<i>C. difficile sigF</i>⁻ sigE⁻, <i>sigG⁻</i>, and <i>sigK⁻</i> sigma factor mutants are defective in mature spore formation.

<p><i>C. difficile</i> strains wildtype (WT), <i>spo0A⁻</i>, <i>sigF⁻</i>, <i>sigE⁻</i>, <i>sigG⁻</i>, and <i>sigK⁻</i> were grown on sporulation media for 18 hrs and evaluated by live phase-contrast and fluorescence microscopy. Phase contrast, FM4-64 membrane staining (red), nucleoid staining with Hoechst (blue), and the merge of these images are shown for each strain. Yellow arrowheads indicate forespore compartments that stain with FM4-64 and Hoechst; green arrowheads indicate phase-dark, immature forespores that stain with FM4-64 but not Hoechst; and pink arrowheads indicate phase-bright mature spores that exclude both the FM4-64 and Hoechst stains. Phase-bright spores were not observed in any of the mutant strains. Circular vesicles that were labeled by FM4-64, but not visible by phase-contrast microscopy (white arrows) were frequently observed in cultures grown on sporulation media, even in the <i>spo0A⁻</i> mutant (data not shown). Scale bars represent 10 µm.