Figure 4
From: CD25890, a conserved protein that modulates sporulation initiation in Clostridioides difficile
Increased expression of spo0A in the \(\Delta\)CD25890 mutant. A: Microscopy analysis of C. difficile cells carrying a PspoOA-SNAPCd transcriptional fusion in the WT and congenic \(\Delta\)CD25890 mutant. The cells were collected after 10 h of growth in SM broth, stained with TMR-Star, and examined by fluorescence microscopy to monitor SNAP production. The panels are representative of the expression patterns observed. The numbers refer to the percentage of cells showing SNAP fluorescence. Data shown are from one experiment and are representative of at least three independent experiments. Scale bar, 1 µm. B: Quantitative analysis of the fluorescence intensity (Fl.) for single cells with no signs of sporulation (n = 100 cells) of the two strains in A. Data shown are from one experiment, representative of at least three independent experiments. The numbers in the legend represent the mean and the SD of fluorescence intensity. ***, p < 0.001. Images were acquired by Metamorph™ (version 5.8; Molecular Devices) C: Samples were collected from the wild type strain (WT), the \(\Delta\)CD25890 mutant and the complementation strain (CD25890C) grown in liquid SM, at the indicated times after inoculation. Extracts were prepared and proteins (15 µg) resolved by SDS-PAGE and subjected to immunobloting using an anti-Spo0A antibody. The position of molecular weight markers (in kDa) is indicated on the left side of the panels, and the arrow on the right indicates the position of Spo0A. D: Spo0A accumulation was assessed by quantification of the intensity of bands in the immunoblots using the Image J software and is shown as the ratio between the \(\Delta\)CD25890 mutant and the WT (red bars) or the complementation strain (green bars) at the indicated times (in hours) after inoculation in liquid SM. All data represent the mean ± SD from three independent experiments.
