Lysis of spheroplasts of Escherichia coli by a non-ionic detergent

Birdsell, Dale C.; Cota-Robles, E. H.

doi:10.1016/0006-291x(68)90496-8

Cited by 36 publications

(23 citation statements)

References 4 publications

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“…These p-strand-rich proteins can produce a hydrophobic exterior only as a completely organized p-barrel structure-i.e., after the complete (or nearly complete) protein has been synthesized. The spheroplasts, however, still have fragments of outer membrane attached (25). Thus at present we cannot exclude the alternative hypothesis that porins are translocated through the membrane fusion sites but fail to become integrated into the outer membrane in the spheroplasts, which may have only limited amounts of LPS.…”

Section: Discussionmentioning

confidence: 75%

In vitro trimerization of OmpF porin secreted by spheroplasts of Escherichia coli.

Sen

Nikaido²

1990

Proc. Natl. Acad. Sci. U.S.A.

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It is not yet clear how bacterial outer membrane proteins reach their correct destination after they are secreted across the cytoplasmic membrane. We show here that porin OmpF is secreted into the medium as a water-soluble monomeric protein by spheroplasts of Escherichia coli. Furthermore, this monomeric porin is taken up by cell envelope preparations or purified lipopolysaccharides in the presence of 0.03% Triton X-100 and is converted correctly into the mature trimeric conformation. These results appear to reproduce a part of the physiological export and targeting steps of this protein.

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Section: Discussionmentioning

confidence: 75%

In vitro trimerization of OmpF porin secreted by spheroplasts of Escherichia coli.

Sen

Nikaido²

1990

Proc. Natl. Acad. Sci. U.S.A.

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“…Spheroplasts were prepared at room temperature by the lysozyme/EDTA method of Birdsell and CotaRobles [8] and re-suspended in 0.5 M sucrose, 50 mM Tris-HC1, 2 mM MgCI:, 1 mM EGTA pH 7.5 (buffer A). They were used immediately following preparation.…”

Section: Preparationsmentioning

confidence: 99%

A Transmembrane location for the proton‐translocating reduced ubiquinone→ nitrate reductase segment of the respiratory chain of Escherichia coli

Boxer

Clegg

1975

FEBS Letters

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“…Our strategy was guided by an earlier report (12) showing that treatment of plasmolyzed E. coli with lysozyme results in cells that, although they are osmotically sensitive, retain their rod shape. When the plasmolyzing sucrose is diluted, such cells round up into spheres.…”

Section: Isolation Of Rod-shaped Membranesmentioning

confidence: 99%

Cell Envelope and Shape of Escherichia coli K12

Henning

Rehn

Hoehn

1973

Proc. Natl. Acad. Sci. U.S.A.

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Rod-shaped "ghosts" that are free of murein have been isolated from E. coli. The shape of these "ghosts" is maintained by a unit membrane soluble in sodium dodecyl sulfate. Ghosts consist of about 20-30% phospholipid (almost exclusively phosphatidylethanolamine) and 50-60% protein; a large fraction of the remaining material is lipopolysaccharide. Sodium -dodecyl sulfate-gel electrophoresis reveals 4-5 different bands corresponding to molecular weights between 10,000 and 40,000. Treatment of ghosts with Pronase reduces this number to 3, and the rod shape still is not lost. Results of treatment of ghosts with a crude extract from Dictyostelium discoideum have supplied tentative evidence that at least one of these proteins is involved in the maintenance of rod shape. It does not appear too unlikely that these polypeptide chains are the final products of the genetic information specifying cellular shape.E. coli is a rod-shaped organism and, as for all other bacteria, it is not known how this shape is determined, i.e., how genetic information is translated into cellular morphology. The expression of specificity for a certain morphology must, of course, involve assembly of the cell envelope. The E. coli cell envelope consists of three layers: cytoplasmic membrane, outer membrane or cell wall, and in between the two, the murein or peptidoglycan (1-3). The murein can be isolated intact and the isolated layer, the "sacculus," retains the shape and dimensions of the cell from which it was made (4).However, the specificity we are looking for cannot reside solely in the sacculus or in its precursors. This covalently closed net is not a self-assembly system. Thus, the information required to produce sacculi of a certain shape can only involve the relevant biosynthetic enzymes in connection with a matrix onto which murein is laid down.In this communication, we report purification and some properties of a membrane from E. coli K12 that is a good candidate for such a matrix. MATERIALS AND METHODSCells, Media, and Growth Conditions. Strain W945-T3282 (subsequently called 3282), which among other markers (5) carries diaminopimelate and lysine auxotrophies, was grown at 300 in Antibiotic Medium no. 3 (Difco) under aerobic conditions. The medium was supplemented with diaminopimelate (20 ,ug/ml) and thymine (50 ,ug/ml).Isolation of Ghosts. All operations were performed at 20-250, and all solutions contained 8 mM MgSO4.Procedure I: Cells grown to 5 to 8 X 108/ml were collected by centrifugation, washed once with 0.85% NaCl, and suspended, per g wet weight, in 10 ml of 20 mM Tris HCl (pH 7.5) containing 40% sucrose. Lysozyme was added (150 2033 Ag/ml) and was allowed to act until >90% of the cells lysed upon dilution into water (about 2 hr). About 0.1 volume of chloroform was added; the suspension was shaken vigorously and then left overnight. Viscosity was broken by forcing the suspension through a hypodermic needle. The cells were centrifuged (10 min, 16,000 X g) and resuspended in the same volume of 20 mM Tris HCl (pH 9). C...

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Lysis of spheroplasts of Escherichia coli by a non-ionic detergent

Cited by 36 publications

References 4 publications

In vitro trimerization of OmpF porin secreted by spheroplasts of Escherichia coli.

In vitro trimerization of OmpF porin secreted by spheroplasts of Escherichia coli.

A Transmembrane location for the proton‐translocating reduced ubiquinone→ nitrate reductase segment of the respiratory chain of Escherichia coli

Cell Envelope and Shape of Escherichia coli K12

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