FIGURE 4.

In vivo assays of DIVa binding by C-terminally truncated LtrA proteins. E. coli DH5α containing pBDG-LtrA expressing wild-type LtrA or the indicated C-terminal truncation mutants was induced with 1% L-arabinose (+Ara) and analyzed for GFP expression by plating and FACS assays and for protein expression by SDS-PAGE and immunoblotting with an anti-LtrA antibody. (A) Schematic of LtrA protein showing the location of C-terminal truncations, and plating assays for GFP fluorescence shown below. The plating assays for different mutants were done in parallel. (B) FACS assays. The FACS profiles show the number of cells in a fluorescence window before (purple) and after (green) induction with L-arabinose. Data are representative of at least two independent repeats for each construct. (C) Immunoblot analysis. SDS-PAGE and immunoblotting with anti-LtrA antibody were done as described in the Materials and Methods. Positions of Precision plus protein standards (Bio-Rad) are shown to the right of the blot. Equal loading was confirmed by Coomassie blue staining of a parallel gel (data not shown).