Reassociation of Purified Lipopolysaccharide and Phospholipid of the Bacterial Cell Envelope: Electron Microscopic and Monolayer Studies

Phosphatidyl ethanolamine and lipopolysaccharide were extracted and purified from the cell envelope fractions of Escherichia coli and Salmonella typhimurium. The two components were studied separately and after recombination, by use of electron microscopy and monolayer techniques, and by measuring their ability to participate in the enzyme-catalyzed uridine diphosphate-galactose:lipopolysaccharide α, 3 galactosyl transferase reaction, which requires a lipopolysaccharide-phospholipid complex as substrate. Electron microscopy of purified lipopolysaccharide showed a uniform population of hollow spheres, with each sphere bounded by a continuous leaflet. The diameter of the spheres was approximately 500 to 1,000 A, and the thickness of the enveloping leaflet was approximately 30 A. Phosphatidyl ethanolamine showed a regular lamellar structure. When lipopolysaccharide and phosphatidyl ethanolamine were mixed under conditions of heating and slow-cooling, the leaflet of the lipopolysaccharide spheroids appeared to extend directly into the phosphatidyl ethanolamine structure, with continuity between the two leaflets. Various stages of penetration were seen. At high concentrations of lipopolysaccharide, there were disruptive changes in phosphatidyl ethanolamine leaflets similar to those seen when saponin acts on cholesterol-lecithin leaflets. Monolayer experiments indicated that lipopolysaccharide penetrated a monomolecular film of phosphatidyl ethanolamine at an air-water interface, as revealed by an increase in surface pressure. The results indicate that a common leaflet structure containing lipopolysaccharide and phosphatidyl ethanolamine may be formed in vitro, and suggest that a similar leaflet may exist in the intact bacterial cell envelope.

[1]  N. Sharon.  The bacterial cell wall. , 1969, Scientific American.

[2]  L. Rothfield,et al.  Role of phospholipids in the enzymatic synthesis of the bacterial cell envelope. , 1966, Federation proceedings.

[3]  K. Knox,et al.  THE CHEMISTRY AND ELECTRON MICROSCOPY OF AN EXTRACELLULAR LIPOPOLYSACCHARIDE FROM ESCHERICHIA COLI , 1966, Annals of the New York Academy of Sciences.

[4]  L. Rothfield,et al.  The role of cell envelope phospholipid in the enzymatic synthesis of bacterial lipopolysaccharide. Structural requirements of the phospholipid molecule. , 1966, The Journal of biological chemistry.

[5]  L. Rothfield,et al.  The role of cell envelope phospholipid in the enzymatic synthesis of bacterial lipopolysaccharide: binding of transferase enzymes to a lipopolysaccharide-lipid complex. , 1965, Biochemical and biophysical research communications.

[6]  S. Mergenhagen,et al.  ULTRASTRUCTURE OF VEILLONELLA AND MORPHOLOGICAL CORRELATION OF AN OUTER MEMBRANE WITH PARTICLES ASSOCIATED WITH ENDOTOXIC ACTIVITY , 1964, Journal of bacteriology.

[7]  L. Rothfield,et al.  THE ROLE OF CELL-WALL LIPID IN THE BIOSYNTHESIS OF BACTERIAL LIPOPOLYSACCHARIDE. , 1964, Proceedings of the National Academy of Sciences of the United States of America.

[8]  A. Grollman,et al.  O-PHOSPHORYLETHANOLAMINE: A COMPONENT OF LIPOPOLYSACCHARIDE IN CERTAIN GRAM-NEGATIVE BACTERIA. , 1964, Biochemistry.

[9]  J. Lucy.  GLOBULAR LIPID MICELLES AND CELL MEMBRANES. , 1964, Journal of theoretical biology.

[10]  L. Rothfield,et al.  BIOSYNTHESIS OF BACTERIAL LIPOPOLYSACCHARIDE. II. INCORPORATION OF GLUCOSE AND GALACTOSE CATALYZED BY PARTICULATE AND SOLUBLE ENZYMES IN SALMONELLA. , 1964, The Journal of biological chemistry.

[11]  L. Rothfield,et al.  Lipopolysaccharide of the Gram-Negative Cell Wall , 1964, Science.

[12]  J. Pieters,et al.  ACCURATE DETERMINATION OF MAGNIFICATION IN THE ELECTRON MICROSCOPE. , 1964, Journal of ultrastructure research.

[13]  Herbert E. Carter,et al.  PURIFICATION AND CHARACTERIZATION OF THE LIPID A COMPONENT OF THE LIPOPOLYSACCHARIDES FROM ESCHERICHIA COLI. , 1964, Biochemistry.

[14]  M. Osborn.  STUDIES ON THE GRAM-NEGATIVE CELL WALL. I. EVIDENCE FOR THE ROLE OF 2-KETO- 3-DEOXYOCTONATE IN THE LIPOPOLYSACCHARIDE OF SALMONELLA TYPHIMURIUM. , 1963, Proceedings of the National Academy of Sciences of the United States of America.

[15]  H FERNANDEZ-MORAN,et al.  Cell‐Membrane Ultrastructure: Low‐Temperature Electron Microsopy and X‐Ray Diffraction Studies of Lipoprotein Components in Lamellar Systems , 1962, Circulation.

[16]  L. Rothfield,et al.  Biosynthesis of bacterial lipopolysaccharide. I. Enzymatic incorporation of galactose in a mutant strain of Salmonella. , 1962, Proceedings of the National Academy of Sciences of the United States of America.

[17]  W. Stoeckenius.  SOME ELECTRON MICROSCOPICAL OBSERVATIONS ON LIQUID-CRYSTALLINE PHASES IN LIPID-WATER SYSTEMS , 1962, The Journal of cell biology.

[18]  S. Fleischer,et al.  The role of soluble lipid in mitochondrial enzyme systems , 1961 .

[19]  V. Luzzati,et al.  La structure des colloïdes d'association. I. Les phases liquide–cristallines des systèmes amphiphile–eau , 1960 .

[20]  V. Vand,et al.  The crystal sructure of lauric acid , 1951 .

[21]  Abraham White,et al.  The Permeability of Natural Membranes , 1944, The Yale Journal of Biology and Medicine.

[22]  D. H. Kay.  Techniques for Electron Microscopy , 1965 .

[23]  A. Bangham,et al.  NEGATIVE STAINING OF PHOSPHOLIPIDS AND THEIR STRUCTURAL MODIFICATION BY SURFACE-ACTIVE AGENTS AS OBSERVED IN THE ELECTRON MICROSCOPE. , 1964, Journal of molecular biology.

[24]  J. Lucy,et al.  STRUCTURE AND ASSEMBLY OF MACROMOLECULAR LIPID COMPLEXES COMPOSED OF GLOBULAR MICELLES. , 1964, Journal of molecular biology.

[25]  D. Dervichian.  The physical chemistry of phospholipids , 1964 .

[26]  A. Bangham.  PHYSICAL STRUCTURE AND BEHAVIOR OF LIPIDS AND LIPID ENZYMES. , 1963, Advances in lipid research.

[27]  Bangham Ad.  PHYSICAL STRUCTURE AND BEHAVIOR OF LIPIDS AND LIPID ENZYMES. , 1963 .

[28]  H. Fernández-morán.  Cell membrane ultrastructure: low-temperature electron microscopy and x-ray diffraction studies of lipoprotein components in lamellar systems. , 1962, Research publications - Association for Research in Nervous and Mental Disease.

[29]  Robertson Jd.  The molecular structure and contact relationships of cell membranes. , 1960 .

[30]  H. K. Schachman.  [2] Ultracentrifugation, diffusion, and viscometry , 1957 .

[31]  J. Finean.  X-ray diffraction studies on the polymorphism of phospholipids. , 1953, Biochimica et biophysica acta.

[32]  T. Malkin,et al.  592. An X-ray examination of synthetic (±)-α- and β-kephalins , 1951 .