SWITCH GOVERNING BACILLUS MOTHER CELL GENE EXPRESSION

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The long-term objective of this research is to understand how gene expression is controlled temporally and spatially during the relatively simple developmental process of Bacillus subtilis sporulation. This process involves a highly ordered program of gene expression and morphological change, and provides an attractive experimental system for elucidating fundamental mechanisms of gene regulation. These studies ar of broad significance and will have important implications for public health, because gene expression is temporally and spatially regulated during the cell cycle, as well as during adaptive and developmental responses, including during the interaction of a pathogen with its host. The proposed research focuses on three key regulators that comprise a switch governing the transition from early to late gene expression in th mother-cell compartment of the developing sporangium. The switch is initiated in response to a signal from the other compartment of the sporangium, the forespore. This step involves processing of an inactive precursor, pro-sigmaK, to sigmaK, a subunit of RNA polymerase that directs late gene transcription in the mother cell. The SpoIVFB protein may be the protease that processes pro-sigmaK, or a regulator of the processing reaction. To elucidate the mechanism of processing, an antibody probe for SpoIVFB will be developed and used to determine its intracellular location, as well as to isolate fractions enriched for SpoIVFB, which will be used to reconstitute pro-sigmaK processing in vitro. Mutations that bypass the need for SpoIVFB in sporulation will be characterized to determine whether SpoIVFB is likely to be the pro- sigmaK processing enzyme and, if not, to identify the protease. The appearance of sigmaK somehow causes a decrease in the level of sigmaE, which directs early mother-cell gene transcription. This halts transcripts of spoIIID, which encodes the second key component of the switch. The SpoIIID protein activates or represses transcription of bot early and late mother-cell genes. Mutations in sigK (encoding sigmaK) will be constructed and used to determine whether sigmaK must be transcriptionally active to negatively regulate the sigmaE and SpoIIID levels. SpoIIID is also subject to a developmentally regulated C- terminal truncation that alters its activity and stability. The resulting 9kDa form of SpoIIID will be analyzed to determine its C- terminus. The effects of truncating the spoIIID gene so that it encodes the 9 kDa protein will be studied. The protein that converts SpoIIID to the 9 kDa form will be identified, the corresponding gene cloned, and th effects of a null mutation determined. SigmaK RNA polymerase transcribe the gene encoding the third component of the switch, GerE, which exerts the opposite effect of SpoIIID on the transcription of several genes. Mutational analyses of the binding sites for SpoIID and GerE in promoter where they activate or repress transcription, and biochemical experiments, will be used to explore how these proteins exert their copious effects on mother-cell gene transcription.