Chemical Composition of Cell Wall Peptidoglycan from Clostridium Saccharoperbutylacetonicum Studied with Phage Endolysins and Gas Chromatography

J. Gen. Appl. Microbiol., 21, 65-74 (1975)

CHEMICAL COMPOSITION OF CELL WALL PEPTIDOGLYCAN FROM CLOSTRIDIUM SACCHAROPERBUTYLACETONICUM STUDIED WITH PHAGE ENDOLYSINS AND GAS CHROMATOGRAPHY

SEIYA OGATA, YASUTAKA TAHARA, AND MOTOYOSHI HONGO

Laboratory of Applied Microbiology, Department of Agricultural Chemistry, Kyushu University, Fukuoka 812

(Received December 6, 1974)

The enzymic digest of cell wall peptidoglycan from Clostridium saccharoperbutylacetonicum by phage HM 7 endolysin (N-acetylmuramyl-L- alanine amidase) was separated into two constituents on ion-exchange chromatography. One was a polysaccharide, which contained N-acetylglucosamine and N-acetylmuramic acid in the molar ratio of 1.00: 0.78. This polysaccharide was digested by phage HM 3 endolysin (N-acetylmuramidase), and the digested product was a saccharide composed of N-acetylglucosamine and N-acetylmuramic acid. The other was a peptide composed of glutamic acid, alanine, and diaminopimelic acid in the molar ratio of 1.00: 2.09: 1.05. Amino acid sequence of the isolated peptide was determined by Edman degradation method, and optical configuration of the component amino acids was confirmed by gas chromatography using their N-trifiuoroacetyl menthyl esters. These analyses indicated that the isolated peptide was composed of a tetrapeptide subunits of NH2-terminal-L-AIa-D-Glu-Dpm-D-Ala. A reasonable structure for the cell wall peptidoglycan was also proposed.

Bacterial cell wall peptidoglycan is a major constituent of the cell wall and maintains the cell rigidity and shape. It is typically composed of an alternating polymer of i3-1,4-linked N-acetylglucosamine and N-acetylmuramic acid. Each muramic acid residue bears a short peptide chain consisting of D-glutamic acid, L- and D-alanine, and either meso-diaminopimelic acid or L-lysine. Little is known, however, about the study of cell wall peptidoglycan of genus Clostridium except for C. botulinum (1, 2) and C. welchii (3). In our previous paper (4), we presented hypothesis for the structure of the cell wall peptidoglycan from C. saccharoper- 65 66 OGATA, TAHARA, and HONGO VoL. 21 butylacetonicum. To gain further detailed structure of the peptidoglycan, we es- pecially sought the sequence and optical configuration of component amino acids of the peptide chain. To resolve racemic alanine and glutamic acid in the peptidoglycan of various bacteria, many different methods have been used; enzymic determination (5), gas chromatographic determination (6), and ion-exchange chromatographic determination (7). All these methods are specific and excellent, but have good and bad points. Recently, HASEGAWAand MATSUBARA(8) reported a simple and rapid gas chromatographic determination of optical isomers of various amino acids using N-trifluoroacetyl menthyl esters. This method made it possible to determine directly and sensitively the optical isomers of amino acids in mixtures without any tedious purifications. Therefore, we used this convenient method for our work. Recently, the possibility of using the lytic enzymes to study the chemical structure of the bacterial cell wall and its peptidoglycan has greatly increased the interest in phage-induced lytic enzymes (phage endolysins) for their substrate specificity and other specificity. In this work, chemical analysis of our peptidoglycan was made with the help of phage HM 7 endolysin (N-acetylmuramyl-L-alanine amidase) and phage HM 3 endolysin (N-acetylmuramidase), whose properties were reported in our previous papers (4, 9,10).

MATERIALSAND METHODS Organisms. The strains used were Nl-4 (ATCC 13564) and N1-504 (ATCC 27022) of Clostridium saccharoperbutylacetonicum (11). Phage HM 3 (ATCC 13564-B2) and phage HM 7 (ATCC 27022--B) were grown on the strains N1-4 and N1-504, respectively (11, 12). Medium and cultural conditions. Growth of the bacterial organisms and pro- pagation of phages were made at 30° under a reduced atmospheric pressure (5 to 10 mmHg) in TYA medium (13), as described previously (11-13). Preparation of cell wall and cell wall peptidoglycan. The cell wall of strain N1-4 was prepared as described previously (4). The cells were harvested in middle logarithmic growth phase, and suspended in cold distilled water. The crude cell wall was prepared by differential centrifugation, after disruption of the cells by sonication for 15 min using insoneter (Model 200 M, Kubota Ltd.). The resulting cell wall was immediately exposed to 1 % SDS solution with shaking at 37° for 15hr. The SDS-treated cell wall was washed three times with distilled water by centrifugation and then suspended in 0.067 M phosphate buffer (pH 7.0) containing trypsin (0.5 mg/ml, Sigma Chemical Co.). After shaking for 4 hr at 37°, the cell wall was sedimented by centrifugation, washed 3 to 5 times with distilled water, and finally suspended in a minimum volume of water and freeze-dried. The cell wall peptidoglycan was prepared from the cell wall, as described previously (4). The cell wall was extracted with formamide for 15 min at 150°. 1975 Chemical Composition of Cell Wall Peptidoglycan of a Clostridium 67

The insoluble residue was treated with acid-ethanol (2 N HC1-ethanol, 1: 9 v/v), and washed twice with the same solution, followed by ethanol and ether. The cell wall peptidoglycan was obtained from the ether solution by evaporation. Preparation of phage endolysin. Partially purified phage endolysins, which were filtered over Sephadex G-75, were used in this work. The preparation of phage lysates was performed as described previously (9,10). The phage endolysin in the lysate was concentrated by ammonium sulfate precipitation. The resulting endolysin solution was dialyzed against 0.067 M phosphate buffer (pH 6.5), and then centrifuged at 55,000 x g for 60 min to remove phage particles. The supernatant was applied to gel filtration over Sephadex G-75 at 4°. Ammonium sulfate precipitation was performed again on the eluted fractions, as described above. The purified endolysin was dissolved in 0.067 M phosphate buffer (pH 6.5). The purification of phage f1M 7 and HM 3 endolysins was described in detail in our previous papers (9,10). Isolation of phage HM 7 endolysin-digested products. The reaction mixture contained 200 mg of the cell wall peptidoglycan and 400 units of phage HM 7 endolysin (N-acetylmuramyl-L-alanine amidase) in 30 ml of 0.067 M phosphate buffer (pH 6.5) at 30°. Units (endolysin activities) were calculated from the following equation : units= 1,000 x (ODo-ODt)/t, where 0D0 is the initial 0D660 of cell wall or cell wall peptidoglycan suspension, and OD t is the terminal OD660 after lysis for t min. The isolation of digestion products from the cell wall peptidoglycan was the same as in our previous work (4). The enzymic digest of the cell wall peptidoglycan was applied on a column (1.5><17 cm) of Dowex 50 x 2 (200-400 mesh, H type, Dow Chemical Co.). The polysaccharide fraction was eluted with water. The peptide fraction was eluted with pyridine-acetate buffer. These fractions were further concentrated in a rotary evaporator at 30°, and applied on a column (1.0 x 100 cm) of Sephadex G-50. The eluted fractions were lyophilized. Purity of the eluted peptide and polysaccharide was described in our previous paper (4). Isolation of phage HM 3 endolysin-digested product. The reaction mixture contained 20 mg of the isolated polysaccharide and 40 units of phage HM 3 endolysin (N-acetylmuramidase) in 10 ml of 0.067 M phosphate buffer (pH 6.5). Further procedure was performed according to the methods of TAKUMIet al. (2). The enzymic digest was applied on a column (1.0><100 cm) of Sephadex G-10, and the digestion product (saccharide) was eluted with water. Chemical analysis of amino acids and amino sugars. For the analysis of amino acids and amino sugars, 2 mg of the isolated polysaccharide or peptide was hydrolyzed with 0.1 ml of 4 N HCl at 105° for 12 hr in a sealed tube. The hydrolysate was dried to remove HCl in vacuo over P2O5and NaOH. The residue was dissolved in 0.20 M citrate buffer (pH 2.2) to a final concentration of 1 mg/ml, and then ap- 68 OGATA, TAHARA, and HONGO VOL. 21 plied to the amino acid analyzer (Model JLC-5 AH, Japan Electron Optics Labora- tory Ltd.). Reduction with NaBH4. Phage HM 3 endolysin-digested product (3 mg) was reduced with 0.6 ml of 0.1 M NaBH4 for 3 hr at room temperature. After reduction, 0.3 ml of conc. HCl was added to it and the mixture was heated at 100° for 3 hr in a sealed tube for hydrolysis of the saccharide (2). The hydrolysate was dried in vacuo over P205 and NaOH, the residue was dissolved in 5 ml of methanol, and this solution was again evaporated. This treatment was repeated seven times to remove boric acid completely. The resulting materials were analysed by the amino acid analyzer. Sequence determination of the isolated peptide. Amino acid sequence of the isolated peptide was determined by EDMAN'smethod (14). A 10-mg amount of the peptide was suspended in 10 ml of distilled water, and 10 ml of dioxane was added. Its pH was adjusted to 9 with 0.01 M NaOH and 0.5 ml of phenylisothio- cyanate (PTC) was added. Further procedure was performed according to the standard manual. Gas chromatographic determination of optical configuration of alanine and glutamic acid. 1) Preparation of alanine derivative. To the acid hydrolyzate (containing 1.0 mg alanine) of the isolated peptide or authentic alanine (D-form, 0.5 mg; L-form, 0.5 mg) 250 mg of 1-menthol was added, and heated at 110° for 1 to 3 hr in an oil bath by FISHER'Smethod (15). Excess HCl and menthol were completely removed by bubbling the solution with dry N2 gas, followed by ad- dition of 0.5 ml of trifluoroacetic anhydride (TFAA). The reaction mixture was shaken by hand for 5 min at room temperature and the excess TFAA was removed by passing gentle stream of dry N2 gas. To N-TFA-l-menthyl ester of alanine so obtained, 0.5 ml of dry ethyl acetate was added and a 1-, tl aliquot of this mixture was applied to gas chromatograph. 2) Preparation of glutamic acid derivative. Acid hydrolyzate (containing 1 mg glutamic acid) of the isolated peptide or authentic glutamic acid (D-form, 1 mg; L-form, 1 mg) was added to 0.5 ml of TFAA and heated at 80° for 1 hr for esteri- fication. After removal of the excess TFAA in vacuum, 2.0 g of l-menthol was added and again heated at 100° for 3 hr, while bubbling with dry HCl gas. Ex- cess HCl and menthol were completely removed by bubbling the mixture with dry N2 gas. To N-TFA-l-dimenthyl ester of glutamic acid so obtained, 0.5 ml of ethyl acetate was added and a 1-,cclaliquot of this mixture was applied to gas chromatograph. 3) Gas chromatography. A JEOL Model JGC-20 K gas chromatograph (Japan Electron Optics Laboratory Ltd.) was used for the alanine derivative. The stainless steel column (4 m><3 mm) was packed with 80-100 mesh polyethylene glycol coated with 5 % adipate. A Hitachi Model 063 gas chromatograph (Hitachi Ltd.) was used for the glutamic acid derivative. The stainless steel column (0.75 m x 3 mm) was packed with 80-100 mesh Chromosorb W coated with 5 1975 Chemical Composition of Cell Wall Peptidoglycan of a Clostridium 69

Apiezon L. Chromatographic conditions are described in the graphs. Other chemical analysis. Determination of NH2-terminal amino acids was also performed by thin-layer chromatography of dinitrophenylated derivatives according to the method of GHUYSENet al. (5), and that of COOH-terminal amino acids by the methods of KATOet al. (16) and of BRAUNand SCHROEDER(17).

RESULTS AND DISCUSSION

Chemical compositions of the isolated polysaccharide and peptide Table 1 shows the components of polysaccharide and peptide fractions isolated from the cell wall peptidoglycan digested with phage HM 7 endolysin. The polysaccharide contained N-acetylglucosamine and N-acetylmuramic acid in the molar ratio of 1.00: 0.78. No other amino sugars were detected. The polysaccharide was digested by phage HM 3 endolysin, and the digestion product was composed of N-acetylglucosamine and N-acetylmuramic acid. The reducing end of the disaccharide was elucidated to be N-acetylmuramic acid by borohydride reduction, as shown in Table 2. This result indicates that the isolated polysaccharide may be composed of alternating N-acetylglucosamine and N-acetylmuramic acid. The peptide contained glutamic acid, alanine, and diaminopimelic acid in the molar ratio of 1.00: 2.09:1.05. No other amino acids were detected in this peptide,

Table 1. Analysis of the isolated polysaccharide and peptide fractions from cell wall peptidoglycan digested with phage HM 7 endolysin.

Table 2. Analysis of the isolated polysaccharide digested with phage HM 3 endolysin. 70 OGATA, TAHARA, and HONGO VOL. 21 and the content of amino sugar was a trace. The components and their molar ratio in the isolated polysaccharide and peptide were identical to those observed for the moiety in the original peptidoglycan (4).

Determination of NH2-terminal or COON terminal amino acids in the isolated peptide The isolated peptide was treated with dinitrofluorobenzene (DNFB) in order to determine NH2-terminal amino acids of the isolated peptide. The DNP-amino acids were extracted in ether phase, and detected by thin-layer chromatography. Chromatographic analysis revealed only DNP-alanine. The DNP method showed alanine as the NH2-terminal. On the other hand, COON-terminal amino acids were hardly detected in the isolated peptide. These results indicate that the isolated peptide has free NH2-terminals but few free COOH-terminals.

Determination of amino acid sequence in the isolated peptide The isolated peptide was subjected to three steps of the Edman degradation. At each step, PTH-amino acids were extracted with organic solvents, and the residues were hydrolyzed and applied to the amino acid analyzer. Step 1: PTH-Ala; residue : Glu, 1.00; Ala, 1.44; Dpm, 0.97. Step 2 : PTH-Glu; residue : Glu, 0.25; Dpm, 0.90; Ala, 1.00. Step 3 : PTH-Dpm; residue: Dpm, 0.52; Ala, 1.00 (Table 3). This result indicates that the three-step Edman degradation yields the sequence Ala-Glu-Dpm-Ala.

Determination of optical configuration of alanine in the isolated peptide Component alanine of the isolated peptide or authentic alanine (containing L-form and D-form in the same molar ratio) was esterified with 1-menthol and tri-

Table 3. Changes in amino acid components in the isolated peptide during Edman degradation. 1975 Chemical Composition of Cell Wall Peptidoglycan of a Clostridium 71 fluoroacetic anhydride (TFAA). The N-TFA-l-menthyl esters of alanine were subjected to gas chromatography. Figure 1 shows that the gas chromatogram gives good separation and complete resolution of enantiomeric pairs of the component alanine in the peptide, as well as those of authentic alanine. This result also indicates that the peptide contained D-alanine and L-alanine in the same molar ratio.

Fig. 1. Analysis of optical configuration of alanine in the peptide on gas chromatography. Conditions : 5 % PEG-adipate (4 m x 3 mm), sample size= 11d, isothermal at 170°, carrier N2 flow= 10 ml/min. The authentic alanine derivatives have the same retention time as those of the component alanine derivatives.

Fig. 2. Analysis of optical configuration of residual alanine in the DNP-peptide on gas chromatography. Conditions are the same as described in Fig. 1. 72 OGATA, TAHARA, and HONGO VOL. 21

Determination of optical configuration of NH2-terminal alanine in the isolated peptide In order to determine the configuration of NH2-terminal alanine, the following procedure was performed. The isolated peptide was treated with DNFB, as described above. The DNP-alanine was extracted in the ether phase, and the residual peptide in the aqueous phase was hydrolyzed with 4 N HC1. The residual alanine was esterified with 1-menthol and TFAA. The N-TFA-l-menthyl esters of alanine were subjected to gas chromatography. As shown in Fig. 2, more than

Fig. 3. Analysis of optical configuration of glutamic acid in the peptide on gas chromatography. Component glutamic acid derivative, ------Authentic glutamic acid derivatives. Conditions : 5 % Apiezon L (0.75 m x 3 mm), sample size=11d, injection=200°, 140 to 240° at 10°/min, carrier He flow= 10 ml/min.

Fig. 4. A possible structure for the cell wall peptidoglycan of Clostridium saccharoperbutylacetonicum. G1cNAc, N-acetylglucosamine; MurNAc, N-acetylmuramic acid ; Ala, alanine; Dpm, diaminopimelic acid; Glu, glutamic acid. 1975 Chemical Composition of Cell Wall Peptidoglycan of a Clostridium 73

70 % of L-alanine was eliminated. Therefore, the residual alanine was D-form, and the DNP-alanine was L-form. It was also revealed that NH2-terminal amino acids of the peptide were detected as L-alanine.

Determination of optical configuration of glutamic acid in the isolated peptide The TFA-l-dimenthyl esters of component glutamic acid or of authentic glutamic acid (containing D-form and L-form in the same molar ratio) were subjected to gas chromatography. As shown in Fig. 3, the component glutamic acid was D-form.

Structure of the cell wall peptidoglycan Digest of the cell wall peptidoglycan of C. saccharoperbutylacetonicum by phage HM 7 endolysin was separated into two constituents; one was a polysaccharide composed of N-acetylglucosamine and N-acetylmuramic acid, and the other was a peptide subunit composed of NH2-terminal-L-AIa-D-Glu-Dpm-D-Ala. The structural scheme of our cell wall peptidoglycan is proposed from the data obtained, and from a schematic structure reported for the cell walls of various bacteria (2,16,18-20), as shown in Fig. 4. The glycan moiety may be composed of alternating glucosamine and muramic acid. Each muramic acid residue is linked by the tetrapeptide subunit of L-AIa-D-Glu-Dpm-D-Ala. The cross-linkage between peptide subunits may be a direct linkage between COOH-terminal D- alanine of one peptide subunit and r-NH2-terminal group located on diaminopimelic acid of another peptide subunit. Further studies on the cell wall structure and the configuration of diaminopimelic acid are now in progress.

The authors thank Y. Kitamura for his technical assistance in some of this work, which was partly supported by a Grant-in-Aid for Scientific Research from the Ministry of Education. Thanks are given to Mr. M. Hasegawa (Tokyo Research Laboratory, Kyowa Hakko Kogyo, Ltd.) for his kind advice in gas chrornatographic techniques.

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