acetate was not observed in a biosynthetic system supplemented with both unlabelled nucleotide and either [acetyl-3H]GlcNAc or [glucosamine-1,6-
3H]GlcNAc.
MATERIALS AND METHODS Bacterial strains. P. mirabilis 19 was kindly provided by J. Gmeiner, Technische Hochschule, Darmstadt, Germany, and a stock laboratory strain was obtained from H. Perkins, Liverpool, United Kingdom. These strains have been extensively characterized (2, 24, 26). Bacillus subtilis W23 was provided by Terry Beveridge, Department of Microbiology, University of Guelph. All bacteria were maintained on nutrient agar slants at 40C. Growth conditions. P. mirabilis strains were cultivated in aerated nutrient broth supplemented with 5.0 g of NaCl, 4.5 g of Na2HPO4, 8.85 mg of GlcNAc, and 3.6 g of glucose per liter on a rotary shaker at 200 rpm and 370C. For the isolation of UDP-N-acetylmuramyl-pentapeptide (UDP-MurNAcpentapeptide), B. subtilis W23 was grown in a Casamino Acids-based medium (15) composed of, per liter, 10.0 g of Casamino Acids, 2.7 g of KH2PO4, 5.0 g of glucose, 0.42 g of MgSO4 7H20, 156 ,ug of sodium citrate dihydrate, and 1.0 ml of a trace metal solution [containing, per liter, 168 ,ug of Fe(NH4)2SO4- 6H20 and 100 ,ug each of (NH4)6Mo7024 4H20, CoCl2 6H20, CUSO4 5H20, MnCl2 .4H20, and ZnSO4 * 7H20] on a rotary shaker at 200 rpm and 37°C. Cell wall fragments. Cell wall fragments of P. mirabilis were prepared by the procedures of Tommassen and Lugtenburg (36) and Whitfield et al. (39). Bacteria grown in the presence of [acetyl-3H]GlcNAc (10 p.Ci/100 ml of culture) were harvested by centrifugation at 8,000 x g for 15 min at 4°C. The cell pellet, following a wash with 10 mM Tris-HCl buffer (pH 8.00) containing 10 puM ZnCl and 1 mM MgCl2, was resuspended in 10 mM Tris-HCl buffer (pH 8.00) containing 25% (wt/vol) sucrose and 5 mM EDTA. The EDTAsucrose-cell suspension was incubated for 15 min at an ambient temperature, and the cells were recovered by centrifugation (10,000 x g, 15 min, 4C). The cells were resuspended in 10 mM Tris-HCl buffer (pH 8.00) and disrupted by two passages through a French pressure cell (Aminco) at 20,000 lb/in2. Undisrupted cells were removed by centrifugation at 3,000 x g and 4°C for 20 min. Cell wall fragments were subsequently sedimented from the supernatant by ultracentrifugation (Beckman L8-55 ultracentrifuge) at 100,000 x g and 4°C for 30 min, washed once with 10 mM Tris-HCl buffer (pH 8.00), and resuspended in 20 mM Tris-HCl buffer (pH 6.80). Permeabilized cells. Cell cultures (160 ml) were grown to the mid-exponential to late exponential phase (A578 = 0.8) and harvested by centrifugation at 8,000 x g for 15 min at 4°C (Sorvall RC-2B centrifuge; DuPont Instruments). Permeabilized cells were prepared by treatment with either diethyl ether by the procedure of Vosberg and HoffmannBerlin (37) and Martin (24) or toluene by the procedure of Schrader and Fan (29). (i) Ether-treated cells. Cells were resuspended in 2.0 ml of basic medium and mixed with an equal volume of diethyl ether by gentle agitation for 1 to 2 min. The solution was
4619
allowed to separate into two phases, and the organic phase was removed and discarded. The aqueous cell suspension was diluted with half its volume of basic medium, layered on a 4.0-mi cushion of 800 mM sucrose, and centrifuged at 8,000 x g for 8 min at 4°C. The ether-treated cells from the pellet were resuspended in 40 mM Tris-HCl buffer (pH 6.8) containing 50 mM ammonium chloride, 20 mM magnesium chloride, and 500 ,uM ,B-mercaptoethanol. (ii) Toluene-treated cells. Cells were resuspended in a minimal volume of 30 mM magnesium chloride, and toluene was added to a final concentration of 1.0% (vol/vol). The cells were stirred vigorously for 30 min at room temperature, harvested by centrifugation at 14,900 x g for 5 min at 4°C, washed with 30 mM magnesium chloride containing 1.0% (vol/vol) toluene, and recentrifuged. The toluene-treated cells from the pellet were resuspended in 100 mM Tris-HCl buffer (pH 6.80) containing 30 mM magnesium chloride and used immediately. In vitro 0 acetylation of PG. Radiolabelled cell wall fragments were incubated in 20 mM Tris-HCI buffer (pH 6.80) containing 1.0 mM MgCl2 at room temperature for 16 h. Insoluble material was collected by ultracentrifugation at 160,000 x g and 4°C for 15 min in an Airfuge ultracentrifuge (Beckman Canada) and washed once with the suspension buffer prior to release and quantitation of 0-linked acetate content. Cell wall fragments heated to 100°C for 15 min were used as controls. In vitro PG synthesis and 0 acetylation. To test the activity of permeabilized cell preparations and optimize assay conditions, we incubated 1- to 2-mg protein equivalents of permeabilized cells in 50 mM Tris-HCl buffer (pH 6.80) containing 30 mM magnesium chloride with 160 nmol of UDP-MurNAc-pentapeptide and 65 nmol (5 ,Ci) of UDP[glucosamine-6-3H]GlcNAc in a total volume of 80 ,ul at 25°C for 30 min. The reactions were stopped by the addition of 80 ,ul of 10% (wtlvol) ice-cold trichloroacetic acid (TCA), and the reaction mixtures were kept at 0°C for a minimum of 1 h. The quenched mixtures were filtered through glass fiber membranes (GF/F; Whatman, Maidstone, United Kingdom), and the TCA precipitates were washed with 10 ml of ice-cold 5% (wt/vol) TCA. The membranes were dried at 60°C for 2 h prior to scintillation counting. Control experiments were done as described above, except that permeabilized cells were incubated at 100°C for 10 min prior to use. The buffers used to determine the optimum composition and pH were 50 mM Tris-maleate (pH 5.73 to 6.90) and 50 mM Tris-HCl (pH 6.80 to 7.20). A typical assay of in vitro PG synthesis and 0 acetylation involved mixing permeabilized cells (1.8 to 2.0 mg of protein) in 50 mM Tris-HCl (pH 6.80)-30 mM magnesium chloride with 160 nmol of UDP-MurNAc-pentapeptide and 62.5 nmol (638 nCi) of UDP-[acetyl-1-1'4C]GlcNAc in a total volume of 80 ,u. After incubation at 25°C for 1 h, the reactions were terminated by the addition of an equal volume of 8% (wt/vol) SDS at 100°C. The temperature was maintained at 60°C for 1 h, and the solutions were allowed to cool to an ambient temperature. SDS-insoluble PG was recovered by centrifugation at 160,000 x g for 15 min at 20°C (Airfuge), and the pellet was washed once with 0.1% (wt/vol) sodium azide. The amounts of radioactivity associated with 6-O-acetyl groups and SDS-insoluble PG were determined as described below. Heat-inactivated (100°C, 15 min), toluene-permeabilized cell wall preparations served as controls. In some experiments, 40 IU of penicillin G was added to the reaction mixtures prior to the addition of the radioactive nucleotide. In other experiments, UDP-[acetyl-1-1'4C]GlcNAc was re-
4620
DUPONT AND CLARKE
J. BACTERIOL.
TABLE 1. PG 0 acetylation in isolated cell wall fragments of P. mirabilis Perkins Radioactivity (%) released after incubation witha:
Radioactivity released as acetate after incubation withb:
Sample (h)
Base
Buffer
Native cell wall fragments 0 16
Heat-inctivated cell wall fragments, 16
NA 11.1 (3.90) 5.52 (2.50)
Base
Buffer (%
pmol . mg of PG-'
NA 56.2 (8.75)
0.00 0.00
18.9 28.2
8.71 13.0
27.6 (8.75)
0.00
13.5
6.23
Values represent the percentage of radioactivity released from cell wall fragments after incubation for 16 h in 20 mM Tris-HCI buffer (pH 6.80) (Buffer) and mild base hydrolysis for 6 h with 20 mM NaOH (Base), relative to the total amount of radioactivity associated with cell wall fragments. Values in parentheses represent standard deviations for five samples. NA, not applicable. b The supernatants of the buffer- or base-treated cell wall fragments were filtered through 0.45-,um-pore-size HA membrane filters, and the [3H]acetate contents were determined by HPLC on an Aminex HPX-87H organic acid column and scintillation counting (11). a
placed with either 65 nmol of UDP-[glucosamine-6-3H] GlcNAc or both 65 nmol of unlabelled UDP-GIcNAc and 2 nmol of either [acetyl-3H]GlcNAc or [glucosamine-1,63H]GlcNAc. Release and quantitation of acetate. Quantitation of radiolabelled 0-linked acetate was achieved by mild base hydrolysis of SDS-insoluble PG (19) in 20 mM NaOH and subsequent analysis of hydrolysates by high-pressure liquid chromatography (HPLC) and liquid scintillation counting as previously described (11, 12). In brief, the mild base-treated PG was collected by centrifugation at 160,000 x g for 15 min at 20°C with an Airfuge, and the pellets were washed once by centrifugation with 200 ,ul of 0.1% sodium azide. The supernatants were pooled, filtered through 0.45-p.m-pore-size HA membrane filters (Millipore Ltd., Mississauga, Ontario, Canada), and injected onto an Aminex HPX-87H (Bio-Rad) organic acid column (7.8 by 300 mm). Fractions (0.5 ml) of the column effluent were collected and counted for levels of radioactivity by liquid scintillation counting. The PG recovered from the pellets of the Airfuge centrifugation was solubilized by treatment with 5 to 10 U of mutanolysin in 10 mM Tris-HCl (pH 6.80) containing 8 mM MgCl2 at 37°C with gentle agitation for 2 h prior to the quantitation of the radioactivity remaining bound. Analytical methods. PG concentrations were determined by amino acid analysis with a Beckman System Gold Amino Acid Analyzer with postcolumn ninhydrin detection. Samples of PG (0.15 mg) were hydrolyzed in vacuo with 4 M HCl at 110°C for 16 h. Acetate quantitation was performed by HPLC with a Beckman system as previously described (11). Measurements of radioactivity were made with a Tri-Carb 2000 liquid scintillation counter (Canberra-Packard Canada, Mississauga, Ontario, Canada) with Ecolume (ICN Biomedicals Canada, Ltd., Montreal, Quebec, Canada) or Liquiscint (National Diagnostics, Manville, N.J.) as the scintillation cocktail. Enzymes and biochemicals. GlcNAc, mutanolysin, and SDS were purchased from Sigma Chemical Co., St. Louis, Mo. Boehringer Mannheim Canada, Laval, Quebec, Canada, supplied pronase, while penicillin G (potassium salt) was a product of Ayerst Laboratories. Ecolume, [acetyl3H]GlcNAc (specific activity, 10 Ci. mmol-1; lots 3305141, 3609124, and 4021162), and UDP-[acetyl-1-14C]GlcNAc (specific activity, 10.2 mCi. mmol-1; lot 3719155) were purchased from ICN Biomedicals, while UDP-[glucosamine6_3H]GlcNAc (specific activity, 26.8 Ci mmol-1; lot 2474-
060) and [glucosamine-1,6-3H]GlcNAc (specific activity, 34.2 Ci- mmol-1; lot 2482-084) were provided by New England Nuclear Research Products, DuPont Canada, Dorval, Quebec, Canada. UDP-MurNAc-pentapeptide was isolated from the supernatant of TCA-treated B. subtilis W23 by gel filtration chromatography by the procedure of Garrett (15). RESULTS In vitro 0 acetylation. Radiolabelled cell wall fragments were prepared from P. mirabilis Perkins by culturing of the cells in the presence of 40 ,uM [acetyl-3H]GlcNAc prior to their disruption in a French pressure cell. The amount of 0-linked [3H]acetate associated with the pelleted insoluble material of the cell wall preparations was calculated as the difference between the amounts liberated by incubation in 20 mM NaOH versus 20 mM Tris-HCI buffer (pH 6.80) which, under these conditions of culturing and preparation, was 18.9 pmol of [3H]acetate mg of PG-1. To investigate whether these cell wall fragments were capable of continuing the process of PG 0 acetylation, we incubated 225-p.l samples for 16 h in 20 mM Tris-HCl buffer (pH 6.80); other samples that were heated to 100°C for 15 min prior to incubation served as controls. The insoluble material was recovered by ultracentrifugation, and the amounts of radioactivity and acetate released both during the 16-h incubation at a neutral pH and from mild base-hydrolyzed PG were determined. A low level of radioactivity was released from both the native insoluble fragments and the boiled control fragments following 16 h of incubation at pH 6.8 (Table 1). However, none of this released radioactivity was determined by HPLC analysis to be acetate (Fig. la and c); it was probably associated with PG fragments released by the action of indigenous heat-stable autolysins. Mild base hydrolysis of the boiled control fragments resulted in the release of 28% of the total initial radioactivity, 23% of which (representing 6.2% of the total) was identified as acetate (Fig. lb). This base-labile [3H]acetate represented that transferred to the C-6 of muramyl residues prior to the isolation of the cell wall fragments from the viable whole cells (Table 1). In contrast, approximately twice as much radioactivity and acetate was detected in the native cell wall fragments treated in a similar manner (Table 1 and Fig. ld), suggesting that [3H]acetate preincorporated as [acetyl-3H]GlcNAc continued to undergo transfer within the isolated PG cell wall
VOL. 173, 1991
IN VITRO PEPTIDOGLYCAN SYNTHESIS AND 0 ACETYLATION
a
b
E
4621
9.0
0
-Y C
EI a
1._
-60
// 0
/
0
8.0p
ct 0
0I1 c
d
C)
0~~ 0
/
7.0 .I/ .....,..,..
z
I
I
0
I
E
S
cLi I
0
5
10 15 20 25 0
Time
5
10
6.0L
i.5 6.0
15 20 25
FIG. 1. HPLC analysis of [3H]acetate released from the in vitro acetylation of P. mirabilis cell wall fragments. Insoluble cell wall fragments (c) and heat-inactivated (100°C, 15 min) control fragments (a) were incubated in 20 mM Tris-HCl buffer (pH 6.80) containing 1.0 mM MgCl2 and 0.1% NaN3 at room temperature for 16 h, and the [3H]acetate released was separated by HPLC. Fractions (0.5 ml) were collected and counted for radioactivity. The recovered insoluble cell wall fragments of the native preparation (d) and the heat-inactivated control preparation (b) were hydrolyzed with 20 mM NaOH (room temperature, 3 h) to release 0-linked [3H]acetate. The retention time for acetic acid is 19.4 min. The solid bars represent 100 cpm and 0.005 absorbance unit.
fragments upon prolonged incubation. The difference in recovered [3H]acetate between the native cell wall fragments at zero time and the heat-inactivated cell wall fragments following 16 h of incubation in Tris-HCl buffer was again likely caused by the loss of insoluble PG because of the action of heat-stable autolysins. In vitro PG synthesis and 0 acetylation. Initial experiments pertaining to the in vitro biosynthesis and 0 acetylation of P. mirabilis PG were done with a protocol involving etherpermeabilized cells (37), adopted by Martin (24) and Brown and Perkins (5) for the cell-free biosynthesis of P. mirabilis PG and N. gonorrhoeae PG, respectively. An average of 62.1 pmol of radiolabel per mg of protein was incorporated into the SDS-insoluble PG fraction of the permeabilized cells, representing an overall efficiency of 0.47%. This low efficiency relative to that previously reported (5, 24), coupled with problems of enzyme stability, prompted the development of an alternative method with toluene-permeabilized
cells. The incorporation of UDP-[glucosamine-6-3H]GlcNAc into TCA-insoluble material isolated from different preparations of toluene-permabilized P. mirabilis cells was monitored to determine the optimum composition of the extraction mixture. The use of 1% (vol/vol) toluene in (i) 100 mM Tris-HCl buffer (pH 6.80), (ii) 100 mM Tris-HCl buffer (pH
70
7.5
pH
(min)
0
6.5
FIG. 2. Dependence on pH of the ability of toluene-permeabilized cells to catalyze the in vitro biosynthesis of P. mirabilis PG. Toluene-permeabilized cells were incubated for 30 min with UDP[glucosamine-6-3H]GlcNAc and UDP-MurNAc-pentapeptide in 50 mM Tris-maleate buffer (pH 5.73 to 6.90) (0) and 50 mM Tris-HCI buffer (pH 6.80 to 7.20) (0). TCA-insoluble material was collected by filtration on GF/F glass fiber membranes and counted for
radioactivity.
6.80) containing 30 mM MgCl2, or (iii) distilled water resulted in permeabilized cells efficient in PG biosynthesis at levels of only 78, 53, and 26%, respectively, compared with cells permeabilized with 1% (vol/vol) toluene in 30 mM
MgCl2. Increasing the concentrations of toluene to 10 and 25% (vol/vol) had deleterious effects, resulting in only 35 and 30% efficiencies, respectively. The optimum composition and optimum pH of the reaction buffer were also determined in a similar manner. While no striking differences were observed between the two buffers investigated (Fig. 2), as previously observed by Brown and Perkins (5) for N. gonorrhoeae, acute maximum activity was observed around pH 6.80. On the basis of the results of these preliminary studies, a protocol of permeabilizing P. mirabilis cells with 1% (vol/vol) toluene in 30 mM MgCl2 and conducting in vitro PG biosynthesis experiments in Tris-HCl buffer (pH 6.80) was adopted for further inves-
tigations.
Studies of PG 0 acetylation were continued by monitoring the migration of [14C]acetate from UDP-[acetyl-114C]GlcNAc in PG synthesized in vitro with toluene-permeabilized cells of P. mirabilis 19. The synthesized material was subjected to hot SDS extraction, and the isolated [14c]PG was examined for migration of the radiolabel. The results of a representative experiment are presented in Table 2. Incorporation of 101 pmol of [14C]acetate mg of pro-
tein-1 into SDS-insoluble PG was achieved after 30 min, representing an increase of 67% over that in ether-treated cell preparations. Mild base hydrolysis of radiolabelled SDS-insoluble PG resulted in the recovery of 21% more
4622
DUPONT AND CLARKE
J. BACTERIOL.
TABLE 2. Transfer of ["4C]acetate from UDP-[acetyl-1-'4C]GlcNAc to muramyl residues in an in vitro biosynthesis system for the 0-acetylated PG of P. mirabilis In vitro biosynthetic system (toluenepermeabilized cells)
UDP-[acetyl-1-14CIGlcNAc incorporated (pmol mg of
protein-') 101 117 19.4 1.46
Cells alone Cells plus chaseb Cell plus Penicillin GC Heat inactivated cellsd
% of radioactivity released from SDS-insoluble materiala Total after incubation with: Buffer
Base
30.9 14.8 38.9 80.5
52.2 43.5 41.0 80.3
Difference
As base-labile acetate
21.3 28.7 2.11 0.00
22.3 28.5 1.87 0.00
Percentages of released radioactivity and acetate were determined as described in Table 1, footnotes a and b. Following the 30-min incubation with [acetate-1-14C]GlcNAc, the reaction mixture was adjusted to contain 130 nM UDP-GlcNAc and incubated for a further 30 min before being quenched with SDS. C Penicillin G (40 IU) was added to toluene-permeabilized cells 5 min prior to the addition of UDP-[acetate-1-14CJGIcNAc. d 100oC, 15 a
b
mim.
radioactivity than in buffer-treated material, and all of this radioactivity was determined to be [14C]acetate (Table 2). Chasing the UDP-[acetyl-1-`4C]GlcNAc initially taken up by the in vitro system in the first 30 min with the addition of 130 nM UDP-GlcNAc and an extra 30 min of incubation appeared to increase the amount of ['4C]acetate released from SDS-insoluble PG by mild base hydrolysis. Control incubations conducted with boiled (100°C, 15 min) permeabilized cells did not lead to incorporation of the radiolabelled nucleotide (less than 1.5 pmol. mg of protein-1). Moreover, no incorporation of radioactivity (less than 0.5 pmol mg of protein-1) was observed when UDP-[acetyl1-`4C]GlcNAc was replaced with both unlabelled UDPGlcNAc and either [acetyl-3H]GlcNAc or [glucosamine-1,63H]GlcNAc. Finally, no radioactive acetate was detected in the mild base hydrolysates of PG synthesized in vitro when UDP-[acetyl-1-`4C]GlcNAc was replaced with UDP-[glucosamine-6-3H]GlcNAc. These latter negative results indicate that the transferred (0-linked) acetate is not derived from the catabolism of supplemented PG precursors but requires the prior incorporation of [acetyl-1-'4CIGlcNAc into PG via the nucleotide. This inference is further supported by the results obtained with the in vitro peptidoglycan biosynthetic system supplemented with penicillin G. With the inclusion of 40 IU of penicillin G in the reaction mixtures, less than 20% of the UDP-[acetate-14C]GlcNAc was taken up by the toluenepermeabilized cells, compared with untreated cells (Table 2). Analysis of the SDS-insoluble PG isolated indicated that no [14Clacetate was released by mild base hydrolysis from the inhibited biosynthesis product. excess
DISCUSSION We have presented evidence to both confirm that 0 acetylation of PG is indeed a process associated with membrane functions outside the cytoplasm and support the hypothesis that PG 0 acetylation occurs via N--0 transacetylation (12). Radioactive acetate was found in a base-labile position within SDS-insoluble PG that had been synthesized in vitro when the acetate was introduced only as UDP-bound [acetyl-1-14C]GlcNAc. It is highly unlikely that this precursor molecule was catabolized within the membrane to liberate [14C]acetate, which was subsequently activated and transferred to PG, because no base-labile radiolabel was detected in PG synthesized by the in vitro system supplemented with
either [acetyl-3H]GIcNAc or [glucosamine-1,6-3H]GlcNAc. In addition, penicillin G inhibited the transfer of ["4C]acetate from the radioactive nucleotide to peptidoglycan. This antibiotic blocks many of the maturation reactions of PG biosynthesis, including transglycosylation and transpeptidation. Hence, the presence of penicillin G in the reaction mixture would cause UDP-[acetyl-1-`4C]GlcNAc derivatives to accumulate in the membrane, and the lack of [14C]acetate transfer implies not only that catabolic activity is absent from the cell wall preparations but also that radioactive GlcNAc derivatives have to be incorporated into PG prior to 0 acetylation. This latter point is further supported by (i) the pulse-chase experiment, in which an increase in the incorporation of the precursor and a concomitant decrease in the amounts of loosely associated (buffer-labile) radiolabel served to elevate the levels of 0-linked ['4C]acetate associated with the synthesized SDS-insoluble PG, and (ii) the significant increase in base-released [3H]acetate observed when cell wall fragments prelabelled with [acetate-3H]GlcNAc were allowed to continue PG biosynthesis and maturation. The degree of 0 acetylation of muramyl residues in PG synthesized in vitro is lower than that observed in living microorganisms (40.2 and 52.8% for P. mirabilis Perkins and 19, respectively [11]). Similar observations were reported by Martin concerning an analogous in vitro biosynthetic system of P. mirabilis 19 PG (24). The extent of 0 acetylation of the synthesized PG was described as resembling the decreased state typically observed in PG of L-form spheroplasts grown in the presence of penicillin. Unfortunately, the specific degrees of PG 0 acetylation were not provided, making a direct comparison with the present data impossible. A thinlayer chromatography analysis of the synthesized PG after hydrolysis by an endo-N,O-diacetylmuramidase from a Chalaropsis sp. did, however, indicate the presence of 0-acetylated monomer and mono- and di-O-acetylated dimer fragments of PG. Taken together, these observations support the previous conclusions that the 0 acetylation of PG is a maturation process (9, 17, 18, 21-23, 33) and that N-linked acetate is transferred to the C-6 position of muramyl residues (12). For N-*O transacetylation to occur within the cell-free system, the autolysins documented to be present (3, 8) would have to be catalytically active since, according to the current hypothesis (12), PG turnover is associated with the process of 0 acetylation. The fact that both PG autolytic and N-0O transacetylase activities were retained in the cell-free system
VOL. 173, 1991
IN VITRO PEPTIDOGLYCAN SYNTHESIS AND 0 ACETYLATION
led to the speculation that, by analogy with some of the penicillin-binding proteins of Escherichia coli, the two enzymatic activities are catalyzed by a single bifunctional enzyme (10). Indeed, three of the high-molecular-weight penicillin-binding proteins, la, lbs, and 3, are bifunctional transglycosidase-transpeptidase enzymes (for a review, see reference 38). Obviously, much more intensive investigation will be required to bear out this postulate. Nevertheless, this study has provided a basis on which to develop an assay for the PG N-*O transacetylase which may be subsequently used to isolate and characterize this interesting and potentially important enzyme. ACKNOWLEDGMENTS This study was supported by an operating grant to A.J.C. from the Medical Research Council and a Natural Sciences and Engineering Research Council postgraduate scholarship to C.D. REFERENCES 1. Abrams, A. 1958. O-Acetyl groups in the cell wall of Streptococcus faecalis. J. Biol. Chem. 230:949-959. 2. Blundeli, J. K., and H. R. Perkins. 1981. Effects of P-lactam antibiotics on peptidoglycan synthesis in growing Neisseria gonorrhoeae, including changes in the degree of O-acetylation. J. Bacteriol. 147:633-641. 3. Blundeli, J. K., and H. R. Perkins. 1985. Selectivity for O-acetylated peptidoglycan during endopeptidase action by permeabilized Neisseria gonorrhoeae. FEMS Microbiol. Lett. 30:6769. 4. Blundell, J. K., G. J. Smith, and H. R. Perkins. 1980. The peptidoglycan of Neisseria gonorrhoeae: O-acetyl groups and lysozyme sensitivity. FEMS Microbiol. Lett. 9:259-261. 5. Brown, C. A., and H. R. Perkins. 1979. In vitro synthesis of peptidoglycan by 13-lactam-sensitive and -resistant strains of Neisseria gonorrhoeae: effects of ,B-lactam and other antibiot-
Antimicrob. Agents Chemother. 16:28-36. Brumfitt, W. 1959. The mechanism of development of resistance to lysozyme by some Gram-positive bacteria and its results. Br. J. Exp. Pathol. 40:441-451. 7. Brumfitt, W., A. C. Wardlaw, and J. T. Park. 1958. Development of lysozyme resistance in Micrococcus lysodeikticus and its association with an increased O-acetyl content of the cell wall. Nature (London) 181:1783-1784. 8. Chapman, S. J., and H. R. Perkins. 1983. Peptidoglycandegrading enzymes in ether-treated cells of Neisseria gonorics.
6.
9. 10. 11.
12. 13.
14.
15. 16.
rhoeae. J. Gen. Microbiol. 129:877-883. Dougherty, T. J. 1983. Synthesis and modification of the peptidoglycan in Neisseria gonorrhoeae. FEMS Microbiol. Lett. 17:51-53. Dupont, C. 1991. Ph.D. thesis. University of Guelph, Guelph, Ontario, Canada. Dupont, C., and A. J. Clarke. 1991. Dependence of lysozymecatalysed solubilization of Proteus mirabilis peptidoglycan on the extent of O-acetylation. Eur. J. Biochem. 195:763-769. Dupont, C., and A. J. Clarke. 1991. Evidence for N- O acetyl migration as the mechanism for 0-acetylation of peptidoglycan in Proteus mirabilis. J. Bacteriol. 173:4318-4324. Fleck, J., M. Mock, R. Minck, and J. M. Ghuysen. 1971. The cell envelope of P. vulgaris P18. Isolation and characterization of the peptidoglycan component. Biochim. Biophys. Acta 233: 489-503.
Fleming, T. J., D. E. Walismith, and R. S. Rosenthal. 1986. Arthropathic properties of gonococcal peptidoglycan fragments: implications for the pathogenesis of disseminated gonococcal disease. Infect. Immun. 52:600-608. Garrett, A. J. 1969. The effect of magnesium ion deprivation on the synthesis of mucopeptide and its precursors in Bacillus subtilis. Biochem. J. 115:419-430. Ghuysen, J. M., and J. L. Strominger. 1963. Structure of the cell wall of S. aureus strain Copenhagen. II. Separation and structure of disaccharides. Biochemistry 2:1119-1125.
4623
17. Gmeiner, J., and H. P. Kroll. 1981. Murein biosynthesis and O-acetylation of N-acetylmuramic acid during the cell division cycle of Proteus mirabilis. Eur. J. Biochem. 177:171-177. 18. Gmeiner, J., and E. Sarnow. 1987. Murein biosynthesis in synchronized cells of Proteus mirabilis. Quantitative analysis of O-acetylated murein subunits and of chain terminators incorporated into the sacculus during the cell cycle. Eur. J. Biochem. 163:389-395. 19. Hoyle, B. D., and T. Beveridge. 1983. Metal binding by the peptidoglycan sacculus of Escherichia coli K-12. Can. J. Microbiol. 30:204-211. 20. Johannsen, L., H. Labischinski, P. Burghaus, and P. Giesbrecht. 1983. Acetylation in different phases of growth of staphylococci and their relation to cell wall degradability by lysozyme, p. 261-266. In R. Hakenbeck, J. Holtje, and H. Labischinski (ed.), The target of penicillin. International FEMS symposium on the murein sacculus of bacterial cell walls. Walter de Gruyter & Co., Berlin. 21. Lear, A. L., and H. R. Perkins. 1983. Degrees of O-acetylation and cross-linking of the peptidoglycan of Neisseria gonorrhoeae during growth. J. Gen. Microbiol. 129:885-888. 22. Lear, A. L., and H. R. Perkins. 1986. O-Acetylation of peptidoglycan in Neisseria gonorrhoeae. Investigation of lipid-linked intermediates and glycan chains newly incorporated into the cell wall. J. Gen. Microbiol. 132:2413-2420. 23. Lear, A. L., and H. R. Perkins. 1987. Progress of O-acetylation and cross-linking of peptidoglycan in Neisseria gonorrhoeae grown in the presence of penicillin. J. Gen. Microbiol. 133:17431750. 24. Martin, H. H. 1984. In vitro synthesis of peptidoglycan by spheroplasts of Proteus mirabilis grown in the presence of penicillin. Arch. Microbiol. 139:371-375. 25. Martin, H. H., and J. Gmeiner. 1979. Modification of peptidoglycan structure by penicillin-action in cell walls of Proteus mirabilis. Eur. J. Biochem. 95:487-495. 26. Martin, H. H., W. Schilf, and P. Gruss. 1975. D-Alanyl-Dalanine carboxypeptidase in the bacterial form and L-form of Proteus mirabilis. Eur. J. Biochem. 55:465-473. 27. Rosenthal, R. S., W. J. Folkening, D. R. Miller, and S. G. Swim. 1983. Resistance of O-acetylated gonococcal peptidoglycan to human peptidoglycan-degrading enzymes. Infect. Immun. 40: 903-911. 28. Rosenthal, R. S., M. A. Gfell, and W. J. Folkening. 1985. Influence of protein synthesis inhibitors on regulation of extent of O-acetylation of gonococcal peptidoglycan. Infect. Immun. 49:7-13. 29. Schrader, W. P., and D. P. Fan. 1974. Synthesis of cross-linked peptidoglycan attached to previously formed cell wall by toluene-treated cells of Bacillus megaterium. J. Biol. Chem. 249: 4815-4818. 30. Seidl, P. H., and K. H. Schleifer (ed.). 1985. Biological properties of peptidoglycan. Walter de Gruyter & Co., New York. 31. Sidow, T., L. Johannsen, and H. Labischinski. 1990. Penicillininduced changes in the cell wall composition of Staphylococcus aureus before the onset of bacteriolysis. Arch. Microbiol. 154:73-81. 32. Smith, T. J., and P. E. Hanna. 1986. N-Acetyltransferase multiplicity and the bioactivation of N-acrylhydroxamic acids by hamster hepatic and intestinal enzymes. Carcinogenesis 7:697-702. 33. Snowden, M. A., H. R. Perkins, A. W. Wyke, M. V. Hayes, and J. B. Ward. 1989. Cross-linking and O-acetylation of newly synthesized peptidoglycan in Staphylococcus aureus H. J. Gen. Microbiol. 135:3015-3022. 34. Swim, S. C., M. A. Gfell, C. E. Wilde III, and R. S. Rosenthal. 1983. Strain distribution in extents of lysozyme resistance and O-acetylation of gonococcal peptidoglycan determined by highperformance liquid chromatography. Infect. Immun. 42:446 452. 35. Tipper, D. J., J. M. Ghuysen, and J. L. Strominger. 1965. Structure of cell wall of Staphylococcus aureus strain Copenhagen. III. Further studies of the disaccharides. Biochemistry 4:468-473. 36. Tommassen, J., and B. Lugtenberg. 1980. Outer membrane
4624
DUPONT AND CLARKE
protein of Escherichia coli K-12 is coregulated with alkaline phosphatase. J. Bacteriol. 143:151-157. 37. Vosberg, H.-P., and H. Hoffmann-Berlin. 1971. DNA synthesis in nucleotide-permeable Escherichia coli cells. I. Preparation and properties of ether-treated cells. J. Mol. Biol. 58:739-753. 38. Waxman, D. J., and J. L. Strominger. 1983. Penicillin-binding
J. BACTERIOL.
proteins and the mechanism of action of P-lactam antibiotics. Annu. Rev. Biochem. 52:825-869. 39. Whitfield, C., R. E. W. Hancock, and J. W. Costerton. 1983. Outer membrane protein K of Escherichia coli: purification and pore-forming properties in lipid bilayer membranes. J. Bacteriol. 156:873-879.
Vol. 173, No. 15
In Vitro Synthesis and 0 Acetylation of Peptidoglycan by Permeabilized Cells of Proteus mirabilis CLAUDE DUPONTt AND ANTHONY J. CLARKE* Guelph- Waterloo Centre for Graduate Work in Chemistry, Department of Microbiology, University of Guelph, Guelph, Ontario NJG 2W1, Canada Received 16 April 1991/Accepted 30 May 1991
The synthesis and 0 acetylation in vitro of peptidoglycan by Proteus mirabilis was studied in microorganisms made permeable to specifically radiolabelled nucleotide precursors by treatment with either diethyl ether or toluene. Optimum synthesis occurred with cells permeabilized by 1% (vol/vol) toluene in 30 mM MgCl2 in in vitro experiments with 50 mM Tris-HCl buffer (pH 6.80). Acetate recovered by mild base hydrolysis from sodium dodecyl sulfate-insoluble peptidoglycan synthesized in the presence of UDP-[acetyl-j-_4CjN-acetyl-Dglucosamine was found to be radioactive. Radioactivity was not retained by peptidoglycan synthesized when UDP-[acetyl-1-_4C]N-acetyl-D-glucosamine was replaced with both unlabelled nucleotide and either [acetyl3H1N-acetyl-D-glucosamine or [glucosamine-1,6-3HJN-acetyl-D-glucosamine. In addition, no radioactive acetate was detected in the mild base hydrolysates of peptidoglycan synthesized in vitro with UDP-[glucosamine-63HJN-acetyl-D-glucosamine as the radiolabel. Chasing UDP-[acetyl-1-_4C]N-acetyl-D-glucosamine with unlabelled material served to increase the yield of 0-linked ['4C]acetate, whereas penicillin G blocked both peptidoglycan synthesis and ['4C]acetate transfer. These results support the hypothesis that the base-labile 0-linked acetate is derived directly from N-acetylglucosamine incorporated into insoluble peptidoglycan via N-*O transacetylation and not from the catabolism of the supplemented peptidoglycan precursors followed by subsequent reactivation of acetate.
The peptidoglycan (PG) of Proteus mirabilis is similar in composition and structure to those of other members of the family Enterobacteriaceae, except for one important distinction, the presence of 0-linked acetyl groups. This modification to PG occurs at the C-6 hydroxyl group of N-acetylmuramyl residues, producing the corresponding 2,6diacetylmuramyl derivative. O-Acetylated PG is resistant to the hydrolytic activity of many lysozymes, including human ones (11 and references therein). Thus, following infection, large fragments of 0-acetylated PG persist and circulate in a host organism, enhancing the induction of the many pathobiological effects of PG (for a review, see reference 30), including rheumatoid arthritis (14). That a number of bacteria, including many pathogenic species (both gram-positive ones, e.g., Staphylococcus aureus [16, 31, 35], and gram-negative ones e.g., Neisseria gonorrhoeae [34] and P. mirabilis [11, 13]) possess 0-acetylated PG makes this an important phenomenon that ironically has received little attention. The PG of some species of bacteria has been reported to be 0 acetylated to the extent of up to 70%, so that it can confer both intrinsic resistance and complete resistance to lysozyme hydrolysis (4, 11, 20, 25, 27). Although 0-acetylated PG was first observed more than 30 years ago (1, 7) and the biological significance of this modification was discerned soon after (6), very little is known about the biosynthetic process involved in PG 0 acetylation. It is known to be stimulated either when cells are in the stationary phase of growth or under conditions which mimic the stationary phase (e.g., under the influence of chloramphenicol [28]). Other studies with P. mirabilis, N. gonorrhoeae, and S. aureus have suggested that PG 0
acetylation occurs within the PG sacculus and outside the cytoplasm (9, 17, 18, 21-23, 33). Indeed, PG 0 acetylation has also been demonstrated to continue in an in vitro P. mirabilis PG biosynthetic system (25), albeit to a lesser extent than that observed in vivo. Furthermore, searches for lipid(bactoprenyl)-linkedN-acetylglucosaminyl-N,O-diacetylmuramyl-pentapeptide precursors in either the cytoplasm or the cytoplasmic membrane have proved futile (22, 33). These observations thus suggest that both an acetyltransferase and a source of transferable acetate must be present outside the cytoplasm. Such conditions would preclude the utilization of the typical activated acetate precursors, such as acetyl coenzyme A or acetyl phosphate, since they are not transported out of the cytoplasm. Through in vivo labelling experiments with P. mirabilis, we recently provided evidence to suggest that acetate is transferred from the N-2 position of PG-bound N-acetylglucosaminyl or N-acetylmuramyl residues to the C6 hydroxyl group of the latter (12). The activity of the putative enzyme responsible for PG N-*O transacetylation was therefore proposed to be analogous to that of aromatic-hydroxylamine acetyltransferase (EC 2.3.1.56), which transfers the N-acetyl group of some aromatic acetohydroxamates to the 0 position of some aromatic hydroxylamines (32). This mechanism of PG 0 acetylation would negate the requirement for readily available activated acetate precursors (e.g., acetyl coenzyme A or acetyl phosphate) in the milieu outside the cytoplasm, while utilizing the conserved bond energies stored within the PG sacculus in a manner analogous to transpeptidation. The fact that free amino sugars were not detected in the PG sacculus but were observed in the spent culture medium as turnover products indicated that the processes of PG turnover and PG N-*O transacetylation are associated (12). In the present study, using an in vitro PG biosynthesis system supplemented with specifically radiolabelled precur-
* Corresponding author. t Present address: Department of Chemistry, Carlsberg Laboratorium, Gamle Carlsbergvej 10, Valby, Copenhagen DK 2500,
Denmark. 4618
VOL. 173, 1991
IN VITRO PEPTIDOGLYCAN SYNTHESIS AND 0 ACETYLATION
sors, we provide further evidence to support the proposal of PG N-+O transacetylation. Sodium dodecyl sulfate (SDS)insoluble PG was found to contain ester-linked [14C]acetate only when synthesized by both ether- and toluene-permeabilized cells of P. mirabilis in the presence of UDP-[acetyl-l"4CIN-acetylglucosamine (UDP-[acetyl-1-"4C]GlcNAc). The transfer of radiolabelled acetate was not observed in a biosynthetic system supplemented with both unlabelled nucleotide and either [acetyl-3H]GlcNAc or [glucosamine-1,6-
3H]GlcNAc.
MATERIALS AND METHODS Bacterial strains. P. mirabilis 19 was kindly provided by J. Gmeiner, Technische Hochschule, Darmstadt, Germany, and a stock laboratory strain was obtained from H. Perkins, Liverpool, United Kingdom. These strains have been extensively characterized (2, 24, 26). Bacillus subtilis W23 was provided by Terry Beveridge, Department of Microbiology, University of Guelph. All bacteria were maintained on nutrient agar slants at 40C. Growth conditions. P. mirabilis strains were cultivated in aerated nutrient broth supplemented with 5.0 g of NaCl, 4.5 g of Na2HPO4, 8.85 mg of GlcNAc, and 3.6 g of glucose per liter on a rotary shaker at 200 rpm and 370C. For the isolation of UDP-N-acetylmuramyl-pentapeptide (UDP-MurNAcpentapeptide), B. subtilis W23 was grown in a Casamino Acids-based medium (15) composed of, per liter, 10.0 g of Casamino Acids, 2.7 g of KH2PO4, 5.0 g of glucose, 0.42 g of MgSO4 7H20, 156 ,ug of sodium citrate dihydrate, and 1.0 ml of a trace metal solution [containing, per liter, 168 ,ug of Fe(NH4)2SO4- 6H20 and 100 ,ug each of (NH4)6Mo7024 4H20, CoCl2 6H20, CUSO4 5H20, MnCl2 .4H20, and ZnSO4 * 7H20] on a rotary shaker at 200 rpm and 37°C. Cell wall fragments. Cell wall fragments of P. mirabilis were prepared by the procedures of Tommassen and Lugtenburg (36) and Whitfield et al. (39). Bacteria grown in the presence of [acetyl-3H]GlcNAc (10 p.Ci/100 ml of culture) were harvested by centrifugation at 8,000 x g for 15 min at 4°C. The cell pellet, following a wash with 10 mM Tris-HCl buffer (pH 8.00) containing 10 puM ZnCl and 1 mM MgCl2, was resuspended in 10 mM Tris-HCl buffer (pH 8.00) containing 25% (wt/vol) sucrose and 5 mM EDTA. The EDTAsucrose-cell suspension was incubated for 15 min at an ambient temperature, and the cells were recovered by centrifugation (10,000 x g, 15 min, 4C). The cells were resuspended in 10 mM Tris-HCl buffer (pH 8.00) and disrupted by two passages through a French pressure cell (Aminco) at 20,000 lb/in2. Undisrupted cells were removed by centrifugation at 3,000 x g and 4°C for 20 min. Cell wall fragments were subsequently sedimented from the supernatant by ultracentrifugation (Beckman L8-55 ultracentrifuge) at 100,000 x g and 4°C for 30 min, washed once with 10 mM Tris-HCl buffer (pH 8.00), and resuspended in 20 mM Tris-HCl buffer (pH 6.80). Permeabilized cells. Cell cultures (160 ml) were grown to the mid-exponential to late exponential phase (A578 = 0.8) and harvested by centrifugation at 8,000 x g for 15 min at 4°C (Sorvall RC-2B centrifuge; DuPont Instruments). Permeabilized cells were prepared by treatment with either diethyl ether by the procedure of Vosberg and HoffmannBerlin (37) and Martin (24) or toluene by the procedure of Schrader and Fan (29). (i) Ether-treated cells. Cells were resuspended in 2.0 ml of basic medium and mixed with an equal volume of diethyl ether by gentle agitation for 1 to 2 min. The solution was
4619
allowed to separate into two phases, and the organic phase was removed and discarded. The aqueous cell suspension was diluted with half its volume of basic medium, layered on a 4.0-mi cushion of 800 mM sucrose, and centrifuged at 8,000 x g for 8 min at 4°C. The ether-treated cells from the pellet were resuspended in 40 mM Tris-HCl buffer (pH 6.8) containing 50 mM ammonium chloride, 20 mM magnesium chloride, and 500 ,uM ,B-mercaptoethanol. (ii) Toluene-treated cells. Cells were resuspended in a minimal volume of 30 mM magnesium chloride, and toluene was added to a final concentration of 1.0% (vol/vol). The cells were stirred vigorously for 30 min at room temperature, harvested by centrifugation at 14,900 x g for 5 min at 4°C, washed with 30 mM magnesium chloride containing 1.0% (vol/vol) toluene, and recentrifuged. The toluene-treated cells from the pellet were resuspended in 100 mM Tris-HCl buffer (pH 6.80) containing 30 mM magnesium chloride and used immediately. In vitro 0 acetylation of PG. Radiolabelled cell wall fragments were incubated in 20 mM Tris-HCI buffer (pH 6.80) containing 1.0 mM MgCl2 at room temperature for 16 h. Insoluble material was collected by ultracentrifugation at 160,000 x g and 4°C for 15 min in an Airfuge ultracentrifuge (Beckman Canada) and washed once with the suspension buffer prior to release and quantitation of 0-linked acetate content. Cell wall fragments heated to 100°C for 15 min were used as controls. In vitro PG synthesis and 0 acetylation. To test the activity of permeabilized cell preparations and optimize assay conditions, we incubated 1- to 2-mg protein equivalents of permeabilized cells in 50 mM Tris-HCl buffer (pH 6.80) containing 30 mM magnesium chloride with 160 nmol of UDP-MurNAc-pentapeptide and 65 nmol (5 ,Ci) of UDP[glucosamine-6-3H]GlcNAc in a total volume of 80 ,ul at 25°C for 30 min. The reactions were stopped by the addition of 80 ,ul of 10% (wtlvol) ice-cold trichloroacetic acid (TCA), and the reaction mixtures were kept at 0°C for a minimum of 1 h. The quenched mixtures were filtered through glass fiber membranes (GF/F; Whatman, Maidstone, United Kingdom), and the TCA precipitates were washed with 10 ml of ice-cold 5% (wt/vol) TCA. The membranes were dried at 60°C for 2 h prior to scintillation counting. Control experiments were done as described above, except that permeabilized cells were incubated at 100°C for 10 min prior to use. The buffers used to determine the optimum composition and pH were 50 mM Tris-maleate (pH 5.73 to 6.90) and 50 mM Tris-HCl (pH 6.80 to 7.20). A typical assay of in vitro PG synthesis and 0 acetylation involved mixing permeabilized cells (1.8 to 2.0 mg of protein) in 50 mM Tris-HCl (pH 6.80)-30 mM magnesium chloride with 160 nmol of UDP-MurNAc-pentapeptide and 62.5 nmol (638 nCi) of UDP-[acetyl-1-1'4C]GlcNAc in a total volume of 80 ,u. After incubation at 25°C for 1 h, the reactions were terminated by the addition of an equal volume of 8% (wt/vol) SDS at 100°C. The temperature was maintained at 60°C for 1 h, and the solutions were allowed to cool to an ambient temperature. SDS-insoluble PG was recovered by centrifugation at 160,000 x g for 15 min at 20°C (Airfuge), and the pellet was washed once with 0.1% (wt/vol) sodium azide. The amounts of radioactivity associated with 6-O-acetyl groups and SDS-insoluble PG were determined as described below. Heat-inactivated (100°C, 15 min), toluene-permeabilized cell wall preparations served as controls. In some experiments, 40 IU of penicillin G was added to the reaction mixtures prior to the addition of the radioactive nucleotide. In other experiments, UDP-[acetyl-1-1'4C]GlcNAc was re-
4620
DUPONT AND CLARKE
J. BACTERIOL.
TABLE 1. PG 0 acetylation in isolated cell wall fragments of P. mirabilis Perkins Radioactivity (%) released after incubation witha:
Radioactivity released as acetate after incubation withb:
Sample (h)
Base
Buffer
Native cell wall fragments 0 16
Heat-inctivated cell wall fragments, 16
NA 11.1 (3.90) 5.52 (2.50)
Base
Buffer (%
pmol . mg of PG-'
NA 56.2 (8.75)
0.00 0.00
18.9 28.2
8.71 13.0
27.6 (8.75)
0.00
13.5
6.23
Values represent the percentage of radioactivity released from cell wall fragments after incubation for 16 h in 20 mM Tris-HCI buffer (pH 6.80) (Buffer) and mild base hydrolysis for 6 h with 20 mM NaOH (Base), relative to the total amount of radioactivity associated with cell wall fragments. Values in parentheses represent standard deviations for five samples. NA, not applicable. b The supernatants of the buffer- or base-treated cell wall fragments were filtered through 0.45-,um-pore-size HA membrane filters, and the [3H]acetate contents were determined by HPLC on an Aminex HPX-87H organic acid column and scintillation counting (11). a
placed with either 65 nmol of UDP-[glucosamine-6-3H] GlcNAc or both 65 nmol of unlabelled UDP-GIcNAc and 2 nmol of either [acetyl-3H]GlcNAc or [glucosamine-1,63H]GlcNAc. Release and quantitation of acetate. Quantitation of radiolabelled 0-linked acetate was achieved by mild base hydrolysis of SDS-insoluble PG (19) in 20 mM NaOH and subsequent analysis of hydrolysates by high-pressure liquid chromatography (HPLC) and liquid scintillation counting as previously described (11, 12). In brief, the mild base-treated PG was collected by centrifugation at 160,000 x g for 15 min at 20°C with an Airfuge, and the pellets were washed once by centrifugation with 200 ,ul of 0.1% sodium azide. The supernatants were pooled, filtered through 0.45-p.m-pore-size HA membrane filters (Millipore Ltd., Mississauga, Ontario, Canada), and injected onto an Aminex HPX-87H (Bio-Rad) organic acid column (7.8 by 300 mm). Fractions (0.5 ml) of the column effluent were collected and counted for levels of radioactivity by liquid scintillation counting. The PG recovered from the pellets of the Airfuge centrifugation was solubilized by treatment with 5 to 10 U of mutanolysin in 10 mM Tris-HCl (pH 6.80) containing 8 mM MgCl2 at 37°C with gentle agitation for 2 h prior to the quantitation of the radioactivity remaining bound. Analytical methods. PG concentrations were determined by amino acid analysis with a Beckman System Gold Amino Acid Analyzer with postcolumn ninhydrin detection. Samples of PG (0.15 mg) were hydrolyzed in vacuo with 4 M HCl at 110°C for 16 h. Acetate quantitation was performed by HPLC with a Beckman system as previously described (11). Measurements of radioactivity were made with a Tri-Carb 2000 liquid scintillation counter (Canberra-Packard Canada, Mississauga, Ontario, Canada) with Ecolume (ICN Biomedicals Canada, Ltd., Montreal, Quebec, Canada) or Liquiscint (National Diagnostics, Manville, N.J.) as the scintillation cocktail. Enzymes and biochemicals. GlcNAc, mutanolysin, and SDS were purchased from Sigma Chemical Co., St. Louis, Mo. Boehringer Mannheim Canada, Laval, Quebec, Canada, supplied pronase, while penicillin G (potassium salt) was a product of Ayerst Laboratories. Ecolume, [acetyl3H]GlcNAc (specific activity, 10 Ci. mmol-1; lots 3305141, 3609124, and 4021162), and UDP-[acetyl-1-14C]GlcNAc (specific activity, 10.2 mCi. mmol-1; lot 3719155) were purchased from ICN Biomedicals, while UDP-[glucosamine6_3H]GlcNAc (specific activity, 26.8 Ci mmol-1; lot 2474-
060) and [glucosamine-1,6-3H]GlcNAc (specific activity, 34.2 Ci- mmol-1; lot 2482-084) were provided by New England Nuclear Research Products, DuPont Canada, Dorval, Quebec, Canada. UDP-MurNAc-pentapeptide was isolated from the supernatant of TCA-treated B. subtilis W23 by gel filtration chromatography by the procedure of Garrett (15). RESULTS In vitro 0 acetylation. Radiolabelled cell wall fragments were prepared from P. mirabilis Perkins by culturing of the cells in the presence of 40 ,uM [acetyl-3H]GlcNAc prior to their disruption in a French pressure cell. The amount of 0-linked [3H]acetate associated with the pelleted insoluble material of the cell wall preparations was calculated as the difference between the amounts liberated by incubation in 20 mM NaOH versus 20 mM Tris-HCI buffer (pH 6.80) which, under these conditions of culturing and preparation, was 18.9 pmol of [3H]acetate mg of PG-1. To investigate whether these cell wall fragments were capable of continuing the process of PG 0 acetylation, we incubated 225-p.l samples for 16 h in 20 mM Tris-HCl buffer (pH 6.80); other samples that were heated to 100°C for 15 min prior to incubation served as controls. The insoluble material was recovered by ultracentrifugation, and the amounts of radioactivity and acetate released both during the 16-h incubation at a neutral pH and from mild base-hydrolyzed PG were determined. A low level of radioactivity was released from both the native insoluble fragments and the boiled control fragments following 16 h of incubation at pH 6.8 (Table 1). However, none of this released radioactivity was determined by HPLC analysis to be acetate (Fig. la and c); it was probably associated with PG fragments released by the action of indigenous heat-stable autolysins. Mild base hydrolysis of the boiled control fragments resulted in the release of 28% of the total initial radioactivity, 23% of which (representing 6.2% of the total) was identified as acetate (Fig. lb). This base-labile [3H]acetate represented that transferred to the C-6 of muramyl residues prior to the isolation of the cell wall fragments from the viable whole cells (Table 1). In contrast, approximately twice as much radioactivity and acetate was detected in the native cell wall fragments treated in a similar manner (Table 1 and Fig. ld), suggesting that [3H]acetate preincorporated as [acetyl-3H]GlcNAc continued to undergo transfer within the isolated PG cell wall
VOL. 173, 1991
IN VITRO PEPTIDOGLYCAN SYNTHESIS AND 0 ACETYLATION
a
b
E
4621
9.0
0
-Y C
EI a
1._
-60
// 0
/
0
8.0p
ct 0
0I1 c
d
C)
0~~ 0
/
7.0 .I/ .....,..,..
z
I
I
0
I
E
S
cLi I
0
5
10 15 20 25 0
Time
5
10
6.0L
i.5 6.0
15 20 25
FIG. 1. HPLC analysis of [3H]acetate released from the in vitro acetylation of P. mirabilis cell wall fragments. Insoluble cell wall fragments (c) and heat-inactivated (100°C, 15 min) control fragments (a) were incubated in 20 mM Tris-HCl buffer (pH 6.80) containing 1.0 mM MgCl2 and 0.1% NaN3 at room temperature for 16 h, and the [3H]acetate released was separated by HPLC. Fractions (0.5 ml) were collected and counted for radioactivity. The recovered insoluble cell wall fragments of the native preparation (d) and the heat-inactivated control preparation (b) were hydrolyzed with 20 mM NaOH (room temperature, 3 h) to release 0-linked [3H]acetate. The retention time for acetic acid is 19.4 min. The solid bars represent 100 cpm and 0.005 absorbance unit.
fragments upon prolonged incubation. The difference in recovered [3H]acetate between the native cell wall fragments at zero time and the heat-inactivated cell wall fragments following 16 h of incubation in Tris-HCl buffer was again likely caused by the loss of insoluble PG because of the action of heat-stable autolysins. In vitro PG synthesis and 0 acetylation. Initial experiments pertaining to the in vitro biosynthesis and 0 acetylation of P. mirabilis PG were done with a protocol involving etherpermeabilized cells (37), adopted by Martin (24) and Brown and Perkins (5) for the cell-free biosynthesis of P. mirabilis PG and N. gonorrhoeae PG, respectively. An average of 62.1 pmol of radiolabel per mg of protein was incorporated into the SDS-insoluble PG fraction of the permeabilized cells, representing an overall efficiency of 0.47%. This low efficiency relative to that previously reported (5, 24), coupled with problems of enzyme stability, prompted the development of an alternative method with toluene-permeabilized
cells. The incorporation of UDP-[glucosamine-6-3H]GlcNAc into TCA-insoluble material isolated from different preparations of toluene-permabilized P. mirabilis cells was monitored to determine the optimum composition of the extraction mixture. The use of 1% (vol/vol) toluene in (i) 100 mM Tris-HCl buffer (pH 6.80), (ii) 100 mM Tris-HCl buffer (pH
70
7.5
pH
(min)
0
6.5
FIG. 2. Dependence on pH of the ability of toluene-permeabilized cells to catalyze the in vitro biosynthesis of P. mirabilis PG. Toluene-permeabilized cells were incubated for 30 min with UDP[glucosamine-6-3H]GlcNAc and UDP-MurNAc-pentapeptide in 50 mM Tris-maleate buffer (pH 5.73 to 6.90) (0) and 50 mM Tris-HCI buffer (pH 6.80 to 7.20) (0). TCA-insoluble material was collected by filtration on GF/F glass fiber membranes and counted for
radioactivity.
6.80) containing 30 mM MgCl2, or (iii) distilled water resulted in permeabilized cells efficient in PG biosynthesis at levels of only 78, 53, and 26%, respectively, compared with cells permeabilized with 1% (vol/vol) toluene in 30 mM
MgCl2. Increasing the concentrations of toluene to 10 and 25% (vol/vol) had deleterious effects, resulting in only 35 and 30% efficiencies, respectively. The optimum composition and optimum pH of the reaction buffer were also determined in a similar manner. While no striking differences were observed between the two buffers investigated (Fig. 2), as previously observed by Brown and Perkins (5) for N. gonorrhoeae, acute maximum activity was observed around pH 6.80. On the basis of the results of these preliminary studies, a protocol of permeabilizing P. mirabilis cells with 1% (vol/vol) toluene in 30 mM MgCl2 and conducting in vitro PG biosynthesis experiments in Tris-HCl buffer (pH 6.80) was adopted for further inves-
tigations.
Studies of PG 0 acetylation were continued by monitoring the migration of [14C]acetate from UDP-[acetyl-114C]GlcNAc in PG synthesized in vitro with toluene-permeabilized cells of P. mirabilis 19. The synthesized material was subjected to hot SDS extraction, and the isolated [14c]PG was examined for migration of the radiolabel. The results of a representative experiment are presented in Table 2. Incorporation of 101 pmol of [14C]acetate mg of pro-
tein-1 into SDS-insoluble PG was achieved after 30 min, representing an increase of 67% over that in ether-treated cell preparations. Mild base hydrolysis of radiolabelled SDS-insoluble PG resulted in the recovery of 21% more
4622
DUPONT AND CLARKE
J. BACTERIOL.
TABLE 2. Transfer of ["4C]acetate from UDP-[acetyl-1-'4C]GlcNAc to muramyl residues in an in vitro biosynthesis system for the 0-acetylated PG of P. mirabilis In vitro biosynthetic system (toluenepermeabilized cells)
UDP-[acetyl-1-14CIGlcNAc incorporated (pmol mg of
protein-') 101 117 19.4 1.46
Cells alone Cells plus chaseb Cell plus Penicillin GC Heat inactivated cellsd
% of radioactivity released from SDS-insoluble materiala Total after incubation with: Buffer
Base
30.9 14.8 38.9 80.5
52.2 43.5 41.0 80.3
Difference
As base-labile acetate
21.3 28.7 2.11 0.00
22.3 28.5 1.87 0.00
Percentages of released radioactivity and acetate were determined as described in Table 1, footnotes a and b. Following the 30-min incubation with [acetate-1-14C]GlcNAc, the reaction mixture was adjusted to contain 130 nM UDP-GlcNAc and incubated for a further 30 min before being quenched with SDS. C Penicillin G (40 IU) was added to toluene-permeabilized cells 5 min prior to the addition of UDP-[acetate-1-14CJGIcNAc. d 100oC, 15 a
b
mim.
radioactivity than in buffer-treated material, and all of this radioactivity was determined to be [14C]acetate (Table 2). Chasing the UDP-[acetyl-1-`4C]GlcNAc initially taken up by the in vitro system in the first 30 min with the addition of 130 nM UDP-GlcNAc and an extra 30 min of incubation appeared to increase the amount of ['4C]acetate released from SDS-insoluble PG by mild base hydrolysis. Control incubations conducted with boiled (100°C, 15 min) permeabilized cells did not lead to incorporation of the radiolabelled nucleotide (less than 1.5 pmol. mg of protein-1). Moreover, no incorporation of radioactivity (less than 0.5 pmol mg of protein-1) was observed when UDP-[acetyl1-`4C]GlcNAc was replaced with both unlabelled UDPGlcNAc and either [acetyl-3H]GlcNAc or [glucosamine-1,63H]GlcNAc. Finally, no radioactive acetate was detected in the mild base hydrolysates of PG synthesized in vitro when UDP-[acetyl-1-`4C]GlcNAc was replaced with UDP-[glucosamine-6-3H]GlcNAc. These latter negative results indicate that the transferred (0-linked) acetate is not derived from the catabolism of supplemented PG precursors but requires the prior incorporation of [acetyl-1-'4CIGlcNAc into PG via the nucleotide. This inference is further supported by the results obtained with the in vitro peptidoglycan biosynthetic system supplemented with penicillin G. With the inclusion of 40 IU of penicillin G in the reaction mixtures, less than 20% of the UDP-[acetate-14C]GlcNAc was taken up by the toluenepermeabilized cells, compared with untreated cells (Table 2). Analysis of the SDS-insoluble PG isolated indicated that no [14Clacetate was released by mild base hydrolysis from the inhibited biosynthesis product. excess
DISCUSSION We have presented evidence to both confirm that 0 acetylation of PG is indeed a process associated with membrane functions outside the cytoplasm and support the hypothesis that PG 0 acetylation occurs via N--0 transacetylation (12). Radioactive acetate was found in a base-labile position within SDS-insoluble PG that had been synthesized in vitro when the acetate was introduced only as UDP-bound [acetyl-1-14C]GlcNAc. It is highly unlikely that this precursor molecule was catabolized within the membrane to liberate [14C]acetate, which was subsequently activated and transferred to PG, because no base-labile radiolabel was detected in PG synthesized by the in vitro system supplemented with
either [acetyl-3H]GIcNAc or [glucosamine-1,6-3H]GlcNAc. In addition, penicillin G inhibited the transfer of ["4C]acetate from the radioactive nucleotide to peptidoglycan. This antibiotic blocks many of the maturation reactions of PG biosynthesis, including transglycosylation and transpeptidation. Hence, the presence of penicillin G in the reaction mixture would cause UDP-[acetyl-1-`4C]GlcNAc derivatives to accumulate in the membrane, and the lack of [14C]acetate transfer implies not only that catabolic activity is absent from the cell wall preparations but also that radioactive GlcNAc derivatives have to be incorporated into PG prior to 0 acetylation. This latter point is further supported by (i) the pulse-chase experiment, in which an increase in the incorporation of the precursor and a concomitant decrease in the amounts of loosely associated (buffer-labile) radiolabel served to elevate the levels of 0-linked ['4C]acetate associated with the synthesized SDS-insoluble PG, and (ii) the significant increase in base-released [3H]acetate observed when cell wall fragments prelabelled with [acetate-3H]GlcNAc were allowed to continue PG biosynthesis and maturation. The degree of 0 acetylation of muramyl residues in PG synthesized in vitro is lower than that observed in living microorganisms (40.2 and 52.8% for P. mirabilis Perkins and 19, respectively [11]). Similar observations were reported by Martin concerning an analogous in vitro biosynthetic system of P. mirabilis 19 PG (24). The extent of 0 acetylation of the synthesized PG was described as resembling the decreased state typically observed in PG of L-form spheroplasts grown in the presence of penicillin. Unfortunately, the specific degrees of PG 0 acetylation were not provided, making a direct comparison with the present data impossible. A thinlayer chromatography analysis of the synthesized PG after hydrolysis by an endo-N,O-diacetylmuramidase from a Chalaropsis sp. did, however, indicate the presence of 0-acetylated monomer and mono- and di-O-acetylated dimer fragments of PG. Taken together, these observations support the previous conclusions that the 0 acetylation of PG is a maturation process (9, 17, 18, 21-23, 33) and that N-linked acetate is transferred to the C-6 position of muramyl residues (12). For N-*O transacetylation to occur within the cell-free system, the autolysins documented to be present (3, 8) would have to be catalytically active since, according to the current hypothesis (12), PG turnover is associated with the process of 0 acetylation. The fact that both PG autolytic and N-0O transacetylase activities were retained in the cell-free system
VOL. 173, 1991
IN VITRO PEPTIDOGLYCAN SYNTHESIS AND 0 ACETYLATION
led to the speculation that, by analogy with some of the penicillin-binding proteins of Escherichia coli, the two enzymatic activities are catalyzed by a single bifunctional enzyme (10). Indeed, three of the high-molecular-weight penicillin-binding proteins, la, lbs, and 3, are bifunctional transglycosidase-transpeptidase enzymes (for a review, see reference 38). Obviously, much more intensive investigation will be required to bear out this postulate. Nevertheless, this study has provided a basis on which to develop an assay for the PG N-*O transacetylase which may be subsequently used to isolate and characterize this interesting and potentially important enzyme. ACKNOWLEDGMENTS This study was supported by an operating grant to A.J.C. from the Medical Research Council and a Natural Sciences and Engineering Research Council postgraduate scholarship to C.D. REFERENCES 1. Abrams, A. 1958. O-Acetyl groups in the cell wall of Streptococcus faecalis. J. Biol. Chem. 230:949-959. 2. Blundeli, J. K., and H. R. Perkins. 1981. Effects of P-lactam antibiotics on peptidoglycan synthesis in growing Neisseria gonorrhoeae, including changes in the degree of O-acetylation. J. Bacteriol. 147:633-641. 3. Blundeli, J. K., and H. R. Perkins. 1985. Selectivity for O-acetylated peptidoglycan during endopeptidase action by permeabilized Neisseria gonorrhoeae. FEMS Microbiol. Lett. 30:6769. 4. Blundell, J. K., G. J. Smith, and H. R. Perkins. 1980. The peptidoglycan of Neisseria gonorrhoeae: O-acetyl groups and lysozyme sensitivity. FEMS Microbiol. Lett. 9:259-261. 5. Brown, C. A., and H. R. Perkins. 1979. In vitro synthesis of peptidoglycan by 13-lactam-sensitive and -resistant strains of Neisseria gonorrhoeae: effects of ,B-lactam and other antibiot-
Antimicrob. Agents Chemother. 16:28-36. Brumfitt, W. 1959. The mechanism of development of resistance to lysozyme by some Gram-positive bacteria and its results. Br. J. Exp. Pathol. 40:441-451. 7. Brumfitt, W., A. C. Wardlaw, and J. T. Park. 1958. Development of lysozyme resistance in Micrococcus lysodeikticus and its association with an increased O-acetyl content of the cell wall. Nature (London) 181:1783-1784. 8. Chapman, S. J., and H. R. Perkins. 1983. Peptidoglycandegrading enzymes in ether-treated cells of Neisseria gonorics.
6.
9. 10. 11.
12. 13.
14.
15. 16.
rhoeae. J. Gen. Microbiol. 129:877-883. Dougherty, T. J. 1983. Synthesis and modification of the peptidoglycan in Neisseria gonorrhoeae. FEMS Microbiol. Lett. 17:51-53. Dupont, C. 1991. Ph.D. thesis. University of Guelph, Guelph, Ontario, Canada. Dupont, C., and A. J. Clarke. 1991. Dependence of lysozymecatalysed solubilization of Proteus mirabilis peptidoglycan on the extent of O-acetylation. Eur. J. Biochem. 195:763-769. Dupont, C., and A. J. Clarke. 1991. Evidence for N- O acetyl migration as the mechanism for 0-acetylation of peptidoglycan in Proteus mirabilis. J. Bacteriol. 173:4318-4324. Fleck, J., M. Mock, R. Minck, and J. M. Ghuysen. 1971. The cell envelope of P. vulgaris P18. Isolation and characterization of the peptidoglycan component. Biochim. Biophys. Acta 233: 489-503.
Fleming, T. J., D. E. Walismith, and R. S. Rosenthal. 1986. Arthropathic properties of gonococcal peptidoglycan fragments: implications for the pathogenesis of disseminated gonococcal disease. Infect. Immun. 52:600-608. Garrett, A. J. 1969. The effect of magnesium ion deprivation on the synthesis of mucopeptide and its precursors in Bacillus subtilis. Biochem. J. 115:419-430. Ghuysen, J. M., and J. L. Strominger. 1963. Structure of the cell wall of S. aureus strain Copenhagen. II. Separation and structure of disaccharides. Biochemistry 2:1119-1125.
4623
17. Gmeiner, J., and H. P. Kroll. 1981. Murein biosynthesis and O-acetylation of N-acetylmuramic acid during the cell division cycle of Proteus mirabilis. Eur. J. Biochem. 177:171-177. 18. Gmeiner, J., and E. Sarnow. 1987. Murein biosynthesis in synchronized cells of Proteus mirabilis. Quantitative analysis of O-acetylated murein subunits and of chain terminators incorporated into the sacculus during the cell cycle. Eur. J. Biochem. 163:389-395. 19. Hoyle, B. D., and T. Beveridge. 1983. Metal binding by the peptidoglycan sacculus of Escherichia coli K-12. Can. J. Microbiol. 30:204-211. 20. Johannsen, L., H. Labischinski, P. Burghaus, and P. Giesbrecht. 1983. Acetylation in different phases of growth of staphylococci and their relation to cell wall degradability by lysozyme, p. 261-266. In R. Hakenbeck, J. Holtje, and H. Labischinski (ed.), The target of penicillin. International FEMS symposium on the murein sacculus of bacterial cell walls. Walter de Gruyter & Co., Berlin. 21. Lear, A. L., and H. R. Perkins. 1983. Degrees of O-acetylation and cross-linking of the peptidoglycan of Neisseria gonorrhoeae during growth. J. Gen. Microbiol. 129:885-888. 22. Lear, A. L., and H. R. Perkins. 1986. O-Acetylation of peptidoglycan in Neisseria gonorrhoeae. Investigation of lipid-linked intermediates and glycan chains newly incorporated into the cell wall. J. Gen. Microbiol. 132:2413-2420. 23. Lear, A. L., and H. R. Perkins. 1987. Progress of O-acetylation and cross-linking of peptidoglycan in Neisseria gonorrhoeae grown in the presence of penicillin. J. Gen. Microbiol. 133:17431750. 24. Martin, H. H. 1984. In vitro synthesis of peptidoglycan by spheroplasts of Proteus mirabilis grown in the presence of penicillin. Arch. Microbiol. 139:371-375. 25. Martin, H. H., and J. Gmeiner. 1979. Modification of peptidoglycan structure by penicillin-action in cell walls of Proteus mirabilis. Eur. J. Biochem. 95:487-495. 26. Martin, H. H., W. Schilf, and P. Gruss. 1975. D-Alanyl-Dalanine carboxypeptidase in the bacterial form and L-form of Proteus mirabilis. Eur. J. Biochem. 55:465-473. 27. Rosenthal, R. S., W. J. Folkening, D. R. Miller, and S. G. Swim. 1983. Resistance of O-acetylated gonococcal peptidoglycan to human peptidoglycan-degrading enzymes. Infect. Immun. 40: 903-911. 28. Rosenthal, R. S., M. A. Gfell, and W. J. Folkening. 1985. Influence of protein synthesis inhibitors on regulation of extent of O-acetylation of gonococcal peptidoglycan. Infect. Immun. 49:7-13. 29. Schrader, W. P., and D. P. Fan. 1974. Synthesis of cross-linked peptidoglycan attached to previously formed cell wall by toluene-treated cells of Bacillus megaterium. J. Biol. Chem. 249: 4815-4818. 30. Seidl, P. H., and K. H. Schleifer (ed.). 1985. Biological properties of peptidoglycan. Walter de Gruyter & Co., New York. 31. Sidow, T., L. Johannsen, and H. Labischinski. 1990. Penicillininduced changes in the cell wall composition of Staphylococcus aureus before the onset of bacteriolysis. Arch. Microbiol. 154:73-81. 32. Smith, T. J., and P. E. Hanna. 1986. N-Acetyltransferase multiplicity and the bioactivation of N-acrylhydroxamic acids by hamster hepatic and intestinal enzymes. Carcinogenesis 7:697-702. 33. Snowden, M. A., H. R. Perkins, A. W. Wyke, M. V. Hayes, and J. B. Ward. 1989. Cross-linking and O-acetylation of newly synthesized peptidoglycan in Staphylococcus aureus H. J. Gen. Microbiol. 135:3015-3022. 34. Swim, S. C., M. A. Gfell, C. E. Wilde III, and R. S. Rosenthal. 1983. Strain distribution in extents of lysozyme resistance and O-acetylation of gonococcal peptidoglycan determined by highperformance liquid chromatography. Infect. Immun. 42:446 452. 35. Tipper, D. J., J. M. Ghuysen, and J. L. Strominger. 1965. Structure of cell wall of Staphylococcus aureus strain Copenhagen. III. Further studies of the disaccharides. Biochemistry 4:468-473. 36. Tommassen, J., and B. Lugtenberg. 1980. Outer membrane
4624
DUPONT AND CLARKE
protein of Escherichia coli K-12 is coregulated with alkaline phosphatase. J. Bacteriol. 143:151-157. 37. Vosberg, H.-P., and H. Hoffmann-Berlin. 1971. DNA synthesis in nucleotide-permeable Escherichia coli cells. I. Preparation and properties of ether-treated cells. J. Mol. Biol. 58:739-753. 38. Waxman, D. J., and J. L. Strominger. 1983. Penicillin-binding
J. BACTERIOL.
proteins and the mechanism of action of P-lactam antibiotics. Annu. Rev. Biochem. 52:825-869. 39. Whitfield, C., R. E. W. Hancock, and J. W. Costerton. 1983. Outer membrane protein K of Escherichia coli: purification and pore-forming properties in lipid bilayer membranes. J. Bacteriol. 156:873-879.
