Fig. 8.

The fmt mutant is oligosporogenous and is defective in membrane fusion. (a) The selected strains were induced to sporulate using a resuspension protocol. Efficiency of spore formation for WT (CU1065) and an isogenic fmt mutant (HB21006) was monitored by plating 3 µl of 10-fold cell dilutions (left to right; 10⁰ to 10⁻⁵) with and without treatment at 80 °C for 30 min to inactivate vegetative cells and was then incubated at 37 °C for 24 h. The photograph is representative of three biological replicates. Additional growth is seen on the fmt plate after 48 h, but there is still a nearly 100-fold reduction in colonies after heating as before, consistent with a low frequency of successful sporulation. (b) Sporulation progress for the WT and fmt mutant as monitored 4 h after resuspension using a membrane fusion assay with staining with FM 4-64 and MitoTracker Green [37]. The arrows (left panel) indicate cells that have completed engulfment and membrane fusion, as indicated by the green staining. In the right panel, cells stain with both dyes, indicative of a block at the membrane fusion step (similar results were seen after 24 h, with cells that had engulfed the forespore but had been blocked in membrane fusion).