Transcriptional regulation of genes encoding spore coat proteins by mother-cell specific sigma(K) RNA polymerase during Bacillus subtilis sporulation

Sporulation of the Gram-positive bacterium Bacillus subtilis is a well established model system to study temporal and spatial gene regulation. Upon starvation, B. subtilis initiates sporulation. Asymmetrical cell division into the larger mother cell and the smaller forespore is the first easily observed morphological change. Each compartment expresses different sets of sporulation specific genes from the identical genome. As sporulation proceeds, the forespore is engulfed by the mother cell, forming a free protoplast inside the mother cell. Maturation of the forespore involves deposition of spore coat proteins, which are encoded in cot genes. Later, the mature spore is released by lysis of the mother cell. These interesting morphological changes are a consequence of cascade activation of RNA polymerase alpha subunits, which allows each cell type to sequentially express specific genes for sporulation. In the mother cell, in addition to a sigmaE and sigmaK, the sequential appearance of two DNA-binding proteins, SpoIIID and GerE, regulates gene expression.;The research presented in this dissertation is focused on understanding the regulatory role of GerE and SpoIIID in transcription by sigma K-containing RNA polymerase during late stages of sporulation in the mother cell. GerE was demonstrated to be a sequence-specific DNA-binding protein by DNase I footprinting, and mapping of these sites revealed a consensus GerE binding sequence. Based on in vitro transcription experiments, GerE directly activates transcription of several cot genes by a sigmaK RNA polymerase, and this activation, in part, involves interaction with the C-terminal domain of the a subunit of RNA polymerase.;In addition to its positive effects, GerE also initiates a negative feedback loop that inhibits transcription of sigK, the gene that encodes a sigmaK. This was demonstrated by comparing expression of the sigK-lacZ fusion in wild-type cells and in a gerE mutant, and by comparing the SigK protein level of these strains by Western blot analysis. GerE was found to bind within the sigK promoter region near the transcriptional start site and repress transcription by sigmaK RNA polymerase.;The combined action of GerE and SpoIIID regulates expression of some cot genes. Analysis of lacZ fusions and mRNA levels showed that cotB is expressed slightly earlier than cotX , whereas cotC expression lags behind that of cotX. This pattern of expression can be explained by different levels of GerE activation and/or SpoIIID repression of the three cot genes, as observed in in vitro transcription experiments. The results support a model in which a decreasing level of SpoIIID and an increasing level of GerE during sporulation set the timing and the level of expression of these cot genes, which may be important for proper assembly of the spore coat.