Sporulation of the bacterium Bacillus subtilis is a relatively simple developmental process that involves a highly ordered program of gene expression and morphological change. The goal of this research is to understand how gene expression is coupled to morphological change during Bacillus sporulation to produce the correct pattern of temporal and spatial gene expression. These studies are of broad significance since all organisms that undergo development must regulate gene expression system to uncover fundamental mechanisms involved in developmental gene regulation. Early in sporulation, the daughter chromosome are partitioned to two compartments, and the subsequent pattern of gene expression in each compartment is distinct. Genetic studies suggest that gene expression in the mother cell compartment is somehow coupled to events occurring in the forespore compartment. The properties of two recently discovered regulators of mother cell-specific gene expression provide tow plausible mechanisms for the coupling phenomenon. One of the regulatory proteins is a sigma subunit of RNa polymerase (called sigmak) that may be synthesized as an inactive precursor. Its processing to the active form could be coupled to forespore events. The other regulatory protein (called the 14kDa protein) is a non-sigma transcription factor whose inactivation has been proposed experiments are designed to test these hypotheses and to define the roles of these two proteins in temporal and spatial gene regulation. Specifically, 1) the binding site of the 14kDa protein in a promoter it represses will be mutated to see if the promoter now becomes active earlier and is uncoupled form forespore events, 2) antibody to the 14kDa protein will be prepared and used to determine its amount and location during sporulation, 3) the sink gene (encoding sigmak) will be fused to an inducible promoter and pro-sigmak will be tested for activity. Antibody to pro-sigmak will be prepared to test directly whether sigmak is made as a precursor during sporulation. If pre-sigmak is inactive, a truncated, active sigk gene will be fused to an induced promoter. The ability to produce pro-sigmak or sigmak at will in cells will permit testing of whether pro-sigmak processing is involved in the coupling between compartments.
