Vol. 35, No. 9

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 1991, p. 1753-1759

0066-4804/91/091753-07$02.00/0 Copyright ) 1991, American Society for Microbiology

Biosynthesis of Peptidoglycan in GafJkya homari: On the Target(s) of Benzylpenicillin RABINDRA K. SINHA AND FRANCIS C. NEUHAUS* Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, Illinois 60208 Received 5 June 1991/Accepted 13 June 1991

The formation of acceptor for the NE-(D-Ala)-acceptor transpeptidase is an essential feature of nascent peptidoglycan processing. In Gaffkya homari the synthesis of cross-bridges in peptidoglycan includes a variety of penicillin-sensitive enzymes, e.g., transpeptidase, DD-carboxypeptidase, and LD-carboxypeptidase. To determine the primary target, we grew cultures in the presence of the MICs of benzylpenicillin (0.2 ,ug/ml), methicillin (10 ,ug/ml), cephalothin (5 ,ug/ml), and cefoxitin (25 ,Lg/ml) and examined the monomer-dimer composition of each peptidoglycan by high-performance liquid chromatography after muramidase digestion. From these studies it was recognized that of all the dimers, the. synthesis of the predominant cross-bridge, diamidated octapeptide (-Ala-4so-D-Gln-Lys-D- Ala -Ala-4so-D-Gln-Lys-D-Ala), is most sensitive to the action of the ,-lactam at its MIC. The enhanced deamidation of the acceptor tetrapeptide, one of the substrates for the transpeptidase, is correlated with the inhibition of this cross-bridge. For example, at the MIC of benzylpenicillin, the ratio of amidated tetrapeptide to nonamidated tetrapeptide decreased from 2.8 in the control to 1.0 in the treated culture. From these results it would appear that a decrease in preferred acceptor for the transpeptidase results in the inhibition of synthesis of this major cross-bridge. Thus, the metabolism of the amide function of the monomer peptides may represent an additional feature of processing in the assembly of cross-bridged dimers in the peptidoglycan of this organism that is sensitive to the action of P-lactam.

Benzylpenicillin is a structural analog of the acyl-D-AlaD-Ala moiety of donor peptide in the reaction catalyzed by the NE-(D-Ala)-acceptor transpeptidase (19). Acylation of this enzyme with penicillin inhibits the cross-linking of peptidoglycan (PG), a feature that is necessary for the structural integrity of this polymer. It has been established that proteins which covalently bind penicillin, i.e., penicillinbinding proteins (PBPs), are essential enzymes in the biosynthesis of PG and are the targets of f3-lactam action. Thus, penicillin is a useful tool for controlling the processing and synthesis of the cross-bridges in PG assembly systems. Reactivated membranes and membrane-walls from Gaffkya homari catalyze the efficient synthesis of nascent PG and its cross-linking (1, 12). It was observed in these systems that N'-(D-Ala)-acceptor transpeptidase is inhibited by high concentrations of penicillin (IC50 [concentration required to inhibit activity by 50%], 630 ,ug/ml) and that the DD-carboxypeptidase is inhibited by low concentrations (IC50, 0.25 ug/Iml) (7). Since the IC50 for the carboxypeptidase is similar to the MIC of the organism, it was concluded that the primary target of penicillin is the DD-carboxypeptidase (7). In contrast, it was established from in vivo penicillin-binding experiments that the DD-carboxypeptidase (PBP-9) is not the primary target. On the basis of these experiments and the MIC, PBP-6, a protein of 116 kDa whose function is not known, appears to be the target in this organism (21). Because of these differing conclusions, it was of interest to examine the composition of the PG from cells grown in the presence of benzylpenicillin in order to identify the features of PG that are sensitive to the action of this P-lactam. From these changes in PG structure, it was our goal to reconcile

*

the conclusions derived from studies of the in vitro and in vivo assembly systems. MATERIALS AND METHODS

Materials. D-[6-3H]glucosamine hydrochloride (20 Ci/ mmol) was purchased from American Radiolabeled Chemicals, Inc., St. Louis, Mo. Benzylpenicillin, methicillin, cephalothin, cefoxitin, muramidase (hen egg white lysozyme), and mutanolysin were purchased from Sigma Chemical Co., St. Louis, Mo. Chalaropsis muramidase was the product of Miles Laboratories Inc., Elkhart, Ind. Yeast extract and Bacto-Peptone were purchased from Difco Laboratories, Detroit, Mich. The sources of other chemicals have been described previously (1, 12). Growth of G. homari in the presence of J8-lactam for PG analysis. G. homari, properly named as Aerococcus viridans subsp. homarus (13), was obtained from the American Type Culture Collection, Rockville Md., as was Aerococcus viridans ATCC 10400. The organism was grown in 1% glycerol-1% yeast extract-1% Bacto-Peptone-0.5% K2HPO4 with shaking at 150 rpm at 32°C. The P-lactam together with D-[6-3H]glucosamine hydrochloride (20 ,uCi) was added to 40 ml of culture (optical density, 0.07; ca. 4 x 107 CFU). Growth was monitored at 585 nm, and at the indicated time the cells were collected, chilled, and washed with 50 mM Tris-hydrochloride (pH 7.8) at 4°C. The cells were extracted with 4% sodium dodecyl sulfate (SDS) at 100°C for 60 min and washed in water (six to eight times). To the washed pellet suspended in 50 mM Tris-hydrochloride (pH 7.8), muramidase (100 ,ug/ml) was added and digested for 16 h at 37°C. After the reaction had been terminated by heating at 100°C, the digest was centrifuged in a Beckman Microfuge and the supernatant fraction was desalted on a Sephadex G-25 column (1.8 by 100 cm). Approximately 80 to 90% of

Corresponding author. 1753

1754

SINHA AND NEUHAUS

the glucosamine-labeled PG was isolated in the supernatant fraction. For the deamidation and high-performance liquid chromatography (HPLC) analyses, the muropeptides were first reduced with NaBH4. The desalted samples were treated with 100 pxg of NaBH4 per fml in 250 mM borate buffer (pH 9.0). After the reaction had continued at 25°C for 20 min, the reduction was terminated by adjUsting the reaction mixture to pH 5.0 with 4 M H3PO4. Preparation of walls from G. homari. The G. homari cell walls were prepared by the method described by Kalomiris et al. (12) with the following modification. Trypsin-treated walls were boiled with 4% SDS for 60 min; after this treatment, the SDS-insoluble wall was thoroughly washed with distilled water and lyophilized. For these preparations the organism was grown on 1% glucose-yeast extractBacto-Peptone-K2HPO4 as described above. These walls were used to obtain monomers and dimers, which, after characterization, were used as standards for the present work. HPLC of the muramidase digest of PG. Chromatographic analysis of the digest was performed by the procedure developed by Glauner et al. (5, 6) for PG from Escherichia coli. The HPLC system consisted of a Waters-600 Multisolvent Delivery System, Waters WISP 712 autoinjector, 490 E Programmable Multiwave Length Detector, and 740 Data Module (Millipore Corp). The separations were accomplished with an ODS-Hypersil (5 ,um) column (Keystone Scientific Inc, State College, Pa.) maintained at 55°C. A linear gradient was established between 50 and 75 mM phosphate (pH 4.30 and 4.95, respectively) in 15% methanol. The flow rate was 0.5 ml/min (run time, 150 min). The muropeptides of the digest were monitored at 208 nm. For each run, approximately 80,000 cpm was chromatographed. To quantify the amount of reduced muropeptide, we established the specific activity of N-acetylglucosamine by determining the molarity of peptide from an amino acid analysis. From the counts per minute and specific activity, the number of moles of monomers and dimer were deterniined. While the muropeptide monomers and dimers were continuously monitored at 208 nm, the radioactivity measurements were performed on column fractions. This procedure for monitoring radioactivity resulted in some apparent loss of resolution. Thus, with the exceptioh of diamidated bis-disaccharide (DS)-tetrapeptide-tetrapeptide (tetra-tetra), the moles percent of the dimers were calculated for the aggregate pool of radioactivity associated with the respective group of muropeptides. The percent cross-linking was calculated by the procedure described by Dezelee and Shockman (3), using only monomers and dimers. Analytical methQds. High-voltage papel electrophoresis to separate non-, tnono- ant diamidated muropeptides was perforfited for 120 mnin at 1,500 V in pyridine-acetic acid-H20 (15:50:2,000). at 38 V/cm. The muropeptides were deamidated by treating the reduced sample at pH 12.5 at 37°C for 1 to 6 h., The time course of deamidation was determined by monitoring the products by the above HPLC procedure. Fast atom bombardme*t mass spectrometry of four of the reduced muropeptide dimers was performed by using the model 70-SE mass spectrometer (VG Instrument Co., Sussex, England) in the Analytical Service Laboratory, Department of Chemistry. The sample matrix was 5% p-toluenesulfonic acid in glycerol. Amino acid analyses of muropeptides were performed on hydrolyzed samples (5.7 N HCI, 10 h at 105°C) by dg1g the phenyl isothiocyanate procedure (10).

ANTIMICROB. AGENTS CHEMOTHER.

REtSULTS Structural analysis of PG from G. homari. A characterization of the PG from G. homari by Nakel et al. (14) revealed a complex mixture of peptides. Two features of this PG are the direct cross-bridge and the variable amidation of the -y-D-glutarnyl residues. To quantify each of the muramidase digestion products of this PG, we have adopted the HPLC procedure designed by Glauner et al. (5, 6) for the PG isolated from E. coli. Because of the -y-D-glutamyl and the iso-D-glutaminyl residues, the number of muramidase cleavage products is large. Thus, it became necessary to group the products according to their amino acid composition and degree of amidation. Walls, which were extracted with 4% $DS at 100°C and thoroughly washed with water, were used as the source of monomer and dimer muropeptides. These walls were digested with either muramidase (hen egg white lysozyme), Chalaropsis muramidase, or mutanolysin. The muropeptide profiles for the Chalaropsis muramidase- and mutanolysindigested walls were essentially identical to that observed for lysozyrme. Thus, this muramidase was used for all the experiments in this study. As summarized in Table 1, three pairs of muropeptides were identified in the monomer region. Each pair had the same amino acid composition but differed only in amide content. Deamidation of muropeptides 2, 4, and 6 (Table 1) yielded the corresponding nonamidated compounds 1, 3, and 5. From deamidation, amino acid composition, and high-voltage electrophoresis studies, it was inferred that compounds 1 and 2 are nonamidated and amidated DS-tripeptide (tri), that compounds 3 and 4 are nonamidated and amidated DS-tetra, and that compounds 5 and 6 are nonamidated and amidated DS-penta, respectively. In the dimer region two groups of muropeptides have been characterized (Table 1). These are bis-DS-heptapeptides and bis-DS-octapeptides. Each group has four compounds, each of which was converted either to the nonamidated bis-DStetra-tri (compound 7) or nonamidated bis-DS-tetra-tetra (comnpound 11). The major dimer (compound 14), with an MH+ of 1,777, is the diamidated bis-DS-tetra-tetra (Fig. 1). Two dimers (compounds 12 and 13) with identical MH+ of 1,778 are monoamidated bis-DS-tetra-tetra (Fig. 1). Since this dimer does not have an axis of symmetry, one stem peptide is not superimposable on the other. Thus, two isomers of the monoamidated bis-DS-tetra-tetra were observed. In addition, two isomers of monoamidated bis-DStetra-tri (compounds 8 and 9) were detected. Deamidation of either pair of monoamidated dimers (i.e., compounds 12 and 13 or compounds 8 and 9) yielded the nonamidated bis-DStetra-tetra (MH+ 1,779) (compound 11) or nonamidated bis-DS-tetra-tri (compounds 7). The muropeptides isolated from the muramidase digest of walls were used as standards for the PG composition studies described in this paper. Composition of PG synthesized in the presence of lbenzylpenicillin. To monitor the PG synthesized during the application of the P-lactam, D-[6-3H]glucosamine was added concurrently to the culture medium. The utilizatidn of the radiolabeled glucosamine was greatly enhanced by changing the carbon source from glucose to glycerol. This change in carbon source resulted in an increase in the doubling time from 70 to 100 min. While the A208 was used to monhior the monomers and dimers in PG, the radiolabeled glucosamine was essential for monitoring the newly synthesized monomers qnd dimers, expressed as moles percent, that were formed ddrin& the presence of the ,-lactam. For the experiments presented in this paper, the labeled cells were ex-

VOL. 35,

1991

TARGET OF BENZYLPENICILLIN IN G. HOMARI

1755

TABLE 1. Composition, deamidation, and mass numbers of monomers and dimers from G. homari PG Structurea

Retention (min)b time

Monomers 1. DS-tri( ) 2. DS-tri(NH2)

3. DS-tetra( ) 4. DS-tetra(NH2) 5. DS-penta( ) 6. t)S-penta(NH2) Dimers: heptapeptidesd 7. bis-DS-tetra( )-tri( ) 8. bis-DS-tetra( )-tri(NH2)e 9. bis-DS-tetra(NH2)-tri( )Y 10. bis-DS-tetra(NH2)-tri(NH2) Dimers: octapeptidesd 11. bis-DS-tetra( )-tetra( )

13. bis-DS-tetra(NH2)-tetra( )e 14. bis-DS-tetra(NH2)-tetra(NH2) a The following examples of abbreviations for reduced

Deamidation (pH 12.5)

Ala:Glu:Lys ratio

28.4 30.5 39.4 42.6 50.3 53.8

No effect 2 -1 No effect 4 -* 3 No effect 6- 5

96.3 99.8 102.1 105.2

No effect 8 7 9- 7 10-7

3:2:2

No effect 12 11 13 11 14 - 11

2:1:1 2:1:1

104.2 109.1 111.5 115.3

12. bis-DS-tetra( )-tetra(NH2)e

MH+'

1,779 1,778 1,778 1,777

1:1:1 1:1:1

2:1:1 2:1:1 3:1:1 3:1:1

3:2:2 3:2:2 3:2:2

2:1:1 2:1:1

muropeptides are used: DS-tetra( ), N-acetylglucosaminyl(GlcNAc)-N-acetyltnuramicitol(MurNAc)

(-Ala--y-D-Glu-Lys-D-Ala); DS-tetra(NH2), N-acetylglucosaminyl-N-acetylmuramicitol (-Ala-iso-D-Gln-Lys-D-Ala); -tri, -Ala--y-D-Glu-Lys; -penta, -Ala-y-DGlu-Lys-D-Ala-D-Ala; bis-DS-tetra(NH2)tetra(NH2); dimer of GIcNAc-MurNAc-tetra(NH2) cross-linked between the lysine of the acceptor stem and D-alanine of the donor stem; bis-DS-tetra(NH2)tri(NH2), dimer of GlcNAc-MurNAc-tetra(NH2) (donor stem) and GIcNAc-MurNAc-tri(NH2) (acceptor stem) cross-linked between the lysine of the acceptor stem and the D-alanine of the donor stem. Unless stated, all abbreviations of amino acid residues denote the L configuration. MurNAc denotes either N-acetylmuramyl or N-acetylmuramicitol. b The reduced muropeptides were isolated from the wall and analyzed by the procedures described in Materials and Methods. The retention times reflect those from the ODS-Hypersil (5-pLm) column. c Mass of the protonated molecular ion. d Only the hepta- and octapeptide dimers have been characterized. The assignment of the amide function to either peptide stem is arbitrary.

tracted with SDS, washed with water, and subjected to muramidase d,igestion as described in Materials and Methods. This method gave the same profile of muropeptides as that determined from isolated cell walls. Since benzylpenicillin does not cause lysis in G. homari (Fig. 2A), the compositional analysis of PG is readily accessible at all levels of P-lactam. For the analyses summarized in Table 2, we have chosen three concentrations of benzylpenicillin: 0.05 ,ug/ml, 0.2 ,ug/ml (the MIC), and 200 ,ug/ml. To maximize the inhibition of cross-linking, we also tested the effect of 200 ,ug/ml, a concentration that is 1,000-fold in excess of the MIC. At the MIC, 100% of the cells survived the 5.5 h of penicillin treatment, whereas at 200 ,ug of benzylpenicillin per ml, at least 33% of the cells survived. Thus, G. homari is tolerant to the MIC during this experimental period. The HPLC profile of the muropeptide fraction from PG G-M

G-M

G-M

A

A

A

E ()

E()

K- A

K- A

A

A

K E

E

K-A

I

A

K

E

A

A

G-M

G-M

(NIi)

(NIt)

E (Nit)

K-

K E()

(Nit) E

A

G-M

i3 12 14 FIG. 1. Schematic structures of Octapeptide dimers (compounds il, 12, 13, and 14). The assignment of the amide function in compounds 12 and 13 is arbitrary. 11

synthesized by G. homari grown in the absence of benzylpenicillin for 2.8 generations is shown in Fig. 3A. The moles percent of the muropeptides are summarized in Table 2. For comparison, the profile of a muramidase digest of PG from a culture treated with 0.2 ,ug of benzylpenicillin per ml is shown in Fig. 31. The amounts of cross-linked dimer were decreased in the penicillin-treated cultures. At this concentration (0.2 ,ug/ml), penicillin inhibited the synthesis of the heptapeptides by 15%, the octapeptides (compounds 11, 12, and 13) by 18%, and the diamidated octapeptide (compound 14) by 59% (Table 2). The PG compositions from cultures grown in the presence of 0.05 and 200 pLg of benzylpenicillin per ml have also been quantified, and the results are presented in Table 2. Clearly, of all the dimers, the synthesis of diamidated bis-DS-tetra-tetra is the most sensitive to inhibition by penicillin. The inhibition of bacterial growth by penicillin requires at least one generation for maximal expression (Fig. 2A). This lag may reflect the time-dependent acylation of the target proteins. Thus, with simultaneous addition of 13-lactam and [3Hlglucosamine, measurements of labeled muropeptides may reflect assembly in both the inhibited and uninhibited syste'ms. To assess the contribution of the uninhibited system, we added the [3H]glucosamine to the culture 1 h atter the app'lication of the 3-lactam. Under these conditions, the synthesis of heptapeptides was inhibited by 15%, that of the octapeptides (compounds 11, 12, and 13) by 35%, and that of the diamidated octapeptide (compound 14) by 58% (Table 2). Since these observations are essentially identical to those observed in cultures with concurrent addition of penicillin (0.2 ,ug/ml) and [3H]glucosamine, it is concluded that the time-dependent inactivation of the target proteins is not a major factor in the interpretation of our results.

ANTIMICROB. AGENTS CHEMOTHER.

SINHA AND NEUHAUS

1756

TABLE 2. Muropeptide composition of PG from G. homari

1.00

grown

Harvest

A

in the presence of benzylpenicillina

Control

-

0.50

Muropeptide

0.05 ,g LO LO

0 -

oi

0.20 6 Or6

6

A

0.20

MAg

Monomers 1. DS-tri( ) 2. DS-tri(NH2)

3.6 0.9

2.9 0.6

3.8 0.4

6.8 3.4

1.8 1.0

3. DS-tetra( ) 4. DS-tetra(NH2)

11.3 31.7

19.2 21.1

18.2 18.5

16.0 16.7

7.5 5.9

5. DS-penta( ) 6. DS-penta(NH2)

2.4 10.8

3.6 21.7

2.8 29.4

4.4 28.2

10.0 56.8

Dimers 7, 8, 9, 10. bis-DS-hepta- 10.5

11.4

8.9

8.9

6.5

15.0

13.0

12.3

9.8

7.4

13.8

6.5

5.7

5.8

3.1

. .g . ;/S~~~~~~~~~~~ 0.200

Penicillin

0.10

g

20

0.05I

0

1

2

3

4

5

6

7

Hours

Composition (mol%)b at benzylpenicillin concn (,ug/ml) of: 0 200 0.2 0.2c 0.05

peptidesd

11, 12, 13. bis-DS-octapeptidese,

14. bis-DS-tetra(NH2)

1.00

tetra(NH2) -

0.50

LO

0..2

LO

0.20 0

0

0

0.10

0.05 0

1

2

3

4 Hours

5

6

7

FIG. 2. (A) Growth of G. homari in the presence of benzylpenicillin. Symbols: 0, control; 0, 0.05 ,ug/ml; A, 0.2 ,ug/ml, A, 200 ,ug/ml. (B) Growth in the presence of 3-lactams. Symbols: 0, 10 ,ug of methicillin per ml; A, 5 p.g of cephalothin per ml; A, 25 ,ug of cefoxitin per ml. At the first arrow, the P-lactam and the radiolabeled glucosamine (20 ,uCi) were added. At the second arrow, the cells were harvested and used for the experiments in Fig. 3 and Tables 2 and 3.

One of the factors which appears to determine the dimer profile in a group of muropeptide-s is.the degree of amidation of the acceptor in the reaction catalyzed by the transpeptidase. It is suggested that a decrease in the amount of amidated DS-tetra will lead to a decrease in the amount of diamidated bis-DS-tetra-tetra (compound 14). In the control culture, 26% of DS-tetra and 80% of DS-tri are not amidated. In contrast, in the culture treated with penicillin at 0.2 ,ug/ml, 50% of DS-tetra and 90% of DS-tri are not amidated (Table 2). The ratios of amidated DS-tetra to nonamidated DS-tetra are 2.8 and 1.0 in the control and penicillin-treated cultures, respectively. Thus, with an amount of benzylpenicillin sufficient for growth inhibition, a shift from more amidated to less amidated DS-tetra was observed. The increase in the amount of nonamidated DS-tetra represents a previously unrecognized response to the action of benzylpenicillin. At a high concentration of penicillin (200 ,ug/ml), the major muropeptide was amidated DS-penta (57%). This observation is consistent with the inhibition of the DD-carboxypeptidase at this concentration, an enzyme that is only partly inhibited at 0.2 ,ug/mi (21). An additional feature of benzylpenicillin action is the

a Cells labeled with [6-3Hlglucosamine for 330 min after addition of benzylpenicillin (Fig. 2A) were treated as described in Materials and Methods. The reduced, radiolabeled muropeptides were separated and quantified as described (Fig. 3A and B for 0 and 0.2 FLg of benzylpenicillin per ml, respectively). b Moles percent based on muropeptides 1 to 14. Compound numbers correspond to peak numbers in Table 1. c The [3H]glucosamine was added 60 min after the addition of the benzylpenicillin. d bis-DS-heptapeptides represent a group of four heptapeptides, 7, 8, 9, and 10, from Table 1. e bis-DS-octapeptides represent three octapeptides, 11, 12, and 13, from Table 1.

formation of two unknown muropeptides at 78 and 82 min (Fig. 3B). These muropeptides were not detected in PG from the control culture. The structures of these compounds are under investigation. Composition of PG synthesized in the presence of other P-lactams. The observations with benzylpenicillin can be the result of inhibition of either transpeptidase, carboxypeptidase, or an enzyme regulating the metabolism of the iso-Dglutaminyl residues. To address these possibilities, we tested three additional 3-lactams, methicillin, cephalothin, and cefoxitin, at their MIC in cultures of G. homari (Fig. 2B) and examined the PG for specific changes in structure. The PG from cultures treated with methicillin showed a similar response to that observed with benzylpenicillin. The amount of nonamidated DS-tetra in a culture grown in the presence of 10 ,ug of methicillin per ml increased from 26 to 47% of the DS-tetrapeptide fraction. The amounts of diamidated bisDS-tetra-tetra (compound 14) and non- and monoamidated bis-DS-tetra-tetra (compounds 11, 12, and 13) were inhibited by 54 and 15%, respectively, of the level in the control culture (Table 3). Thus, as in the case of benzylpenicillin, the synthesis of the diamidated bis-DS-tetra-tetra would appear to be more sensitive to P-lactam action than is the synthesis of dimers 11, 12, and 13. In contrast to the other P-lactams tested, cephalothin at its MIC inhibited the synthesis of all cross-linked dimers. bis-DS-tetra-tri (compounds 7, 8, 9, and 10) was inhibited by 58%, while bis-DS-tetra-tetra (compounds 11, 12, and 13) and the diamidated bis-DS-tetra-tetra (compound 14) were inhibited by 77 and 91%, respectively. These results suggested that the mechanism of growth inhibition by cephalo-

VOL. 35, 1991

TARGET OF BENZYLPENICILLIN IN G. HOMARI

1757

-

at

c0o 0)

0 C\2

a)

Q)

.0 co v0U)

C.)

U C..

.0a .0

-o

4

3

co0

co0 x

2

X- 2

1

1

C)

0

0

0

20

40

60

80

100

120

140

Time (min)

0

20

40

60

80

100

120

140

Time (min)

FIG. 3. Separation of muropeptides from PG of control cells (A) and from PG of cells grown in the presence of 0.2 ,ug of benzylpenicillin ml (B). The cells from each of the cultures in Fig. 2 were extracted with SDS and digested with muramidase, and the digest was desalted by the procedures described in Materials and Methods. In each case, 80,000 cpm was chromatographed. In the top profile, the AUFS (Absorbance Unit Full Scale) at 208 nm is shown; in the bottom profile, the solid bars represent the labeled monomers and dimers synthesized during the time (330 min) between the addition of r-lactam and harvesting (arrows in Fig. 2). per

thin may be different from that by methicillin and benzylpenicillin. Cefoxitin caused a significant decrease (60%) in the amount of amidated DS-tetra. This inhibition is the result of the ability of cefoxitin to inhibit DD-carboxypeptidase (21). The low level of nonamidated DS-tetra (8.5%) is associated with the low level of amidated DS-tetra. No inhibition of the bis-DS-tetra-tri group or bis-DS-tetra-tetra (compounds 11, 12, and 13) was observed. However, diamidated bis-DStetra-tetra (compound 14) decreased from 13.8 to 9.7 mol%. This decrease appears to be correlated with the decrease in amidated DS-tetra acting as acceptor in the reaction catalyzed by the transpeptidase. DISCUSSION

Analyses of PG from ,-lactam-inhibited cultures of G. homari have provided the basis for defining a number of unique features that are essential to our understanding of cross-bridge formation in this organism. One of these is the multiplicity of cross-bridged products which result from the variable amidation of the donor and acceptor stem peptides in the reaction catalyzed by the transpeptidase. The finding that the synthesis of the diamidated bis-DS-octapeptide is more sensitive to the action of 1-lactam than is the formation of the nonamidated and monoamidated peptide dimers was unexpected. Our observations suggest that G. homari has a variety of ,-lactam-sensitive enzymes that function in the assembly of cross-linked PG. It is our hypothesis that these enzymes include both transpeptidase(s) and DD-carboxypeptidase with different levels of sensitivity. In addition, it appears that a penicillin-sensitive amidohydrolase, which converts amidated stem peptides to nonamidated ones, is

involved in the metabolism of the iso-D-glutaminyl residues of PG. G. homari is one of a variety of bacteria that contain a significant amount of amidated -y-D-glutamyl residues in their wall PG (14). These residues were first described for Staphylococcus aureus and Arthrobacter crystallopoietes (18). Virtually all of the -y-D-glutamyl residues are amidated in S. aureus. Siewert and Strominger (17) established that the amidation of these residues involves the participation of the lipid intermediate, undecaprenyl diphosphate-N-acetylmuramyl(pentapeptide)-N-acetylglucosamine in an ATP, glutamine(NH4')-dependent reaction. In G. homari, the highest degree of amidation is found in the pentapeptide fraction whereas the lowest degree is found in the tripeptide fraction. In the tetrapeptide fraction, the ratio of amidated to nonamidated monomer is 2.8 in the control culture. Since the peptides in nascent PG are incorporated as the amidated pentapeptide, one may conclude that an amidohydrolase progressively removes the amide function in the processing of penta-, tetra-, and tripeptides. Our data indicate that the deamidation of the tetra- and tripeptides is more prevalent than that of the pentapeptide. Although the function of these amide groups in the assembly of PG has not been established (16), Nakel et al. (14) proposed that at least one of the peptide stems is required to be amidated in G. homari for cross-linking to occur. In the present study, growth in the presence of penicillin at the MIC (0.2 Fig/ml) resulted in a 60% increase in nonamidated DS-tetra and a 42% decrease in amidated DS-tetra in the assembled PG when compared with the control culture. Thus, the ratio of amidated to nonamidated DS-tetra decreased from 2.8 in the control to 1.0 at this concentration of 3-lactam. With these changes in monomer composition, a 32% reduction in cross-linked dimers was

1758

ANTIMICROB. AGENTS CHEMOTHER.

SINHA AND NEUHAUS

TABLE 3. Muropeptide composition of PG from G. homari grown in the presence of methicillin, cephalothin, and cefoxitina Composition (mol%)b in presence of: Muropeptide

Monomers 1. DS-tri( ) 2. DS-tri(NH2)

Control (no Methicillin Cephalothin Cefoxitin addition) (10 Fg/ml) (5 p.g/ml) (25 iig/ml)

3.6 0.9

2.8 0.9

3.0 5.6

3.4 2.4

3. DS-tetra( ) 4. DS-tetra(NH2)

11.3 31.7

18.2 20.9

19.3 31.3

8.5 12.9

5. DS-penta( ) 6. DS-penta(NH2)

2.4 10.8

3.0 26.6

5.3 26.3

11.1 23.9

Dimers 7, 8, 9, 10. bis-DSheptapeptidesc 11, 12, 13. bis-DS-

10.5

8.5

4.4

11.8

15.0

12.7

3.5

16.3

octapeptidesd 14. bis-DS-tetra

13.8

6.4

1.3

9.7

(NH2)tetra(NH2) a Cells labeled with [6_3Hlglucosamine for 330 min after addition of 13-lactam (Fig. 2B) were treated as described in Materials and Methods. The reduced, radiolabeled muropeptides were separated and quantified as described in Table 2. b Moles percent based only on muropeptides 1 to 14. Compound numbers correspond to peak numbers in Table 1. I bis-DS-heptapeptides represent a group of four heptapeptides, 7, 8, 9, and 10, from Table 1. d bis-DS-octapeptides represent three octapeptides, 11, 12, and 13, from Table 1.

observed. Of all the dimers in the heptapeptide and octapeptide groups, the synthesis of the diamidated octapeptide was the most sensitive to the action of penicillin. At 0.2 ,ug of penicillin per ml, conversion of amidated tetrapeptide to its nonamidated form appears to lead to a decrease of preferred acceptor in the reaction catalyzed by the transpeptidase. Since the diamidated octapeptide is the predominant crosslinked dimer, it is proposed that the amidated tetrapeptide is the preferred acceptor. The other dimer (compound 13) that requires this tetrapeptide will also be affected by this change in acceptor. Thus, the regulated action of the proposed enzyme, -iso-D-Gln-amidohydrolase, may play an indirect role in controlling cross-linking in G. homari by determining the concentration of the preferred acceptor. Although the proposal for a penicillin-stimulated amidohydrolase is consistent with the observations in this paper, alternative explanations may be considered. For example, the altered amidation levels could be the result of decreased cross-linking rather than the cause of it. However, it is not clear how a decrease in the percent cross-linking triggers deamidation. Another explanation is the suggestion that penicillin enhances the conversion of the nonamidated pentapeptide to the nonamidated tetra- and tripeptides. This explanation has the advantage that it would explain both the observed accumulation of amidated pentapeptide relative to the nonamidated pentapeptide and the deficiencies of the amidated tetra- and tripeptides. These alternatives represent clear possibilities that will require further analyses in in vitro membrane and membrane-wall PG assembly systems. Thus, the hypothesis of a penicillin-sensitive amidohydrolase is considered to be one of several explanations for the observations reported in this paper. If the concentration of penicillin is increased 1,000-fold (to

200 ,ug/ml), a majority (57%) of the monomer peptide is amidated pentapeptide. In this culture, both amidated and

nonamidated tetrapeptides are greatly decreased. Thus, at high concentrations an appreciable amount of the DD-carboxypeptidase activity is inhibited. These decreases are associated with the inhibition of all dimers, in particular the diamidated octapeptide. In contrast, at 0.2 ,tg of penicillin per ml (the MIC), DD-carboxypeptidase is only slightly inhibited, as reflected in the amount of tetrapeptide (36.7%) compared with the control (43%). At 0.05 ,ug of penicillin per ml, the amount of tetrapeptide is 40.3%. These results strongly support the proposal of Wrezel et al. (21) that the DD-carboxypeptidase is not the primary target of penicillin at the MIC of this P-lactam. With the ultimate goal of identifying the targets of penicillin action, we have investigated the synthesis and processing of PG in membranes and membrane-walls from G. homari (15). These systems require both NH4' and ATP for amidation of the -y-D-glutamyl residues (2, 8, 12). In the membrane system from this organism, the synthesis of dimers by the transpeptidase utilizes amidated monomers in preference to nonamidated monomers (1). The observations of Hammes (7) and Hammes and Seidel (9) with membrane-walls strongly suggest that the monomer acceptors are processed prior to their utilization by the transpeptidase. Processing includes the action of DD-carboxypeptidase and LD-carboxypeptidase. Their action produces a population of penta-, tetra-, and tripeptides (48:23:29) which are required for the successful cross-linking and assembly of PG (9). Since the in vitro transpeptidase activity is relatively penicillin insensitive, it was proposed by Hammes (7) that the DD-carboxypeptidase (PBP-9) is the target of penicillin. However, binding experiments with labeled penicillin identified PBP-6 as the primary target. The function of this protein in PG assembly has not yet been established. Since G. homari has nine PBPs (PBP-1 to PBP-6, >200 to 112 kDa; PBP-7 to PBP-9, 95 to 53 kDa) with B50s (concentration of P-lactam which acylates 50% of the available PBP) from