Abstract
Cross-polarization magic-angle spinning 13C and 15N NMR, rotational-echo double resonance 13C NMR, and delays alternating with nutation for tailored excitation-difference 13C NMR spectra have been obtained from lyophilized cell walls of Bacillus subtilis grown on a synthetic medium containing D,L-[2-13C, 15N]aspartate and D-[1-13C]alanine. Label from aspartate is incorporated into D-glutamic acid and m-diaminopimelic acid of cell-wall peptidoglycan, while label from alanine appears in the C-1 positions of both D- and L-alanyl residues. The cross-link index (the fraction of peptide stems joined by an isopeptide covalent bond) is obtained directly from analysis of the results of the 13C NMR experiments. However, specific isotopic enrichments of cell-wall components cannot be obtained from NMR data alone. The latter are determined either from a gas chromatographic-mass spectrometric analysis of the amino acids derived from hydrolysis of cell-wall peptidoglycan, or from a combination of NMR and gas chromatographic-mass spectrometric results. The combined analysis is overdetermined and so involves the least error for evaluations of both the cross-link index and the isotopic enrichments. The cross-link index is 0.33 +/- 0.03 for cell walls of B. subtilis grown in the presence of the antibiotic, cephalothin.
Highlights
Cross-polarizationmagic-angle spinning 13Cand 16N L-lysine or m-diaminopimelic acid (Dprn).’In some cases the NMR, rotational-echo double resonance 13CNMR, and peptidoglycan is covalently linked to polyribitol or polyglydelays alternating with nutation for tailored excita- cero1 phosphate polymers known as teichoic acids
Tion-difference “C NMR spectra have been obtained For different bacteria the sugar dimer is conserved, but’the from lyophilized cell walls of Bacillus subtilis grown peptide stems and cross-links provide a source of diversity on a synthetic medium containing D, L - [ ~ - ’ ~ C, ’ ~ N ]a~nSd -a basis for classification (3, 4)
Label from aspartate is subtilis, classified as Aly, is derived from the pentapeptide incorporated into D-glutamic acid and m-diaminopimelic acid of cell-wall peptidoglycan, while label from alanine appears in the C-1 positions of both D- and Lalanyl residues
Summary
The abbreviations used are: Dpm, diaminopimelic acid; DANTE, delays alternating with nutation for tailored excitation; CPMAS, cross-polarization magic-angle spinning; REDOR, rotational-echo double-resonance; BSSM, B. subtilis synthetic medium; MC/MS, mass chromatography-mass spectrometry. Quantification of Alanine, Diaminopimelic Acid, and Glutamic Acid-Cell-wall amino acid compositions were obtained by mass spectroscopic monitoring of the gas chromatographic separations of the N-t~fluoroacetynl -propyl ester derivatives of hydrolyzed bacterial cell walls (21).The G1u:D-A1a:Dpm ratios were obtained from the total ion current by comparisons to mixtures of known composition. Intensities of the p, p+l, and p+2 peaks obtained inscan mode, after application of a natural abundance correction, could be solved for the isotopomeric specific activities E-I, Table I
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