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Bacillus subtilis, a benign gram-positive bacterium, utilizes the strategy of sporulation, which enables it to survive stresses such as starvation, desiccation, and UV irradiation. The spore provides greatly heightened safety to heat and noxious chemicals and remains dormant until conditions become favorable to growth. Sporulation by Bacillus subtilis is a primitive example of cell differentiation. The study of sporulation by Bacillus subtilis has become a paradigm for the study of differentiation in prokaryotes. Central to this process is the establishment of distinct patterns of gene expression in the cell types involved. Our laboratory has developed a two-part sacB/SacY probe to study the temporal and spatial compartmentalization of gene expression. It utilizes the anti-terminator protein SacY to control the transcription of reporter lacZ, (cloned downstream of the sacB gene,) which is regulated by anti-termination. Expression of sacB and SacY is regulated by a pair of promoters specific for sF (prespore specific) and sE (mother cell specific.) Both SacY and sacB must be in the same compartment of the sporulating cell in order to obtain ß-galactosidase activity. Mutagenesis of Bacillus subtilis was employed to identify determinants of compartmentalization of gene expression during sporulation. Mutants were screened for loss of compartmentalization using the two-part probe. In addition to the two-part sacB/SacY probe, a second method was developed; transposon mutagenesis was performed on strains where expression of gfp was regulated by promoters recognized by either sF or sE. Cells deficient in sporulation were isolated and evaluated by fluorescence microscopy for uncompartmentalized gfp expression. A rescue vector was developed that allowed for efficient cloning of Tn10 insertions. This plamid, pJP17, proved to be an essential tool. Mutations causing uncompartmentalized sF activity were identified in spoIIIE, spoIIIAA, spoIIIAB, spoIIIJ, spoIIE, spoIIAA, spoIID, spoIIM, kinA and ald. The spoIIIE mutation provides the most dramatic phenotype, and was the only mutation, that resulted in 100% loss of compartmentalization during stage II of sporulation. In contrast to all other mutants, the dramatic stage II loss of compartmentalized activity of sF indicates a regulatory role for SpoIIIE, which has yet to be elucidated. Taken together, these results indicate a central role for SpoIIIE in preventing activation of sF in the mother cell in addition to its DNA translocation activity. / Microbiology and Immunology