The bacterial response regulator ArcA uses a diverse binding site architecture to regulate carbon oxidation globally

Despite the importance of maintaining redox homeostasis for cellular viability, stress responses and the production of various biocommodities, how cells control redox balance globally is poorly understood. The work in this thesis provides new mechanistic insight into how redox couples are regulated at the level of gene expression by mapping the regulon of the bacterial response regulator ArcA from Escherichia coli, which responds to the quinone/quinol redox couple via its membrane-bound sensor kinase, ArcB. This genome-wide analysis reveals that ArcA reprograms metabolism under anaerobic conditions to maintain homeostasis of redox carriers through the transcriptional repression of genes associated with carbon oxidation pathways that recycle redox carriers via respiration. This strategy favors use of catabolic pathways, which, in the absence of respiration, recycle redox carriers via fermentation. Unexpectedly, bioinformatic analysis revealed that most ArcA target genes contained additional direct repeat elements beyond the two required for binding of an ArcA dimer. Nuclease protection assays suggest that non-canonical arrangements of cis-regulatory modules dictate both the length and concentration-sensitive occupancy of DNA sites. To verify the functionality of additional DR elements in transcriptional regulation, we analyzed point mutations in each of three DR elements within the icdA (isocitrate dehydrogenase) promoter. We found that all three DR elements are involved in ArcA DNA binding and repression of icdA. Our results suggest that an ArcA dimer bound to DR1 and DR2 stabilizes binding of a second ArcA dimer to DR3. Although in vivo occupancy of DR1 and DR2 is sufficient to direct a moderate amount of icdA repression, additional occupancy of DR3 is required for maximal repression. This binding site architecture broadens the concentration-sensitive occupancy of the icdA promoter by ArcA. Furthermore, a certain level of degeneracy in these DR elements is critical for maintaining a balance between high affinity binding and signal dependent regulation of binding. We propose that this plasticity in ArcA binding site architecture provides an effective mechanism for global control of carbon metabolism to maintain redox homeostasis.