Abstract
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 pap 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.
Highlights
The pyelonephritis-associated pili operon in Escherichia coli is regulated by an epigenetic mechanism involving the formation of specific DNA methylation patterns characteristic of transcriptionally active and inactive cells
To determine if binding of leucine-responsive regulatory protein (Lrp) at GATCprox was necessary for repression of papBA transcription, we analyzed a pap mutation that reduced the affinity of Lrp for the GATCprox region
Lrp blocked methylation of GATCdist in the pap-13 mutant, but did not repress papBA transcription. These results show that binding of Lrp to the GATCprox region is sufficient for the establishment of the phase OFF DNA methylation pattern and repression of papBA transcription
Summary
Lrp IS SUFFICIENT FOR THE ESTABLISHMENT OF THE PHASE OFF pap DNA METHYLATION PATTERN AND REPRESSION OF pap TRANSCRIPTION IN VITRO*. Lrp blocked methylation of GATCdist in the pap-13 mutant, but did not repress papBA transcription Together, these results show that binding of Lrp to the GATCprox region is sufficient for the establishment of the phase OFF DNA methylation pattern and repression of papBA transcription. Binding of Lrp-PapI to pap Lrp binding sites 4, 5, and 6 was blocked when GATCdist was fully methylated, providing a means by which DNA methylation could control the Pap phase variation switch [10]. Lrp was shown to bind to pap regulatory DNA encompassing the GATCdist and GATCprox sites in vitro, it was not clear if this was sufficient for the formation of the DNA methylation patterns observed for phase ON and phase OFF cells in vivo, or how this binding affected pap transcription [14]. These studies provide an in vitro system for the biochemical analysis of Pap phase variation
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