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.