Fig 6.

PBP1 and PBP3 localize to the site of asymmetric division. C. difficile strains harboring either mScI3-pbp1 (a–d) or mScI3-pbp3 (e and f) expression constructs under an aTc-inducible promoter were grown on sporulating-inducing plates containing 10 ng/mL aTc for 12 h. Cells were then labeled with HADA and fixed for fluorescence microscopy. A representative cell undergoing asymmetric division is shown in the inset for each genotype. The mScI3 and HADA fluorescence along the medial axis of a cell was quantified for 20 individual cells from two independent experiments, and the fluorescent signal was normalized to the maximum fluorescence for each cell before generating aggregate curves. The mean normalized fluorescence ± standard deviation is graphed along the normalized cell distance. The level of induction by aTc on agar medium was variable, likely due to altered diffusion through the bacterial lawn on the agar surface, resulting in some bacteria showing a decreased mScI3 signal (insets in a–d). To facilitate comparison between images, the insets in panels a and d were adjusted to the same brightness/contrast, and those in panels e and f were adjusted to the same brightness/contrast. Yellow arrowheads point to the site of asymmetric division. Scale bars, 2 µm. Images are representative of at least two independent experiments.