Investigation of the transcription activating properties of Spo0A from Bacillus subtilis

• Main Author: Bird, Terry H.
• Format: Others
• Language: English
• Published: 2009
• Online Access: http://hdl.handle.net/2429/8739

SpoOA is a key regulator of sporulation in Bacillus subtilis. Genetic investigations have indicated that SpoOA activates or represses transcription of various loci which ultimately commit the cell to spore development under poor nutrient conditions. The transcription regulating properties of SpoOA are believed to be controlled by protein phosphorylation through a signal transduction system termed a ‘phosphorelay’. The spoIL4 and spoliG operons are two targets for activation by SpoOA-P that are essential to the induction of a sporulation response because each encodes a sporulation specific sigma factor. An in vitro transcription assay was used to examine the effect of SpoOA and its phosphorylation on transcription initiation at the spollA and spoliG promoters (P[sub spoIlA] and P[sub spoIIG]). Phosphorylation of SpoOA dramatically enhanced its ability to stimulate the level of transcription initiation at both promoters. A kinetic analysis of the initiation process at P[sub spoIIG] demonstrated that the rate of initiation was independent of the concentration of RNA polymerase and promoter template. In contrast, increased concentrations of SpoOA-P accelerated the initiation reaction at P[sub spollG] by affecting a rate limiting step that occurred after RNA polymerase bound to the promoter. Initiation rates at P[sub spollG] were also stimulated by a truncated form of the SpoOA protein (SpoOA[sup BD]) which contained the DNA binding domain but not the phospho-acceptor site. While transcription stimulation by SpoOA or SpoOA[sup BD] was observed to be extremely salt sensitive, the effect of SpoOA-P was insensitive to salt concentration if the predominant anion was acetate. DNaseI protection experiments demonstrated that both SpoOA and SpoOA-P catalyzed structural changes in ternary complexes formed between RNA polymerase and P[sub spollG]. These isomerizations appeared to correlate with the modest and dramatic effects of SpoOA and SpoOA-P, respectively, on P[sub spollG] transcription. DNaseI protection patterns obtained at various temperatures showed that although protein binding appeared normal, low temperature prohibited the structural transformations attributed to SpoOA(-P). Footprinting experiments carried out on a mutant promoter construct(Psp0llG[sup M94/84])indicated that SpoOA binding sites located at -94 and -84 relative to the transcription start site, are dispensable to the effect of SpoOA(-P) on ternary complex isomerization. However, these sites may function to enhance factor binding to what seem to be the crucial OA boxes at -50 and -40. The biochemical analysis presented in this thesis supports the hypothesis that the phosphorelay functions as a signal transduction system that can induce a sporulation response through activation of the transcription regulating properties of SpoOA. === Science, Faculty of === Microbiology and Immunology, Department of === Graduate