JOURNAL

OF

Vol. 135, No. 1

BACTERIOLOGY, July 1978, p. 71-77

0021-9193/78/0135-0071$02.00/0 Copyright i 1978 American Society for Microbiology

Printed in U.S.A.

Site of Inhibition of Peptidoglycan Biosynthesis During the Stringent Response in Escherichia coli WILLIAM D. RAMEY AND EDWARD E. ISHIGUROt* Department of Microbiology, University of British Columbia, Vancouver, British Columbia, V6T I W5, Canada Received for publication 26 January 1978

The site of inhibition of peptidoglycan synthesis during the stringent response in Escherichia coli was determined in strains which were auxotrophic for both lysine and diaminopixnelic acid (DAP). Cells were labeled with [3H]DAP for 30 to 60 min in the presence and absence of required amino acids, and the cellular distribution of [3H]DAP was determined. In both stringent (relt) and relaxed (relA) strains, amino acid deprivation did not inhibit the incorporation of [3H]DAP into the nucleotide precursor and lipid intermediate fractions. The amount of [3H]DAP incorporated into the peptidoglycan fraction by the amino aciddeprived relA strain was over 70% of the amount incorporated in the presence of required amino acids. In contrast, the amounts of labeled peptidoglycan in amino acid-deprived relt strains were only 20 to 44% of the amounts synthesized in the presence of amino acids. These results indicate that a late step in peptidoglycan synthesis is inhibited during the stringent response. The components of the lipid intermediate fraction synthesized by relt strains in the presence and absence of required amino acids were quantitated. Amino acid deprivation did not inhibit the synthesis of either the monosaccharide-pentapeptide or the disaccharidepentapeptide derivatives of the lipid intermediate. Thus, the reaction which is most likely inhibited during the stringent response is the terninal one involving the incorporation of the disaccharide-pentapeptide into peptidoglycan.

The morphology and size of a bacterium are largely determined by the cell wall polymer known as peptidoglycan. Some of the major steps in the biosynthesis of peptidoglycan in Escherichia coli are outlined in Fig. 1. Detailed information is available in a review by Ghuysen and Shockman (7). A series of soluble enzymes (steps 1 to 8) are involved in the synthesis of the nucleotide precursors, uridine 5'-diphospho-Nacetylglucosamine (UDP-GlcNAc) and uridine 5' -diphospho-N-acetylmuramyl-L-Ala-D-Glumeso-diaminopimelic acid-D-Ala-D-Ala (UDPMurNAc-pentapeptide). Particulate enzymes catalyze the translocation of MurNAc-pentapeptide (step 9) and GlcNAc (step 10) to a membrane glycosyl carrier lipid (GCL-P) to yield the lipid intermediates, GCL-P-P-MurNAc-pentapeptide and GCL-P-P-MurNAc (-GlcNAc)-pentapeptide. MurNAc (-GlcNAc)-pentapeptide is then transferred to an acceptor site in nascent peptidoglycan (step 11). The resulting GCL-P-P is dephosphorylated (step 12) to yield GCL-P. The mass of a bacterial cell is dependent on growth rate; the size and macromolecular con-

tent of bacteria increase as the growth rate is increased (5, 15). Therefore, bacteria undoubtedly possess a means of regulating both the rate and extent of peptidoglycan synthesis during growth because this.polymer is the major determinant of cell size. The regulatory mechanisms involved are largely unknown. We have shown that the synthesis of peptidoglycan in E. coli is under stringent control during amino acid deprivation (8). When stringent (rel) strains were deprived of a required amino acid, the rate of incorporation of radioactive diaminopimelic acid (DAP) into peptidoglycan was significantly reduced. In contrast, a relaxed (reM) mutant incorporated radioactive DAP at comparable rates in the presence or absence of a required amino acid. Guanosine 5'-diphosphate 3'-diphosphate (ppGpp) accumulates during amino acid deprivation in relt strains but not in relA strains (see reference 4 for a recent review). This nucleotide may be the mediator of stringent control. We have demonstrated that the synthesis of lipid intermediates and peptidoglycan by an in vitro enzyme system could be inhibited by physiological concentrations of ppGpp (8). In this study, we have attempted to identify the site of inhibition of peptidoglycan synthesis

t Present address: Department of Biochemistry and Microbiology, University of Victoria, Victoria, B.C., V8W 2Y2, Canada. 71

72

RAMEY AND ISHIGURO

J. BACTERIOL. Cell WallAssociated

t"

1

-Abl

UDP-MurN4Ac-L-Aia

(3 e,0-GkE

LXUOP-MurNAc-L-ala-D-Glu -ggDap-D-Ala-D-AIa

(UDP-MurNAc-pwt$e) UVP-MurNAc-L-Al&-D-Glu

l

UDPlMurNAc-L-Ala-D-Gluhmnso-DapZ 1)

L-Ala

(

D-Ala

(0

D-Aba-DAla

FIG. 1. Major steps in peptidoglycan biosynthesis in E. coli.

during the stringent response. Strains of E. coli which were auxotrophic for both lysine and DAP (lysA dapD) were labeled with [3H]DAP. The cellular distributions of [3H]DAP incorporated in the presence and absence of a required amino acid were compared. During amino acid deprivation of rel+ strains, the incorporation of [3H]DAP into the nucleotide precursor and the lipid intermediate fractions was not inhibited, whereas the incorporation of [3H]DAP into the peptidoglycan fraction was inhibited by 56 to 80%. Quantitation of the components of the lipid intermediate fraction indicated that amino acid deprivation did not inhibit the synthesis of either GCL-P-P-MurNAc-pentapeptide or GCL-P-PMurNAc (-GlcNAc)-pentapeptide. These results suggest that the incorporation of MurNAc (-GlcNAc)-pentapeptide into acceptor peptidoglycan is inhibited during the stringent response. MATERIALS AND METHODS Bacterial strains. The bacteria used in this study derivatives of E. coli K-12. Strains LD5456 (relA IysA dapD), LD5 (rel lysA dapD), and LD52 (relt IysA dapD thr) have been described (8). Strain LD2 (retl lysA dapD metE) was constructed by mating strains AT980 and X478 by methods described in detail elsewhere (8). Culture conditions. Bacteria were grown in M9 minimal medium with 0.4% glucose and required growth factors (8).Cultures were incubated in a 37°C water bath shaker, and growth was followed with a were

Klett-Summerson colorimeter (blue filter). After the culture had undergone four doublings (to a density of 2 x 108 to 3 x 108 cells per ml), the cels were harvested and washed (8). The washed cells were inoculated into 18 ml of either complete M9 or M9 lacking one or more required amino acids. The media contained [G3H]DAP (1.1 uCi/g, Amersham/Searle) at a concentration of 0.2 iLg/ml. The cultures were incubated at 37°C with shaking, and incorporation of [3H]DAP into trichloroacetic acid-insoluble material was followed as previously described (8). Cellular distribution of ['H]DAP. Bacteria were labeled with [3H]DAP in the presence and absence of required amino acids as described above. The amounts of [3H]DAP incorporated into the peptidoglycan, nucleotide precursor, and lipid intermediate fractions were determined by paper chromatography of intact cells as described by Lugtenberg and De Haan (11). At designated intervals, 8-ml samples of the cultures were removed and immediately centrifuged for 3 min at 35,000 x g in a Sorvall RC2B centrifuge operating at 2MC. The culture supernatants were saved for analysis of autolytic products. The cell pellets were resuspended in 60 pd of ice-cold distilled water, and the samples were applied quantitatively as 1-cm streaks to Whatman 3MM chromatography paper (57 cm long). The chromatogram was developed in isobutyric acid-i M NH40H (5:3) for 19 to 22 h. Radioactive spots were detected and counted as previously described (8). The Rf values for the various fractions were: (i) peptidoglycan, R, of 0.0; (ii) nucleotide precursors (mixture of UDP-MurNAc-tripeptide and UDP-MurNAc-pentapeptide), Rf of 0.1 to 0.2; (iii) [3H]DAP, Rf of 0.3 to 0.4; and (iv) lipid intermediates, Rf of 0.9. The results were normalized to cell mass and are presented as counts

VOL. 135, 1978

CONTROL OF PEPTIDOGLYCAN SYNTHESIS

minute per milligram of dry weight of cells. The dry weight of cells applied to the chromatogram was determined turbidimetricaily from a standard curve (8). Analysis of culture supernatants. The culture supernatants were concentrated by lyophilization and desalted by gel filtration on a column (2.5 by 50 cm) of Sephadex G-25. Gel filtration also removed most of the [3H]DAP in the samples. The desalted samples were lyophilized and analyzed for possible labeled autolytic products by paper chromatography as described above. Analysis of lipid intermediate fractions. The components of the lipid intermediate fraction were quantitated by a modified version of the method of Braun and Bosch (2). A suspension of cells labeled for 60 min with [3H]DAP was prepared as described above. A portion of this material, designated as the untreated sample, was directly applied to Whatman 3MM paper. The remainder of the material was boiled for 4 min. The sample was then centrifuged for 3 min at 36,000 X g. The supernatant was discarded, and the pellet was washed once with 0.40 ml of boiling, distilled water. A portion of this material, designated as the boiled sample, was applied to Whatman 3MM paper. The remainder was transferred to a glass vial, and acetic acid was added to a final concentration of 10%. The vial was sealed, and the sample was heated for 60 min in an oven at 105°C. This material, designated as the acid-hydrolyzed sample, was applied to Whatman 3MM paper. The chromatogram was developed and counted as described above. The total radioactivity in the various fractions was determined from the untreated sample. The lipid intermediates were hydrolyzed by mild acid treatment, and the resulting labeled products, MurNAc-pentapeptide and MurNAc (-GlcNAc)-pentapeptide, were quantitated from the hydrolyzed sample. The presence of [3H]DAP and labeled nucleotide precursors interferes with the determination of the acid hydrolysis products of lipid intermediates.The boiling treatment quantitatively extracted [3H]DAP and nucleotide precursors but had no effect on the lipid intermediate fraction (W. D. Ramey, Ph.D. thesis, University of British Columbia, Vancouver, B. C., 1977). This was demonstrated with the boiled sample. In some experiments, the boiled cells were treated with a protease. A mixture containing boiled cells, 2 mg of Streptomyces griseus protease (Sigma Chemical Co.) per ml, 5.5 mM potassium penicillin G (to inhibit possible D-alanine carboxypeptidase activity), and 200 mM tris(hydroxymethyl)aminomethane-hydrochloride (pH 7.0) was incubated at 37°C for 30 min. The protease-treated sample was analyzed by paper chromatography with or without mild acid hydrolysis. Determination of [Hlysine. All bacterial strains used in this study were DAP decarboxylase mutants (QysA). The degree of leakiness of this mutation (i.e., the degree to which [3H]DAP was converted to ['H]lysine) was determined in the following manner. Cells labeled with [3H]DAP for 60 min were divided into two portions. One portion was boiled as described above. Samples of the boiled cells and the intact cells were hydrolyzed with 6 N HCI in sealed vials at 105°C for 24 h. The hydrolysates were dried in vacuo over

per

73

NaOH pellets and analyzed by paper chromatography in n-butanol-acetic acid-water (2:1:1). Only two radioactive compounds were detected, and these corresponded to DAP and lysine. The identities of the compounds were established by chromatography in three other solvent systems: (i) isobutyric acid-I M NH40H (5:3), (ii) ethanol-NH4OH-water (180:10:10), and (iii) pyridine-acetic acid-water (50:35:5).

RESULTS Distribution of ['HIDAP incorporated during amino acid deprivation. Bacteria were labeled with [3H]DAP in the presence and absence of required amino acids. To localize the site of stringent control, the amounts of label incorporated into the peptidoglycan, nucleotide precursor, and lipid interinediate fractions were compared. The results obtained with strains LD5456 (relA), LD5 (relt), LD52 (relt) and LD2 (relt) are shown in Table 1. The following is a summary of these results. (i) Amino acid deprivation resulted in a decrease in the amount of [3H]DAP incorporated into trichloroacetic acid-insoluble material by rel strains but had little effect on strain LD5456 (relA). (ii) The synthesis of nucleotide precursors was not inhibited during amino acid deprivation. After 60 min, the amount of labeled nucleotide precursors was 1.3- to 5-fold higher in amino acid-deprived bacteria than in untreated bacteria. UDP-

MurNAc-pentapeptide accounted for approximately 95% of the labeled nucleotide precursor pool in both relt and reLA strains (manuscript in preparation). (iii) Amino acid deprivation did not inhibit the incorporation of [3H]DAP into the lipid intermediate fraction. It should be noted that lysine deprivation resulted in an increase in the amount of [3H]DAP incorporated into this fraction, but that this increase was not observed in bacteria deprived of either threonine (strain LD52) or methionine (strain LD2). This phenomenon is discussed below. (iv) The amount of [3H]DAP incorporated into peptidoglycan by amino acid-deprived cells of strain LD5456 (reLA) was over 70% of the amount incorporated by untreated cells. In contrast, the amount of labeled peptidoglycan synthesized by amino acid-deprived rel+ strains was only 20 to 44% of the amount found in the control cells. These observed differences were not due to autolysis; under all conditions, the culture supernatants of both rel and reL4 strains contained insignificant quantities of material which may have resulted from autolysis (Ramey, Ph.D. thesis). The results suggest that the basis for stringent control is not the limitation of either nucleotide precursors or GCL-P-P-MurNAc-pentapeptide. The site of inhibition is almost certainly one, or both, of the enzymes catalyzing

74

RAMEY AND ISHIGURO

J. BACTrERIOL.

the last two reactions in peptidoglycan synthesis (steps 10 and 11, Fig. 1). Composition of the lipid intermediate fractions. The rel+ strains, LD5, LD2, and LD52, were labeled with [3H]DAP for 60 min in

the presence and absence of a required amino acid. The components of the lipid intermediate fractions were quantitated after mild acid hydrolysis. The results are presented in Table 2. Acid hydrolysis did not release any radioactivity

TABLE 1. Distribution of [3H]DAP incorporated by rel+ and relA strains in the presence and absence of required amino acids 104 cpm/mg of cell dry wta Strain

LD5456 (relA)

Sample time (mm)

Growth medium

30

60

LD5 (rel+)

30 60

Total trichloroacetic acid insoluble

Nucleotide

Lipid interme-

precursors

diate

Peptidoglycan

Complete -Lysine Complete -Lysine

10.04 11.84 20.01 18.30

(100) (118) (100) (91)

0.58 2.13 0.63 3.28

(100) (367) (100) (521)

0.09 0.53 0.28 2.26

(100) (588) (100) (807)

8.74 8.43 17.77 12.93

Complete -Lysine Complete -Lysine

10.44 3.12 20.84 6.02

(100) (30) (100) (29)

0.62 (100) 0.90 (145) 0.72 (100) 1.00 (139)

0.08 0.19 0.23 0.82

(100) (238) (100) (357)

10.74 (100)

(100) (96) (100) (73)

3.18 (30) 21.92 (100) 4.46 (20)

15.94 (100) 15.09 (100) 0.12 (100) 0.48 (100) Complete 5.06 (34) 0.35 (292) 5.20 (33) 0.44 (92) -Lysine 6.64 (44) 0.11 (92) 6.41 (40) 0.89 (185) -Threonine 6.45 (43) 0.40 (333) 7.45 (47) 0.64 (133) -Threonine and lysine 0.96 (100) 0.24 (100) 60 37.60 (100) 37.89 (100) LD2 (rel+) Complete 8.97 (24) 0.94 (392) 1.31 (136) 12.76 (34) -Lysine 10.05 (27) -Methionine 11.45 (30) 1.40 (146) 0.22 (92) 10.32 (27) 1.58 (165) 0.96 (400) -Methionine and 10.23 (27) lysine Figures in parentheses represent percentage of control. b Counts per minute in this column before normalization to cell mass were 18,949; 16,198; 44,267; 22,387; 22,379; 5,850; 62,307; 9,968; 34,808; 9,032; 13,212; 11,819; 72,859; 15,751; 18,695; and 19,527, respectively.

LD52 (rel+)

60

TABLE 2. Analysis of components of lipid intermediate fractionsa 103 cpm/mg of cell dry weight Culture

-Lysine -Threonine

componentd

0.51 0.68

0.80 2.03

0.21 Nil

0.32 3.44

0.49 0.47

0.71 0.78

0.32 0.21

0.25 0.15

256.3 46.7

1.79 5.97

161.6 70.9

1.72 1.62

b

LD52 (rel+)

Complete medium

Unidentified

(-GIcNAc)-

GCL-P-PMurNAc-penta-

can

LD5 (rel+) Complete medium

GCL-P-PMurNAc

Total lipid inemdae

Peptidogly-

peptide

LD2 (rel+)

Ai-eit Acid-resist

nentcmo pentapeptiden

0.31 0.63 0.91 0.24 2.04 204.2 4.88 0.65 1.27 0.14 6.91 57.2 0.30 1.01 0.21 2.11 0.68 49.3 -Methionine a Bacteria were labeled for 60 min in the presence and absence of a required amino acid. Peptidoglycan and total lipid intermediates were determined in samples which were not subjected to boiling and acetic acid treatments. b Counts per minute in this column before normalization to cell mass were 62,807; 10,279; 60,046; 21,537; 66,738; 14,014; and 12,680, respectively. 'Counts per minute in this column before normalization to cell mass were 1,239; 1,204; 638; 637; 1,319; 1,206; and 1,252,

Complete medium -Lysine

respectively. d Hydrolysis product with Rf of 0.52 in isobutyric acid-I M NH4OH (5:3). ' Rf of 0.9 after treatment with 10% acetic acid.

VOL. 135, 1978

75

CONTROL OF PEPTIDOGLYCAN SYNTHESIS

from the peptidoglycan fraction, and the boiling treatment which preceded acid hydrolysis quantitatively removed [3H]DAP and labeled nucleotide precursors (Ramey, Ph.D. thesis). By comparing the total radioactivity of the lipid intermediate fractions in unhydrolyzed samples with the total radioactivity of the acid-hydrolyzed samples, it is clear that labeled components were quantitatively recovered after acid hydrolysis. The synthesis of lipid intermediates was not inhibited during amino acid deprivation. The amount of [3H]DAP incorporated into GCL-PP-MurNAc-pentapeptide was the same in the presence and absence of required amino acids. The amount of labeled GCL-P-P-MurNAc (-GlcNAc)-pentapeptide in amino acid-deprived cells was equal to, or higher than (1.4- to 2.5fold), the amount in untreated cells. However, amino acid deprivation resulted in a 56 to 82% inhibition in the incorporation of [3H]DAP into peptidoglycan. These results indicate that the terminal reaction involving the incorporation of MurNAc (-GlcNAc)-pentapeptide into peptidoglycan (step 11, Fig. 1) is inhibited during the stringent response. Two other components were present in the acid-hydrolyzed lipid intermediate fraction (Table 2). The presence of these components does not seriously affect the conclusion made above. One of these components was an unidentified product of acid hydrolysis with an Rf of 0.53 in isobutyric acid-i M NH40H (5:3). This component was present in approximately equivalent amounts in amino acid-deprived and untreated cells of all strains except LD5. It represented 11 to 18% of the total radioactivity in the lipid intermediate fraction, but for unknown reasons, it was not detectable in lysine-deprived cells of strain LD5. The Rf of the second component was identical to the Rf of the unhydrolyzed lipid intermediate fraction (0.9). Thus, it apparently was resistant to mild acid hydrolysis. This component consistently represented 9 to 17% of the total radioactivity in the lipid intermediate fraction of cells grown in the presence of amino acids and of cells deprived of a required amino acid other than lysine. When cells were deprived of lysine, the acid-resistant component represented 50 to 80% of the total radioactivity in the lipid intermediate fraction. As previously mentioned (Table 1), the amount of [3H]DAP incorporated into the lipid intermediate fraction by rel+ strains deprived of lysine was three- to fourfold higher than the control value (in 60 min); lysine deprivation of strain LD5456 (reLA) resulted in an eightfold increase. Furthermore, this increased amount of [3H]DAP incorporation was not observed if cells were deprived of an amino acid other than lysine. The results in Table 2

suggest that this "excess" [3H]DAP incorporated into the lipid intermediate fraction during lysine deprivation represented material which was resistant to mild acid hydrolysis. This acid-resistant material was probably not a lipid intermediate because lipid intermediates are known to be sensitive to mild acid hydrolysis (1, 2). The experiments described below suggest that the material was labeled protein. The strains used in this study were DAP decarboxylase mutants (lysA). The lysA mutation was leaky, and the bacteria were able to convert some [3H]DAP to [3H]lysine, particularly when they were deprived of lysine. This was demonstrated in 6 N HCl hydrolysates of cells labeled for 60 min with [3H]DAP (Table 3). When cells were labeled in the presence of amino acids or in the absence of an amino acid other than lysine, [3H]lysine was present in amounts less than 0.3% of the total trichloroacetic acidinsoluble radioactivity. Lysine-deprived cells contained 7 to 11 times more [3H]lysine, and this represented about 2 and 6% of the total trichloroacetic acid-insoluble radioactivity in the relA and relr strains, respectively. The [3H]DAP used for labeling was chromatographically homogeneous. Furthermore, no [3H]lysine was reTABLE 3. Determination of [3Hflysine in cells labeled with [3H]DAPa 10' cpm/mg of cell dry wt Intact cells

Culture

LD5456 (relA) +Lysine

-Lysine

Acid-re-

Trichlo-

[I3H]lysine" LD5 (rel+) +Lysine -Lysine

Boiled cells

roacetic acid-insoluble

[:'H]I ysine'

sistant

inlipid terme-

diate

0.36 4.12

226.42 59.86

0.34 3.92

0.32 3.46

0.43 3.16

179.17 149.36

0.44 2.97

0.30 3.22

LD2 (rel+) +Amino 0.39 0.33 230.21 0.36 acids 3.97 56.32 3.62 3.84 -Lysine -Methionine 0.10 0.31 58.13 0.11 a Bacteria were labeled with [ 'H]DAP for 60 min. A portion of the cells, designated as intact cells, was hydrolyzed with 6 N HCl for determination of ['H]Ilysine. Another portion, designated as boiled cells, was boiled for4 min. Half of the boiled cells was used for [ 'H]lysine determination, and the other half was used for determination of the acid-resistant component in the lipid intermediate fraction. h Counts per minute in this column before normalization to cell mass were 296; 2,862; 382; 2,948; 414; 3,216; and 112, respectively. 'Counts per minute in this column before normalization to cell mass were 302; 2,672; 344; 2,731; 386; 3,142; and 106, respectively.

76

RAMEY AND ISHIGURO

leased by boiling the cells or by hydrolyzing samples of the nucleotide precursor fraction with 6 N HCl (Ramey, Ph.D. thesis). As shown in Table 3, the quantities of [3H]lysine closely approximated the quantities of the acid-resistant component in the lipid intermediate fractions. Additional experiments indicated that the acidresistant material could be quantitatively eliminated from the lipid intermediate fraction by treatment with S. griseus protease (Ramey, Ph.D. thesis). Collectively, these results suggest that the acid-resistant material was [3H]lysinelabeled protein. Furthermore, this material probably represented the excess radioactivity incorporated into the lipid intermediate fraction during lysine deprivation. We have shown that this excess radioactivity did not appear if cells were deprived of lysine in the presence of chloramphenicol (Ramey, Ph.D. thesis).

J . BACTrERIOL.

study was leaky. During the labeling of the bacteria, small quantities of [3H]DAP were converted to [3H]lysine. Lysine-deprived bacteria contained up to 11 times more [3H]lysine than bacteria labeled in the presence of lysine. Thus, the absence of lysine may have resulted in the derepression of the mnutant IysA gene (10) and the elimination of feedback inhibition of DAP decarboxylase (17). In addition, the [3H]lysine synthesized would be more readily utilized for protein synthesis because it would not be diluted by unlabeled lysine. It should be emphasized that the quantities of [3H]lysine present were not sufficient to affect the conclusion that the synthesis of peptidoglycan was stringently controlled in E. coli. The lipid intermediate fraction was contaminated with two unidentified components: (i) a component which was sensitive to mild acid hydrolysis; and (ii) a component which was protease-sensitive but resistant to mild acid hydrolDISCUSSION ysis. The protease-sensitive material was present The utilization of lipid intermediates for pep- in significant amounts only when bacteria were tidoglycan synthesis was inhibited during amino labeled in the absence of lysine. The composition acid deprivation in rel+ strains of E. coli but not of this material is under investigation. Proteins in a relA strain. The proposed site of inhibition such as the peptidoglycan lipoprotein are known is depicted in Fig. 1 (step 11) as the reaction to comigrate with the lipid intermediate fraction involving the addition of a disaccharide-peptide (3, 16). It is of interest that most of the [3H]unit to a peptidoglycan acceptor site. This stage lysine incorporated into trichloroacetic acid-inin peptidoglycan synthesis has not been fully soluble material apparently could be accounted established, and the reaction may actually be for by this material. The presence of the contammore complex than shown in Fig. 1. For example, inants in the lipid intermediate fraction does not disaccharide-peptide units may be polymerized affect the conclusion that the synthesis of lipid on the lipid carrier, and the resulting disacchar- intermediates was not inhibited during amino ide-peptide oligomer may then be transferred to acid deprivation. The synthesis of nucleotide precursors was acceptor peptidoglycan (7, 13). Although a lipidlinked disaccharide-peptide oligomer was re- not inhibited during amino acid deprivation, and cently isolated from Bacillus megaterium (6), equivalent amounts of [3H]DAP were incorpoour attempts to demonstrate such an interme- rated in the presence and absence of a required amino acid by rel+ bacteria. However, nucleotide diate in E. coli have so far been unsuccessful. The synthesis of lipid intermediates was not precursors did not accumulate significantly in inhibited during amino acid deprivation. One amino acid-deprived relt bacteria despite the attractive feature of this is that the synthesis of fact that peptidoglycan synthesis was inhibited peptidoglycan could proceed immediately upon by as much as 80%. It was previously demonresumption of growth, and the prior synthesis of strated that nucleotide precursors also did not lipid intermediates would not be necessary. Fur- accumulate if the synthesis of peptidoglycan was thermore, a full supply of lipid internediates inhibited by either antibiotic treatment or by would be available during amino acid depriva- exposing temperature-sensitive mutants to a tion for the purpose of repair and maintenance nonpermissive temperature (12). It was sugof the peptidoglycan layer. It was previously gested that the synthesis of UDP-MurNAc-penshown that physiological concentrations of tapeptide was regulated by feedback inhibition ppGpp inhibited the in vitro synthesis of lipid (12). Our results indicate that feedback inhibiintermediates (8). The activities of the enzymes tion alone is not sufficient to account for the involved in the synthesis of lipid internediates regulation of nucleotide precursor synthesis. are dependent on Mg2" (9). Because ppGpp is Amino acid deprivation of the reA strain, known to chelate divalent cations (14), it is LD5456, resulted in a fivefold accumulation of possible that the inhibitory effects of ppGpp in nucleotide precursors in 60 min. Thus, the relA gene product is involved in preventing the acvitro were due to chelation of Mg2e. The lysA mutation in the strains used in this cumulation of nucleotide precursors. We will

CONTROL OF PEPTIDOGLYCAN SYNTHESIS

VOL. 135, 1978

substantiate this elsewhere (manuscript in preparation). ACKNOWLEDGM ENI W.D.R. was the recipient of a Frank Wesbrook predoctoral fellowship from the University of British Columbia. This investigation was supported by grant A6606 from the National Research Council of Canada.

LITERATURE CITED 1. Anderson, J. S., M. Matsuhashi, M. A. Haskin, and J. L Strominger. 1967. Biosynthesis of the peptidoglycan

2. 3.

4.

5. 6.

7.

8.

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Site of inhibition of peptidoglycan biosynthesis during the stringent response in Escherichia coli.

JOURNAL OF Vol. 135, No. 1 BACTERIOLOGY, July 1978, p. 71-77 0021-9193/78/0135-0071$02.00/0 Copyright i 1978 American Society for Microbiology Pr...
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