A Thermosensitive, Phase-Variable Epigenetic Switch: pap Revisited

Abstract

It has been more than a decade since the last comprehensive review of the phase-variable uropathogen-associated pyelonephritis-associated pilus (pap) genetic switch. Since then, important data have come to light, including additional factors that regulate pap expression, better characterization of H-NS regulation, the structure of the Lrp octamer in complex with pap regulatory DNA, the temperature-insensitive phenotype of a mutant lacking the acetyltransferase RimJ, evidence that key components of the regulatory machinery are acetylated, and new insights into the role of DNA binding by key regulators in shaping both the physical structure and regulatory state of the papI and papBA promoters. This review revisits pap, integrating these newer observations with older ones to produce a new model for the concerted behavior of this virulence-regulatory region.

Keywords: acetylation; bacteria; epigenetics; gene silencing; genetic models; histone; nucleoid-associated protein; phase variation; pili; protein acetylation; temperature; transcription.

FIG 1

pap promoter region. The 330-bp regulatory region between the divergent papI and papBA promoters contains binding sites for PapB (light blue), CRP (orange), and Lrp (green), shown above the DNA and numbered as in the text. One GATC methylation site (black) is present within the center-most site of each set of Lrp binding sites.

FIG 2

General overview of the silenced, inactive, and active states of pap. (A) The fully silenced state is induced at low (nonhost) temperatures and is characterized by an H-NS filament covering the entire pap DNA region. Here, neither papI nor papBA is expressed. (B) In the inactive state, the Lrp octamer is bound to the three proximal Lrp sites (sites 1 to 3) and the distal GATC site in Lrp site 5 is fully methylated. Basal transcription of papBA can occur, and a molecule of PapB, together with CRP, recruits RNAP and activates transcription of papI. PapI levels are high but papBA transcription is still low, causing the system to remain Phase OFF with regard to pilus expression. (C) In the active state, the Lrp octamer is bound to the three distal sites (sites 4 to 6), while PapI helps to stabilize Lrp in this position. The proximal GATC site within Lrp site 2 is fully methylated. This new positioning of Lrp allows it to bend the DNA to recruit CRP to activate transcription of papBA. Both papI and papBA levels now are high, and the cells are phenotypically Phase ON.

FIG 3

Lrp binding to the pap promoter DNA. (A) General crystal structure of the Lrp octamer generated from PDB entry 2GQQ in the absence of DNA. The green regions correspond to the regulator of amino acid metabolism (RAM) domain, which also allows for Lrp oligomerization, and the blue regions correspond to the helix-turn-helix (HTH) region of Lrp that is responsible for binding to DNA. (B) The Lrp octamer cooperatively binds to sites 1, 2, and 3 in the pap promoter region, causing DNA bending. When in this position, pap is in its inactive, or Phase OFF, state. (C) The Lrp octamer cooperatively binds to sites 4, 5, and 6 in the pap promoter region, causing DNA bending again, but now leaves pap in the active, or Phase ON, state.

FIG 4

Molecular model for pap regulation. (A) Beginning from the fully silenced state, the H-NS nucleoprotein complex with the pap DNA is destabilized as the temperature increases upon host entry and is replaced by binding of CRP and/or Lrp, depending on nutrient conditions. Both papBA and papI transcription are off. (B) An Lrp octamer binds to the three proximal Lrp sites, while Dam methylates the distal GATC site. Basal levels of transcription of papBA allow for the production of a few molecules of PapB. The pap promoter is in the inactive state and still phenotypically Phase OFF. (C) PapB binds to its highest affinity site (PapB binding region 1) near the papI promoter and bends the DNA such that CRP can recruit RNAP for transcription of papI. PapI levels increase. The cells are still Phase OFF. Upon cell division, the fully methylated distal GATC site becomes hemimethylated, and Lrp may translocate along the DNA and transiently bind the three distal binding sites. PapI binds to the Lrp-distal site complexes and stabilizes Lrp in this position. (D) The movement of Lrp to the distal sites and stabilization therein permits Dam to access and methylate the proximal GATC site. Because Lrp is no longer obstructing the papBA promoter, the papBA levels increase and additional PapB begins to form a filament in PapB binding region 1. This state marks a transition point between the inactive and active states from the standpoint of regulatory logic. (E) Increased levels of PapB allow it to form a filament near the papI promoter, effectively shutting off the expression of papI and locking the cells in the active state. Lrp bound at the distal sites allows for bending of the DNA such that CRP can recruit RNAP for activation of papBA. The papBA expression is now at its highest, and the cells are phenotypically Phase ON. (F) PapB levels continue to rise until PapB binds to PapB binding regions 2 and 3, effectively shutting off papBA expression.