PhD

dissertationThe pyelonephritis-associated pili (pap) operon in Escherichia coli is regulated by an epigenetic mechanism involving the formation of specific DNA methylation patterns characteristic of transcriptionally active (phase ON) and inactive (phase OFF) cells. The binding site for cyclic AMP receptor protein (CAP) regulates the switch between phase ON and phase OFF states. Activating region 1 of CAP was shown to activate papBA transcription by a Class I-like mechanism from a distance -215.5) greater than any other that has been found in E coli. CAP does not act as an antirepressor, as was previously suggested. Instead, CAP plays an active role in stimulating papBA transcription, possibly by interacting with the C-terminal domain of the alpha subunit of RNA polymerase. An in vitro transcription system and methylation protection assay were developed using supercolied plasmid DNA to study the phase OFF (repression) and phase ON (activation) transcription states of papBA. Binding of Lrp to a Dam target site proximal to the papBA promoter (designated GATC[prox]) established a DNA methylation pattern and transcription state identical to that observed in vivo in phase OFF cells. Two pap regulatory mutations were used (pap-13 and pap-1) to study the phase ON state of pap transcription because they are constitutively (locked ON) transcribed in vivo. Binding of Lrp to a Dam target site distal to the papBA promoter (designated GATC[dis]) blocked methylation of this site and correlated with activation of transcription. Lrp is sufficient to activate transcription from pap-13 but not pap-1 template. CAP further stimulates pap-13 template transcription in an Lrp dependent manner. This thesis establishes an in vitro transcription system that has allowed the study of repression and activation of pap transcription by two global regulators of E. coli gene regulation, CAP and Lrp