||The expression of Pap pili alternates between ON (pili+) and OFF (pili-) states, a process called phase variation. Pap phase variation is controlled by Leucine responsive regulatory protein (Lrp), PapI, and Deoxyadenosine methylase (Dam). Methylation patterns of two GATC sites in the pap regulatory region are established by competition between Dam methylation and Lrp binding. In the phase OFF state, Lrp binds cooperatively to promoter proximal binding sites (1,2,3) and protects GATC-II from methylation whereas GATC-I is methylated. In the phase ON state Lrp binds to distal binding sites (4,5) protecting GATC-I from methylation whereas GATC-II is methylated. Lrp binding to GATC-I required PapI, suggesting that PapI is required for switching to the phase ON state. PapI bound specifically to Lrp-pap DNA complexes, but not Lrp-ilvIH, Lrp in solution or pap DNA. PapI reduced the affinity of Lrp for sites (1,2,3) and increased the affinity for sites (4,5) resulting in translocation of Lrp from the pap GATC-II region to the pap GATC-I region. These observations suggested that Lrp and PapI may directly interact in switching to phase ON. To identify PapI-Lrp binding sites, mutagenesis of papI and lrp was performed. Mutagenesis of papI yielded mutations that resulted in unstable proteins. A novel two color genetic screen was performed to identify pap-specific mutations in lrp. papBA-phoA (blue indicator) and ilvIH-lacZYA (red indicator) fusions generated purple colonies in the presence of wild type lrp. Red colonies were chosen because they contained lrp mutations that could activate ilvIH but not papBA transcription. One mutant, designated Lrp[E133G failed to translocate to GATC-I but was competent for pap and ilvIH DNA binding and PapI-pap DNA binding. Another pap-specific mutation, LrpY115C] may be defective in PapI binding. Furthermore, radiolabeled PapI bound to an Lrp peptide spanning amino acids 105-118. Together, these data indicate that Lrp contains a PapI binding site and a region required for translocation to GATC-I. A model for PapI mediated translocation of Lrp in the phase OFF to phase ON switch is presented.