A Spo0A box is important for high affinity binding by <i>C. difficile</i> Spo0A. A.

<p>Domain organization of Spo0A. The site of the duplication in strain 630Δerm identified in this study is indicated by an arrow. <b>B.</b> Sequence alignment of the C-terminal regions of the Spo0A proteins of <i>B. subtilis</i> 168 and <i>C. difficile</i> strains 630 and 630Δerm. Residues identified in structural studies using <i>Bacillus</i> Spo0A as involved in backbone interactions are indicated in yellow, residues forming base-specific contacts are indicated in red <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048608#pone.0048608-Zhao1" target="_blank">[13]</a>. The region of the 6aa duplication and the helix-turn-helix motifs are boxed in gray and the duplication in strain 630Δerm is underlined. <b>C.</b> PCR showing the presence of the duplication near the DNA binding domain in <i>C difficile</i> 630Δerm compared to 630. <b>D.</b> Electrophoretic mobility shift assay using purified <i>C. difficile</i> Spo0A-DBD-6xHis and a radiolabeled P<i>abrB</i> 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. <b>E.</b> Electrophoretic mobility shift assays without (−) or with (+) 150 nM Spo0A-DBD-6xHis added to radiolabeled P<i>abrB</i> fragments carrying mutations in the consensus Spo0A box (in red). Arrows indicate DNA:protein complexes. The negative control is P<i>citG</i> from <i>B. subtilis</i>.</p>