A Spo0A box is important for high affinity binding by C. difficile Spo0A. A.

Domain organization of Spo0A. The site of the duplication in strain 630Δerm identified in this study is indicated by an arrow. B. Sequence alignment of the C-terminal regions of the Spo0A proteins of B. subtilis 168 and C. difficile strains 630 and 630Δerm. Residues identified in structural studies using Bacillus Spo0A as involved in backbone interactions are indicated in yellow, residues forming base-specific contacts are indicated in red [13]. The region of the 6aa duplication and the helix-turn-helix motifs are boxed in gray and the duplication in strain 630Δerm is underlined. C. PCR showing the presence of the duplication near the DNA binding domain in C difficile 630Δerm compared to 630. D. Electrophoretic mobility shift assay using purified C. difficile Spo0A-DBD-6xHis and a radiolabeled PabrB DNA fragment. X  =  no protein control, the triangle indicates 1.3-fold serial dilutions of protein to the indicated concentrations. The arrow indicates a DNA:protein complex. E. Electrophoretic mobility shift assays without (−) or with (+) 150 nM Spo0A-DBD-6xHis added to radiolabeled PabrB fragments carrying mutations in the consensus Spo0A box (in red). Arrows indicate DNA:protein complexes. The negative control is PcitG from B. subtilis.