Primers and probes used in this study.

<p>Primers and probes used in this study.</p

Characterization of the <i>C. difficile tcdC</i> mutant.

<p>(<b>A</b>) Schematic representation of 3 different domains of TcdC and the intron insertion site for the inactivation of TcdC. The arrows in the putative repressor domain represent the locations and orientation of the primers used in the RT-q-PCR and conventional control PCRs. (<b>B</b>) PCR confirmation of the wild type strain and the CT::<i>tcdC</i> mutant. The primer EBS universal and tcdC2 generated a PCR product of 302 bp for the CT::<i>tcdC</i> strains. Primers tcdC1 and tcdC2 generated a 699 bp PCR product for the wild type and for the CT::<i>tcdC</i> strain a PCR product of circa 2800 bp. (<b>C</b>) Southern blot analysis of EcoRV digested genomic DNA of wild type and CT::<i>tcdC</i> strains with a Group II intron, <i>ermB</i> gene and <i>tcdC</i> specific probes. Note that probing with the <i>ermB</i> probe results in 2 bands for the CT::<i>tcdC</i> strains, since wild type already carries a copy of the <i>ermB</i> gene in the genome <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043247#pone.0043247-Sebaihia1" target="_blank">[35]</a>. (<b>D</b>) Western blot analysis of TcdC production in wild type and CT::<i>tcdC</i> strain 8 hours post inoculation. The arrow indicates the location of TcdC protein based on MW and absence of the protein in the PaLoc negative Type 035 strain. Note that cross-reaction of TcdC antibody with a protein of similar MW was also observed in Carter et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043247#pone.0043247-Carter1" target="_blank">[30]</a>. (<b>E</b>) Growth curves of <i>C. difficile</i> 630ΔErm and <i>C. difficile</i> CT::<i>tcdC</i> mutant strains. The absorbance (OD₆₀₀) was measured over 48 hrs of growth in TY medium. The error bars indicate the standard error of the mean of six experiments.</p

The toxin production profiles of wild type and CT::<i>tcdC</i> mutant strains in time.

<p>Wild type corresponds to black bars, CT::<i>tcdC</i>1 mutant strains to gray bars, CT::<i>tcdC</i>2 to the white bars. Total toxin amounts were quantified by using two independent assays. (<b>A</b>) The supernatants were incubated in a ten fold dilutions series on Vero cell monolayers. After 24 hrs the cytotoxic effects were quantified by determing the toxin end point titer. Values are given as means (n = 6). (<b>B</b>) An enzyme immunoassay was used for direct quantification of the secreted toxins according manufacters protocol. The supernatants of 12 and 24 hours post inoculation were 10 times diluted. The supernatants of 48 hours post inoculation were diluted 10 and 100 times. Values are given as means ± standard error of the mean (n = 6).</p

Strains and plasmids used in this study.

<p>Strains and plasmids used in this study.</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

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

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

<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