Vol. 125. No. 2
JOURNAL OF BACTERIOLOGY, Feb. 1976. p. 626-634 Copyright ( 1976 American Society for Microbiology
Printed
in
U.S.A.
Biosynthesis of Peptidoglycan in Staphylococcus aureus: Incorporation of the Nc-Ala-Lys Moiety into the Peptide Subunit of Nascent Peptidoglycan Department of Biochemistry
J. C. SWENSON AND F. C. NEUHAUS* and Molecular Biology, Northwestern University, Evanston, Illinois 60201
Received for publication 22 September 1975
UDP-MurNAc-Ala-DGlu-Lys(N'-Ala)-DAla-DAla was isolated from extracts of Staphylococcus aureus Copenhagen. This nucleotide accumulated in media deficient in glycine. To establish its role in peptidoglycan biosynthesis, the nucleotide-hexapeptide was compared with UDP-MurNAc-Ala-DGlu-LysDAla-DAla in the reaction catalyzed by phospho-MurNAc-pentapeptide translocase and in the membrane-catalyzed nascent peptidoglycan-synthesizing system. In the exchange reaction catalyzed by the translocase, the Rmax and RmaJKm are 1.79 ,uM/min and 4.47 x 10-/min, respectively, for UDP-MurNAcpentapeptide and 1.81 AM/min and 4.46 x 10-2/min, respectively, for UDP-MurNAc-hexapeptide. In the synthesis of nascent peptidoglycan, the Vmax is 1.8 AM/min x 102 for both the nucleotide-hexapeptide and -pentapeptide. The Vmax/Km is 5.6 x 10-' and 4.3 x 10-4/min for the nucleotide-pentapeptide and -hexapeptide, respectively. Schleifer, Hammes, and Kandler (Adv. Microb. Physiol., in press) observed that growth of S. aureus Copenhagen on a glycinepoor medium results in a peptidoglycan structure in which 20% of the lysine residues are substituted at the e-amino group by L-alanine residues that do not participate in interpeptide bridge formation. The in vitro studies demonstrate that UDP-MurNAc-Ala-DGlu-Lys(N-Ala)-DAla-DAla is a possible precursor of the NE-Ala-Lys moiety. In Staphylococcus aureus Copenhagen, the interpeptide bridge of the peptidoglycan consists of five glycine residues (1, 19) when the organism is cultured in a glycine-rich medium. When it is grown in a glycine-poor medium, the walls show a decrease in cross-linking (K. H. Schleifer, Proc. Soc. Gen. Microbiol. 57:xiv, 1969; K. H. Schleifer, W. P. Hammes, and 0. Kandler, Adv. Microb. Physiol., in press). Under these conditions a significant percentage (20%) of the lysine residues of the peptidoglycan are substituted at the e-amino group by alanine residues that do not participate in interpeptide bridge formation. Since the presence of N'-Ala-Lys dipeptide moieties in the peptidoglycan can be correlated with the degree of cross-linking in S. aureus Copenhagen, the mechanism by which this dipeptide moiety is introduced into this polymer is of interest. The biosynthesis of peptidoglycan is catalyzed by a series of membraneassociated enzymes that utilize two nucleotideactivated precursors, UDP-N-acetylglucosamine and UDP-MurNAc-Ala-DGlu-Lys-DAlaDAla. A possible precursor of the N'-Ala-Lys moiety is the UDP-MurNAc-hexapeptide:
UDP - MurNAc - Ala - DGlu - Lys(N' - Ala) DAla-DAla. Nucleotide-activated hexapeptides have been found in two different species of' bacteria, Lactobacillus viridescens (11) and Streptococcus faecalis (9). Membrane preparations from L. viridescens utilize the nucleotideactivated hexapeptide for the synthesis of peptidoglycan (11). In contrast to S. aureus Copenhagen, the L-alanine residue linked to the e-amino group of' lysine derived from UDPMurNAc-hexapeptide forms an integral part of the interpeptide bridge of L. viridescens (13) and S. faecalis. In S. aureus Copenhagen it is N terminal. It is the purpose of this paper to describe the isolation of' UDP-MurNAc-Ala-DGlu-Lys(N'Ala)-DAla-DAla from S. aureus Copenhagen and to define conditions for its optimal accumulation. Since this nucleotide represents the possible precursor of the NE-Ala-Lys dipeptide moiety observed by Schleifer et al. (in press), we present in vitro experiments to compare its activity in the reaction catalyzed by phosphoMurNAc-pentapeptide translocase and the peptidoglycan-synthesizing system from S. aureus.
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MATERIALS AND METHODS
UDP-MurNAc-hexapeptide was isolated from the UDP-MurNAc-peptide fraction by chromatography Materials. S. aureus Copenhagen was a gift from J. on DEAE-cellulose. This fraction was applied to a L. Strominger and was maintained as a lyophilized column (0.9 by 57 cm) of DEAE-cellulose, and the culture in vacuo at -25 C. S. aureus H (ATCC 13801) column was then developed with a linear gradient of was purchased from the American Type Culture Col- ammonium bicarbonate buffer (pH 8.5; from 0.1 to lection. UDP- [U- 14C ]GlcNAc was purchased from 0.5 M). A typical elution pattern is shown in Fig. 1. ICN Isotope and Nuclear Division and Amersham/ The partially resolved contaminant in the hexapepSearle. The plastic beads (styrene-divinylbenzene tide fraction was not included when the fractions were copolymer, 20 to 50 mesh, 8% cross-linked) were the pooled. The contaminant was resolved from UDPkind gift of Richard Reitz of the Dow Chemical Co. MurNAc-hexapeptide by rechromatography, as deWhatman DE-52 microgranular diethylaminoethyl scribed above, with a linear gradient of 0.15 to 0.3 M (DEAE)-cellulose was purchased from H. Reeve ammonium bicarbonate. The UDP-MurNAc-hexapeptide was characterized Angel Co. [5-3HJUMP was purchased from Schwarz/ Mann. Peptone and yeast extract were products of as follows. With paper chromatography the hexapepthe Difco Laboratories. Levigated alumina (15 um), tide has R, values of 0.26 and 0.39 in solvents A and B manufactured by Buehler Ltd., was purchased from (see below), respectively. For comparison, the penFisher Scientific Co. The sources of other chemicals tapeptide has R, values of 0.22 and 0.46 and the were described previously (3, 4). tripeptide has R, values of 0.17 and 0.60 in solvents A Isolation of UDP-MurNAc-Ala-DGlu-Lys(N and B, respectively. Hydrolysis of the nucleotide with Ala)-DAla-DAla. For the preparation of the nucleo- 0.1 M HCl at 100 C for 15 min results in the formation tide-activated hexapeptide, S. aureus Copenhagen of UMP and UDP. The amino acid composition of the was grown aerobically in the medium described by hexapeptide is: glutamic acid-alanine-lysine-uridine Heydanek et al. (5) at 37 C. The cells were harvested (1.00:3.95:0.98:1.10). Small amounts of glycine are and transferred to accumulation medium (80 ml/g consistently detected in this fraction. UDP-MurNAc[wet weight] of cells) at 37 C. This medium contained pentapeptide has the ratio 1:3:1:1. Thus, the nucleoper liter: DL-alanine, 400 mg; D-glutamic acid, 200 mg; tide-hexapeptide differs from UDP-MurNAc-pentalysine (monohydrochloride), 200 mg; uracil, 150 mg; peptide by one additional residue of alanine. Since glucose, 10 g; and K,HPO4, 5 g. Penicillin G (1,600 the additional alanine is not sensitive to the action, U/mg) was added as indicated. After incubation at of D-amino acid oxidase, it is concluded that the 37 C in the accumulation medium (2 h), the cells were extra residue is L-alanine. Thus, the nucleotide has harvested and extracted with cold 10'S trichloroacetic two residues of D-alanine and two of L-alanine. acid (3 ml/g [wet weight] of cells). Partial removal of Reaction of the nucleotide with 1-dimethylaminothe trichloroacetic acid was accomplished by extrac- naphthalene-5-sulfonyl-chloride (dansyl-chloride) foltion with diethyl ether. After the pH was adjusted to lowed by hydrolysis results in the release of dansyl7.8, the UDP-MurNAc-peptide fraction was isolated alanine, indicating that this residue is N terminal. from the other components of the cell extract by the To determine the location of the N-terminal alanine, the sequence analysis procedure of Gray and Smith method of Stickgold and Neuhaus (14).
I
5.0
Upentopeptide DP-MurNAc-
4..0
E
R x 10-8
48
40 NH4HCO3 -0.5M
3..0
01-z
40
c
2..0
UDP-MurNAc-
hexapeptide
32 ±- 0.4M ± 0.3 M 24 - -0.2M +-0.1 M 16
1..0 _
n
8 -a_d
1
_"
30
60
90
120 150 180 Volume (ml)
210
240
0
FIG. 1. Separation of UDP-MurNAc-hexapeptide and UDP-MurNAc-pentapeptide by ion-exchange chromatography. The UDP-MurNAc-peptide fraction, isolated by the method of Stickgold and Neuhaus (14), was applied to a column of DEAE-cellulose, and the nucleotides were eluted with the linear gradient described in the text. The absorbance at 260 nm (0), the exchange activity (R) (A) observed with a 30-Ml sample, and the molarity of the NH4HCO3 (-) are presented.
628
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SWENSON AND NEUHAUS
peptide translocase. For measuring the reaction catalyzed by phospho-MurNAc-pentapeptide translocase, the exchange assay described by Hammes and Neuhaus (3) was used. This assay measures the rate of' exchange of' [3HJUMP with the UMP moiety of UDP-MurNAc-pentapeptide. Exchange activity was calculated by the method of' Struve et al. (18). The; rate of exchange, R, is reported as moles exchanged per liter per minute. The reaction mixture contained: 0.05 M Tris-hydrochloride (pH 7.8); 0.21 M KClI 0.042 M MgCl2; membrane f'ragments (41.3 ug of protein); and [3H]UMP and UDP-MurNAc-peptide UDP-MurNAc-Ala-DGlu-Lys(N '-Ala)-DAla-DAla. Preparation of membrane fragments. Membrane as indicated, in a total volume of' 60 pl. 'I'he incubafragments from S. aureus were prepared bv two tion was carried out at 25 C f'or 10 min. Af'ter the different procedures. For specificity studies with phos- reaction was terminated by placing the mixture in pho-MurNAc-pentapeptide translocase, S. aureus boiling water for 2 min, the amount of' exchange was Copenhagen (late log phase) was disrupted with determined as described previously (3). Peptidoglycan synthesis assay. Peptidoglycan plastic beads, and the membrane f'ragments were isolated by the procedure of' Struve et al. (18). For synthesis was assayed by determining the incorspecificity studies with the peptidoglycan-synthesiz- poration of NAc-["C]glucosamine from UDP-["4C ]ing system, membrane fragments were prepared by GlcNAc into a lysozyme-sensitive, chromatographthe method of Strominger et al. (16), using levigated ically immobile f'raction. That this labeled f'racalumina to disrupt the cells. Membrane fragments tion is peptidoglycan was concluded f'rom its sensitivwere suspended in 0.05 M tris(hydroxymethyl)- ity to lysozyme and its dependence for synthesis on aminomethane (Tris)-hydrochloride (pH 7.8) con- the second substrate UDP-MurNAc-peptide. The retaining 10-4 M magnesium acetate and 10'- M action mixture contained: 0.10 M Tris-hydrochloride, 2-mercaptoethanol and stored at - 196 C. As illus- pH 8.5; 3 mM magnesium acetate; 10 mM NH4CI; 1 trated in Fig. 2, the maximal rate of' peptidoglycan mM ATP; 1 mM 2-mercaptoethanol; 98 MM UDPsynthesis was attained with early-log-phase cells. ["4C]GlcNAc (25 to 35 counts/min per pmol); 2)0 to A fourf'old-higher rate of' synthesis was achieved with 200 Mg of membrane protein; and the indicated S. aureus H than with S. aureus Copenhagen. Thus, concentration of UDP-MurNAc-peptide, in a total f'or the studies with the peptidoglycan-synthesizing volume of 25 pl. Duplicate incubations were carried system, membrane f'ragments f'rom S. aureus H out at 20 C for 1 h. The reactions were terminated by placing the incubation mixtures into a boiling-water were used. Exchange assay for phospho-MurNAc-penta- bath for 2 min. One of the samples was incubated for an additional 30 min at 37 C with 150 Mg of lysozyme. The reaction mixtures were then quantitatively applied to Whatman 3 MM paper, and the paper chromatograms were developed by descending chromatography in solvent A (see below) for about 20 to 22 h. The origins were assayed for chromatographically immobile 14C-labeled material. Peptidoglycan synthesis was found to continue linearly for at least 110 min under the conditions I described. A slight drop f'rom linearity was observed as the concentration of' membrane fragments was 1.0 increased. When UDP- [14C ]GlcNAc was used to 0.5 label the immobile product, signif'icant amounts ot origin material were found to be lysozyme insensitive. This lysozyme-insensitive product also was formed in the absence of UDP-MurNAc-peptide. All measurements of peptidoglycan synthesis with ["'C]GlcNAc have been corrected for lysozyme-insensitive mateHours rial. When incorporation of MurNAc-Ala-DGlu-LysFIG. 2. Specific activities of peptidoglycan synthe- D[14C]Ala-D['4C]Ala was measured. the quantity sis by membrane fragments from S. aureus Copenha- of' the peptidoglycan produced was the same as the amount of lysozyme-sensitive product when [ 4Cgen (A) and S. aureus H (0). The organisms were GlcNAc was the label. grown aerobically in the medium described by HeydaAnalytical methods. For the determination of' nek et al. (5) at 37 C. Samples of cells were harvested radioactivity in aqueous samples, the scintillation at the indicated times, disrupted by the method of Strominger et al. (16), and assayed for peptidoglycan fluid described by Patterson and Greene (10) was synthesis by the procedure described in the text. used; for paper chromatograms the fluid contained Growth of the cells was determined by turbidity at 650 0..3'7( 2,5-diphenyloxazole and 0.025%'( 1,4-bis-[2](5-phenyloxazolyl)benzene in toluene. Descending nm. (2) was used. Treatment of' the nucleotide-hexapeptide by this procedure results in the liberation of' the e-amino group of' lysine. Thus, the L-alanine residue is covalently linked to the e-amino group. The existence of' muramic acid is indicated by a peak, shown on the amino acid analyzer. that has this characteristic elution volume. The similarity of' this nucleotide to UDP-MurNAc-pentapeptide and its identity with that synthesized by L. viridescens and that isolated from S. faecalis suggest that the nucleotide-hexapeptide f'rom S. aureus Copenhagen is
0
W)
PEPTIDOGLYCAN BIOSYNTHESIS IN S. AUREUS
VOL. 125, 1976
629
UDP-MurNAc-pentapeptide(Ne-Ala). The ratio of UDP-MurNAc -Ala-DGlu -Lys(N Ala)-DAla-DAla to UDP-MurNAc-Ala-DGluLys-DAla-DAla increases from 0.06 at mid-log to approximately 0.15 at the transition from the late log to the stationary phase. The effect of penicillin in the accumulation medium was tested to optimize conditions for the production of UDP-MurNAc-Ala-DGluLys(N E-Ala)-DAla-DAla. Cells were harvested from the growing culture at the time of maximal accumulation of the two nucleotides (Fig. 3A, 3.5 h). They were transferred to accumulation medium modified in the amount of penicillin present. As shown in Fig. 3B, the concentration of penicillin in the accumulation medium has no effect on the accumulation of UDP-MurNAc-hexapeptide. The presence of penicillin results in a small decrease in the accumulation of UDP-MurNAc-pentapeptide. Strominger and Threnn (17) reported a significant accumulation of N-acetylamino sugar nucleotides in S. aureus in the absence of penicillin (6.2 umol/liter of culture at half-maximal growth) in a medium similar to the accumulation medium used in the present work. When penicillin (100 /g/ml) was added to the accumulation medium, only a 2.5-fold increase (15.5 gmol) in the amount of these nucleotides was observed. Thus, the lack of effect of penicillin in the accumulation medium in the present work can be compared with the low increase (2.5-fold) observed by Strominger and Threnn. Since the accumulation of UDP-MurNAcpeptides in S. aureus Copenhagen is not greatly affected by the addition of penicillin to the accumulation medium, the presence or absence
paper chromatography was performed with Whatman 3 MM paper. Solvent A contains isobutyric acid-concentrated NH4OH-water (66:2:33, vol/vol/vol). Solvent B contains 0.1 M phosphate buffer (pH 6.8)-(NH4) 2S04-n-propanol (100:60:2, vol/wt/vol). Amino acids were analyzed on a Durrum amino acid analyzer (model D-500) after hydrolysis of the samples with 5.7 N HCl for 12 h at 100 C. Protein was determined by the method of Lowry et al. (7), with bovine serum albumin as the standard.
RESULTS Accumulation of UDP-MurNAc-AlaDGlu-Lys(N e-Ala)-DAla-DAla. In preparing UDP-MurNAc-Ala-DGlu-Lys-DAla-DAla and UDP - MurNAc - Ala - DGlu - Lys(N"-Ala) DAla-DAla from S. aureus Copenhagen, it was observed that the quantities of these nucleotides varied with the growth phase of the bacteria at the time of harvest for transfer to the accumulation medium. To determine the influence of the growth phase on the accumulation of these nucleotides, samples of S. aureus from the growth medium were harvested at different times and transferred to the accumulation medium for 2 h. In Fig. 3A the amount of each nucleotide per 1012 cells is presented as a function of the time of harvest before transfer to the accumulation medium. The incubation in this medium (2 h) does not allow growth of the cells but does result in maximal accumulation of nucleotides (8, 15). Therefore, this plot represents the potential (or capacity) of the cells to synthesize these nucleotides at the time of their harvest from the growing culture. During the late log phase of growth, there is a large increase in the capacity for synthesis of UDP-MurNAcpentapeptide followed by a smaller increase in
B 6
0
H 5 >
0
N 0
4
2
S4 1
2
3
4 5 Hours
6
7
8
0 25 50
100
150
200
[Penicillin] (pg/mi)
FIG. 3. Accumulation potentials (capacities) for the synthesis of UDP-MurNAc-pentapeptide (0) and UDP-MurNAc-hexapeptide (A) as a function of growth (A). Effect of increasing concentrations of penicillin on the accumulation of UDP-MurNAc-pentapeptide (0) and UDP-MurNAc-hexapeptide (A) (B). The growth (-) was measured by turbidity at 650 nm. (A) Cells harvested at the indicated time were suspended in accumulation medium containing 25 ,g of penicillin G per ml. (B) Cells were harvested at 3.5 h and transferred to the accumulation media containing the indicated concentration of penicillin G.
630
SWENSON AND NEUHAUS
J. BACTERIOL.
of another component in the medium might be nificant factor in determining the amount of responsible for the accumulation that is ob- UDP-MurNAc-hexapeptide. Effect of UDP-MurNAc-Ala-DGlu-Lysserved. Glycine is a major component of the interpeptide bridge of S. aureus. Its deficiency (N -Ala)-DAla-DAla on phospho-MurNAcin the accumulation medium might result in an pentapeptide translocase. The initial meminhibition of cross-linked peptidoglycan synthe- brane reaction in the biosynthesis of peptidoglysis and accumulation of nucleotide precursors. can is catalyzed by phospho-MurNAc-penThus, addition of glycine to the acccumulation tapeptide translocase (UDP - MurNAc - Ala medium might reduce the accumulation of DGlu - Lys - DAla - DAla:undecaprenyl-phosUDP-MurNAc-pentapeptide and -hexapeptide. phate phospho-MurNAc-pentapeptide transAs shown in Table 1, the addition of 0.7 g of ferase). In addition to the transfer of phosphoglycine (9.3 mM) per liter results in a signifi- MurNAc-pentapeptide from UMP to undecacant decrease in the accumulation of UDP- prenyl-phosphate, this enzyme catalyzes the MurNAc - pentapeptide, and UDP- MurNAc- exchange of [3H]UMP with the UMP moiety Ala-DGlu-Lys(N -Ala)-DAla-DAla is not ob- of UDP-MurNAc-pentapeptide according to served. When penicillin G (250 mg/liter) was the reaction: added to the accumulation medium with gly- [3H]UMP + UDP-MurNAc-peptide cine, the quantity of UDP-MurNAc-pentapep[3H]UDP-MurNAc-peptide + UMP tide was similar to that observed in the accumulation medium without penicillin. UDP- This exchange reaction has been employed MurNAc-hexapeptide, on the other hand, was extensively as a routine assay because of its not detected in the accumulation medium con- sensitivity. In addition, a single labeled subtaining glycine and penicillin. From the above strate [3H ]UMP can be used in the presence of results it is concluded that the presence of unlabeled analogues of UDP-MurNAc-penglycine in the accumulation medium is a sig- tapeptide. As shown recently, there was good correlation between the results obtained from the exchange and transfer reactions (3). It was TABLE 1. Accumulation of concluded that results from the exchange reacUDP-MurNAc-pentapeptide and UDP-MurNAc-hexapeptide in the presence ofglycine tion provide an index of specificity for comparing UDP - MurNAc - Ala - DGlu - Lys - DAla Nucleotide DAla with other analogues. accumulated' In Fig. 4, the activity of UDP-MurNAc-Ala-` (gmol) DGlu-Lys(N'-Ala)-DAla-DAla is compared with UDPthat of UDP-MurNAc-pentapeptide. From UDPAdditions to mediaa MurNAc- MurNActhe Lineweaver-Burk plots (Fig. 4A, B), values hexapentafor Rmax are determined at different concenpeptide pept ide trations of UDP-MurNAc-peptide. From the intercept replot (Fig. 4C, D), values for Km Series A 1.5 13.6 of the UDP-MurNAc-peptides are established. Complete 0 7.5 Complete t 9.3 mM glycine Within experimental error, the Michaelis-Men0 13.1 Complete + 9.3 mM glycine ten constants for the two UDP-MurNAc-pepand 0.25 g of penicillin tides are identical. A useful index for comparing G per liter nucleotides in this assay is the ratio Rmax/Km (3). This ratio represents the apparent firstSeries B order rate constant for the reaction of substrate 1.8 10.0 Complete at low substrate concentrations. Under these 0 4.3 Complete + 9.3 mM glycine conditions, Rmax /Km reflects the effectiveness of 0 11.9 Complete + 9.3 mM glycine an analogue in the exchange assay. The values and 0.25 g of penicillin of Rmax are used to compare the effectiveness of G per liter the substrates at high concentration. As summaa The accumulation medium used in series A is the rized in Table 2, the ratios are almost identical standard accumulation medium as described in Ma- for UDP-MurNAc-pentapeptide and -hexapepterials and Methods. The medium used for series B is tide. the same medium to which 40 g of sucrose has been Biosynthesis of peptidoglycan(NE-Ala) added per liter. ' Cells for nucleotide accumulation were grown to with UDP MurNAc Ala DGlu Lys(N'membranes isolated Since Ala)-DAla-DAla. an optical density of 3 and transferred to the appropriate accumulation medium. Nucleotides were iso- from S. aureus Copenhagen prepared by several different procedures do not catalyze a high rate lated and quantitated.
PEPTIDOGLYCAN BIOSYNTHESIS IN S. AUREUS
VOL. 125, 1976
631
E I-I
a
10
2
10
3X.0
O ,'lE
_ 1.0
^
O
..
S
10
1-
15
(I/[UDP-MurNAc-hepepfide]) 104 FIG. 4. Exchange of [3H]UMP with the UMP moiety of UDP-MurNAc-pentapeptide (A) and UDPMurNAc-hexapeptide (B). Intercept replots for the determination of the Km and Rmax values of UDP-MurNAcpentapeptide (C) and of UDP-MurNAc-hexapeptide (D). (A) Concentrations of UDP-MurNAc-pentapeptide are: 1.33 x 10-5 M (0); 3.33 x 10-' M (A); 6.77 x 10-' M (0); 1.33 x 10- M (0). (B) Concentrations of UDP-MurNAc-hexapeptide are: 7.8 x 10-6 M (0); 1.3 x 10-5 M (A); 2.6 x 10-5 M (U); 3.9 x 10-5 M (0). All assays were performed with 41.3 ,ug of membrane protein prepared from S. aureus Copenhagen. (I/[UDP-MurNAc-Pentopeptkdq]) l0e x
of peptidoglycan synthesis, we used the system from S. aureus H, prepared by the method of Strominger et al. (16) (Fig. 2). The requirements for the synthesis of nascent peptidoglycan are summarized in Table 3. The incorporation of [4C ]GlcNAc from UDP- [4C ]GlcNAc into peptidoglycan requires the addition of UDP-MurNAc-peptide and is stimulated by the addition of NH4+. In this preparation, ATP also stimulates peptidoglycan synthesis. Lysozyme renders approximately 90% of the product from UDP-MurNAc- [14C ]pentapeptide soluble, whereas about 40% of the labeled material from UDP- ["C ]GlcNAc is lysozyme sensitive. When incorporation of ["4C]GlcNAc is corrected for the lysozyme-insensitive incorporation, the rates of GlcNAc and MurNAc-pentapeptide incorporation into peptidoglycan are equal. To compare the effectiveness of each nucleotide in the nascent peptidoglycan-synthesizing system, the values of Vmax and Km were established from Lineweaver-Burk plots (Fig. 5). The values obtained from these plots are compiled in Table 2. Comparison of values for Vmax
x
indicates that UDP- MurNAc- pentapeptide and -hexapeptide are used equally well at high substrate concentrations. The values of Vmax/Km are also similar; the Vmax/Km for UDP-MurNAc-pentapeptide is 5.6 x 10-'/min, and the Vmax/Km for UDP-MurNAc-hexapeptide is 4.3 x 10-4/min. The difference between the two nucleotides as determined in these kinetic studies is the presence of a second line segment (Fig. 5A, dotted line) in the Lineweaver-Burk plot of UDP-MurNAc-pentapeptide at low substrate concentrations. In the substrate range from Km/2 to 5 Km, UDPMurNAc-pentapeptide and UDP-MurNAc-hexapeptide are equally effective for the synthesis of peptidoglycan by the membrane system from S. aureus H.
DISCUSSION The composition and degree of cross-linking of peptidoglycan from S. aureus Copenhagen is partially determined by the amount of glycine in the growth medium (Schleifer et al., in press). For example, when this organism is
632
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SWENSON AND NEUHAUS
TABLE 2. Comparison of kinetic data for UDP-MurNAc-pentapeptide and UDP-MurNAc-hexapeptidea
Km
Substrate
A. Exchange activity catalyzed by phospho - MurNAc - pentapeptide translocaseb UDP-MurNAc-pentapeptide UDP-MurNAc-hexapeptide
Rmax
(MM)
(MM/min)
40.0 40.6
1.79 1.81
Rmax/Km x 10-2
(min-'
(
M/min
(MM/m2 x 10-2)
Vm.a/K. (min-I x 10-4)
4.47 4.46
B. Peptidoglycan synthesis 32c UDP-MurNAc-pentapeptide 1.8c 5.6 42 1.8 UDP-MurNAc-hexapeptide 4.3 a Data in A and B were established from the Lineweaver-Burk plots presented in Fig. 4 and 5, respectively. b Values for the indicated substrate are extrapolated to infinite UMP concentration. cThese values were determined from data in the concentration range 13 to 160 MM (solid line, Fig. 5A inset).
in a glycine-poor medium, 20% of the lysine residues are substituted with N-terminal alanyl residues and 20% of the lysine residues are unsubstituted. At most, 60% of the e-amino groups can be substituted with pentaglycine. In glycine-sufficient media almost all of the e-amino groups of lysine are linked to pentaglycine. Thus, the degree of L-alanine substitution reflects the amount of glycine available to the organism and may play an important role in determining the degree of cross-linking. In this paper we describe a possible precursor of the N'-alanyl-lysine dipeptide moiety, i.e., UDP MurNAc Ala DGlu Lys-(Ne-Ala)DAla-DAla. The utilization of this nucleotide by phospho-MurNAc-pentapeptide translocase and its incorporation into peptidoglycan by systems from S. aureus are established. Several reports describing the accumulation of nucleotide-activated MurNAc-peptides in various strains of S. aureus have been published (6, 12, 15, 17). None of these reports suggested the presence of UDP-MurNAc-Ala-DGlu-Lys(N'-Ala)-DAla-DAla in this organism. In the present experiments, the accumulation of UDPMurNAc-hexapeptide by resting cells was not affected by the presence of penicillin in the accumulation medium. The addition of glycine to the accumulation medium eliminates the accumulation of UDP-MurNAc-hexapeptide. Since glycine is required for the formation of the interpeptide bridge, it might be expected that a glycine deficiency could lead to a reduced rate of peptidoglycan synthesis with an accompanying accumulation of UDP-MurNAcpeptides. This requirement for glycine is reflected by the accumulation of UDP-MurNAcpentapeptide in a glycine-deficient medium and the reduction of accumulation when glycine grown
-
-
-
-
TABLE 3. Requirements for the synthesis of peptidoglycan Peptidoglycan synthesis (pmol/h) UDPAdditions
UDP-
I14C IGlcNAc MurNAc-AlaDGlu-LysDI14C JAlaD 14C ]Ala
Expt Ia A. Complete Complete UDP-MurNAcpentapeptide B. Complete Complete -
24.5 0
24.0 0
UDP-G1cNAc Expt Ilb
Complete Complete -ATP Complete - NH4+ Complete UDP-MurNAcpentapeptide
46.7 27.3 31 8 6.6
a In experiment I the complete reaction mixture contained 98 MM UDP-MurNAc-Ala-DGlu-Lvs-DAla-DAla and 98 MM UDP-[14CGlccNAc (28.8 counts/min per pmol) in A or 98 MM UDP-GlcNAc and 98 MM UDP-MurNAcAla-DGlu-Lys-D["ClAla-D[l14C]Ala (23.6 counts/min per pmol) in B, with 117 gg of membrane protein (preparation A) in addition to the standard reaction components. The reaction was carried out, and peptidoglycan synthesis was determined as a chromatographically immobile. lysozymesensitive product. The amount of labeled material incorporated into lysozyme-insensitive material was 38.8 pmol from UDP-[4"C]GlcNAc and 4.2 pmol from UDP-MurNAc-[14C]Cpentapeptide. 'In experiment II all conditions and concentrations are the same as in experiment I, except that the reaction mixture contained 100 Ag of membrane protein (preparation B). The amount of [14C IGlcNAc incorporated into lysozyme-insensitive material was 34.9 pmol.
VOL. 125, 1976
PEPTIDOGLYCAN BIOSYNTHESIS IN S. AUREUS
[UDP-MurNAc-hexopeptide]
x
633
MurNAc-pentapeptide formation (11). Thus, acylation of residue 3 with L-alanine does not result in a discrimination against this nucleotide in the reaction catalyzed by the initial membrane-associated enzyme in peptidoglycan synthesis. Since isolated membranes from S. aureus Copenhagen do not catalyze a high rate of nascent peptidoglycan synthesis, the membrane preparation from S. aureus H prepared by alumina grinding (16) has been used for comparing UDP-MurNAc-pentapeptide and -hexapeptide. In the concentration range of Km/2 to 5 K., UDP-MurNAc-hexapeptide is as effective as UDP-MurNAc-pentapeptide in the biosynthesis of peptidoglycan. The parameters Vmax and Vmax/Km are similar for both nucleotides. With UDP-MurNAc-pentapeptide the incorporation is characterized by a second line segment in the Lineweaver-Burk plot at low substrate concentrations. The second line segment is not observed with the hexapeptide. The results presented in this paper and those described by Schleifer et al. (in press) suggest that in S. aureus UDP-MurNAc-hexapeptide is utilized effectively for synthesis of nascent peptidogly-
10-4
FIG. 5. Incorporation of ['4C]GlcNAc from UDP- can. In L. viridescens, the N-terminal alanine of ["C]GIcNAc into peptidoglycan as a function of UDP-MurNAc-pentapeptide (A) and UDP-MurNAc- the hexapeptide serves as an acceptor at the hexapeptide (B). Membrane fragments from S. aureus lipid intermediate stage for an additional serine H (68.5 gg of protein) were incubated with the indi- residue required for the formation of the intercated concentrations of UDP-MurNAc-peptide as peptide bridge. In contrast, on the basis of the described in the text. The insets in A and B show in vivo results described by Schleifer et al., the Lineweaver-Burk analyses of the data from which the N-terminal alanine of the hexapeptide the values for K. and Vmax were determined. of S. aureus Copenhagen does not function as
an acceptor for the glycine residues required is added. The relationship between glycine de- for interpeptide bridge formation. The presence ficiency and UDP-MurNAc-hexapeptide pro- of the N-terminal alanine decreases the number duction is not well understood. Since N- of potential interpeptide bridges for cross-linkterminal alanine in the peptide moiety of the ing the glycan strands. Thus, growth in a peptidoglycan is not further substituted in this glycine-poor medium indirectly affects the deorganism, we might view the formation of the gree of cross-linking of the peptidoglycan. hexapeptide as a mechanism for conserving the glycine pool of the growing bacterium. ACKNOWLEDGMENTS Acylation of the c-amino group of the lysine We are grateful to Walter P. Hammes for helpful discusresidue with L-alanine does not affect the activ- sions of these results. This investigation was supported by Public Health Service ity catalyzed by phospho-MurNAc-pentapeptide translocase. From the results with the ex- grant AI-04615 from the National Institute of Allergy and change reaction, it was concluded that the rate Infectious Diseases. of undecaprenyl diphosphate MurNAc hexa LITERATURE CITED peptide formation is similar at both high and 1. D. J. Tipper, C. H. Birge, and J. L. Ghuysen, J.-M., low substrate concentrations when compared Strominger. 1965. Structure of the cell wall of Staphylowith UDP-MurNAc-pentapeptide. With memcoccus aureus strain Copenhagen. VI. The soluble brane preparations from L. viridescens, the rate glycopeptide and its sequential degradation by peptidases. Biochemistry 4:2245-2254. of undecaprenyl diphosphate - MurNAc - hexa W. R., and J. F. Smith. 1970. Rapid sequence peptide formation from UDP-MurNAc-Ala- 2. Gray, analysis of small peptides. Anal. Biochem. 33:36-42. DGlu-Lys(Ne-Ala)-DAla-DAla is similar to that 3. Hammes, W. P., and F. C. Neuhaus. 1974. On the specificity of phospho-N-acetylmuramyl-pentapeptide for described undecaprenyl-diphosphate-
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translocase. The peptide subunit of uridine diphosphate-N-acetylmuramyl-pentapeptide. J. Biol. Chem. 249:3140-3150. Hammes, W. P., and F. C. Neuhaus. 1974. Biosynthesis of peptidoglycan in Gaffkya homari: role of the peptide subunit of uridine diphosphate-N-acetylmuramyl-pentapeptide. J. Bacteriol. 120:210-218. Heydanek, M. G., Jr., R. Linzer, D. D. Pless, and F. C. Neuhaus. 1970. Initial stage in peptidoglycan synthesis. Mechanism of activation of phospho-N-acetylmuramyl-pentapeptide translocase by potassium ions. Biochemistry 9:3618-3623. Ito, E., and M. Saito. 1963. Time course of accumulation of UDP-N-acetylamino sugar derivatives in Staphylococcus aureus. Biochim. Biophys. Acta 78:237-247. 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. Mandelstam, J., and H. J. Rogers. 1959. The incorporation of amino acids into the cell-wall mucopeptide of staphylococci and the effect of antibiotics on the process. Biochem. J. 72:654-662. Mandelstam, P., R. Loercher, and J. L. Strominger. 1962. A uridine diphosphoacetylmuramyl hexapeptide from penicillin-treated Streptococcus faecalis. J. Biol. Chem. 237:2683-2688. Patterson, M. S., and R. C. Greene. 1965. Measurement of low energy beta-emitters in aqueous solution by liquid scintillation counting of emulsions. Anal. Chem. 37:854-857. Plapp, R., and J. L. Strominger. 1970. Biosynthesis of the peptidoglycan of bacterial cell walls. XVII. Biosynthesis of the peptidoglycan and of interpeptide bridges in Lactobacillus viridescens. J. Biol. C hem. 245:3667-3674. Saito, M., N. Ishimoto, and E. Ito. 1963. Uridine diphos-
phate N-acetylamino sugar derivatives in penicillintreated Staphylococcus aureus. J. Biochem. 54:273-278. 13. Schleifer, K. H., and 0. Kandler. 1972. Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bacteriol. Rev. 36:407-477. 14. Stickgold, R. A., and F. C. Neuhaus. 1967. On the initial stage in peptidoglycan synthesis. Effect of 5-fluoroura-
15.
16.
17.
18.
19.
cil substitution on phospho-N-acetylmuramyl-pentapeptide translocase (uridine 5'-phosphate). J. Biol. Chem. 242:1331-1337. Strominger, J. L. 1957. Microbial uridine-5'-pyrophosphate N-acetylamino sugar compounds. I. Biology of the penicillin-induced accumulation. J. Biol. Chem. 224:509-523. Stroininger, J. L., M. Matsuhashi, J. S. Anderson, C. P. Dietrich, P. M. Meadow, W. Katz, G. Siewert, and J. M. Gilbert. 1966. Glycopeptide synthesis in Staphylococcus aureus and Micrococcus lysodeikticus, p. 473486. In S. P. Colowick and N. 0. Kaplan (ed.), Methods in enzymology, vol. 8. Academic Press Inc., New York. Strominger, J. L., and R. H. Threnn. 1959. Accumulation of a uridine nucleotide in Staphyloccus aureus as the consequence of lysine deprivation. Biochim. Biophys. Acta 36:83-92. Struve, W. G., R. K. Sinha, and F. C. Neuhaus. 1966. On the initial stage in peptidoglycan synthesis. PhosphoN-acetylmuramyl-pentapeptide translocase (uridine monophosphate). Biochemistry 5:82-93. Tipper, D. J., J. L. Strominger, and J. C. Ensign. 1967. Structure of the cell wall of Staphylococcus aureus, strain Copenhagen. VII. Mode of action of the bacteriolytic peptidase from Myxobacter and the isolation of intact cell wall polysaccharides. Biochemistry 6:906920.
JOURNAL OF BACTERIOLOGY, Feb. 1976. p. 626-634 Copyright ( 1976 American Society for Microbiology
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Biosynthesis of Peptidoglycan in Staphylococcus aureus: Incorporation of the Nc-Ala-Lys Moiety into the Peptide Subunit of Nascent Peptidoglycan Department of Biochemistry
J. C. SWENSON AND F. C. NEUHAUS* and Molecular Biology, Northwestern University, Evanston, Illinois 60201
Received for publication 22 September 1975
UDP-MurNAc-Ala-DGlu-Lys(N'-Ala)-DAla-DAla was isolated from extracts of Staphylococcus aureus Copenhagen. This nucleotide accumulated in media deficient in glycine. To establish its role in peptidoglycan biosynthesis, the nucleotide-hexapeptide was compared with UDP-MurNAc-Ala-DGlu-LysDAla-DAla in the reaction catalyzed by phospho-MurNAc-pentapeptide translocase and in the membrane-catalyzed nascent peptidoglycan-synthesizing system. In the exchange reaction catalyzed by the translocase, the Rmax and RmaJKm are 1.79 ,uM/min and 4.47 x 10-/min, respectively, for UDP-MurNAcpentapeptide and 1.81 AM/min and 4.46 x 10-2/min, respectively, for UDP-MurNAc-hexapeptide. In the synthesis of nascent peptidoglycan, the Vmax is 1.8 AM/min x 102 for both the nucleotide-hexapeptide and -pentapeptide. The Vmax/Km is 5.6 x 10-' and 4.3 x 10-4/min for the nucleotide-pentapeptide and -hexapeptide, respectively. Schleifer, Hammes, and Kandler (Adv. Microb. Physiol., in press) observed that growth of S. aureus Copenhagen on a glycinepoor medium results in a peptidoglycan structure in which 20% of the lysine residues are substituted at the e-amino group by L-alanine residues that do not participate in interpeptide bridge formation. The in vitro studies demonstrate that UDP-MurNAc-Ala-DGlu-Lys(N-Ala)-DAla-DAla is a possible precursor of the NE-Ala-Lys moiety. In Staphylococcus aureus Copenhagen, the interpeptide bridge of the peptidoglycan consists of five glycine residues (1, 19) when the organism is cultured in a glycine-rich medium. When it is grown in a glycine-poor medium, the walls show a decrease in cross-linking (K. H. Schleifer, Proc. Soc. Gen. Microbiol. 57:xiv, 1969; K. H. Schleifer, W. P. Hammes, and 0. Kandler, Adv. Microb. Physiol., in press). Under these conditions a significant percentage (20%) of the lysine residues of the peptidoglycan are substituted at the e-amino group by alanine residues that do not participate in interpeptide bridge formation. Since the presence of N'-Ala-Lys dipeptide moieties in the peptidoglycan can be correlated with the degree of cross-linking in S. aureus Copenhagen, the mechanism by which this dipeptide moiety is introduced into this polymer is of interest. The biosynthesis of peptidoglycan is catalyzed by a series of membraneassociated enzymes that utilize two nucleotideactivated precursors, UDP-N-acetylglucosamine and UDP-MurNAc-Ala-DGlu-Lys-DAlaDAla. A possible precursor of the N'-Ala-Lys moiety is the UDP-MurNAc-hexapeptide:
UDP - MurNAc - Ala - DGlu - Lys(N' - Ala) DAla-DAla. Nucleotide-activated hexapeptides have been found in two different species of' bacteria, Lactobacillus viridescens (11) and Streptococcus faecalis (9). Membrane preparations from L. viridescens utilize the nucleotideactivated hexapeptide for the synthesis of peptidoglycan (11). In contrast to S. aureus Copenhagen, the L-alanine residue linked to the e-amino group of' lysine derived from UDPMurNAc-hexapeptide forms an integral part of the interpeptide bridge of L. viridescens (13) and S. faecalis. In S. aureus Copenhagen it is N terminal. It is the purpose of this paper to describe the isolation of' UDP-MurNAc-Ala-DGlu-Lys(N'Ala)-DAla-DAla from S. aureus Copenhagen and to define conditions for its optimal accumulation. Since this nucleotide represents the possible precursor of the NE-Ala-Lys dipeptide moiety observed by Schleifer et al. (in press), we present in vitro experiments to compare its activity in the reaction catalyzed by phosphoMurNAc-pentapeptide translocase and the peptidoglycan-synthesizing system from S. aureus.
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MATERIALS AND METHODS
UDP-MurNAc-hexapeptide was isolated from the UDP-MurNAc-peptide fraction by chromatography Materials. S. aureus Copenhagen was a gift from J. on DEAE-cellulose. This fraction was applied to a L. Strominger and was maintained as a lyophilized column (0.9 by 57 cm) of DEAE-cellulose, and the culture in vacuo at -25 C. S. aureus H (ATCC 13801) column was then developed with a linear gradient of was purchased from the American Type Culture Col- ammonium bicarbonate buffer (pH 8.5; from 0.1 to lection. UDP- [U- 14C ]GlcNAc was purchased from 0.5 M). A typical elution pattern is shown in Fig. 1. ICN Isotope and Nuclear Division and Amersham/ The partially resolved contaminant in the hexapepSearle. The plastic beads (styrene-divinylbenzene tide fraction was not included when the fractions were copolymer, 20 to 50 mesh, 8% cross-linked) were the pooled. The contaminant was resolved from UDPkind gift of Richard Reitz of the Dow Chemical Co. MurNAc-hexapeptide by rechromatography, as deWhatman DE-52 microgranular diethylaminoethyl scribed above, with a linear gradient of 0.15 to 0.3 M (DEAE)-cellulose was purchased from H. Reeve ammonium bicarbonate. The UDP-MurNAc-hexapeptide was characterized Angel Co. [5-3HJUMP was purchased from Schwarz/ Mann. Peptone and yeast extract were products of as follows. With paper chromatography the hexapepthe Difco Laboratories. Levigated alumina (15 um), tide has R, values of 0.26 and 0.39 in solvents A and B manufactured by Buehler Ltd., was purchased from (see below), respectively. For comparison, the penFisher Scientific Co. The sources of other chemicals tapeptide has R, values of 0.22 and 0.46 and the were described previously (3, 4). tripeptide has R, values of 0.17 and 0.60 in solvents A Isolation of UDP-MurNAc-Ala-DGlu-Lys(N and B, respectively. Hydrolysis of the nucleotide with Ala)-DAla-DAla. For the preparation of the nucleo- 0.1 M HCl at 100 C for 15 min results in the formation tide-activated hexapeptide, S. aureus Copenhagen of UMP and UDP. The amino acid composition of the was grown aerobically in the medium described by hexapeptide is: glutamic acid-alanine-lysine-uridine Heydanek et al. (5) at 37 C. The cells were harvested (1.00:3.95:0.98:1.10). Small amounts of glycine are and transferred to accumulation medium (80 ml/g consistently detected in this fraction. UDP-MurNAc[wet weight] of cells) at 37 C. This medium contained pentapeptide has the ratio 1:3:1:1. Thus, the nucleoper liter: DL-alanine, 400 mg; D-glutamic acid, 200 mg; tide-hexapeptide differs from UDP-MurNAc-pentalysine (monohydrochloride), 200 mg; uracil, 150 mg; peptide by one additional residue of alanine. Since glucose, 10 g; and K,HPO4, 5 g. Penicillin G (1,600 the additional alanine is not sensitive to the action, U/mg) was added as indicated. After incubation at of D-amino acid oxidase, it is concluded that the 37 C in the accumulation medium (2 h), the cells were extra residue is L-alanine. Thus, the nucleotide has harvested and extracted with cold 10'S trichloroacetic two residues of D-alanine and two of L-alanine. acid (3 ml/g [wet weight] of cells). Partial removal of Reaction of the nucleotide with 1-dimethylaminothe trichloroacetic acid was accomplished by extrac- naphthalene-5-sulfonyl-chloride (dansyl-chloride) foltion with diethyl ether. After the pH was adjusted to lowed by hydrolysis results in the release of dansyl7.8, the UDP-MurNAc-peptide fraction was isolated alanine, indicating that this residue is N terminal. from the other components of the cell extract by the To determine the location of the N-terminal alanine, the sequence analysis procedure of Gray and Smith method of Stickgold and Neuhaus (14).
I
5.0
Upentopeptide DP-MurNAc-
4..0
E
R x 10-8
48
40 NH4HCO3 -0.5M
3..0
01-z
40
c
2..0
UDP-MurNAc-
hexapeptide
32 ±- 0.4M ± 0.3 M 24 - -0.2M +-0.1 M 16
1..0 _
n
8 -a_d
1
_"
30
60
90
120 150 180 Volume (ml)
210
240
0
FIG. 1. Separation of UDP-MurNAc-hexapeptide and UDP-MurNAc-pentapeptide by ion-exchange chromatography. The UDP-MurNAc-peptide fraction, isolated by the method of Stickgold and Neuhaus (14), was applied to a column of DEAE-cellulose, and the nucleotides were eluted with the linear gradient described in the text. The absorbance at 260 nm (0), the exchange activity (R) (A) observed with a 30-Ml sample, and the molarity of the NH4HCO3 (-) are presented.
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peptide translocase. For measuring the reaction catalyzed by phospho-MurNAc-pentapeptide translocase, the exchange assay described by Hammes and Neuhaus (3) was used. This assay measures the rate of' exchange of' [3HJUMP with the UMP moiety of UDP-MurNAc-pentapeptide. Exchange activity was calculated by the method of' Struve et al. (18). The; rate of exchange, R, is reported as moles exchanged per liter per minute. The reaction mixture contained: 0.05 M Tris-hydrochloride (pH 7.8); 0.21 M KClI 0.042 M MgCl2; membrane f'ragments (41.3 ug of protein); and [3H]UMP and UDP-MurNAc-peptide UDP-MurNAc-Ala-DGlu-Lys(N '-Ala)-DAla-DAla. Preparation of membrane fragments. Membrane as indicated, in a total volume of' 60 pl. 'I'he incubafragments from S. aureus were prepared bv two tion was carried out at 25 C f'or 10 min. Af'ter the different procedures. For specificity studies with phos- reaction was terminated by placing the mixture in pho-MurNAc-pentapeptide translocase, S. aureus boiling water for 2 min, the amount of' exchange was Copenhagen (late log phase) was disrupted with determined as described previously (3). Peptidoglycan synthesis assay. Peptidoglycan plastic beads, and the membrane f'ragments were isolated by the procedure of' Struve et al. (18). For synthesis was assayed by determining the incorspecificity studies with the peptidoglycan-synthesiz- poration of NAc-["C]glucosamine from UDP-["4C ]ing system, membrane fragments were prepared by GlcNAc into a lysozyme-sensitive, chromatographthe method of Strominger et al. (16), using levigated ically immobile f'raction. That this labeled f'racalumina to disrupt the cells. Membrane fragments tion is peptidoglycan was concluded f'rom its sensitivwere suspended in 0.05 M tris(hydroxymethyl)- ity to lysozyme and its dependence for synthesis on aminomethane (Tris)-hydrochloride (pH 7.8) con- the second substrate UDP-MurNAc-peptide. The retaining 10-4 M magnesium acetate and 10'- M action mixture contained: 0.10 M Tris-hydrochloride, 2-mercaptoethanol and stored at - 196 C. As illus- pH 8.5; 3 mM magnesium acetate; 10 mM NH4CI; 1 trated in Fig. 2, the maximal rate of' peptidoglycan mM ATP; 1 mM 2-mercaptoethanol; 98 MM UDPsynthesis was attained with early-log-phase cells. ["4C]GlcNAc (25 to 35 counts/min per pmol); 2)0 to A fourf'old-higher rate of' synthesis was achieved with 200 Mg of membrane protein; and the indicated S. aureus H than with S. aureus Copenhagen. Thus, concentration of UDP-MurNAc-peptide, in a total f'or the studies with the peptidoglycan-synthesizing volume of 25 pl. Duplicate incubations were carried system, membrane f'ragments f'rom S. aureus H out at 20 C for 1 h. The reactions were terminated by placing the incubation mixtures into a boiling-water were used. Exchange assay for phospho-MurNAc-penta- bath for 2 min. One of the samples was incubated for an additional 30 min at 37 C with 150 Mg of lysozyme. The reaction mixtures were then quantitatively applied to Whatman 3 MM paper, and the paper chromatograms were developed by descending chromatography in solvent A (see below) for about 20 to 22 h. The origins were assayed for chromatographically immobile 14C-labeled material. Peptidoglycan synthesis was found to continue linearly for at least 110 min under the conditions I described. A slight drop f'rom linearity was observed as the concentration of' membrane fragments was 1.0 increased. When UDP- [14C ]GlcNAc was used to 0.5 label the immobile product, signif'icant amounts ot origin material were found to be lysozyme insensitive. This lysozyme-insensitive product also was formed in the absence of UDP-MurNAc-peptide. All measurements of peptidoglycan synthesis with ["'C]GlcNAc have been corrected for lysozyme-insensitive mateHours rial. When incorporation of MurNAc-Ala-DGlu-LysFIG. 2. Specific activities of peptidoglycan synthe- D[14C]Ala-D['4C]Ala was measured. the quantity sis by membrane fragments from S. aureus Copenha- of' the peptidoglycan produced was the same as the amount of lysozyme-sensitive product when [ 4Cgen (A) and S. aureus H (0). The organisms were GlcNAc was the label. grown aerobically in the medium described by HeydaAnalytical methods. For the determination of' nek et al. (5) at 37 C. Samples of cells were harvested radioactivity in aqueous samples, the scintillation at the indicated times, disrupted by the method of Strominger et al. (16), and assayed for peptidoglycan fluid described by Patterson and Greene (10) was synthesis by the procedure described in the text. used; for paper chromatograms the fluid contained Growth of the cells was determined by turbidity at 650 0..3'7( 2,5-diphenyloxazole and 0.025%'( 1,4-bis-[2](5-phenyloxazolyl)benzene in toluene. Descending nm. (2) was used. Treatment of' the nucleotide-hexapeptide by this procedure results in the liberation of' the e-amino group of' lysine. Thus, the L-alanine residue is covalently linked to the e-amino group. The existence of' muramic acid is indicated by a peak, shown on the amino acid analyzer. that has this characteristic elution volume. The similarity of' this nucleotide to UDP-MurNAc-pentapeptide and its identity with that synthesized by L. viridescens and that isolated from S. faecalis suggest that the nucleotide-hexapeptide f'rom S. aureus Copenhagen is
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PEPTIDOGLYCAN BIOSYNTHESIS IN S. AUREUS
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UDP-MurNAc-pentapeptide(Ne-Ala). The ratio of UDP-MurNAc -Ala-DGlu -Lys(N Ala)-DAla-DAla to UDP-MurNAc-Ala-DGluLys-DAla-DAla increases from 0.06 at mid-log to approximately 0.15 at the transition from the late log to the stationary phase. The effect of penicillin in the accumulation medium was tested to optimize conditions for the production of UDP-MurNAc-Ala-DGluLys(N E-Ala)-DAla-DAla. Cells were harvested from the growing culture at the time of maximal accumulation of the two nucleotides (Fig. 3A, 3.5 h). They were transferred to accumulation medium modified in the amount of penicillin present. As shown in Fig. 3B, the concentration of penicillin in the accumulation medium has no effect on the accumulation of UDP-MurNAc-hexapeptide. The presence of penicillin results in a small decrease in the accumulation of UDP-MurNAc-pentapeptide. Strominger and Threnn (17) reported a significant accumulation of N-acetylamino sugar nucleotides in S. aureus in the absence of penicillin (6.2 umol/liter of culture at half-maximal growth) in a medium similar to the accumulation medium used in the present work. When penicillin (100 /g/ml) was added to the accumulation medium, only a 2.5-fold increase (15.5 gmol) in the amount of these nucleotides was observed. Thus, the lack of effect of penicillin in the accumulation medium in the present work can be compared with the low increase (2.5-fold) observed by Strominger and Threnn. Since the accumulation of UDP-MurNAcpeptides in S. aureus Copenhagen is not greatly affected by the addition of penicillin to the accumulation medium, the presence or absence
paper chromatography was performed with Whatman 3 MM paper. Solvent A contains isobutyric acid-concentrated NH4OH-water (66:2:33, vol/vol/vol). Solvent B contains 0.1 M phosphate buffer (pH 6.8)-(NH4) 2S04-n-propanol (100:60:2, vol/wt/vol). Amino acids were analyzed on a Durrum amino acid analyzer (model D-500) after hydrolysis of the samples with 5.7 N HCl for 12 h at 100 C. Protein was determined by the method of Lowry et al. (7), with bovine serum albumin as the standard.
RESULTS Accumulation of UDP-MurNAc-AlaDGlu-Lys(N e-Ala)-DAla-DAla. In preparing UDP-MurNAc-Ala-DGlu-Lys-DAla-DAla and UDP - MurNAc - Ala - DGlu - Lys(N"-Ala) DAla-DAla from S. aureus Copenhagen, it was observed that the quantities of these nucleotides varied with the growth phase of the bacteria at the time of harvest for transfer to the accumulation medium. To determine the influence of the growth phase on the accumulation of these nucleotides, samples of S. aureus from the growth medium were harvested at different times and transferred to the accumulation medium for 2 h. In Fig. 3A the amount of each nucleotide per 1012 cells is presented as a function of the time of harvest before transfer to the accumulation medium. The incubation in this medium (2 h) does not allow growth of the cells but does result in maximal accumulation of nucleotides (8, 15). Therefore, this plot represents the potential (or capacity) of the cells to synthesize these nucleotides at the time of their harvest from the growing culture. During the late log phase of growth, there is a large increase in the capacity for synthesis of UDP-MurNAcpentapeptide followed by a smaller increase in
B 6
0
H 5 >
0
N 0
4
2
S4 1
2
3
4 5 Hours
6
7
8
0 25 50
100
150
200
[Penicillin] (pg/mi)
FIG. 3. Accumulation potentials (capacities) for the synthesis of UDP-MurNAc-pentapeptide (0) and UDP-MurNAc-hexapeptide (A) as a function of growth (A). Effect of increasing concentrations of penicillin on the accumulation of UDP-MurNAc-pentapeptide (0) and UDP-MurNAc-hexapeptide (A) (B). The growth (-) was measured by turbidity at 650 nm. (A) Cells harvested at the indicated time were suspended in accumulation medium containing 25 ,g of penicillin G per ml. (B) Cells were harvested at 3.5 h and transferred to the accumulation media containing the indicated concentration of penicillin G.
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of another component in the medium might be nificant factor in determining the amount of responsible for the accumulation that is ob- UDP-MurNAc-hexapeptide. Effect of UDP-MurNAc-Ala-DGlu-Lysserved. Glycine is a major component of the interpeptide bridge of S. aureus. Its deficiency (N -Ala)-DAla-DAla on phospho-MurNAcin the accumulation medium might result in an pentapeptide translocase. The initial meminhibition of cross-linked peptidoglycan synthe- brane reaction in the biosynthesis of peptidoglysis and accumulation of nucleotide precursors. can is catalyzed by phospho-MurNAc-penThus, addition of glycine to the acccumulation tapeptide translocase (UDP - MurNAc - Ala medium might reduce the accumulation of DGlu - Lys - DAla - DAla:undecaprenyl-phosUDP-MurNAc-pentapeptide and -hexapeptide. phate phospho-MurNAc-pentapeptide transAs shown in Table 1, the addition of 0.7 g of ferase). In addition to the transfer of phosphoglycine (9.3 mM) per liter results in a signifi- MurNAc-pentapeptide from UMP to undecacant decrease in the accumulation of UDP- prenyl-phosphate, this enzyme catalyzes the MurNAc - pentapeptide, and UDP- MurNAc- exchange of [3H]UMP with the UMP moiety Ala-DGlu-Lys(N -Ala)-DAla-DAla is not ob- of UDP-MurNAc-pentapeptide according to served. When penicillin G (250 mg/liter) was the reaction: added to the accumulation medium with gly- [3H]UMP + UDP-MurNAc-peptide cine, the quantity of UDP-MurNAc-pentapep[3H]UDP-MurNAc-peptide + UMP tide was similar to that observed in the accumulation medium without penicillin. UDP- This exchange reaction has been employed MurNAc-hexapeptide, on the other hand, was extensively as a routine assay because of its not detected in the accumulation medium con- sensitivity. In addition, a single labeled subtaining glycine and penicillin. From the above strate [3H ]UMP can be used in the presence of results it is concluded that the presence of unlabeled analogues of UDP-MurNAc-penglycine in the accumulation medium is a sig- tapeptide. As shown recently, there was good correlation between the results obtained from the exchange and transfer reactions (3). It was TABLE 1. Accumulation of concluded that results from the exchange reacUDP-MurNAc-pentapeptide and UDP-MurNAc-hexapeptide in the presence ofglycine tion provide an index of specificity for comparing UDP - MurNAc - Ala - DGlu - Lys - DAla Nucleotide DAla with other analogues. accumulated' In Fig. 4, the activity of UDP-MurNAc-Ala-` (gmol) DGlu-Lys(N'-Ala)-DAla-DAla is compared with UDPthat of UDP-MurNAc-pentapeptide. From UDPAdditions to mediaa MurNAc- MurNActhe Lineweaver-Burk plots (Fig. 4A, B), values hexapentafor Rmax are determined at different concenpeptide pept ide trations of UDP-MurNAc-peptide. From the intercept replot (Fig. 4C, D), values for Km Series A 1.5 13.6 of the UDP-MurNAc-peptides are established. Complete 0 7.5 Complete t 9.3 mM glycine Within experimental error, the Michaelis-Men0 13.1 Complete + 9.3 mM glycine ten constants for the two UDP-MurNAc-pepand 0.25 g of penicillin tides are identical. A useful index for comparing G per liter nucleotides in this assay is the ratio Rmax/Km (3). This ratio represents the apparent firstSeries B order rate constant for the reaction of substrate 1.8 10.0 Complete at low substrate concentrations. Under these 0 4.3 Complete + 9.3 mM glycine conditions, Rmax /Km reflects the effectiveness of 0 11.9 Complete + 9.3 mM glycine an analogue in the exchange assay. The values and 0.25 g of penicillin of Rmax are used to compare the effectiveness of G per liter the substrates at high concentration. As summaa The accumulation medium used in series A is the rized in Table 2, the ratios are almost identical standard accumulation medium as described in Ma- for UDP-MurNAc-pentapeptide and -hexapepterials and Methods. The medium used for series B is tide. the same medium to which 40 g of sucrose has been Biosynthesis of peptidoglycan(NE-Ala) added per liter. ' Cells for nucleotide accumulation were grown to with UDP MurNAc Ala DGlu Lys(N'membranes isolated Since Ala)-DAla-DAla. an optical density of 3 and transferred to the appropriate accumulation medium. Nucleotides were iso- from S. aureus Copenhagen prepared by several different procedures do not catalyze a high rate lated and quantitated.
PEPTIDOGLYCAN BIOSYNTHESIS IN S. AUREUS
VOL. 125, 1976
631
E I-I
a
10
2
10
3X.0
O ,'lE
_ 1.0
^
O
..
S
10
1-
15
(I/[UDP-MurNAc-hepepfide]) 104 FIG. 4. Exchange of [3H]UMP with the UMP moiety of UDP-MurNAc-pentapeptide (A) and UDPMurNAc-hexapeptide (B). Intercept replots for the determination of the Km and Rmax values of UDP-MurNAcpentapeptide (C) and of UDP-MurNAc-hexapeptide (D). (A) Concentrations of UDP-MurNAc-pentapeptide are: 1.33 x 10-5 M (0); 3.33 x 10-' M (A); 6.77 x 10-' M (0); 1.33 x 10- M (0). (B) Concentrations of UDP-MurNAc-hexapeptide are: 7.8 x 10-6 M (0); 1.3 x 10-5 M (A); 2.6 x 10-5 M (U); 3.9 x 10-5 M (0). All assays were performed with 41.3 ,ug of membrane protein prepared from S. aureus Copenhagen. (I/[UDP-MurNAc-Pentopeptkdq]) l0e x
of peptidoglycan synthesis, we used the system from S. aureus H, prepared by the method of Strominger et al. (16) (Fig. 2). The requirements for the synthesis of nascent peptidoglycan are summarized in Table 3. The incorporation of [4C ]GlcNAc from UDP- [4C ]GlcNAc into peptidoglycan requires the addition of UDP-MurNAc-peptide and is stimulated by the addition of NH4+. In this preparation, ATP also stimulates peptidoglycan synthesis. Lysozyme renders approximately 90% of the product from UDP-MurNAc- [14C ]pentapeptide soluble, whereas about 40% of the labeled material from UDP- ["C ]GlcNAc is lysozyme sensitive. When incorporation of ["4C]GlcNAc is corrected for the lysozyme-insensitive incorporation, the rates of GlcNAc and MurNAc-pentapeptide incorporation into peptidoglycan are equal. To compare the effectiveness of each nucleotide in the nascent peptidoglycan-synthesizing system, the values of Vmax and Km were established from Lineweaver-Burk plots (Fig. 5). The values obtained from these plots are compiled in Table 2. Comparison of values for Vmax
x
indicates that UDP- MurNAc- pentapeptide and -hexapeptide are used equally well at high substrate concentrations. The values of Vmax/Km are also similar; the Vmax/Km for UDP-MurNAc-pentapeptide is 5.6 x 10-'/min, and the Vmax/Km for UDP-MurNAc-hexapeptide is 4.3 x 10-4/min. The difference between the two nucleotides as determined in these kinetic studies is the presence of a second line segment (Fig. 5A, dotted line) in the Lineweaver-Burk plot of UDP-MurNAc-pentapeptide at low substrate concentrations. In the substrate range from Km/2 to 5 Km, UDPMurNAc-pentapeptide and UDP-MurNAc-hexapeptide are equally effective for the synthesis of peptidoglycan by the membrane system from S. aureus H.
DISCUSSION The composition and degree of cross-linking of peptidoglycan from S. aureus Copenhagen is partially determined by the amount of glycine in the growth medium (Schleifer et al., in press). For example, when this organism is
632
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TABLE 2. Comparison of kinetic data for UDP-MurNAc-pentapeptide and UDP-MurNAc-hexapeptidea
Km
Substrate
A. Exchange activity catalyzed by phospho - MurNAc - pentapeptide translocaseb UDP-MurNAc-pentapeptide UDP-MurNAc-hexapeptide
Rmax
(MM)
(MM/min)
40.0 40.6
1.79 1.81
Rmax/Km x 10-2
(min-'
(
M/min
(MM/m2 x 10-2)
Vm.a/K. (min-I x 10-4)
4.47 4.46
B. Peptidoglycan synthesis 32c UDP-MurNAc-pentapeptide 1.8c 5.6 42 1.8 UDP-MurNAc-hexapeptide 4.3 a Data in A and B were established from the Lineweaver-Burk plots presented in Fig. 4 and 5, respectively. b Values for the indicated substrate are extrapolated to infinite UMP concentration. cThese values were determined from data in the concentration range 13 to 160 MM (solid line, Fig. 5A inset).
in a glycine-poor medium, 20% of the lysine residues are substituted with N-terminal alanyl residues and 20% of the lysine residues are unsubstituted. At most, 60% of the e-amino groups can be substituted with pentaglycine. In glycine-sufficient media almost all of the e-amino groups of lysine are linked to pentaglycine. Thus, the degree of L-alanine substitution reflects the amount of glycine available to the organism and may play an important role in determining the degree of cross-linking. In this paper we describe a possible precursor of the N'-alanyl-lysine dipeptide moiety, i.e., UDP MurNAc Ala DGlu Lys-(Ne-Ala)DAla-DAla. The utilization of this nucleotide by phospho-MurNAc-pentapeptide translocase and its incorporation into peptidoglycan by systems from S. aureus are established. Several reports describing the accumulation of nucleotide-activated MurNAc-peptides in various strains of S. aureus have been published (6, 12, 15, 17). None of these reports suggested the presence of UDP-MurNAc-Ala-DGlu-Lys(N'-Ala)-DAla-DAla in this organism. In the present experiments, the accumulation of UDPMurNAc-hexapeptide by resting cells was not affected by the presence of penicillin in the accumulation medium. The addition of glycine to the accumulation medium eliminates the accumulation of UDP-MurNAc-hexapeptide. Since glycine is required for the formation of the interpeptide bridge, it might be expected that a glycine deficiency could lead to a reduced rate of peptidoglycan synthesis with an accompanying accumulation of UDP-MurNAcpeptides. This requirement for glycine is reflected by the accumulation of UDP-MurNAcpentapeptide in a glycine-deficient medium and the reduction of accumulation when glycine grown
-
-
-
-
TABLE 3. Requirements for the synthesis of peptidoglycan Peptidoglycan synthesis (pmol/h) UDPAdditions
UDP-
I14C IGlcNAc MurNAc-AlaDGlu-LysDI14C JAlaD 14C ]Ala
Expt Ia A. Complete Complete UDP-MurNAcpentapeptide B. Complete Complete -
24.5 0
24.0 0
UDP-G1cNAc Expt Ilb
Complete Complete -ATP Complete - NH4+ Complete UDP-MurNAcpentapeptide
46.7 27.3 31 8 6.6
a In experiment I the complete reaction mixture contained 98 MM UDP-MurNAc-Ala-DGlu-Lvs-DAla-DAla and 98 MM UDP-[14CGlccNAc (28.8 counts/min per pmol) in A or 98 MM UDP-GlcNAc and 98 MM UDP-MurNAcAla-DGlu-Lys-D["ClAla-D[l14C]Ala (23.6 counts/min per pmol) in B, with 117 gg of membrane protein (preparation A) in addition to the standard reaction components. The reaction was carried out, and peptidoglycan synthesis was determined as a chromatographically immobile. lysozymesensitive product. The amount of labeled material incorporated into lysozyme-insensitive material was 38.8 pmol from UDP-[4"C]GlcNAc and 4.2 pmol from UDP-MurNAc-[14C]Cpentapeptide. 'In experiment II all conditions and concentrations are the same as in experiment I, except that the reaction mixture contained 100 Ag of membrane protein (preparation B). The amount of [14C IGlcNAc incorporated into lysozyme-insensitive material was 34.9 pmol.
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x
633
MurNAc-pentapeptide formation (11). Thus, acylation of residue 3 with L-alanine does not result in a discrimination against this nucleotide in the reaction catalyzed by the initial membrane-associated enzyme in peptidoglycan synthesis. Since isolated membranes from S. aureus Copenhagen do not catalyze a high rate of nascent peptidoglycan synthesis, the membrane preparation from S. aureus H prepared by alumina grinding (16) has been used for comparing UDP-MurNAc-pentapeptide and -hexapeptide. In the concentration range of Km/2 to 5 K., UDP-MurNAc-hexapeptide is as effective as UDP-MurNAc-pentapeptide in the biosynthesis of peptidoglycan. The parameters Vmax and Vmax/Km are similar for both nucleotides. With UDP-MurNAc-pentapeptide the incorporation is characterized by a second line segment in the Lineweaver-Burk plot at low substrate concentrations. The second line segment is not observed with the hexapeptide. The results presented in this paper and those described by Schleifer et al. (in press) suggest that in S. aureus UDP-MurNAc-hexapeptide is utilized effectively for synthesis of nascent peptidogly-
10-4
FIG. 5. Incorporation of ['4C]GlcNAc from UDP- can. In L. viridescens, the N-terminal alanine of ["C]GIcNAc into peptidoglycan as a function of UDP-MurNAc-pentapeptide (A) and UDP-MurNAc- the hexapeptide serves as an acceptor at the hexapeptide (B). Membrane fragments from S. aureus lipid intermediate stage for an additional serine H (68.5 gg of protein) were incubated with the indi- residue required for the formation of the intercated concentrations of UDP-MurNAc-peptide as peptide bridge. In contrast, on the basis of the described in the text. The insets in A and B show in vivo results described by Schleifer et al., the Lineweaver-Burk analyses of the data from which the N-terminal alanine of the hexapeptide the values for K. and Vmax were determined. of S. aureus Copenhagen does not function as
an acceptor for the glycine residues required is added. The relationship between glycine de- for interpeptide bridge formation. The presence ficiency and UDP-MurNAc-hexapeptide pro- of the N-terminal alanine decreases the number duction is not well understood. Since N- of potential interpeptide bridges for cross-linkterminal alanine in the peptide moiety of the ing the glycan strands. Thus, growth in a peptidoglycan is not further substituted in this glycine-poor medium indirectly affects the deorganism, we might view the formation of the gree of cross-linking of the peptidoglycan. hexapeptide as a mechanism for conserving the glycine pool of the growing bacterium. ACKNOWLEDGMENTS Acylation of the c-amino group of the lysine We are grateful to Walter P. Hammes for helpful discusresidue with L-alanine does not affect the activ- sions of these results. This investigation was supported by Public Health Service ity catalyzed by phospho-MurNAc-pentapeptide translocase. From the results with the ex- grant AI-04615 from the National Institute of Allergy and change reaction, it was concluded that the rate Infectious Diseases. of undecaprenyl diphosphate MurNAc hexa LITERATURE CITED peptide formation is similar at both high and 1. D. J. Tipper, C. H. Birge, and J. L. Ghuysen, J.-M., low substrate concentrations when compared Strominger. 1965. Structure of the cell wall of Staphylowith UDP-MurNAc-pentapeptide. With memcoccus aureus strain Copenhagen. VI. The soluble brane preparations from L. viridescens, the rate glycopeptide and its sequential degradation by peptidases. Biochemistry 4:2245-2254. of undecaprenyl diphosphate - MurNAc - hexa W. R., and J. F. Smith. 1970. Rapid sequence peptide formation from UDP-MurNAc-Ala- 2. Gray, analysis of small peptides. Anal. Biochem. 33:36-42. DGlu-Lys(Ne-Ala)-DAla-DAla is similar to that 3. Hammes, W. P., and F. C. Neuhaus. 1974. On the specificity of phospho-N-acetylmuramyl-pentapeptide for described undecaprenyl-diphosphate-
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5.
6. 7.
8.
9.
10.
11.
12.
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translocase. The peptide subunit of uridine diphosphate-N-acetylmuramyl-pentapeptide. J. Biol. Chem. 249:3140-3150. Hammes, W. P., and F. C. Neuhaus. 1974. Biosynthesis of peptidoglycan in Gaffkya homari: role of the peptide subunit of uridine diphosphate-N-acetylmuramyl-pentapeptide. J. Bacteriol. 120:210-218. Heydanek, M. G., Jr., R. Linzer, D. D. Pless, and F. C. Neuhaus. 1970. Initial stage in peptidoglycan synthesis. Mechanism of activation of phospho-N-acetylmuramyl-pentapeptide translocase by potassium ions. Biochemistry 9:3618-3623. Ito, E., and M. Saito. 1963. Time course of accumulation of UDP-N-acetylamino sugar derivatives in Staphylococcus aureus. Biochim. Biophys. Acta 78:237-247. 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. Mandelstam, J., and H. J. Rogers. 1959. The incorporation of amino acids into the cell-wall mucopeptide of staphylococci and the effect of antibiotics on the process. Biochem. J. 72:654-662. Mandelstam, P., R. Loercher, and J. L. Strominger. 1962. A uridine diphosphoacetylmuramyl hexapeptide from penicillin-treated Streptococcus faecalis. J. Biol. Chem. 237:2683-2688. Patterson, M. S., and R. C. Greene. 1965. Measurement of low energy beta-emitters in aqueous solution by liquid scintillation counting of emulsions. Anal. Chem. 37:854-857. Plapp, R., and J. L. Strominger. 1970. Biosynthesis of the peptidoglycan of bacterial cell walls. XVII. Biosynthesis of the peptidoglycan and of interpeptide bridges in Lactobacillus viridescens. J. Biol. C hem. 245:3667-3674. Saito, M., N. Ishimoto, and E. Ito. 1963. Uridine diphos-
phate N-acetylamino sugar derivatives in penicillintreated Staphylococcus aureus. J. Biochem. 54:273-278. 13. Schleifer, K. H., and 0. Kandler. 1972. Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bacteriol. Rev. 36:407-477. 14. Stickgold, R. A., and F. C. Neuhaus. 1967. On the initial stage in peptidoglycan synthesis. Effect of 5-fluoroura-
15.
16.
17.
18.
19.
cil substitution on phospho-N-acetylmuramyl-pentapeptide translocase (uridine 5'-phosphate). J. Biol. Chem. 242:1331-1337. Strominger, J. L. 1957. Microbial uridine-5'-pyrophosphate N-acetylamino sugar compounds. I. Biology of the penicillin-induced accumulation. J. Biol. Chem. 224:509-523. Stroininger, J. L., M. Matsuhashi, J. S. Anderson, C. P. Dietrich, P. M. Meadow, W. Katz, G. Siewert, and J. M. Gilbert. 1966. Glycopeptide synthesis in Staphylococcus aureus and Micrococcus lysodeikticus, p. 473486. In S. P. Colowick and N. 0. Kaplan (ed.), Methods in enzymology, vol. 8. Academic Press Inc., New York. Strominger, J. L., and R. H. Threnn. 1959. Accumulation of a uridine nucleotide in Staphyloccus aureus as the consequence of lysine deprivation. Biochim. Biophys. Acta 36:83-92. Struve, W. G., R. K. Sinha, and F. C. Neuhaus. 1966. On the initial stage in peptidoglycan synthesis. PhosphoN-acetylmuramyl-pentapeptide translocase (uridine monophosphate). Biochemistry 5:82-93. Tipper, D. J., J. L. Strominger, and J. C. Ensign. 1967. Structure of the cell wall of Staphylococcus aureus, strain Copenhagen. VII. Mode of action of the bacteriolytic peptidase from Myxobacter and the isolation of intact cell wall polysaccharides. Biochemistry 6:906920.
