Rapid method for isolation of large quantities of outer membrane from Escherichia coli K-12 and its application to the study of envelope mutants.
A rapid method for the isolation of large quantities of bacterial outer membrane is described. This cell envelope component was removed from plasmolyzed cells of Escherichia coli K-12 by lysozyme-ethylenediaminetetra-acetic acid treatment, aggregated by lowering the pH to 5.0, and recovered by centrifugation. Aggregates of membrane fragments were clearly identified in an electron microscope. A criterion of homogeneity of the preparation was obtained by isopycnic sucrose gradient centrifugation. A single band appeared at a density of 1.24 g/cc. The cytoplasmic membrane marker, succinate dehydrogenase activity, was 40 times lower in the outer membrane preparation than in complete cell envelope preparations. A rich activity was, however, found for the outer membrane marker, phospholipase A. The compositions of outer membranes from a transductant pair were compared. One transductant was a chain-forming, antibiotic-supersensitive envA strain, whereas the other contained the envA(+) allele. The envA strain showed a slightly modified protein pattern and a lower relative content of phosphatidylglycerol.
D001426:Bacterial Proteins / Q000032:analysis; D002247:Carbon Isotopes; D002462:Cell Membrane; D002473:Cell Wall / Q000032:analysis / Q000201:enzymology; D002499:Centrifugation, Density Gradient; D003593:Cytoplasm; D004492:Edetic Acid; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000032:analysis / Q000166:cytology* / Q000201:enzymology; D008055:Lipids / Q000032:analysis; D008070:Lipopolysaccharides / Q000032:analysis; D008722:Methods; D009113:Muramidase; D009154:Mutation; D009995:Osmosis; D010740:Phospholipases / Q000378:metabolism; D010743:Phospholipids / Q000032:analysis; D010759:Phosphorus Isotopes; D011135:Polysaccharides, Bacterial / Q000032:analysis; D014316:Tritium
D001426:Bacterial Proteins; D002247:Carbon Isotopes; D008055:Lipids; D008070:Lipopolysaccharides; D010743:Phospholipids; D010759:Phosphorus Isotopes; D011135:Polysaccharides, Bacterial; D014316:Tritium; D004492:Edetic Acid; D010740:Phospholipases; D009113:Muramidase