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 sac- charoperbutylacetonicum 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-acetyl- glucosamine and N-acetylmuramic acid in the molar ratio of 1.00: 0.78. This polysaccharide was digested by phage HM 3 endolysin (N-acetyl- muramidase), 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 chro- matography using their N-trifiuoroacetyl menthyl esters. These analyses indicated that the isolated peptide was composed of a tetrapeptide sub- units 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 pepti- doglycan of various bacteria, many different methods have been used; enzymic determination (5), gas chromatographic determination (6), and ion-exchange chro- matographic 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 peptidogly- can 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 centrifuga- tion 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 pre- viously (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 hydro- lyzed 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).