©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Effect of the FruR Regulator on Transcription of the pts Operon in Escherichia coli(*)

(Received for publication, September 23, 1994; and in revised form, November 8, 1994)

Sangryeol Ryu (1) Tom M. Ramseier (2) Valerie Michotey (2) Milton H. Saier Jr. (2) Susan Garges (1)(§)

From the  (1)Laboratory of Molecular Biology, NCI, National Institutes of Health, Bethesda, Maryland 20892-4255 and the (2)Department of Biology, University of California at San Diego, La Jolla, California 92093-0116

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The promoters of the pts operon of Escherichia coli are controlled by the cyclic AMP receptor protein (CRP) complexed with cAMP (CRPbulletcAMP). In addition, glucose stimulates pts operon expression in vivo. The pts promoter region has a fructose repressor (FruR)-binding site (the FruR box) that partially overlaps with one of the CRPbulletcAMP-binding sites. The effects of the pleiotropic transcriptional regulator FruR on pts operon expression were studied to determine whether the in vivo glucose effect on pts operon expression is mediated by FruR. In vitro, FruR can repress P1b transcription, which is activated by CRPbulletcAMP, and restore P1a transcription, which is repressed by CRPbulletcAMP. FruR can displace CRPbulletcAMP from its binding site in the presence of RNA polymerase even though FruR and CRPbulletcAMP can bind simultaneously to their partially overlapping binding sites in the absence of RNA polymerase. FruR had very little effect on the transcription of the P0 promoter, which is most important for regulation by glucose. Consistent with the in vitro results, pts P0 transcription did not increase as much in cells grown in the presence of fructose or in fruR mutant cells as in cells grown in the presence of glucose. These results suggest that FruR alone does not mediate the in vivo glucose effect on pts operon expression.


INTRODUCTION

The phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) (^1)catalyzes the phosphorylation and transport of its sugar substrates and acts as a major signal transduction system in bacterial cells(1, 2, 3) . The PTS also regulates the transport and metabolism of a variety of non-PTS sugars as well as the activity of adenylate cyclase(2, 4, 5) . In Escherichia coli, the first two steps of the PTS phosphoryl transfer chain are catalyzed by two general cytoplasmic proteins, Enzyme I, encoded by the ptsI gene, and HPr, encoded by the ptsH gene. In addition, PTS sugar-specific membrane complexes are needed for the transport of individual PTS sugars. Enzyme IIA, a protein needed for the transport of glucose, is encoded by the crr gene. The ptsH, ptsI, and crr genes constitute the pts operon, located at 52 min on the E. coli chromosome(6, 7, 8, 9) .

The pts operon is regulated in a complex fashion by at least five promoters, P1a, P1b, P0a, P0b, and Px, which cluster into two groups, P1 and P0 (see Fig. 1)(10) . P1a, P1b, P0a, and P0b are -RNA polymerase-dependent. P1a and P1b partially overlap and initiate transcription in vitro from positions +1 and +8, respectively. P1a and P1b are regulated inversely by CRPbulletcAMP: the CRPbulletcAMP complex stimulates P1b and inhibits P1a (see Fig. 1B). P1a is active only when the template is supercoiled. P0a initiates transcription at position -100 and also needs supercoiled DNA as template. P0a is marginally stimulated by CRPbulletcAMP. When the template is linear, transcriptional initiation switches from P0a to P0b, 3 base pairs farther upstream from P0a, and becomes more CRPbulletcAMP-dependent. Px is recognized by -RNA polymerase and initiates transcription from position -94(10) . The regulatory circuits of pts promoters are summarized in Fig. 1B.


Figure 1: A, nucleotide sequence of the promoter region of the pts operon. The last codon of the cysK gene is boxed with an asterisk, and the first codon of the ptsH gene is boxed. The -10 and -35 regions of the P0a and P1a promoters are underlined. The -10 and -35 regions of the P0b and P1b promoters are overlined. The transcription start site of each promoter is boxed with an arrow. The CRP0 and CRP1 sites are boxed and labeled. The FruR-binding site is labeled as FruR box. B, schematic diagram showing the regulatory circuit of promoters of the pts operon by CRPbulletcAMP and FruR. Drawing is not to scale. All numbering is relative to the transcription initiation site of P1b. +, activation; -, repression.



pts operon expression is regulated both by CRPbulletcAMP and glucose in vivo(7, 11, 12) . Both CRPbulletcAMP and glucose have stimulatory effects on pts expression in vivo. Transcription increases in the presence of CRPbulletcAMP as well as glucose(10, 12) . The glucose-mediated and the cAMP-mediated activation occur independently of each other(7, 10, 12) , even though glucose lowers the level of cAMP in the cell(4, 5) . There is no P0 expression in the absence of CRP or cAMP in the cell, unless glucose is present. P1b expression needs both CRPbulletcAMP and glucose in the cell(10) . These observations suggest the presence of a repressor that is sensitive to glucose.

The fructose repressor, FruR, plays a pleiotropic role in the transcriptional regulation of many genes encoding enzymes of carbon and energy metabolism(13, 14, 15, 16) . For example, the expression of many enzymes including phosphoenolpyruvate synthase, phosphoenolpyruvate carboxykinase, fructose-1,6-diphosphatase, isocitrate lyase, and malate synthase is activated by FruR. In addition, FruR represses the expression of the fructose (fru) operon, which encodes the diphosphoryl transfer protein, fructose-1-phosphate kinase, and Enzyme II. The synthesis of some glycolytic enzymes is also subject to repression.

FruR consists of 334 amino acid residues and contains an amino-terminal helix-turn-helix DNA-binding motif, similar to other members of the GalR-LacI family of proteins(15, 16, 17, 18, 19, 20) . The fruR genes of E. coli and Salmonella typhimurium show 99% sequence identity, with no sequence differences in the proposed helix-turn-helix DNA-binding motif(15, 19) . Ramseier et al.(16) have identified many FruR-binding sites in E. coli and S. typhimurium, one of which is in the pts operon. In the pts operon, the center of the FruR-binding site is between the two promoter clusters P1 and P0, centered at 55.5 bp upstream from the P1b start site. The FruR-binding site partially overlaps the CRP1-binding site, which is centered at 42.5 bp upstream from P1b (see Fig. 1).

In this work, we studied the effect of FruR on pts operon expression in order to determine if FruR mediates the positive glucose effect on pts operon expression observed in vivo. We investigated the effect of FruR on transcription of the pts operon both in vitro and in vivo. We found that FruR repressed P1b transcription. However, FruR had almost no effect on the activity of the P0 promoter, which is regulated by glucose in vivo(10, 12, 21) . The significance of our findings is discussed.


EXPERIMENTAL PROCEDURES

Materials

Fructose 1-phosphate and fructose 6-phosphate were obtained from Sigma. RNA polymerase and nucleotide triphosphates were purchased from Pharmacia (Uppsala, Sweden). The cycle sequencing system was from Life Technologies, Inc. [-P]ATP and [alpha-P]UTP were from Amersham Corp. The polymerase chain reaction kit was from Perkin-Elmer. Klenow polymerase was from New England Biolabs Inc. (Beverly, MA).

Strains

The fruR strain SR500 was derived from SA2600 (wild type; F his rpsL relA) by transferring the fruR mutation from strain HK1543 (kindly provided by H. L. Kornberg) (22) using P1-mediated phage transduction (23) with selection for resistance to tetracycline and then screening for xylitol-sensitive transductants(24) .

Plasmid Construction and Preparation

The basic cloning protocols used were as described by Sambrook et al.(25) . Polymerase chain reaction cloning of the pts promoters was done using primers that have unique restriction sites in their sequences. All clones were verified by DNA sequencing. The plasmid pHX, which contains both the P0 and P1 promoters, was made by inserting a DNA segment from base pairs 8 to 290 (all these numberings are based on the pts sequence in Fig. 1) between the EcoRI and PstI sites in front of a rpoC terminator in plasmid pSA600(10) . Supercoiled DNA was prepared using a Plasmid Maxi kit (QIAGEN Inc., Chatsworth, CA), and linear DNA was prepared by cutting each plasmid with the restriction enzyme ApaI. DNA was verified by agarose gel electrophoresis.

Preparation of FruR

Hyperexpression of the fruR gene was performed basically as described by Ramseier et al.(16) , except that the incubation at 42 °C following temperature shift was for 30 min. Cells were washed three times in 3 mM potassium phosphate buffer, pH 7.2, containing 70 mM NaCl and 10% glycerol and ruptured by three passages through an Aminco French pressure cell at 10,000 pounds/square inch. A high speed supernatant fraction was prepared by ultracentrifugation at 120,000 times g for 90 min. For the purification of FruR, the method of Burgess (26) was followed. DNA-binding proteins were precipitated with polyethyleneimine, which was added to the supernatant fraction to a final concentration of 1%. The mixture was incubated on ice for 10 min and centrifuged at 7000 times g for 10 min. The pellet was resuspended in 3 mM potassium phosphate buffer, pH 7.2, containing 220 mM NaCl and 10% glycerol. Ammonium sulfate was added to a final concentration of 70%. After an overnight incubation, proteins were pelleted by centrifugation at 25,000 times g for 20 min. The protein fraction was finally resuspended in 3 mM potassium phosphate buffer, pH 7.2, containing 140 mM NaCl and 10% glycerol. The FruR protein was estimated to be 90% pure.

In Vitro Transcription

Reactions were performed as described by Ryu et al.(27) in a 50-µl volume containing the following: 20 mM Tris acetate, pH 8.0, 3 mM magnesium acetate, 200 mM potassium glutamate, 1 mM dithiothreitol, 0.2 mM ATP, 0.2 mM GTP, 0.2 mM CTP, 0.02 mM UTP, 10 µCi of [alpha-P]UTP (800 Ci/mmol), 2 nM DNA template, 40 nM CRP, 100 µM cAMP, 20 nM RNA polymerase holoenzyme, and 5% glycerol. The concentration of FruR was varied between 0 and 160 µM as indicated. All components except nucleotides were incubated at 37 °C for 10 min. Transcriptions were started by the addition of nucleotides and terminated after 10 min by the addition of 50 µl of formamide loading buffer (80% formamide, 89 mM Tris borate, 89 mM boric acid, 2 mM EDTA, 0.05% bromphenol blue, 0.05% xylene cyanol). RNA was resolved by electrophoresis on an 8 M urea, 8% polyacrylamide gel. Gels were dried, and the transcripts were visualized by autoradiography using Kodak XAR-2 film. The amounts of transcripts were measured using an AMBIS beta-scanner. The 106/107-base rep RNA from the plasmid origin of replication was used as an internal control when comparing the amounts of transcripts.

DNase I Footprinting

DNase I protection experiments were done as described by Ryu et al.(28) . 2 nM DNA, 80 nM FruR, 40 nM CRP, and 100 µM cAMP were mixed in transcription buffer in the combinations indicated in Fig. 5and incubated at 37 °C for 10 min. RNA polymerase was added and incubated for 10 min at 37 °C before DNase I treatment. The DNase I treatment was terminated by adding an equal volume of 20 mM EDTA and heating at 85 °C for 3 min. The DNase I-treated DNA was cleaned with a Wizard DNA cleanup kit (Promega, Madison, WI). The DNA was denatured with 1 mM NaOH and hybridized with P-labeled primer in extension buffer (50 mM Tris-HCl, pH 7.4, 10 mM MgSO(4), and 0.2 mM dithiothreitol) at 75 °C for 3 min. 0.1 volume of dNTP solution (0.5 mM final concentration) containing 3 units of Klenow polymerase was added after the above mixture was cooled in ice. The reaction was incubated at 50 °C for 10 min and stopped by adding an equal volume of stop solution (1 M Tris-HCl, pH 7.4, 30 mM EDTA, and 10 µg/ml poly(dI-dC)). DNA was precipitated and dissolved in a loading dye (98% (v/v) formamide, 10 mM EDTA, 1% (v/v) xylene cyanol, 1% (v/v) bromphenol blue) and analyzed on an 8% sequencing gel.


Figure 5: DNase I protection of pts DNA by FruR (80 nM), CRP (40 nM), and RNA polymerase (20 nM). The supercoiled DNA was treated with DNase I in the presence of various combinations of proteins as indicated at the top. Each strand of treated DNA was probed with two different P-end-labeled primers. Numbering is relative to the P1a transcription initiation site. The results are summarized on C. R (box), DNA region protected by RNA polymerase; F (&cjs2113;), DNA region protected by FruR; C (&cjs2112;), DNA region protected by CRPbulletcAMP; CR (&cjs2108;), region protected by CRPbulletcAMP and RNA polymerase; FC (), DNA region protected by FruR and CRPbulletcAMP; FCR (&cjs2110;), DNA region protected by FruR, CRPbulletcAMP, and RNA polymerase; , hypersensitive site.



DNA Band Migration Retardation Assay

Proteins and P-endlabeled 640-bp DNA fragments were incubated at room temperature for 10 min in the same buffer used for the in vitro transcription assay. The final reaction contained 20 mM Tris acetate, pH 8.0, 3 mM magnesium acetate, 200 mM potassium glutamate, 1 mM dithiothreitol, 1 mg/ml bovine serum albumin, 5% sucrose, 5 ng DNA, 0.2 mM cAMP, and various amounts of proteins. The electrophoresis was done with a 5% polyacrylamide gel at room temperature in 50 mM Tris borate/EDTA buffer containing 0.2 mM cAMP in the top reservoir.

Primer Extension

Cells were grown in Tryptone broth (1% Bacto-Tryptone, 0.8% NaCl) with or without 0.2% glucose or 0.2% fructose. Total E. coli RNA was purified using Tri reagent (Molecular Research Center, Inc., Cincinnati, OH). The RNA was resuspended in sterile distilled water. Purified -P-end-labeled primer P11 (complementary to DNA sequence 326 to 297 in Fig. 1, 5`-GCCAGTTTTTAACAGACGCGACGCACGAAG-3`) was coprecipitated with 30 µg of total cell RNA, and the pellet was resuspended in 20 µl of 250 mM KCl, 2 mM Tris, pH 7.9, and 0.2 mM EDTA. The mixture was heated to 60 °C and then allowed to cool to room temperature over a period of 1 h. After annealing, 50 µl of reaction solution was added, which contained 5 µg of actinomycin D, 700 µM dNTPs, 10 mM MgCl(2), 5 mM dithiothreitol, 20 mM Tris, pH 8.7, 30 units of RNasin (Promega), and 150 units of Superscript reverse transcriptase (Life Technologies, Inc.). The mixture was incubated at 40 °C for 60 min and treated with ribonuclease T1 (Life Technologies, Inc.). The DNA was precipitated; washed with 70% ethanol; and resolved on an 8 M urea, 8% polyacrylamide gel. The same primer was also used for sequencing pHX using a double-stranded DNA cycle sequencing system (Life Technologies, Inc.).


RESULTS

Effects of FruR on Transcription of the pts Operon in Vitro

In vitro transcription assays were performed to elucidate the effects of FruR on pts gene expression. The single FruR-binding site in the pts operon is between the two promoter clusters P0 and P1 (Fig. 1). Both supercoiled and linear pHX DNA containing all promoters and associated DNA-controlling elements (bases 8-290 in Fig. 1) was used as template (Fig. 2). Transcription from P1b was activated by CRPbulletcAMP, whereas it was repressed by CRPbulletcAMP from P1a in a supercoiled template. FruR inhibited P1b, but could restore the activity of P1a (Fig. 2, A, lanes 3-7; and C). The activity of P0 was inhibited slightly at high FruR concentration (Fig. 2C). The transcription pattern was similar with a linear DNA template (Fig. 2, B and D), except that higher concentrations of FruR were needed for P1b repression. This suggests that FruR binds better to supercoiled DNA. Similar results were obtained when the P0 promoter cluster was separated from the P1 promoter cluster (data not shown), indicating that P1 and P0 do not interfere with each other's activities.


Figure 2: Effects of FruR on the activity of the pts promoter in vitro. The plasmid pHX, which has both P0 and P1 promoters, was used. A 85-base transcript from P1a and a 78-base transcript from P1b are indicated as P1a and P1b, respectively. The 185- and 183-base transcripts from P0a and P0b were not separated from each other well on this 8% sequencing gel and are marked as P0. The transcripts from plasmid origin of replication (106/107 bases) are marked as rep. Lane1, no CRPbulletcAMP; lanes 2-7, 40 nM CRPbulletcAMP with the following concentrations of FruR, respectively: 0, 5, 10, 20, 40, and 80 nM. A, supercoiled DNA templates used for in vitro transcription assay; B, linear DNA used for in vitro transcription assay; C, scanning results of A; D, scanning results of B. C and D cannot be compared directly because rep activity used as an internal control varies with the superhelical density of template DNA.



Fructose 1-phosphate inhibits FruR binding to the fruB regulatory region in vitro and thus has been identified as the inducer of the fructose operon(16) . Induction of transcription by fructose 1-phosphate by neutralization of the FruR-mediated repression of the P1b promoter was tested by comparing the effects of fructose 1-phosphate and fructose 6-phosphate on FruR in a transcription assay (Fig. 3). Fructose 1-phosphate at 1 mM inhibited FruR repression of P1b almost completely, while fructose 6-phosphate had no effect under these conditions. The slight inhibitory effect of fructose 6-phosphate at 100 mM may be due to contaminating fructose 1-phosphate in the preparation of fructose 6-phosphate.


Figure 3: Effects of fructose 1-phosphate and fructose 6-phosphate on FruR function in in vitro transcription assay. 40 nM FruR was used as explained under ``Experimental Procedures,'' and the results were quantitated with an AMBIS beta-scanner.



The FruR-binding site resides at position -55.5 from the transcription start site of P1b and partially overlaps the CRP-binding site of P1 (Fig. 1)(16) . FruR could repress P1b transcription by displacing CRPbulletcAMP from its binding site, thus restoring P1a activity from CRPbulletcAMP inhibition. This possibility was tested by studying the binding of FruR and CRPbulletcAMP to the pts promoter region by DNA band mobility shift and DNase I protection assays.

FruR and CRPbulletcAMP Binding

The binding of FruR to its binding site was studied by DNA band mobility shift assay using a P-end-labeled 640-bp DNA fragment containing all of the pts promoters, the two CRPbulletcAMP-binding sites, and the FruR box (Fig. 4). The complex with one dimeric CRPbulletcAMP molecule bound to the CRP0 site (CD; Fig. 4, lane2) formed and migrated as a sharp band at low concentrations of CRPbulletcAMP. The complex with two dimeric CRPbulletcAMP molecules, each bound to the CRP0 and CRP1 sites, respectively (C(2)D; Fig. 4, lane5), was formed at higher concentrations of CRPbulletcAMP and yielded a more diffuse band. Weak binding of CRPbulletcAMP to the CRP1 site may be the cause of band broadening as shown for the C(2)D complex. The affinity of CRPbulletcAMP for the CRP1 site is at least 10 times weaker than that for the CRP0 site (12, 20) .


Figure 4: Gel shift assay using the 640-bp DNA fragment containing the pts promoter region. D, free DNA; CD, DNA complexed with one molecule of CRPbulletcAMP at the CRP0 site; C(2)D, DNA complexed with two molecules of CRPbulletcAMP at the CRP0 and CRP1 sites; FD, DNA complexed with FruR; FCD, DNA complexed with FruR and one molecule of CRPbulletcAMP; FC(2)D, DNA complexed with FruR and two molecules of CRPbulletcAMP. The slowest migrating species seen when CRP concentration is high is CRP bound nonspecifically.



Fig. 4shows that the complex containing one FruR molecule and one CRPbulletcAMP molecule (FCD; Fig. 4, lane8) formed at low concentrations of CRPbulletcAMP in the presence of 5 nM FruR. It seems that one more CRPbulletcAMP molecule could bind to the FCD complex without displacing FruR, as shown by the formation of a FC(2)D band (Fig. 4, lane10). This FC(2)D band began to form at lower CRPbulletcAMP concentrations than C(2)D (Fig. 4, compare lanes3 and 9) and was sharper than C(2)D. This suggests that FruR may assist CRPbulletcAMP binding to its weak CRP1 site. In other words, there may be a cooperative interaction between CRPbulletcAMP and FruR at this site. FruR and CRPbulletcAMP seem to bind to their partially overlapping DNA-binding sites perhaps on opposite faces without displacing each other. Such bindings were further tested by DNase I footprinting experiments employing supercoiled DNA because FruR, as suggested above, may bind more tightly to supercoiled DNA, and P1a is not active when the template DNA is linear (Fig. 2).

DNase I Footprinting Analysis of the Promoter Region of the pts Operon

The binding of FruR, CRPbulletcAMP, and RNA polymerase to the pts promoter was studied by DNase I footprinting analysis. Supercoiled pHX DNA was treated with DNase I in the presence of different combinations of proteins, and the protection pattern from DNase I of each DNA strand was probed with P-end-labeled primers (Fig. 5). The binding of CRPbulletcAMP to the CRP0 site (Fig. 5A, lane5) was not affected by FruR. The hypersensitive band near position -162 on the lower strand caused by the binding of CRPbulletcAMP to the CRP0 site was maintained in the presence of FruR (Fig. 5A, lanes5, 6, and 8). The binding of CRPbulletcAMP to the CRP1 site was highlighted with the appearance of a unique band around position -33 (P1a start site referred to as position +1) as shown in Fig. 5A (lanes 5-7). FruR binding was manifested by the protection of two bands around positions -55 and -60 (Fig. 5A, lane3). The unique band pattern resulting from the binding of each protein was maintained in the presence of both proteins (Fig. 5A, lane7; and C). This agrees well with the DNA band mobility shift assays, which showed that CRPbulletcAMP and FruR could bind simultaneously to their partially overlapping binding sites. The DNase I digestion pattern, however, was changed in the presence of RNA polymerase. When CRPbulletcAMP, FruR, and RNA polymerase were all present (Fig. 5A, lane8), the DNase I digestion pattern near position -33 changed to resemble that when CRPbulletcAMP was absent (Fig. 5A, lanes2 and 4). These results suggest that FruR displaces CRPbulletcAMP from the CRP1 site in the presence of RNA polymerase. This would explain the restoration of P1a transcription in the presence of FruR.

There were slight differences in the DNase I digestion pattern in the P0 region around positions -115 and -135 caused by FruR when CRPbulletcAMP and RNA polymerase were present (Fig. 5A, compare lanes2, 4, and 6 with lane8). These differences may be responsible for the slight repression of P0 by high concentrations of FruR as seen in Fig. 2. It is likely that a change in DNA topology caused in part by DNA bending due to CRPbulletcAMP binding to the CRP0 site and FruR binding to the FruR box has an effect on DNase I activity. Similar changes may repress P0 transcription.

Effects of FruR on pts Expression in Vivo

There was almost no repression of P0 transcription by FruR in vitro (Fig. 2). This suggests that FruR may not be the mediator of the glucose effect on pts expression observed in vivo because P0 is the target of the glucose effect in vivo(10, 12, 21) . The in vitro results were confirmed in vivo by studying the effect of FruR on pts expression in fruR (SA2600) and fruR (SR500) strains. Total RNA was isolated from these cells grown in the absence and presence of glucose or fructose, and the activity of each promoter was analyzed using the primer extension assay. Neither fruR cells nor cells grown in the presence of fructose showed a significant increase in P0 activity compared with cells grown in the presence of glucose (Fig. 6, compare lanes2 and 5 with lanes3, 4, and 6). Glucose, but not fructose, increased P0 activity regardless of the fruR allele. We could not study the effects of FruR on P1b transcription in vivo because P1b is not expressed well under the experimental conditions used. Neither the cells grown in the presence of fructose or glucose nor fruR cells showed a significant increase in transcription from P1b (Fig. 6, lanes2, 3, 5, and 6).


Figure 6: Transcription from pts promoters in wild-type (SA2600) and fruR (SR500) strains. Total RNA was isolated from cells grown in Tryptone broth with glucose or fructose as indicated at the top. Primer extension analysis of 30 µg of total RNA/reaction using primers complementary to sequence 297-326 in Fig. 1was done as described under ``Experimental Procedures.'' Lanes 1-3, wild-type strain (SA2600); lanes4-6, strain fruR (SR500).



FruR may be involved in the regulation of pts expression in an alternative way. Cells do not need to increase pts operon expression in the presence of fructose because fructose has its own specific HPr-like protein(13, 14) . Consistently, the P1a activity was repressed in the presence of fructose in the wild-type strain (Fig. 6, lane3). P1a activity was, nevertheless, constitutive in a fruR strain under the same conditions (Fig. 6, lane6).


DISCUSSION

The pts operon of E. coli is regulated in a complex fashion by at least five promoters, P1a, P1b, P0a, P0b, and Px, that cluster into two groups, P1 and P0. Transcription from each promoter is modulated by several factors such as CRPbulletcAMP, superhelical density of the DNA, and -RNA polymerase (10) . In addition, glucose positively controls pts operon expression in vivo by an unknown mechanism(10, 12, 21) . Our previous study on the transcriptional regulation of pts promoters indicated the presence of a glucose-inducible repressor(10) . Knowledge of the mechanism of pts operon regulation by glucose is essential for a better understanding of this complex regulatory system.

FruR is a DNA-binding protein with 334 amino acid residues and contains a typical amino-terminal helix-turn-helix DNA-binding motif. FruR plays a pleiotropic role in the transcriptional regulation of many genes encoding enzymes of carbon and energy metabolism(14, 16) . We have tested (i) whether glucose induction is mediated by FruR, whose binding site is between the two pts promoters P0 and P1(16) , and (ii) whether glucose or one of its metabolic products acts as an allosteric modifier of FruR action. We used the S. typhimurium FruR protein for in vitro studies of pts promoters of E. coli because the fruR genes of E. coli and S. typhimurium are almost identical, with 99% sequence identity(17, 19, 20) . Furthermore, there is no difference in the helix-turn-helix DNA-binding motif of the FruR proteins (20) and in the FruR-binding sites of ptsH genes from E. coli and S. typhimurium(29) . As expected, the protein from S. typhimurium has been shown to bind to the FruR-binding site of the E. coli pts promoter region(16) .

In vitro transcription results showed that FruR repressed P1b transcription, which is activated in the presence of CRPbulletcAMP (Fig. 2). FruR had no effect on P1a transcription in the absence of CRPbulletcAMP, but it could restore P1a activity, which is repressed by CRPbulletcAMP. FruR slightly repressed P0 transcription at high concentrations. The mechanism of FruR repression of P1b in vitro was studied by DNA band mobility shift assay and DNase I footprinting experiments. FruR may repress P1b either by displacing CRPbulletcAMP from its overlapping binding site or by another unknown mechanism while maintaining CRPbulletcAMP at its binding site. The weak affinity of CRPbulletcAMP for the CRP1 site and the restoration of P1a transcription, which is repressed by CRPbulletcAMP in the presence of FruR, support the first possibility (Fig. 2).

The DNA band mobility shift assay, however, revealed that CRPbulletcAMP and FruR could bind to their overlapping binding sites simultaneously in the absence of RNA polymerase even though CRPbulletcAMP bound poorly to the CRP1 site (Fig. 4). The simultaneous binding to overlapping regions is possible when two proteins bind to opposite faces of the DNA. It is interesting to note that DNA band mobility shift assays showed that CRPbulletcAMP binds better to the CRP1 site in the presence of FruR. FruR binding may change the DNA conformation to one to which CRPbulletcAMP binds better. Alternatively, FruR may increase the local CRPbulletcAMP concentration near the binding site by directly interacting with CRPbulletcAMP. An example of a protein-protein interaction between two proteins has been reported in the deoCp2 promoter, where CytR interacts with CRPbulletcAMP, forming a bridge between the two DNA-bound CRPbulletcAMP complexes(30) .

DNase I footprinting using supercoiled DNA was employed for further binding experiments in which RNA polymerase was included. The DNA band mobility shift assay in which linear DNA was used could not be used for the binding experiment in the presence of RNA polymerase because the affinity of FruR and the activity of pts promoters are influenced by the superhelical density of the DNA(10) . The DNase I digestion pattern unique to CRPbulletcAMP binding was maintained in the presence of FruR, in agreement with DNA band mobility shift assay results, but was absent in the presence of both FruR and RNA polymerase (Fig. 5). The footprinting results suggest that FruR and CRPbulletcAMP bind simultaneously to their overlapping binding sites, but FruR displaces CRPbulletcAMP from its binding site in the presence of RNA polymerase. In other words, FruR represses P1b by displacing CRPbulletcAMP from its binding site. This model also explains the mechanism of restoration of P1a by FruR in the presence of CRPbulletcAMP.

It is not known why P1b is poorly expressed in vivo, even in a fruR strain. The predominant P1b is activated easily by CRPbulletcAMP in vitro in the absence of FruR. Perhaps growth conditions for optimal expression of P1b have not been achieved. The large difference in affinity between CRPbulletcAMP and FruR for their overlapping binding sites may be important for the modulation of P1a and P1b transcription in vivo. FruR binds to its overlapping binding site much better than CRPbulletcAMP to its site (Fig. 4). Cells grown in the presence of both glucose and exogenous cAMP express P1b most, but even under these conditions, P1b is not very active(10) . P1a is the major promoter in vivo. The main reason for the incomplete pts promoter switch from P1a to P1b in vivo is not competition between CRPbulletcAMP and FruR binding to their overlapping binding sites because the effects are similar in a fruR strain (Fig. 6).

We confirmed that fructose 1-phosphate is the inducer of FruR by in vitro transcription assay (Fig. 3). Fructose 1-phosphate is produced from fructose via the fructose-specific PTS(31) . There is no known mechanism for producing fructose 1-phosphate when glucose is metabolized in the cell. It is not clear whether fructose 1,6-bisphosphate can be converted into fructose 1-phosphate or how efficient it is as an inducer. Glucose may provide partial inactivation of FruR, or cells may not have enough CRPbulletcAMP for binding to the CRP1 site even when exogenous cAMP is provided. Cells may need a high concentration of CRPbulletcAMP and inactivation of FruR for CRPbulletcAMP to bind to the very weak CRP1 site in order to express P1b in vivo. This is unrealistic for normal growth conditions because the conditions for inactivation of FruR (for example, growth in the presence of glucose or fructose) normally depress cAMP production in the cell. Cells may not have enough CRP for P1b expression because we could not get full P1b expression even with fruR cells (SR500) grown in the presence of glucose and exogenous cAMP (data not shown). The level of CRP may be low under these conditions. crp gene expression is autoregulated and is reduced by glucose(32) . Note that the incomplete promoter switch in vivo was also noted in the E. coli gal operon(33, 34) , which has two promoters inversely regulated by CRPbulletcAMP as in pts P1a and P1b. The CRP-binding site of the gal promoter is also relatively weak.

Cells may maintain a certain level of pts operon expression by steady expression of P1a and may modulate different levels of pts expression mainly by changing P0 activity. In support of this speculation, we have found that P1a mRNA is more stable than P0 mRNA. (^2)The importance of the modulation of P1a and P1b activity by CRPbulletcAMP in vivo in the regulation of pts expression remains to be studied.

De Reuse et al.(12) have found that there is very little CRPbulletcAMP modulation of P1 promoter activity in vivo and that P0 is important for pts regulation by glucose in vivo, in agreement with our present results. We found that FruR did not significantly repress P0 transcription. There was only slight repression of P0 activity at high concentrations of FruR in vitro (Fig. 2). This may be due to partial termination of transcription through blockage of elongation by FruR bound at its binding site 55 bp downstream from P0a(35) . It is also possible that the change in DNA topology caused by CRPbulletcAMP binding to the CRP0 site and FruR binding to the FruR box as shown by DNase I footprinting (near positions -115 and -135; Fig. 5A, lane8) may have an effect on P0 transcription at different levels. In any case, that effect was not sufficient to account for the glucose effect on pts expression (2.5-fold increase in pts expression) seen in vivo(12) .

Fig. 6shows that glucose primarily increased P0 expression, whereas the fruR mutation could not increase P0 transcription in vivo. Furthermore, glucose could activate P0 transcription in a fruR strain. These results suggest that FruR does not play an important role in the induction of pts expression by glucose. However, fructose reduced overall pts transcription in wild-type cells (Fig. 6, lane 3) in a FruR-dependent fashion since that effect disappeared in a fruR background. Interestingly, it has been shown that the activities of HPr and Enzyme I were induced to a maximal extent of 3-fold by either glucose or fructose(36) . These observations suggest that FruR and fructose may have effects on pts expression by still unknown mechanisms.

It is possible that the glucose-induced changes (37) in the superhelical density of chromosomal DNA near the pts promoters have an important function in the modulation of P0 activity. It has been shown that the activity of P0 is sensitive to the superhelical density of the DNA template in vitro(10) .


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Lab. of Molecular Biology, Bldg. 37, Rm. 2E06, NCI, National Institutes of Health, Bethesda, MD 20892-4255. Tel.: 301-496-1019; Fax: 301-402-1344.

(^1)
The abbreviations used are: PTS, phosphotransferase system; CRP, cyclic AMP receptor protein; bp, base pair(s).

(^2)
S. Ryu and S. Garges, unpublished data.


ACKNOWLEDGEMENTS

We thank Dr. Hans Kornberg for providing strains and Dr. Sankar Adhya for helpful discussions and comments on the manuscript.


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