Procedure for isolation of bacterial lipopolysaccharides from both smooth and rough Pseudomonas aeruginosa and Salmonella typhimurium strains

Abstract

Lipopolysaccharide (LPS) is a major component of the outer membrane of gram-negative bacteria. Within a single organism, size heterogeneity of this molecule can exist. A LPS isolation procedure which is effective in extracting smooth and rough LPS in high yields (51-81% of the LPS present in whole cells as quantitated by using hydroxy fatty acid, heptose and 2-keto-3-deoxyoctonate yields) and with a high degree of purity was developed. The contamination by protein (0.1% by weight of LPS), nucleic acids (1%), lipids (2-5%) and other bacterial products was low. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the LPS demonstrated the presence of a high degree of size heterogeneity in the isolated smooth LPS and the presence of significant amounts of rough-type LPS. The P. aeruginosa LPS interacted well with a monoclonal antibody in a variety of immunochemical analyses. The usefulness of the procedure was demonstrated by comparing LPS preparations obtained from wild-type and mutant strains of P. aeruginosa and S. typhimurium. The LPS of an antibiotic supersusceptible mutant Z61 of P. aeruginosa, which was previously characterized as identical to wild type with respect to the ratio of smooth to rough LPS molecules isolated by the phenol-water procedure, actually contained only a small proportion of O-antigenic side chains.