Release of Cell Wall Peptides into Culture Medium by Exponentially Growing Escherichia coli

Transcription

1 JOURNAL OF BACTERIOLOGY, Apr. 1985, p /85/ $02.00/0 Copyright 1985, American Society for Microbiology Vol. 162, No. 1 Release of Cell Wall Peptides into Culture Medium by Exponentially Growing Escherichia coli E. WILLIAM GOODELLt* AND ULI SCHWARZ Abteilung Biochemie, Max-Planck-Institut fur Entwicklungsbiologie, D-7400 Tubingen, Federal Republic of Germany Received 23 July 1984/Accepted 16 January 1985 Escherichia coli W7 cells were found to release three different muropeptides into the culture medium: tetrapeptide (L-Ala-D-Glu-meso-diaminopimelic acid-d-ala), tripeptide (L-Ala-D-Glu-meso-diaminopimelic acid), and a previously undescribed dipeptide (meso-diaminopimelic acid-d-ala). From the rate of release of these three peptides, it was calculated that 6 to 8% of the murein in the sacculus was lost per generation. Bacteria which have a rigid murein cell wall commonly possess one or more murein hydrolases which are potentially capable of lysing the cell (22). In gram-positive rods these murein hydrolases seem to be active during growth, since fragments are released from the murein as the cells elongate and expand their surface area (1, 3, 15). In contrast, gram-negative bacteria, with the exception of Neisseria gonorrhoeae (9, 10, 23), have not yet been clearly demonstrated to release muropeptides from their cell walls. One group (4) has reported that Escherichia coli sheds muropeptides into the medium at a slow rate; however, some of the cells were undergoing lysis, making the results difficult to interpret. Thus, the action of the murein hydrolases of gram-negative bacteria has remained uncertain, and several authors (5, 11, 24, 30, 31) have proposed that some of these enzymes might not function as hydrolases in vivo but as transferases and that, as such, they may be involved in the remodelling and randomization of the murein in the sacculus. In this communication, we describe the loss of muropeptides from the murein of exponentially growing E. coli cells. The nature of the muropeptides released indicates that the N-acetylmuramyl-L-alanine amidase (20, 30) and probably one or both lytic transglycosylases (11, 18) are involved and that in vivo these enzymes do function, at least in part, as hydrolases. MATERIALS AND METHODS Bacterial strains and culture conditions. E. coli W7 (Dap- Lys-; 24) was grown in Penassay broth (PB; Difco Laboratories, Detroit, Mich.) containing 2,ug of diaminopimelic acid (A2pm) per ml (2, 6) at 30 C with vigorous aeration. Growth of the cultures was periodically assayed by measuring the turbidity of the culture at A578. Labeling with [3H]A2pm. Typically, a 1-ml culture of exponentially growing bacteria (0.15 A578 units) was labeled with 0.2 mci of [3,4,5-3H]A2pm (25 Ci/mmol; Commisariat a l'energie atomique, Gif-sur Yvette, France) for 60 min. The cells were washed free of the [3H]A2pm by centrifuging them into a linear 6-ml Ficoll (Ficoll 400; Pharmacia, Uppsala, Sweden) gradient (0 to 25%). The Ficoll gradient was prepared in a cellulose nitrate tube (16 by 102 mm) by using 3 ml of 25% Ficoll (dissolved in PB containing 400,ug of unlabeled A2pm per ml) and 3 ml of PB (also containing 400,ug of * Corresponding author. t Present address: Natural Sciences Department, State College of New York College of Technology, Utica, NY unlabeled A2pm per ml). The gradient was prewarmed to 30 C, overlayered with the 1-ml culture, and centrifuged at 2,500 x g for 3 min at 30 C in a swinging bucket rotor. A band of cells formed in the lower half of the gradient; it was collected by puncturing the side of the tube with a syringe and cultured in 7 ml of PPB (containing 400,ug of unlabeled A2pm per ml) at 30 C. Samples (1 ml) were removed from the culture after 0, 15, 30, 60, 90, and 120 min; at 120 min when the culture reached 0.5 to 0.6 A578 units, it was diluted 16-fold with fresh prewarmed medium supplemented with 400 F.g of A2pm per ml, and 16-ml samples were removed after 180 and 240 min. After collection each sample was immediately centrifuged in an Eppendorf centrifuge (9,980 x g) for 1 min to pellet the bacteria. The culture fluid was collected and frozen for further analysis. The cell pellet was resuspended in 1 ml of boiling 4% sodium dodecyl sulfate (SDS) and boiled for an additional 5 min; unlabeled murein (0.1 mg) was then added to the samples, and they were centrifuged (100,000 x g, 60 min, room temperature) to pellet the murein sacculi. The SDS supernatant was collected and frozen. The sacculi were washed twice in distilled water and resuspended in 1 ml of water plus 10 ml of scintillation fluid, and the radioactivity was measured. Addition of the excess unlabeled murein to the [3H]murein (see above) insured that the quenching did not vary during the course of the experiment due to increasing cell numbers. Suspension of the sample in a relatively large volume of scintillation fluid minimized the amount of quenching; the counting efficiency was not increased by prior digestion with Chalaropsis muramidase (7). Separation and characterization of [3HJA2pm-labeled compounds. The samnples of culture medium and SDS supernatant were first placed on' a Fractogel TSK HW-40-S (E. Merck AG, Darmstadt, Federal Republic of Germany) column (2.5 by 60 cm) equilibrated with 0,02 M ammonium acetate buffer (ph 7.5) containing 0.02% sodium azide, The samples were eluted with the same buffer (30 ml/h); 1-ml fractions were collected. Paper electrophoresis in 0.1 M formic acid on Whatmann 3 MM paper (20 V/cm; 90 min) was performed as described by Tomioka et al. (27), Descending paper chromatography with pyridine-water (4:1) and n-butanol-acetic water-water (4:1:5; upper phase) was done on SS 2040b paper (Schleicher & Schull, Inc., Keene, N.H.) as described by Schwarz and Weidel (25). Murein synthesis in ether-treated cells was performed essentially as described by Maass and Pelzer (14). After permeabilization with ether (14), the cells (40 Ixl; 0.6 mg)

2 392 GOODELL AND SCHWARZ were added to 160,ul of incubation buffer. The reaction mixture contained (final concentration) 50 mm Tris (ph 8.3), 50 mm NH4Cl, 20 mm MgCl2, 0.5 mm mercaptoethanol, 8 nmol of [3H]UDP-pentapeptide, and 4 nmol of UDP-N-acetylglucosamine (GlcNAc) (Sigma Chemical Co., St. Louis, Mo.). The samples were incubated for 60 min at 300; the reaction was stopped by boiling in 4% SDS for 30 min. The murein was collected on a membrane filter (0.2-,um pore size) and washed three times with 5 ml of distilled water. The filter was then dried and counted. High-pressure liquid chromatography (HPLC) of GIcNAc- (,11,4)1,6-anhydro-N-acetylmuramic acid (MurNAc)- tripeptide (X) and GlcNAc(P1,4)1,6-anhydro-MurNActetrapeptide (X') was done as described by Glauner and Schwarz (7). Samples (in 100,ul of 50 mm phosphate buffer [ph 4.69]) were applied to a reverse-phase column (Li- Chrosorb RB-18; 250 by 4 mm; 5-.m particle size; E. Merck) which had been equilibrated with 50 mm phosphate buffer (ph 4.69). The compounds were eluted with a linear gradient of methanol (0 to 15%) in 50 mm phosphate (ph 4.69) at a flow rate of 0.5 ml/min; 0.1-ml fractions were collected. X was eluted after 67 min; X' was eluted after 80 min (7). Preparation of [3HJA2pm-labeled standards. X and X' were gifts of Bernd Glauner. They were prepared by digestion of E. coli murein with lytic transglycosylase isolated from E. coli (11); 50 U of enzyme (11) and 5 mg of murein (containing 0.5 mci of [3H]A2pm) were incubated in 10 ml of 10 mm Tris-maleate buffer (ph 6.0) containing 10 mm MgSO4 and 0.2% Triton X-100 at 370C for 12 h. X and X' were separated from the larger cross-linked muropeptides by chromatography on the Fractogel column and then separated from each other on the HPLC column (see above). The tetrapeptide and tripeptide were obtained from X' and X, respectively, by digestion with human N-acetylmuramyl- L-alanine amidase (29; S. Mollner, Ph.D. thesis, University of Tubingen, Tubingen, Federal Republic of Germany, 1984). The enzyme (0.01 U) and substrate (0.5 mg) were incubated in 1 ml of 50 mm Tris-hydrochloride buffer (ph 7.9) containing 5 mm MgCl2 and 0.05% sodium azide for 2 h at 37. The sample was then chromatographed on the Fractogel column to remove any undigested substrate. The bisdissacharide muropeptide obtained from the Fractogel column was used to prepare cross-linked 'octapeptide. The disaccharide moiety was removed by amidase digestion (see above), and the sample was again applied to the Fractogel column to isolate the octapeptide. Dipeptides were prepared by partial acid hydrolysis of the purified tetrapeptide and tripeptide; the samples were treated with 6 N HCl at 800 for 30 min and then dried several times over NaOH pellets and phosphorous pentoxide to remove the HCl. UDP-MurNAc-L-Ala-D-Glu-meso-[3H] A2pm-D-Ala-D-Ala ([3H]UDP-pentapeptide) was obtained from E. coli W7. The bacteria were grown in 10 ml of PB containing 2,ug of unlabeled A2pm and 0.2 mci of [3H]A2pm. When the culture had reached an A578 value of 0.6, the cells were harvested and boiled in 1 ml of 4% SDS for 5 min. The murein was removed by centrifugation at 100,000 x g for 60 min at room temperature, and the SDS supernatant was placed on the Fractogel column. The UDP-pentapeptide peak was eluted shortly after the exclusion volumn; this material was pooled, lyophilized, and chromatographed on the HPLC column as described above. The UDP-pentapeptide was eluted after 17 min. MurNAc-pentapeptide was prepared from UDP-pentapeptide by boiling for 10 min in 0.1 N HCl (17). Lactyl-pentapeptide was prepared from MurNAc-pentapeptide by 13 elimination (26). RESULTS Release of muropeptides into the culture medium by E. coli cells. Loss of murein fragments from exponentially growing gram-positive rods occurs at a rate of 15 to 50% per generation (1, 3, 15). It is easily detected by labeling the murein with an appropriate radioactive amino acid or amino sugar and then measuring the loss of label from the murein after transferring the cells to a medium without label. Usually it is sufficient to measure the decline in acid-precipitable counts in the culture (1, 3). Earlier studies with E. coli from this laboratory (unpublished data) and elsewhere (4) had indicated that the loss of label from E. coli murein, if it existed, would be low and difficult to detect in the above manner. It appeared more likely that one could accurately determine the rate of murein loss in E. coli by measuring the rate of release of muropeptides from the murein into the culture medium. The results of such an experiment are described below. An exponential culture of E. coli (Dap- Lys-) in PB was grown at 300 for 1 h (two generations) in the presence of [3H]A2pm to label the murein. The bacteria were then washed and transferred to fresh unlabeled medium. Samples of the culture were removed during the next 4 h (eight generations), and the cells and culture medium were separated by centrifugation. The medium was collected, and the bacteria in the pellet were immediately resuspended in boiling 4% SDS and later centrifuged to separate the murein sacculi and the SDS-soluble components. The amount of 3H-label recovered in the culture medium, in the murein, and in the SDS supernatant for each of the samples is given in Table 1; the proportion of label in the culture medium increased steadily from 8% of the total to 51% during this chase period, suggesting that the cells were releasing muropeptides from their murein into the culture medium. To determine which compounds were being released, the medium samples were placed on a Fractogel TSK HW-40 column, and the compounds were separated according to size. Figure 1 shows the distribution of 3H-label for a typical sample (90 min) and the positions of several reference muropeptides of known molecular weight. Three peaks TABLE 1. Chase Chase Distribution of radioactive label in the culture during the chasea Total cpm (%) in: J. BACTERIOL. duration (min) Murein SDS Medium supernatant 0 66,339 (51) 52,980 (11) 10,275 (8) 129, ,571 (63) 34,052 (25) 16,335 (12) 134, ,445 (71) 22,644 (16) 19,950 (14) 142, ,954 (65) 22,040 (15) 27,900 (20) 142, ,969 (66) 17,024 (12) 32,462 (22) 145, ,581 (63) 13,310 (10) 37,391 (27) 137, ,109 (61) 10,680 (8) 43,219 (31) 138, ,964 (54) 9,426 (7) 52,440 (39) 133, ,004 (44) 6,963 (5) 73,382 (51) 141,459 a Given are the total counts per minute recovered from the fractions of each sample. Al-ml culture of E, coli W7 was labeled for 60 min with [3H]A2pm and after being washed was transferred to fresh medium (final volume, 8 ml) containiq$ 400 F.g of unlabeled A2 pm per ml. Samples (1 ml) were then removed during the next 2 h; at 120 min, when the culture reached an A578 value of 0.6, it was diluted 16-fold with fresh, prewarmed medium to allow continued exponential growth, and 16-ml samples were taken. ~~~~~~~~~~~~~Total

3 VOL. 162, 1985 were obtained. The smallest (peak A) cochromatographed with unlabeled A2pm, and the largest (peak C) chromatographed with a mixture of tetrapeptide (L-Ala-D-Glu-meso- A2pm) and tripeptide (L-Ala-D-Glu-meso-A2pm) obtained from E. coli murein. The third substance (peak B) was of intermediate size and appeared to be a dipeptide. An examination of the kinetics of release of these compounds revealed that the compounds in peaks B and C were released at a constant rate throughout the chase period (6,300 cpm per generation), whereas nearly all of the compound in peak A (A2pm) was lost from the cells within the first 15 min (Fig. 2). Characterization of the 3H-muropeptides in the culture medium. The size of the compounds present in peak C (Fig. 1) suggested that they were free tetrapeptide or tripeptide, or both, perhaps formed by the E. coli N-acetyl-muramyl-Lalanine amidase (20). To determine whether this was the case, the C peaks obtained from the various medium samples were pooled and further analyzed. Paper chromatography in pyridine-water (4:1) was found to separate tripeptide and tetrapeptide; purified tetrapeptide from E. coli murein had an RA2pm (Rf with respect to A2pm) of 2.7 in this system, and tripeptide had an RA2Pm of 1.0 in this system. The substances in peak C were separated into two peaks when chromatographed in this system, with mobilities identical to those of the tetrapeptide and tripeptide. Most of the label (70%) was recovered in the tetrapeptide fraction; the rest was recovered in the tripeptide fraction. Further characterization by chromatography in butanol-acetic acid-water and Fraction No FIG. 1. Fractionation of 3H-compounds released by E. coli W7 into the culture fluid. The medium sample, after centrifugation to remove the bacteria, was chromatographed on a Fractogel column, and the labeled compounds were separated according to size. Three 3H-labeled Peaks were obtained (A, B, and C). The relative positions of several standards are indicated by arrows: A2pm, tripeptide and tetrapeptide, X', and octapeptide. B E RELEASE OF MUROPEPTIDES BY E. COLI 393 s mmn FIG. 2. Kinetics of release of 3H-compounds into the culture medium. The figures shows the amount of [3H]A2pm (X), 3H-tripeptide and 3H-tetrapeptide (U), and 3H-dipeptide (0) which had been lost by the cells into the culture medium at various times during the 4-h chase. The labeled compounds in each sample were fractionated by size on the Fractogel column; the amount of label recovered in each fraction was then plotted against the sample time. electrophoresis in 0.1 N formic acid (25) also indicated that these compounds were tripeptide and tetrapeptide. The material in peak B (Fig. 1), from its behavior on the Fractogel column, appeared to be a dipeptide. Two A2pmcontaining dipeptides are possible in E. coli murein: D-Glumeso-A2pm and meso-a2pm-d-ala. To determine which of these compounds was present in peak B, purified tetrapeptide and tripeptide were both subjected to partial acid hydrolysis; only the tetrapeptide should release meso-a2pm- D-Ala. When both digests were fractionated on the Fractogel column, only the tetrapeptide digest contained a compound with the same mobility as the compound in peak B, indicating that the compound in peak B was meso-a2prp-d-ala. The mobility of the compound in peak B in pyridine-water (RA2pm = 2.6) and in butanol-acetic acid-water (RA2pm = 1.3) was the same as meso-a2pm-d-ala obtained from the tetrapeptide. In contrast, D-Glu-A2pm from the tripeptide h4d a lower mobility (RA2pm = 1) in these systems. The cells thus appeared to be releasing three 3H-peptides into the medium: tetrapeptide (L-Ala-D-Glu-meso-A2pm-D- Ala), tripeptide (L-Ala-D-Glu-meso-A2pm), and dipeptide (meso-a2pm-d-ala); 68% of the 3H-label was in the dipeptide, 22% was in the tetrapeptide, and 10% was in the tripeptide. Retention of larger muropeptides within the cell. The outer membrane of E. coli restricts the passage of oligopeptides and oligosaccharides with a molecular weight larger than 600 (21). The tetrapeptide from murein has a molecular weight of 461 and should pass through the pores in the outer membrane; however, if any larger murein fragments are produced, they may be retained within the periplasm. To determine whether larger muropeptides were inside the

4 394 GOODELL AND SCHWARZ ' X 0 40 A.2~~~~~~~~~~~~~~~~~~~~-1 _ 0 Z~~~~~~~ E* E 2 '~500 - 'U ~ ~ ~ ~ Fato No Fraction No FIG. 3. Separation of 3H-substances retained within the bacteria. The cells were boiled in SDS, and the murein sacculi were removed by centrifugation. The SDS supernatant was then placed on the Fractogel column. Five peaks (A through E) were obtained; from 'their elution positions, they' were provisionally identified as A2pm (A), dipeptide (B), tripeptide and tetrapeptide (C), X and X' (D), and UDP-pentapeptide (E). Also indicated, with arrows, are the relative elution positions of unlabeled A2pm, tripeptide and tetrapeptide, and X'. cells, the SDS supematant samples were also chromatographed on the Fractogel column. Two substances of larger molecular weight (Fig. 3, peaks D and E), were found as well as the three smaller components already found in the culture fluid. From their sizes, peaks E and D were provisionally identified as UDP-MurNAc-L-Ala-D-Glu-meso-A2pm-D-Ala- D-Ala (UDP-pentapeptide) and GlcNAc(31,4)1,6-anhydro- MurNAc-peptide. The latter material is produced when murein is digested with lytic transglycosylase from E. coli (11). Figure 4 shows the kinetics of labeling of the intracellufar pools of the components in peaks A through E (Fig. 3); the label in the murein is also shown for comparison. As expected, the amount of label in peaks A and E dropped rapidly during the initial phase of the chase period, indicating that they were murein precursors; peaks B, C, and D, in contrast, increased slowly during the chase. Characterization of the intracellular 3H-labeled components. From its size and labeling kinetics, the material in peak E appeared to be UDP-pentapeptide; however, the amount of label in this peak remained surprisingly high in the longer chase times (Fig. 4), suggesting that some of the label might be in another compound, perhaps the cross-linked J. BACTERIOL. dimer of the tetrapeptide (octapeptide) which migrates at the same position on the column (Fig. 1 and 3). To test this possibility, each of the peak E samples (Fig. 3) was subjected to paper electrophoresis in 0.1 N formic acid (ph 2.5). At this ph, murein precursors with phosphate groups such as UDP-pentapeptide still have a net negative charge and move towards the anode, whereas muropeptides without phosphate groups have a net positive charge and move in the opposite direction (19, 25). When tested, all 3H-label in the samples was found to move towards the anode; electrophoresis of control substances, UDP-pentapeptide, octapeptide, and tetrapeptide showed that UDP-pentapeptide migrated in the same position as peak E and that octapeptides and tetrapeptides moved in the opposite direction. We also tested the ability of UDP-pentapeptide (Fig. 3, peak E) from pulse-chased cells (180 and 240 min) to support murein synthesis in ether-permeabilized E. coli cells. The [3H]UDP-pentapeptide (8 nmol) plus 4 nmol UDP-GlcNAc was mixed with 0.6 mg of ether-treated cells, and the mixture was incubated for 60 min at 30 C; as a control, [3H]UDP-pentapeptide purified by HPLC (see above) was also used. In both cases, a similar amount of murein (0.08 nmol) was synthesized, indicating that the material in peak E supported a normal quantity of murein synthesis; we concluded that this material was UDP-pentapeptide and not a min FIG. 4. Kinetics of labeling of the intracellular 3H-compounds. The amount of label recovered in murein (A) and in peaks A through E during the chase period is given: A2pm (peak A; X), dipeptide (peak B; 0), tripeptide and tetrapeptide (peak C; U), X and X' (peak D; O), and UDP-pentapeptide (peak E; 0).

5 VOL. 162, 1985 murein breakdown product (an intermediate in the formation of tripeptide and tetrapeptide). Additional evidence in support of this conclusion is given in the discussion. To identify the substances present in peak D (Fig. 3), this material from the various SDS samples was pooled, lyophilized, and chromatographed in butanol-acetic acid-water (4:1:5). The label was separated into three components (D1, D2, and D3); D1 and D2 were tentatively identified as X and X', respectively, by comparison with the RA2pm of purified X and X' obtained from E. coli murein. To obtain more positive identification of these two compounds, they were eluted from the paper and chromatographed on reversephase HPLC (7). Two peaks were also obtained in this system with retention times (67 and 80 min) identical to those of X and X', respectively (7). D3 moved in butanol-acetic acid-water as a single sharp peak in the same position as lactyl-pentapeptide obtained from UDP-pentapeptide by mild acid hydrolysis to remove the UDP moiety, followed by P elimination of the sugar at ph 11. The identity of D3 was confirmed by its binding to the antibiotic vancomycin (6). Thus, D3 seems to be a degradation product of UDP-pentapeptide, perhaps formed when the cells were boiled in 4% SDS. A total of 8% of the label in peak D was recovered in X, 22% was recovered in X', and 69% was recovered in the lactyl-pentapeptide. The material in the C peaks (Fig. 1 and 3) was also pooled and chromatographed in pyridine-water. All of the label moved as tripeptide, suggesting that the cells were preferentially retaining this compound over the tetrapeptide. Thus, three muropeptides which appeared to be degradation products of murein were isolated from the cells: tripeptide, X and X'. Most of the label in the muropeptides was in the tripeptide (90%), followed by X' (7%) and X (3%). The retention of X and X' inside the cells appears to be due to their size (850 and 921 daltons), which prevents their movement across the outer membrane. However, the presence of more tripeptide in the cells than tetrapeptide cannot be ascribed to size, but suggests that the cells are actively retaining tripeptide, perhaps in the cytoplasm, to reuse it to synthesize UDP-pentapeptide (see below). Evidence for viable, intact cells. The release of muropeptides into the culture medium could be an artifact of cell lysis, perhaps induced by washing and transferring them to fresh medium. We indeed found that when E. coli cells were washed by the usual filtration method (24), they released some UDP-pentapeptide into the filtrate. The method used here to wash the cells was therefore designed to minimize lysis. The culture was layered over a 0 to 25% linear Ficoll gradient (made with PB containing 400,ug of unlabeled A2pm per ml and prewarmed to 30 C) and centrifuged, also at 30 C, for 3 min. Cells near the bottom of the gradient were then transferred to prewarmed PB containing 400,ug of A2pm per ml. These cells immediately continued their exponential growth (30-min generation time), as determined by increases in both optical density and viable counts. Examination of the cells in the light microscope shortly after transfer also did not reveal any lysis. These measurements indicate that most of the cells were viable and growing; however, they do not exclude the existence of a small population (5 to 10%) of lysing cells. Stronger evidence that the muropeptides were being released by intact, functional cells is provided by the nature of the released substances. First, the cells released no [3H]UDPpentapeptide into the medium, indicating that they were not lysing and that the cytoplasmic membrane was intact. Second, the size of the escaping muropeptides indicates that the RELEASE OF MUROPEPTIDES BY E. COLI 395 outer membrane was intact and functioning. No compounds larger than 461 daltons were lost into the culture fluid; X and X' remained within the cell. In contrast, lysing E. coli cells, as well as osmotically protected spheroplasts, release larger muropeptides, including X, X', and crosslinked dimers, into the culture medium (13, 25; U. Schwarz, Ph.D. thesis, University of Tubingen, Tubingen, Federal Republic of Germany, 1963). To further document that growing E. coli cells do release peptides into the medium, an exponentially growing culture of E. coli W7 was labeled for two generations with [3H]A2pm (0.2 mci). At an A578 value of 0.6, the cells were removed by centrifugation, and the culture medium was analyzed by gel filtration; peaks B and C were found, as well as a large amount of unincorporated [3H]A2pm. DISCUSSION This study reveals that exponentially growing E. coli cells do release [3H]A2pm-labeled compounds into the growth medium. Three different compounds were identified: a tetrapeptide (L-Ala-D-Glu-meso-A2pm-D-Ala), a tripeptide (L-Ala-D-Glu-meso-A2pm), and a previously undescribed dipeptide (meso-a2pm-d-ala). Several features of their formation indicate that the murein is the source of these muropeptides (and not UDP-pentapeptide or some other murein precursor). (i) Both the intracellular and extracellular pools of these muropeptides were labeled after murein itself, as expected for a degradation product of murein. (ii) The muropeptides are released at a constant rate throughout the chase period (ca. 6,000 cpm per generation). The intracellular pools of A2pm and UDP-pentapeptide are large in E. coli (3, 16), and initially the rate of decline of the label in these pools could account for the increase in extracellular label. However, after 60 min the decline in the combined [3H]A2pm and [3H]UDP-pentapeptide pools dropped below 4,000 cpm per generation and could no longer explain the increase in 3H-label in the medium. This was especially apparent during the last 2 h of the chase, when the [3H]A2pm pool had essentially dropped to zero (1% of the original level), and the rate of decline of the [3H]UPD-pentapeptide pool was only 1/10 as rapid as the rate of increase in the extracellular 3H-peptides. The murein showed, as expected, a net decline of label during this period, since label was no longer being replaced from the precursor pool (iii) Finally, the rapid loss of muropeptides into the culture medium indicates that they were being produced outside the cytoplasmic membrane, i.e., from the murein in the periplasm. We therefore used the rate of release of muropeptides into the medium to estimate the rate of murein loss; in the experiment illustrated in Fig. 2, we found a rate of 6.2% per generation. In a separate experiment, a somewhat higher value of 8.4% was obtained. The constant release of muropeptides, without an apparent lag, suggests that new and old murein lose label at the same rate. It will be important to label murein with shorter pulses to establish this point. A surprising result for us was the large amount of intracellular [3H]UDP-pentapeptide in the cells even after prolonged chase periods and after the intracellular [3H]A2pm pool had been depleted. This result suggested that [3H]UDPpentapeptide was being formed from some other compound, perhaps 3H-muropeptides. We have since found (E. W. Goodell, submitted for publication) that E. coli W7 cells will rapidly take up and incorporate 3H-tetrapeptides and 3Htripeptides into their murein. No free [3H]A2pm was found in these cells, only 3H-peptides and [3H]UDP-pentapeptide, suggesting that the cells were converting the peptides di-

6 396 GOODELL AND SCHWARZ rectly to UDP-pentapeptide without breaking them down to individual amino acids. Thus muropeptides can apparently be recycled by the cells, and it therefore seems that the loss rate of 6 to 8% obtained above is an estimate of only the net loss of muropeptides from the cell and that the actual rate of cleavage of murein fragments from the sacculus is probably higher. That muropeptides are released from the murein sacculus indicates that one or more of the murein hydrolases isolated from E. coli do act in vivo as hydrolases. An N-acetyl-muramyl-L-alanine amidase has been isolated from E. coli (20, 30) which could, in principle, be acting alone to release peptide side chains from the murein. However, despite extensive searching (20), no evidence has been obtained for MurNAc residues in the murein of E. coli cells which lack peptide side chains. In addition, the purified amidase has no activity against intact sacculi, but acts only on MurNAc peptides (20). Thus, other enzymes seem to be involved. The isolation of small amounts of X and X' from the cells suggests that one or both of the lytic transglycosylases (11, 18) are also active. These enzymes can degrade murein sacculi in vitro; the X and X' so formed can also be degraded to tripeptide and tetrapeptide by crude extracts of E. coli (S. Klencke, Ph.D. thesis, University of Tubingen, Tubingen, Federal Republic of Germany, 1980). However, X and X' are poor substrates for the isolated amidase (20). Thus an additional enzyme, perhaps the P-N-acetylglucosamidase (12, 31), may remove the GlcNAc residues from X and X' and convert them into amidase substrates. We were, however, unable to detect these intermediates in our SDS supernatant fractions, indicating that if they are formed they must be rapidly degraded by the amidase to free peptides. Thus, the sequence of reactions which leads to the formation of tripeptide and tetrapeptide remains to be determined. A peptidase able to form the dipeptide meso-a2pm-d-ala has not yet been isolated from E. coli, so the mechanism of formation of this compound is also uncertain. It may be produced from free tetrapeptide; however, Gmeiner and Kroll (8) have reported that Proteus mirabilis murein contains a significant amount of L-Ala-D-Glu dipeptide side chains and that the amount increases as the murein ages. They also reported that E. coli murein has these dipeptide side chains. Thus, meso-a2pm-d-ala may be cleaved directly from the murein sacculus. Though this study shows that release of murein fragments from the sacculus of E. coli does occur, it does not indicate what physiological function such turnover has. Several possibilities exist. From a theoretical point of view both the enlargement of the sacculus during cell elongation and the formation of the septum during cell division require hydrolysis of covalent bonds in the sacculus. It should be possible to determine whether turnover is associated with either or both of these processes by measuring the loss of label from murein in synchronized cells. Repair of the sacculus or correction of its shape are additional possibilities. For example, Trueba and Woldringh (28) reported that the diameter of the cell decreases as the cell elongates, suggesting that the cell continuously adjusts the shape of the sacculus as it grows. The decrease in diameter reported by these authors was 8%, which agrees well with the 6 to 8% loss of label which we found. ACKNOWLEDGMENTS We thank Bernd Glauner and Werner Kraus for help in characterizing the muropeptides and Marion Huber for providing the space and equipment which made this work possible. J. BACTERIOL. LITERATURE CITED 1. Boothby, D., L. Daneo-Moore, M. L. Higgins, J. Coyette, and G. D. Shockman Turnover of bacterial cell wall peptidoglycans. J. Biol. Chem. 248: Burman, L. G., J. Raichler, and J. T. Park Evidence for diffuse growth of the cylindrical part of the Escherichia coli murein sacculus. J. Bacteriol. 155: Chaloupka, J., and P. Kteekov Turnover of mucopeptide during the life cycle of Bacillus megaterium. Folia Microbiol. 16: Chaloupka, J., and M. Strnadov Turnover of murein in a diaminopimelic acid dependent mutant of Escherichia coli. Folia Microbiol. 17: Chaterjee, A. N., R. J. Doyle, and U. N. Streips A proposed functional role for bacterial N-acetylmuramyl-L-alanine amidases. J. Theor. Biol. 68: de Pedro, M. A., and U. Schwarz Heterogeneity of newly inserted and preexisting murein in the sacculi of Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 78: Glauner, B., and U. Schwarz The analysis of murein composition with high-pressure-liquid chromatography, p In R. Hackenbeck, J.-V. Holtje, and H. Labischinski (ed.), The target of penicillin. Walter de Gruyter, Berlin. 8. Gmeiner, J., and H.-P. Kroll N-acetylglucosaminyl-Nacetyl-muramyl-dipeptide, a novel murein building block formed during the cell division cycle of Proteus mirabilis. FEBS Lett. 129: Goodell, E. W., M. Fazio, and A. Tomasz Effect of benzylpenicillin on the synthesis and structure of the cell envelope ofneisseria gonorrhoeae. Antimicrob. Agents Chemother. 13: Hebeler, B. H., and F. E. Young Chemical composition and turnover of peptidoglycan in Neisseria gonorrhoeae. J. Bacteriol. 126: Holtje, J. V., D. Mirelman, N. Sharon, and U. Schwarz Novel type of murein transglycosylase in Escherichia coli. J. Bacteriol. 124: Hrebenda, J Mutants of Escherichia coli with altered level of,-n-acetylglucosaminidase activities. Acta Microbiol. Polon. 28: Lubitz, W., and R. Plapp Murein degradation in Escherichia coli infected with bacteriophage 4)X174. Curr. Microbiol. 4: Maass, D., and H. Pelzer Murein biosynthesis in ether permeabilized Escherichia coli starting from early peptidoglycan percursors. Arch. Microbiol. 130: Mauck, J., L. Chan, and L. Glazer Turnover of the cell wall of gram-positive bacteria. J. Biol. Chem. 246: Mengin-Lecreulx, D., B. Flouret, and J. van Heienoort Cytoplasmic steps of peptidoglycan synthesis in Escherichia coli. J. Bacteriol. 151: Mett, H., R. Bracha, and D. Mirelman Soluble nascent peptidoglycan in growing Escherichia coli cells. J. Biol. Chem. 255: Mett, H., W. Keck, A. Funk, and U. Schwarz Two different species of murein transglycosylase in Escherichia coli. J. Bacteriol. 144: Nguyen-Distkche, M., J.-M. Ghuysen, J. J. Pollock, P. Reynolds, H. R. Perkins, J. Coyette, and M. R. J. Salton Enzymes involved in wall peptide crosslinking in Escherichia coli K12, strain 44. Eur. J. Biochem. 41: Parquet, C., B. Flouret, M. Leduc, Y. Hirota, and J. van Heienoort N-acetylmuramoyl-L-alkaline amidase ofescherichia coli K12. Possible physiological functions. Eur. J. Biochem. 133: Payne, J. W Transport and utilization of peptides by bacteria, p In J. W. Payne (ed.), Microorganisms and nitrogen sources. John Wiley & Sons, Ltd., London. 22. Rogers, H. J., H. R. Perkins, and J. B. Ward Microbial cell walls and membranes. Chapman and Hall, Ltd., London. 23. Rosenthal, R. S Release of soluble peptidoglycan from growing gonococci: hexaminidase and amidase activities. Infect. Immun. 24:

7 VOL. 162, 1985 RELEASE OF MUROPEPTIDES BY E. COLI Ryter, A., Y. Hirota, and U. Schwarz Process of cellular division in Escherichia coli. Growth pattern of E. coli murein. J. Mol. Biol. 78: Schwarz, U., and W. Weidel Zum Wirkungsmechanismus von Penicillin. I. Isolierung and Characterisierung 2.6- Diaminopimelinsaure enthaltender, niedermolekularer Peptide aus Penicillin-spharoplasten von Escherichia coli B. Z. Naturforsch. 20b: Tipper, D. J Alkali-catalyzed elimination of D-lactic acid from muramic acid and its derivatives and the determination of muramic acid. Biochemistry 7: Tomioka, S., T. Nikaido, T. Miyakawa, and M. Matsuhashi Mutation of the N-acetylmuramyl-L-alanine amidase gene of Escherichia coli K-12. J. Bacteriol. 156: Trueba, F. J., and C. L. Woldringh Changes in cell diameter during the division cycle of Escherichia coli. J. Bacteriol. 142: Valinger, Z., B. Ladegic, and J. TomaNik Partial purification and characterization of N-acetylmuramyl-L-alanine amidase from human and mouse serum. Biochim. Biophys. Acta 701: van Heienoort, J., C. Parquet, B. Flouret, and Y. van Heienoort Envelope-bound N-acetylmuramyl-L-alanine amidase of Escherichia coli K12. Purification and properties of the enzyme. Eur. J. Biochem. 58: Yem, D. W., and H. C. Wu Purification and properties of P-N-acetylglucosaminidase from Escherichia coli. J. Bacteriol. 125: