INTRODUCTION

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The global regulator leucine-responsive regulatory protein (Lrp) plays an essential and central role in regulating the phase variation of pyelonephritis-associated pili (Pap) in Escherichia coli (4). Pap expression is regulated in part by the binding of Lrp to two sets of DNA target sites located in the pap upstream regulatory region (11). The binding of Lrp at pap DNA target sites 1 to 3, which overlap the papBA pilin promoter, blocks transcription and Pap fimbrial expression, resulting in the off expression phase (Fig. 1A). Under conditions of high levels of cyclic AMP (cAMP), the PapI coregulatory protein is expressed from the divergent papI promoter. PapI causes an increase in the affinity of Lrp for pap DNA sites 4 and 5, located over 100 bp upstream from sites 1 to 3 (11). Transcription from the papBA promoter is activated by Lrp bound at sites 4 and 5 and by cAMP receptor protein (CRP), which binds about 30 bp upstream of pap site 4 (7) (Fig. 1A). Analysis of Lrp activation mutants has shown that binding of Lrp to pap DNA sites 4 and 5 is necessary but not sufficient for transcription activation (17). Lrp participates in activating papBA transcription, since lrp null mutants do not express Pap fimbriae (2, 3). Thus, Lrp may act as either a repressor or an inducer of transcription of the pap operon, depending on which set of pap DNA binding sites Lrp occupies (17).

View larger version (15K): [in this window] [in a new window]   FIG. 1.   Isolation and identification of mutations in lrp that specifically block transcription of the papBA operon. (A) Two-color genetic screen. The lacZ and phoA reporter genes were used to simultaneously detect transcription from ilvIH and papBA as described in Materials and Methods. Base pair locations of the regulatory regions of papBA and ilvIH are shown relative to the transcription start site at +1. Lrp binding sites are depicted as open rectangles (1, 18). The GATC-I and GATC-II sites are depicted as black boxes within Lrp binding sites 5 and 2, respectively. These GATC sites are substrates for Dam and are differentially methylated in Pap phase-on and -off states (3). (B) Functional map of Lrp. Amino acid regions of Lrp which are required for DNA binding, activation, and leucine responsiveness are based on an lrp mutational analysis by Platko and Calvo (13). Lrp mutations at amino acids 126, 133, or 134 that block PapI-dependent pap transcription are shown by arrows.

Pap phase variation is also regulated epigenetically via deoxyadenosine methylation of two pap DNA GATC sites designated GATC-I and GATC-II (16). GATC-I is located within Lrp binding site 5, whereas GATC-II is located within Lrp binding site 2 (Fig. 1A). Methylation of the pap DNA GATC-I site reduces the affinity of Lrp for sites 4 and 5 and blocks transition to the phase-on state. It is likely that this process serves to lock cells in the off state until DNA replication occurs and a hemimethylated GATC-I site is generated (12). In vitro binding analyses have shown that Lrp binds to pap regulatory DNA containing a hemimethylated GATC-I site with significantly higher affinity than to pap DNA containing a fully methylated GATC-I site (12). In contrast to the negative role that methylation of GATC-I plays in Pap phase variation, genetic evidence indicates that methylation of GATC-II is required for the transition from the off to the on state (3). It is possible that methylation of GATC-II reduces the affinity of Lrp for pap DNA sites 1 to 3, freeing the papBA promoter to bind RNA polymerase (3).

The pap operon is a member of the methylation-dependent fimbrial operons which are characterized by the following: first, they require Lrp for activation and repression but are not modulated by leucine; second, they contain GATC-box DNA regions which are contained within the Lrp binding sites and are substrates for deoxyadenosine methylase (Dam); and third, they express PapI or coregulatory proteins homologous to PapI. Nonfimbrial operons of the Lrp regulon such as ilvIH do not contain a PapI-like coregulator or GATC-box regulatory sequences, and they do not require Dam for transcription activation (5). To learn more about the roles that Lrp plays in coordinating Pap phase variation, we developed a two-color genetic screen to identify mutations in lrp that blocked Pap phase variation but had little to no effect on ilvIH transcription. Notably, these lrp mutants map within a region, between codons for amino acids 126 and 134 inclusive, which lies outside of the DNA binding and transcription activation regions of Lrp identified by Platko and Calvo (13). Our data suggest that these mutant Lrp's are defective in a step in the switch from Pap phase off to phase on which has not yet been identified.

	MATERIALS AND METHODS

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Two-color genetic screen. The two-color genetic screen was carried out with E. coli DL2561 (phoA lacZ lrp-402) containing a chromosomal copy of ilvIHp-lacZ and plasmid-borne papBAp-phoA (Table 1). This E. coli isolate was constructed as follows. First, the Mu dI1734 lac insertion within ilvIH in E. coli CV975 was transduced into E. coli mPh2phoA by phage P1 transduction (14). Second, a papBAp-phoA operon fusion was constructed in which a promoterless phoA gene was inserted downstream and tandem rrnB1 transcription terminators were inserted upstream of the papBA promoter. The E. coli K-12 phoA gene was amplified by PCR with oligonucleotides 5'-CACTCGTCGACCGGTTTTATTTCAGCCCCAG-3' and 5'-CTCGGATCCACAGATCTGTACATGGAGAAATAAAGT-3'. The resulting DNA fragment was digested with restriction endonucleases BamHI and SalI and ligated to a plasmid vector pACYC184 derivative lacking most of tet (bp 2544 to 2820) to construct plasmid pDAL446. The 1.76-kb pap regulatory region, which includes the papBA promoter, was amplified by PCR with plasmid pDAL354 (11) and oligonucleotides 5'-GGAAGATCTCAGCGGATCAATTCCCAATTC-3' and 5'-GGAAGATCTCGCATTTCCTGACCGACTGA-3'. The resulting pap DNA fragment contained tandem rrnB1 transcription terminators, derived from plasmid pRS551 (15), between the vector sequence and the papBA promoter. This pap DNA fragment was digested with BglII and ligated to the BglII-digested plasmid vector pDAL446 to construct the pap-phoA fusion vector pDAL454. Plasmid pDAL454 was transformed into E. coli mPh2phoA containing ilvIH-lacZ to construct the double-fusion isolate DL2561.

Mutagenesis of lrp. The low-copy-number plasmid pMBF1 containing lrp (2) was mutagenized by passage through the mutagenic strain XL1-Red (Stratagene). DL2561 was transformed with mutagenized plasmid pMBF1 by electroporation (Electroporator II; Invitrogen). E. coli transformants were screened on M9 minimal medium containing 5-bromo-4-chloro-3-indolyl phosphate (X-Phos) and 6-chloro-3-indolyl--D-galactoside (Red-Gal) indicators (Research Organics). Red colonies containing lrp mutations that specifically reduced expression of papBA but not ilvIH were picked for further analysis. Plasmids were isolated by Qiaprep (Qiagen) and transformed back into DL2561. Strains which still maintained the mutant phenotype were chosen for further study. Sequencing of the mutations was performed at the sequencing facility at the University of Utah.

-Galactosidase and phase variation assays. -Galactosidase (1) and phase variation analyses (3) were performed as previously described. Plating of 10⁵ E. coli DL2746 cells (expressing the T134A Lrp mutant [Lrp^T134A]), did not yield any blue (Lac⁺) colonies (see Table 2). Blue DL2746 colonies were obtained by picking blue sectors, present in some colonies, and streaking onto M9 minimal medium until nonsectored blue colonies were obtained. Such colonies were assumed to have arisen from a single phase-on cell (1) and were used to obtain the rate of switching from phase on to phase off (see Table 2). For E. coli containing ilvIH-lacZ and papBA-lacZ operon fusions, the level of transcription was assumed to be equal to the -galactosidase activity. The standard deviations from the means of -galactosidase activities were calculated with data from two separate colonies with three replicates each for Tables 2 to 5.

Preparation of cell extracts and DNA probes. Cell extracts were prepared by growing 200-ml bacterial cultures at 37°C to an optical density at 590 nm of 0.5 and centrifuging the cultures at 2,000 × g for 30 min at 4°C. Cells were washed two times in dialysis buffer (40 mM Tris HCl [pH 7.5], 0.1 mM EDTA, 1 mM dithiothreitol, 60 mM KCl) and resuspended in 5 ml of dialysis buffer. Next, 10 µl of 0.5 M EDTA and 5 µl of 50-mg/ml lysozyme were added to the cells, which were held on ice for 30 min prior to sonication in Falcon 2057 tubes in a cup horn sonicator at 4°C. Cell breakage was checked by phase-contrast microscopy. Cell debris was separated by centrifugation at 15,000 × g for 20 min at 4°C. Protein levels in the supernatant solution were quantitated by the Bradford assay (Bio-Rad), and extracts were flash frozen in liquid nitrogen and stored at 70°C.

DNA mobility shift assay and immunoblotting. DNA mobility shift assays were performed with high-ionic-strength gels as described previously (12) except that reaction mixtures (20 µl in dialysis buffer) contained NaCl (100 mM), poly(dI-dC) (2 µg), and acetylated bovine serum albumin (1 µg). Protein-DNA complexes were quantitated on a model GS-250 phosphorimager (Bio-Rad). PapI was purified by thrombin cleavage of a purified glutathione S-transferase-PapI fusion as described previously except that storage buffer was 50 mM Tris-Cl (pH 8.0)-0.15 M NaCl-2.5 mM CaCl₂-0.1% Triton X-100-10 mM dithiothreitol. PapI was used within 1 week (8). The Lrp protein levels in the extracts were quantitated by immunoblotting onto polyvinylidene difluoride membranes. Lrp was detected with rabbit polyclonal anti-Lrp antiserum followed by chemiluminescence detection (Super Signal; Pierce) and phosphorimager analysis (Bio-Rad).

	RESULTS

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Isolation of pap-specific lrp mutants by a two-color genetic screen. The goal of this study was to identify the region(s) of Lrp that is specifically required for regulation of Pap phase variation. This goal was carried out by simultaneously detecting transcription from the ilvIH and papBA operons by a two-color genetic screen. Since the ilvIH operon lacks a PapI-like coregulator and is not controlled by the methylation state of its DNA, we reasoned that Lrp mutants showing reduced pap transcription but normal ilvIH transcription might have mutations within the PapI binding domain (8) or another Lrp region required specifically for pap gene regulation.

phoA lacZ lrp-402

4

Characterization of the Lrp^T134A and Lrp^E133G mutants. (i) DNA binding and PapI response. Previous work investigating the interactions of Lrp with ilvIH identified DNA binding, leucine response, and transcription activation regions of Lrp (13). To determine if the Lrp mutants identified here were phenotypically defective in any of these functions, we first measured the affinities of the Lrp mutants with both pap and ilvIH DNAs. Lrp levels in the extracts were measured by immunoblotting and equalized (Materials and Methods). DNA mobility shift analyses showed that Lrp^T134A and Lrp^E133G bound to ilvIH DNA with affinities similar to that of wild-type Lrp (Fig. 2B). Lrp appears to bind to the pap GATC-II region containing sites 1 to 3 in phase-off cells (3). Gel shift analysis showed that Lrp^T134A (Fig. 2A) and Lrp^E133G (not shown) bound to pap sites 1 to 3 with affinities similar to that of wild-type Lrp.

View larger version (12K): [in this window] [in a new window]   FIG. 2.   Measurement of the affinities of wild-type and mutant Lrp's for pap and ilvIH DNAs. The relative affinities of wild-type and mutant Lrp's for papBA and ilvIH DNAs were analyzed by mobility shift analysis as described in Materials and Methods. Results obtained with wild-type Lrp are shown by circles, those obtained with LrpE133G are shown by squares, and those obtained with LrpT134A are shown by triangles. Measurements obtained in the absence of PapI are shown as open symbols, whereas those obtained in the presence of PapI (120 nM) are shown as filled symbols. The Lrp levels in extracts were equalized by immunoblot analysis and are shown as relative values. The percentage of DNA bound was calculated based on the level of free DNA remaining at each Lrp level. (A) Nonmethylated pap site 1 to 3 DNA (bp 118 to +49); (B) nonmethylated ilvIH DNA (bp 311 to +2); (C) hemimethylated pap site 4 to 6 DNA (bp 278 to 112). The base pair numbering is relative to the papBAp transcription start site (Fig. 1).

View larger version (38K): [in this window] [in a new window]   FIG. 3.   PapI increases the affinities of wild-type and E133G mutant Lrp's for pap DNA sites 4 to 6. (A) Mobility shift analysis. Mobility shifts were carried out with LrpE133G and 32P-labeled pap site 4 to 6 DNA (40,000 cpm, 0.5 nM) as described in Materials and Methods. An LrpE133G level sufficient for a shift of about 15% of pap DNA probe was added to each sample (3 µg of DL3255 cell extract) except that the sample analyzed in lane 2 contained 3 µg of DL1784 (Lrp) extract instead of LrpE133G extract. Various amounts of PapI were added to each sample as indicated below in a total volume of 20 µl. The positions of unbound and bound pap DNAs are shown at the left. Lane 1, 120 nM PapI; lane 2, 120 nM PapI (Lrp extract); lane 3, 120 nM PapI plus 125 nM unlabeled pap sites 4 to 6 (250-fold molar excess over the molar concentrations of labeled pap DNA sites); lane 4, no PapI addition; lane 5, 1.2 nM PapI; lane 6, 2.4 nM PapI; lane 7, 6 nM PapI; lane 8, 12 nM PapI; lane 9, 60 nM PapI; lane 10, 120 nM PapI. (B) Summary of mobility shift data. Data obtained with wild-type Lrp (circles) and LrpE133G (squares) are shown. Results are expressed as fractions of free pap DNA probe remaining compared with the level observed in the absence of PapI (Fig. 3A, lane 4).

(ii) Leucine response. Certain operons within the Lrp regulon are affected by leucine addition and are considered leucine responsive. The transcriptional activities of operons, such as ilvIH, which are positively regulated by Lrp are decreased by leucine. The addition of leucine reduces transcriptional activation of ilvIH 10-fold in E. coli containing wild-type Lrp and 16-fold in E. coli containing Lrp^E133G (Table 3). These data show that the leucine response region of Lrp^E133G remains fully functional.

Analysis of the effects of Lrp^T134A and Lrp^E133G on pap transcription in vivo. The in vitro analyses shown above indicated that the Lrp^T134A and Lrp^E133G mutants retained intact DNA binding and PapI responsiveness. In addition, Lrp^E133G was still responsive to leucine (Table 3). Because these in vitro assays did not provide a clue as to the possible defect(s) of the Lrp mutants, we explored Lrp function in vivo. Previously we showed that an A-to-C transversion mutation at GATC-I (GCTC-I) resulted in a phase-on-locked phenotype (3). Although this mutation does not disrupt binding of Lrp to pap sites 4 and 5, the ability of Dam to methylate the GATC-I site is precluded. Methylation of this site serves to keep cells in the off transcription state by blocking binding of Lrp in the presence of PapI (12). Further analysis of the GCTC-I mutant showed that in the absence of papI, cells remained locked in phase on with only about a twofold reduction in pap transcription, which was dependent on the presence of Lrp (Table 4). Thus, pap transcription from the GCTC-I mutant occurs in the absence of PapI.

	DISCUSSION

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The goal of this study was to identify a region(s) of the global regulator Lrp that is specifically required for Pap phase variation to help define the mechanisms by which Lrp responds to PapI and the pap DNA methylation state. We identified lrp mutations that differentially inhibited papBA transcription and ilvIH transcription. Our data show that mutations at codons for amino acids 126, 133, or 134 of Lrp greatly reduced the rate of switching of pap from off to on (20- to more than 50-fold) but had much smaller effects on activation of ilvIH (less than 4-fold [Table 2]). These Lrp mutations overlap a region of Lrp that is associated with leucine response and is adjacent to a transcription activation region (Fig. 1B) (13). In vivo analyses showed that Lrp^E133G remained responsive to leucine (Table 3). Moreover, Lrp^T134A and Lrp^E133G activated pap transcription to 55 and 94% of wild-type levels, respectively, in the pap Lrp binding site 3 mutant background (Table 5). Thus, these Lrp mutants retained the ability to activate pap transcription.

Our data indicate that the E133G and T134A Lrp mutants were not altered in their affinities for either ilvIH or pap DNA sequences, consistent with previous data showing that the DNA binding domain of Lrp spans amino acids 13 through 70, well beyond the area of the mutations identified here (13). Moreover, Lrp^E133G and Lrp^T134A maintained responsiveness to PapI, as evidenced by an increase in their affinities for both nonmethylated and hemimethylated pap DNA sites 4 to 6 (Fig. 2C and 3). These results were puzzling, since we expected to find alterations in either responses to PapI or the ability to bind hemimethylated DNA, both hallmarks of Pap phase variation that are not involved in ilvIH gene regulation (3-5, 11, 16).

Further in vivo analysis of the Lrp mutants was carried out with two types of phase-on-locked pap mutants which are both PapI independent. Using the GCTC-I background and Lrp^T134A, we found that pap transcription was reduced over 60-fold compared to that of wild-type Lrp (Table 4). These results are consistent with the >50-fold decrease in the rate of switching of pap from phase off to phase on measured with this Lrp mutant (Table 2). The Lrp^T134A mutant was unable to activate pap transcription under conditions in which wild-type Lrp activated transcription in the absence of PapI. These results indicate that Lrp^T134A is defective in a step in Pap phase variation that does not involve PapI. Lrp^E133G, in contrast, showed less than a 2-fold decrease in pap transcription in the GCTC-I background even though it displayed a 20-fold decrease in the rate of switching of pap from phase off to phase on compared with that of wild-type pap (Tables 2 and 4). The reason for this difference in the abilities of the Lrp mutants to activate pap transcription in the GCTC-I background is not clear, but it correlates with the severity of the Pap phase variation phenotype (Table 2).

In contrast to results obtained with the GCTC-1 background, Lrp^T134A activated pap transcription to near wild-type levels in the pap Lrp binding site 3 mutant background (Table 5). These results indicate that the Pap phase variation step blocked in the Lrp^T134A mutant is prior to activation of pap transcription. Possible locations for a block are where Lrp dissociates from pap DNA sites 1 to 3 or binds to sites 4 to 6. DNA mobility shift analyses indicated that the affinity of Lrp^T134A for pap DNA sites 1 to 3 and 4 to 6 appeared normal in vitro (Fig. 2A and data not shown). It is therefore likely that there is an additional factor(s) required for the Pap switch from phase off to phase on in vivo that we have not yet identified. Such a factor(s) might be identified by isolating second-site mutations that suppress the Lrp^T134A pap transcription phenotype.

Why is Lrp^T134A unable to activate pap transcription in the pap GCTC-I background but able to activate transcription of the pap Lrp binding site 3 mutant (Tables 4 and 5)? Previous results showed that the affinity of wild-type Lrp for pap-13 DNA (the pap site 3 DNA) decreased threefold for sites 2 and 3 but increased for sites 5, 6, and 1 in the absence of PapI (11). Thus, the phase on state of the pap site 3 mutant may differ from that of wild-type pap in that Lrp may be bound to pap sites 5, 6, and 1 in the former and sites 4 and 5 in the latter, based on in vitro analyses (14). In contrast, Lrp bound to the GCTC-I mutant DNA with an affinity similar to that of wild-type pap (5). Unlike the pap site 3 mutant, the pap GCTC-I mutant is locked in phase on because it cannot be methylated at GATC-I. Presumably, this methylation is required to reduce the affinity of Lrp for the GATC-I region and to maintain cells in the off state until sufficient PapI is present to facilitate binding of Lrp to pap sites 4 and 5. The processes involved in the transition from phase off to phase on with the GCTC-I mutant may thus more closely parallel those of wild-type pap than those of the site 3 mutant, even though both pap mutants are locked in phase on and PapI independent. This hypothesis is supported by additional data indicating that at 23°C the pap site 3 mutant remains locked in phase on but that transcription from the GCTC-I mutant, like that of wild-type pap, is shut off by the normal thermoregulatory mechanism (unpublished data). It is therefore possible that a critical step(s) in pap gene regulation is bypassed in the pap site 3 mutant, enabling Lrp^T134A to activate transcription under these conditions.

Previous results showed that the rate of switching of pap from phase off to phase on for cells grown in glucose is 35-fold lower than that for cells grown in glycerol, yet the on-to-off switch rates for cells grown on either one of the two carbon sources are similar (1). It is likely that the decrease in the rates of switching by glucose from off to on is the result of lowered PapI levels, which are under cAMP-catabolite activator protein control. These results suggest that the on-to-off switch is independent of PapI levels and occurs by a pathway distinct from the off-to-on switch. The data presented here support this hypothesis, since although the three Lrp mutations analyzed greatly reduced the rate of switching from off to on, they had little or no effect on the rate of switching from on to off, which occurs at a 100-fold higher rate (Table 2). Further work is needed to understand the mechanism by which Lrp dissociates from pap DNA sites 4 to 6 and how the transition to the off transcription phase occurs.

The two-color genetic screen described here is an extension of previous work on CRP by Zhou et al. (19). In their study, crp mutants specifically defective in activation of lac transcription but retaining DNA binding activity were isolated by simultaneously screening ribose (Rbs) and lactose (Lac) fermentation on tetrazolium indicator media. For that screen a lacUV5-O^CRP promoter in which CRP represses transcription was used. Thus, CRP activation mutants gave a Rbs Lac phenotype since they failed to activate rbs transcription and bind to the lacUV5-O^CRP promoter to repress lac transcription. The advantage of the approach used in the present study is that since each reporter gene yields a different colony color, mutations that affect only one operon can easily be detected. This method should be widely applicable for the identification of specific functional regions of global regulators.