SYNTHESIS OF ESCHERICHIA COLI CELL WALLS IN THE PRESENCE OF PENICILLIN*

BY RAUL E. TRUCCOt AND ARTHUR B. PARDEE

(From the Virus Laboratory, University of California, Berkeley, California)

(Received for publication, August 19, 1957)

The mechanism of action of penicillin at the cellular level has been con- siderably clarified recently. It seems clear that penicillin damages the surface of the bacteria, as first suggested by Cooper (l), and this damage leads to leakage of the cell contents, alteration of the internal environment, and death of the bacteria. On the biochemical level, the evidence is still indirect. Lederberg (a), because of observations of creation of spherical forms of Escherichia coli by penicillin in hypertonic solution, has suggested that synthesis of some component of the cell wall is inhibited preferentially by the antibiotic to such an extent that continued growth of the cytoplasm brings about extrusion of the cell contents as a protoplast (3). Park and Strominger (4) favor this hypothesis since it is consistent with their observa- tion that compounds in part similar in composition to cell wall are released into the medium by Staphylococcus aureus in the presence of penicillin, and these compounds may be precursors of the unfinished walls. An alternative hypothesis has been put forth by Prestidge and Pardee (5). It was suggested that penicillin causes formation of an enzyme that attacks the cell membrane (rather than the cell wall) and that disruption of the mem- brane results in escape of the cell contents. A further test of this hy- pothesis has not yet been devised. However, it seemed feasible to test directly the Lederberg hypothesis, and the results of such tests are the subject of the present communication. Measurements were made of cell wall synthesis by E. coli in the presence and the absence of a lethal level of penicillin, and in hypo- and hypertonic media. No significant difference in the rates of synthesis of cell wall, relative to the rates of cytoplasmic synthesis, was observed.

Methods

E. coli strain B, grown at 37” with aeration by swirling, were used in all the experiments. For studies on the effects of penicillin in hypotonic medium, the bacteria were grown in the glucose-salts medium of Davis (6).

* Aided by a grant from the University of California Cancer Research Funds and by a grant from the Rockefeller Foundation.

t Present address, Oklahoma Medical Research Foundation, Oklahoma City, Okla- homa.

435

by guest on July 30, 2018http://w

ww

.jbc.org/D

ownloaded from

43.3 E. COLI CELL WALLS AND PENICILLIN

When the bacteria were to be subsequently exposed to penicillin in t,he presence of sucrose, the glucose-salts medium devised by Hook et al. (7) was employed with the addition of 1 mg. of FeC13 per liter. Magnesium sulfate was added after sterilization to avoid precipitate formation. Tur- bidity, measured with a Klett-Summerson calorimeter and green filter, was used to follow growth. Counts of total bacteria were made with a Petroff-Hausser bacterial counting chamber and phase contrast microscope. Viable counts were made by spreading suitably diluted aliquots on the surface of tryptone-agar plates.

Crystalline ribonuclease (RNase) was prepared in this laboratory; deoxyribonuclease (DNase) was obtained from the Worthington Bio- chemical Corporation. Crystalline potassium penicillin G, obtained from Eli Lilly and Company, was dissolved in water immediately before use. Uniformly labeled CY4-glucose was in part a gift from Dr. W. Z. Hassid and Dr. E. W. Putman (specific activity, 200 PC. per mg.) and in part was purchased from the California Foundation for Biochemical Research (12 pc. per mg.).

Protein and ribonucleic acid (RNA) were determined by the Folin (8) and orcinol (9) methods, respectively. Samples for radioactive counting were evaporated as very thin films on nickel planchets and counted with a thin window automatic gas flow counter (Nuclear Instrument and Chemical Corporation, Chicago). Specific activity is defined as the ratio of counts per minute to mg. of protein. Optical density of the medium was meas- ured at 260 rnp with a Beckman model DU spectrophotometer.

Cell walls were prepared by the method of Salton and Horne (10) after disruption with the Mickle disintegrator. These walls were then treated further to free them from nucleic acids by suspension in 10 ml. of 0.05 M

phosphate buffer, pH 7.5, and 0.01 M MgCl, at 37” and incubation for 5 hours with 20 y of RNase and 20 y of DNase. By repeating this proce- dure, preparations containing less than 1 mg. of RNA per 100 mg. of protein were obtained. The cell walls were finally washed three times with distilled water and were suspended in distilled water for counting and analysis.

To prepare radioautograms of cell walls, the purified walls were hydro- lyzed under conditions described by Salton (11) for identification of amino acids from cell walls. Aliquots of the hydrolysate containing the same total amount of amino acids as estimated by the ninhydrin method (12) were deposited on Whatman No. 1 filter paper, and chromatograms were run in two dimensions (phenol-water, 80:20, followed by n-butanol-acetic acid-water, 52 : 13 : 35). The dried chromatogram was placed next to Eastman “non-screen” x-ray film for several days, and the developed films were examined visually for exposed areas. The original chroma- tograms were subsequently sprayed with ninhydrin to locate the amino acids, etc.

by guest on July 30, 2018http://w

ww

.jbc.org/D

ownloaded from

R. E. TRUCCO AND A. I). PARDEE 437

Results

Experiments in Hypotonic Medium-These experiments were designed to determine the relative amounts of cell wall and cytoplasm synthesized by E. coli in the presence and the absence of penicillin. The conditions were similar to those used in previous studies from this laboratory, except that glucose was used in place of glycerol as a carbon source. In this medium 150 y per ml. of penicillin killed over 99 per cent of the bacteria in 1 hour. Lower concentrations were also lethal; in the same period 15 y per ml. killed over 90 per cent and 5 y per ml. killed 50 per cent of the bacteria. At 2 y per ml. a bacteriostatic action for 2 hours was noted, and a large proportion of long forms of the bacteria was observed. The relatively high concentration of 150 y per ml. was used in the following experiments in order to determine whether a measurable inhibition of cell wall synthesis occurred when penicillin rapidly inactivated (5) the bacteria in hypotonic medium.

To measure incorporation of C14-glucose into the components of E. coli, the bacteria were grown from IO8 to about 1.5 X log cells per ml. in four 1 liter Florence flasks, each containing 435 ml. of culture. The bacteria were centrifuged and again suspended in the same volume of fresh, warm medium lacking glucose (initial turbidity, 109). Glucose at a final con- centration of 1.75 mg. per ml. and 18 PC. of CY4-glucose were then added to each flask. Immediately afterwards, 150 y per ml. of Genicillin were added to two of the flasks, and all four flasks were swirled for 30 minutes at 37”. Subsequent results indicated that the glucose was completely utilized and growth stopped at about 20 minutes. Duplicate flasks were combined and put on ice. The cultures were centrifuged, and the pellets were washed three times with cold distilled water. Aliquots of the washed cells and media were kept for protein and radioactivity determinations. The bacteria were treated to obtain cell walls, as described under “Methods.” Four fractions were separated from the broken cells: (a) the material, mainly unbroken cells, deposited by initial centrifugation at 3000 r.p.m. (1000 X g) in 10 minutes; (b) the cytoplasm, obtained as material not sedimented at 10,000 r.p.m. (11,000 X g) in 30 minutes; (c) the cell walls, sedimented between 3000 and 10,000 r.p.m. and further purified as described above; and (d) the combined washings of the cell wall fraction. Protein was determined after trichloroacetic acid (TCA) precipitation, and radioactivity was measured without further treatment. The results from one of two such experiments are shown in Table I. It is seen from the specific activities that there was no specific inhibition of cell wall synthesis by penicillin, within an experimental error of about 10 per cent. The second experiment gave similar results, but the recovery of material was poorer.

by guest on July 30, 2018http://w

ww

.jbc.org/D

ownloaded from

438 E. COLI CELL WALLS AND PENICILLIN

Portions (1.5 mg.) of the preparations from the above experiment were hydrolyzed, and 0.52 mg. was applied to paper for chromatography and radioautography, as described under “Methods” (Fig. 1). The expected pattern of ninhydrin-positive spots was found, and similar radioactivity was associated with each major spot in the radioautograms from walls of control and penicillin-treated bacteria (as judged by visual inspection). Rut several faint, ninhydrin-negative spots were found in the control

TABLE I

Incorporation oj 0.Glucose into Components of E. coli in Hypotonic Medium

At 30 min.

Klett reading.. Viable cells X 10-r per ml.. Optical density of medium.. Radioactivity in total culture X lo+

c.p.m..

Fractions

Cells (washed) ........................ Deposited at 3000 r.p.m ................ Cytoplasm. ............................ Combined washings .................... Cell wall. ............................

Sum. . .

.

__ 1

_

3 -

Control culture Penicillin culture

131 116 227 58

0.165 0.367

6.16 , I

5.96

Pro- ;z& az;i$cX Protein,’ kin,* mg.

/I I

10-s 10’ WT. c.p.m. c.p.m.

198.0 6.4 3.2 198.0 28.6 1.0 3.5 34.5 83.0 2.9 3.4 72.5

9.4 0.5 (5.3)t 8.9 8.0 0.2 3.0 9.7

129.0 4.6 3.5 125.6

Ra+- Specific ,$$“;( activity X

10-e 10-4

c.p.m. c.p.m.

6.6 3.3 0.9 2.6 2.3 3.2 0.4 (4.5)? 0.3 2.9

~~

3.9 3.1

* From entire culture. t The values in parentheses are of questionable accuracy owing to difficulty of

complete TCA precipitation.

radioautograms and were absent in the samples from penicillin-treated bacteria. The significance of these is uncertain.

Experiments in Hypertonic Medium-The concept that penicillin specifi- cally inhibits cell wall synthesis is derived from experiments of Lederberg which were performed in a hypertonic medium. Therefore, it seemed especially pertinent to measure cell wall synthesis directly in such a medium. However, the actual medium used by Lederberg was an extremely rich one, so that determinations of small amounts of de novo synthesis were not possible with C14-glucose as a substrate. Instead, the conditions of Hahn and Ciak (13) were employed. These authors utilized a hypertonic synthetic medium and obtained results similar to those of Lederberg in

by guest on July 30, 2018http://w

ww

.jbc.org/D

ownloaded from

R. E. TBUCCO AND A. B. PARDEE 439

that penicillin brought about the production of a series of abnormal forms of E. COG. It is likely that somewhat greater stability can be achieved in other media.

Some consequences of the action of penicillin under these conditions were determined. The bacteria were grown to lo9 cells per ml. in 360 ml. of the medium of Hook et al. (7). Then 100 ml. of sucrose (0.5 M final concentration) were added, and the culture was further incubated with swirling for 30 minutes. Penicillin (30 y per ml. of final concentration)

FIG. 1, Radioautogram of a cell wall acid hydrolysate from penicillin-treated E. co&i.

was added to half of the culture, incubation was continued without agita- tion, and the cultures were handled carefully. Growth was much slower after sucrose was added. Aliquots were removed at once and at hourly intervals for the determinations shown in Fig. 2. By 3 hours after the addition of penicillin, growth had ceased, as shown by the absence of tur- bidity increase or protein synthesis. Essentially all of the bacteria were unable to form colonies on tryptone-agar plates. Leakage of material absorbing at 260 rnp commenced at the time of addition of penicillin and continued for the duration of the experiment. Observations were also made with a Petroff-Hausser counter; the total number of bacteria closely followed the turbidity changes. The number of abnormal forms was also

by guest on July 30, 2018http://w

ww

.jbc.org/D

ownloaded from

440 E. COLI CELL WALLS AND PENICILLIN

noted; after approximately 1.5 hours a few abnormal forms were observed, and these increased to 16 per cent in 2 hours, 54 per cent in 3 hours, and 86 per cent in 4 hours. Most of the forms observed in the first 3 hours showed emerging protrusions, although types with well developed globular portions predominated at later times (see Hahn and Ciak (13) for photo- graphs). Mixtures of shapes were always present. The time selected for subsequent experiments was 3 hours after addition of penicillin, for

o-

-c >lOO- iL k - 8 ;50-

0

/LEAKAGE /-----I

FIG. 2. Changes during growth of E. coli in hypertonic medium in and the absence of penicillin. 0.1). = optical density.

the presence

there was then a definite action on cell morphology, synthetic abilities, leakage, and viability under these hypertonic conditions (but little decrease in bacterial number, unlike later times).

Experiments on the incorporation of Cl4 from uniformly labeled glucose were next performed by using the conditions of the experiment described immediately above and with samples taken at 3 hours. Two such experi- ments were performed and are described together below. The bacteria were grown in two cultures, each consisting of 1440 ml. distributed in four 1 liter Florence flasks, with 0.75 mg. per ml. of glucose (Experiment 1) or 0.35 mg. per ml. of glucose (Experiment 2). When the bacterial density reached 7 X lo8 cells per ml., 0.5 M sucrose (final concentration) was added,

by guest on July 30, 2018http://w

ww

.jbc.org/D

ownloaded from

R. l!J. TRUCCO AND A. B. PARDEl2 441

and growth was continued for 30 minutes. Then 94 PC. of CY4-glucose were added to each culture (Experiment I), or 224 PC. (Experiment 2). 10 ml. of aliquots were removed from each flask, and the four samples from each culture were pooled and used for the initial analyses. Immediately after sampling, penicillin (30 y per ml. of final concentration) was added to one set of four flasks, and both cultures were incubated without shaking for 3 hours. Aliquots were removed as before for analyses, and the bulk of the cultures was placed on ice. Bacteria were removed from the media by centrifugation for 15 minutes in a Spinco preparative refrigerated centri- fuge at 10,000 r.p.m. (11,000 X g). These many centrifugations required 6 hours; the pellets were removed immediately after each centrifugation and suspended in cold water. Cell walls and other fractions were prepared as before, including two treatments with nucleases. Both protein and radioactivity determinations of the fractions were performed on pre- cipitates, made in 10 per cent TCA. For counting, the precipitates were washed three times with 5 per cent TCA resuspended in water, plated for counting, and dried carefully. The results of the two experiments are seen in Table II. As in hypotonic medium, no significant specific inhibi- tion of cell wall synthesis was observed.

Electron micrographs were made of the cell wall preparations, both in order to check purity and to observe consequences of penicillin action, if possible. Suspensions of the walls in distilled water at approximately the same protein concentrations were mixed with known concentrations of polystyrene latex particles. Specimens were prepared by the spray droplet technique (14), so that the ratio of cell walls to polystyrene latex particles could be determined and from this, the number of walls per ml. Repre- sentative sections of micrographs are shown in Fig. 3. Cell walls isolated from the penicillin culture looked very similar in shape and structure to walls from the control, but seemed larger, on the average. No indication of damage or abnormal structure caused by penicillin could be found. The number of mg. of protein per apparently complete cell wall was 2.6 X lo-l1 in the penicillin preparation and 1.6 X lo--” in the control; thus there was 1.6 times more protein per wall after penicillin treatment. Little contaminating material was seen.

An unexpected finding not directly connected to the action of penicillin, but which greatly diminished the uptake of C4-glucose, was that E. coli strain B metabolized sucrose from 0.5 M solution readily. This became apparent when it was found that the bacteria in hypertonic medium took up only about 1 per cent of the anticipated quantity of radioactive glucose. A simple calculation shows that sucrose must have been used extensively for growth (rather than that an impurity diluted the specific activity of the C4-glucose). The extent to which the glucose added to the medium is

by guest on July 30, 2018http://w

ww

.jbc.org/D

ownloaded from

TABLE II

Incorporation of 04-Glucose into Component of E’. coli in Hypertonic Medium -

-

Experiment 1

Penicillin culture

80 47

0 0.460

75

.80 0 73 34 35 81

0.230 0.194 !28 106

47.5

Time, min. .......................... 0 Klett reading ........................ 40 Viable cells X 10e7 per ml ............ 80 Optical density of medium. .......... 0.165 Protein, mg .......................... 122 Added radioactivity X 10m6 c.p.m .... 47.5

Fractions rot&,* WT.

Radio- activity*

x 10-4 C.p.lll.

182.0 49.6 9.3 2.6 7.9 1.8

101.0 33.7 12.4 4.9 14.1 4.5

136.0 44.9

Radio- activity x 10-4 c.p.m.

Specific activity x 10-t C.P.lIl.

27.7 0.31 11.2 0.13

1.9 0.31 15.8 0.32

1.0 (0.26) 3.5 0.30

22.2 0.32

a?$; F;g

C.P.lll. mg.

0.27 90.0 (0.3)t87.4 0.23 6.2 0.33 48.7 0.39 3.8 0.31 11.8

__~

0.33 70.5

Cells Medium (TCA-precipitable) Deposited at 3000 r.p.m.. Cytoplasm. Combined washings.. Cell wall.

Sum.

Experiment 2

Control culture Penicillin culture

Time, min. .......................... 0 Klett reading, ...................... 38 Viable cells X 10-T per ml ............ 75 Optical density of medium. .......... 0.180 Protein, mg .......................... 125 Added radioactivity X 1O-6 c.p.m .... 113.5

80 0 68 31 51 75

0.240 0.165 12 93.5

113.5

80 46

0 0.380

90

Radio- activity

Specific tctivity

x 10-a x 10-4 c.p.n-. C.P.JXl.

Fractions I ‘rotein, mg.

Radio- activity

x 10-4 c.p.m.

Cells ................................ Medium (TCA-precipitable). ......... Deposited at 3000 r.p.m .............. Cytoplasm. .......................... Combined washings. ................. Cell wall, ..........................

155.0 274.0 9.1 5.9

11.3 20.2 86.0 120.0

7.5 11.6 17.8 29.5

Sum. .............................. 122.6 181.3

Sp+fic yv;v; ;ga,

c.p.m. WC.

1.7 77.2 (0.7) 51.6 1.8 7.5 1.4 43.0 1.5 2.6 1.6 12.0

____

1.5 65.1

132.0 41.6 10.7 54.5

0.7 18.5

1.7 0.8 1.4 1.3

(2.7) 1.5

84.4 1.3

* From entire culture. t The values in parentheses are of questionable accuracy owing to difficulty of

complete TCA precipitation.

442

by guest on July 30, 2018http://w

ww

.jbc.org/D

ownloaded from

R. E. TRUCCO AND A. B. PARDEE 443

diluted by the other utilizable carbon source determines the specific activity of the incorporated carbon, relative to that of the original glucose. From such data it was calculated that the medium contained 70 gm. of utilizable sugar per liter, and, since 171 gm. of sucrose were actually added, the sucrose must have been used nearly as effectively as glucose. This strain of E. coli was incapable of growth with dilute sucrose as a sole carbon source.

FIG. 3. Electron micrographs of E. coli cell walls. A, control culture (magnifica- tion 10,500 X); B, penicillin culture (magnification 10,500 X).

DISCUSSION

Penicillin clearly had no specific inhibitory effect on cell wall synthesis in hypotonic medium. The ratio of the specific activity of wall to specific activity of cytoplasm was 0.90 in the penicillin-treated cells, and for the control of normal cells it was 0.88, well within experimental error. Also, the quantities of protein recovered were similar for both samples. The recoveries of about 65 per cent are quite satisfactory, since the experiments were designed to furnish reasonably pure samples of cell walls rather than to obtain complete recoveries of material. The conditions (medium, penicillin concentration, and time of sampling) were selected to reproduce those used in previous studies (5). Samples consisted of bacteria which had been exposed to penicillin for a sufficient time so that the majority (75 per cent) were irreversibly damaged and were leaky, while secondary effects which might have altered the specific activity ratios had not become prominent. It appears, then, that in hypotonic medium penicillin can irreversibly destroy the bacterial barriers with no appreciable gross inhibi- tion of cell wall synthesis during a period in which the increase of cell mass is only about 20 per cent (or surface area 13 per cent). A splitting

by guest on July 30, 2018http://w

ww

.jbc.org/D

ownloaded from

444 E. COLI CELL WALLS AND PENICILLIN

of the surface by increased internal pressure seems unlikely, under these conditions, especially for an organism as resistant to physical mistreatment as E. coli.

It is important to note that the specific activities (counts per mg. of protein) provide a measure of the total material synthesized after addition of C14-glucose, irrespective of losses during purification. This is true be- cause little total protein synthesis occurred, and therefore the specific activity was essentially the ratio of incorporated radioactivity to original protein (proportionate losses of radioactivity and protein during purifica- tion being assumed). Also, when the various fractions are synthesized in proportion, their specific activities should be similar, since their protein cont)ents are each about ‘70 per cent of the dry weight.

The observation that hydrolyzed cell wall preparations of penicillin- treated bacteria revealed only minor deficiencies of newly formed com- ponents, if any, and showed a pattern similar to that of normal cells, rules out the possibility that the radioactivity is taken up into materials such as polysaccharides which are unlike the normal cell wall, but which upon purification are measured as wall. Also, it argues against specific inhibi- tion of a unique wall component, unless that component occurs in small amounts.

Bacteria exposed to penicillin in hypertonic medium produced abnormal forms, including rounded bodies, as described by others (2, 13). However, under these conditions, and at a time when more than 99 per cent of the bacteria were sufficiently damaged to be non-viable upon plating, no signifi- cant difference in specific activity of cell wall and cytoplasm was observed (ratio 0.94, Experiment 1; 1 .I 5, Experiment 2). The preparations from the controls, made from normal cells grown in sucrose, gave a ratio of 0.94, Experiment 1; 1.14, Experiment 2. Conditions of these experiments were again chosen so that penicillin had definitely set up alterations in the great majority of the bacteria, but damage had not progressed so far as to confuse seriously the interpretation of the results (as would be the case at later times when a considerable disappearance of the cells had occurred).

The electron micrographs revealed not only that the material being analyzed as walls had the appearance of the usual preparation (lo), but also that the control and penicillin-treated cell walls were similar in appear- ance, with no evidence of artifacts in the latter that could confuse the analyses. The protein content per countable wall was 1.6 times greater after penicillin treatment, as would be expected, since the antibiotic in- hibited cell division (as shown by direct counts) but not wall synthesis. Scrutiny of the walls did not reveal any unusual formations or cracks or defects in the penicillin-treated samples.

In both of the experiments performed in hypertonic medium, the TCA- insoluble material that leaked from the penicillin-treated bacteria was of a

by guest on July 30, 2018http://w

ww

.jbc.org/D

ownloaded from

R. E. TRUCCO AND A. B. PARDEE 445

specific activity about half that of the cells. This means that much of this material leaked from the cells early in the experiment (also shown by the curve of leakage versus time) and that the leaky cells must have continued to assimilate C14-glucose. These results demonstrate a large (50 per cent) but very gradual leakage, presumably through some minor defects in the cell surface. The odd shaped bacteria do not appear to have the same permeability properties as the original cells. The observations on “proto- plast” formation by yeast after damage to the cell by an added enzyme (15) might well serve as a model for the type of lesion involved.

The results of the present experiments do not explain how penicillin causes a defect in the cell barrier or whether the damage is to the wall or membrnnc. However, the results apparently leave the hypothesis of inhibition of wall synthesis (2, 4, 13) untenable and suggest that alternative mechanisms should be investigated. The former ideas possibly could be modified to fit the present, observations by assuming a highly specialized or localized inhibition of cell wall synthesis by penicillin, resulting in local weakness that is exploited by random strains (rather than by the small growth of the cytoplasm). However, proof for such a scheme is entirely lacking, and it would seem desirable to provide direct evidence.

We are indebted to Mr. Louis Davis for taking the electron micrographs, and to Mrs. Louise Prestidge for assistance with certain experiments.

SUMMARY

1. Direct measurements have been mndc of cell wall and cytoplasm syntheses by Escherichia coli grown on glucose-C14 in the presence and absence of penicillin, in hypotonic and hypertonic media, by determination of the incorporated radioactivity of isolated cell walls and cytoplasm.

2. The data showed no inhibition by penicillin of cell wall formation relative to cytoplasmic syntheses. Hydrolysis and chromatography of the wall preparations did not reveal any major differences of radioactive components.

3. Electron micrographs of cell wall preparations from E. coli rendered non-viable by penicillin appeared the same as walls made from normal bacteria. The ratio of gm. of protein per number of walls was 1.6 times higher in the former preparations, indicating continued wall synthesis without cell division.

4. Penicillin does not selectively halt all cell wall synthesis. It probably causes, directly or indirectly, some minute lesion in the wall or mem- brane through which the cytoplasm escapes. In spite of the leakage of approximately half of the cell protein contents, protein synthesis con- tinues.

by guest on July 30, 2018http://w

ww

.jbc.org/D

ownloaded from

446 E. COLI CELL WALLS AND PENICILLIN

BIBLIOGRAPHY

1. Cooper, I’. D., Bact. Rev., 20, 28 (1956). 2. Lederberg, J., Proc. Nat. A cad. SC., 42, 574 (1956). 3. Weibull, C., in Spooner, E. T. C., and Stocker, B.A. D., Bacterial anatomy, Cam-

bridge, 111 (1956). 4. Park, J. T., and Strominger, J. I,., Science, 126, 99 (1957). 5. Prest,idge, L. S., and Pardee, A. B., J. Bact., 74, 49 (1957). 6. Lederberg, J., in Gerard, R. W., Methods in medical research, Chicago, 3,5 (1950). 7. Hook, A. E., Beard, D., Taylor, A. R., Sharp, I>. G., and Beard, J. W., J. Biol.

Chem., 166, 241 (1946). 8. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J., J. Biol. Chem.,

193, 265 (1951). 9. Schneider, W. C., J. Biol. Chem., 161, 293 (1945).

10. Salton, M. R. J., and Horne, R. W., Biochim. et biophys. acta, 7, 177 (1951). 11. Salton, M. R. J., Biochim. et biophys. acta, 10, 512 (1953). 12. Moore, S., and Stein, W. H., J. Biol. Chem., 211, 907 (1954). 13. Hahn, F. E., and Ciak, J., Science, 126, 119 (1957). 14. Backus, R. C., and Williams, R. C., J. Appl. Phys., 21, 11 (1950). 15. I:ddy, A. A., and Williamson, D. H., Nature, 179, 1252 (1957).

by guest on July 30, 2018http://w

ww

.jbc.org/D

ownloaded from

Raul E. Trucco and Arthur B. PardeePENICILLIN

CELL WALLS IN THE PRESENCE OF SYNTHESIS OF ESCHERICHIA COLI

1958, 230:435-446.J. Biol. Chem. 

  http://www.jbc.org/content/230/1/435.citation

Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

alerts to choose from all of JBC's e-mailClick here

  tml#ref-list-1

http://www.jbc.org/content/230/1/435.citation.full.haccessed free atThis article cites 0 references, 0 of which can be

by guest on July 30, 2018http://w

ww

.jbc.org/D

ownloaded from