Solution structure of Escherichia coli PapI, a key regulator of the pap pili phase variation

Pyelonephritis-associated pili (pap) allow uropathogenic Escherichia coli to bind to epithelial cells and play an important role in urinary tract infection. Expression of pap is controlled by a phase-variation mechanism, based on the two distinct heritable states that are the result of adenine N6-methylation in either of the two GATC sequences in its regulatory region. The methylation status of these two sequences is sensed by the action of two proteins, Lrp and PapI, and they play a central role in determining pap gene expression in both phase-ON and phase-OFF cells. We used modern NMR techniques to determine the solution structure and backbone dynamics of PapI. We found its overall fold resembles closely that of the winged helix-turn-helix family of DNA-binding proteins. We determined that PapI possesses its own DNA-binding activity, albeit non-sequence-specific, independent of Lrp. PapI appears to bind to DNA with a K(d) in the 10 microM range. Possible mechanisms by which PapI might participate in the regulation of the pap operon are discussed in light of these new findings.     

Stochastic modeling of the phase-variable pap operon regulation in uropathogenic Escherichia coli

Regulation of the pap operon in uropathogenic Escherichia coli is phase variable. This phase variation arises from competition between regulatory proteins at two sites within the regulatory region, GATC(dist) and GATC(prox). We have used the available literature data to design a stochastic model of the molecular interactions of pap regulation and expression during growth in a non-glucose environment at 37 degrees C. The resulting wild-type model is consistent with reported data. The wild-type model served as a basis for two "in silico" mutant models for investigating the role of key regulatory components, the GATC(dist) binding site and the PapI interaction with Lrp at the GATC(prox) site. Our results show that competition at GATC(dist) is required for phase variation, as previously reported. However, our results suggest that removal of competition at GATC(dist) does not affect initial state dependence. Additionally, the PapI involvement in Lrp translocation from GATC(prox) to GATC(dist) is required for the initial state dependence but not for phase variation. Our results also predict that pap expression is maximized at low growth rates and minimized at high growth rates. These predictions provide a basis for further experimental investigation.     

The mechanism by which DNA adenine methylase and PapI activate the pap epigenetic switch

The expression of pyelonephritis-associated pili (Pap) in uropathogenic Escherichia coli is epigenetically controlled by a reversible OFF to ON switch. In phase OFF cells, the global regulator Lrp is bound to pap sites proximal to the pilin promoter, whereas in phase ON cells, Lrp is bound to promoter distal sites. We have found that the local regulator PapI increases the affinity of Lrp for the sequence "ACGATC," which contains the target "GATC" site for DNA adenine methylase (Dam) and is present in both promoter proximal and distal sites. Mutational analyses show that methylation of the promoter proximal GATC(prox) site by Dam is required for transition to the phase ON state by specifically blocking PapI-dependent binding of Lrp to promoter proximal sites. Furthermore, our data support the hypothesis that PapI-dependent binding of Lrp to a hemimethylated GATC(dist) site generated by DNA replication is a critical component of the switch mechanism.     

Competitive Lrp and Dam assembly at the pap regulatory region: implications for mechanisms of epigenetic regulation

Escherichia coli DNA adenine methyltransferase (Dam) and Leucine-responsive regulatory protein (Lrp) are key regulators of the pap operon, which codes for the pilus proteins necessary for uropathogenic E. coli cellular adhesion. The pap operon is regulated by a phase variation mechanism in which the methylation states of two GATC sites in the pap regulatory region and the binding position of Lrp determine whether the pilus genes are expressed. The post-replicative reassembly of Dam, Lrp, and the local regulator PapI onto a hemimethylated pap intermediate is a critical step of the phase variation switching mechanism and is not well understood. We show that Lrp, in the presence and in the absence of PapI and nonspecific DNA, specifically protects pap regulatory GATC sites from Dam methylation when allowed to compete with Dam for assembly on unmethylated and hemimethylated pap DNA. The methylation protection is dependent upon the concentration of Lrp and does not occur with non-regulatory GATC sites. Our data suggest that only at low Lrp concentrations will Dam compete effectively for binding and methylation of the proximal GATC site, leading to a phase switch resulting in the expression of pili.