Escherichia coli tol-pal mutants form outer membrane vesicles

Abstract

Mutations in the tol-pal genes induce pleiotropic effects such as release of periplasmic proteins into the extracellular medium and hypersensitivity to drugs and detergents. Other outer membrane defective strains such as tolC, lpp, and rfa mutations are also altered in their outer membrane permeability. In this study, electron microscopy and Western blot analyses were used to show that strains with mutations in each of the tol-pal genes formed outer membrane vesicles after growth in standard liquid or solid media. This phenotype was not observed in tolC and rfaD cells in the same conditions. A tolA deletion in three different Escherichia coli strains was shown to lead to elevated amounts of vesicles. These results, together with plasmid complementation experiments, indicated that the formation of vesicles resulted from the defect of any of the Tol-Pal proteins. The vesicles contained outer membrane trimeric porins correctly exposed at the cell surface. Pal outer membrane lipoprotein was also immunodetected in the vesicle fraction of tol strains. The results are discussed in view of the role of the Tol-Pal transenvelope proteins in maintaining outer membrane integrity by contributing to target or integrate newly synthesized components of this structure.

FIG. 1

Immunoblot analyses of tolA (JC7782), tolB pal (JC7752), and wild-type (wt) parent (1292) strains. About 10⁸ cells and the supernatant of 5 × 10⁸ cells were analyzed after heat denaturation. S1 and S2 correspond to the supernatants resulting from centrifugation (20,000 × g) and ultracentrifugation (250,000 × g), respectively. Three immunodetections were carried out sequentially with antiporin (cross-reacting with OmpA), anti-β-lactamase (β-Lac), and anti-TolAIII antibodies.

FIG. 2

(a) Immunodetection of OmpFC and OmpA in tolRA (JC8031) cells, supernatants, and vesicles. Cells (lane 1), supernatant (lane 2), and resuspended vesicle pellet of ultracentrifugation (lane 3) of 5 × 10⁸ cells are shown. (b) Electron micrograph showing negative staining of the vesicle suspension (same sample as analyzed in lane 3).

FIG. 3

Thin sections of Epon-embedded tolA cells (LG10).

FIG. 4

Negative staining of tol cells and vesicles. Electron micrographs are representative of the vesicle amounts: high (a; JC7782 cells), low (b; JC7752 cells), and none (c; 1292 cells).

FIG. 5

Immunodetection of TolB and Pal in the supernatants and vesicle fractions of tolQ (TPS13) and tolR (TPS300) cells. Cells, vesicles (Ves), cell supernatant (S1), and ultracentrifuged supernatant (S2) were loaded in the same amounts as for Fig. 1 and subjected to immunodetection with anti-TolB-Pal antibodies.