PaLoc in <i>C. difficile</i>.

<p><b>A.</b> Schematic diagram of PaLoc in toxigenic <i>C. difficile</i> strains. In non-toxigenic strains this region is replaced by a short 115 bp sequence. Arrows indicates the positive regulation of <i>tcdR</i>, <i>tcdB</i> and <i>tcdA</i> by σ^TcdR. <b>B</b> Predicted topology of TcdE and. λS holin Horizontal black bars represent the relative position of Cys 51 in S¹⁰⁵ and Cys 29, 56, 79, 111 and 113 in TcdE.</p

Localization of TcdE in <i>E. coli</i> and <i>C. difficile</i>.

<p><b>A.</b> Cytoplasmic and membrane proteins analysis of <i>E. coli</i> lysogens of λCmrΔ(SR) carrying pBR322 (control) or pCD463 (+<i>tcdE</i>-6xHis). <b>B.</b> Cytoplasmic and membrane proteins analysis of <i>C. difficile</i> strain carrying either pMTL84151 (control) or pRG46 (+<i>tcdE</i>-6xHis). (1) SDS-PAGE coomassie stained gel. Western blots probed with 6XHis Tag antibody (2), ATPase Beta subunit antibody (3), and Ribosomal subunits LI/L2 monoclonal antibody (4). <b>C.</b> Membrane protein samples from bacterial cells expressing TcdE-6His resuspended in denature or native sample buffers and analyzed by Western blot using His-Tag antibody. <b>D.</b> Membrane proteins of JIR8094, TcdE mutant and complemented TcdE mutant strains were harvested from bacterial cultures induced with 20 ng/ml of ATc for 2 hours, separated in 16% Tris-Glycine gel and transferred into PVDF membrane. Panels 1. Ponceau stained membrane; 2. Probed with TcdE antibody.</p

FACS analysis of <i>C. difficile</i> cells for membrane permeability through propidium iodide (PI) and SYTO staining.

<p><b>A.</b> The viability standard samples containing the heat killed and actively growing <i>C. difficile</i> cells at 1/100, 50/50 and 100/1 ratio, respectively. <b>B.</b> The <i>tcdE</i> mutant and the parent JIR8094 cells collected from the overnight (16 h) cultures, were subjected to FACS analysis following propidium iodide (PI) and SYTO staining.</p

Construction and characterization of the <i>tcdE</i> mutant in <i>C. difficile</i>.

<p><b>A.</b> PCR verification of the intron insertion using gene-specific primers OBD231 and OBD232 or the intron-specific primer EBS(U) or ERM in association with primers OBD231 or OBD232. <b>B.</b> Southern blot analysis of genomic DNA from <i>C. difficile</i> JIR8094 and <i>tcdE</i> mutant strains with an intron probe. Chromosomal DNA was digested by either <i>Eco</i>RV or <i>Hin</i>dIII. <b>C.</b> Growth curves of JIR8094 and <i>tcdE</i> mutant in TY medium. <b>D.</b> TcdA Dot blot analysis. The crude lysates prepared by sonication of cells with their supernatants (200 ng proteins) were probed with TcdA monoclonal antibody (PCG4).</p

Cell lysis in <i>C. difficile</i> A⁻B⁻ double mutant strain.

<p><b>A.</b> Growth curves of strains, 630A⁻B⁻ (<i>C. difficile tcd</i>AB double mutant), 630E (wild type) and a PaLoc negative strain. <i>C. difficile</i> strains were grown in TY medium in a 100 ml Erlenmeyer flask and the optical density at 600 nms was recorded at regular time interval. <b>B.</b> Bacterial cultures were harvested at a 30 hour time point for FACS analysis after propidium iodide (PI) and SYTO staining.</p

Bacterial strains, plasmids and phages used in this study.

<p>Bacterial strains, plasmids and phages used in this study.</p

Oligonucleotides used in this study.

<p>Oligonucleotides used in this study.</p

Complementation of TcdE mutant.

<p><b>A</b> Growth curve of parent, the TcdE mutant and the complemented TcdE mutant strains. A. The inducer ATc (20 ng/ml) was added to bacterial cultures at 4 hrs after inoculation, indicated by an arrow. The star * indicates the time point when the cultures were harvested for toxin release analysis. <b>B.</b> Toxins were quantified by ELISA from supernatants of bacterial cultures induced by 20 ng/ml ATc for 2 hours. The signal from the test was recorded as absorbance at 450 nm. The data shown are the mean +/− standard error of three replicative samples. <b>C.</b> Dot blots of culture supernatants of the parental, the TcdE mutant and the complemented TcdE mutant, induced or not induced by ATc (0 and 20 ng/ml), with monoclonal anti-TcdA. <b>D.</b> Dot blots of samples in B with monoclonal antibodies against L7/L12 ribosomal subunits.</p

Under-expressed sporulation genes in R20291::<i>sinRR’</i>.

<p>Under-expressed sporulation genes in R20291::<i>sinRR’</i>.</p

Characterization of <i>C</i>. <i>difficile</i> R20291::<i>sinR’</i>.

<p><b>(A)</b><i>C</i>. <i>difficile</i> cultures were grown in 70:30 medium for 30 h under anaerobic conditions and Sporulation frequency (CFU/ml of ethanol resistant spores) of R20291, <i>sinR’</i> mutant was determined. The data shown are means ± standard errors of three biological replicates. <b>(B)</b> Motility assays of the <i>C</i>. <i>difficile</i> R20291, <i>sinR’</i> mutant and complemented <i>sinR’</i> mutant. The experiments were repeated at least three times independently (*, <i>P</i>≤0.05 by a two-tailed Student's <i>t</i>-test). <b>(C)</b> Toxin production measured by ELISA. Statistical analysis was performed using one way-ANOVA with Dunnett’s multiple comparisons test comparing values to the average of the parent with vector control (***<0.0005, *< 0.05 <i>p</i>-value).</p