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Methylation patterns in pap regulatory DNA control pyelonephritis-associated pili phase variation in E. coli

Abstract We have examined the roles of pap DNA methylation patterns in the regulation of the switch between phase ON and OFF pyelonephritis-associated pili (Pap) expression states in E. coli. Two Dam methyltransferase sites, GATC 1028 and GATC 1130 , were shown previously to be differentially methylated in phase ON versus phase OFF cells. In work presented here, these sites were mutated so that they could not be methylated, and the effects of these mutations on Pap phase variation were examined. Our results show that methylation of GATC 1028 blocks formation of the ON state by inhibiting the binding of Lrp and Papl regulatory proteins to this site. Conversely, methylation of GATC 1130 is required for the ON state. Evidence indicates that this occurs by the inhibition of binding of Lrp to sites overlapping the pilin promoter. A model describing how the transition between the phase ON and OFF methylation states might occur is presented.

Epigenetic phase variation of the pap operon in Escherichia coli

Expression of the pyelonephritis-associated pilus (pap) operon in Escherichia coli is regulated by a complex epigenetic phase-variation mechanism involving the formation of differential DNA-methylation patterns. This review discusses how DNA-methylation patterns are formed by protein-DNA interactions and how methylation patterns, in turn, control pap gene expression.

Evidence for global regulatory control of pilus expression in Escherichia coli by Lrp and DNA methylation: model building based on analysis of pap.

Pyelonephritis‐associated pilus (Pap) expression is regulated by a phase variation control mechanism involving PapB, Papl, catabolite activator protein (CAP), leucine‐responsive regulatory protein (Lrp) and deoxyadenosine methylase (Dam). Lrp and Papl bind to a specific non‐methylated pap regulatory DNA region containing the sequence ‘GATC’ and facilitate the formation of an active transcriptional complex. Evidence indicates that binding of Lrp and Papl to this region inhibits methylation of the GATC site by Dam. However, if this GATC site is first methylated by Dam, binding of Lrp and Papl is inhibited. These events lead to the formation of two different pap methylation states characteristic of active (ON) and inactive (OFF) pap transcription states. The fae (K88), daa (F1845) and sfa (S) pilus operons share conserved ‘GATC‐box’ domains with pap and may be subject to a similar regulatory control mechanism involving Lrp and DNA methylation.

Differential binding of Lrp to two sets of pap DNA binding sites mediated by Pap I regulates Pap phase variation in Escherichia coli.

Pyelonephritis‐associated pili (Pap) expression in Escherichia coli is subject to a phase variation control mechanism that is regulated by the leucine‐responsive regulatory protein (Lrp), PapI, and deoxyadenosine methylase (Dam). In previous work, we found that the differential Dam methylation of two target sites in pap regulatory DNA, GATC‐I and GATC II, is essential for the transition between active and inactive pap transcriptional states. Here, we identify six Lrp binding sites within the pap regulatory DNA, each separated by about three helical turns. Lrp binds with highest affinity to three sites (1, 2 and 3) proximal to the papBAp promoter. A mutational analysis indicates that the binding of Lrp to sites 2 and 3 inhibits pap transcription, which is consistent with the fact that Lrp binding site 3 is located between the −35 and −10 RNA polymerase binding region of papBAp. The addition of PapI decreases the affinity of Lrp for sites 1, 2 and 3 and increases its affinity for the distal Lrp binding sites 4 and 5. Mutations within Lrp binding sites 4 and 5 shut off pap transcription, indicating that the binding of Lrp to this pap region activates pap transcription. The pap GATC‐I and GATC‐II methylation sites are located within Lrp binding sites 5 and 2, respectively, providing a mechanism by which Dam controls Lrp binding and Pap phase variation.

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

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.

Thermoregulation of Escherichia coli pap transcription: H‐NS is a temperature‐dependent DNA methylation blocking factor

The expression of Pap pili that facilitate the attachment of Escherichia coli to uroepithelial cells is shut off outside the host at temperatures below 26°C. Ribonuclease protection analysis showed that this thermoregulatory response was rapid as evidenced by the absence of papBA transcripts, coding for Pap pilin, after only one generation of growth at 23°C. The histone‐like nucleoid structuring protein H‐NS and DNA sequences within papB were required for thermoregulation, but the PapB and PapI regulatory proteins were not. In vivo analysis of pap DNA methylation patterns indicated that H‐NS or a factor regulated by H‐NS bound within the pap regulatory region at 23°C but not at 37°C, as evidenced by H‐NS‐dependent inhibition of methylation of the pap GATC sites designated GATC‐I and GATC‐II. These GATC sites lie upstream of the papBAp promoter and have been shown previously to play a role in controlling Pap pili expression by regulating the binding of Lrp, a global regulator that is essential for activating papBAp transcription. Competitive electrophoretic mobility shift analysis showed that H‐NS bound specifically to a pap DNA fragment containing the GATC‐I and GATC‐II sites. Moreover, H‐NS blocked methylation of these pap GATC sites in vitro : H‐NS blocked pap GATC methylation at 1.4 μM but was unable to do so at higher concentrations at which non‐specific binding occurred. Thus, non‐specific binding of H‐NS to pap DNA was not sufficient to inhibit methylation of the pap GATC sites. These results suggest that the ability of H‐NS to act as a methylation blocking factor is dependent upon the formation of a specific complex of H‐NS with pap regulatory DNA. We hypothesize that a function of H‐NS such as oligomerization was altered at 23°C, which enabled H‐NS to repress pap gene expression through the formation of a specific nucleoprotein complex.

Leucine‐responsive regulatory protein and deoxyadenosine methylase control the phase variation and expression of the sfa and daa pili operons in Escherichia coli

The Escherichia coli operons daa and sfa encode F1845 and S pill, respectively. In this paper we show that the expression of these operons is under phase variation control at a transcriptional level. The transcription of both operons is dependent on the global regulator leucine‐responsive regulatory protein (Lrp) and deoxyadenosine methylase (Dam). Lrp is required for methylation protection of two GATC sites located within conserved DNA sequences in the regulatory regions of these operons. These GATC sites are differentially methylated, establishing a methylation pattern which is characteristic of either the phase ON or phase OFF state. We also show that Lrp binds to the daa and sfa regulatory regions and that this binding is modulated by the methylation of the GATC sites. These results indicate that the phase variation of the daa and sfa operons is regulated by a mechanism involving differential binding of Lrp owing to methylation of GATC sites in the regulatory region, which is similar to the mechanism that controls phase variation of the pap operon.

LEUCINE-RESPONSIVE REGULATORY PROTEIN PLAYS DUAL ROLES AS BOTH AN ACTIVATOR AND A REPRESSOR OF THE ESCHERICHIA COLI PAP FIMBRIAL OPERON

The expression of the pap pilus operon of Escherichia coli is under a phase‐variation control mechanism in which cells undergo a reversible transition between transcriptionally active (phase ON) and inactive (phase OFF) states. In this study, we explore the roles of leucine‐responsive regulatory protein (Lrp) and the histone‐like protein H‐NS in the regulation of pap phase variation. Our data indicate that the phase OFF state results from repression of the intrinsically active papBA promoter by Lrp and H‐NS, each of which can act independently as transcriptional repressors. Lrp requires pap DNA sequences upstream of the papBA promoter for its repressor activity whereas H‐NS does not. In contrast, in the ON state, Lrp, in conjunction with Papl, activates pap transcription. This activation is not merely a result of alleviating the H‐NS mediated repression, but induces a level of transcription that is eightfold higher than the basal level of transcription from the papBA promoter measured in the absence of both H‐NS and Lrp. Analysis of Lrp activation mutants indicates that binding of Lrp to pap DNA sequences is not sufficient for transcription activation, consistent with a model in which an additional domain of Lrp interacts with the transcriptional apparatus. Together, our results show that Lrp functions as a transcriptional activator in phase‐ON cells and as a repressor of basal transcription in phase‐OFF cells. Because pap phase variation occurs in the absence of H‐NS, it is not clear what role this regulatory protein plays in pap gene regulation.

The essential role of the promoter‐proximal subunit of CAP in pap phase variation: Lrp‐ and helical phase‐dependent activation of papBA transcription by CAP from −215

Catabolite gene activator protein (CAP) is essential for the expression of Pap pili by uropathogenic Escherichia coli. Both in vitro and in vivo analyses indicate that binding of cAMP–CAP centred at 215.5 bp upstream of the papBA promoter is essential for activation of transcription. CAP‐dependent activation of papBA requires binding of the leucine‐responsive regulatory protein (Lrp) at binding sites that extend from −180 to −149 relative to the start site of papBA. Our data indicate that CAP and Lrp bind independently to their respective pap DNA sites. Activation of papBA transcription was eliminated by mutations in the activating region 1 (AR1) of CAP, but not in the AR2 region, similar to class I CAP‐dependent promoters. Also, like class I promoters, the C‐terminal domain of the α‐subunit of RNA polymerase appears to play a role in transcription activation. Moreover, phase variation is strictly dependent upon the helical phase of the CAP DNA binding site with respect to the papBA transcription start site. Using an ‘oriented heterodimer’ approach with wild‐type and AR1 mutant CAPs, it was shown that the AR1 region of the CAP subunit proximal to papBA is required for stimulation of papBA transcription, whereas AR1 of the promoter‐distal subunit is not. Previously, CAP was hypothesized to activate pap transcription indirectly by disrupting repression mediated by H‐NS. The results presented here show that AR1 of the promoter‐proximal CAP subunit was required for papBA transcription even in the absence of the histone‐like protein H‐NS. These results show that the promoter‐proximal subunit of CAP, bound 215.5 bp upstream of the papBA transcription start site, plays an active role in stimulating papBA transcription, possibly by interacting with the C‐terminal domain of the α‐subunit of RNA polymerase.

The frequency of expression of pyelonephritis‐associated pili is under regulatory control

The Bscherfchia coli urinary tract Isolate C1212 contains two pyelonephritis‐associated pili (pap) DNA sequences designated here as pap‐17 and pap‐21. Each of these pap sequences encodes antigenically‐distinct pilin monomers, pllin‐17 and pilin‐21, respectively. Most individual strain C1212 cells isolated from a single bacterial colony expressed pilin‐21. Only a small fraction (5%) of strain C1212 cells expressed pilin‐17. Most of the latter population simultaneously expressed pilin‐21, but a low percentage of cells expressed pill composed of pilin‐17 alone. In contrast, almost every E. coli K‐12 cell containing multicopy pap‐17 expressed pilin‐17 at the ceil surface. These results Indicated that the regulation of pilin‐17 expression observed for strain C1212 was lost when pap‐17 was in the multicopy state. Transfer of pap‐17 to a single copy vector resulted in a pilin‐17 expression frequency lower than strain C1212 (1%). Using E. coli K‐12 containing single copy pap‐17, we found that the frequency of piiin‐17 expression increased about 15‐foid when pap‐21 was present in multiple copies in trans. Subcloning of pap‐21 showed that a 2.2 kilobase‐pair DNA sequence adjacent to, but not including, the pilin‐21 structural gene was sufficient for activation of pilin‐17 expression.