Spore resistance to heat treatment is dependent on the formation of a thick layer of cell wall cortex and thus heat-resistance is a useful assay to detect sporulation efficiency. Engulfment has also been recently shown to depend on cell wall synthesis and polymerization and can be used to measure the temporal dynamics of sporulation (Meyer et al., 2010). When the genomic murG allele was deleted, serious defects in localization, engulfment, and heat resistance were observed during sporula-tion in cells expressing delocalized murG-gfp point mutants (Fig. 2C and D and Table S1). Wild type murG-gfp (JDB2501) and murG(M75E)-gfp (JDB2542) strains showing MurG localization to the forespore sporulated normally in DSM medium and their spores were heat-resistant (see Table S1). A dramatic reduction in heat resistance and engulfment was detected in V74E and F77E mutants (JDB552, JDB2559) as cells were blocked in septation. V74E and F77E show 1000-fold fewer CFU/mL postheat compared to heat resistant strains which maintain similar CFU/mL preheat and postheat. (Fig. 2B, S5 and Table S1). Finally, V81E and S67E mutants (JDB2602, JDB2601) showed a lower frequency of sporulation (see Table S1) and decreased heat resistance in DSM, while engulfment was delayed when compared to wild type cells (Fig. 2B, S5).