Characterization of lipopolysaccharide from a heptoseless mutant of Escherichia coli by carbon 13 nuclear magnetic resonance

Lipopolysaccharide (LPS) isolated from Escherichia coli D31m4, a heptoseless mutant, was studied by ¹³C and ³¹P NMR spectroscopy. Modified isolation and purification procedures are described which permitted high resolution NMR spectra to be obtained from samples of intact LPS. ³¹P NMR was used to monitor the purity and native heterogeneity of LPS samples. The anomeric carbon region of the ¹³C NMR spectrum taken at pH 7 contained five resonances that were assigned on the basis of chemical shift correlation, ¹³C-¹H couplings, and T₁ relaxation times. Two resonances, at 99.9 and 100.8 ppm, were attributed to two residues of 3-deoxy-D-manno-octulosonate (KDO) of which both were tentatively assigned to the α configuration. The Lipid A moiety gave rise to resonances at 94.0 and 94.9 ppm, both assigned to GlcN(I), and a resonance at 103.1 ppm, both assigned to glcN(II). The two anomeric carbon resonances observed for GlcN(I) reflected the variable substitution of C-1 with monophosphate or diphosphate groups. GlcN(I) and GlcN(II) were ascertained to be of the α and β anomeric configuration, respectively, through chemical shift comparisons with model saccharides. The accepted KDO linkage site at C-3' of GlcN(II) appears not to be supported by the ¹³C chemical shift data.