Pal lipoprotein of Escherichia coli plays a major role in outer membrane integrity

Abstract

The Tol-Pal system of gram-negative bacteria is composed of five proteins. TolA, TolQ, and TolR are inner membrane proteins, TolB is a periplasmic protein, and Pal, the peptidoglycan-associated lipoprotein, is anchored to the outer membrane. In this study, the roles of Pal and major lipoprotein Lpp were compared in Escherichia coli. lpp and tol-pal mutations have previously been found to perturb the outer membrane permeability barrier and to cause the release of periplasmic proteins and the formation of outer membrane vesicles. In this study, we showed that the overproduction of Pal is able to restore the outer membrane integrity of an lpp strain but that overproduced Lpp has no effect in a pal strain. Together with the previously reported observation that overproduced TolA complements an lpp but not a pal strain, these results indicate that the cell envelope integrity is efficiently stabilized by an epistatic Tol-Pal system linking inner and outer membranes. The density of Pal was measured and found to be lower than that of Lpp. However, Pal was present in larger amounts compared to TolA and TolR proteins. The oligomeric state of Pal was determined and a new interaction between Pal and Lpp was demonstrated.

FIG. 1.

Comparison of production levels of Lpp and Pal. lpp⁺ KS272 (wt1), lpp KS303, pal⁺ JC8056 (wt2), and pal JC892 cells were used in the presence of the indicated plasmids. (A) Autoradiogram of immunoprecipitated Pal and Lpp from labeled membrane extracts using anti-Pal antibodies. (B) Western blot immunodetection of Pal and Lpp. Prestained (A) and unstained (B) size markers are indicated in kilodaltons.

FIG. 2.

In vivo and in vitro oligomerization of Pal. In vivo experiments were carried out with the lpp-ompA JC8963 strain. Western blotting using anti-Pal antibodies or Coomassie blue staining are shown. (A) In vivo and in vitro cross-linking with FA cross-linked samples were treated at 37°C (FA) and 96°C (FA96). (B) In vivo cross-linking of lpp-ompA cells with FA, EGS, or DST. Control cells were treated at 37°C (C) and 96°C (C96). Pal multimers (P2 and P3) and size markers (in kilodaltons) are indicated.

FIG. 3.

Purified Pal forms dimers. Serial dilutions of native Pal (indicated in μM) were cross-linked with FA, DSP, and EGS, and samples were analyzed by Western blot immunodetection with anti-Pal antibody in the absence of heat denaturation. Size markers and Pal multimers (P2 and P3) are indicated.

FIG. 4.

Pal interacts in vivo with Lpp and OmpA. (A) In vivo cross-linking of JC8056 cells (wt) treated with FA, EGS, or DST. (B) In vivo cross-linking of the indicated strains with FA. The antibodies used for immunodetections and the different proteins and complexes are indicated.