Isolation and characterization of the outer membrane and lipopolysaccharide from Eikenella corrodens

Abstract

The chemical composition of the outer membrane fractions (OMF) of E. corrodens strains 23834 and 470 and the strain 23834 lipopolysaccharide (LPS) was determined. The OMF were obtained by Triton X-100 treatment of the heavier membrane fraction from sucrose density centrifugation of the total membrane fraction. The resulting OMF of strains 23834 and 470, free of cytoplasmic membrane components, contained 69.6 and 75.0% (wt/wt) protein, 4.8 and 9.2% lipid, 4.6 and 4.7% carbohydrate and 2.0 and 4.6% muramic acid, respectively. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE] both OMF contained major peptide determined to be 33,500 daltons for the strain 23834 OMF and 37,500 daltons for the strain 470 OMF. Analysis of the OMF fatty acids revealed hexadecanoic, hexadecenoic, octadecenoic and lesser amounts of octadecanoic acids. Transmission electron microscopic [TEM] examination of the OMF revealed typical large sheets of membrane. Structures (10 nm in diameter) resembling pores were also evident. The E. corrodens LPS was composed of 34.5% (wt/wt) carbohydrate and 25.0% lipid A. Only minute amounts of 2-keto-3-deoxyoctonate and heptose were detected. Fatty acid analysis revealed primarily octadecanoic and hexadecanoic acids, with lesser amounts of octadecenoic acid. No hydroxy fatty acids were detected. SDS-PAGE analysis showed the E. corrodens LPS to resemble other smooth-type LPS. TEM examination revealed a vesicle-like morphology. The E. corrodens LPS appears not to be a classical, i.e., enteric, type of LPS.