RELATIONS BETWEEN BACTERIAL CELL WALL SYNTHESIS, GROWTH PHASE, AND AUTOLYSIS*

BY GERALD D. SHOCKMAN, JOSEPH J. KOLB, AND GERRIT TOENNIES

(From the Lankenau Hospital Research Institute and the Institute for Cancer Research, Philadelphia, Pennsylvania)

(Received for publication, August 15, 1957)

When microorganisms are allowed to grow under conditions whereby a single amino acid is the growth-limiting factor, the extent of the ensuing growth response is a measure of the amount of the particular amino acid (microbiological assay). Our previous studies, in which we used Strep- tococcus faecalis 9790, revealed characteristic differences in the patterns of the growth responses evoked by different growth-limiting amino acids. For instance, in the case of valine limitation the increase in optical density ceases after 17 hours of incubation, although in the analogous case of threonine it continues for several days, and in the case of lysine limitation a brief period of growth is followed by autolysis in less than 24 hours (3, 4). Further investigation (5) showed that the end of the exponential growth phase (5 to 6 hours of growth) is marked by the depletion of the limiting amino acid from the medium and its complete incorporation into the cells, and that this point is followed by further slow increases in optical density of the order of 50 per cent for valine and 100 per cent for threonine, or, in the case of lysine, by immediate lysis. The data to be presented now partially clarify these phenomena; they indicate that termination of the exponential growth phase and termination of the assimilation of amino acids required for cytoplasmic synthesis are normally followed by a stage of metabolism during which a large increase in the amount of cell wall sub- stance takes place. Recent investigations (6-11) have shown that rela- tively few amino acids are components of streptococcal cell walls and that a prominent member of this group is lysine. Our findings suggest that, when exponential growth is terminated by depletion of lysine, the absence of this amino acid for postexponential cell wall synthesis is causally related to the ensuing autolysis.

Methods

Growth Conditions-S. faecalis (ATCC 9790) was grown on a highly buffered medium (5). In order to increase the growt’h yield of the medium,

* This work was done under a research contract with the Office of Naval Research. It was also aided by an institutional grant of the American Cancer Society. The results have been presented in part at the 1957 meetings of the American Society of Biological Chemists (1) and the Society of American Bacteriologists (2).

961

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962 CELL WALL SYNTHESIS AND AUTOLYSIS

the previously employed quantities of adenine, guanine, and uracil were tripled and the quantity of pantothenic acid was doubled. The previously used nn-amino acids were replaced by the L compounds, with the exception of alanine. Only minor alterations of the amino acid response curves resulted from this change. In the improved culture medium duplication at constant rate (the exponential growth phase) extends to an adjusted optical density (AOD, adjusted to agree with Beer’s law (12)). of 3000, measured on a Coleman model 14 spectrophotometer in 19 X 150 mm. tubes at 675 rnp. This is equivalent to a population of about 3 X log cells per ml. or 1.2 gm. of cellular dry weight per liter. Since growth to these high densities involves the formation of lactic acid beyond the buffering capacity of the 0.3 M sodium phosphate buffer, a procedure of continuous neutralization with NaOH, with use of a Beckman automatic titrator, was employed to maintain the pH at 6.5. The glucose level was maintained above 1 per cent by periodic additions, based on the amount of alkali consumed. Alkali consumption was translated into glucose utili- zation on the basis of the formation of 2 moles of lactic acid from 1 mole of glucose.

6 liter batches were grown at 38”, in a water bath, and density measure- ments were made by removing small samples and diluting them with deficient culture medium to suitable optical densities. The cultures were inoculated with exponentially growing cells (5), at a level that would give an initial AOD of 1 (about lo6 cells per ml.). Since, under our conditions, exponential phase cells seem to be of constant composition (5), it was only necessary to be certain that such cultures were harvested at a point that was well within the exponential growth phase. “Non-limited” stationary phase cells were harvested from non-limited medium at a point at which the optical density had leveled off despite continuous neutralization and maintenance of an adequate supply of glucose. This occurred at about 5 hours after, and at a level of optical density about 50 per cent higher than that at the end of the exponential phase. Cessation of growth of these stationary cells, “non-limited” by amino acids or glucose, may be due to depletion of some other nutritional factor or factors or to accumulation of lactate. These cultures reached a density of about 2 gm. per liter. Stationary phase cells whose growth had ceased because of the depletion of valine or lysine were also grown at constant pH and harvested at growth levels of about 1 gm. per liter. Another crop of stationary phase, valine- limited cells was grown without neutralization, but was harvested at about one-half the growth density of the neutralized cultures (final pH 5.4). Histidine-limited cells, in smaller quantities, were similarly grown.

Harvesting-Growth was stopped at the desired level by pouring the whole culture onto an equal weight of ice, and the cells were harvested in

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G. D. SHOCKMAN, J. J. KOLB, AND G. TOENNIES 963

the cold on a Westphalia Universal centrifuge (model LWA 205, Centrico, Inc., 75 West Forest Avenue, Englewood, New Jersey). The cells were suspended twice in ice-cold distilled water equal to one-half the volume of the culture, and separated again on the Westphalia centrifuge. During this entire process the temperature was kept near zero by maintaining ice (distilled water) in the cell suspension. Four or five additional washings were carried out immediately in a refrigerated centrifuge with much smaller volumes of ice water, and during this procedure small amounts of foreign matter, picked up during growth and centrifugation, were removed by discarding the black material that settled to the bottom of the centri- fuge tubes. The harvesting and washing operations took a total of 7 to 9 hours; they should not be interrupted, since upon continued suspension

TABLE I

Bacterial Preparations

Prepa- ratwn

NO.

Y-

Growth phase

Exponential I‘ “

Stationary, non-limited “ ‘I ‘I valine-limited “ lysine-limited ‘I valine-limited

Continuous neutralization ‘I I‘ “ ‘I “ ‘I I‘ “ “ “ “ “

No neutralization

T-

Yield Nitrogen

sm. per cent 3.4 13.3 f 0.1 4.8 13.1 f 0.3 3.9 13.3 zlz 0.2 3.9 11.0 f 0.1 7.1 11.0 f 0.1 3.1 10.7 f 0.2 2.5 11.9 f 0.2 3.6 11.5 f 0.2

in ice-cold water the cells begin to release significant quantities of nin- hydrin-reactive, and 260 rnp-absorbing, materials.’ The final products were lyophilized and stored in vacua. Their net yield and nitrogen con- tent are listed in Table I.

Disruption of Cells-The lyophilized cells were quantitatively separated into soluble (protoplasm) and insoluble (cell wall) fractions by shaking with glass beads and centrifugation at 20,000 X g, all carried out in the cold. The apparatus and procedures have been previously described (14). A clean separation seems to be achieved, as indicated by the absence of protoplasmic constituents in the cell wall preparations and the lack of cell wall constituents in the soluble fraction.

Electron Microscopy-The purity of the cell wall preparations was confirmed by electron microscopy of chromium-shadowed preparations.

1 Stegemann, H., and Toennies, G., unpublished studies; cf. also Salton (13).

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964 CELL WALL SYNTHESIS AND AUTOLYSIS

There was little evidence of cytoplasmic contamination (electron dense material) in any of the well washed preparations.

Analytical Methods-Total nitrogen was estimated by a semimicro- modification of the nesslerization method of Miller and Miller (15). For the determination of bacterial dry weights, aliquots of cultures were thor- oughly washed with the aid of high speed centrifugation and brought to constant weight at 105“. Amino acids were assayed microbiologically with S. faecalis (3, 5). Rhamnose was estimated by the method of Dische and Shettles (16), and hexosamine by a modification (17) of the Elson and Morgan reaction (18).

Paper Chromatography of Cell Wall Hydrolysates-5 to 10 mg. samples of cell wall were hydrolyzed in 0.5 to 1.0 ml. of 3 N HCl at 120” for 8 hours in evacuated sealed tubes. The tubes were cooled to room temperature and carefully opened (19). The tops of the tubes were cut off and the hydrolysates were evaporated to dryness in 2racuo over NaOH and HzS04. The evaporated residues were taken up in 0.2 ml. of water per mg. of cell wall. Excellent separation of the amino acids in the cell wall hydrolysate was obtained on ascending one-dimensional paper chromatograms run on Schleicher and Schiill No. 507 paper, a completely miscible solvent system of water, glacial acetic acid, and n-butanol (30: 15.5: 55 by weight) being used. If the solvent is mixed in the order indicated and held below 20”, it will keep for several days without separation. The solvent front ad- vanced 25 to 30 cm. in 17 to 20 hours at 25”. After drying in air overnight, the chromatograms were dipped in a ninhydrin solution (0.1 per cent ninhydrin in 95 per cent acetone) (20), air-dried, and heated to 68” for 10 minutes for color development. The spots were identified by ap- propriate reference standards and confirmation runs with several other solvent systems (see Table VII).

Results

Relations between Optical Density, Dry Weight, and Nitrogen Content during Growth-Our previous experiments (4, 5) indicated that the relative optical density and the amino acid composition of S. faecalis remain constant throughout the exponential phase of growth, and that most of the essential amino acids are utilized and retained without metabolic losses. Therefore, the optical density level at which a given quantity of an essential amino acid has been removed from the medium could be determined from the rela- tive content of the particular amino acid in exponential phase bacteria (5). It was found that this optical density level, termed “depletion point,” coincides closely with the end of the exponential phase.

Table II summarizes data on the optical density, dry weight, and nitrogen content of cells at different stages of growth, when cultured under conditions

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of histidine (A) and valine (B) restriction. It is apparent that the post- exponential increase in optical density is accompanied by marked increases in dry weight but not by significant gains in cellular nitrogen. In these experiments optical density seems to increase far more than the dry weight.

TABLE II Optical Density, BzGcterial Dry Weight, and Nitrogen Content during Growth

(Alcohol-Washed Cells)

Growth phase

Change f;~;tdepletion

Age Density Celteyg\tdry Cellular nmogen . Dry DensltY weight

I I Nitro- &Ten

Restriction A, growth limited by L-histidine (13.5 X 10e6 M)

Exponential phase at deple- tion pointt.. . . .

Phase of decreasing growth rate. .

Stationary phase. . . Declining “

hrs.* AOD y per ml. y per ml.

5.5

9 17 44

500

585 775 590

190 25.6

223 26.6 232 26.6 192 21.4

er cent

+17 +55 +I8

+17 +4 +22 +4

+1 -16

Restriction B, growth limited by L-valine (103 X lo+ M)

Exponential phase at deple- tion pointt.. 5.5

Phase of decreasing growth rate.. . . . 9

Stationary phase. . . . 17 Declining “ . . . . 40

+14 +3 +15 -4 +8 -12

* Hours from inoculation at AOD 1; division time during exponential phase 0.6 hour.

t Calculated from the assimilation factors (millimicromoles per ml. per AOD) 0.227 for histidine and 0.133 for valine; these differ somewhat from those reported previously (5), probably because of the omission of n-amino acids from the medium.

In order to verify these findings and to study the fate of cellular amino acids during the postexponential optical density increase, new crops of exponential and valine-limited 17 hour cells were grown and analyzed. In these preparations only cold water was used for washing. The results are shown in Table III. Here, as in the experiments of Table II, the ex- ponential cells were harvested before the depletion point. Columns 1 and 3, Table III, show the composition of the isolated bacterial substance, whereas Columns 2, 4, 5, and 6 refer to the total quantities contained in the cells present at the growth levels indicated by the AOD values of the

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966 CELL WALL SYNTHESIS AND AUTOLYSIS

first line. The postexponential increase in dry weight is substantially larger than in the comparable experiment in Table II, and total nitrogen shows a gain of 10 per cent, which is about one-quarter of the density and weight increases. We attribute the lower values in Table II to the extrac- tion of various cellular components (free amino acids, nucleic acid deriva- tives) by washing with alcohol (21).

TABLE III Analysis of Exponential and Valine-Limited Cells (Water Washed)

-

-

I <

Optical density

Dry weight Total nitrogen Amino acids1

Histidine Arginine Threonine Isoleucine Valine Leucine Lysine

Exponential cells

(1)

%action of Iry weight

9er cent

-

.-

(

(2) (3) (4)

:oncentra- tion

-

.-

(

.-

hc tion of Iry weight

:oncentra- tion

(5) (6)

Concentration

13.2

AOD

775* mg. per 1. 310 41

per Gent

10.5

AOD

11OOt mg. #er 1.

430 45

AOD

325 mg. per 1.

+120 +4

1.18 3.7 0.94 4.0 +0.3 2.71 8.4 1.98 8.5 +0.1 2.82 8.7 2.11 9.1 +0.4 3.52 10.9 2.57 11.1 +0.2 3.92 12.1 2.72 11.7 -0.4 4.11 12.7 2.96 12.7 0 4.9 15.2 4.8 20.6 +5.4

Change (from exponen- tial cells at depletion oint to stationary cells) P’

.-

-

per cent

+42

+39 +10

+8 +1 +5 +a -3

0 +36

* Depletion point. t Harvesting point. $ The average precision of these values, in terms of the average deviation of repli-

cates from the mean, is about f0.06.

Of the six essential amino acids examined, all but lysine show only minor changes after the end of exponential growth, but bacterial lysine increased nearly as much as optical density and dry weight. These observations suggested to us that the postexponential increase in cell substance involves formation of carbohydrate associated with the amino acid lysine. It be- came apparent that the cell wall substance of X. fueculis has these attributes (6-9).

Rhamnose and Hexosamine Content of Cells-Both rhamnose and hexos- amine have been shown to be important constituents of the cell wall of streptococci (6-11, 22, 23). If the postexponential weight increases were the result of the formation of additional cell wall substance, this should

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G. D. SHOCKMAN, J. J. KOLB, AND G. TOENNIES 967

be reflected in corresponding increases in cellular rhamnose and hexosa- mine contents. According to the data of Table IV, both the “non-limited” and the valine-limited stationary cells, but apparently not the lysine- limited cells, show such increases, of the order of 30 to 40 per cent.

TABLE IV Rhamnose and Hexosamine Content of Different Cell Types

Stationary phase cells Exponential phase cells

“Non-limited” Valine-limited Lysine-limited

Crop No. “$$- Hexosamine Crop No. Rn2sF- “$Tea- Crop No. Rhamnose Crop No. “;&y- ____-- --

per cent per cent per Gent per cent per Gent per cent 1 3.6 3.9 4 4.6 6 5.0 7 3.7 2 3.5 5 4.5 5.2 8 5.1 3 3.5

Crop numbers as in Table I. The precision of these determinations, in terms of the average deviation of replicates from the mean, is ~0.1 to 0.2.

100 0

a---- ----- --- ---@

EXPONENTIAL CELLS

STATIONARY CELLS

I I I I I IO 20 30 40

MINUTES

FIG. 1. Disruption of exponential and stationary cells. Abscissa, time of shak- ing; ordinate, soluble part of total cellular nitrogen.

Study of Cell Wall Substance-In order to verify the presumed increase in cell wall substance, the mechanical disruption (14) of both exponential and stationary phase cells was studied. Fig. 1 shows that the share of the total nitrogen which remains insoluble is nearly twice as large in the valine- limited stationary cells as in the exponential ones, but that the time-course

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968 CELL WALL SYNTHESIS AND AUTOLYSIS

of making bacterial substance soluble is essentially the same in both kinds of cells. Further confirmation was obtained when additional cell prepara- tions were tested by shaking with glass beads for 20 minutes (14), with the results shown in Table V. The nitrogen figures for the insoluble fractions

TABLE V Mechanical Disruption of Cells

Cell crop’

Exponential phase 1 2 2 3

Average. . . . . . . . . . Average by difference. . . .

Stationary phase Valine-limited

6 8 8

“Non-limited’! 4 5 5

Average. . . . . . . . . . . . . . . Average by difference. . . . . . .

Lysine-limited 7

* Numbers as in Table I.

-

--

-_

-

Share of cellular nitrogen in

Soluble fraction Insoluble fraction

per cenf

88 89 88 90

89

78 26.5 79 22 78 20

80 80 81

21 21.5

_- per cent

15 11.5

16.5 _-

14 11

_-

. -

79 22 21

81 17

- :

-

--

.-

.-

-

Share of absorption at 260 nyl in soluble

fraction

per cent

94 96 97 95

96

90 92

91 89 92

91

94

are erratic but tend to confirm the more accurate values obtained on the soluble fractions. The absorption curve of the wall material was obtained directly on cell wall suspensions, and also calculated from the difference of the readings of the original cell suspension and the supernatant liquid obtained after disruption (14). The same type of smooth scatter curve as reported by Salton and Horne (24), without any indication of a peak at 260 rnp, was obtained by either procedure. Absence of an absorption peak at 260 rnp in the wall substance indicates that all of the cellular nucleic acid

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G. D. SHOCKMAN, J. J. KOLB, AND G. TOENNIES 969

substances are in the soluble fraction. The values of 89 to 97 per cent in the last column of Table V result from comparisons between the extinc- tions of the clear soluble fractions and the total suspensions after disrup- tion, and therefore must be considered as minimal values because of non- specific absorption in the suspension (25).

TABLE VI

Data Indicating That Growth after Depletion Point Is Due to Cell Wall Formation

Whole cell substance Optical density, AOD Weight, mg. per 1. Nitrogen, mg. per 1. Lysine, mg. per 1. Rhamnose, mg. per 1. Hexosamine, mg. per I.

Wall substance Nitrogen

As fraction of wall, per cent

As fraction of whole cell N, per cent

As amount, mg. per 1. Weight

As fraction of whole cell (calcu- lated), per cent

As amount, mg. per 1. Rhamnose

As fraction of wall, per cent As amount, mg. per 1.

Exponential ~11s at deple-

tion point

V

775 1100 310 430

41 45 15.2 20.6 10.8 21.9 12.1 22.4*

5.7 11

5.8 21

4.5 9.5

25.5 38

79 164

14 14 11.5 22

aline-limited stationary

cells

I

Gain (of valine-limited stationary cells over exponential cells at

depletion point)

Amount

mg. per 1.

120 4 5.4

11.1 (10.3)

5

85

10.5

Fraction

per cent

42 39 10 36

103

(85)

111

108

91

* Value from “non-limited” stationary cells (see Table IV).

The resulting wall preparations were analyzed for nitrogen and rhamnose content. For technical reasons their weight cannot easily be determined directly, but it can be calculated from the cellular nitrogen content (Table III), the insoluble share of the cellular nitrogen (Table V), and the nitrogen content of the wall substance. Table VI is assembled from the resulting data and, for comparison, relevant results of the analysis of whole cell substance. In addition, the wall preparations were investigated by chromatography of hydrolysates; the results are described in Fig. 2 and Table VII. It should be noted that the major cell wall amino acids of this

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970 CELL WALL SYNTHESIS AND AUTOLYSIS

strain of S. faecalis include aspartic acid, in addition to glutamic acid, alanine, and lysine as found by other investigators (6-11). All preparations examined showed evidence for the presence of trace amounts of other amino acids. The consistency of their appearance in our preparations and in those of others (6-11) may indicate that they are not merely contami- nants.

The amount of additional weight, nitrogen, rhamnose, and hexosamine found in the postexponential cells (Table VI) and the similarity of the increases, whether obtained from the analysis of whole cell substance or

FIG. 2. Paper chromatograms of cell wall hydrolysates. 15 W, cell wall from “non-limited” stationary phase cells; 16 W, cell wall from exponential phase cells; ALL, mixture of lysine, glutamic acid, aspartic acid, and alanine; NO ALA, same without alanine; ASP, without aspartic acid; GLU, without glutamic acid; LYS, without lysine; G, with glucosamine. Solvent, water-acetic acid-n-butanol.

wall, indicate that a large quantity of new wall substance has been formed. The similarity of the two types of wall preparation in nitrogen and rham- nose content, and the resemblances of the percentage gains of wall nitro- gen, wall weight, and wall rhamnose, together with the chromatographic results, suggest that the postexponential formation of cell wall substance is not accompanied by major changes in its composition.

Autolytic Properties of Exponential Cells in Comparison with Lysine- and Valine-Limited Postexponential Cells-If cellular lysis upon lysine deple- tion is a consequence of the limited amount of cell wall substance possessed by exponential phase cells (see under “Discussion”), the lysis phenomenon should not be confined to cells which have depleted the medium of lysine, but upon transfer to a lysine-free medium it should be exhibited by ex- ponential cells, whether or not they were grown in a lysine-limited medium.

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G. D. SHOCKMAN, J. J. KOLB, AND G. TOENNIES 971

Fig. 3 summarizes the results of a series of experiments in which cells grown under specified conditions were transferred to other media. The exponential cells were grown in a non-limited medium to approximately AOD 600. In the growth of the lysine- and valine-limited cells the levels

TABLE VII Chromatographic Identijkation of Cell Wall Components

The solvents used were as follows: (a) phenol-isopropanol-water (70:5:25 by weight), (b) water-acetic acid-n-butanol (30:15.5:55 by weight), (c) methyl ethyl ke- tone-tert-butanol-water-formic acid (8:8:9:1 by volume), (d) methyl ethyl ketone- tert-butanol-water-diethylamine (8:8: 9: 1 by volume). One-dimensional runs in solvents (a) and (5) gave the following Rp values:

Preparation

Alanine. . . . 0.20 0.22 0.22 0.24 0.25 0.25 Aspartic acid. 0.05 0.06 0.07 0.12 0.14 0.15 Glutamic “ 0.11 0.10 0.10 0.19 0.20 0.21 Lysine. . 0.06 0.06 0.07 0.07 0.07 0.08 Glucosamine 0.08 0.10 0.10 0.10 0.12 0.12

-

Solvent (a) I

Solvent (a)

Markers 1.5 W 16 W Markers 15 w 16 W

The markers were identified by single omissions from the mixture of four amino acids and glucosamine. Preparation 15 W represents the walls of stationary phase cells, 16 W those of exponential phase cells. Glucosamine was further identified by the lack of a ninhydrin-reacting spot after preheating the paper to 120” for 20 minutes, and by one-dimensional chromatography in solvent (c), where its RF was 0.74, compared with 0.73 for both cell wall hydrolysates. Further evidence for the identity of the five principal constituents of the cell wall hydrolysates was obtained by two-dimensional small scale paper chromatography (26, 27) with two solvent pairs, viz. (A), solvent (b) in the first and solvent (d) in the second dimension; and (B), solvent (d) in the first and solvent (c) in the second dimension. In solvent pair (A) color differentiation of the ninhydrin spots aids in identification. The same color differentiation can be obtained with the other solvents by dipping the papers in a 5 per cent solution of cyclohexylamine or dicyclohexylamine in acetone (28) before ninhydrin color development. For the amino acids listed above this is par- ticularly valuable for the identification of aspartic acid (light blue).

of the limiting amino acid used corresponded to depletion points of AOD 600 to 700; the valine-limited cells were harvested after 20 hours of growth and the lysine-limited ones about 2 hours past the depletion point when the optical density had declined by 15 to 20 per cent. The cells were centri- fuged in the cold, washed once, and then resuspended in sterile water for redistribution to fresh valine- or lysine-limited medium. The data show that in regard to lysis in lysine-free medium the exponential cells and the lysine-limited cells resemble one another and are unlike the valine-limited

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972 CELL WALL SYNTHESIS AND AUTOLYSIS

cells. They differ, however, in their response to a valine-free medium. In the exponential cells a density increase of nearly 50 per cent occurs which we can now interpret as being due to cell wall synthesis (which does not depend on valine). However, the cells harvested some time after lysine depletion show a net decrease of nearly 30 per cent in the valine-free medium. Inasmuch as these cells decrease by 80 per cent in a lysine-free medium, they may respond to the valine-free medium by cell wall synthesis, but this response may be only a partial one because some of the cells are already irreversibly committed to lysis.

EXPONENTIAL CELLS

150

100

\

50

\

LYSINE LIMITED VALINE LIMITED CELLS CELLS

HOURS

FIG. 3. Behavior of different cell types upon transfer to different media. A, valine-free medium; 0, lysine-free medium. The ordinate represents AOD in per cent of the initial value, the abscissa hours of incubation at 38”.

In order to explore this question further, a series of “lysinelimited” tubes was inoculated and incubated. At different periods, as lysis was beginning and as it progressed, individual tubes were supplemented with an additional portion of lysine which by itself would be capable of pro- ducing a maximal growth response of AOD 700. A net response of this magnitude should be obtained if the cells which have not yet lysed could be salvaged by a new supply of lysine. The results, presented schemati- cally in Fig. 4, revealed a steady decrease in the response potential of the remaining cells with progressive lysis, as shown by an increasing lag period. Furthermore, in the earliest stage of lysis the net response to supplemen- tation was of the expected magnitude, decreasing rapidly as lysis pro- gressed; as lysis approaches completion the net response tends to go up again, while the lag period continues to lengthen, presumably because of the

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G. D. SHOCKMAN, J. J. KOLB, AND G. TOENNIES 973

dwindling number of responsive cells. The experiments of Figs. 3 and 4 differ in that the response of lysine-limited cells to lysine takes place in a valine-free medium in the one case and in the presence of valine in the other. In the former only cell wall formation, in the presence of valine exponential growth, is possible. The observations may reflect a rapid increase, after onset of lysine deficiency, in the proportion of cells irre- versibly committed to lysis. The process is being studied further.

An aspect of the lysis phenomenon which should be mentioned here is the occurrence of a lysis-resistant variant which was obtained by subculturing lysine-limited growth tubes after autolysis. After several passages,

1200-

I I I I I I I I I I I I 2 4 6 8 IO 12

HOURS FIG. 4. Response of cells which are lysing because of lysine depletion, to replenish-

ment of the medium with lysine. In different tubes identical amounts of new lysine were added when the optical density had reached the levels indicated by arrows; see the text.

characterized by decreasing rates of autolysis, a strain results which shows essentially no lysis. The properties of this variant, in terms of cell wall proportion and composition, remain to be studied.

DISCUSSION

The evidence presented indicates that the increase in optical density after the end of the exponential growth phase and after an essential amino acid is depleted from the culture medium is not due to balanced cellular growth but can largely be attributed to an increase in the amount of cell wall present per unit weight of cells. The substantiating data are sum- marized in Table VI. It may be noted that wall nitrogen, wall weight, and wall rhamnose all increase about 100 per cent. Analyses of the soluble (non-wall) substance showed practically no rhamnose. Reflecting

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974 CELL WALL SYNTHESIS AND AUTOLYSIS

the specificity of rhamnose as a component of the wall, its percentage increase, obtained from analysis of the whole cell substance, resembles the increase obtained from the analysis of wall substance. The net gain in lysine (5.4 mg.) calculated as per cent of the net gain in wall substance (85 mg.) gives a value of 6.3 per cent for the lysine content of the wall. Ikawa and Snell (11) reported approximately 4.7 per cent lysine for S. fczecalis wall substance, but their strain apparently differs from ours in that aspartic acid is not a major wall component. The net gain in cell wall nitrogen (5 mg.) seems to account fully for the gain in total cellular nitrogen (4 mg.), but it should not be overlooked that the estimated weight of the new wall substance (85 mg.) accounts only for about 70 per cent of the gain in whole cell substance. The cause of this discrepancy remains to be clarified.

Although our data (Fig. 2, Tables VI and VII) are consistent in sug- gesting that the composition of the cell wall remains the same during its postexponential increase, minor changes such as those involved in anti- genicity are certainly not ruled out.

According to the data of Table VI, dry weight and lysine increase ap- proximately in proportion to the increase in AOD, but the increase in nitrogen is much less. Accordingly, AOD, but not nitrogen, may be used as an approximate measure of the weight of cells per ml. of culture, at least through this part of the growth cycle.

Autolysis in the absence of an adequate supply of lysine seems to be intimately related to the limited amount of cell wall substance possessed by exponential and lysine-limited postexponential cells. The observed doubling of the cell wall substance, with little increase in cytoplasmic substance, in the conversion of exponential cells to valine-limited stationary cells, may reflect the presence of more cell wall per surface area (i.e., a thicker or denser wall), but it could also reflect a difference in cell size. Halving of the cell diameter would double the surface to volume ratio, and the proportion of cell wall substance would double even if the amount per surface area remained the same. However, since electron microscopy of either the whole cells or the cell walls showed no major difference in the size of exponential and postexponential cells, the postexponential cells would seem to have more wall substance per area. It may well be that during the exponential growth phase the formation of new cell wall lags slightly behind cell division and that the weakest point is at the place of incipient division. Evidences for this are the hemispherical shells ob- tained by Mitchell and Moyle from autolyzed cultures (29) and the cy- tological studies of Bisset (30). When in exponential cells further wall synthesis is halted nutritionally (e.g. by lysine deficiency) or by a specific inhibitor of cell wall synthesis (e.g. penicillin) (31, 32), one apparently

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G. D. SHOCKMAN, J. J. KOLB, AND G. TOENNIES 975

obtains either the release of protoplasts or autolysis, depending upon the osmotic environment. Treatment of exponentially growing S. faecalis cells with penicillin causes autolysis which resembles that obtained under conditions of lysine limitation.2

Autolytic phenomena have recently been studied by Mitchell and Moyle (29) and by Meadow, Hoare, and Work (33). The latter observed lysis upon depletion of diaminopimelic acid which is essential for cell wall synthesis in the Escherichia wli mutants studied. The former relate the production of protoplasts to autolysis. They found that rapidly growing cells were much more prone to the development of osmotic fragility than stationary phase cells. These same authors carefully point out that, unless practically no cell wall material is present, care should be taken in calling osmotically fragile bodies “protoplasts.”

Since glutamic acid and alanine resemble lysine in being essential for growth and at the same time being components of the cell wall, their restriction may also be expected to give rise to lysis phenomena. Experi- ments with L-glutamic acid showed no evidence of lysis, but in the case of low levels of n-alanine (vitamin Bs-deficient medium) initial growth was followed by lysis, which in turn was followed by a new wave of growth to a level which was stable and much higher than that of the initial response. Obviously, undisclosed factors, including adaptative processes (as shown above in the case of lysine), are involved in the lysis phenomenon.

The stability of the over-all composition of the cell wall during the growth cycle is in good accord with the evidence reviewed by Cummins (34) which demonstrates that the composition of the cell wall is less de- pendent on cultural conditions than that of other cellular components in a particular species or strain. However, the change in amount of cell wall present per unit weight, with the age of the culture, may help to explain the wide variation (16 to 76 per cent of the cell weight) of the estimates of cell wall percentage in different species (35-38). Another contributing factor to these variable results may be the particular growth medium em- ployed, and our values of 26 per cent cell wall for the exponential phase cells and 38 per cent for stationary phase valine-limited cells may well be dependent on such factors. From the dependence of the extent of the optical density increase on the limitation of specific amino acids (3, 5) one might expect, for example, that threonine-limited cells, which show an optical density increase after the depletion point of nearly 100 per cent, would have an even higher percentage of wall substance than valine-limited cells. Such variations may have considerable influence on the function of the cell wall or cell wall components. Rigidity of the cells, resistance to environmental changes, as well as immunochemical properties may be

2 Phillips, P., Shockman, G. D., and Toennies, G., unpublished observations.

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976 CELL WALL SYNTHESIS AND AUTOLYSIS

quantitatively affected. It may be well to keep changes of this kind in mind when applying the composition of the bacterial cell wall to bacterial taxonomy (34, 9).

The generally greater sensitivity of physiologically young cells (39) to a variety of adverse conditions such as cold shock (40), osmotic salt treatment (41), heat (42), ultraviolet irradiation (43), and antibiotics may be attributed, at least in part, to differences in amount of cell wall.

We would like to thank Mr. Jack Kelsch of the RCA Laboratories and Dr. Roger Simard and Mr. Alfred Baltz of the Atlantic Refining Company Research and Development Laboratories for their kind cooperation in preparing and observing specimens in the electron microscope.

SUMMARY

Cells of Streptococcus jaecalis 9790, harvested in the exponential growth phase and in the postexponential states resulting from valine, histidine, or lysine depletion of the medium, were analyzed in terms of optical density of the culture, dry weight, nitrogen, and amino acid content, and in terms of the amount and composition of the cell wall. Among several amino acids studied, only lysine continued to be incorporated significantly into the cells after the depletion of valine. The accompanying increase in weight and optical density was accounted for, for the most part, by an approximately 100 per cent gain in cell wall substance, of which lysine, but not valine, is an essential component. As shown earlier, depletion of lysine is followed by autolysis. This process may be attributed to the prevention of postexponential cell wall synthesis, which requires lysine. The observation that exponential cells resemble lysine-limited cells in regard to autolytic propensity suggests that in the absence of continued renewal the amount of cell wall possessed by exponential cells is inadequate for their maintenance. No evidence was found for significant changes in the composition of the cell wall during its postexponential increase. The formation of a lysis-resistant variant of S. jaecalis was observed.

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Gerrit ToenniesGerald D. Shockman, Joseph J. Kolb and

PHASE, AND AUTOLYSISCELL WALL SYNTHESIS, GROWTH RELATIONS BETWEEN BACTERIAL

1958, 230:961-977.J. Biol. Chem. 

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