USE OF GREEN FLUORESCENT PROTEIN FOR DETECTION OF CELL-SPECIFIC GENE-EXPRESSION AND SUBCELLULAR PROTEIN LOCALIZATION DURING SPORULATION IN BACILLUS-SUBTILIS

Wild-type and mutant forms of the gene encoding green fluorescent prot ein (GFP) from Aequorea victoria have been introduced into Bacillus su btilis as translational fusions to the prespore-specific and mother-ce ll-specific genes dacF and spoIVA. In both cases, the protein was read ily detected by fluorescence microscopy, and its synthesis was correct ly localized. The S65T substitution, which improves the quantum yield and rate of development of fluorescence, also produced a spectral shif t that allowed the protein to be colocalized with DNA, after staining with 4',6-diamidino-2-phenylindole. Three different translational fusi ons to the N-terminal region of GFP all produced active protein. Moreo ver, a full-length SpoIVA-GFP fusion showed proper targeting to the su rface of the spore, albeit at low temperature and in the presence of w ild-type SpoIVA protein. A mutation in the gfp gene which changes the light emitted by the protein from green to blue was found not to be us eful because of the intrinsic autofluorescence of a. subtilis in the b lue part of the spectrum.