MATURATION AND LOCALIZATION OF THE TOLB PROTEIN REQUIRED FOR COLICIN IMPORT

The tolB gene has been shown previously to encode two proteins of 47.5 kDa (TolB) and 43 kDa (TolB). To explain the presence of these two f orms, two hypotheses have been proposed: TolB might be posttranslation ally processed to TolB8, or an internal in-frame translation initiatio n resulting in TolB may occur (S. K. Levengood and R. E. Webster, J. Bacteriol. 171:6600-6609, 1989). To address this question, TolB was ta gged by inserting in its C-terminal region an epitope recognized by mo noclonal antibody 1C11 without altering the function of TolB. It was t hen demonstrated that the functional protein corresponded to TolB, th e mature periplasmic protein, and that TolB was its precursor form, wh ich was observed only when the protein was overexpressed. These two fo rms were purified by immunoprecipitation, and their N-terminal sequenc es were determined. An antibody directed against TolB was raised, whic h confirmed the results obtained with the tagged TolB.