JouRNAL OF BACTERIOLOGY, Sept. 1976, p. 1119-1126 Copyright © 1976 American Society for Microbiology

Vol. 127, No. 3 Printed in U.S.A.

Stringent Control of Peptidoglycan Biosynthesis in Escherichia coli K-12 EDWARD E. ISHIGURO* AND WILLIAM D. RAMEY Department of Microbiology, University ofBritish Columbia, Vancouver, B.C., V6T 1 W5 Canada

Received for publication 10 May 1976

[3H]Diaminopimelic acid (Dap) was incorporated exclusively into peptidoglycan by Escherichia coli strains auxotrophic for both lysine and Dap. The rate of [3H]Dap incorporation by stringent (rel+) strains was significantly decreased when cells were deprived of required amino acids. The addition of chloramphenicol to amino acid-starved rel+ cultures stimulated both peptidoglycan and ribonucleic acid synthesis. In contrast, a relaxed (relA) derivative incorporated [3H]Dap at comparable rates in the presence or absence of required amino acids. Physiologically significant concentrations of guanosine 5'-diphosphate 3'-diphosphate (ppGpp) inhibited the in vitro synthesis of both carrier lipid-linked intermediate and peptidoglycan catalyzed by a particulate enzyme system. The degree of inhibition was dependent on the concentration ofppGpp in the reaction mixture. Thus, the results of in vivo and in vitro studies indicate that peptidoglycan synthesis is stringently controlled in E. coli. Peptidoglycan is the major constituent found in the rigid layer of all bacterial cell walls, and the structural integrity of this polymer is normally essential for the maintenance of cell viability. Although the key steps in the biosynthesis of peptidoglycan have been described (6, 13, 14), the means by which bacteria regulate this important biosynthetic process remain largely unknown. In many bacterial species, the synthesis of peptidoglycan continues after cell growth is arrested by inhibition of protein synthesis. This phenomenon has been demonstrated in amino acid auxotrophs deprived of required amino acids (35) as well as in cells subjected to chloramphenicol (CAM) treatment (8, 27, 30). The continued synthesis of peptidoglycan in the absence of growth results in cell wall thickening (35). We have investigated various aspects of peptidoglycan synthesis in nongrowing cells of Escherichia coli. The synthesis of stable ribonucleic acid (RNA) usually ceases abruptly when E. coli is deprived of required amino acids. This regulatory mechanism is referred to as the stringent response, and the organism is designated rel+ (7). In contrast, relaxed (rel-) mutants continue to accumulate stable RNA during amino acid deprivation. The relaxed phenotype is the result of a mutation in any one of three genes (12, 20, 37) designated as relA, relB, and relC (12). The rel genes are also involved in regulating the biosynthesis of other cellular constituents which include phospholipids (15, 31, 36),

ribosomal proteins (11), and polyamines (18). Two novel nucleotides, guanosine 5'-diphosphate 3'-diphosphate (ppGpp) and guanosine 5'-triphosphate 3'-diphosphate (pppGpp), rapidly accumulate during amino acid deprivation in rel+ strains but not in rel- strains (7). These nucleotides are synthesized by an enzyme which is believed to be the product of relA (4). The stringent response is apparently mediated by ppGpp and pppGpp. For example, ribosomal RNA transcription is inhibited by ppGpp in vitro (34). Furthermore, the inhibition of key enzymes involved in phospholipid (25, 31) and polyamine (18) synthesis by ppGpp has been demonstrated. In this paper, we present the following in vivo and in vitro evidence for stringent control of peptidoglycan synthesis in E. coli. (i) Upon amino acid starvation, the rate of peptidoglycan synthesis is significantly decreased in rel+ cells but not in relA- cells. (ii) Physiologically significant concentrations of ppGpp inhibit peptidoglycan synthesis catalyzed by an in vitro particulate enzyme system.

MATERIALS AND METHODS Bacterial strains and strain construction. The E. coli K-12 strains used in this study are listed in Table 1. Strain LD5 (dap- lys-) was constructed by crossing AT980 with X478. Donor and recipient strains were grown in LB broth (32) at 37°C to approximately 2 x 108 cells/ml. For AT980, the medium was supplemented with 150 Ag of DL-2,6-diaminopimelic acid (Dap) per ml. The conjugal cross was initiated 1119

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TABLE 1. E. coli K-12 strains Strain Relevant characteristicse Source F- thi leu trpE purE proC metE lysA str B. J. Bachmann X478 AT980 Hfr thi dapD relA origin 56 min, counterclockwise B. J. Bachmann transfer E177 F- thi thr leu thyA B. J. Bachmann CP78 F- thi thr leu his arg B. J. Bachmann CP79 Same as CP78 except relA B. J. Bachmann F- thi lysA dapD str LD5 Conjugal cross: AT980 x X478 LD51 Same as LD5 except leu PlvirA (CP78) transductant of LD5 LD52 Same as LD5 except thr PlvirA (CP78) transductant of LD5 LD54 Same as LD5 except thyA PlvirA (E177) transductant of LD5 LD5456 Same as LD5 except relA PlvirA (CP79) transductant of LD54 VC10 str Derived from W3110 (reference 3) a The genotype designations are those of Taylor and Trotter (38) with the exception of relA. The relA designation is according to the proposal of Friesen et al. (12).

by mixing the donor and recipient strains at a ratio of 1:10 in LB supplemented with Dap. After 2 h of incubation at 37°C, the cells were harvested by centrifugation and washed with sterile saline. Serial dilutions of the washed cells were plated on glucoseDavis minimal agar (Difco) supplemented with (per milliliter) 50 ,ug of L-lysine, 50 ,ug of Dap, 1 gg of thiamine, and 100 ,ug of streptomycin. Colonies were screened for Dap and lysine requirements. Derivatives of LD5 were constructed by transduction with PlvirA. The bacteriophage was obtained from Christoph Beck (Department of Molecular Biology, University of California, Berkeley). The methods described by Miller (32) were used for the preparation of lysates and for transduction. Strains LD51 (leu-) and LD52 (thr-) were isolated after one cycle of penicillin enrichment (32). The relA derivative, LD5456, was constructed by cotransduction of relA with thyA. This was accomplished in two steps. In the first step, a thyA transductant, LD54, was isolated by trimethoprim selection (10). In the second step, LD54 was transduced with a PlvirA lysate prepared on CP79 (relA), and thy+ lys- transductants were selected. LD5456 was isolated after screening the thy+ lys- transductants for ability to accumulate [14C]uracil into trichloroacetic acid-precipitable material during lysine starvation, as outlined below. Cultural conditions. The dap- lys- strains were grown in M9 minimal medium (32) with (per milliliter) 4 mg of glucose, 1 ,ug of thiamine, 5 Ag of Dap, and 50 gg of L-lysine. When required, other amino acids were added as L-isomers at 50 ,ug/ml. Cultures were incubated in 37°C water bath shakers. Effects of amino acid deprivation and CAM treatment on peptidoglycan synthesis. A 40- to 50-ml culture of a dap- lys- strain was grown for about four generations to a density of 2 x 108 to 3 x 108 cells/ml. The cells were then harvested by one of two methods. In the first method, cells were collected on a 0.45-Am membrane filter (Millipore) and washed with 50 ml of sterile saline. Alternatively, the culture was centrifuged at 27,000 x g for 2 min, and the cell pellet was washed with 40 ml of sterile saline. The washed cells obtained by either method were resuspended in saline and inoculated into flasks containing 10 ml of medium to a density of about 108

cells/ml. Four flasks were usually used: flask A contained complete M9; flask B contained M9 without a required amino acid (usually L-lysine); flask C contained complete M9 with 200 gg of CAM per ml; flask D contained M9 without a required amino acid but with 200 ,ug of CAM per ml. Media in all flasks were supplemented with 0.2 jig of [G-3H]Dap (1.1 ACi4tg) per ml. Under our experimental conditions, this quantity of Dap was sufficient to support at least 2.5 exponential doublings in flask A. Cultures were incubated in a 37°C water bath shaker, and peptidoglycan synthesis was determined by following the incorporation of [3H]Dap into trichloroacetic acid-precipitable material. At designated intervals, 100-t1I samples were removed from each flask and applied to Whatman 3MM filter paper disks (2.4-cm diameter). The disks were immersed in cold 5% trichloroacetic acid for at least 30 min. This treatment was repeated with fresh 5% trichloroacetic acid. The disks were then rinsed twice in 95% ethanol and once in ether. After drying, the disks were counted in toluene-based scintillation fluid with a NuclearChicago Isocap 300 liquid scintillation counter. The results have been normalized to cell mass and are presented as counts per minute incorporated per milligram of dry weight of cells. Cell dry weight was deternined turbidimetrically from a standard curve.

The effectiveness of the various treatments was evaluated as follows. Culture turbidity was monitored on a Klett-Summerson colorimeter with a blue filter. Viable cell counts were determined at intervals by plating on nutrient agar (Difco) supplemented with 50 ,ug of Dap per ml. Total cell counts were determined with a Coulter counter model B coupled to a Nuclear Data 256-channel pulse-height analyzer. The Coulter counter was fitted with a 30,um orifice tube (Particle Data, Inc.). Samples to be counted were diluted in saline containing 4% formalin. Particles were removed from the formalinsaline by filtration through a 0.22-,Im membrane filter (Millipore) prior to use. To determine the fate of the incorporated [3H]Dap, 6 N HCl hydrolysates of labeled cells or cell envelopes were analyzed by paper chromatography (22). RNA synthesis. The protocol described above for

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the determination of peptidoglycan synthesis was used to determine RNA synthesis with the following modification. The medium in each flask contained (per milliliter) 5 ug of unlabeled Dap and 10 ,ug of [2'4C]uracil (0.01 uCi). Utilization of intracellular peptidoglycan precursors. The effect of lysine deprivation on utilization of intracellular peptidoglycan precursors was determined as follows. Intracellular precursors were labeled by growing dap- Iys- strains in 40 ml of M9 supplemented with 5 ,ug of [3H]Dap (0.22 ,uCi/,ug) per ml. After four generations (at a density of 3 x 108 cells/ml), the cells were harvested by centrifugation and washed twice with sterile saline. The utilization of labeled precursors was determined by following the incorporation of radioactivity into trichloroacetic acid-insoluble material in media lacking Dap. The washed cells were inoculated into 10 ml of: (i) M9 without Dap and lysine and (ii) M9 without Dap but with lysine. A flask containing 10 ml of complete M9 was included as a control. Preparation of UDP-N-acetylmuramyl-peptides. Uridine diphospho-N-acetylmuramyl-LAla-DGlumeso-Dap (UDP-MurNAc-tripeptide) was obtained from trichloroacetic acid extracts of i-cycloserinetreated Bacillus cereus cells (29). The crude extracts were fractionated by Dowex-1 column chromatography (16). UDP-MurNAc-tripeptide was purified by preparative paper chromatography of the appropriate Dowex-1 column fraction (29). UDP-MurNAc-LAla-DGlu-meso-Dap-D ['4C]AlaD['4C]Ala ('4C-labeled UDP-MurNAc-pentapeptide) was synthesized by incubating D-[U-14C]alanine (36 Ci/mol) and UDP-MurNAc-tripeptide with an enzyme preparation from E. coli VC10 containing both DAla-DAla synthetase and UDP-MurNAc-tripeptide: DAla-DAla ligase activities (9). The enzyme mixture was a dialyzed preparation taken from the first (NH4)2SO4 precipitation step in the purification procedure described by Comb (9). Potassium penicillin G was added to the reaction mixture at a concentration of 1 mg/ml to inhibit a n-alanine carboxypeptidase present in the enzyme preparation. The 14Clabeled UDP-MurNAc-pentapeptide was purified by preparative paper chromatography (19). Effect of ppGpp on in vitro peptidoglycan synthesis. A particulate enzyme fraction for in vitro peptidoglycan synthesis was prepared from E. coli VC10 as described by Izaki et al. (19). The preparation contained 15 mg of protein per ml as determined by the method of Lowry et al. (24) with bovine serum albumin as a standard. The effect of ppGpp was tested in an incubation mixture which contained (i) 5 ,ul of a stock solution of tris(hydroxymethyl)aminomethane-hydrochloride (1 M, pH 8.2), MgCl2 (20 mM), and UDP-N-acetylglucosamine (2 mM); (ii) 5 .al of particulate enzyme; (iii) 5 ,ul of ppGpp at the appropriate concentration or distilled water; and (iv) 5 ,ul of potassium penicillin G (1.1 mM). The mixtures were preincubated at 37°C for 10 min. The reactions were then initiated by the addition of 5 ,ul of '4C-labeled UDP-MurNAc-pentapeptide (0.75 mM, 18 mCi/mmol) to each incubation mixture. After 10 min, the reactions were terminated by chilling on ice and adding 10 ,ul of isobutyric acid.

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The radioactive products were separated by paper chromatography and counted as previously described (19). Penicillin was added to the incubation mixtures to inhibit D-alamnne carboxypeptidase and peptidoglycan transpeptidase activities (19). Omission of the antibiotic did not alter the general trend of the experimental results presented below although, as noted by others (17, 19), the amount of radioactivity present in peptidoglycan (Rf = 0) and in carrier lipid-linked intermediate (Rf = 0.9) was decreased in the absence of penicillin due to enzymatic removal of the terminal -[P4C]alanine residues. Materials. All radiochemicals were purchased from Amersham/Searle Corp. Antibiotics, UDP-Nacetylglucosamine, Dap, and other amino acids were obtained from Sigma Chemical Co. Michael Cashel kindly provided the ppGpp.

RESULTS Bacterial strains. Radioactive Dap was incorporated exclusively into peptidoglycan by the dap- lys- auxotrophs used in this study. All strains lacked Dap decarboxylase activity and were unable to convert Dap to lysine. This was corifirmed by the following observations made on [3H]Dap-labeled cells. (i) All of the trichloroacetic acid-precipitable radioactivity was recovered in the cell envelope fraction. (ii) Virtually all of the labeled material in the cell envelope fraction was solubilized by lysozyme. (iii) At least 98% of the radioactivity in a 6 N HCl hydrolysate of labeled cells was recovered as Dap after paper chromatography, and no other labeled compounds were detected. Two dap- lys- strains, designated LD5 and LD5456, were used in most experiments. Strain LD5 was rel+ as evidenced by the abrupt cessation of RNA synthesis when cells were deprived of lysine (Fig. 1A). The stringent response was abolished when CAM was added to lysine-deprived cultures. Strain LD5456 was a relA derivative of LD5. LD5456 continued to accumulate RNA during lysine deprivation (Fig. 1B). Peptidoglycan synthesis in vivo. Peptidoglycan synthesis was compared in the presence and absence of protein synthesis. Protein synthesis was inhibited by amino acid deprivation, by CAM treatment, or by a combination of amino acid deprivation and CAM treatment. The effects of these treatments on growth of strain LD5 are shown in Fig. 2. Growth of washed cells resumed immediately upon resuspension in complete medium (Fig. 2). In contrast, the optical densities of CAM-treated and lysine-deprived cultures remained essentially constant for 120 min (Fig. 2A). The total cell numbers in these treated cultures continued to increase for approximately 40 min and then remained constant for the duration of the ex-

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periment (Fig. 2B). Cell numbers increased 40 and 20% in lysine-deprived and CAM-treated cultures, respectively. A lysine-deprived culture simultaneously treated with CAM gave the same results as a culture treated with CAM alone. Cell viability was not affected by inhibition of protein synthesis. Viable cell counts always closely paralleled the total cell counts obtained with the Coulter counter (Fig. 2C). Strain LD5456 behaved similarly under these experimental conditions. The effect of inhibition of protein synthesis on peptidoglycan synthesis in strain LD5 is shown in Fig. 3A. The rate of [3H]Dap incorporation by a lysine-deprived culture gradually decreased during the initial 30 to 40 min of incubation. Thereafter, a low linear rate of incorporation was established which was maintained for at least an additional 60 min. The initial rates of [3H]Dap incorporation were approximately equal in CAM-treated lysine-deprived and lysine-supplemented cultures. Furthermore, the addition of CAM stimulated peptidoglycan synthesis in the lysine-deprived culture. The stimulatory effect of CAM on peptidogly-

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FIG. 1. RNA synthesis in strains LD5 rel+ (A) and LD5456 reU- (B). Exponential-phase cells were harvested by centrifugation, washed, and resuspended in M9 with (0) and without (-) L-lysine. For LD5, 200 pg of CAM per ml was added to a lysinedeprived culture (A). All media were supplemented with 10 pg (0.01 ,uCi) of [4C]uracil per ml, and trichloroacetic acid-precipitable radioactivity was determined at designated intervals. FIG. 2. Effect of amino acid deprivation and CAM treatment on growth of strain LD5 (rel+). Washed exponential phase cells were resuspended in M9 (0), M9 minus lysine (-), M9 plus 200 pg ofCAMper ml

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FIG. 3. (A) Effect of amino acid deprivation and CAM treatment on [3H]Dap incorporation into peptidoglycan by strain LD5 (rel+). Washed cells were inoculated into M9 (0), M9 minus lysine (a), M9 plus 200 pg ofCAM per ml (A), and M9 minus lysine plus 200 pg of CAM per ml (A). The radioactivity incorporated has been normalized to cell mass. (B) Effect of addition of CAM on [3H]Dap incorporation by a lysine-deprived culture of LD5 (rel+). Washed cells were inoculated into M9 (0), M9 minus lysine (a), and M9 plus 200 pg of CAM per ml (A). After 30 min (arrow), the lysine-deprived culture was divided in half, and 200 pg of CAM per ml was added to one half (A.

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intracellular precursors for peptidoglycan synthesis in rel+ and relA- strains during lysine deprivation. Cultures of LD5 and LD5456 were labeled with [3H]Dap for four generations. Under these conditions, part of the radioactivity associated with the cells was incorporated in peptidoglycan; the remainder represented soluble precursors. The labeled cells were washed and inoculated into the various nonradioactive media described in Fig. 5. The following observations are noteworthy. (i) For both strains, the growth rates of cells in complete medium and in medium lacking Dap were almost identical for at least two doublings (Fig. 5A and 5B). The Dap-deprived cultures utilized labeled Dap present in intracellular pools for growth, and lysis probably coincided with the depletion of this Dap supply (Fig. 5C and 5D). Thus, the intracellular level of Dap is clearly sufficient to support considerable growth, and Dap-deprived cultures are not initially dependent on exogenous Dap. (ii) In complete medium, intracellular [3H]Dap continued to be incorporated into peptidoglycan (Fig. 5C and 5D). In this case,

8 can synthesis in rel+ strains was not specifically associated with lysine deprivation. Identical results were obtained with leucine-deprived and threonine-deprived cultures of strains LD51 and LD52, respectively. Furthermore, ,) CAM stimulated peptidoglycan synthesis even when it was added some time after the onset of amino acid deprivation. In the experiment described in Fig. 3B, a culture of LD5 was divided into two parts after 30 min of lysine depriva- C) 4 tion. The addition of CAM to one part resulted in an increased rate of [3H]Dap incorporation within 10 min. 2 Since CAM stimulated both RNA and pepti- 0L doglycan synthesis in amino acid-starved cul2 tures of LD5, it was possible that a common regulatory element was involved in the synthesis of these macromolecules, i.e., the rel gene. To test this, peptidoglycan synthesis was examined in the relA derivative, LD5456 (Fig. 4). 0 30 60 Peptidoglycan synthesis was initially stimulated when cells were deprived of lysine. The M inutes effects of CAM on LD5456 were similar to those FIG. 4. Effect of amino acid deprivation and CAM described for LD5. These results indicate that treatment on [3H]Dap incorporation into peptidoglyrelA is involved in regulating peptidoglycan can by strain LD5456 (relA-). Washed cells were synthesis. inoculated into M9 (0), M9 minus lysine (-), M9 Utilization of soluble intracellular precur- plus 200 pg of CAM per ml (A), and M9 minus lysine sors. We compared the utilization of soluble plus 200 pg of CAM per ml (A).

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J. BAcTrziu. ate, i.e., the first step in the synthesis of peptidoglycan (6, 13, 14). It is possible that ppGpp also inhibited enzymes catalyzing later reactions. The formation of peptidoglycan was more strongly inhibited by ppGpp than the formation of lipid-linked internediate; e.g., 2 mM ppGpp inhibited peptidoglycan formation by over 50% but had little effect on the synthesis of lipid intermediate.

DISCUSSION The rel+ strain LD5 was constructed by conjugation using a relA donor (AT980). LD5 presumably did not inherit relA because this marker is located 3 min from the origin of transfer in AT980. Markers close to the origin at low frequencies (23). recombine N6 1m 60 1m It was previously reported (27, 30) that peptiMinutes doglycan synthesis continued in CAM-treated FIG. 5. Effect of lysine deprivation on utilization E. coli. In addition to confirming this, we have of soluble intracellular peptidoglycan precursors. now shown that CAM actually stimulated pepo

0

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Cells labeled for four generations with [3H]Dap were washed and inoculated into the following nonradioactive media complete M9 (0); M9 without Dap and lysine (A); and M9 without Dap but with lysine (0). Growth ofcultures ofLD5 rel+ (A) and LD5456 relA (B) was determined turbidimetrically. Utilization of labeled intracellular precursors in cultures of LD5 rel+ (C) and LD5456 relA- (D) was determined by 'following the incorporation of radioactivity into trichloroacetic acid-insoluble fractions. The radioactivity present in peptidoglycan at 0 min has been subtracted from the data (6,820 and 7,130 cpmlml for LD5 and LD5456, respectively).

the inability of exogenous Dap to prevent furM I NUTE S ther incorporation of [3H]Dap was probably due to the large size of the Dap pool in E. coli (5). FIG. 6. Utilization of labeled intracellular pepti(iii) The rate of [3H]Dap utilization by a Dap- doglycan precursors by Dap-deprived cultures in the deprived culture is an indication of the rate of presence and absence of lysine. Experiments with soluble precursor utilization. When this was LD5 rel+ (A) and LD5456 relA (B) were performed compared in the presence and absence of lysine, as described in the legend to Fig. 5. Radioactivity utilization of intracellular precursors appeared present in peptidoglycan at 0 min has been subto be stringently controlled in the rel+ strain tracted (99,5(0 and 93,400 cpmlmg for LD5 and respectively). The data have been normal(Fig. 5C), whereas the control was relaxed in LD5456, ized to cell mass. Symbols: M9 without Dap and the relA- strain (Fig. 5D). Additional data nor- lysine (0); M9 without Dap but with lysine (0). malized to cell mass are presented in Fig. 6. Effect of ppGpp on in vitro peptidoglycan of various concentrations ofppGpp synthesis. Physiologically significant concen- TABLz 2.onEffect in vitro peptidoglycan synthesis trations of ppGpp inhibited the utilization of Lipid-linked 14C-labeled UDP-MurNAc-pentapeptide by a Peptidoglycan intermediate formed particulate enzyme preparation. ppGpp informed Concn of ppGpp formed hibited the formation of carrier lipid-linked in(mM) termediate as well as the formation of peptidoRel( act Rel%act cpm cpm glycan (Table 2). Furthermore, the degree of inhibition was dependent on the concentrationx 100 0 850 1,198 100 1 87 of ppGpp. These results indicate that phospho791 740 66 2 97 824 553 46 N-acetylmuramyl-pentapeptide translocase is 3 378 32 64 540 ppGpp sensitive. This enzyme catalyzes the 4 180 47 15 402 synthesis of the carrier lipid-linked intermedi-

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tidoglycan synthesis when added to amino aciddeprived cultures of rel+ strains. This result suggested that the synthesis of peptidoglycan may be stringently controlled, since CAM is known to antagonize the stringent control of stable RNA (1, 2) and phospholipid (31, 36) synthesis. This antagonistic effect has been correlated to the lack of accumulation of ppGpp in the presence of CAM (7). Parallel experiments with a reLA derivative, LD5456, clearly supported the above proposal. The relA gene product, therefore, regulates the synthesis of two major cell surface components, phospholipid and peptidoglycan. This may represent one means of coordinating the synthesis of the various cell envelope layers in E. coli. Under our experimental conditions, cell division continued for 30 to 40 min in amino aciddeprived cultures. In rel+ cultures, this residual division occurred in the absence of detectable mass increase and resulted in the production of cells which were smaller than nonnal (our unpublished observation). One intriguing possibility is that the synthesis of the peptidoglycan portion of septa is not subject to stringent control in cells committed to cell division at the time of amino acid deprivation. Residual division in amino acid-deprived rel+ cultures may account for the higher rate of [3H]Dap incorporation observed during the initial 20 to 40 min of our experiments. It should also be noted that although CAM stimulated peptidoglycan synthesis, it had an inhibitory effect on cell diyision. Peptidoglycan synthesis was not as stringently controlled as stable RNA synthesis. Incorporation of [3H]Dap continued at a low linear rate well after cell division had come to a complete halt in amino acid-deprived rel+ cultures. Phospholipid synthesis appears to behave similarly (15, 31, 33, 36). The purpose for the residual synthesis of peptidoglycan by amino acid-deprived rel+ cells is not known. It could be related to the essential nature of the cell envelope. It may be necessary for nongrowing cells to retain at least partial capacity to synthesize peptidoglycan, e.g., for the purpose of repair and maintenance of the cell envelope. The peptidoglycan synthesized during the stringent response may account for the observation that cell wall thickening occurs in both rel+ and relh strains during amino acid deprivation (unpublished data cited in reference 15). Our data suggest that the extent of wall thickening should be greater in rel- strains, but this difference may not be readily detected by electron microscopy. The fate of the peptidoglycan synthesized during amino acid deprivation is also not known, although our preliminary experi-

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ments indicate that this material does not undergo turnover in either rel+ or reUA- strains upon resumption of growth. Thus, this "excess" peptidoglycan may be redistributed over the growing cell surface. From the experiments on utilization of intracellular precursors (Fig. 5 and 6), it is clear that the effects of amino acid deprivation on peptidoglycan synthesis by rel+ and relA- strains are not due to differences in the ability of these organisms to transport Dap. Dap-deprived cells were not immediately dependent on exogenous Dap for growth. In fact, in agreement with previous observations (5), Dap-deprived cultures grew almost as well as Dap-supplemented cultures for at least two doublings. Moreover, the control of intracellular Dap utilization appeared to be stringent in the rel+ strain and relaxed in the relA - strain. A particulate enzyme system incorporates precursors into acceptor peptidoglycan (6, 13, 14). The basis for stringent control appears to be the inhibition of an enzyme(s) in this system by ppGpp. Our data implicate phospho-N-acetyl-muramyl-pentapeptide translocase as being ppGpp sensitive. However, as noted above, other enzymes may also be inhibited. We are in the process of definitively identifying the ppGpp-sensitive site(s) in the particulate system by employing specific assays for the individual enzymes. Several soluble enzymes are involved in the synthesis of peptidoglycan precursors (6, 13, 14). In our continuing studies, we have thus far been unable to demonstrate a ppGpp-sensitive enzyme in this system. Therefore, peptidoglycan synthesis appears to be regulated in two ways: (i) the intracellular levels of precursors may be controlled mainly by feedback inhibition as proposed by others (13, 26, 27); (ii) the rate at which precursors are utilized by the particulate enzymes is controlled by ppGpp. Although our studies have been confmed to nongrowing cells, it is worthwhile to consider the possibility that these same mechanisms govern the rate of peptidoglycan synthesis during growth. This idea is especially appealing if the intracellular concentration of ppGpp varies inversely with growth rate as reported by Lazzarini et al. (21). ACKNOWLEDGMENTlS W. Ramey is the recipient of a Frank Wesbrook predoctoral fellowship from the University of British Columbia. We thank Suzanne Jansen and Mike Morley for technical assistance, Barbara J. Bachmann for bacterial strains, Christoph Beck for the PlvirA phage, Mike Cashel for ppGpp, and J. J. R. Campbell for critical comments. This investigation was supported by grant A6606 from the No-

tional Research Council of Canada.

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LITERATURE CITED 1. Aronson, A. I., and S. Spiegelman. 1961. On the nature of the ribonucleic acid synthesized in the presence of chloramphenicol. Biochim. Biophys. Acta 53:84-95. 2. Aronson, A. I., and S. Spiegelman. 1961. Protein and ribonucleic acid synthesis in a chloramphenicol-inhibited system. Biochim. Biophys. Acta 53:70-84. 3. Bachmann, B. J. 1972. Pedigrees of some mutant strains of Escherichia coli K-12. Bacteriol. Rev. 36:525-557. 4. Block, R., and W. A. Haseltine. 1973. Thermolability of the stringent factor in rel mutants ofEscherichia coli. J. Mol. Biol. 77:625-629. 5. Braun, V. 1975. Covalent lipoprotein from the outer membrane of Escherichia coli. Biochim. Biophys. Acta 415:335-377. 6. Braun, V., and K. Hantke. 1974. Biochemistry of bacterial cell envelopes. Annu. Rev. Biochem. 43:89-121. 7. Cashel, M. 1975. Regulation of bacterial ppGpp and pppGpp. Annu. Rev. Microbiol. 29:301-318. 8. Chung, K. L. 1967. Autoradiographic studies of bacterial cell wall replication. I. Cell wall growth ofBacillus cereus in the presence of chloramphenicol. Can. J. Microbiol. 13:341-350. 9. Comb, D. G. 1962. The enzymatic addition of D-alanylD-alanine to a uridine nucleotide-peptide. J. Biol. Chem. 237:1601-1604. 10. Dale, B. A., and G. R. Greenberg. 1972. Effect of the folic acid analogue, trimethoprim, on growth, macromolecular synthesis, and incorporation of exogenous thymine in Escherichia coli. J. Bacteriol. 110:905916. 11. Dennis, P. P., and M. Nomura. 1974. Stringent control of ribosomal protein gene expression in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 71:3819-3823. 12. Friesen, J. D., N. P. Fiil, J. M. Parker, and W. A. Haseltine. 1974. A new relaxed mutant ofEscherichia coli with an altered 50S ribosomal subunit. Proc. Natl. Acad. Sci. U.S.A. 71:3465-3469. 13. Ghuysen, J. M., and G. D. Shockman. 1973. Biosynthesis of peptidoglycan, p. 37-130. In L. Lieve (ed.), Bacterial membranes and walls. Marcel Dekker, Inc., New York. 14. Glaser, L. 1973. Bacterial cell surface polysaccharides. Annu. Rev. Biochem. 42:92-112. 15. Golden, N. G., and G. L. Powell. 1972. Stringent and relaxed control of phospholipid metabolism in Escherichia coli. J. Biol. Chem. 247:6651-6658. 16. Good, C. M., and D. J. Tipper. 1972. Conditional mutants of Staphylococcus aureus defective in cell wall precursor synthesis. J. Bacteriol. 111:231-241. 17. Hammes, W. P., and F. C. Neuhaus. 1974. On the mechanism of action ofvancomycin: inhibition of peptidoglycan synthesis in Gaftlya homari. Antimicrob. Agents Chemother. 6:722-728. 18. Holttia, E., J. Janne, and J. Pispa. 1974. The regulation of polyamine synthesis during the stringent control inEscherichia coli. Biochem. Biophys. Res. Commun. 59:1104-1111. 19. Izaki, K., M. Matsuhashi, and J. L. Strominger. 1968. Biosynthesis of the peptidoglyean of bacterial cell walls. XIII. Peptidoglycan transpeptidase and D-alanine carboxypeptidase: penicillin-sensitive enzymatic reaction in strains ofEscherichia coli. J. Biol. Chem. 243:3180-3192. 20. Lavalle, R. 1965. Nouveaux mutants de regulation de la synthese de I'ARN. Bull. Soc. Chim. Biol. 47:1567-

J. BACTERIOL. 1570. 21. Lazzarini, R. A., M. Cashel, and J. Gallant. 1971. On the regulation of guanosine tetraphosphate levels in stringent and relaxed strains of Escherichia coli. J. Biol. Chem. 246:4381-4385. 22. Leutgeb, W., and U. Schwarz. 1967. Abbau des Mureins als erster Schritt beim Wachstum des Sacculus. Z. Naturforschg. Teil B 22:545-549. 23. Low, B. 1965. Low recombination frequency for markers very near the origin in conjugation in E. coli. Genet. Res. 6:469473. 24. 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. 25. Leuking, D. R., and H. Goldfine. 1975. The involvement of guanosine 5'-diphosphate-3'-diphosphate in the regulation of phospholipid biosynthesis in Escherichia coli. J. Biol. Chem. 250:4911-4917. 26. Lugtenberg, E. J. J. 1972. Studies on Escherichia coli enzymes involved in the synthesis of uridine diphosphate-N-acetyl-muramyl-pentapeptide. J. Bacteriol. 110:26-34. 27. Lugtenberg, E. J. J., L. de Haas-Menger, and W. H. M. Ruyters. 1972. Murein synthesis and identification of cell wall precursors of temperature-sensitive lysis mutants ofEscherichia coli. J. Bacteriol. 109:326-335. 28. Lugtenberg, E. J. J., and P. G. De Haan. 1971. A simple method for following the fate of alanine-containing components in murein synthesis in Escherichia coli. Antonie van Leeuwenhoek J. Microbiol. Serol. 37:537-552. 29. Lugtenberg, E. J. J., A. van Schbndel-Van Dam, and T. H. M. van Bellegem. 1971. In vivo and in vitro action of new antibiotics interfering with the utilization of N- acetyl-glucosamine-N-acetyl-muramyl-pentapeptide. J. Bacteriol. 108:20-29. 30. Matsuzawa, H., M. Matauhashi, A. Oka, and Y. Sugino. 1969. Genetic and biochemical studies on cell wall peptidoglycan synthesis inEscherichia coli K-12. Biochem. Biophys. Res. Commun. 36:682-689. 31. Merlie, J. P., and L. I. Pizer. 1973. Regulation of phospholipid synthesis in Escherichia coli by guanosine tetraphosphate. J. Bacteriol. 116:355-366. 32. Miller, J. H. 1972. Experimental molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 33. Nunn, W. D., L. A. Meade, and B. E. Tropp. 1975. Lipid synthesis in stringent Escherichia coli: an artifact in acetate labeling of phospholipids during a shiftdown in growth rate. J. Bacteriol. 121:396-399. 34. Reiness, G., H. L. Yang, G. Zubay, and M. Cashel. 1975. Effects of guanosine tetraphosphate on cell-free synthesis of Escherichia coli ribosomal RNA and other gene products. Proc. Natl. Acad. Sci. U.S.A. 72:2881-2885. 35. Shockman, G. D. 1965. Symposium on the fine structure and replication of bacteria and their parts. IV. Unbalanced cell-wall synthesis: autolysis and cellwall thickening. Bacteriol. Rev. 29:345-358. 36. Sokawa, Y., E. Nakao, and Y. Kaziro. 1968. On the nature of the control by RC Gene in E. coli: amino acid-dependent control of lipid synthesis. Biochem. Biophys. Res. Commun. 33:108-112. 37. Stent, G. S., and S. Brenner. 1961. A genetic locus for the regulation of ribonucleic acid synthesis. Proc. Natl. Acad. Sci. U.S.A. 47:2005-2014. 38. Taylor, A. L., and C. D. Trotter. 1972. Linkage map of Escherichia coli K-12. Bacteriol. Rev. 36:504-524.

Stringent control of peptidoglycan biosynthesis in Escherichia coli K-12.

JouRNAL OF BACTERIOLOGY, Sept. 1976, p. 1119-1126 Copyright © 1976 American Society for Microbiology Vol. 127, No. 3 Printed in U.S.A. Stringent Con...
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