Protein Composition of the Cell Wall and Cytoplasmic Membrane of Escherichia coli
01 Nov 1970-Journal of Bacteriology (American Society for Microbiology)-Vol. 104, Iss: 2, pp 890-901
TL;DR: Envelope preparations obtained by passing Escherichia coli cells through a French pressure cell were separated by sucrose density gradient centrifugation into two distinct particulate fractions, and one of the proteins which is clearly localized in the cell wall is the protein with a molecular weight of 44,000, which is the major component of the envelope.
Abstract: Envelope preparations obtained by passing Escherichia coli cells through a French pressure cell were separated by sucrose density gradient centrifugation into two distinct particulate fractions. The fraction with the higher density was enriched in fragments derived from the cell wall, as indicated by the high content of lipopolysaccharide, the low content of cytochromes, and the similar morphology of the fragments and intact cell walls. The less-dense fraction was enriched in vesicles derived from the cytoplasmic membrane, as indicated by the enrichment of cytochromes, the enzymes lactic and succinic dehydrogenase and nitrate reductase, and the morphological similarity of the vesicles to intact cytoplasmic membrane. Both fractions were rich in phospholipid. The protein composition was compared by mixing the cytoplasmic membrane-enriched fraction from a (3)H-labeled culture with the cell wall-enriched fraction from a (14)C-labeled culture and examining the resulting mixture by gel electrophoresis. Thirty-four bands of radioactive protein were resolved; of these, 27 were increased two- to fourfold in the cytoplasmic membrane-enriched fraction, whereas 6 were similarly increased in the cell wall-enriched fraction. One of the proteins which is clearly localized in the cell wall is the protein with a molecular weight of 44,000, which is the major component of the envelope. This protein accounted for 70% of the total protein of the cell wall, and its occurrence in the envelope from spheroplasts suggests that it is a structural protein of the outer membranous component of the cell wall.
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TL;DR: The cell envelope of Enterobacteriaceae consists of two membranes separated by a peptidoglycan layer, and methods have been developed to separate the cytoplasmic membrane from the outer membrane.
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TL;DR: In this paper, the sensitivity of the outer and cytoplasmic membranes of Escherichia coli to detergent was examined by isopycnic sucrose density gradient centrifugation.
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TL;DR: The sensitivity of the method for detection of changes both in soluble proteins and the insoluble membrane proteins of the cell is shown and the major membrane proteins observed and their relationship to proteins described in the literature are discussed.
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References
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TL;DR: The inner membrane-matrix fraction retained a high degree of morphological and biochemical integrity and exhibited a high respiratory rate and respiratory control when assayed in a sucrose-mannitol medium containing EDTA.
Abstract: Treatment of rat liver mitochondria with digitonin followed by differential centrifugation was used to resolve the intramitochondrial localization of both soluble and particulate enzymes. Rat liver mitochondria were separated into three fractions: inner membrane plus matrix, outer membrane, and a soluble fraction containing enzymes localized between the membranes plus some solublized outer membrane. Monoamine oxidase, kynurenine hydroxylase, and rotenone-insensitive NADH-cytochrome c reductase were found primarily in the outer membrane fraction. Succinate-cytochrome c reductase, succinate dehydrogenase, cytochrome oxidase, beta-hydroxybutyrate dehydrogenase, alpha-ketoglutarate dehydrogenase, lipoamide dehydrogenase, NAD- and NADH-isocitrate dehydrogenase, glutamate dehydrogenase, aspartate aminotransferase, and ornithine transcarbamoylase were found in the inner membrane-matrix fraction. Nucleoside diphosphokinase was found in both the outer membrane and soluble fractions; this suggests a dual localization. Adenylate kinase was found entirely in the soluble fraction and was released at a lower digitonin concentration than was the outer membrane; this suggests that this enzyme is localized between the two membranes. The inner membrane-matrix fraction was separated into inner membrane and matrix by treatment with the nonionic detergent Lubrol, and this separation was used as a basis for calculating the relative protein content of the mitochondrial components. The inner membrane-matrix fraction retained a high degree of morphological and biochemical integrity and exhibited a high respiratory rate and respiratory control when assayed in a sucrose-mannitol medium containing EDTA.
1,324 citations
"Protein Composition of the Cell Wal..." refers methods in this paper
...Total protein, radioactive protein, phospholipid, succinic dehydrogenase, and lactic dehydrogenase were assayed as previously described (19, 20)....
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...The dehydration was continued, and the pellets were embedded in Epon 812, sectioned, and stained with lead citrate and uranyl acetate as previously described (19)....
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TL;DR: It seems that cleavage of only one peptide bond adjacent to the lysine link between the lipoprotein and the murein causes the rapid decrease of the absorbance and as shown by electron microscopic exmination of ultrathin sections of trypsin treated cell walls, two separated membrane structures appear which otherwise are closely adjacent to one another.
Abstract: A decrease of the absorbance at 578 nm of a cell wall suspension of mid log phase E. coli occurs when the suspension is incubated with trypsin. The reaction is so rapid that 55% of the total decrease is obtained within the first 2 min [ratio of enzyme to total cell wall protein = 1: 50 (w/w), room temperature]. The rate of the reaction is specific for trypsin when compared with other proteases, different lipases, lysozyme and other glycosidases. A peptide bond especially sensitive to trypsin could be localized within the complex cell wall by the demonstration that the decrease of the absorbance is paralleled by the splitting of the protein from the murein.
This protein could be shown to be a lipoprotein with a part of the lipid probably covalently bound to the protein. It is called murein-lipoprotein. The link between the lipoprotein and the murein is -lysine. After trypsin digestion lysine is the only additional amino acid remaining at the murein. The ratio of the amount of lysine to the known constituents of the murein demonsstrates that on the average one lipoprotein molecule is covalently bound to every tenth repeating unit of the murein (N-acetylglucosamine–N-acetylmuramic acid–l-alanine-d-glutamic acid–meso-diaminopimelic acid–d-alanine). After 3 min incubation with trypsin, the isolated lipoprotein molecules have a lysine to arginine ratio of 4:4 as compared with 5:4 in the native molecule. The lipoprotein has an unusual amino acid comosition since it contains about 65% polar amino acids and no glycine, cysteine, proline, phenylalanine, and histidine could be found. On a weight basis the lipoprotein accounts for more than 40% of the rigid layer. Since the murein is held together exclusively by covalent bonds one can get a fairly accurate idea of the distribution of the lipoprotein molecules in the cell wall. About 105 lipoprotein molecules per cell should be distributed 103 A apart along the glycosidic chains of the murein.
The lipoprotein has an important function in stabilizing the total structure of the cell wall. It seems that cleavage of only one peptide bond adjacent to the lysine link between the lipoprotein and the murein causes the rapid decrease of the absorbance and as shown by electron microscopic exmination of ultrathin sections of trypsin treated cell walls, two separated membrane structures appear which otherwise are closely adjacent to one another.
513 citations
"Protein Composition of the Cell Wal..." refers background in this paper
...(2-4) is responsible for the "beaded" appearance of this layer (Fig....
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...(2-4), who found this to be a protein of low molecular weight (approximately 7,000) which is covalently bound to diaminopimelic acid residues of the murein....
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...Braun and Rehn (2) demonstrated that this protein contains tightly bound lipid and suggested that it serves to bind the murine layer to the other outer constituents of the cell wall....
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TL;DR: Electron micrographs of sections of Escherichia coli have shown that the wall has an extra component 20–30 A in thickness on the inside of the usual double-track profile, which is considered to contain the mucopeptide characteristic of bacteria.
Abstract: Electron micrographs of sections of Escherichia coli have shown that the wall has an extra component 20–30 A in thickness on the inside of the usual double-track profile. Demonstration was aided by...
345 citations
TL;DR: The structure conferring rigidity and shape on the complex cell wall of Escherichia coli strain B has been isolated in a state virtually free from other wall material and shows a characteristic surface pattern which indicates the fairly simple principles of its construction.
Abstract: SUMMARY: The structure conferring rigidity and shape on the complex cell wall of Escherichia coli strain B has been isolated in a state virtually free from other wall material. It shows a characteristic surface pattern which, together with observations on its mode of disintegration by phage enzyme or lysozyme, indicates the fairly simple principles of its construction.
285 citations
TL;DR: The chemical nature of the layers constituting the wall of E. coli was studied by examining the modifications induced in the ultrastructure of the wall by several enzymatic and chemical treatments, which could account for the available electron microscopic and chemical data.
256 citations