The overall goal of these studies is to determine the mechanisms whereby gene expression is regulated during the process of spore formation in the Gram-positive bacterium Bacillus subtilis. Specifically, we propose to manipulate the exprerssion of genes whose transcription is induced at the onset of sporulation or at intermediate stages of development through the isolation of promoter mutations, regulatory mutations and extragenic suppressor mutations. The effects of these mutations will be analyzed in vivo by employing gene and operon fusions to the lacZ gene of E. coli and in vitro by means of transcription studies with purified forms of E. coli and in vitro by means of transcription studies with purified forms of B. subtilis RNA polymerase holoenzyme. A principal aim of this project is to understand the role of spoO gene products in the initiation phase of sporulation, a problem that will be addressed through studies on the promoters for two genes whose transcription is induced at the onset of sporulation, and through studies on the novel RNA polymerase signa factors that determine their recognition. We willalso examine the regulation of a third gene whose induction is coupled to genetic and morphological events occurring at an intermediate stage of sporulation. In addition, we will develop fusion-generating derivatives of the transposon Tn917 that will create transcription fusions in vivo of B. subtilis genes to the lacZ gene of E. coli or to the luxA and luxB genes of Vibrio fischeri as new tools for the rapid analysis of sporulation genes of many types. Finally, we propose to test genetically the model that RNA polymerase sigma factors are sequence-specific DNA binding proteins that recognize conserved nucleotide sequence signals in the """"""""-35"""""""" and """"""""-10"""""""" regions of their cognate promoters through the isolation of change-of-specificity mutants of the O-37 species of B. subtilis sigma factor. It is anticipated that these studies on cellular differentiation in a highly accessible procaryotic model system may provide significant insights into problems of normal and abnormal differentiation in higher cells.
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