Mutagenesis of Peb1 as well as analysis of Peb1 expression and secretion in representative mutant library clones. (A) Distribution of the five-residue MGS-generated insertions in Peb1 and schematic presentation of Peb1 with the transport-affecting regions TAR1 (pink) and TAR2 (yellow) identified on the basis of the transposon mutagenesis indicated. (B) SDS-PAGE analysis of cell-free culture supernatant of MKS12 carrying the plasmids indicated (top panel). Western blot analysis with anti-Peb1 antibodies shows the level of Peb1 expression in whole bacterial cells of the various clones (second panel from the top) and verifies that the secreted protein is Peb1 (third panel from the top). Western blot analysis with anti-GroEL antibodies in cells and supernatants was used for assessment of cell lysis (two bottom panels). Mutants p9G2 and p4G8 are examples of clones that secrete Peb1 well (neutral mutants) and carry the insertions at positions H25 and G106, respectively. Mutants p4G10 and p4B7 have the insertion in the TAR1 region, whereas the insertion is in the TAR2 region in mutants p3D9 and p8C3. Mutant p1B6 has an insertion in the promoter region (at -87 nt) of the fliC-peb1 construct, which abolishes Peb1 expression. Wt Peb1 is encoded by plasmid pLA25 and pMCS3'UTR is the vector control. Cell-free culture supernatant (S) and whole bacterial cell samples (C) corresponding to 100 μl of bacterial culture were analyzed. Molecular weight markers in kDa are indicated on the left.