31P Nuclear magnetic resonance and freeze-fracture electron microscopy studies on Escherichia coli. II. Lipopolysaccharide and lipopolysaccharide-phospholipid complexes

Abstract

1. 1. Freeze-fracture electron microscopy and 31P-NMR spectroscopy on native and electrodialyzed lipopolysaccharide from Escherichia coli K12 cells, both above and below the phase transition temperature, are described.

2. 2. Freeze-fracture electron microscopy of native lipopolysaccharide shows ribbon-like structures below (0 and 22°C) and large vesicles above (37°C) the phase transition temperature. Electrodialyzed lipopolysaccharide (sodium salt) occurs in ribbon-like structures at 0, 22 and 37°C if sodium lipopolysaccharide is hydrated in water. If sodium lipopolysaccharide is hydrated in Tris-HCl/NaCl buffer these ribbon-like structures occur only below the phase transition temperature. Above the phase transition temperature stacked sheets are observed. Moreover, in the latter case, the fracture planes contain particles and pits. Upon etching, sodium lipopolysaccharide when hydrated in water appears to form rods and when hydrated in buffer appears to form mainly stacked lamellae both above (37°C) and below (0°C) the phase transition temperature.

3. 3. High resolution 31P-NMR spectra show that the chemical shifts of the phosphorus atoms in native lipopolysaccharide differ from those in electrodialyzed lipopolysaccharide, probably due to conformational and compositional (the disappearance of ions and (poly)electrolytes) changes.The 31P-NMR spectra of native lipopolysaccharide dispersed in Tris-HCl/NaCl buffer are very broad at 20 and at 40°C indicating little motion. At 22°C electrodialyzed lipopolysaccharide also gives a broad spectrum; at 40°C the spectrum is narrower, indicating more motion, and two peaks are visible. After dispersion in H2O and subsequent addition of buffer, the spectrum of electrodialyzed lipopolysaccharide is narrow both at 20 and 40°C, which can be correlated with the rods observed in freeze etching.After treatment with Ca2+, electrodialyzed lipopolysaccharide shows a very broad spectrum at 40°C probably due to immobilization of the lipopolysaccharide.

4. 4. Freeze-fracture electron microscopy and 31P-NMR spectroscopy of liposomes consisting of native lipopolysaccharide and total phospholipids indicate that the phospholipids and the lipopolysaccharide are mainly organized in bilayers. Lipopolysaccharide in such liposomes undergoes more motion than in the absence of phospholipids. Ca2+ does not influence this behaviour.