The composition of the cell envelope of a heptose-deficient lipopolysaccharide mutant of Escherichia coli, GR467, was studied after fractionation into its outer and cytoplasmic membrane components by means of sucrose density gradient centrifugation. The outer membrane of GR467 had a lower density than that of its parent strain, CR34. Analysis of the fractionated membranes of GR467 indicated that the phospholipid-to-protein ratio had increased 2.4-fold in the outer membrane. The ratio in the mutant cytoplasmic membrane was also increased, although to a lesser extent. By employing a third parameter, the lipid A content of the outer membrane, it was found that the observed phospholipidto-protein change in the outer membrane was due predominantly to a decrease in the relative amount of protein. This decrease in protein was particularly significant, since it was concomitant with a 68% decrease in the lipid A recovered in the outer membrane of GR467 relative to the lipid A recovered in the outer membrane of CR34. Similar findings were observed in a second heptose-deficient mutant of E. coli, RC-59. The apparent protein deficiency in GR467 was further studied by subjecting solubilized envelope proteins to sodium dodecyl sulfatepolyacrylamide gel electrophoresis. It was found that major envelope proteins which were localized in the outer membrane were greatly diminished in GR467. Two revertants of GR467 with the wild-type amounts of heptose had wild-type relative levels of protein in their outer membranes. A partial heptose revertant had a relative level of protein in its outer membrane between those of the mutant and wild type. The organization of the gram-negative bacterial cell envelope may be considered on a number of levels: (i) the chemical composition and linear structure of the major macromolecules, e.g., lipopolysaccharide, lipoprotein, protein, phospholipid, and peptidoglycan; (ii) the stereochemistry of these macromolecules; (iii) the cross-linking of repeating units of a given macromolecule; and (iv) the bonds linking different macromolecules. These factors all contribute to the formation of a multilayered structure which supports such phenomena as active transport of small molecules, enzymatic reactions, and, indeed, the growth of existing envelope structures involving translocation of preformed subunits or in situ synthesis. The cell envelope is generally thought to consist of three distinct layers-the outermost layer containing lipopolysaccharide (LPS), protein, and phospholipid, a rigid peptidoglycan layer, and the cytoplasmic membrane consisting mainly of protein and phospholipid (7, 22, 24, 28, 37, 44). Extensive literature exists describing the chemical composition and linear structure of the LPS in gram-negative bacteria. A large portion of the information available has been derived from analyses of Salmonella strains (19). These analyses indicate that the LPS structure can be divided into three regions, designated as 0-antigen polysaccharide, core polysaccharide, and lipid A. The 0-antigen sugars, wh...

The surface of freeze-etched E. coli strain GR467, a heptose-deficient ("deep rough") mutant derived from CR34, was studied by electron microscopy. The outer membrane of GR467 has an increased ratio of phospholipid to protein, mainly due to a decreased protein content. Freeze-etched CR34 showed structural features indistinguishable from wild-type E. coli, i.e., the primary cleavage occurring in the inner membrane with only minor appearances of cleavage within the outer membrane. In contrast to this, in mutant GR467 most of the freeze-cleavages had taken place along a new plane, presumably in a hydrophobic region of the outer membrane. In this cleavage plane numerous particles were seen. Often the cleavage extended over the entire exposed cell surface; occasionally only a few large plateaus were visible, around which the next deeper cleavage plane, that of the protoplasmic or inner membrane, was discernible. Two spontaneous revertants (R11 and R16) with protein and lipid A levels similar to wild-type cells showed mostly freeze fractures with wild-type characteristics, and only a few cells had retained fracturing properties of GR467. A partial revertant revealed intermediate characteristics.Thus, there appears to be a morphological correlation with the chemical data relating the amount of outer membrane protein with the heptose content of the lipopolysaccharide.The components of the cell envelope of Gram negative bacteria, such as Escherichia coli, are arranged in two composite layers: (i) the outer membrane (OM) to which is attached the "rigid" peptidoglycan, and, still deeper in the envelope, (ii) the inner membrane (IM), surrounding the protoplasmic contents of the cell. In E. coli the OM is composed of lipopolysaccharide (LPS), proteins, and phospholipid, whereas the IM is largely composed of phospholipid and protein.The 15. The medium used for growth was Trypticase soy broth (BBL) supplemented with 10 ,g of thymine per ml. The cells were grown at 30' in tubes with aerators and harvested in the logarithmic state of growth at densities of 2 to 4 X 108 cells per ml. The cells were sedimented by spinning for 10 min at 6000 X g, and portions of the pellets were quickly frozen on gold grids in Freon at liquid nitrogen temperature. Care was taken that all preparations were handled at the same temperatures. The specimens were subsequently freeze-etched at -100°in a Balzer BM 360 unit and shadowed with platinum-carbon before carbon replication (see ref. 13). Micrographs of the replicas were taken in a Siemens 101 electron microscope using Kodak electron microscope film. Negative copies were printed, so that in the final micrograph the shadows are black and the source of platinum appears as a source of illumination. RESULTSThe results of this study can best be followed by referring to the diagrammatic representation of the E. coli cleavage planes (Fig. 1). After freeze-fracture, cells of strains CR34 are structurally indistinguishable from other wild-type E. coli strains, such as E. coli B or Cla. The contour ...