Isolation, purification, and chemical analysis of the lipopolysaccharide and lipid A of Acinetobacter calcoaceticus NCTC 10305.

The lipopolysaccharide of Acinetobacter calcoaceticus NCTC 10305 (London) was obtained by a modified phenol/chloroform/light petroleum method from the bacterial cells and from the culture medium in yields of 1.6% and 2.2% respectively (based on the bacterial dry weight). On chemical analysis, both preparations proved to be identical. The lipopolysaccharide obtained from the cells was purified by repeated ultracentrifugation, electrodialysis, and precipitation with sodium chloride. It was free of nucleic acids, proteins, and glycans. In the analytical ultracentrifuge, the triethylamine and sodium salt forms of the lipopolysaccharide showed a s20 value of 8.9 S and 51 S, respectively. The lipopolysaccharide consisted of glucosamine, 3-deoxy-D-manno-octulosonic acid, D-glucose, fatty acids, and phosphate in a molar ratio of 2:1:7:6:4. The fatty acids were predominantly lauric acid, 2-hydroxy, and 3-hydroxylauric acid in a molar ratio of 1:1:2. Only 3-hydroxylauric acid was found in amide linkage. On mild acid hydrolysis of the lipopolysaccharide, 65% lipid A were obtained, to which glucosamine was retained quantitatively. It still contained 50% of the original glucose, while one third (15%) of the liberated glucose was in monomeric form.