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

More documents

Recommendations

Info

VOL 36, 1972 PEPTIDOGLYCAN TYPES OF BACTERIAL CELL WALLS 413 c CD r- Ln u c 0 m 0 tn m 90 Ala 120 150 180 210 240 min FIG. 3. Elution profile of a partial acid hydrolysate (4 N HCl, 100 C, 2 hr) of cell walls of Sarcina lutea A TCC 383 separated on a model 120C Beckman amino acid analyzer. Conditions: one-column procedure. First, buffer A (0.2 M sodium citrate buffer, pH 3.24); after 100 min, buffer B (0.7 M sodium citrate buffer, pH 4.18). Temperature change from 30 to 55 C after 15 min. 1, L-Ala-D-Glu; 2, D-Ala-L-Ala; 3, L-Ala-L-Ala; 4, L-Lys-D-Ala; 5, 'y-D-Glu- L-Lys; 6, unidentified peptide; 7, N6-L-Ala-L-Lys. R F - - - - - - --s w~~~ '-8 1 94t 4 4 15v 16 17 18 6 46 M. Luteus hI 100 9_ 4 4 11 6 B. breve FIG. 4. Two-dimensional chromatograms of partial acid hydrolysates (4 N HCI, 100 C, 1.5 hr) of cell walls of Micrococcus luteus (A2, Fig. 7) and Bifidobacterium breve (A3a, Fig. 23). I, isopropanol-acetic acid-water (75:10:15 v/v/v); II, a-picoline-25% NH4OH-water (70:2:28 v/v/v). 1, Lys; 2, Glu; 3, Ala; 4, Gly; 5, Mur; 6, GlcNH2; 7, L-Ala-D-Glu; 8, -y-D-Glu- L-Lys; 9, L-Lys-D-Ala; 10, N6-Gly- L-Lys; 11, N6-Gly- L-Lys-D- Ala; 12, D-Ala-Gly; 13, D-Ala-L-Ala; 14, D-Glu-Gly; 15, N6-D-Ala-L-Lys; 16, N6-D-Ala-L-Lys-D-Ala; 17, Lys- D-Ala-L-Ala; 18, Mur-GlcNH2. precursors of the peptidoglycan, accumulating when growing bacteria were poisoned by vancomycin (336) or D-cycloserine (250, 341), were extracted. In some cases the precursors were 14 2 7 3 5 ._t 13 isolated from noninhibited bacteria in the stationary-growth phase (81). The nucleotide precursors were extracted with cold trichloroacetic acid from the cells, and the neutralized extract 2 12 7 3 5 Downloaded from http://mmbr.asm.org/ on December 3, 2012 by guest
414 was subjected either to column chromatography on Dowex-1 (300) or to column chromatography on Sephadex G-25 (81, 323). Fractions absorbing at 260 nm and containing bound Nacetylamino sugar (313) were pooled and chromatographed one-dimensionally on Whatman no. 3MM in isobutyric acid-0.5 M ammonia (5:3 v/v) and afterwards in 1 M ammonium acetate-ethanol (5: 2 v/v). The ultraviolet (UV)absorbing and ninhydrin-positive bands were eluted, and the amino acid sequence was elucidated by applying the previously discussed methods. We also isolated muropeptides from lysozyme lysates of cell walls (403) and determined their amino acid sequence (346). Rapid screening method. To use the peptidoglycan type as a criterion in the classification of gram-positive bacteria, it would be valuable to have a simple and rapid procedure. Especially the preparation of the cell walls is very time-consuming. Several extraction procedures were tried to prepare a relatively pure cell wall preparation from whole cells without disintegrating the cells by mechanical means. Some authors have suggested NaOH for extracting whole cells (45, 172). This method may be useful with certain organisms but had the disadvantage that it hydrolyzes some peptide linkage and can lead to a complete dissolution of the cells (17, 18). In our hands a modification of the original method of Park and Hancock (280) was most successful and will be discussed in some detail. All strains were grown in 30- to 50-ml amounts and harvested after an overnight incubation. The sediment was resuspended in 10% trichloroacetic acid. The suspension should have at 1:10 dilution an optical density of about 1.0 measured at 650 nm. The suspension was placed in a boiling-water bath for 20 min and was then centrifuged. The sedimented material was carefully rinsed with distilled water and resuspended in trypsin-phosphate buffer (2 mg trypsin/10 ml of 0.1 M phosphate buffer, pH 7.9). This suspension was incubated at 37 C on a shaker for about 2 hr and was centrifuged, and the pellet washed two times with distilled water. A sample of the cell wall preparation was hydrolyzed with 6 N HCl at 105 C for 6 hr to determine the quantitative amino acid content, and another sample was hydrolyzed with 4 N HCI in a boiling-water bath for 45 to 60 min. This partial acid hydrolysate was spotted on a two-dimensional paper chromatogram (Schleicher and Schiill 2043b, 29 by 30 cm) and was separated by descending chromatography in the solvent systems, isopropanol and a-picoline, in each direction over- SCHLEIFER AND KANDLER BACTERIOL. REV. night (about 14 hr). In some cases it was necessary to run each direction twice. The "finger prints" were compared with those of known peptidoglycan types and this comparison, together with the molar ratios of the amino acids, made it possible to recognize the peptidoglycan type. The trichloroacetic acid-extracted, trypsintreated cells yielded relatively clean cell wall preparations. In Table 4 the quantitative amino acid compositions of these preparations are compared with those of conventionally isolated and purified cell walls. The cell wall preparations obtained by the short procedure contained minor contaminating amino acids. But the contamination was usually so low that it did not interfere with the determination of the peptidoglycan type. In particular the "finger prints" were not changed by these contaminating amino acids. In most cases it was sufficient to make only the finger prints to establish the peptidoglycan type. This short procedure is very reliable and has been useful for the screening of a great number of organisms, whereby known peptidoglycan types were easily recognized. This method also can be used as a routine laboratory procedure to establish the peptidoglycan type of gram-positive organisms when it is necessary as a criterion for the classification of these organisms. Variation of the Peptide Moiety In contrast to the uniform structure of the glycan, the peptide moiety reveals considerable variation. The variety in the amino acid composition of the cell walls of gram-positive organisms has been demonstrated by the pioneering work of Cummins and Harris (75). Both qualitative and quantitative modifications were found. More detailed studies especially by Ghuysen and his co-workers, Perkins and our own group led to knowledge of how both the peptide subunit and the interpeptide bridges can vary in their composition. Variation of the peptide subunit. The peptide subunits are bound by their N-terminal amino acid to the carboxyl group of muramic acid. The amino acid sequence of the peptide subunit and its variations are depicted in Fig. 5. The amino acid linked to muramic acid is usually L-Ala, but in some cases it can be replaced by Gly or L-Ser (129, 249, 250, 288, 289, 335, 341). D-Glu in position 2 can be hydroxylated in a few coryneform bacteria and then threo-3hydroxy-glutamic acid (3-Hyg) is found instead of D-Glu (342). The hydroxylation of Glu to 3-Hyg depends very much on the oxygen supply Downloaded from http://mmbr.asm.org/ on December 3, 2012 by guest
Page 1 and 2: CONTENT ALERTS implications. Peptid
Page 3 and 4: 408 SCHLEIFER AND KANDLER BACTERIOL
Page 5 and 6: 410 glycan of gram-positive bacteri
Page 7: 412 SCHLEIFER AND KANDLER BACTER0i,
Page 11 and 12: 416 peptide bond can be substituted
Page 13 and 14: 418 SCHLEIFER AN4D KANDLER BACTERIO
Page 15 and 16: 420 SCHLEEFER AND KANDLER BACTERIOL
Page 17 and 18: 422 SCHLEIFER AND KANDLER B can be
Page 21 and 22: 426 D-alanine and through their a-a
Page 23 and 24: 428 SCHLEIFER AND KANDLER TABLE 13.
Page 25 and 26: 430 Staphylococci are usually separ
Page 31 and 32: 436 SCHLEIFER AND KANDLER antigens.
Page 33 and 34: 438 S Sero- No. of Culture SCHLEIFE
Page 35 and 36: 440 the exception of L. plantarum v
Page 37 and 38: 442 SCHLEIFER AND KANDLER L-Ala, L-
Page 41 and 42: 446 depicted in Fig. 24b. The inter
Page 43 and 44: TABLE 31. Coryneform organisms with
Page 57 and 58: 462 SCHLEIFER AND KANDLER BACTERIMI
Page 59 and 60:
464 SCHLEIFER AND KANDLER BAC-rERIO
Page 61 and 62:
466 SCHLEIFER AND KANDLER tion of l
Page 63 and 64:
468 SCHLEIFER AND KANDLER BACTrERIO
Page 65 and 66:
470 SCHLEIFER AND KANDLER BACTERIOL
Page 67 and 68:
Page 69 and 70:
Page 71 and 72:
show all

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

Create successful ePaper yourself

Delete template?

Save as template?