In response to nutritional stress, Bacillus subtilis bacteria activate numerous specialized, stationary-phase-specific sets of genes, and initiate a series of complex physiological and morphological changes, as they differentiate into a dormant cell-type called the """"""""endospore"""""""". Equally complicated gene expression and biological changes accompany the germination of endospores, when the vegetative phase is regenerated. New gene-manipulation techniques, many of which exploit the ability of Streptococcus transposon, Tn917 to function efficiently in a Bacillus host, will be applied to the systematic identification and isolation of the genes whose products are involved in these developmental transitions and to the analysis of their regulation. Derivatives of Tn917 that carry a promoterless copy of the Escherichia coli laz gene (conceptually similar to the Mudlac elements of E. coli) will be used to isolate developmental mutations (spo and ger mutations) in such a way that transcriptional fusions of the lazZ coding sequence to the developmentally regulated genes are an automatic consequence of the mutational event. Derivatives of Tn917 that generate simultaneous transcriptional fusions to lacZ and to the cat-86 gene of Bacillus pumilus will be used in a way that permits the identification of stationary-phase-specific genes even when their disruption does not cause an obvious developmental phenotype. Use of a highly sensitive indicator substrate (4-methylumbelliferyl-Beta-D-galactoside) which yields a fluorescent hydrolysis product, affords still other ways of identifying the regulated genes of interest and perhaps even ways of actually isolating bacteria by means of a fluorescence-activated cell sorter on the basis of their expression of lacZ fusions. The expression patterns of these lacZ-fusions and the responses of fused genes to mutations in other genes will be used to define the regulated sets that comprise the overall program of sporulation-related gene expression. Individual genes representative of different sets will then be analyzed in detail to determine the molecular basis of their regulation. In addition, attempts will be made to investigate the mechanisms that control the commitment of bacteria to the sporulation pathway through the isolation of conditional constitutive sporulation mutations and extragenic suppressors of such mutations.
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