Plasmids used in this study.

*<p>Bsu  =  DNA derived from <i>B. subtilis</i> JH642. Cdi  =  DNA derived from <i>C. difficile</i> 630Δerm.</p

<i>C. difficile</i> Spo0A binds to predicted and expected target sequences.

<p>Electrophoretic mobility shift assay using purified <i>C. difficile</i> Spo0A-DBD-6xHis and a radiolabeled DNA fragments. X  =  no protein control, the triangle indicates 1.3-fold serial dilutions of protein to the indicated concentrations. The arrows indicate DNA:protein complexes. <b>A. </b><i>In silico</i> predicted target sequences upstream of the genes encoding Spo0A (<i>spo0A</i>), σH (<i>sigH</i>), a lipoate ligase (<i>lplA</i>) and an aliphatic sulphonates ABC transporter (<i>ssuA</i>). <b>B.</b> Target sequences predicted on the basis of findings in other organisms: <i>spoIIAA</i>, <i>spoIIE</i> and <i>spoIIGA</i>. The DNA upstream of <i>spoVG</i> serves as a negative control.</p

<em>C. difficile</em> 630Δerm Spo0A Regulates Sporulation, but Does Not Contribute to Toxin Production, by Direct High-Affinity Binding to Target DNA

<div><p><em>Clostridium difficile</em> is a Gram positive, anaerobic bacterium that can form highly resistant endospores. The bacterium is the causative agent of <em>C. difficile</em> infection (CDI), for which the symptoms can range from a mild diarrhea to potentially fatal pseudomembranous colitis and toxic megacolon. Endospore formation in Firmicutes, including <em>C. difficile</em>, is governed by the key regulator for sporulation, Spo0A. In <em>Bacillus subtilis</em>, this transcription factor is also directly or indirectly involved in various other cellular processes. Here, we report that <em>C. difficile</em> Spo0A shows a high degree of similarity to the well characterized <em>B. subtilis</em> protein and recognizes a similar binding sequence. We find that the laboratory strain <em>C. difficile</em> 630Δerm contains an 18bp-duplication near the DNA-binding domain compared to its ancestral strain 630. <em>In vitro</em> binding assays using purified C-terminal DNA binding domain of the <em>C. difficile</em> Spo0A protein demonstrate direct binding to DNA upstream of <em>spo0A</em> and <em>sigH</em>, early sporulation genes and several other putative targets. <em>In vitro</em> binding assays suggest that the gene encoding the major clostridial toxin TcdB may be a direct target of Spo0A, but supernatant derived from a <em>spo0A</em> negative strain was no less toxic towards Vero cells than that obtained from a wild type strain, in contrast to previous reports. These results identify for the first time direct (putative) targets of the Spo0A protein in <em>C. difficile</em> and make a positive effect of Spo0A on production of the large clostridial toxins unlikely.</p> </div

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

Purification and detection of <i>C. difficile</i> Spo0A. A.

<p>Heterologous overproduction of Spo0A-6xHis and Spo0A-DBD-6xHis in <i>E. coli</i> Rosetta(DE3) pLysS. M  =  molecular weight marker, numbers indicate hours after induction with 1 mM IPTG. P  =  metal affinity purified protein. Lysates were separated on a 12% SDS-PAGE. <b>B </b><i>and </i><b>C.</b> Immunoblot detection of Spo0A in total cell lysates of a <i>C. difficile spo0A</i> mutant (CT::spo0A) and a wild type strain (630Δerm). Times indicated range from early exponential (3 h post inoculation) to late stationary growth phase (48 h post inoculation). Sample volumes were corrected for OD600 to ensure loading of similar amounts of total cell lysate in each lane. For details see Materials and Methods. M  =  molecular weight marker. <b>B.</b> ECL+ detection. <b>C.</b> Fluorescent detection. Y-axes show peak volumes normalized to values at 48 hours post inoculation (closed diamonds; left axis) and optical density readings at 600 nM (open squares; right axis). Inset shows the blot on which the curve is based. Vertical dashed line indicates the moment Spo0A levels increase sharply (6 hours post inoculation).</p