Cell Envelope and Shape of Escherichia coli K12. Crosslinking with Dimethyl Imidoesters of the Whole Cell Wall

Abstract

E. coli cells treated with the bifunctional crosslinking reagents dimethyl malonimidate, succinimidate, adipimidate, suberimidate, and sebacinimidate served for the isolation of rod-shaped "ghosts." These ghosts proved to be crosslinked over their entire surface; i.e., a macromolecule (resistant to boiling 1% Na dodecyl sulfate) the size of the cell had been created. Also, ghosts could similarly be crosslinked. In both cases, the final "sacs" contained about 60-70% protein, and very little or no lipopolysaccharide. When ghosts from which phospholipid had been removed were crosslinked, the covalently closed ghosts were almost pure protein; 80-90% of their dry mass was accounted for by protein. Ammonolysis of the crosslinked material (whether stemming from crosslinked cells or ghosts) showed that the same four proteins (Na dodecyl sulfate gel bands) had been crosslinked that are found in normally prepared ghosts. These observations practically exclude the hypothesis that a fluid mosaic model of membrane structure can be applied to the outer membrane of the E. coli cell envelope; rather, extensive protein-protein interactions must exist over the whole surface of this membrane. These findings are consistent with the possibility that the ghost polypeptide chains are involved in the determination of cellular shape.We have shown that rod-shaped "ghosts," which are surrounded by the outer membrane of the cell envelope, devoid of murein, and free from all cytoplasmic material except for remaining fragments of the cytoplasmic membrane, can be isolated from Escherichia coli cells (1, 2). These ghosts consist of about 25% phospholipid, 25-30% lipopolysaccharide, and 45-50% protein. We have shown that the protein of ghosts is separable into four main bands (I, II, III, and IV) in Na dodecyl sulfate-polyacrylamide gel electrophoresis (see Fig. 2). We have speculated that one or more of these polypeptide chains, i.e., presumably by their self assembly, could be the final products of the genetic information specifying cellular shape. One prediction following from this hypothesis is that protein-protein interactions should be existent over the whole cell envelope between one or more of the proteins mentioned. We show in this communication that such appears, indeed, to be the case. MATERIALS AND METHODSCells, Media, Growth Conditions, and Preparation of Ghosts. The E. coli K12 strain W945-T3282 [a diaminopimelate plus lysine auxotroph (3)] was used in the same way as described (1, 2). Ghosts were isolated following the recently described (1) procedure II. In brief, it involves treatment of cells with Triton X-100 in 40% sucrose, urea, trypsin, and finally lysozyme.Crosslinking. All diimidoesters were prepared essentially according to Davies' and Stark's (4) version of the method of McElvain and Schroeder (5), and all dinitriles were purchased from Schuchardt (Mfinchen, Germany). Whole cells for crosslinking were, after harvesting, washed once with 150 mM NaCl and once with 1 M triethanolamine, pH 8.5. They were suspended ...

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