Regulation of pyelonephritis‐associated pili phase‐variation in Escherichia coli: binding of the Papl and the Lrp regulatory proteins is controlled by DNA methylation

Abstract

Expression of pyelonephritis‐associated pili (Pap) in Escherichia coli is under a phase‐variation control mechanism in which individual cells alternate between pili⁺ (ON) and pili⁻ (OFF) states through a process involving DNA methylation by deoxyadenosine methylase (Dam). Methylation of two GATC sites (GATC₁₀₂₈ and GATC₁₁₃₀) within the pap regulatory region is differentially inhibited in phase ON and phase OFF cells. The GATC₁₀₂₈ site of phase ON cells is non‐methylated and the GATC₁₁₃₀ site is fully methylated. Conversely, in phase OFF cells the GATC₁₀₂₈ site is fully methylated whereas the GATC₁₁₃₀ site is non‐methylated. Two transcriptional activators, Papl and Lrp (leucine‐responsive regulatory protein), are required for this specific methylation inhibition. DNA footprint analysis using non‐methylated pap DNAs indicates that Lrp binds to a region surrounding the GATC₁₁₃₀ site, whereas Papl does not appear to bind to pap regulatory DNA. However, addition of Lrp and Papl together results in an additional DNasel footprint around the GATC₁₀₂₈ site. Moreover, Dam methylation inhibits binding of Lrp/Papl near the GATC₁₀₂₈ site and alters binding of Lrp at the GATC₁₁₃₀ site. Our results support a model in which Dam and Lrp/Papl compete for binding near the GATC₁₀₂₈ site, regulating the methylation state of this GATC site and, consequently, the pap transcription state.