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Determining the In-Vitro Transcriptional Regulon of SigG in Nostoc punctiforme
Nostoc punctiforme is a cyanobacterial species that is capable of adapting its morphology into several different states of which the spore-like akinetes are the least understood. A time-course array previously revealed that Npun_F4153, a putative ECF sigma-encoding gene, was significantly upregulated upon akinete induction along with Npun_F4154, the cognate anti-sigma factor to Npun_F4153. Based on sequence alignments, Npun_F4153 was initially designated as putative RpoE-like protein, SigG, with subsequent experiments having since supported this identification. In this thesis, bioinformatics analysis suggests that Npun_F4154, designated SapG, shares homology with RseA-like sequences in organisms both from its own phylum and others. A knockout mutant strain of sigG, previously thought to not show any difference in phenotype from a WT strain, was discovered to exhibit a more buoyant cell pellet compared to WT N. punctiforme in log-phase vegetative cells. Furthermore, nitrogen deprivation in an attempt to induce heterocysts resulted in a massive rate of hormogonia formation in sigG mutant relative to WT. Attempts to complement the mutant were unsuccessful. Notably, sigG mutant showed increased susceptibility to lysozyme stress in both vegetative and akinete-induced strains, while akinete-induced mutant showed increased susceptibility to cold stress relative to akinete-induced WT cells. Once these phenotypic findings were established, N. punctiforme RNA polymerase core enzyme, as well as hexahistidine-tagged SigG protein were expressed and purified. Selected promoter templates based on previous in-vivo regulon results were also amplified, of which one was shown to be a direct target of SigG under heterocyst-inducing conditions. Encouraging preliminary results using these purified products with selected promoter templates and optimized in-vitro reaction conditions culminated in a runoff-transcription microarray-analysis (ROMA) using sheared, N. punctiforme genomic DNA templates. The in-vitro regulon generated from this experiment was in turn analyzed to develop a consensus sequence logo and for in-silico SigG regulon determination. Finally, regulons from all three approaches were compared to determine the most likely gene promoter targets of N. punctiforme SigG.