The pap operon in Esherichia coli consists of genes that code for the production and assembly of P pili, protein structures on the cell surface that adhere to human urinary tract cells causing infection. Regulation of the pap operon involves a phase variation mechanism where cells either express (phase ON) or do not express (phase OFF) pili. The regulatory mechanism is epigenetic in that it involves the differential methylation by DNA adenine methyltransferase (Dam) of two GATC sequences (GATC ^prox and GATC^dist) in the pap regulatory region. Leucine-responsive regulatory protein (Lrp) is an essential regulator of the pap operon and binds cooperatively to specific sites in the regulatory region. A phase ON phenotype occurs when GATC ^prox is methylated by Dam and Lrp is bound to sites 4, 5 and 6, preventing methylation of GATC^dist. A phase OFF phenotype occurs when GATC^dist is methylated and Lrp is bound to sites 1, 2, and 3, preventing methylation of GATC^prox.

The pap phase OFF to ON switching mechanism is not well understood but has been proposed to involve two rounds of DNA replication to remove the methylated adenine of GATC^dist. The questions persist whether Dam or Lrp initiates the phase switch immediately after replication and how each protein contributes to the mechanism. By kinetic and mechanistic studies, we showed that the non-processive methylation of the pap GATC sites by Dam is dependent on the three-basepair sequences surrounding each pap GATC site. These pap-specific GATC flanking sequences decrease the methyltransfer kinetics of Dam. Dam's poor preference for pap GATC sites provides a basis for Lrp to gain access and bind to specific sites following replication. Nevertheless, in vitro binding assays revealed that Lrp has a low discrimination between specific and non-specific DNA sequences suggesting that Dam may initiate the post-replicative switch while Lrp is sequestered elsewhere in the genome. In vitro kinetic and equilibrium competition assays revealed that the concentration of Lrp is a major factor in determining which protein initiates the switch, leading to a model where Dam initiates the phase OFF to ON switch at low Lrp concentrations.