Peptidoglycan .Types of Bacterial Cell Walls and their Taxonomic ...

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VOLX 36, 1972 PEPTIDOGLYCAN TYPES OF BACTERIAL CELL WALLS ./*-* Mur x Glu -aLys E /-*--: 0----L-Ala-L-Ala °=° c . / ; o-- o L-Ala-D-Glu o1 - N6 -L-Ala-L-Lys ur 1.0 U~~~~~~ C .0 zto.s .0. '°" \a . D-Ala-L-Ala -o0y D-Glu-L-Lys 1 2 3 4 5hrs FIG. 2. Quantitative release of various amino acids and peptides from cell walls of Sarcina lutea ATCC 383 during hydrolysis with 4 N HCI at 100 C as a function of time. TABLE 2. RAIG values of various amino acids and amino sugars after two-dimensional descending separationa RAI. values Amino acid or amino sugar Isopro- a-Picopanol line Alanine ...................... 1.0 1.0 Aspartic acid ................. 0.55 0.28 Diaminobutyric acid .......... 0.28 0.58 Diaminopimelic acid .......... 0.11 0.14 Galactosamine ............... 0.57 1.68 Glucosamine ................. 0.60 1.80 Glutamic acid ................ 0.81 0.29 Glycine ...................... 0.60 0.66 Homoserine .................. 0.83 1.01 Threo-3-hydroxyglutamic acid 0.43 0.27 Lysine ....................... 0.30 0.42 Mannosamine ................ 0.71 1.80 Muramic acid ................ 1.01 1.70 Ornithine .................... 0.27 0.41 Serine ...................... 0.59 0.81 Threonine ................... 0.84 1.02 aTwo-dimensional descending separation in solvent systems isopropanol (machine direction) and a-picoline on Schleicher-Schiill 2046b Mgl paper. Running time: each direction 2 x 24 hr. Temperature of chromatography chamber: 27 to 28 C. tri-L-alanyl peptides. The dipeptides L-Ala-D- Glu, y->-Glu-L-Lys, and L-Lys-D-Ala are derived from the peptide subunit, whereas N6-L-Ala-L-Lys, L-Ala-L-Ala, and D-Ala-L- 411 Ala are typical for the interpeptide bridge and its connection to the peptide subunit. Typical pictures of two-dimensional paper chromatograms of partial acid hydrolysates of cell walls are given in Fig. 4. The cell walls of these two organisms have almost identical amino acid composition, but the "finger prints" are quite different. This indicates that the amino acid sequences of the two peptidoglycans are unlike. The detailed analysis showed that the peptidoglycan of Micrococcus luteus belongs to subgroup A2 (see below), whereas that of Bifidobacterium breve belongs to subgroup A3 (vide infra). If some peptides were not sufficiently well resolved in the two standard solvent systems (isopropanol and a-picoline), we used other systems, especially n-butanol-acetic acid-pyridine-water (420:21:280:210, v/v/v/v) and nbutanol-propionic acid-water (750: 352:498 v/v/v) (344). For the separation of diaminopimelic acid (Dpm)-containing peptides, we applied the modified solvent system of Rhuland et al. (315), methanol-pyridine: formic acid: water (80:10: 1: 19 v/v/v/v), or high-voltage electrophoresis on Whatman no. 3MM paper (39). The peptides were isolated by repeated onedimensional paper chromatography (336) or by developing a two-dimensional chromatogram with ninhydrin and cutting out the corresponding areas from other unsprayed parallel twodimensional chromatograms. In the case of Downloaded from http://mmbr.asm.org/ on December 3, 2012 by guest
412 SCHLEIFER AND KANDLER BACTER0i,I. REV. TABL.E 3. RALa values of various peptides from partial acid hydrolysates of cell walls after two-dimensional descending separationa R5,, values R,,,, values PepideIsopro) a panol -Picoline Isolor5-Pcs panol line Peptide Isopro- a-Pico- Color' Peptide Isopro aPic olor D-Ala- L-Ala . . . . .. D-Ala-D-Ala or L-Ala- L-Ala L-Ala-L-Ala- i -Ala D-Ala-Gly D-Ala-Gly-Gly ... L-Ala-Thr. L-Ala-D-Glu or D-Ala-L-Glu D-Ala- D-Asp D-Ala-D-G.lu L-Ala-ay-D-GIU-L-Lys D-Ala- -y- -Glu-Gly D-Ala-7- L-GIU-L-Ala D-Ala-L-Ala-D-Glu . .. D-Ala-Dpm ........ D-Asp-L-Ala. Dpm-D-Ala y-D-GlU-L-Lys -y-L-Glu-Gly -y-L-GlU-L-Ala ........ a-n-Glu-Gly ........ D-Glu-Dpm-D-Ala Gly-Gly .... Gly-Gly-Gly. Gly-Gly-Gly-Gly Gly-L-Ser .......... Gly-L-Ala Gly-D-Glu Gly-Gly-L-Ala Gly-a-Hyg-Gly Gly-y-Hyg-Hsr Gly- L, L-Dpm- )-Ala L-Lys-D-Ala . L-Lys-D-Ala-D-Ala ..... 1.17 1.26 1.30 0.93 0.78 1.05 0.95 0.95 1.05 0.19 0.67 0.92 1.05 0.28 0.55 0.31 0.20 0.56 0.92 0.77 0.15 0.61 0.51 0.39 0.50 0.90 0.72 0.71 0.35 0.36 0.21 0.39 0.52 1.20 1.25 1 .32 0.91 0.83 1.27 0.23 0.23 0.29 0.32 0.31 (.41 0.54 0. 134 0. 33 0.40 0.23 0.27 0.40 0.20 0.08 0.59 0.62 0.64 0.74 0.85 0.17 0.83 0.15 0.20 0.14 0.60 0.87 Steel blue Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Brownish t-Lys-D-Ala-i,-Glu Mur-L-Ala..... Mur-Gly Mur-L-Ala-D-Glu Mur-GlcNH, N 2-Gly-L-Lys N 2 D-Ala-D-Lys N'-Gly-L-Lys ... N 6-L-Ser-L-LyS ..... N 6-L-Ala-L-Lys N -Thr-L-Lvs . N 6-Gly-L-Lys-D-Ala N 6-L-Ser-L-Lys-D-Ala N 6-L-Ala-L-Lys-D-Ala N 6-a-D-Glu-D-Lys N 6--y-D-GlU-L-Lys e-(Aminosuccinyl-) lysine N 6-(L-Ala-L-Ala)-L-Lys N 6_( D-Ala-D-Asp)-L-Lys N 6_(L-Ala-Thr)-L-Lys N 6_(D-Asp-I,-Ala)-L-Lys N 2-D-Ala-D-Orn N 5-Gly-L-Orn N -L-Ser-L-Orn N5-Gly, N 2-D-Ala-D-Orn N '-L-Ala-L-Orn L-Orn-D-Ala . . L-Ser-L-Ala .......... L-Ser-D-Glu L-Ser-Gly L-Ser-L-Ser Thr-L-Ala 0.:37 1.20 0.98 1.08 0.30 0.27 0.39 0.22 0.24 0.40 0.32 0.33 0.33 0.47 0.36 0.30 0.32 0.46 0.39 0.40 0.17 0.37 0.20 0.22 0.29 0.40 0.37 0.93 0.70 0.66 0.55 1.16 0.27 1.60 1.39 0.81 1.36 0.53 0.60 0.62 0.7. 0.87 0.91 0.76 0.85 0.95 0.27 0.26 0. 77I 1.0 0.97 1.02 0.60 0.58 0.58 0.75 0.72 0.87 0.58 1.01 0.2,3 0.80 0.90 1.36 Brownish Brownish Brownish Yellow Brownish Brownish Yellow Yellow Yellow Yellow Yellow Yellow aTwo-dimensional descending separation in solvent systems isopropanol (machine direction) and a-picoline on Schlescher-Schiill 2043b Mgl paper. Running time: each direction 2 x 24 hr. Temperature of chromatography chamber: 27 to 28 C. bMost spots give the usual violet color with ninhydrin; only the unusual colors are specified. Dpm-peptides preparative electrophoresis was applied (39). The isolated dipeptides were identified by determining the quantitative amino acid composition and the configuration of the amino acids and the N-terminal amino acid (336). The tri- and higher oligo-peptides were again subjected to partial acid hydrolysis, and the resulting smaller peptides were identified. The involvement of the,-carboxyl group of Asp or the -y-carboxyl group of Glu in a peptide bond was demonstrated by photolysis of the corresponding DNP-peptides (271, 288). -- -1 In many cases these four steps (quantitative amino acid composition, configuration of' the amino acids, N- and C-terminal amino acids, and isolation and identification of oligopeptides after partial acid hydrolysis of cell walls) were sufficient to establish the amino acid sequence of' the peptidoglycan. Only in the case of' very complicated structures were additional data necessary to decide which component belongs to the peptide subunit and which to the interpeptide bridge. To obtain this additional information, nucleotide Downloaded from http://mmbr.asm.org/ on December 3, 2012 by guest
Page 1 and 2: CONTENT ALERTS implications. Peptid
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466 SCHLEIFER AND KANDLER tion of l
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