stearothermophilus: Mode of Action of a

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1 JOURNAL OF BACTERIOLOGY, Sept. 1971, p Vol. 17, No. 3 Copyright 1971 American Society for Microbiology Printed in U.S.A. Structure of the Cell Wall of Bacillus stearothermophilus: Mode of Action of a Thermophilic Bacteriophage Lytic Enzyme N. E. WELKER Department of Biological Sciences, Northwestern University, Evanston, Illinois 621 Received for publication 26 March 1971 The mode of action of a bacteriophage lytic enzyme on cell walls of Bacillus stearothermophilus (NCA 153-R) has been investigated. The enzyme is an endopeptidase which catalyzes the hydrolysis of the L-alanyl-D-glutamyl linkage in peptide subunits of the cell wall peptidoglycan. Preliminary studies on the soluble components in lytic cell wall digests indicate that the glycan moiety is composed of alternating glucosamine and muramic acid; one half of the muramic acid residues contain the tripeptide, L-alanyl-D-glutamyldiaminopimelic acid, and the remaining residues contain the tetrapeptide, L-alanyl-D-glutAmyldiaminopimeyl-D-alanine. Almost one half of the peptide subunits are involved in cross-linkages of chemotype I. A structure for the cell wall peptidoglycan is proposed in the light of these findings. A bacteriophage-induced lytic enzyme of Bacillus stearothermophilus (NCA 1 53-R) has been purified and characterized (21). The lytic enzyme is isolated from mitomycin C lysates and lyses whole cells or cell walls of only a few strains of B. stearothermophilus. The lytic enzyme was purified about 2,-fold and was shown to be free from protein contaminants. Preliminary studies on the mode of action of this enzyme have revealed that lysis of cell walls of strain NCA 153-R is accompanied by a release of soluble amino groups. The gross chemical composition of the cell walls of this strain was reported by Sutow and Welker (17). The purpose of this paper is to define the linkages which are hydrolyzed by this enzyme and to examine the resulting cell wall fragments. A preliminary report on the mode of action of the phage lytic enzyme has been presented previously (Mollman and Welker, Bacteriol. Proc., p. 58, 1969). MATERIALS AND METHODS Hydrolysis of cell walls by lytic enzyme. Cell walls of B. stearothermophilus (NCA 1 53-R) were prepared as described by Sutow and Welker (17) and the purification and properties of the phage lytic enzyme were described by Welker (21). Cell walls were heated in a boiling water bath for 1 min to inactivate residual autolytic enzyme activity (21 ). Cell walls (87.5 mg) were suspended in 33 ml of.5 M sodium phosphate-1- M MgCI2 buffer, ph (PM buffer) and incubated with 556 ug of phage lytic enzyme (11, units) at 55 C for hr. The cell wall suspension was water clear after 15 min of incubation and the release of soluble NH2-terminal groups was complete after 1.5 hr of incubation. A control consisting of cell walls (75.6 mg) suspended in 28 ml of PM buffer without lytic enzyme was treated in an identical manner. The cell wall suspensions were heated in a boiling water bath for 1 min and stored at -2 C. Gel filtration. The soluble components of a cell wall suspension (25 to 35 mg) digested with lytic enzyme were fractionated by filtration through a Sephadex G- 5 fine-grade column (2.5 by 8 cm; Pharmacia Inc.). The column was run at room temperature; the eluting solvent was water; and the eluate was collected in 2.6- ml fractions. The flow-rate through the column was 26 ml per hr. Separation of soluble cell wall components. The soluble components in the lytic enzyme digest were separated by a two-dimensional combination of thin-layer electrophoresis and chromatography. A sample (25 to 5 Mliters) was applied in a spot (.5 cm in diameter), on a Silica Gel G plate (2 by 2 cm), 6 cm from the cathode edge and air dried. The plate was sprayed evenly with.2 M pyridine-acetic acid buffer, ph 3.9, and immediately placed in a Desaga Migration Chamber. The temperature of the chamber was maintained at 5 C and electrophoresis was carried out at 6 to 7 v/cm for 1.5 to 2 hr. The plate was heated at 1 C for 1 to 15 min. Ascending thin-layer chromatography was run in 1-butanol-pyridine-acetic acid-water (6:6:6:8) for 2 to 2.5 hr at room temperature. The plate was heated at 1 C for 1 to 15 min and immediately sprayed either with ninhydrin (.2%) in n-butanol to detect free amino groups or with.5 N NaOH

2 698 WELKER J. BACTERIOL: in ethanol-l-propanol (6:) for the detection of oligosaccharides (16). Plates sprayed with ninhydrin or NaOH were heated at 9 C for 5 min and 12 C for 1 min, respectively. Analytical procedures. Reducing power was determined by a modification (18) of the ferricyanide procedure of Park and Johnson (15), by using N-acetylglucosamine as a standard. Total free amino groups were measured with fluoro-2,-dinitrobenzene (5) by using D-alanine as the standard. Determination and quantitation of NH2-terminal amino acids were carried out on hydrolysates (6 N HCI; 2,, 6, 8, 1, and 12 hr at 15 C) of dinitrophenylated samples of cell walls and lytic enzyme-digested cell walls (5). Controls were a mixture of a, E-diaminopimelic acid, DL-alanine, and D-glutamic acid and ly- L-glutamyl-L-alanine. Determination and quantitation of COOH-terminal amino acids were carried out by a modification of the hydrazinolysis procedure described by Braun and Schroeder (2). Cell wall samples (5 to 7 mg) were placed into acid-washed Pyrex tubes (18 by 15 mm), lyophilized, and placed over P2O, in a vacuum dessicator. To each tube was added 5 mg of Amberlite CG-5 (Mallinckrodt Chemical Works) and 2 ml of redistilled hydrazine (City Corp., New York). The tubes were evacuated and sealed, followed by heating at 1 C for 1, 2, 3,, 5, and 6 hr. The samples were lyophilized and stored over P25 in a vacuum dessicator. Each sample was suspended in I ml of water, placed in an ice bath, and mixed with 1.3 ml of isovaleraldehyde (1). After standing for 3 to 6 min, the contents of each tube were filtered through a sintered-glass filter (medium porosity). The filtered samples were washed five to six times with 5-ml volumes of ethyl acetate, adjusted to ph 2 with 2 N HCI, and the volume was adjusted to 5 ml with distilled water. Controls were a mixture of a, E-diaminopimelic acid, DLalanine, and D-glutamic acid; L-alanyl-L-alanine; L- alanyl-l-glutamic acid. The samples (2 to 5 ml) were subjected to chromatographic analysis on a Spinco model 12 B amino acid analyzer (Beckman Instruments, Inc., Fullerton, Calif.) which had been modified for accelerated analysis. The column (.9 by 62 cm) was packed with Spinco UR-3 resin (Beckman Instruments, Inc.) and the paths in the photometer were 2.2 and 6.6 mm. The temperature of the columns was maintained at 5 C; the flow rate through the column was ml per hr; and the buffer was.2 N sodium citrate, ph.26. Amino acid and amino sugar analyses were carried out by a modification of the procedure described by Sutow and Welker (17). Samples (.5 to 3. mg) of a cell wall or lytic enzyme-digested cell wall suspension were placed into acid-washed Pyrex tubes (18 by 15 mm), lyophilized, and suspended in.5 ml of 6 N HCI. The tubes were evacuated and sealed, followed by heating at 15 C for 2,, 6, 8, 1, and 12 hr. The contents of each tube were lyophilized, suspended in 5 ml of distilled water, and again lyophilized. Samples were placed over P25 in a vacuum dessicator for 2 hr, taken up in 1 ml of.2 N sodium citrate buffer (ph 2.2, sample diluter), and filtered through a sinteredglass filter (fine porosity). On the 62-cm columns, the change from the ph 3.28 to.25 buffer was made be- fore the elution of valine. Under these conditions, muramic acid elutes between serine and glutamic acid, diaminopimelic acid between valine and methionine, and glucosamine after phenylalanine. Samples of unhydrolyzed, lytic enzyme-digested cell walls were filtered through a sintered-glass filter (ultrafine porosity) before chromatographic analysis. All analyses were performed in duplicate. The amount of each dinitrophenyl-amino acid, amino acid, or amino sugar and ammonia in each sample was determined by comparing the analytical values from each hydrolysis time and selecting the values which indicated a complete libration for each component. The stereochemical configuration of alanine and glutamic acid was determined by chromatographic separation of the diastereoisomeric dipeptides obtained after derivitization with L-leucine-N-carboxyanhydride [Lleucine-NCA (12)]. Samples containing 2 to 8 Mmoles of either alanine or glutamic acid were lyophilized and suspended in 2 ml of cold.5 M sodium borate buffer, ph 1.3. Each sample was filtered through a sinteredglass filter (ultrafine porosity). One milliliter of filtrate was mixed with I ml of sodium borate buffer in a screw-cap tube (2 by 13 mm). The tube was immersed in an ice bath and L-leucine-NCA (2% molar excess) was added. After vigorous agitation on a Vortex mixer for min,.8 ml of I N HCI was added. The peptide solutions were stored at -2 C. Samples (2 Mumoles) were mixed with an equal volume of sample diluter and analyzed on the amino acid analyzer. Controls used were D- and L-alanine and D- and L-glutamic acid. The dipeptides L-leucyl-L-alanine, L-Ieucyl-D-alanine and L-leucyl-L-glutamic acid, L-leucyl-D-glutamic acid were eluted from a column (.9 by 62 cm) with ph.25 and 3.1 buffer (.2 N sodium citrate), respectively. The coupling was complete in min with a 9 to 9% yield. The L-amino acids were obtained from Calbiochem, D-amino acids and dipeptides from Cyclo Chemical Corp., and DL-amino acids from Mann Research Laboratories. Diaminopimelic acid was obtained from Sigma Chemical Co. L-Leucine-NCA was obtained from Miles Laboratories, Inc. RESULTS The major components of B. stearothermophilus (NCA 153-R) cell walls were glutamic acid, diaminopimelic acid, alanine, muramic acid, and glucosaniine in the molar ratio 1.: 1.2:1.5:1.5:1.3 (Table 1). The proportion of L- and D-alanine was 1.:.53 and all of the glutamic acid was of the D-configuration. The configuration of diaminopimelic acid was not determined. The content of ammonia was 2 moles per mole of glutamic acid. Although the conditions of hydrolysis described by Sutow and Welker (17) were different than those used in this investigation, similar mole ratios of glutamic acid, diaminopimelic acid, and alanine (1.: 1.25: 1.5 5) were reported.

3 VOL. 17, 1971 CELL WALL STRUCTURE OF B. STEAROTHERMOPHILUS 699 Liberation of N H2- and COOH-terminal groups from cell walls by the lytic enzyme. The digestion of cell walls by lytic enzyme results in the concomitant release of.59,umoles per mg of cell walls of COOH-terminal L-alanine and.57 tsmoles per mg of cell walls of NH2-terminal glutamic acid (Table 2). Ninety-seven per cent of the cell wall L-alanine and 92% of the glutamic acid residues have a COOH-terminal and NH2- terminal group, respectively, after digestion with lytic enzyme. The amounts of COOH-terminal D-alanine and NH2- and COOH-terminal diaminopimelic acid were unchanged. Free amino acid, amino sugars, or ammonia were not detected in unhydrolyzed, lytic enzyme digested cell walls. A small fraction (9.%) of the D-alanine residues have a COOH-terminal group. Fractionation, separation, and analyses of soluble components in cell wall digests. The soluble components in a cell wall suspension that was incubated with lytic enzyme for hr were fractionated by filtration through a Sephadex G-5 column. A typical elution profile is shown in Fig. 1. The soluble cell wall components were distributed over 12 tubes with a 98% recovery of re- TABLE 1. Analyses of cell walls of Bacillus stearothermophilus NCA 153R Micromoles Component per mg of cell wall Molea ratios Muramic acid Glucosamine Alanine L-Alanine D-Alanine D-Glutamic acid Diaminopimelic acid Ammonia a Expressed as moles per mole of glutamic acid in cell wall. TABLE 2. COOH-terminal and NH2-terminal amino acids in cell walls before and after digestion with phage lytic enzymea Component Cell walls Digested cell walls C-ter- N-ter- C-ter- N-terminal minal minal minal L-Alanine D-Alanine D-Glutamic acid Diaminopimelic acid.. i a Values expressed as micromoles per milligram of cell wall. z (I) c TUBE NUMBER FIG. 1. Gel filtration of lytic enzyme digested cell walls on Sephadex G-5. Reducing power (absorbancy at 7 nm), ; amino groups (absorbancy at 2 nm),. ducing power and amino groups. The eluate was combined into three main fractions. The material in fraction I (tubes 2 to 35) was excluded from Sephadex G-5 and contains 8 to 85% of the reducing groups. Fraction II (tubes 36-81) and fraction III (tubes 82-12) consist of a mixture of components containing mainly amino groups. Low-molecular-weight material having reducing groups is present in fraction 11 (tubes 38 to 2, 8 to 52, and 76 to 81 ) and fraction III (tubes 8 to 87). Separation of the soluble components was accomplished by a two-dimensional combination of thin-layer electrophoresis (TLE) and thinlayer chromatography (TLC), and the location of each component is illustrated in Fig. 2. The unfractionated cell wall digest contains 12 components (Fig. 2A). The stippled and clear areas represent components with a ninhydrin blue and a bright pink color, respectively, after spraying with ninhydrin. The areas surrounded by a dashed line were arbitrarily judged to be minor components on the basis of their color intensity (ninhydrin spray). Oligosaccharides were detected in components 1 (fraction 1, Fig. 2B), 5, 6, and 7 (fraction 11, Fig. 2C), and (fraction III, Fig. 2D). Component I exhibits the greatest intensity of fluorescence. Since muramidase or glucosaminadase activity could not be detected in

4 7 WELKER J. BACTERIOL. I ; 6 a. (. I 2l2 : ( ow a-. I. (. 2_.1 Iv- c-~ A 1., 2 2 ELECTROPHORESIS, CM -, ; r:! 8(B = L 6 I. (.9 < - C.) I I <. 6 C at2 I~~~~~~~ D ELECTROPHORESIS, CM I. I I I 2 6 8G ELECTROPHESIS, CM 12 8.'73)9, II 5S k l..> a ELECTROPHORESIS, CM FIG. 2. Separation of lytic enzyme-digested cell walls by a two-dimensional combination of thin-layer electrophoresis and thin-layer chromatography. Unfractionated cell wall digest (A) and fractions I (B), II (C), and III (D) from a Sephadex G-5 column (Fig. 1). pure lytic enzyme preparations (21), the lowmolecular-weight oligosaccharides (Fig. 1) present in fractions II and III probably reflect the presence of residual autolytic enzyme(s) in the cell wall or the release of low-molecularweight glycan present in native cell wall. No new components were found when TLE was run at ph 3., 5.6, 7., or 8. or when a variety of developing solvents were used for TLC. The electrophoretic mobilities of components 2, 3, 5-7, 9, and II appear to be greater in fraction II (Fig. 2C) than in fraction III (Fig. 2D) or the unfractionated cell wall digest (Fig. 2A). When samples of fraction II and III were combined and the soluble components were separated by the same procedure, the electrophoretic mobility of each component was identical to that observed for the same components in the unfractionated cell wall digest. The observed variability in electrophoretic mobility is probably a result of an interaction between charged components. Fraction I is composed of material that does not move from the origin (Fig. 2B) and contains muramic acid, glucosamine, and L-alanine in the molar ratio 1.: 1.1: 1.1 (Table 3). All of the 7 B (D2 I L-alanine residues have a COOH-terminal group. These results indicate that fraction I contains a major portion of the glycan moiety of the cell wall peptidoglycan with L-alanine attached. The amount of glutamic acid and diaminopimelic acid detected probably represents the presence of some undigested peptidoglycan. Fraction II is composed of six components (components 2, 3, 5-7, and 11; Fig. 2C) which migrate toward the anode. Components I and 9 are carry-over from fractions I and III, respectively (see Fig. I ). Fraction III is composed of five components (components, 8-1, and 12; Fig. 2D) with components 1 and 12 migrating toward the anode; component 8 migrates toward the cathode, and components and 9 have no electrophoretic mobility. Components 5 and 11 are carry-over from fraction II (see Fig. 1). The amino acid and amino sugar composition of the material in each fraction is shown in Table 3. Fraction II contains alanine (99% D-), glutamic acid, diaminopimelic acid, and ammonia in the molar ratio.78:1.:1.5:2.1. All of the glutamic acid residues have a NH2-terminal group and 19% of the D-alanine residues have a

5 VOL. 17, 1971 CELL WALL STRUCTURE OF B. STEAROTHERMOPHILUS 71 TABLE 3. Analyses offractions 1, II, and III from Sephadex G-5 fractionation Component Diamino- Muramic acid Glucosamine Alanine Glutamic acid pimelic Ammonia acid Fraction I Amounta Mole ratiob... (1I) Per cent of total' Fraction 11 Amounta Mole ratiod (1.) Per cent of totalc Fraction III Amounta... _e e Mole ratiod (1.) Per cent of totalc a Total micromoles in fraction. Expressed as moles per mole of muramic acid. c Amount of component detected per amount of component in cell wall digest applied to Sephadex G-5 column. d Expressed as moles per mole of glutamic acid. e None detected. COOH-terminal group. Fraction III contains D- alanine, glutamic acid, diaminopimelic acid, and ammonia in the molar ratio of.31:1.:.96: All of the glutamic acid residues and 81% of the D-alanine residues contain an NH2-terminal group and a COOH-terminal group, respectively. All of the cell wall components, with the exception of glucosamine (86.%) and muramic acid (86.1%), were quantitatively recovered (Table 3). The amount of ammonia detected in fractions II and III cannot be accounted for by the destruction of glucosamine or muramic acid becuase these fractions contain a small portion of the total glycan. The ammonia is probably from amide groups on the carboxyl groups of glutamic acid and diaminopimelic acid. DISCUSSION The phage-induced lytic enzyme is an endopeptidase which catalyzes the hydrolysis of the L- alanyl-d-glutamyl linkage in peptide subunits of the cell wall peptidoglycan of B. stearothermophilus (NCA 153-R). The data obtained in these studies indicate that the glycan moiety is composed of alternating glucosamine and muramic acid. Each muramic acid residue contains a peptide subunit consisting of L-alanine, D-glutamic acid, and diaminopimelic acid, and one half of these tripeptide subunits contain D-alanine. The cross-linkage between peptide subunits is of chemotype I () with a direct linkage between the COOH-terminal D-alanine of one peptide subunit and the NH2-terminal group located on a diaminopimelic acid residue of another peptide subunit. The extent of cross-linking between peptide subunits is not clear from the present study. However, the number of peptide subunits containing D-alanine without a COOH-terminal group (7%) indicates that almost one half of the subunits are involved in cross-linkages (Tables I and 2). It is possible that the recoveries of COOH-terminal D-alanine are low, in which case the estimated extent of cross-linking between peptide subunits will be reduced. The proposed structural scheme of the cell wall peptidoglycan and the site of action of the phage lytic enzyme is shown in Fig. 3. Fractionation of lytic enzymedigested peptidoglycan on Sephadex G-5 separates the glycan moiety plus L-alanine (fraction I) from the peptide fractions. The remaining peptide material consists of dipeptide (d) and tripeptide (a) monomers, tripeptide dimers (c), and dipeptide-tripeptide dimers (b). Since more than four components were detected (see Fig. 2 C and D) and only % of the diaminopimelic acid residues have a free NH2-terminal group, fractions II and III probably also contain tripeptide oligomers. A value of 5% would be obtained if all of the peptide subunits are present as dimers. A similar type of structure has been proposed by Grant and Wicken (6) for the cell wall peptidoglycan of B. stearothermophilus (B65). The cell walls of this strain, however, contain glutamic acid, diaminopimelic acid, alanine, muramic acid, and glucosamine in the molar ratio 1.:.85:2.3:.6:.6, glycerol teichoic acid substituted with glucose and alanine (3, 22), and aspartic acid, glycine, and serine in the molar

6 72 WELKER LYTIC ENZYME G 'I Froction I G G G G G G / / / / / / M M M M M M /1 /1 /1 /1 /1 /1 o i) 3 G) (E (E G) (E G) a b c d Froctions 3 ond m * L-Ala D-Glu DAP O D-Ala FIG. 3. Proposed structural scheme of the cell wall peptidoglycan of Bacillus stearothermophilus (NCA 153R) and the site of action of the phage lytic enzyme. Tripeptide monomer, a; tripeptide-dipeptide dimer, b; tripeptide dimer, c; dipeptide monomer, d. ratio. II:.13:.31 (relative to glutamic acid). Since strain B65 will grow at 37 and 55 C (facultative thermophile) and strain NCA 153-R will grow only at 55 C (obligate thermophile), the variation in cell wall composition is probably due to strain differences. From the data presented here, the cell wall peptidoglycan of B. stearothermophilus (NCA 153-R) has a general structure similar to that reported for cell walls of Escherichia coli (19, 2), Bacillus megaterium KM (19), and B. subtilis [23; A. D. Warth and J. L. Strominger, Bacteriol. Proc., p. 6, 1968; see review by Ghuysen (5)], Corynebacterium diphteriae (1), Bacillus licheniformis (7, 8, 9), and Lactobacillus plantarum. ATCC 81 (13, 1). The isolation of each component in the cell wall digest is necessary before a detailed analysis of the cell wall can be made. Preliminary experiments with Sephadex G-25 and G-1, Dowex-5, and CM-cellulose have shown that all 12 components can be isolated in good yields. The analysis of these components is underway in this laboratory to reconstruct the actual nature of the cell wall peptidoglycan. This is the first report of a phage lytic enzyme which hydrolyzes an internal linkage of the cell wall peptide subunit. The specificity of the phage-induced endopeptidase is identical to one of the autolytic enzymes found in sporulating cells of Bacillus thuringiensis (1 1). The phage endopeptidase will prove to be a valuable tool in studies on the structure of cell walls of thermo- philes. J. BACTERIOL. ACKNOWLEDGMENTS I am indebted to Jack L. Strominger and Donald Tipper for their assistance with the early phases of this investigation. This investigation was supported by Public Health Service research grant Al 6382 from the National Institutes of Allergy and Infectious Diseases. LITERATURE CITED 1. Akabori, S., K. Ohno, T. Ikenaka, Y. Okada, H. Hanafusa, 1. Haruna, A. Tsugita, K. Sugae, and T. Matsushima Hydrazinolysis of peptides and proteins. 11. Fundamental studies on the determination of the carboxyl-ends of proteins. Bull. Chem. Soc. Japan 29: Braun, V., and W. A. Schroeder A reinvestigation of the hydrazinolytic procedure for the determination of C-terminal amino acids. Arch. Biochem. Biophys. 118: Forrester, 1. T., and A. J. Wicken The chemical composition of the cell walls of some thermophilic bacilli. J. Gen. Microbiol. 2: Ghuysen, J. M Use of bacteriolytic enzymes in determination of wall structure and their role in cell metabolism. Bacteriol. Rev. 32: Ghuysen, J. M., D. J. Tipper, and J. L. Strominger Enzymes that degrade bacterial cell walls, p In S. P. Colowick and N.. Kaplan (ed.), Methods in enzymology, vol. 8. Academic Press Inc., New York. 6. Grant, W. D., and A. J. Wicken Autolysis of cell walls of Bacillus stearothermophilus B65 and the chemical structure of the peptidoglycan. Biochem. J. 118: Hughes, R. C The cell wall of Bacillus licheniformis N.C.T.C. 636: composition of the mucopeptide component. Biochem. J. 16: Hughes, R. C The cell wall of Bacillus licheniformis N.C.T.C. 636: isolation of low-molecular-weight fragments from the soluble mucopeptide. Biochem. J. 16: Hughes, R. C., J. G. Pavlik, H. J. Rogers, and P. J. Tanner Organization of polymers in the cell walls of some bacilli. Nature (London) 219: Kata, K., J. L. Strominger, and S. Kotani Structure of the cell wall of Corynebacterium diphtheriae. 1. Mechanism of hydrolysis by the L-3 enzyme and the structure of the peptide. Biochem. 7: Kingan, S. L., and J. C. Ensign Isolation and characterization of three autolytic enzymes associated with sporulation of Bacillus thuringiensis var. thuringiensis. J. Bacteriol. 96: Manning, J. M.. and S. Moore Determination of D- and L-amino acids by ion exchange chromatography as L-D and L-L dipeptides. J. Biol. Chem. 23: Matsuda, T., S. Kotani, and K. Kato Structure of the cell walls of Lactobacillus plantarum, ATCC Isolation and identification of the peptides released from cell wall peptidoglycans by Streptomyces L-3 enzyme. Biken J. 11: Matsuda, T., S. Kotani, and K. Kato Structure of the cell walls of Lactobacillus plantarum, ATCC Cross linkage between D-alanine and a.a'-diaminopimelic acid in the cell wall peptidoglycans studies with an L- Il enzyme from Flavobacterium sp. Biken J. 11: Park, J. T., and M. J. Johnson A submicrodetermination of glucose. J. Biol. Chem. 181: Sharon, N., and S. Seifter A transglycosylation reaction catalyzed by lysozyme. J. Biol. Chem. 239: PC2398.

7 VOL. 17, 1971 CELL WALL STRUCTURE &F B. STEAROTHEMOPHILUS Sutow, A. B., and N. E. Welker Chemical composition of the cell walls of Bacillus stearothermophilus. J. Bacteriol. 93: Thompson, J. S.. and G. D. Shockman A modification of the Park and Johnson reducing sugar determination suitable for the assay of insoluble materials: its application to bacterial cell walls. Anal. Biochem. 22: van Heijenoort, J., L. Elbaz, P. Deztlee, J. F. Petit, E. Bricas, and J. M. Ghuysen Structure of the mesodiaminopimelic acid containing peptidoglycans in Escherichia coli B and Bacillus megaterium KM. Biochemistry Weidel, W., and H. Peizer Bagshaped macromolecules: a new outlook on bacterial cell walls. Advan. Enzymol. 26: Welker, N. E Purification and properties of a thermophilic bacteriophage lytic enzyme. J. Virol. 1: Wicken, A. J The glycerol teichoic acid from the cell wall of Bacillus stearothermophilus B65. Biochem. J. 99: Young, F. E., J. Spizizen, and 1. P. Crawford Biochemical aspects of competence in Bacillus subtilis transformation system 11. Chemical composition of cell walls. J. Biol. Chem. 238: Downloaded from on November 16, 218 by guest