Regulation of pap phase variation -: Lrp is sufficient for the establishment of the phase off pap DNA methylation pattern and repression of pap transcription in vitro

The 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 formation of pap DNA methylation patterns in vivo was previously shown to require the leucine-responsive regulatory protein (Lrp) and DNA adenine methylase (Dam). To monitor the binding of Lrp to pap DNA, an in vitro methylation protection assay was developed. Binding of Lrp to a Dam target site proximal to the papBA promoter (designated GATC(prox)) blocked methylation of this site and specifically repressed transcription. The DNA methylation pattern and transcription state are identical to those observed in vivo in phase OFF cells. To determine if binding of Lrp at GATC(prox) was necessary for repression of papBA transcription, we analyzed a pnp mutation (pap-13) that reduced the affinity of Lrp for the GATC(prox) region. Binding of Lrp to pap-13 DNA was shifted to a promoter distal Dam target site (designated GATC(dist)). Lrp blocked methylation of GATC(dist) in the pap-13 mutant, but did not repress papBA transcription. Together, these results show that binding of Lrp to the GATC(prox) region is sufficient for the establishment of the phase OFF DNA methylation pattern and repression of papBA transcription.