RNA POLYMERASE AND BACTERIAL DIFFERENTIATION

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As the bacterium Bacillus subtilis differentiates from the vegetative form into a dormant endospore, complex morphological changes occur that require the expression of many genes. During the process, new RNA polymerase sigma subunits appear, displacing one another and conferring on the RNA polymerase different specificities for the recognition of different classes of promoters. The activity of each sigma factor appears to be regulated in response to morphological cues that signal the completion of specific stages in the differentiation process, and in several instances intercellular signaling between the forespore and mother cell is required. Elucidation of the mechanisms that synchronize morphological transformations and gene expression in Bacillus subtilis may lead to the discovery of novel mechanisms that regulate gene expression in a wide variety of microorganisms. Therefore, we will study the mechanisms that synchronize the activation of two different sigma factors with two different morphological transformations. SapA protein acts in the early stage of spore coat assembly and may be proteolytically processed by the same machinery that processes pro- to activate thereby coordinating the activation of sigma K with the completion of the foundation for the spore coat. We will test this hypothesis, and determine how SapA is targeted to the interface region between the undercoat and cortex peptidoglycan. Forespore-specific transcription is initiated by sigma F-RNA polymerase, and results in the forespore-specific production of sigmaG. However, transcription of spoIIIG, the structural gene for , is delayed relative to other sigmaF-dependent genes, and requires an unidentified intercellular signal. We will characterize this mechanism of intercellular regulation by identifying cis-acting elements in the spoIIIG promoter, and trans-acting factors that regulate spoIIIG promoter activity. sigmaG accumulates after transcription of spoIIIG but does not become fully active until engulfment of the forespore is completed. We will study the function of two proteins that appear to affect sigmaG activation. Transcription of the orfD gene, which encodes one of these proteins, is directed from a promoter unlike any other known to be active during sporulation. Therefore, we will elucidate the mechanism that regulates orfD transcription. Mutations in spoIIIJ prevent sigmaG activation. We will test the hypothesis that spoIIIJ encodes a pheromone-like peptide that plays a role in signaling sigmaG activation.