In vitro characterization of peptidoglycan-associated lipoprotein (PAL)-peptidoglycan and PAL-TolB interactions

Abstract

The Tol-peptidoglycan-associated lipoprotein (PAL) system of Escherichia coli is a multiprotein complex of the envelope involved in maintaining outer membrane integrity. PAL and the periplasmic protein TolB, two components of this complex, are interacting with each other, and they have also been reported to interact with OmpA and the major lipoprotein, two proteins interacting with the peptidoglycan. All these interactions suggest a role of the Tol-PAL system in anchoring the outer membrane to the peptidoglycan. Therefore, we were interested in better understanding the interaction between PAL and the peptidoglycan. We designed an in vitro interaction assay based on the property of purified peptidoglycan to be pelleted by ultracentrifugation. Using this assay, we showed that a purified PAL protein interacted in vitro with pure peptidoglycan. A peptide competition experiment further demonstrated that the region from residues 89 to 130 of PAL was sufficient to bind the peptidoglycan. Moreover, the fact that this same region of PAL was also binding to TolB suggested that these two interactions were exclusive. Indeed, the TolB-PAL complex appeared not to be associated with the peptidoglycan. This led us to the conclusion that PAL may exist in two forms in the cell envelope, one bound to TolB and the other bound to the peptidoglycan.

FIG. 1

The purified PAL lipoprotein interacts in vitro with the peptidoglycan. Purified PAL or purified TolRIIHis were incubated with peptidoglycan (+ Peptidoglycan) as described in Materials and Methods. A control incubation was carried out with PAL without peptidoglycan (PAL). After ultracentrifugation and washing as described in Materials and Methods, the Supernatant (S), wash (W), and pellet (P) fractions were loaded on an SDS–15% PAGE gel and electrotransfered to a nitrocellulose membrane, and proteins were detected with antiserum directed against PAL or TolR (Ab anti-PAL or Ab anti-TolR). The positions of the molecular mass markers are indicated on the left.

FIG. 2

The PAL peptide competes with PAL for binding to the peptidoglycan. (A) Different amounts of the PAL peptide (0, 20, and 100 μg) were added to the PAL-peptidoglycan binding assay. The different fractions were analyzed by SDS–15% PAGE by immunoblotting with antibodies directed against PAL. (B) One hundred micrograms of the PAL peptide or 250 μg of the TolR peptide was added to the PAL-peptidoglycan binding assay. The different fractions were analyzed on an SDS–15% PAGE gel by immunoblotting with antibodies directed against PAL. The rest of the procedure was as described in the legend to Fig. 1.

FIG. 3

The PAL peptide competes with PAL for binding to TolB. (A) Purified PAL and TolBHis proteins can be cross-linked in vitro. Purified PAL and TolBHis proteins were mixed (PAL + TolB) or treated independently with 1% formaldehyde (see Materials and Methods). An in vivo cross-linking was performed as a control to localize the Tol-PAL complex on whole cells overproducing the TolB and PAL proteins (JC7752 pBP). The samples were analyzed by SDS–12% PAGE and by immunoblotting with antibodies directed against TolB and PAL (Ab anti-TolB and Ab anti-PAL). (B) Purified PAL, purified TolBHis, and increasing amounts of the PAL peptide (0 to 100 μg) or 250 μg of the TolR peptide were treated with 1% formaldehyde as described in Materials and Methods. The samples were analyzed by SDS–12% PAGE and by immunoblotting with an antiserum directed against TolB (Ab anti-TolB). The positions of the TolB and PAL proteins, of the TolB-PAL complex, and of the molecular mass markers are indicated.

FIG. 4

The TolB-PAL complex does not interact in vivo with the peptidoglycan. Whole cells overproducing the TolB and PAL proteins (JC7752 transformed with pBP) were treated with (F) or without (C) 1% formaldehyde, and after the treatment the cell envelopes were prepared and the cell lysate was kept (Lysate). The non-peptidoglycan-associated fraction (37°C) and the peptidoglycan-associated fraction (60°C) were prepared as described in Materials and Methods. In parallel, a sample was treated directly at 60°C (60°C direct). The different fractions (equivalent to 0.3 OD₆₀₀ unit) were analyzed by SDS–12% PAGE and by immunoblotting with antibodies directed against TolB and PAL. The positions of the TolB and PAL proteins, of the TolB-PAL complex, and of the molecular mass markers are indicated.