| The regulation of expression of pyelonephritis associated pili (Pap) in Escherichia coli is under off-on switch control. The Pap switch is controlled at the most basic level by the methylation states of two GATC sequences, which affect binding of the global regulator Lrp to the pap regulatory region that controls pap transcription. In the phase OFF state the GATC sequence located distal to the pap pillin promoter (GATCdist) is methylated and Lrp is bound to three sites proximal to the pap pillin promoter, sites 1-3. In the phase ON state the GATC sequence located proximal to the promoter (GATCprox) is methylated and Lrp is bound to sites 4-6 distal to the pap pillin promoter. The transition from the phase OFF state to the phase ON state involves a complex series of events and is aided by the local pap-specific regulators PapI and PapB. PapB activates transcription of PapI by binding to Site1PapB, a site near the papI promoter. PapI, in turn, increases the affinity of Lrp for sites 4-6 to facilitate transition to the ON state. PapB also binds to a second site with lower affinity, Site 2PapB, which overlaps the papBA promoter and has previously been suggested to be involved in a negative autoregulatory loop. The results presented here indicate that PapB does not repress papBA transcriptional activity, acting only as a positive regulator of the Pap switch. A FACS-based assay to rapidly and quantitatively measure pap expression using a green fluorescent protein reporter is also described in this dissertation. The effects of varying the concentrations of the core switch regulatory proteins Lrp, Dam and PapI on the pap switch frequency was determined using the FACS-based assay and dose-response curves were generated. The data presented in this dissertation has led to a more detailed understanding of the Pap switch, particularly the roles of PapB in pap regulation. The FACS-based assay provides a powerful tool for testing systems models for Pap phase variation. |
