JOURNAL OF BACTERIOLOGY, Feb. 1976, p. 509-517 Copyright 0 1976 American Society for Microbiology
Vol. 125, No. 2 Printed in U.S.A.
Peptidoglycans Synthesized by a Membrane Preparation of Micrococcus luteus G. PELLON, C. BORDET,
AND
G. MICHEL*
Laboratoire de Biochimie Microbienne, Universite Claude Bernard Lyon, 69621 Villeurbanne, France Received for publication 7 October 1975
By incubation of cell-free particulate preparations from Micrococcus luteus with nucleotidic precursors uridine 5'-diphosphate-N-acetylglucosamine and uridine 5'-diphosphate-N-acetylmuramic acid-L-Ala-D-iso-Glu-L-Lys-D-Ala-DAla, several types of peptidoglycans were obtained: soluble peptidoglycan, insoluble peptidoglycan bound to the membrane and solubilized by trypsin, and peptidoglycan, which remained insoluble after the action of trypsin. The structure of each type of peptidoglycan was studied by action of lytic enzymes and separation of the fragments on Sephadex. Soluble peptidoglycans consist of a mixture of un-cross-linked polymers of various molecular weights. Trypsinsolubilized peptidoglycans are also a mixture of polymers of various sizes. They contain a preponderance of un-cross-linked material and some bridges with dimer peptides. Insoluble peptidoglycans, after the action of trypsin, contain about 50% of un-cross-linked peptide residues; in the other moiety, peptide units are cross-linked by D-Ala--L-Lys and D-Ala-L-Ala bonds which characterize the natural peptidoglycan. Therefore, the cell-free particulate preparation possesses the whole enzymatic system necessary for synthesis of cross-linked peptidoglycan.
A particulate preparation from Micrococcus luteus NCTC 2665 has been described which catalyzes peptidoglycan synthesis in vitro by incubation with the nucleotide precursors uridine 5'-diphosphate (UDP)-N-acetylglucosamine (UDPGlcNAc) and UDP-N-acetylmuramic acid-L-Ala-D-iso-Glu-L-Lys-D-Ala-DAla (UDPMurNAc-pentapeptide) (1, 2). The peptidoglycan products are characterized as chromatographically immobile material. More recently, other enzymatic systems have been prepared from Staphylococcus aureus (13), Micrococcus luteus (12), and Bacillus licheniformis (19, 20). These systems contain membrane material which brings the enzymes necessary to the biosynthesis of peptidoglycan and cell walls which are acceptors in the last step of this biosynthesis. In the presence of such acceptors, cross-linked peptidoglycans are formed. With a cell wall-free membrane preparation from M. luteus, a mixture of soluble and insoluble lysozyme-sensitive peptidoglycans was obtained in the presence and in the absence of penicillin (3). A recent study showed that a major part of the insoluble polymer obtained in the presence of penicillin is solubilized by trypsin digestion, so its insolubility is due to an association with the membrane (14). In this paper, we report a study of the
structures of the soluble and insoluble peptidoglycans obtained in the absence of penicillin from a cell wall-free preparation from M. luteus. These structures had not previously been investigated, and no evidence for transpeptidation had been reported with cell wall-free preparations from M. luteus (2, 9).
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MATERIALS AND METHODS
Paper and thin-layer chromatography. Paper chromatography was carried out using the following solvents: (A) isobutyric acid-0.5 M NH4OH (5:3, vol/vol); (B) ethanol-1 M ammonium acetate (5:2, vol/vol) (pH 7.4). Whatman 3MM paper was used for precursor purification and soluble peptidoglycan preparation; it was first washed with 1 M ammonium acetate and water. Thin-layer chromatography of monomer and dimer peptides was performed on cellulose MN 300 G in butanol-pyridine-acetic acid-water (30:20:6:24). The separation of e-N-dinitrophenylated lysine was carried out by thin-layer silica gel chromatography in chloroform-methanol-acetic acid-water (65:25:13:9). Preparation of UDPMurNAc-L-Ala--y-D-Glu-LLys-D-Ala-D-Ala and UDPMurNAc-L-Ala--y-DG1U-L- ['4C]Lys-D-Ala-D-Ala. Unlabeled nucleotide muramyl-pentapeptide was prepared by accumulation in cells of M. luteus grown in a medium containing ethylenediaminetetraacetate as described by Garrett (4). The radioactive precursor was obtained by suc-
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PELLON, BORDET, AND MICHEL
cessive additions of L- [4C]Lys and D-Ala-D-Ala to the nucleotide-dipeptide extracted from lysine-deprived cells of S. aureus (17). These additions were carried out by incubating suitable substrates with a crude extract from S. aureus (6). The method described by Ito and Strominger (6-8) was used with some modifications. L- ['4C ]lysine (specific activity, 255 mCi/mmol) was supplied by the C.E.A. (Saclay, France); D-Ala-D-Ala was commercially available (Cyclo Chemical Corp.). Crude enzyme protein concentration was estimated by the method of Lowry et al. (10). For L-lysine addition, the reaction mixture consisted of 16.5 nmol of L- [14C ]lysine (2.5 x 106 counts/ min), 16 nmol of nucleotide muramyl-dipeptide, 200 nmol of adenosine 5'-triphosphate, 400 nmol of MgCl,, 20 nmol of KF, and 20 Ml of crude enzyme preparation in 85 Ml of 0.05 M tris(hydroxymethyl)aminomethane (Tris)-hydrochloride buffer, pH 8.9 (final volume). Incubations were carried out at 37 C for 2.5 h. The reaction was stopped by boiling for 2 min, and then the incubation mixture was applied to Whatman 3MM paper and developed for 24 h in solvent A. The radioactive nucleotide muramyl-tripeptide product was identified using an authentic sample obtained from S. aureus by accumulation with D-Cycloserine (18) and purified by chromatography on Whatman 3MM paper in solvent B. For D-Ala-D-Ala addition, incubation mixtures contained 5 nmol of nucleotide muramyl- ["4C tripeptide (106 counts/min), 6 nmol of D-Ala-D-Ala, 200 nmol of adenosine 5'-triphosphate, 100 nmol of MnCl,, 20 nmol of KF, and 20 Mil of crude enzyme preparation in 70 Ml of 0.05 Tris-hydrochloride buffer, pH 8.7 (final volume). They were incubated for 2.5 h at 37 C. After boiling for 2 min, the reaction mixture was applied to Whatman 3MM paper and chromatographed repeatedly in solvent B until the nucleotide muramyltripeptide and the nucleotide muramyl-pentapeptide were well separated. The nucleotide muramyl["IC ]pentapeptide was isolated and diluted with unlabeled precursor to a final specific activity of 28 mCi/mmol. Particulate enzyme preparation. M. luteus NCTC 2665 was grown at 35 C in a rotatory shaker on brain heart infusion medium (B. D. Merieux, 69 Marcy L'Etoile, France). Cells were harvested at mid-log phase and washed with 0.02 M Tris-hydrochloride buffer (pH 8.0). All the following operations were carried out at 4 C. Particulate enzyme was prepared by grinding the cells with three times their wet weight of levigated alumina as previously described (3). The broken cells were suspended into 50 mM Tris-hydrochloride buffer, pH 7.5, containing 0.1 M MgCl, and 1 mM 2-mercaptoethanol (15 ml/g of cells, wet weight) and centrifuged twice at 10,000 x g for 10 min. The pellet was discarded, and the supernatant was centrifuged at 105,000 x g for 1 h. The membrane pellet was washed twice with buffer and finally suspended to give a protein concentration of 7 mg/ml as estimated by the method of Lowry et al. (10). Particulate enzyme stability during the storage could be improved by the
J. BACTERIOL.
addition of UDP-GlcNAc to the preparation (27 nmollmg of protein). Preparation of labeled peptidoglyeans. Reaction mixtures contained the following components: 9 nmol of UDPMurNAc- [14C ]pentapeptide (1.3 x 101 counts/ min), 13.5 nmol of UDPGlcNAc, 6 umol of MgCl2, 7.5 Amol of Tris-hydrochloride buffer (pH 8.6), and about 500 Mg of particulate enzyme protein in a final volume of 140 Ml. After incubation for 2 h at 37 C, the reaction was stopped by boiling for 2 min. Incubation mixtures were centrifuged at 24,000 x g for 10 min. The pellet, washed three times with incubation buffers, was then used as the "crude insoluble peptidoglycan." The soluble fraction was applied to Whatman 3MM paper and developed in solvent A for 24 h. By autoradiography, the chromatogram showed three radioactive spots: a peptidoglycan product (R,, 0 to 0.15), the residual nucleotide muramyl-pentapeptide precursor (R,, 0.26), and an unidentified compound, possibly a nucleotide muramyl-tetrapeptide (R,, 0.35). The product with an R, of 0 to 0.15 was eluted and used as the "soluble peptidoglycan." The crude insoluble peptidoglycan was digested for 4 h at 37 C with 50 Mg of trypsin in 150 Ml of 0.1 Tris-hydrochloride buffer (pH 8.4). After boiling for 5 min, the reaction mixture was centrifuged at 24,000 x g for 10 min. Radioactive material was found in the supernatant and in the pellet. The pellet was suspended with 50 Mg of trypsin and incubated again in a Tris-hydrochloride buffer (pH 8.4) for 3 h. After centrifugation, the supernatant contained a negligible amount of radioactive product. The pellet, washed three times, consisted of a radioactive product sensitive to lysozyme. It was used as the "insoluble peptidoglycan." The supernatant consisted of radioactive trypsin-solubilized polymers. Degradation of labeled peptidoglycans. Four different peptidoglycan hydrolases have been used during this study: lysozyme, Myxobacter AL-1 protease, N-acetylmuramyl-L-alanine amidase, and MLendopeptidase from Streptomyces albus G. The amidase and endopeptidase from S. albus G were obtained from J. M. Ghuysen (Liege, Belgium), and Myxobaeter AL-1 protease was obtained from J. F. Petit (Orsay, France). The activities of these enzymes were tested on specific substrates. Incubations were carried out on samples containing 3,000 to 5,000 counts/min as described below. (i) Digestion by lysozyme and Streptomyces amidase. Incubations were performed at 37 C for 24 h with shaking. The reaction mixtures contained the radioactive peptidoglycan and 40 Mg of lysozyme in 500 Ml of 0.01 M sodium acetate buffer (pH 5.4). In some experiments, after incubation the mixture was completed with 3 gl of Streptomyces amidase preparation and 10 Mmol of sodium acetate buffer (pH 5.4) in a final volume of 560 MlA and incubated for 6 h at 37 C. (ii) Digestion by ML-endopeptidase and lysosyme. The radioactive peptidoglycan was incubated for 5 h at 37 C with 3 Ml of ML-endopeptidase preparation in 200 Ml of 0.01 M veronal buffer (pH 9.0). The reaction mixture was neutralized with acetic acid and incubated at 37 C for 24 h with 40 Mg of
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VOL. 125, 1976
lysozyme and 5 Mmol of sodium acetate buffer (pH 5.4) in a final volume of 500 MIl. (iii) Digestion by Myxobacter AI-i protease. The radioactive polymers were digested by incubation for 24 h at 37 C with shaking with 10 Il of Myxobacter AL-1 protease preparation (5) in 475 ul of 0.01 M veronal buffer (pH 9). Fractionation and identification of radioactive compounds. Filtration of soluble peptidoglycans and peptidoglycan fragments was carried out in 0.1 M acetic acid on two linked Sephadex G-50 and Sephadex G-25 columns (VO = 19 ml, V, = 48 ml) at a flow rate of 5 ml/h. The volume of the fractions was 0.3 ml, and the radioactivity of every second fraction was measured in a Packard Tri-Carb 2001 liquid scintillation spectrometer. Samples were mixed with 5 ml of the following scintillation liquid: 7 g of 2,5diphenyloxazole, 0.15 g of 1,4-bis-(5-phenyloxazol-2yl)-benzene, and 50 g of naphthalene in 1,030 ml of toluene-dioxane-ethanol (20:80:3). In some experiments, activity of the products was rather low. To distinguish radioactive material from the background and obtain reliable results, every fraction was counted four times for 10 min. RESULTS
Properties of soluble peptidoglyeans. Soluble peptidoglycans were obtained after a 2-h incubation. The time course of the biosynthesis of soluble and insoluble polymers has been previously studied (3), and a greater amount of polymers was not found when an incubation longer than 2 h was performed. Soluble peptidoglycans were purified from the residual precursor by paper chromatography (see above). To check their molecular size, they were fractionated on two linked Sephadex G-50 and G-25 columns; soluble peptidoglycans were shown to be a mixture of polymers with various molecular
weights (KD 0.1 to 0.8). These polymers were treated by lysozyme, and the lysozyme digest was fractionated on Sephadex G-50 and G-25; 95% of the radioactive product was found in a single peak (KD = 0.7). This is a disaccharidepeptide monomer (GlcNAc-MurNAc-tetra- or pentapeptide). A radioactive standard monomner gives a single peak (KD = 0.7) by gel filtration in the same experimental conditions. Thus, soluble peptidoglycans appear to be polydisperse, un-cross-linked polymers. Occurrence of a membrane-bound fraction in the crude insoluble peptidoglycan. The crude insoluble peptidoglycan still contained protein components derived from particulate preparation. This peptidoglycan was treated by trypsin, centrifuged, and washed three times. Radioactivity was found in the supernatant and in the pellet (Table 1). A further treatment of the pellet by trypsin gave a small amount of radioactive soluble product (< 3%). The pellet consisted of insoluble peptidoglycan and was almost entirely degraded by lysozyme. The radioactive product solubilized by trypsin treatment and found in the supernatant after centrifugation was shown to be a membrane-bound peptidoglycan (14). When the peptidoglycan was prepared in the presence of 10 gg of benzylpenicillin per ml added to the incubation medium, both soluble and crude insoluble polymers were synthesized in about the same proportions as in the absence of antibiotic (3), but the amount of radioactivity solubilized by treatment of the crude insoluble peptidoglycan with trypsin increased to a higher value, about 80% (Table 1). These two components (insoluble peptidogly=
TABLE 1. Composition of the crude insoluble peptidoglycana Expression of radioactivity
Peptidoglycan fraction
Assay no. lb % dpm
Assay no. 2° % dpm
Assay no. 3c dpm
%
100 100 100 10,300 34,000 Crude insoluble peptidoglycan 8,234 78 51 60 8,010 17,600 4,950 Membrane-bound peptidoglycan solubilized by trypsin 12 39 35 1,200 13,400 2,910 True insoluble peptidoglycan solubilized by lysozyme aProportions of membrane-bound soluble peptidoglycan and insoluble peptidoglycan in the crude insoluble peptidoglycan. The crude insoluble peptidoglycan was prepared as described. In assay 3, 10 gg of benzylpenicillin per ml was added to the incubation medium. It was digested by trypsin to solubilize the membrane-bound fraction; the insoluble residue was then treated by lysozyme, giving soluble fragments. These trypsin-soluble and lysozyme-soluble radioactivities were respectively used as estimations for the membranebound soluble peptidoglycan and for the true insoluble peptidoglycan. They were expressed as disintegrations per minute and as an average percentage of the initial radioactivity. For conditions, see the text. 'No penicillin added. cPenicillin added at a concentration of 10 gg/ml.
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J. BACTERIOL.
can and membrane-bound peptidoglycan) were prepared on a large scale, and their structures were determined. Nature of membrane-bound peptidoglycans. The trypsin-solubilized fraction was submitted to filtration on two linked Sephadex G-50 and Sephadex G-25 columns. As indicated by the gel filtration profile, it consists of a polydisperse polymer containing a high percentage of large-molecular-size fractions (Fig. 1). Then the peptidoglycan was hydrolyzed by lysozyme. The resulting disaccharide-peptide fragments were incubated with S. albus G amidase which splits MurNAc - L-Ala linkages, giving rise to peptides (Fig. 2). These degradation products were fractionated by gel filtration and characterized essentially as peptide monomer (KD = 0.8) (major product) and peptide dimer (KD = 0.58) (Fig. 3). These peptides have the same KD values and the same R, values in thin-layer chromatography as the peptide monomer (R, = 0.29) and the peptide dimer (R, = 0.17) obtained from the natural peptidoglycan after treatment with Myxobacter enzyme. Thus the membrane-bound peptidoglycan contains a large majority of un-cross-linked peptides and some interpeptide bridges in a lesser amount. Structural investigations on the insoluble peptidoglycan. The insoluble peptidoglycan
0
0.4
was degraded by lysozyme and then by Streptomyces amidase. The resulting peptide fragments were resolved by gel filtration which gave a major product (KD = 0.8) characterized as the peptide monomer, a dimer (KD = 0.6), and several oligomers of higher molecular weight (Fig. 4.). Thus the insoluble peptidoglycan appears to be a polymer which contains cross-linked residues. However, the degree of cross-linking is much smaller than that of the natural peptidoglycan from the cell walls of M. luteus. This peptidoglycan is known to contain three classes of peptide arrangements: a small proportion of monomers and dimers (D-Ala L-Lys) and a major proportion of oligomers, mostly hexamers with D-Ala - L-Ala linkages (head-to-tail structure) (5). The structure of the insoluble peptidoglycan formed in vitro was further investigated with special attention to the occurrence of such head-to-tail oligomers. First, the peptidoglycan was incubated with Streptomyces ML-endopeptidase which specifically cleaves D-Ala _ L-Lys linkages (15). The glycan moiety was then degraded by lysozyme, and the following products were separated by gel filtration: a disaccharide-peptide monomer (KD = 0.68), a dimer (KD = 0.48), and highermolecular-weight oligomers (Fig. 5). With these conditions of degradation, only dimer and higher oligomers containing -AlaL-Ala cross-links should persist in the digest; other oligomers with -Ala _ L-Lys should have
0.0
o.s
KD
FIG. 1. Sephadex filtration of the membrane-bound peptidoglycan solubilized by trypsin.
IN VITRO SYNTHESIS OF PEPTIDOGLYCANS
VOL. 125, 1976
-(a) s GloNAc
9
-
(a)
(a) MurNAc r-
(b),(c)-
T
V
l
*
(a) GIcNAc
-
MurNAc
(b).(c) --1-
t
D-Glu
O-GIU (d)
(c)
L- Lys - D-Ala
T
L-Ala
L-AlI |
513
T
L-Ala
-
D-Clu
- L-Lys - D-Ala
I
+ L-Lys - D-Ala
* 9~~~~~~~~~~ FiG, 2. A portion of cross-linked peptidoglycan strand. (a) Site of action of lysozyme; (b) site of action of amidase; (c) site of action of MyxobacterAL-I protease; (d) site of action of ML-endopeptidase.
0
0.4
0.6
0.6
KD
FiG. 3. Sephadex filtration of the membrane-bouwnd peptidoglycan degraded by lysozyme and Streptomyces amidase.
been hydrolyzed into monomers. Thus the dimer (KD = 0.48) should consist of a disaccharide-peptide dimer with a D-Ala _ L-Ala linkage derived from a head-to-tail trimer by cleavage of the D-Ala -- L-Lys bond (Fig. 2). The occturrence of peptide oligomers with D-Ala D L-Lys bonds was studied by incubation of the insoluble peptidoglycan with Myxobacter AL-1 enzyme. This enzyme splits MurNAc L.Ala and D-Ala -- L-Ala linkages; thus, oligomers with such linkages give imonomers and dimners with D-Ala , L-Lys linkage (Fig. 2). After incubation with Myxobacter protease and gel filtration of the peptidoglycan digest, peptide monomer (KD = 0.78) and peptide dlimer (Ka = 0,6) were chaTacteriwed (Fig. 6). The peptidases which werp used for the structural determination of peptidoglycan have complementary activities. A control of the complete hydrolysis of D-Ala -- L-Lys linkages by ML
endopeptidase and D-Ala - L-Ala linkages by Myxobacter endopeptidase was carried out. An insoluble peptidoglycan was first hydrolyzed by ML-endopeptidase and then by Myxobacter AL-1 endopeptidase. These hydrolyses were performed under the same conditions as those used previously (data shown in Fig. 5 and 6). The products were studied by gel filtration on Sephadex G-50 and G-25 columns as described above. The radioactivity was located in a single peak (KD = 0.8) as shown in Fig. 7. It is obvious that dimers and higher peptides oligomers resulting from ML-endopeptidase and lysozyme hydrolysis (Fig. 5) contain D-Ala _ L-Ala linkages which have been split by Myxobacter endopeptidase hydrolysis. The presence of D-Ala - L-Ala linkages was confirmed by analysis of peptides dimers obtained after hydrolysis of the insoluble peptidoglycan by ML-endopeptidase, lysozyme, and
514
J. BACTERIOL.
PELLON, BORDET, AND MICHEL Radloactivity
cpmnml
FIG. 4. Sephadex filtration of the insoluble peptidoglycan degraded by lysozyme and Streptomyces amidase. Radioactivity cpm/ml
150
100I
\
50
0 0
0.2
0.4
0.6
0.6
K
FIG. 5. Sephadex filtration of the insoluble peptidoglycan degraded by ML-endopeptidase and lysozyme.
amidase. The dimer peptides were separated by gel filtration and submitted to dinitrophenylation. A control was carried out on the monomer peptide. The dinitrophenylated peptides were hydrolyzed for 15 h by 6 N HCl at 110 C, and the products were studied by thin-layer chromatography. In both cases, in monomer and dimer peptides 85% of the radioactivity was located as E-2,4-dinitrophenyl-Lys; thus, the interpeptide
linkage in the dimer must be a D-Ala linkage.
-
L-Ala
DISCUSSION Upon incubation with nucleotide precursors, a cell wall-free membrane preparation from M. luteus gives rise to a soluble polymer and an insoluble material (3). The soluble polymer consists of a mixture of un-cross-linked peptido-
IN VITRO SYNTHESIS OF PEPTIDOGLYCANS
VOL. -125, 1976
glycans with various molecular weights which give only monomer units after lysozyme hydrolysis. It can be assumed that they are either precursors in the biosynthesis of insoluble polymers or degradation products resulting from the action of lytic enzymes and particularly of endopeptidases contained in membrane preparations. This last hypothesis seems likely since the formation of soluble peptidoglycans increases more rapidly than the formation of insoluble peptidoglycans with the time of incubation (3). The insoluble materials consists of a mixture of two types of polymers: a membrane-bound peptidoglycan solubilized by treatment with trypsin and an insoluble peptidoglycan.
515
The membrane-bound peptidoglycan consists of polymers with a small percentage of bridges, since essentially monomer units and dimer units in a lesser amount were found after lysozyme degradation and treatment with amidase. This material could be a precursor of the cross-linked insoluble peptidoglycan still attached to the active site of the membrane where the cross-linkings are realized by transpeptidation. Polymers which remain insoluble after digestion with trypsin consist of partially crosslinked peptidoglycans. Evidence for transpeptidation in M. luteus has been found with a cell wall preparation, and an explanation of the biosynthetic process has been proposed: the
R-dloectivity cpm ml
100
50
0
0.4
0.2
0.8
0.6
KD
FIG. 6. Sephadex filtration of the insoluble peptidoglycan degraded by Myxobacter AL-I protease.
0.2
0.4
0.6
0
%
Fr.. 7. Sephadex filtration of the insoluble peptidoglycan degraded by ML-endopeptidase and Myxobacter AL-I protease.
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PELLON, BORDET, AND MICHEL
newly synthetized strands of peptidoglycan are attached to the pre-existing cell wall present in the medium by transpeptidation and in part by transglycosylation (11). Our membrane preparation is quite different from the cell wall preparation of Mirelman et al. (12). Our results indicate that pre-existing cell walls are not essential for the formation of peptide crossbridges. A part (about 20%) of the synthetized peptidoglycan is insoluble after a trypsin treatment, and this peptidoglycan was found to contain both types of interpeptide linkages: D-Ala - L-Lys and D-Ala - L-Ala. The comparison of the natural peptidoglycan of M. luteus with the peptidoglycans synthetized in vitro by membrane preparation shows large differences in the degree of cross-linking. Ghuysen et al. found a wall structure in which 5% of peptide units occur as monomers, 44% as dimers, 15% as trimers, and 60% as hexamers (5). The peptidoglycan synthetized in vitro contains 50% soluble un-cross-linked polymers, and 30% of the polymers are associated with the membrane and solubilized by trypsin digestion; they consist of poorly cross-linked peptidoglycans and give essentially monomers and some dimers after enzyme hydrolysis. Only 20% of total peptidoglycan occurs as insoluble, crosslinked polymers whose proportions of crossbridges were determined by gel fractionation of the fragments obtained after specific cleavages. Fifty percent of the peptide fragments are not cross-linked, 10% are linked by a D-Ala _ L-Lys bridge and give dimers by lysozyme and amidase treatment, and 40% are linked by D-Ala _ L-Ala linkages and give oligomers after hydrolysis by lysozyme and amidase. The formation of oligomers with D-Ala L-Ala linkages results from the sequential action of an amidase and a transpeptidase (16). Moreover, two distinct transpeptidases with different sensitivity to penicillin are involved in the synthesis of the two types of interpeptides bridges: D-Ala _ L-Ala and D-Ala - L-Lys (11). These enzymes must be present in our particulate preparation from M. luteus, which contains the whole system for the peptidoglycan biosyn-
2.
3.
4.
5.
6.
7.
8.
9.
10. 11.
12.
13.
14.
thesis.
ACKNOWLEDGMENTS We are indebted to J. M. Ghuysen, Departement de Botanique, Universite de Liege, Belgium, for a gift of Streptomyces amidase and ML-endopeptidase, to J. F. Petit, Institut de Biochimie, Orsay, France, for a gift of Myxobacter AL-1 protease, and to the Commissariat a l'Energie Atomique, Departement de Biologie, Saclay, France, for financial aid.
15.
16. 17.
LITERATURE CITED 1.
Anderson, J. S.. M. Matsuhashi, M. A. Haskin, and J. L.
18.
Strominget. 1965. Lipid-phosphoacetyltnutainyl-pentapeptide and lipid-phosphbdlsacchatide-pentapeptide: presunied membrane transport interthediates in cell-wall synthesis. Proc. NatI. Acad. Sci. U.S.A. 53:881-889. Anderson, J. S., P. M. Meadow, M. A, Haskih, and J, L. Strominger. 1966. Biosynthesis of the peptidoglycan of bacterial cell walls. 1. Utilization of uridine diphosphate acetylmuramyl pentapeptide and uridine diphosphate atetylglucosamine for peptidoglycan synthesis by particulate enzymes ftom Staphylococcus aureus and Micrococcus lysodeikticus. Arch. Biochem. Biophys. ll6!487-515. Bordet, C., and H. R. Perkins. 1970. lodinated vancomycin and mucopeptide biosynthesis by cell-free preparations fronV Micrococcus lysodeikticus. Biochem. J. 119:877-883. Garrett A. J. 1969. The effect of magnesium Ion deprivation on the synthesis of nucopeptide and its precursbrs in Bacillus subtilis. Biochem. J. 115'419-430. Ghuysen, J. M., E. Bricas, M. Lache, and M. LeyhBouille. 1968. Structure of the cell walls of Micrococcus lysodeikticus. III. Isolation of a new peptide dimer. Biochemistry 7:1450-1460. Ito, E., and J. L. Strominger. 1962. Enzymatic sy,nthesis of the peptide in bacterial uridine nucleotides. 1. Enzymatic addition of L-alaninf, D-glutamic acid and L-lysine. J. Biol. theni. 237:2689-269&. Ito, E., and J. L. Strominger. 1962. Enzymatic synthesis of the peptide in bacterial uridine nucleotides. II. Enzymatic synthesis and addition of D-alanyl-D-alanine. J. Biol. Chem. 231:2696L-2703. Ito, E., and J. L. Strominger. 1964. Enzymatic synthesis of the peptide in bacterial utidine nucl5otides. III. Purification and properties of L-lysine-adding enzyme. J. Biol. Chem. 239:210-214. Katz, W., M. Matsuhashi. C. P. Dietrich, and J. L. Strominger. 1967. Biosynthesis of the peptidoglyean of bacterial cell walls. IV. Incorporation of glycine in Micrococcus lysodeikticus. J. Biol. Chem. 242:3207-3217. Lowry, 0. H., N. J. Rosebrough, A. L. Farr. and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. Mirelman, D., and R. Bracha. 1974. Effect of penicillin on the in vivo formation of the D-alanyl-L-alanine peptide cross-linkage in cell walls of Micrococcus luteus. Antimicrob. Ageits Chemother. 5:663-B66. 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. Mirelman, D., and N. Sharon. 1972. Biosynthesis of peptidoglycan by a cell wall preparation of Staphylococcus dureus and its inhibition by penicillin. Biochem. Biophys. Res. Commun. 46:1909-1917. Pellon, G., C. Bordet, and G. Michel. 1974. Membranepeptidoglycan association in the in vitro biosynthesis of the peptidoglycan of Micrococcus luteus. Ann. Microbiol. (Paris) 125B:149-158. Petit, J. F., E. Munoz, and J. M. Ghuysen. 1966. Peptide cross-links in bacterial cell wall peptidoglycans studied with specific endopeptidases from Streptomyces albtis G. Biochemistry 5:2764-2776. Schleifer, K. H., and 0. Kandler. 1967. Micrococcus lysodeikticus: a new type of cross-linkage of the murein. Biochem. Biophys. Res. Commun. 28:965-972. Strominger, J. L., and R. H. Threnn. 1959. Accumulation of a uridine nucleotide in Staphylococcus aureus as the consequence of lysine deprivation. Biochim. Biophys. Acta 36:83-92. Strominger, J. L., R. H. Threnn, and S. S. Scott. 1959.
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Oxamyfin, a competitive antagonist of the incorporation of D-alanine into a uridine nucleotide in Staphylocofc4s aure4s. J. Am. Chem. Soc. 81:3803-3804. 19. Ward, J. B. 1974. The synthesis of peptidoglycan in an autolysindeficient mutant of Bacillus licheniformis NCTC 6346 and the effect of ,-lactam antibiotics,
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bacitracin and vancomycin. Biochem. J. 141:227-241. 20. Ward, J. B., and H. R. Perkins. 1974. Peptidoglycans biosynthesis by preparations from Bacillus licheniformis: cross-linking of newly synthesized chains to preformed cell wall. Biochem. J. 139:781-784.
Vol. 125, No. 2 Printed in U.S.A.
Peptidoglycans Synthesized by a Membrane Preparation of Micrococcus luteus G. PELLON, C. BORDET,
AND
G. MICHEL*
Laboratoire de Biochimie Microbienne, Universite Claude Bernard Lyon, 69621 Villeurbanne, France Received for publication 7 October 1975
By incubation of cell-free particulate preparations from Micrococcus luteus with nucleotidic precursors uridine 5'-diphosphate-N-acetylglucosamine and uridine 5'-diphosphate-N-acetylmuramic acid-L-Ala-D-iso-Glu-L-Lys-D-Ala-DAla, several types of peptidoglycans were obtained: soluble peptidoglycan, insoluble peptidoglycan bound to the membrane and solubilized by trypsin, and peptidoglycan, which remained insoluble after the action of trypsin. The structure of each type of peptidoglycan was studied by action of lytic enzymes and separation of the fragments on Sephadex. Soluble peptidoglycans consist of a mixture of un-cross-linked polymers of various molecular weights. Trypsinsolubilized peptidoglycans are also a mixture of polymers of various sizes. They contain a preponderance of un-cross-linked material and some bridges with dimer peptides. Insoluble peptidoglycans, after the action of trypsin, contain about 50% of un-cross-linked peptide residues; in the other moiety, peptide units are cross-linked by D-Ala--L-Lys and D-Ala-L-Ala bonds which characterize the natural peptidoglycan. Therefore, the cell-free particulate preparation possesses the whole enzymatic system necessary for synthesis of cross-linked peptidoglycan.
A particulate preparation from Micrococcus luteus NCTC 2665 has been described which catalyzes peptidoglycan synthesis in vitro by incubation with the nucleotide precursors uridine 5'-diphosphate (UDP)-N-acetylglucosamine (UDPGlcNAc) and UDP-N-acetylmuramic acid-L-Ala-D-iso-Glu-L-Lys-D-Ala-DAla (UDPMurNAc-pentapeptide) (1, 2). The peptidoglycan products are characterized as chromatographically immobile material. More recently, other enzymatic systems have been prepared from Staphylococcus aureus (13), Micrococcus luteus (12), and Bacillus licheniformis (19, 20). These systems contain membrane material which brings the enzymes necessary to the biosynthesis of peptidoglycan and cell walls which are acceptors in the last step of this biosynthesis. In the presence of such acceptors, cross-linked peptidoglycans are formed. With a cell wall-free membrane preparation from M. luteus, a mixture of soluble and insoluble lysozyme-sensitive peptidoglycans was obtained in the presence and in the absence of penicillin (3). A recent study showed that a major part of the insoluble polymer obtained in the presence of penicillin is solubilized by trypsin digestion, so its insolubility is due to an association with the membrane (14). In this paper, we report a study of the
structures of the soluble and insoluble peptidoglycans obtained in the absence of penicillin from a cell wall-free preparation from M. luteus. These structures had not previously been investigated, and no evidence for transpeptidation had been reported with cell wall-free preparations from M. luteus (2, 9).
509
MATERIALS AND METHODS
Paper and thin-layer chromatography. Paper chromatography was carried out using the following solvents: (A) isobutyric acid-0.5 M NH4OH (5:3, vol/vol); (B) ethanol-1 M ammonium acetate (5:2, vol/vol) (pH 7.4). Whatman 3MM paper was used for precursor purification and soluble peptidoglycan preparation; it was first washed with 1 M ammonium acetate and water. Thin-layer chromatography of monomer and dimer peptides was performed on cellulose MN 300 G in butanol-pyridine-acetic acid-water (30:20:6:24). The separation of e-N-dinitrophenylated lysine was carried out by thin-layer silica gel chromatography in chloroform-methanol-acetic acid-water (65:25:13:9). Preparation of UDPMurNAc-L-Ala--y-D-Glu-LLys-D-Ala-D-Ala and UDPMurNAc-L-Ala--y-DG1U-L- ['4C]Lys-D-Ala-D-Ala. Unlabeled nucleotide muramyl-pentapeptide was prepared by accumulation in cells of M. luteus grown in a medium containing ethylenediaminetetraacetate as described by Garrett (4). The radioactive precursor was obtained by suc-
510
PELLON, BORDET, AND MICHEL
cessive additions of L- [4C]Lys and D-Ala-D-Ala to the nucleotide-dipeptide extracted from lysine-deprived cells of S. aureus (17). These additions were carried out by incubating suitable substrates with a crude extract from S. aureus (6). The method described by Ito and Strominger (6-8) was used with some modifications. L- ['4C ]lysine (specific activity, 255 mCi/mmol) was supplied by the C.E.A. (Saclay, France); D-Ala-D-Ala was commercially available (Cyclo Chemical Corp.). Crude enzyme protein concentration was estimated by the method of Lowry et al. (10). For L-lysine addition, the reaction mixture consisted of 16.5 nmol of L- [14C ]lysine (2.5 x 106 counts/ min), 16 nmol of nucleotide muramyl-dipeptide, 200 nmol of adenosine 5'-triphosphate, 400 nmol of MgCl,, 20 nmol of KF, and 20 Ml of crude enzyme preparation in 85 Ml of 0.05 M tris(hydroxymethyl)aminomethane (Tris)-hydrochloride buffer, pH 8.9 (final volume). Incubations were carried out at 37 C for 2.5 h. The reaction was stopped by boiling for 2 min, and then the incubation mixture was applied to Whatman 3MM paper and developed for 24 h in solvent A. The radioactive nucleotide muramyl-tripeptide product was identified using an authentic sample obtained from S. aureus by accumulation with D-Cycloserine (18) and purified by chromatography on Whatman 3MM paper in solvent B. For D-Ala-D-Ala addition, incubation mixtures contained 5 nmol of nucleotide muramyl- ["4C tripeptide (106 counts/min), 6 nmol of D-Ala-D-Ala, 200 nmol of adenosine 5'-triphosphate, 100 nmol of MnCl,, 20 nmol of KF, and 20 Mil of crude enzyme preparation in 70 Ml of 0.05 Tris-hydrochloride buffer, pH 8.7 (final volume). They were incubated for 2.5 h at 37 C. After boiling for 2 min, the reaction mixture was applied to Whatman 3MM paper and chromatographed repeatedly in solvent B until the nucleotide muramyltripeptide and the nucleotide muramyl-pentapeptide were well separated. The nucleotide muramyl["IC ]pentapeptide was isolated and diluted with unlabeled precursor to a final specific activity of 28 mCi/mmol. Particulate enzyme preparation. M. luteus NCTC 2665 was grown at 35 C in a rotatory shaker on brain heart infusion medium (B. D. Merieux, 69 Marcy L'Etoile, France). Cells were harvested at mid-log phase and washed with 0.02 M Tris-hydrochloride buffer (pH 8.0). All the following operations were carried out at 4 C. Particulate enzyme was prepared by grinding the cells with three times their wet weight of levigated alumina as previously described (3). The broken cells were suspended into 50 mM Tris-hydrochloride buffer, pH 7.5, containing 0.1 M MgCl, and 1 mM 2-mercaptoethanol (15 ml/g of cells, wet weight) and centrifuged twice at 10,000 x g for 10 min. The pellet was discarded, and the supernatant was centrifuged at 105,000 x g for 1 h. The membrane pellet was washed twice with buffer and finally suspended to give a protein concentration of 7 mg/ml as estimated by the method of Lowry et al. (10). Particulate enzyme stability during the storage could be improved by the
J. BACTERIOL.
addition of UDP-GlcNAc to the preparation (27 nmollmg of protein). Preparation of labeled peptidoglyeans. Reaction mixtures contained the following components: 9 nmol of UDPMurNAc- [14C ]pentapeptide (1.3 x 101 counts/ min), 13.5 nmol of UDPGlcNAc, 6 umol of MgCl2, 7.5 Amol of Tris-hydrochloride buffer (pH 8.6), and about 500 Mg of particulate enzyme protein in a final volume of 140 Ml. After incubation for 2 h at 37 C, the reaction was stopped by boiling for 2 min. Incubation mixtures were centrifuged at 24,000 x g for 10 min. The pellet, washed three times with incubation buffers, was then used as the "crude insoluble peptidoglycan." The soluble fraction was applied to Whatman 3MM paper and developed in solvent A for 24 h. By autoradiography, the chromatogram showed three radioactive spots: a peptidoglycan product (R,, 0 to 0.15), the residual nucleotide muramyl-pentapeptide precursor (R,, 0.26), and an unidentified compound, possibly a nucleotide muramyl-tetrapeptide (R,, 0.35). The product with an R, of 0 to 0.15 was eluted and used as the "soluble peptidoglycan." The crude insoluble peptidoglycan was digested for 4 h at 37 C with 50 Mg of trypsin in 150 Ml of 0.1 Tris-hydrochloride buffer (pH 8.4). After boiling for 5 min, the reaction mixture was centrifuged at 24,000 x g for 10 min. Radioactive material was found in the supernatant and in the pellet. The pellet was suspended with 50 Mg of trypsin and incubated again in a Tris-hydrochloride buffer (pH 8.4) for 3 h. After centrifugation, the supernatant contained a negligible amount of radioactive product. The pellet, washed three times, consisted of a radioactive product sensitive to lysozyme. It was used as the "insoluble peptidoglycan." The supernatant consisted of radioactive trypsin-solubilized polymers. Degradation of labeled peptidoglycans. Four different peptidoglycan hydrolases have been used during this study: lysozyme, Myxobacter AL-1 protease, N-acetylmuramyl-L-alanine amidase, and MLendopeptidase from Streptomyces albus G. The amidase and endopeptidase from S. albus G were obtained from J. M. Ghuysen (Liege, Belgium), and Myxobaeter AL-1 protease was obtained from J. F. Petit (Orsay, France). The activities of these enzymes were tested on specific substrates. Incubations were carried out on samples containing 3,000 to 5,000 counts/min as described below. (i) Digestion by lysozyme and Streptomyces amidase. Incubations were performed at 37 C for 24 h with shaking. The reaction mixtures contained the radioactive peptidoglycan and 40 Mg of lysozyme in 500 Ml of 0.01 M sodium acetate buffer (pH 5.4). In some experiments, after incubation the mixture was completed with 3 gl of Streptomyces amidase preparation and 10 Mmol of sodium acetate buffer (pH 5.4) in a final volume of 560 MlA and incubated for 6 h at 37 C. (ii) Digestion by ML-endopeptidase and lysosyme. The radioactive peptidoglycan was incubated for 5 h at 37 C with 3 Ml of ML-endopeptidase preparation in 200 Ml of 0.01 M veronal buffer (pH 9.0). The reaction mixture was neutralized with acetic acid and incubated at 37 C for 24 h with 40 Mg of
511
IN VITRO SYNTHESIS OF PEPTIDOGLYCANS
VOL. 125, 1976
lysozyme and 5 Mmol of sodium acetate buffer (pH 5.4) in a final volume of 500 MIl. (iii) Digestion by Myxobacter AI-i protease. The radioactive polymers were digested by incubation for 24 h at 37 C with shaking with 10 Il of Myxobacter AL-1 protease preparation (5) in 475 ul of 0.01 M veronal buffer (pH 9). Fractionation and identification of radioactive compounds. Filtration of soluble peptidoglycans and peptidoglycan fragments was carried out in 0.1 M acetic acid on two linked Sephadex G-50 and Sephadex G-25 columns (VO = 19 ml, V, = 48 ml) at a flow rate of 5 ml/h. The volume of the fractions was 0.3 ml, and the radioactivity of every second fraction was measured in a Packard Tri-Carb 2001 liquid scintillation spectrometer. Samples were mixed with 5 ml of the following scintillation liquid: 7 g of 2,5diphenyloxazole, 0.15 g of 1,4-bis-(5-phenyloxazol-2yl)-benzene, and 50 g of naphthalene in 1,030 ml of toluene-dioxane-ethanol (20:80:3). In some experiments, activity of the products was rather low. To distinguish radioactive material from the background and obtain reliable results, every fraction was counted four times for 10 min. RESULTS
Properties of soluble peptidoglyeans. Soluble peptidoglycans were obtained after a 2-h incubation. The time course of the biosynthesis of soluble and insoluble polymers has been previously studied (3), and a greater amount of polymers was not found when an incubation longer than 2 h was performed. Soluble peptidoglycans were purified from the residual precursor by paper chromatography (see above). To check their molecular size, they were fractionated on two linked Sephadex G-50 and G-25 columns; soluble peptidoglycans were shown to be a mixture of polymers with various molecular
weights (KD 0.1 to 0.8). These polymers were treated by lysozyme, and the lysozyme digest was fractionated on Sephadex G-50 and G-25; 95% of the radioactive product was found in a single peak (KD = 0.7). This is a disaccharidepeptide monomer (GlcNAc-MurNAc-tetra- or pentapeptide). A radioactive standard monomner gives a single peak (KD = 0.7) by gel filtration in the same experimental conditions. Thus, soluble peptidoglycans appear to be polydisperse, un-cross-linked polymers. Occurrence of a membrane-bound fraction in the crude insoluble peptidoglycan. The crude insoluble peptidoglycan still contained protein components derived from particulate preparation. This peptidoglycan was treated by trypsin, centrifuged, and washed three times. Radioactivity was found in the supernatant and in the pellet (Table 1). A further treatment of the pellet by trypsin gave a small amount of radioactive soluble product (< 3%). The pellet consisted of insoluble peptidoglycan and was almost entirely degraded by lysozyme. The radioactive product solubilized by trypsin treatment and found in the supernatant after centrifugation was shown to be a membrane-bound peptidoglycan (14). When the peptidoglycan was prepared in the presence of 10 gg of benzylpenicillin per ml added to the incubation medium, both soluble and crude insoluble polymers were synthesized in about the same proportions as in the absence of antibiotic (3), but the amount of radioactivity solubilized by treatment of the crude insoluble peptidoglycan with trypsin increased to a higher value, about 80% (Table 1). These two components (insoluble peptidogly=
TABLE 1. Composition of the crude insoluble peptidoglycana Expression of radioactivity
Peptidoglycan fraction
Assay no. lb % dpm
Assay no. 2° % dpm
Assay no. 3c dpm
%
100 100 100 10,300 34,000 Crude insoluble peptidoglycan 8,234 78 51 60 8,010 17,600 4,950 Membrane-bound peptidoglycan solubilized by trypsin 12 39 35 1,200 13,400 2,910 True insoluble peptidoglycan solubilized by lysozyme aProportions of membrane-bound soluble peptidoglycan and insoluble peptidoglycan in the crude insoluble peptidoglycan. The crude insoluble peptidoglycan was prepared as described. In assay 3, 10 gg of benzylpenicillin per ml was added to the incubation medium. It was digested by trypsin to solubilize the membrane-bound fraction; the insoluble residue was then treated by lysozyme, giving soluble fragments. These trypsin-soluble and lysozyme-soluble radioactivities were respectively used as estimations for the membranebound soluble peptidoglycan and for the true insoluble peptidoglycan. They were expressed as disintegrations per minute and as an average percentage of the initial radioactivity. For conditions, see the text. 'No penicillin added. cPenicillin added at a concentration of 10 gg/ml.
512
PELLON, BORDET, AND MICHEL
J. BACTERIOL.
can and membrane-bound peptidoglycan) were prepared on a large scale, and their structures were determined. Nature of membrane-bound peptidoglycans. The trypsin-solubilized fraction was submitted to filtration on two linked Sephadex G-50 and Sephadex G-25 columns. As indicated by the gel filtration profile, it consists of a polydisperse polymer containing a high percentage of large-molecular-size fractions (Fig. 1). Then the peptidoglycan was hydrolyzed by lysozyme. The resulting disaccharide-peptide fragments were incubated with S. albus G amidase which splits MurNAc - L-Ala linkages, giving rise to peptides (Fig. 2). These degradation products were fractionated by gel filtration and characterized essentially as peptide monomer (KD = 0.8) (major product) and peptide dimer (KD = 0.58) (Fig. 3). These peptides have the same KD values and the same R, values in thin-layer chromatography as the peptide monomer (R, = 0.29) and the peptide dimer (R, = 0.17) obtained from the natural peptidoglycan after treatment with Myxobacter enzyme. Thus the membrane-bound peptidoglycan contains a large majority of un-cross-linked peptides and some interpeptide bridges in a lesser amount. Structural investigations on the insoluble peptidoglycan. The insoluble peptidoglycan
0
0.4
was degraded by lysozyme and then by Streptomyces amidase. The resulting peptide fragments were resolved by gel filtration which gave a major product (KD = 0.8) characterized as the peptide monomer, a dimer (KD = 0.6), and several oligomers of higher molecular weight (Fig. 4.). Thus the insoluble peptidoglycan appears to be a polymer which contains cross-linked residues. However, the degree of cross-linking is much smaller than that of the natural peptidoglycan from the cell walls of M. luteus. This peptidoglycan is known to contain three classes of peptide arrangements: a small proportion of monomers and dimers (D-Ala L-Lys) and a major proportion of oligomers, mostly hexamers with D-Ala - L-Ala linkages (head-to-tail structure) (5). The structure of the insoluble peptidoglycan formed in vitro was further investigated with special attention to the occurrence of such head-to-tail oligomers. First, the peptidoglycan was incubated with Streptomyces ML-endopeptidase which specifically cleaves D-Ala _ L-Lys linkages (15). The glycan moiety was then degraded by lysozyme, and the following products were separated by gel filtration: a disaccharide-peptide monomer (KD = 0.68), a dimer (KD = 0.48), and highermolecular-weight oligomers (Fig. 5). With these conditions of degradation, only dimer and higher oligomers containing -AlaL-Ala cross-links should persist in the digest; other oligomers with -Ala _ L-Lys should have
0.0
o.s
KD
FIG. 1. Sephadex filtration of the membrane-bound peptidoglycan solubilized by trypsin.
IN VITRO SYNTHESIS OF PEPTIDOGLYCANS
VOL. 125, 1976
-(a) s GloNAc
9
-
(a)
(a) MurNAc r-
(b),(c)-
T
V
l
*
(a) GIcNAc
-
MurNAc
(b).(c) --1-
t
D-Glu
O-GIU (d)
(c)
L- Lys - D-Ala
T
L-Ala
L-AlI |
513
T
L-Ala
-
D-Clu
- L-Lys - D-Ala
I
+ L-Lys - D-Ala
* 9~~~~~~~~~~ FiG, 2. A portion of cross-linked peptidoglycan strand. (a) Site of action of lysozyme; (b) site of action of amidase; (c) site of action of MyxobacterAL-I protease; (d) site of action of ML-endopeptidase.
0
0.4
0.6
0.6
KD
FiG. 3. Sephadex filtration of the membrane-bouwnd peptidoglycan degraded by lysozyme and Streptomyces amidase.
been hydrolyzed into monomers. Thus the dimer (KD = 0.48) should consist of a disaccharide-peptide dimer with a D-Ala _ L-Ala linkage derived from a head-to-tail trimer by cleavage of the D-Ala -- L-Lys bond (Fig. 2). The occturrence of peptide oligomers with D-Ala D L-Lys bonds was studied by incubation of the insoluble peptidoglycan with Myxobacter AL-1 enzyme. This enzyme splits MurNAc L.Ala and D-Ala -- L-Ala linkages; thus, oligomers with such linkages give imonomers and dimners with D-Ala , L-Lys linkage (Fig. 2). After incubation with Myxobacter protease and gel filtration of the peptidoglycan digest, peptide monomer (KD = 0.78) and peptide dlimer (Ka = 0,6) were chaTacteriwed (Fig. 6). The peptidases which werp used for the structural determination of peptidoglycan have complementary activities. A control of the complete hydrolysis of D-Ala -- L-Lys linkages by ML
endopeptidase and D-Ala - L-Ala linkages by Myxobacter endopeptidase was carried out. An insoluble peptidoglycan was first hydrolyzed by ML-endopeptidase and then by Myxobacter AL-1 endopeptidase. These hydrolyses were performed under the same conditions as those used previously (data shown in Fig. 5 and 6). The products were studied by gel filtration on Sephadex G-50 and G-25 columns as described above. The radioactivity was located in a single peak (KD = 0.8) as shown in Fig. 7. It is obvious that dimers and higher peptides oligomers resulting from ML-endopeptidase and lysozyme hydrolysis (Fig. 5) contain D-Ala _ L-Ala linkages which have been split by Myxobacter endopeptidase hydrolysis. The presence of D-Ala - L-Ala linkages was confirmed by analysis of peptides dimers obtained after hydrolysis of the insoluble peptidoglycan by ML-endopeptidase, lysozyme, and
514
J. BACTERIOL.
PELLON, BORDET, AND MICHEL Radloactivity
cpmnml
FIG. 4. Sephadex filtration of the insoluble peptidoglycan degraded by lysozyme and Streptomyces amidase. Radioactivity cpm/ml
150
100I
\
50
0 0
0.2
0.4
0.6
0.6
K
FIG. 5. Sephadex filtration of the insoluble peptidoglycan degraded by ML-endopeptidase and lysozyme.
amidase. The dimer peptides were separated by gel filtration and submitted to dinitrophenylation. A control was carried out on the monomer peptide. The dinitrophenylated peptides were hydrolyzed for 15 h by 6 N HCl at 110 C, and the products were studied by thin-layer chromatography. In both cases, in monomer and dimer peptides 85% of the radioactivity was located as E-2,4-dinitrophenyl-Lys; thus, the interpeptide
linkage in the dimer must be a D-Ala linkage.
-
L-Ala
DISCUSSION Upon incubation with nucleotide precursors, a cell wall-free membrane preparation from M. luteus gives rise to a soluble polymer and an insoluble material (3). The soluble polymer consists of a mixture of un-cross-linked peptido-
IN VITRO SYNTHESIS OF PEPTIDOGLYCANS
VOL. -125, 1976
glycans with various molecular weights which give only monomer units after lysozyme hydrolysis. It can be assumed that they are either precursors in the biosynthesis of insoluble polymers or degradation products resulting from the action of lytic enzymes and particularly of endopeptidases contained in membrane preparations. This last hypothesis seems likely since the formation of soluble peptidoglycans increases more rapidly than the formation of insoluble peptidoglycans with the time of incubation (3). The insoluble materials consists of a mixture of two types of polymers: a membrane-bound peptidoglycan solubilized by treatment with trypsin and an insoluble peptidoglycan.
515
The membrane-bound peptidoglycan consists of polymers with a small percentage of bridges, since essentially monomer units and dimer units in a lesser amount were found after lysozyme degradation and treatment with amidase. This material could be a precursor of the cross-linked insoluble peptidoglycan still attached to the active site of the membrane where the cross-linkings are realized by transpeptidation. Polymers which remain insoluble after digestion with trypsin consist of partially crosslinked peptidoglycans. Evidence for transpeptidation in M. luteus has been found with a cell wall preparation, and an explanation of the biosynthetic process has been proposed: the
R-dloectivity cpm ml
100
50
0
0.4
0.2
0.8
0.6
KD
FIG. 6. Sephadex filtration of the insoluble peptidoglycan degraded by Myxobacter AL-I protease.
0.2
0.4
0.6
0
%
Fr.. 7. Sephadex filtration of the insoluble peptidoglycan degraded by ML-endopeptidase and Myxobacter AL-I protease.
516
J. BACTERIOL.
PELLON, BORDET, AND MICHEL
newly synthetized strands of peptidoglycan are attached to the pre-existing cell wall present in the medium by transpeptidation and in part by transglycosylation (11). Our membrane preparation is quite different from the cell wall preparation of Mirelman et al. (12). Our results indicate that pre-existing cell walls are not essential for the formation of peptide crossbridges. A part (about 20%) of the synthetized peptidoglycan is insoluble after a trypsin treatment, and this peptidoglycan was found to contain both types of interpeptide linkages: D-Ala - L-Lys and D-Ala - L-Ala. The comparison of the natural peptidoglycan of M. luteus with the peptidoglycans synthetized in vitro by membrane preparation shows large differences in the degree of cross-linking. Ghuysen et al. found a wall structure in which 5% of peptide units occur as monomers, 44% as dimers, 15% as trimers, and 60% as hexamers (5). The peptidoglycan synthetized in vitro contains 50% soluble un-cross-linked polymers, and 30% of the polymers are associated with the membrane and solubilized by trypsin digestion; they consist of poorly cross-linked peptidoglycans and give essentially monomers and some dimers after enzyme hydrolysis. Only 20% of total peptidoglycan occurs as insoluble, crosslinked polymers whose proportions of crossbridges were determined by gel fractionation of the fragments obtained after specific cleavages. Fifty percent of the peptide fragments are not cross-linked, 10% are linked by a D-Ala _ L-Lys bridge and give dimers by lysozyme and amidase treatment, and 40% are linked by D-Ala _ L-Ala linkages and give oligomers after hydrolysis by lysozyme and amidase. The formation of oligomers with D-Ala L-Ala linkages results from the sequential action of an amidase and a transpeptidase (16). Moreover, two distinct transpeptidases with different sensitivity to penicillin are involved in the synthesis of the two types of interpeptides bridges: D-Ala _ L-Ala and D-Ala - L-Lys (11). These enzymes must be present in our particulate preparation from M. luteus, which contains the whole system for the peptidoglycan biosyn-
2.
3.
4.
5.
6.
7.
8.
9.
10. 11.
12.
13.
14.
thesis.
ACKNOWLEDGMENTS We are indebted to J. M. Ghuysen, Departement de Botanique, Universite de Liege, Belgium, for a gift of Streptomyces amidase and ML-endopeptidase, to J. F. Petit, Institut de Biochimie, Orsay, France, for a gift of Myxobacter AL-1 protease, and to the Commissariat a l'Energie Atomique, Departement de Biologie, Saclay, France, for financial aid.
15.
16. 17.
LITERATURE CITED 1.
Anderson, J. S.. M. Matsuhashi, M. A. Haskin, and J. L.
18.
Strominget. 1965. Lipid-phosphoacetyltnutainyl-pentapeptide and lipid-phosphbdlsacchatide-pentapeptide: presunied membrane transport interthediates in cell-wall synthesis. Proc. NatI. Acad. Sci. U.S.A. 53:881-889. Anderson, J. S., P. M. Meadow, M. A, Haskih, and J, L. Strominger. 1966. Biosynthesis of the peptidoglycan of bacterial cell walls. 1. Utilization of uridine diphosphate acetylmuramyl pentapeptide and uridine diphosphate atetylglucosamine for peptidoglycan synthesis by particulate enzymes ftom Staphylococcus aureus and Micrococcus lysodeikticus. Arch. Biochem. Biophys. ll6!487-515. Bordet, C., and H. R. Perkins. 1970. lodinated vancomycin and mucopeptide biosynthesis by cell-free preparations fronV Micrococcus lysodeikticus. Biochem. J. 119:877-883. Garrett A. J. 1969. The effect of magnesium Ion deprivation on the synthesis of nucopeptide and its precursbrs in Bacillus subtilis. Biochem. J. 115'419-430. Ghuysen, J. M., E. Bricas, M. Lache, and M. LeyhBouille. 1968. Structure of the cell walls of Micrococcus lysodeikticus. III. Isolation of a new peptide dimer. Biochemistry 7:1450-1460. Ito, E., and J. L. Strominger. 1962. Enzymatic sy,nthesis of the peptide in bacterial uridine nucleotides. 1. Enzymatic addition of L-alaninf, D-glutamic acid and L-lysine. J. Biol. theni. 237:2689-269&. Ito, E., and J. L. Strominger. 1962. Enzymatic synthesis of the peptide in bacterial uridine nucleotides. II. Enzymatic synthesis and addition of D-alanyl-D-alanine. J. Biol. Chem. 231:2696L-2703. Ito, E., and J. L. Strominger. 1964. Enzymatic synthesis of the peptide in bacterial utidine nucl5otides. III. Purification and properties of L-lysine-adding enzyme. J. Biol. Chem. 239:210-214. Katz, W., M. Matsuhashi. C. P. Dietrich, and J. L. Strominger. 1967. Biosynthesis of the peptidoglyean of bacterial cell walls. IV. Incorporation of glycine in Micrococcus lysodeikticus. J. Biol. Chem. 242:3207-3217. Lowry, 0. H., N. J. Rosebrough, A. L. Farr. and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. Mirelman, D., and R. Bracha. 1974. Effect of penicillin on the in vivo formation of the D-alanyl-L-alanine peptide cross-linkage in cell walls of Micrococcus luteus. Antimicrob. Ageits Chemother. 5:663-B66. 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. Mirelman, D., and N. Sharon. 1972. Biosynthesis of peptidoglycan by a cell wall preparation of Staphylococcus dureus and its inhibition by penicillin. Biochem. Biophys. Res. Commun. 46:1909-1917. Pellon, G., C. Bordet, and G. Michel. 1974. Membranepeptidoglycan association in the in vitro biosynthesis of the peptidoglycan of Micrococcus luteus. Ann. Microbiol. (Paris) 125B:149-158. Petit, J. F., E. Munoz, and J. M. Ghuysen. 1966. Peptide cross-links in bacterial cell wall peptidoglycans studied with specific endopeptidases from Streptomyces albtis G. Biochemistry 5:2764-2776. Schleifer, K. H., and 0. Kandler. 1967. Micrococcus lysodeikticus: a new type of cross-linkage of the murein. Biochem. Biophys. Res. Commun. 28:965-972. Strominger, J. L., and R. H. Threnn. 1959. Accumulation of a uridine nucleotide in Staphylococcus aureus as the consequence of lysine deprivation. Biochim. Biophys. Acta 36:83-92. Strominger, J. L., R. H. Threnn, and S. S. Scott. 1959.
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IN VITRO SYNTHESIS OF PEPTIDOGLYCANS
Oxamyfin, a competitive antagonist of the incorporation of D-alanine into a uridine nucleotide in Staphylocofc4s aure4s. J. Am. Chem. Soc. 81:3803-3804. 19. Ward, J. B. 1974. The synthesis of peptidoglycan in an autolysindeficient mutant of Bacillus licheniformis NCTC 6346 and the effect of ,-lactam antibiotics,
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