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Rapid Method for Isolation of Large Quantities of Outer Membrane from Escherichia coli K-12 and Its Application to the Study of Envelope Mutants

¹ Departments of Microbiology and Histology, University of Umeå, S-901 87 Umeå, Sweden

ABSTRACT

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.