Live-cell fluorescence imaging was performed by placing cells on a 2% (wt/vol) agarose pad prepared in resuspension medium and set using a Gene Frame (Bio-Rad). When sporulating cells reached the desired time point, 200 μL of the culture was pelleted by centrifugation and then resuspended in 10 μL of resuspension medium containing the membrane dye TMA-DPH [1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate] (0.05 mM). After gentle vortexing, 2 μL of the cell suspension was spread on the agarose pad, and a coverslip was placed on top of the Gene Frame. Cells were imaged by standard epifluorescence using a Zeiss Axioplan 2 microscope equipped with 100× objective NA 1.4. Membrane fluorescence from the TMA-DPH dye was captured using an exposure time of 400 ms. Cyan fluorescent protein (CFP) and GFP images were each acquired with an acquisition time of 800 ms, except GFP-SpoIIP, which had an acquisition time of 2,000 ms.
When required, sporulating cells were treated with 50 μg/mL of cephalexin at T2 (2 h after the onset of sporulation) and then incubated for a further 1 h before preparation for imaging as described above.
When required, sporulating cells were treated with 50 μg/mL of cephalexin at T2 (2 h after the onset of sporulation) and then incubated for a further 1 h before preparation for imaging as described above.