Dependencies for the activation of the cell type-specific sporulation sigma factors.

<p>(A) qRT-PCR analysis of <i>gpr</i>, <i>spoIIIAA</i>, <i>sspA</i> and <i>cotE</i> gene transcription in strain 630Δ<i>erm</i> (wt), and in congenic <i>sigF</i>, <i>sigE</i>, <i>sigG</i> and <i>sigK</i> mutants. RNA was extracted from cells collected 14 hours (<i>gpr</i> and <i>spoIIIAA</i>), 19 hours (<i>sspA</i>) and 24 hours (<i>cotE</i>) after inoculation in SM liquid medium. Expression is represented as the fold ratio between the wild type (wt) and the indicated mutants. Values are the average ± SD of two independent experiments. (B) Cell type-specific expression of transcriptional fusions of the <i>gpr</i>, <i>spoIIIAA</i>, <i>sspA</i> and <i>cotE</i> promoters to <i>SNAP^Cd</i> in the wild type and in the indicated congenic mutants. For each of the strains, expressing the indicated fusions, cells were collected from SM cultures 24 h after inocculation and labeled with TMR-Star (red) and with the membrane dye MTG (green). Following labeling, the cells were observed by phase contrast (PC) and fluorescence microscopy. Merge is the overlap between the TMR-Star (red) and MTG (green) channels. The images are ordered, and the morphological classes defined as in the legend for <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003782#pgen-1003782-g004" target="_blank">Figure 4</a>. The numbers refer to the percentage of cells at the represented stage showing SNAP fluorescence. The data shown are from one experiment, representative of three independent experiments. The number of cells analysed for each class, n, is as follows: class <i>a</i>, 30–50; class <i>b</i>, n = 50–60; class <i>c</i>, n = 30–40; class <i>d</i>, n = 15–25; for <i>sigF/E</i> mutants, n = 80–120; for <i>sigG</i> and <i>sigK</i> mutants, n = 40–50. Scale bar: 1 µm.</p

Temporal and cell type-specific expression of <i>sigF</i>, <i>sigE</i>, <i>sigG</i> and <i>sigK</i> during sporulation.

<p>Microscopy analysis of <i>C. difficile</i> cells carrying fusions of the <i>sigF</i>, <i>sigE</i>, <i>sigG</i> and <i>sigK</i> promotors to <i>SNAP^Cd</i> in strain 630Δ<i>erm</i> (wt) and in the indicated mutants. The cells were collected after 24 h of growth in SM liquid medium, stained with TMR-Star and the membrane dye MTG, and examined by phase contrast (PC) and fluorescence microscopy to monitor SNAP production. The merged images shows the overlap between the TMR-Star (red) and MTG (green) channels. The panels are representative of the expression patterns observed for different stages of sporulation, ordered from early to late for the wild type strains according to the morphological classes <i>a</i>-<i>d</i> defined in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003782#pgen-1003782-g001" target="_blank">Figure 1</a>, as indicated. For the <i>sig</i> strains, the morphological stage characteristic of each mutant is indicated. An extra class that accounts for pre-divisional cells (PD) was introduced for the analysis of both <i>sigF</i> and <i>sigE</i> transcription. The numbers refer to the percentage of cells at the represented stage showing SNAP fluorescence. The data shown are from one representative experiment, of three performed independently. The number of cells analysed for each class, n, is as follows: PD, n = 100–150; class <i>a</i>, n = 30–50; class <i>b</i>, n = 50–60; class <i>c</i>, n = 30–40; class <i>d</i>, n = 15–25; for <i>sigF/E</i> mutants, n = 80–120; for <i>sigG</i> and <i>sigK</i> mutants, n = 40–50. Scale bar: 1 µm.</p

Quantitative analysis of <i>sigF</i>, <i>sigE</i>, <i>sigG</i> and <i>sigK</i> activities during sporulation.

<p>(A) SDS-PAGE gel and Western Blot analysis of extracts from 630Δ<i>erm</i> carrying fusions of <i>gpr</i>, <i>spoIIIAA</i>, <i>sspA</i> and <i>cotE</i> to SNAP. Black arrows point to unlabeled SNAP^Cd protein, while red arrows point to SNAP after TMR-Star labeling (distinguishable in the WB from the unlabeled form by a shift in protein migration). The TMR-Star fluorescent signal from the SDS-PAGE gel was obtained using a fluorimager. TMR-Star incorporation as well as the amount of protein loaded is indicated for each lane. 630Δ<i>erm</i> carrying pFT47 empty vector was used as a negative control of SNAP production. (B) Quantitative analysis of the SNAP fluorescence (Fl.) signal in different cell types of the reporter strains for σ^F, σ^E, σ^G and σ^K activity, as indicated. The numbers in the legend represent the average ± SD of fluorescence intensity for the cell class considered (NB: 50 cells were analysed for each cell type). The average fluorescence intensity (all classes included) from P<i>_cotE</i>-<i>SNAP^Cd</i> is 1.9±1.3 for the wild type, 1.3±0.8 for a <i>sigG</i> mutant and 0.3±0.1 for the <i>sigK</i> mutant. Data shown are from one experiment, representative of three independent experiments. Schematic representation of the deduced spatial and temporal pattern of transcription is shown for the different fusions (with darker red denoting increased transcription). The cell membrane is represented in green. No activity was seen in predivisional cells for any of the σ factors (not represented). PD, pre-divisional cell; MC, mother cell; FS, forespore; B, phase bright spore; <i>a</i> to <i>d</i>: sporulation classes ordered and defined as in the legend for <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003782#pgen-1003782-g004" target="_blank">Figure 4</a>.</p

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.</p

Quantitative analysis of <i>sigF</i>, <i>sigE</i>, <i>sigG</i> and <i>sigK</i> expression during sporulation.

<p>(A) Whole cell extracts were prepared from derivatives of strain 630Δ<i>erm</i> bearing the indicated plasmids or fusions, immediately after labeling with TMR-Star, indicated by the “+” sign (the “−” sign indicates control, unlabeled samples). The indicated amount of total protein was resolved by SDS-PAGE, and the gels scanned on a fluorimager (top) or subject to immunoblotting with anti-SNAP antibodies (bottom). Black and red arrows point to unlabeled or TMR-Star-labeled, respectively, SNAP. Strain 630Δ<i>erm</i> carrying pFT47 (empty vector) was used as a negative control for SNAP production. The position of molecular weight markers (in kDa) is indicated. (B) Quantitative analysis of the fluorescence (Fl.) intensity in different cell types of the reporter strains for <i>sigF</i>, <i>sigE</i>, <i>sigG</i> and <i>sigK</i> transcription, as indicated. The numbers in the legend represent the average ± SD of fluorescence intensity for the cell class considered (n = 50 cells analysed for each morphological cell class). The data shown are from one experiment, representative of three independent experiments. Schematic representation of the deduced spatial and temporal pattern of transcription (with darker red denoting increased transcription) is shown for each transcriptional fusion. The cell membrane is represented in green. PD, pre-divisional cell; MC, mother cell; FS, forespore; B, phase bright spore; <i>a</i> to <i>d</i>: sporulation classes 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>.</p

Total and heat resistant (Heat^R) cell counts (CFU/ml) for the wild type strain (630Δ<i>erm</i>) and congenic <i>sigF</i>, <i>sigE</i>, <i>sigG</i> and <i>sigK</i> mutants in SM.

<p>Note: values represent the average±SD of three independent experiments.</p

Functional analysis of the <i>sigK</i> gene.

<p>(A) Live/dead assay for the wild type (630Δ<i>erm</i>) and the <i>sigK</i> mutant. Shown are phase contrast and the merge between syto 9- (live cells stain; green) and propidium iodide- (dead cells stain; red) stained cells collected at 24 h of growth in SM broth. In the wild type, but not in the <i>sigK</i> mutant, development of spore refractility is accompanied by loss of mother cell viability. (B) Percentage of the sporulating cells of the wild type and <i>sigK</i> mutant strains (i.e., with visible spores) showing signs of mother cell lysis (red; propidium iodide staining) as scored by direct microscopic observation, 24 hours after inoculation in SM medium. Values are the average ± SD of three independent experiments; “n” represents the total number of cells analyzed. (C) The <i>sigK-skin</i> region of the 630Δ<i>erm</i> chromosome and plasmids used to complement <i>sigK</i> mutant strain. Replicative plasmid pFT38 carries <i>sigK</i> interrupted by a shorter version of the <i>skin</i>^Cd element, which includes the gene (CD12310) for the recombinase (in orange). Replicative plasmid pFT42 carries an uninterrupted <i>sigK</i> gene. The coding sequences are numbered according to the reanotation of the <i>C.difficile</i> genome <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003782#pgen.1003782-Monot1" target="_blank">[91]</a>. (D) Percentage of sporulation for strains 630Δ<i>erm</i> (wt), <i>sigK</i> and <i>sigK</i> bearing either pFT38 or pFT42. The indicated percentages are the ratio between the titer of heat resistant spores and the total cell titer, measured 72 h following inoculation in SM medium. Values are the average ± SD of three independent experiments. (E) Fluorescence microscopy showing the phenotype of <i>sigK</i> bearing pFT42. Cells were collected at 72 h of growth in SM broth, stained with DAPI and FM4-64, and viewed by phase contrast (PC) and fluorescence microscopy. Scale bar in (A) and (E), 1 µm.</p

Stages and cell of σ^F, σ^E, σ^G and σ^K activity.

<p>The figure compares the main periods of activity of the 4 cell type-specific sigma factors of sporulation in <i>B. subtilis</i> (A) and <i>C. difficile</i> (B). The figure incorporates data on the morphological analysis of sporulation in the <i>sigF</i>, <i>sigE</i>, <i>sigG</i> and <i>sigK</i> mutants (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003782#pgen-1003782-g002" target="_blank">Figure 2</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003782#pgen-1003782-g004" target="_blank">4</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003782#pgen-1003782-g006" target="_blank">6</a>) and on the stage, dependencies and cell where σ^F, σ^E , σ^G and σ^K are active. Solid or broken arrows represent dependencies or partial dependencies, respectively. The representation of the <i>C. difficile</i> sigma factors indicates activity; black indicates the main period of activity. Possible cell-cell signaling pathways are show by both a broken line and a question mark. The SpoIIIA-SpoIIQ/CD0125 channel is represented in yellow. PD: predivisional cell; MC: mother cell; FS: forespore. The red horizontal broken line distinguishes early (prior to engulfment completion) from late (post-engulfment completion) development.</p

Oligonucleotides for expression analysis used in this study.

<p>Oligonucleotides for expression analysis used in this study.</p

Virulence of the <i>Bacillus cereus</i> bv <i>anthracis</i> and CA derivative strains by the subcutaneous route in guinea pigs.

<p>Guinea pigs were inoculated with graded spore inocula of each strain by subcutaneous route in the flank (four animals per dose). The presence of pBCXO1 and PBCXO2 and the gene inactivated on pBCXO1 is specified where applicable. Results are expressed as mean lethal dose (LD50) and mean time to death in days (MTD, mean ± SD). Each experiment was performed at least twice.</p><p>Virulence of the <i>Bacillus cereus</i> bv <i>anthracis</i> and CA derivative strains by the subcutaneous route in guinea pigs.</p