Alterations in envelope structure of heptose-deficient mutants of Escherichia coli as revealed by freeze-etching.

Abstract

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 ...

Bacteriophage-resistant mutants of Salmonella typhimurium deficient in two major outer membrane proteins

“…We would also like to point out that the frequent fracturing of the cells through the OM differs from that found by other workers (3,26) and is caused by the high concentration of glycerol (30%, vol/vol) used in their preparation (Lounatmaa and Nanninga, in preparation). With this method, the cell wall of smooth S. typhimurium fractures through the OM much more easily than when lower concentrations of glycerol are used.…”

A Complete Lipopolysaccharide Inner Core Oligosaccharide Is Required for Resistance of Burkholderia cenocepacia to Antimicrobial Peptides and Bacterial Survival In Vivo

“…This could account for the low level of complementation with HldA alone. SAL1 was also more sensitive that K56-2 to treatment with novobiocin and SDS (data not shown), which indicated that this mutant exhibits a deeprough LPS phenotype (2,51).…”

Analysis of Borrelia burgdorferi membrane architecture by freeze-fracture electron microscopy

“…When overlying lipid bilayers are freeze cleaved, the fracture plane passes preferentially through the membrane with the lower protein/phospholipid ratio. For this reason, fractures in typical gram-negative bacteria rarely occur through the protein-rich, phospholipid-poor OM (9,32). In contrast to the OMs of gram-negative bacteria, the B. burgdorferi OM contained the preferred plane of cleavage, and this was explained by the significantly higher particle density of the B. burgdorferi CM.…”