The sporulation pathway in <i>C. difficile</i> 630Δ<i>erm</i>, and the role of <i>sigF</i>, <i>sigE</i>, <i>sigG</i> and <i>sigK</i>.

<p>Phase contrast (PC) and fluorescence microscopy analysis of spore morphogenesis in the following strains: (A) 630Δ<i>erm</i> (wild type, wt) and the congenic <i>sigF</i>, <i>sigE</i>, <i>sigG</i> and <i>sigK</i> mutants; (B) the <i>sig</i> mutants bearing the indicated plasmids or the wt strain carrying the empty vector pMTL84121. Cells were collected 24 (A) or 48 h (B) after inoculation in SM broth, and stained with DAPI and FM4-64, prior to microscopic examination. (C) Quantification of the cells in each morphological class, as defined in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003782#pgen-1003782-g001" target="_blank">Figure 1</a>, in the experiment documented in panel A, for the wt strain and the <i>sig</i> mutants. The data represent the average ± SD of three independent experiments. The total number of cells analysed (n) is indicated for each strain. (D) TEM images of <i>sigF</i>, <i>sigE</i>, <i>sigG</i> and <i>sigK</i> mutant cells. The Images are representative of the most common morphological phenotype observed for each mutant. (E) Fluorescence microscopy of 630Δ<i>erm</i> (wt) and <i>sigG</i> and <i>sigK</i> strains carrying CotE- and CotB-SNAP fusions. Cells were collected 24 h after inoculation in SM medium and labeled with the SNAP substrate TMR-Star (red channel) and the membrane dye MTG (green channel), with which a membrane-staining pattern similar to FM4-64 was obtained. The numbers on the bottom panel represent the percentage of cells which have completed the engulfment process that show localization of the protein fusions around the forespore. Data shown are from one representative experiment in which 80–100 cells were analysed for each strain. Scale bar in panels (A, B, D, E), 1 µm. Total cell extracts were prepared from 24 h SM cultures of derivatives of the 630Δ<i>erm</i>, <i>sigG</i> and <i>sigK</i> strains producing the CotE- (left) and CotB-SNAP (right) fusions, immediately after labeling with TMR-Star. Proteins (30 µg) were resolved by SDS-PAGE and the gel scanned using a fluorimager. Production of the SNAP protein in the background of 630Δ<i>erm</i> strain from the P<i>_tet</i> promoter (P<i>_tet</i>-<i>SNAP^Cd</i>, in pFT46; see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003782#pgen.1003782.s013" target="_blank">Text S1</a>), was used as a control. The position of the SNAP or SNAP fusion proteins is indicated by arrowheads. Asteriks indicate possible degradation products.