THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 238, No. 9, September 1963

Printed in U.S. A.

Biochemical Aspects of Competence in the Bacillus subtilis Transformation System

I. CHEMICAL COMPOSITION OF CELL WALLS*

F. E. TonNc,t JOHN SPIZIZEN,$ AND I. P. CRAWFORDS

From the Departments of Microbiology and Pathology, Western Reserve University, Cleveland 6, Ohio

(Received for publication, April 1, 1963)

Populations of Pneumococcus (1)) Haemophilus inJEuenxae (2)) and Bacillus subtilis (3, 4) develop the capacity to incorporate and to be modified genetically by deoxyribonucleic acid during the latter portion of the logarithmic phase of growth. In 1946 McCarty, Taylor, and Avery (5) suggested that this rapid de- velopment of sensitivity to transforming DNA might be related to a reversible alteration of the surface of pneumococcal cells by an enzyme which would make possible the absorption or pene- tration of DNA. Since the development of sensitivity to trans- forming DNA or competence in B. subtilis required a period of unbalanced growth and was inhibited by excessive amounts of cell wall precursors, Spieizen (3) also postulated that an alteration of the cell wall might be necessary for the development of com- petence in this transformation system. Recent studies have indirectly supported this hypothesis. For instance, the poorly transformable mutant strains of B. subtilis isolated to date do not incorporate DNA irreversibly (6). However, if the DNA was introduced by a phage (transduction) the frequency of ge- netic modification was similar in both the poorly and highly transformable strains (7, 8). Finally, protoplasts of Xhigella paradysenteriae (9) and Escherichia coli (10-12) but not intact bacteria can be infected by phage DNA.

This morphological and chemical study was initiated in order further to investigate the role of the cell wall in competence. The evidence presented below demonstrates that there are quan- titative chemical differences between cell walls obtained from poorly and highly transformable strains of B. subtilis.

* This investigation was aided by United States Atomic Energy Contract AT (11.1)-944 with the Division of Biology andMedicine, by Grant E 288 from the American Cancer Society, and by a grant for a Faculty Research Associate from the American Cancer Society.

t United States Public Training Fellow in Pathology 1958 to 1962. These studies constitute a portion of the thesis submitted to the Department of Microbiology in partial fulfillment of the requirements for the degree of Doctor of Philosophy, Western Reserve University. Present address, Department of Pathology, Western Reserve University.

$ Present address, Department of Microbiology, University of Minnesota, Minneapolis, Minnesota.

$ This investigation was supported in part by United States Public Health Service Research Career Program Award 5-K& GM-15411 from the Division of General Medical Sciences.

EXPERIMENTAL PROCEDURE

Materials

Most of the strains of B. subtilis employed in these experi- ments were obtained from the collection of Burkholder and Giles (13) through the courtesy of Dr. Charles Yanofsky. The indole- requiring mutant, B. subtilis 168 I-C+, was the standard recipi- ent. The I- refers to the auxotrophic character of the strain, either indole or tryptophan being necessary to permit growth on minimal medium. The C+ refers to the ability to bind DNA’ irreversibly. The frequency of transformation in the poorly transformable mutant strain B. subtiilis 168 I-C- is lO,oOO- to 50,000-fold lower than in the transformable strain (6). All of the strains were maintained on potato agar (14) and tryptose blood agar base (TBAB, Difco). The DNA was prepared from the wild strain of B. subtilis and was partially purified by treat- ment with RNase, trypsin, and deoxycholate (6).

The amino acids and amino sugars utilized as standards in paper chromatography were obtained from Nutritional Biochem- icals Corporation and Mann Research Laboratories. A sample of muramic acid was kindly provided by Dr. James Park. Ga- lactosamine and glucosamine were obtained from Mann Research Laboratories, Inc. The amino acids employed for the calibra- tion of the Beckman/Spinco automatic amino acid analyzer were obtained from the Spinco Division, Beckman Instruments, Inc. Crystalline pancreatic DNase and RNase (twice crystallized), trypsin, and crystalline lysozyme were obtained from Worthing- ton Biochemical Corporation, Nutritional Biochemicals Corpora- tion, and Armour and Company, respectively.

Methods

Transformation Assay-A sample of cells was assayed for transformation from indole auxotrophy to prototrophy after a 15-minute contact with donor DNA as described previously (6).

Preparation of Cell Walls-The highly transformable strain, B. subtilis 168 I-C+, was grown in the following fashion to ob- tain samples of cells with different degrees of competence. An

1 The compet.ent recipient, strain was designated Sp+ in a previous publication (6). This strain snorulates when crown on minimal medium containing ferric chlbride (50 rg pei ml) or manganese chloride (10 pg per ml). The poorly transformable strain (C-) did not sporulate under those conditions and was consequently designated Sp-. Both strains sporulate on potato agar.

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3120 Biochemical Aspects of Competence in Transformation. I Vol. 238, No. 9

inoculum of B. subtilis 168 I-C+ was added to 1.5~liter aliquots of the usual growth medium (6) in Q-liter Erlenmeyer flasks. The initial optical density of all cultures at 660 mp was 18 f 4 Klett units. The cultures were incubated for l*, 5, and 12 hours at 37” on a rotary shaker. The cells from each culture were then harvested by centrifugation in sterile containers, sus- pended in 1.5 liters of the usual transformation medium (6), and incubated in a 4-liter Erlenmeyer flask for an additional 2 hours at 37” on a rotary shaker. An aliquot was assayed for transformability of the indole marker. The remaining cells were harvested by centrifugation and stored for 1 to 2 weeks at -60”. B. subtilis 168 I-C- was grown for 5 hours in the growth medium and 2 hours in the transformation medium. These conditions produced the maximal degree transformation that was attainable in this strain.

Eight- to fourteen-gram aliquots of cells were thawed slowly to 1” and transferred to a 75-ml Duran 50 flask containing an amount of acid-washed, O.l-mm glass beads (Minnesota Mining and Manufacturing Company) that equaled 3 times the wet weight of the cells. Sufficient cold distilled water was added to produce a 50% suspension of bacteria (w/v). The mixture was shaken for two l-minute intervals in the Braun cell homogenizer (Braun Company, Melsungen, Germany) and cooled with inter- mittent jets of COZ. The slurry was diluted immediately with 250 ml of cold distilled water, transferred to cold centrifuge tubes, and centrifuged for 10 minutes at 1000 X g at 1” to sedi- ment glass beads and intact bacteria. Complete disruption of the remaining bacteria was achieved by subjecting this residue to two more treatments in the Braun cell homogenizer.

The 1,000 x g supernatant fractions were centrifuged two more times at 1,000 x g for 10 minutes at 1” and then at 10,000 x g for 10 minutes at lo. The 10,000 x g sediments were pooled, suspended in cold distilled water, and centrifuged at 10,000 X g for 10 minutes at 1”. After this washing procedure was repeated 12 times, an aliquot was removed for electron microscopy. The remainder of the sample was lyophilized. The frequency of transformation obtained in the various populations of cells uti- lized for the preparations of cell walls is summarized in Table I. The italic number in parentheses after the strain refers to the duration of incubation in the growth medium. Thus, “B. subtilis

TABLE I Samples of cell walls employed in chemical analyses

Strain

168 I-C+ 168 I-C+ 168 I-C+ 168 I-C+ 168 I-C+ 168 I-C- 168 I-C- 168 I-C-

Duration of growth’ Transformationt

hrs %

14 0.003 5 1.1 5 1.1 5 0.7

12 0.03 5 0.00064 5 0.00005 5 0.00006

* Duration of incubation in the growth medium. This period will be indicated immediately after the strain (italic figure in parentheses) in all subsequent tables. Thus, “168 I-C+ (6)” indicates that the strain has grown for 5 hours in the growth medium.

t The frequency of transformation of the population of cells utilized for the preparation of cell walls.

168 I-C+ (la)” was incubated for 13 hours in the growth me- dium and 2 hours in the transformation medium. “B. subtilis 168 I-C+ (5)” was incubated for 5 hours in the growth medium and 2 hours in the transformation medium. Identical samples of walls which contained less than 0.01% intact bacteria were pooled, stored at -2O”, and utilized for chemical studies.

Electron Microscopy-Droplets of washed cell walls were placed on copper grids covered with a film of Formvar and dried at room temperature. The proportion of bacteria contained in a sample of cell walls was estimated by direct examination of the grids with electron microscopy with the assistance of Dr. Melvin Schoenberg and Mr. V. Mumaw. For morphological study, the specimens were shadowed with chromium.

Hydrolysis of Cell Wall Samples---Samples of cell walls were dried for 24 hours over P,Os in a vacuum desiccator at room temperature. Samples (10 mg) were placed in acid-washed Pyrex tubes (8 X 125 mm) and dissolved in 0.7 ml of 6 N HCI. The sealed tubes were hydrolyzed in an autoclave at 105” f 1” for 8, 16, and 24 hours. The contents of each tube were filtered through acid-resistant filter paper, transferred to a loo-ml beaker, evaporated to dryness in a vacuum desiccator at room tempera- ture, and then rehydrated and evaporated three times to remove the HCl.

Partial hydrolysis was produced by dissolving 5- to IO-mg samples of dried cell walls in 1 ml of 0.5 N HzS04 in acid-washed Pyrex tubes (8 X 125 mm). The sealed samples were hydro- lyzed at 100” for various intervals of time and then cooled rapidly to 0”. The contents of each tube were neutralized to pH 4.5 j= 0.2 with saturated Ba(OH)z, centrifuged, filtered, and the su- pernatant liquid was diluted to 5 ml with deionized water.

Chromatography-Single or two-dimensional descending paper chromatography of the cell wall hydrolysates was carried out on. Whatman No. 1 chromatography paper with pyridine-water (4: 1, v/v) and with tert-amyl alcohol-water-formic acid (15) solvents.

Carbohydrates were detected on paper chromatograms by spraying with aniline phthalate (16), p-anisidine (17), and silver nitrate (18) reagents. The sensitivity and range of compounds detected by the aniline phthalate reagent were increased by examining the chromatogram under ultraviolet light. Amino acids and amino sugars were detected by ninhydrin spray re- agent obtained from Sigma Chemical Company.

Quantitative determinations of the amino acids of the cell wall were obtained by chromatographic analysis of 2-mg samples on the Beckman/Spinco model 120 amino acid analyzer (19, 20) with the technical assistance of H. Stewart. On standard chro- matograms muramic acid elutes between serine and glutamic acid, diaminopimelic acid between valine and methionine, and glucosamine and galactosamine after phenylalanine. Since glucosamine and galactosamine can be separated and quantitated on the 15- and 150-cm columns, the values reported for glucosa- mine and galactosamine represent the average of these two de- terminations. All analyses were performed in duplicate.

Chemical DeterminationsThe methods employed for the analysis of DNA, RNA, protein, nitrogen, phosphorus, and hexuronic acids were described by Burton (21), Ashwell (22), Lowry et al. (23), Kabat and Mayer (24), Fiske and SubbaRow (25), and Dische (26), respectively. Glucose was estimated calorimetrically by the methods of Dische (26) and Ghuysen (27) and enzymatically by the glucose oxidase assay (28). The alanine liberated during partial hydrolysis was measured by the

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September 1963 F. E. Young, J. Xpixizen, and I. P. Crawford 3121

method of Rosen (29). Unless otherwise indicated analyses B. subtilis phage examined to date. It is unlikely that free were performed in triplicate. phage would sediment under the conditions employed for the

RESULTS isolation of cell walls. Therefore, these particles are probably debris or dirt and not phage.

Morphology-The cell walls of B. sub&s are blunted ellipsoids Chemical Composition of Cell Walls of B. subtilis-The amount (Fig. 1). The length of the chromium shadows indicates that of nitrogen in lo-mg samples of the various preparations of cell the cell walls are 50 i 10 A in thickness. Although the surface walls was essentially constant (46.5 i 2.4 pg per mg). An of the cell walls from B. subtilis 168 I-C+ (13) was slightly more estimate of the rate of degradation of the various amino sugars granular than that of the other cell walls, there were no striking was obtained by hydrolyzing lo-mg samples for 8, 16, and 24 or consistent morphological differences among the various prep- hours in 6 N HCl at 105” f lo. As shown in Table II, the rate arations. In some samples there were irregular electron-dense of degradation was different for each amino sugar. By extrapo- particles on the film and associated with the cell wall (Fig. 2). lation to zero time, the amount of degradation of the amino These were smaller and more irregular than the preparations of sugars after 16 hours was 13%, and 9% for muramic acid

FIG. 1. Electron micrograph of cell walls of B. subtilis 168 I-C- (5)

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3122 Biokhemical Aspects of Competence in Transformation. I Vol. 238, No. 0

FIG. 2. Electron micrograph of cell walls of B. subtilis 168 I-C+ (5). The irregular electron-dense particles on the grid and walls were not observed in all preparations.

(3-carboxyethyl glucosamine), galactosamine,2 and glucosamine, HCl the extent of degradation of standard nonacetylated mu- respectively. In contrast, after 16 hours of hydrolysis in 6 N ramic acid, galactosamine, and glucosamine was identical (28%).

e The values obtained for the degradation of galactosamine Regardless of the explanations for this discrepancy, the extrapo-

(Table II) do not fall on a straight line when they are plotted lated values were used to correct for degradation.

graphically. Since standard solutions of galactosamine elute in As shown in Table III, alanine, glutamic acid, diaminopimelic a broader peak than glucosamine and muramic acid, this variation acid, muramic acid, glucosamine, galactosamine, and ammonia may be in part due to the greater difficulty encountered in calcu- are the major components of the cell wall of B. subtilis. These lating the galactosamine. Even though the standard solutions of galactosamine and the compound identified as galactosamine in

compounds constitute approximately 40% of the cell wall. The

the cell walls elute in the same position, it is still possible that C- strain contained lower amounts of alanine, galactosamine,

the galactosamine peak is inhomogenous. Only the S-hour and and most of the minor components (Table IV), but more dia- the 24-hour points were utilized for extrapolation to calculate the minopimelic acid and glucosamine than the Cf strain grown extent of degradation of galactosamine. under the same conditions. Despite these quantitative differ-

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September 1963 F. E. Young, J. Xpizizen, and I. P. Crawford 3123

ences, the total amounts of amino acids and amino sugars in these strains were relatively constant (2.99 f 0.09 pmoles per 2 mg and 1.71 + 0.04 pmoles per 2 mg, respectively). During growth of the transformable strain the total major components (excluding ammonia) increased by 0.26 pmole per 2 mg whereas the minor components decreased by 0.29 pmole per 2 mg.

The studies of Armstrong, Baddiley, and Buchanan (3032) have demonstrated that the teichoic acid of B. subtilis consists of approximately 9 alanyl glucosyl ribitol residues linked by phosphodiester bonds through the l- and 5-positions of ribitol. The P-n-glucose is linked to the 4-position and the n-alanine is linked by a “labile” ester bond to the 2- or a-position of ribitol. In the present investigation, the phosphorus, glucose, and “la- bile” alanine contents of the cell walls were utilized as indices of the teichoic acid content. As shown in Table V, the phosphorus content of comparable samples of cell walls of the transformable and poorly transformable strains of B. subtilis was similar. Dur- ing the growth of the transformable strain there was a slight de- crease in total phosphorus content. In this study and in those of Salton and Pavlik (33) and Ghuysen (27), there was a dis-

TABLE II Effect of hydrolysis time on amino sugar content of cell walls

Two-milligram samples of cell walls which were hydrolyzed for 8,16, and 24 hours in 6 N HCl at 105” were analyzed on the Beck- man/Spinco amino acid analvzer.

TABLE IV Chemical composition of cell walls of B. subtilis: minor components

Conditions were similar to those of Table III.

Component Strain

168 I-C+ (I*) ( 168 I-C+ (5) 1168 I-C+ (12) / 168 I-C- (j)

Lysine 0.08 Histidine 0.01 Arginine 0.03 Aspartic acid. 0.07 Threonine . . 0.06 Serine 0.06 Glycine . 0.07 Half cystine 0.04 Valine, 0.05 Methionine *

Isoleucine 0.06 Leucine 0.05 Tyrosine 0.02 Phenylalanine 0.02

p??&&?/Z mg

0.05 0.01 0.01 0.03 0.05 0.03 0.04

*

0.03 *

0.03 0.03 0.01 0.01

0.06 0.01 0.01 0.05 0.06 0.04 0.05 0.03 0.04

*

0.03 0.04 0.01 0.01

0.44

0.06 0.01 0.01 0.04 0.07 0.03 0.05 0.03 0.03

*

0.02 0.04 0.01 0.01

Total 0.62 0.41

* Less than 0.01 rmole.

TABLE V Phosphorus, glucose, and “labile” alanine content of cell walls component

Time of hydrolysis

T

Phosphorus* Glucoset -

1 -

Uuramic acid Glucosamine falactosamine

hrs

8 16 16 24 24

0.54 0.39 0.37 0.32 0.34

$mole/2 ?ng

0.63 0.59 0.62 0.59 0.57

0.49 0.38 0.38 0.38 0.44

8 0.54 0.79 0.34 8 0.56 0.74 0.32

16 0.47 0.76 0.21 16 0.45 0.72 0.23 24 0.39 0.66 0.24 24 0.35 0.66 0.26

“Labile” alanine +

diaminco$nelic

j.lmolelmg

0.38 0.38 0.33 0.27

I’ 168 1-c+ (6)

168 I-C- (5)

168 I-C+ (f;) 0.96 0.40 0.64 168 1-c+ (6) 0.86 1.2 0.48 0.68 168 I-C+ (12) 0.81 0.50 0.73 168 I-C- (6) 0.81 1 1.1 0.54 0.68

i - * Phosphorus content of the IO-mg samples of cell walls hy-

drolyzed in 6 N HCl for 16 and 24 hours at 105” f 1”. t Glucose content of samples of cell walls hydrolyzed in 0.5 N

HzSOa for 3 hours at 98”. 1 The amino acid content of samples of cell walls hydrolyzed

for 15 minutes in 0.5 N H&0( at 98”. Under these conditions the “labile” alanine was completely hydrolyzed whereas the only other amino acid released was a trace of diaminopimelic acid.

TABLE III Chemical composition of cell walls of B. subtilis: major componenta

Ten-milligram aliquots of cell walls were hydrolyzed for 16 hours at 105” f 1” in 6 N HCI. Two-milligram aliquots were analyzed on the Beckman/Spinco amino acid analyzer.

Strain component

168 I-C+ (If) I I I

168 I-C+ (5) 168 I-C+ (12) 168 I-C- (5)

crepancy between the enzymatic and calorimetric analyses for glucose. According to Salton and Pavlik (33), this is related to an enhancement of the calorimetric reaction by amino acids. The “labile” alanine released during partial hydrolysis of the samples of cell walls would produce an unreliable calorimetric reaction. Thus, the enzymatic reaction was considered to be a more specific method for determining glucose. As shown in Table V, the glucose content of the transformable strain increases slightly with growth. The glucose content of the poorly trans- formable strain, as measured by the enzymatic reaction, was consistently higher than that of the transformable strain at a comparable period of growth. The amount of “labile” alanine liberated from cell walls decreased with growth of the trans- formable strain. The amount of “labile” alanine was lowest in the poorly transformable strain. Therefore, if the phosphorus

pmoles/Z mg

Alanine. 1.50 Diaminopimelic

1.50 1.53 1.27

acid............. 0.73 0.77 0.89 1.03 Glutamic acid. 0.62 0.63 0.73 0.77 Muramic acid. 0.56 0.57 0.60 0.68 Glucosamine. 0.68 0.67 0.65 0.81 Galactosamine.. 0.44 0.43 0.39 0.25 Ammonia. 1.17 1.24 1.44 1.22

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TABLE VI Chemical composition of cell walls of B. subtilis:

recovery of nitrogen and mass

Wall component

Alanine ................ Diaminopimelic acid .... Glutamic acid. ......... Muramic acid ........... Glucosamine ........... Galactosamine. ......... NH3, ................... Minor components. ..... Ribitol phosphate. ..... Glucose ................

Total recovery. 94.1 76.4

-

I -

168 I-C+ (5) 168 I-C- (5)

Vitrogen Cell lnass

% 21.6 6.7 18.4 5.7 22.2 7.3 29.8 9.8 9.1 4.7 11.1 5.7 8.2 7.2 9.8 8.5 9.7 6.0 11.7 7.3 6.4 3.8 3.6 2.2

17.9 1.8 17.6 1.7 9.0 4.2 4.8 2.3 0 26.0* 0 24.8 0 8.7 0 9.8

77.8

litrogen Cell mass

%

* The ribitol phosphate content of the cell walls was calculated from the phosphorus content. The cell walls of B. subtilis con- tain approximately 1 mole of ribitol phosphate per mole of phos- phorus (30-32).

content is a reliable index of the teichoic acid content of the cell wall, the amount of substituted glucose appears to be lower and the amount of “labile” alanine higher in the transformable strain of B. subtilis.

As shown in Table VI, the nitrogen recovery is essentially complete; however, 23% of the mass of the cell walls has not been accounted for. Even if all of the known hexosamines were acetylated as postulated by Salton in Micrococcus lysodeikticus, this would only increase the total recovery to approximately 86%.

DISCUSSION

Bacterial transformation involves the penetration of the re- cipient cell by DNA and subsequent replication, function, and integration of the newly acquired segment of DNA. Since transformation and incorporation of DNA were proportional under diverse conditions of environment, Lerman and Tolmach (34) suggested that competence is related to the cells’ capacity to incorporate DNA. Two hypotheses have been proposed to explain this process: (a) competence requires the development of specific adsorptive sites foi- DNA and (b) competence is depend- ent upon an alteration of the cell surface which permits the highly negatively charged DNA to become more accessible to the cell (35). Detection of such changes on the surface of the cell wall necessitates that a significant proportion of the popula- tion be modified and that the chemical structure be unaltered by the isolation procedure. Although the maximal frequency of transformation for a single gene in Pneumococcus, H. iv@uenzae, and B. subtilis is usually 1 to 5%, indirect evidence indicates that a majority of the cells in the population are competent (2, 36). Since in the present study the cell walls were isolated at 0” and were washed in distilled water, it is unlikely that the autolytic enzymes altered the cell wall (37). Furthermore, the cell walls were not treated with enzymes during the purification procedure.

Previous investigations on the chemical composition of the cell walls of B. subtilis have demonstrated that this structure may be composed of at least four heteropolymers: (a) a muco-

peptide composed of glucosamine, muramic acid, alanine, glu- tamic acid, and diaminopimelic acid (38); (b) teichoic acid, con- sisting of 9 alanylglucosylribitol residues (30-32) ; (c) teichuronic acid, a mucopolysaccharide consisting of equimolar amounts of N-acetylgalactosamine and glucuronic acid (39); and (d) a pro- tein fraction (39). Teichuronic acid has not been found in all strains of B. subtilis (40, 41). Although the cell walls of B. subtilis 168 contained galactosamine, glucuronic acid was not identified with the analytical methods employed by Janczura et al. (39). In addition to this qualitative variation, there were slight differences in the quantitative amounts of nitrogen, phos- phorus, and major components among the various strains of B. subtilis studied to date (33, 3841). Similar differences were observed among strains of Staphylococcus and Micrococcus (42). These discrepancies could be related to physiological conditions of growth as well as genetic variation.

In the present investigation, only 77% of the mass of the cell wall was recovered. Three compounds which were not deter- mined could contribute to this deficit. First, not all of the com- ponents of teichoic acid were determined. Instead, the amount of ribitol phosphate was calculated on the assumption that ribitol and phosphorus occur in approximately equimolar amounts in the cell walls of B. subtilis (30-32). I f this assumption is not valid or if all the phosphorus was not released, this would greatly influence the theoretical recovery. Second, hexosamines were not calculated as the N-acetyl derivatives. Finally, the lipid content of the cell wall was not determined. However, the investigations of Salton indicate that lipid comprises less than 3% of the cell wall of B. subtilis (40). On the other hand, es- sentially all of the nitrogen was recovered. Since only 25% of the total ammonia in the hydrolyzed samples could be accounted for by degradation of known hexosamines, it is probable that some of the ammonia was derived from the breakdown of un- identified compounds.

Quantitative differences exist among preparations of cell walls derived from the transformable strain during growth. Fur- thermore, differences exist between the transformable strain and a poorly transformable strain at a comparable period of growth. The nature of these changes is more apparent in the fractions of cell walls obtained after autolysis of the purified cell walls. Therefore, the discussion of these data will be deferred to the second paper of this series (37).

SUMMARY

The morphology and chemical composition of the cell wall of the highly and poorly transformable strains of Bacillus subtilis were studied. The major components of both strains are alanine, diaminopimelic acid, glutamic acid, muramic acid, glucosamine, galactosamine, and ammonia. The cell walls of the poorly transformable strain contain more glutamic acid, diaminopimelic acid, and glucosamine, but less galactosamine, alanine, and minor components than the transformable strain at a comparable period of growth. Quantitative chemical changes also occur in the cell wall of the transformable organism during growth.

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