FIG 5 .

The aspartic acid residue found in the predicted EscR TMD3 sequence is critical for TMD self-oligomerization. (A) Helical wheel representation of the EscR TMD3 sequence demonstrating that three polar residues (Y164, D171, and S175) localized to one interface. The polar residues are marked with *. The EscR WT TMD3 sequence inserted between ToxR and MBP, as well as the point mutation sequences, is presented in a table. (B) LacZ activity of FHK12 bacteria expressing the ToxR-TMD-MBP chimeras. The activities of well-characterized dimerizing (GpA and Tar-1) and nondimerizing (A16 and 7L9A) TMDs are also shown. A major reduction in the oligomerization activity of EscR TMD3 was observed for the point mutation D171A, while the point mutations Y164A and S175A showed similar oligomerization activity as EscR TMD3 WT sequence. Bars represent the average (+standard deviation) from at least three independent experiments. Statistical significance was determined by Student’s t test (**, P < 0.005). (C) Correct integration of the ToxR-TMD-MBP chimera proteins was examined as described in the Fig. 3D legend. PD28 bacteria were transformed with plasmids expressing chimera proteins containing the EscR TMD3 WT sequence or the Y164A, S175A, or D171A mutation or in the absence of a TMD (ΔTM) and were grown in a minimal medium containing maltose. The ΔTM negative control showed no growth, whereas all the other constructs showed similar growth curves, indicating proper membrane integration. (D) Samples of FHK12 cells containing the ToxR-TMD-MBP chimera protein with WT EscR TMD3 and single mutations were lysed, separated, and immunoblotted using an anti-MBP antibody. The ToxR-TMD-MBP chimera protein (65 kDa) is marked with **, and the endogenous MBP (40 kDa) is marked with *.