CRP Modulates fis Transcription by Alternate Formation of Activating and Repressing Nucleoprotein Complexes*

William NasserDagger , Robert SchneiderDagger §, Andrew Travers, and Georgi MuskhelishviliDagger ||

From the Dagger  Institut für Genetik und Mikrobiologie, Ludwig-Maximilians-Univesitaet, Maria-Ward-Strasse 1a, 80638 München, Germany and the  Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, United Kingdom

Received for publication, January 23, 2001, and in revised form, February 22, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The DNA architectural proteins FIS and CRP are global regulators of transcription in Escherichia coli involved in the adjustment of cellular metabolism to varying growth conditions. We have previously demonstrated that FIS modulates the expression of the crp gene by functioning as its transcriptional repressor. Here we show that in turn, CRP is required to maintain the growth phase pattern of fis expression. We demonstrate the existence of a divergent promoter in the fis regulatory region, which reduces transcription of the fis promoter. In the absence of FIS, CRP activates fis transcription, thereby displacing the polymerase from the divergent promoter, whereas together FIS and CRP synergistically repress fis gene expression. These results provide evidence for a direct cross-talk between global regulators of cellular transcription during the growth phase. This cross-talk is manifested in alternate formation of functional nucleoprotein complexes exerting either activating or repressing effects on transcription.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The rapid reorganization of cellular metabolism in response to changing growth conditions is a hallmark of bacterial cells. These metabolic reorganizations involve global regulators, many of which serve as DNA architectural factors associated with bacterial chromatin (1, 2). The abundant DNA bending chromatin protein FIS is a global regulator of bacterial metabolism facilitating the adjustment of cells to rapid growth conditions (3-6). The expression of fis positively correlates with the richness of the medium (5), such that the amount of FIS protein produced is commensurate with the available metabolic potential. FIS increases the synthesis of stable RNA (tRNA and rRNA) species (7-9), adjusting the capacity of the translational machinery to changes in the nutritional supply. FIS is thought to activate the transcription of stable RNA genes by stabilizing local DNA architectures in their promoter regions (10). However, at many other gene promoters, including the fis promoter, FIS acts as a transcriptional repressor (11-18).

The transcription of the fis operon sharply increases on subculturing stationary cells in rich medium, then steeply decreases because of autoregulation by FIS, and ceases in late exponential phase (11, 16). This pattern of expression during the growth phase is probably optimized, because constitutive fis expression negatively affects the survival of cells under stress conditions (19). The expression of fis is largely regulated at the transcriptional level without any significant contribution of fis mRNA decay rates (11, 16, 20). As expected for a gene involved in the modulation of cellular physiology, the expression of fis is subject to a plethora of control mechanisms. The activity of the fis promoter responds both to the growth rate-dependent and stringent control systems (16). This latter abrogates stable RNA production on limitation of amino acid availability and is mediated by the nucleotide ppGpp (21). However, the role of ppGpp in the shut-off of fis expression has been questioned in the study that showed that deletion of the genes for both ppGpp synthases (relA and spoT) does not change the growth phase expression pattern of fis (11). The fis promoter is also exquisitely sensitive to changes in the superhelical density of DNA and is thought to respond to growth phase-dependent fluctuations in DNA topology (22). In addition, the fis promoter activity is dependent on the availability of the initiating nucleoside triphosphates (23) and is positively regulated by the DNA bending chromatin protein IHF (20).

The CRP protein is another global regulator that, on exhaustion of glucose or its analogues, switches the cellular metabolism to the utilization of alternative carbon sources, such as lactose, maltose, and arabinose (24, 25). As glucose is consumed the intracellular levels of cAMP rise, and an active cAMP-CRP complex forms, which then modulates the activity of numerous genes at the transcriptional level (24, 26, 27). In growing bacterial cells the level of cAMP sharply increases on transition to stationary phase (28). Thus, the majority of the effects of CRP and FIS on gene expression are probably exerted under different growth conditions. Nevertheless, there are many examples of genes regulated by both FIS and CRP (13, 18, 29, 30). The expression of most of the FIS-regulated genes involved in the catabolism of sugars and nucleic acids is also dependent on the cAMP-CRP complex (4). Notably, the activity of the crp promoter itself is modulated by both the cAMP-CRP complex and FIS during the growth phase (15, 31, 32). This control of crp expression is thought to operate by changing the composition of transcriptional complexes formed in the crp promoter region in response to the physiological state of the cell (15).

In this study we investigated the effect of CRP on the expression of the fis gene. We demonstrate that CRP modulates the fis promoter activity, supporting the transient mode of fis expression during the growth phase. In analogy to crp regulation, this modulation involves alterations in the composition of functional nucleoprotein complexes formed by FIS, CRP, and RNA polymerase in the fis regulatory region.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Chemicals and Enzymes-- Chemicals and enzymes used in this work were obtained from commercial sources.

Bacterial Strains, Growth Conditions, and Plasmids-- Bacterial strains used in this study were Escherichia coli K12 derivatives. The genotype of CSH50 is ara Delta (lac pro) thi rpsL (33). The construction of CSH50Delta fis is described elsewhere (34). This strain carries a kanamycin resistance gene substituted for fis. CSH50Delta crp is described in González-Gil et al. (15). This strain carries a streptomycin resistance determinant. All strains were grown in 2× YT medium (16 g of tryptone, 10 g of yeast extract, 5 g of NaCl/liter, pH 7.4). Single colonies were isolated on YT plates (8 g of tryptone, 5 g of yeast extract, 5 g of NaCl, 15 g of agar/liter).

The construct pFP2 is described by Ninnemann et al. (16). For construction of pWN1 (fis promoter lacZ fusion), a fis promoter fragment (positions -308 to +106) was PCR1 amplified with the primers LfispRI5' (5'-CGGAATTCCGGTCGTAAGAATTAACCTTC-3') and ORF13' (5'-AGGTCTGTCTGTAATGCCAG-3') using E. coli CSH50 chromosomal DNA as a template. For the construction of pWN3 (divergent promoter lacZ fusion), a fis promoter fragment encompassing positions -308 to +106 was PCR amplified with the primers pfis-308op-NruI (5'- GCCTATCGCGACGTAAGAAATTAACCTTCGCAT-3') and ORF1-EcoRI (5'-CGGAATTCAGGTCTGTCTGTAATGCCAG-3') using E. coli chromosomal DNA as a template. These fragments were cloned in EcoRI/NruI sites of ptyrTlac (17), and thereby the tyrT promoter was replaced by these fis promoter DNA fragments.

A two-step PCR method using mutagenic oligonucleotides and Pfu DNA polymerase (Stratagene) was employed for site-directed mutagenesis (35). To construct pWN-75A/C, the primer pfis-87op comprising positions -87 to -64 and containing an A-to-C base substitution at -75 on the top strand (5'-CATTTTAAATGCCATTCTTTGATC-3') and primer ORF13' were used to initiate the first round PCR on the pWN1 template. The resulting PCR product was then used as a "megaprimer" together with the primer LfispRI5' for the second round PCR on the pWN1 template and the product cloned in EcoRI/NruI sites of ptyrTlac. For construction of pWN3-75A/C, the pWN1-75A/C DNA was PCR amplified with the primers pfis-308op-NruI and ORF1-EcoRI and cloned in EcoRI/NruI sites of ptyrTlac.

Proteins-- FIS and RNA polymerase were isolated as described previously (36, 37). Purified CRP was kindly provided by Dr. A. Kolb.

RNA Isolation and Northern Analysis-- Northern analyses were performed as described in Schneider et al. (17). The concentration of RNA in different samples was detected spectrophotometrically, normalized for each sample, and verified on a reference gel, using rRNA as loading control. For Northern analysis 15 µg of RNA/lane was separated on a 1% agarose gel in the presence of glyoxal and transferred to Hybond N+ filters (Amersham Pharmacia Biotech). Filters were hybridized overnight with [alpha -32P]dCTP-labeled probes made by Megaprime DNA labeling system (Amersham Pharmacia Biotech). As template DNA for the Megaprime reactions served a BglII/HindIII fis fragment cut out of pADfis (38) and an eno fragment, prepared by PCR amplification of a 1283-bp internal fragment of the E. coli enolase gene, using the primer eno5 (5'-AGTCTTATGCCTGGCCTTG-3') and eno3 (5'-CATCGGTCGTGAAATCATCG-3'). The signals obtained in three separate experiments were detected by phosphorimaging (PhosphorImager Storm 840, Molecular Dynamics) and quantified using the IMAGE-QUANT software, averaged, and normalized to the value obtained for the 5-min time point for one of the strains.

Western Analysis-- Western blotting was carried out essentially as described earlier (39), except that 18% SDS-polyacrylamide gel electrophoresis was used for the detection of FIS and methanol (10%) was included in the buffers during electrotransfer of proteins onto polyvinylidendifluoride membranes (Immobilon-P, Millipore). FIS was detected by using the FIS-specific polyclonal (rabbit) antibody (39). The beta  subunit of RNAP was detected by using a RNAP beta  subunit-specific polyclonal (goat) antibody (a generous gift of Hermann Heumann). Horseradish peroxidase-conjugated goat (Promega) and rabbit (Sigma) IgG-specific secondary antibodies were used for detection with an ECL+Plus kit (Amersham Pharmacia Biotech). The autoradiographs were scanned and quantified by NIH-IMAGE software.

DNase I Footprinting-- The conditions of DNase I footprinting were as described earlier (39). The fis promoter region (-185 to +65) was PCR-amplified using the primers fis3 (5'-GATTTCTGAGCTGATATTGTC-3') and fis5 (5'-AAAGAAAAATTGAGAACTTACTC-3') and the pFP2 DNA as a template. The primer fis3 was uniquely end-labeled by [gamma -32P]ATP and T4 polynucleotide kinase. The fragments obtained were purified by polyacrylamide gel electrophoresis using a neutral 0.5× TBE gel. The incubation mixture contained 10 mM Tris-HCl, pH 7.9, 75 mM NaCl, 1 mM dithiothreitol, 0.2 mM cAMP (whenever necessary), and proteins as indicated in a 20-µl volume. After incubation for 30 min at 37 °C, DNase I and MgCl2 were added to 2 µg/ml and 10 mM final concentrations, respectively. The reaction was terminated after 10 s by adding 80 µl of the solution containing 0.5% SDS and 50 mM EDTA. After digestion by proteinase K for 45 min at 45 °C and phenol extraction, the aqueous phase was precipitated with ethanol. The pellets were washed with 70% ethanol, dried, dissolved in loading dye, and analyzed on a 6% denaturing polyacrylamide gel. Protected and hypersensitive bands were identified by using the Maxam-Gilbert G-ladder (40) of the same DNA fragment as reference.

Potassium Permanganate Reactivity Assay-- The reactions on linear templates were assembled and processed similarly to those used for DNase I footprinting. The reactions were carried out as described by Schneider et al. (17), except that 100 µM each of CTP and GTP were included in the incubation mixtures.

For the reactions with supercoiled templates, the plasmid DNA (500 ng) and proteins as indicated were incubated in 50 µl of a buffer containing 10 mM Tris-HCl, pH 8, 200 mM NaCl, 0.2 mM dithiothreitol, and 100 µM each of CTP and GTP. After footprinting the plasmid DNA was used for five cycles of PCR (41) with the 5'-radiolabeled primer ORF1. The reaction products were analyzed on 6% sequencing gels. The signals caused by permanganate reactivity of bases were visualized and quantitated as described above.

In Vitro Transcription-- Supercoiled plasmid pWN1 and pWN-75A/C were used for in vitro transcription and primer extension reactions according to Lazarus and Travers (42). The mRNA obtained after in vitro transcription was divided in three parts and used for primer extension by reverse transcriptase (Moloney murine leukemia virus reverse transcriptase, RNase H minus, Fermentas) with radioactively end-labeled primers ORF1 for fis mRNA, -257op (5'-GAGCAAGCTCACAAAAGGC-3') for divergent transcript and bla3B4 (5'-CAGGAAGGCAAAATGCCGC-3') for the bla transcript. The extension with primers ORF1, -257op and bla3B4 yields 106-, 149-, and 100-bp fragments, respectively. After primer extension the samples were loaded on 6% denaturing polyacrylamide gels and analyzed by phosphorimaging. The length of the transcripts was estimated by using corresponding dideoxy sequencing reactions as a reference. The amount of the fis and div transcript produced was quantitated and normalized to that of bla.

DMS Footprinting-- In vivo DMS footprinting was performed essentially as described by Schneider et al. (43). DMS (0.2 or 0.4%) was added in 1 ml of cell cultures on transition to stationary phase (A600 = 1.5-2) grown at 37 °C and incubation continued at 32 °C for 5 min. The purified plasmid DNA (500 ng) was used as a template for five cycles of PCR (41) with 5'-radiolabeled primer 5'-CTGAGCTGATATTGTCCG-3' priming about 60 bp downstream of the fis operon transcription initiation site.

The DMS footprinting reaction in vitro was performed by adding 1 µl of DMS to supercoiled pWN1 plasmid DNA (500 ng) and proteins as indicated in a buffer containing 10 mM Tris-HCl, pH8.0, 100 mM NaCl, 0.2 mM dithiothreitol, 0.2 mM cAMP for 1 min at room temperature in a 100-µl volume. The DNA pellets were dried and used for PCR as described above. The PCR products were analyzed on 6% sequencing gels and visualized by autoradiography. A dideoxy sequencing ladder was used to identify the position of protected guanines.

beta -Galactosidase Determinations-- Overnight cultures were diluted 1:30 in fresh 2× YT medium. Samples taken at the indicated times were assayed for beta -galactosidase activity following the protocol of Sadler and Novick (44). beta -Galactosidase units were multiplied by 1000 to make them equivalent to those of Miller (33).

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of crp Deletion on Cell Growth-- In our experiments we used the E. coli strain CSH50 and its derivative CSH50Delta crp lacking the crp gene. After subculturing cells in rich medium (a procedure that hereafter we term a nutritional shift-up) the wild type and crp cells grew similarly until the late exponential phase. Although the transition to stationary growth phase occurred at lower optical densities in the crp mutant, both cultures achieved similar densities in late stationary phase (Fig. 1A). However, when the mutant and wild type strains were grown in mixed cultures, the wild type demonstrated a clear advantage, because the crp mutant cells were rapidly selected out (Fig. 1B).


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Fig. 1.   Growth characteristics of CSH50 and CSH50Delta crp strains. A, growth curves of CSH50 and CSH50Delta crp cells. The overnight cultures were diluted 1:100 in fresh 2× YT medium, and the absorbance at 600 nm (ordinate, arbitrary units) was measured after the time intervals indicated. The zero point on the abscissa indicates that the measurement was done immediately after the dilution. B, the growing CSH50Delta crp cells are rapidly lost from mixed cultures. The overnight cultures of CSH50 and CSH50Delta crp cells were diluted 1:10.000 in fresh 2× YT medium and mixed (zero point on the abscissa). After 24, 48, 72, and 96 h, aliquots were plated on streptomycin-containing medium, and the number of survivors was scored (see "Materials and Methods" for details). wt, wild type.

Deletion of crp Alters fis Expression-- To evaluate the effect of crp on fis expression, total cellular RNA was isolated at intervals after the shift-up from wild type and crp cell cultures having similar optical densities. The amount of fis mRNA was quantitated by Northern analyses. In wild type cells the typical pattern of fis expression was observed, with an initial sharp increase followed by a steep decrease and an almost complete shut-off in 120 min (Fig. 2, A and B). In crp mutant cells the amount of fis message was elevated at the earliest time point measured (5 min) but achieved its maximum levels later than in wild type. Most remarkably, in crp cells substantial amounts of fis transcript were detectable in late exponential phase (after 120 min), suggesting that the down-regulation of fis is impaired. During the analyzed time period both strains grew equally well (Fig. 1A), and in addition, we did not observe any significant differences between the strains in the expression pattern of eno mRNA (Fig. 2A, lower panel). Consistent with the Northern data, the crp cells transformed with the fis promoter-lacZ fusion construct pWN1 (containing the extended fis promoter region from position -308 to +106) reproducibly demonstrated a slow decrease of beta -galactosidase activity during the growth phase (see Fig. 7C).


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Fig. 2.   Effect of crp deletion on fis expression. A, a representative example of Northern hybridization analysis of fis mRNA is shown. eno RNA is shown as a control. Total RNA was isolated from CSH50 and CSH50Delta crp cells at intervals after shift-up as indicated. We note that the ratio of the two fis transcripts changes in crp cells, but we did not explore this phenomenon further. B, quantitative Northern analysis of fis expression in CSH50 and CSH50Delta crp cells. The data were derived from values obtained in three independent experiments. C, Western analysis of FIS content in CSH50 and CSH50Delta crp cells is shown. The amount of total protein was 500 ng for 15 and 120 min and 200 ng for 60 min. The immunodecoration of the beta  subunit of RNAP was carried out with the same filter. wt, wild type.

To test whether the impaired down-regulation of fis expression is due to a lower concentration of FIS in mutant cells, we compared the content of FIS in wild type and crp cell extracts. However, by quantitative Western analyses we detected increased amounts of FIS protein in crp cell extracts 60 and 120 min after the shift-up (Fig. 2C). Thus higher fis mRNA and higher FIS protein levels persist in exponentially growing crp cells. Taken together, these data suggest that crp is involved in the maintenance of the growth phase expression pattern of fis.

CRP and FIS Compete with RNAP for Binding at the fis Promoter-- The fis promoter regulatory region contains sequences matching the CRP binding site consensus (11, 24). To test whether CRP directly binds at the fis promoter, we carried out DNase I footprinting studies. Addition of CRP to linear fis promoter DNA fragments protected the positions -57/-58 from DNase I cleavage and increased DNase I cleavage around positions -60, -70, and -110 with respect to the start point of transcription at +1 (Fig. 3A, lanes 5 and 6). Inspection of this region revealed two sequences matching the CRP binding site consensus (24), one (site I) centered at position -62.5 (5'-TTTGAtccatcTCAGA-3') and another (site II) at position -103.5 (5'-TATGAgtaattaTCGCA-3') (Fig. 3C). Because only DNase I hypersensitivity indicative of DNA distortion but no extensive protection of these putative sites by CRP was observed, it appears that CRP does not bind tightly. However, we confirmed CRP binding in both these regions by DMS footrpinting experiments, showing a CRP-dependent modification of Gs at -67, -77, -97, -106, and -108 (Fig. 3C; data not shown).


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Fig. 3.   Footprinting analysis of the fis promoter. A, DNase I footprinting of FIS, CRP and RNAP binding at the fis promoter. The used 250-bp fis promoter fragment (~5 nM) was uniquely 5' end-labeled at the bottom strand. The protein concentrations used are indicated. The regions binding FIS, CRP, and RNAP are indicated by thick, thin, and dashed lines, respectively. The stars indicate hypersensitivities induced by binding of CRP. The arrowheads indicate hypersensitivities induced by binding of both FIS and CRP or both CRP and RNAP. B, KMnO4 reactivity of the fis promoter (bottom strand) and the divergent promoter (top strand) on linear templates (0.2 nM). The concentration of RNAP was 20 nM; the concentration of FIS was 20 nM in lanes 2 and 6 and 100 nM in lanes 7 and 8; the concentration of CRP was 20 nM in lanes 2 and 3 and 10 nM in lane 4. Lane 5, free DNA. C, sequence of the fis promoter. The binding sites for proteins are indicated as in A. The RNA polymerase binding elements (-10 and -35 hexamers) of the fis and divergent promoters are boxed, and the start points of transcription are shown by arrows. The down mutation introduced in the -35 hexamer of the divergent promoter is indicated by C above the substituted A in the -35 region. The thick, thin, and dotted lines indicate the binding regions for FIS, CRP, and RNAP, respectively. The filled and open circles mark the G residues whose methylation by DMS is diminished in the presence of low (10 nM) and high (100 nM) concentrations of cAMP-CRP complex, respectively.

To investigate the effect of CRP binding on the interaction of RNAP with the fis promoter, we first footprinted the fis promoter with RNAP alone. Binding of RNAP increased DNase I cleavage at two positions (-37 and -46) near the -35 element of the fis promoter and protected from DNase I cleavage the upstream region between -65 and -125. An additional DNase-hypersensitive site was induced around position -88 (Fig. 3A, lane 7). The observed pattern confirms a previous report on the binding of two closely spaced RNAP molecules with different affinities at the fis promoter (11).

Addition of CRP abolished both the DNase I cleavage around -88 and the protection of the upstream region by RNAP (Fig. 3A, compare lanes 7-10), indicating that CRP precludes the binding of RNAP to the upstream DNA. By contrast, the protection of the fis core promoter by RNAP was enhanced (Fig. 3A, compare lanes 7-9), suggesting that binding of CRP stabilizes RNAP at the fis core promoter. However, at higher CRP concentrations the protection of the fis promoter by RNAP was abolished (compare lanes 9 and 10). This latter effect of high CRP concentrations is most probably due to nucleation of binding in the region overlapping the -10 hexamer of the fis promoter, as observed by DMS footprinting (Fig. 3C; data not shown).

To gain more information on the interactions between CRP, FIS, and polymerase at the fis promoter, we footprinted FIS alone and also in combination with CRP and RNAP. On addition of FIS the first site occupied was the high affinity binding site II centered at -42 and overlapping the -35 region of the fis promoter (Fig. 3A, lane 1). When FIS and CRP were added in combination, the DNase I hypersensitivity around positions -38/-39 induced by binding of FIS at site II and the protection of FIS site III were noticeably enhanced (Fig. 3A, compare lanes 13-15). However, CRP did not increase the FIS protection in the downstream region (e.g. position -31) observed at high FIS concentrations (compare lanes 1-3 with lanes 13-15). This suggests that CRP may specifically facilitate the binding of FIS at sites II and III. When FIS and RNAP were added in combination, FIS prevented the binding of polymerase at the fis promoter, as judged by the disappearance of the DNase I hypersensitive sites at -37 and -46 attributable to RNAP binding (Fig. 3A, compare lanes 7 and 11). However, the RNAP-dependent protection of the upstream region and the DNase I hypersensitive site around -88 remained largely unaffected.

Similar experiments were carried out using potassium permanganate as a chemical probe. Potassium permanganate preferentially targets the pyrimidine residues in the untwisted regions of DNA and thus allows the extent of promoter opening to be measured. On addition of RNAP, we observed two regions of enhanced KMnO4 reactivity, one within the -10 element of the fis promoter and another upstream at positions -101 and -102 (Fig. 3B, lane 1). We attribute this latter to the binding of the second RNAP molecule, because deletion of the fis core promoter region to position -42 does not affect this upstream reactivity pattern (data not shown). Addition of CRP substantially decreased the KMnO4 reactivity of the bases at -101 and -102, whereas the reactivity in the -10 region of the fis promoter was noticeably increased (Fig. 3B, compare lanes 1, 3, and 4). By contrast, addition of FIS reduced more strongly the KMnO4 reactivity of the fis promoter -10 region than that of the bases at -101 and -102 (compare lanes 1 and 6). This latter reactivity was substantially reduced only at higher FIS concentration (compare lanes 6 and 7). When FIS and CRP were added in combination, the KMnO4 reactivity of the fis promoter -10 element and especially that of bases at -101 and -102 was strongly reduced, suggesting a cooperative effect (Fig. 3B, compare lanes 2, 3, and 6).

From these data we infer that binding of CRP in the fis regulatory region displaces RNAP from upstream DNA site but facilitates the polymerase binding and open complex formation at the fis promoter. By contrast, binding of FIS predominantly displaces RNAP from the fis promoter. Furthermore, CRP appears to stabilize the interaction of FIS with binding site II implicated in repressing fis promoter activity (20). Finally, FIS and CRP cooperate in repressing the interaction of RNAP with the upstream binding site.

CRP Activates the fis Promoter in the Absence of FIS-- To evaluate the functional importance of the observed interactions, we first investigated the effect of CRP on the open complex formation at the fis promoter. KMnO4 footprinting was carried out using linear fis promoter fragments with RNAP and different CRP concentrations. Addition of CRP enhanced the reactivity of the bases within the -10 region of the fis promoter to permanganate, but as observed by DNase I footprinting experiments described above, increasing CRP concentrations abolished this enhancing effect (Fig. 4A), probably because of binding of CRP at a low affinity site overlapping the -10 region (Fig. 3C). Next we performed in vitro transcription reactions using CRP with supercoiled pWN1 DNA. The results demonstrated a similar dependence of fis promoter activity on CRP concentration (Fig. 4B, upper panel), whereas the transcription of the bla promoter located on the same plasmid was not noticeably affected (Fig. 4B, lower panel). We thus conclude that in the absence of FIS, CRP can activate the fis promoter by facilitating open complex formation.


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Fig. 4.   CRP modulates the fis promoter opening and transcriptional activity in vitro. A, potassium permanganate footprinting of the 250-bp fis promoter fragment (~0.5 nM) incubated with RNAP (30 nM) and CRP. The concentrations of CRP (dimer) in lanes 2, 3, 4, 5, and 6 were 10, 20, 40, 100, and 100 nM, respectively. B, in vitro transcription from supercoiled pWN1 (0.5 µg) in the presence of RNAP (10 nM) and CRP. The results of primer extension reactions of the generated fis mRNA are shown. Halves of the same mRNA preparations were used to measure bla transcription as an internal control (lower panel). The concentrations of CRP (dimer) in lanes 2, 3, 4, and 5 were 4, 10, 15, and 80 nM, respectively.

FIS and CRP Synergistically Repress the fis Promoter-- We next asked how the simultaneous addition of CRP and FIS affects the fis promoter activity. For this purpose we monitored fis transcription using pWN1 DNA with FIS and CRP proteins added either alone or in combination. As expected, addition of increasing FIS concentrations gradually decreased the transcription of the fis promoter (Fig. 5, upper panel, compare lanes 1-4), whereas under the reaction conditions CRP alone had no detectable effect (Fig. 5, compare lanes 1, 7, and 8). However, when these same CRP concentrations were combined with a low FIS concentration (corresponding to that in lane 2), which alone had only marginal effect on transcription, the activity of the fis promoter was strongly inhibited (Fig. 5, lanes 5 and 6). Under the same conditions the transcription of the reference bla promoter was not noticeably affected (Fig. 5, lower panel). Thus, a combination of FIS and CRP at concentrations that themselves have little, if any, effect on transcription, strongly inhibit fis promoter activity. We infer that FIS and CRP synergistically repress the fis promoter in vitro.


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Fig. 5.   Effect of CRP and FIS on transcription of the fis promoter in vitro. The result of primer extension of the fis mRNA generated by in vitro trancription from plasmid pWN1 in the presence of 25 nM RNAP is shown. The used concentrations of CRP and FIS are indicated. Halfs of the same mRNA preparations were used to measure bla transcription as an internal control (lower panel). The data were normalized to the bla transcript.

CRP Facilitates FIS Binding at Site II both in Vitro and in Vivo-- To clarify the mechanism of synergy between FIS and CRP in repressing the fis promoter activity, we investigated the effect of CRP on the binding of FIS at site II by DMS footprinting. To obtain signatures for site II occupation by FIS, the DMS footprinting reactions were carried out in vitro with the fis promoter construct pWN1. Most characteristic signatures obtained for FIS binding at site II were an increased reactivity of G -37 relative to G -38 and strongly decreased reactivities of G -48 and G -49 (Fig. 6, A and B, compare lanes 1 and 2). Addition of CRP alone caused only slight decrease in the reactivity of G -48 (compare lanes 1 and 10), whereas simultaneous addition of FIS and CRP yielded essentially the same pattern as with FIS alone. However, consistent with DNase I footprinting data in the presence of CRP, the reactivity signatures attributable to FIS binding at site II were substantially increased (Fig. 6, A and B, compare lanes 2 and 3). Addition of polymerase did not change the pattern observed with FIS and CRP alone (compare lanes 3 and 4).


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Fig. 6.   CRP facilitates the interaction of FIS with site II at the fis promoter. A, the DMS reactivity of the fis promoter construct pWN1 treated either in vitro or in vivo is shown. The positions of modified guanines and the concentration of proteins used for the in vitro reactions are indicated. The DMS concentration used for the in vivo modification was 0.2% in lanes 5-7 and 0.4% in lanes 11-13. The strains are indicated above. B, densitometric scans of the FIS binding site II. The numbers 1-4 correspond to the lanes in A. Arrows indicate the positions of guanines whose DMS reactivity is modified by FIS.

We next investigated whether CRP similarly modulates the interaction of FIS with binding site II in living cells. In vivo DMS footprinting was carried out with wild type, crp, and fis cells containing pWN1 and grown to similar optical densities. The footprinting of the fis promoter was performed on transition to stationary growth phase, i.e. under the conditions of a shut-off of fis expression in wild type cells. The comparison of the DMS footprint obtained in crp mutant cells with that obtained in cells lacking fis (used as a negative control) revealed in the former a shift in the reactivity of G -37 relative to G -38, and a suppression of the reactivities of G -48 and G -49 (Fig. 6, A, compare lane 5 with lane 7 or lane 11 with lane 13; B, compare fis and crp, right panel). The pattern obtained is almost identical to that observed in vitro and indicates that FIS interacts with the binding site II in crp mutant cells. However, in wild type cells the interaction of FIS with site II was notably increased in comparison to crp mutant cells, as judged by the same reactivity signatures (Fig. 6, A, compare lane 5 with lane 6 or lane 11 with lane 12; and B, compare crp and wt, right panel). We thus infer that CRP is required to stabilize the interaction of FIS with binding site II in vivo.

The Upstream RNAP Binding Site Represents a Divergent Promoter-- Finally, we addressed the question on the role of the upstream polymerase binding site in the regulation of fis. Previous studies could not detect any transcription driven by RNAP from this site. To test whether this RNAP molecule transcribes in the direction opposite to that of the fis operon, we carried out primer extension reactions with total RNA isolated from CSH50 cells. Two divergent transcripts were detected with start points at -108 and around -87 (Fig. 7A, lane 1). To discriminate whether these divergent transcripts result from true initiation events or represent processing products, we carried out in vitro transcription reactions with pWN1. Three divergent transcripts were produced in vitro only one of which (initiating at -108) coincided with that observed in vivo (Fig. 7A, lanes 2-4). This latter is the only transcript reproducibly observed with different fis promoter constructs in vitro (data not shown). Inspection of the region around the common initiation site at -108 identified both in vivo and in vitro, revealed two sequences 5'-TTGCAT-3' and 5'-GATAAT-3' with reasonable matches to the sigma 70 consensus -35 and -10 elements, respectively (Fig. 3C).


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Fig. 7.   A divergent promoter is located in the fis regulatory region. A, primer extension analysis of total RNA (10 µg) extracted from CSH50 cells (lane 1) and of the transcripts produced from pWN1 in vitro (lanes 2-4) with the primer pfis-257op. The concentration of RNAP in lanes 2-4, was 15, 30, and 50 nM, respectively. The dideoxy sequencing ladder performed with the same primer on pWN1 is shown. B, effect of the down mutation in the -35 element of the divergent promoter. In the left panel the result of primer extension of RNAs produced in vitro from the divergent, fis, and bla (used as an internal control) promoters on pWN1 and pWN-75A/C is shown. The amount of the fis and div transcript produced was quantitated and normalized to that of bla. In the right panel the effect of the mutation on the KMnO4 reactivity of the divergent promoter (bottom strand) and fis promoter (top strand) after binding of RNAP is shown. Lanes 1, 2, 5, and 6 show the reactions with pWN1. Lanes 3, 4, 7, and 8 show the reactions with pWN-75A/C. C, the time course of beta -galactosidase expression driven from the fis and divergent promoters using the pWN1 and pWN3 constructs in growing wild type and crp cells. All values were normalized by setting the amount of beta -galactosidase produced from pWN1 in each overnight (time 0) wild type and crp cell cultures at 100%. The data were derived from values obtained in three independent experiments.

We generated the construct pWN-75A/C in which a "down" mutation was introduced impairing the match of the putative -35 element of the divergent promoter to the consensus sequence (TTGCAT right-arrow TgGCAT, see legend to Fig. 3C) and investigated the effect of the mutation on promoter function in vitro. Consistent with the prediction, in pWN-75A/C both the initiation of the divergent transcript at -108 and the permanganate reactivity of bases at -101 and -102 were substantially reduced (Fig. 7B, compare lanes 1 and 2 with lanes 3 and 4 in the left panel, and lanes 5 and 6 with lanes 7 and 8 in the right panel). Furthermore, the -35 down mutation increased the activity of the fis promoter on the pWN-75A/C template about 5-fold with 20 nM and 2-fold with 50 nM RNAP, respectively (Fig. 7B, compare lane 1 with lane 3 and lane 2 with lane 4). This mutation also increased 2-fold the beta -galactosidase expression of the fis promoter from pWN-75A/C measured in 3 h after the shift-up (2150 Miller units for pWN-75A/C versus 1025 Miller units for pWN1). The activity of the divergent promoter construct pWN3 containing the extended fis promoter region from position -308 to +106 in an inverted orientation with respect to lacZ reporter gene was also about 2-fold higher (262 Miller units) than that of the mutant construct pWN3-75A/C (150 Miller units). From these data we conclude that the upstream polymerase binding site in the fis regulatory region acts as a divergent promoter that interferes with transcription initiation at the fis promoter.

To evaluate the effect of CRP on the activity of the divergent promoter in vivo, we measured the beta -galactosidase activity produced from pWN3 and the fis promoter construct pWN1 in wild type and crp cells. We observed that the beta -galactosidase activity of cells containing pWN3 rapidly decreased during first 15 min after the shift up in both strains, whereas the beta -galactosidase expression from pWN1 increased as expected (Fig. 7C). In 3 h after the shift-up the beta -galactosidase levels decreased further 4-fold in comparison to the 15 min value in wild type but only slightly in crp cells. Thus, slower decrease in the activities of both the fis and divergent promoters are observed in crp cells in comparison with wild type. These results are consistent with the cooperation of FIS and CRP in repressing the function of both the fis and divergent promoters observed in vitro (Figs. 3B and 5).

    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The central finding of this study is that the cAMP-CRP complex is required to maintain the growth phase pattern of fis expression. We demonstrate that CRP is involved in the formation of distinct nucleoprotein complexes that can either activate or repress fis transcription. More specifically, we show that a synergy between FIS and CRP in repressing fis promoter activity enables an efficient shut-off of fis expression in mid exponential phase. Because FIS is itself regulating crp transcription (15), these data indicate a direct cross-talk between two global transcriptional regulators in coordinating cellular gene expression with varying growth conditions. RNAP holoenzyme is also implicated as a potential regulator of fis expression.

The Regulatory Role of the Divergent Promoter-- We have shown here that the upstream polymerase binding site in the fis regulatory region represents a promoter transcribing in a direction opposite to fis operon. We could not identify any meaningful ORF downstream of the divergent promoter, and no genes transcribed in this direction have been reported so far. The adjacent panF/prmA operon located upstream is transcribed in the same direction as fis (45). In agreement with earlier observations (11), our in vitro data indicate that polymerase binds more tightly at the divergent than at the fis promoter. In addition, polymerase forms open complexes readily at the divergent promoter, whereas detectable open complex formation at the fis promoter requires magnesium ions and high concentrations of initiating nucleoside triphosphates.2 Furthermore, the inactivation of the divergent promoter leads to an activation of the fis promoter in vitro, the effect being more pronounced at low RNAP concentrations. One implication of these observations is that under conditions of RNAP limitation, the divergent promoter is competing with the fis promoter for RNAP binding. Thus, it is possible that the divergent promoter has primarily a regulatory role. Whether this competition for RNAP binding is involved in the control of fis gene expression is not clear at present. The reported activation of the fis promoter after the deletion of the region comprising the divergent promoter (20) would be consistent with the competition model. Morever, we observe a 2-fold increase of beta -galactosidase production from the fis promoter when the divergent promoter is inactivated by mutation. The inverse correlation of changes in the fis and divergent promoter activities observed during the first 15 min after the nutritional shift-up (Fig. 7C) provides further circumstantial evidence for the regulatory role of the divergent promoter. However, the interpretation is complicated by the fact that the data were obtained with fis promoter constructs located on multi-copy plasmids. A more thorough analysis is required to understand the link between the growth phase regulation of fis expression and divergent promoter activity. A divergent promoter has also been implicated in the regulation of crp expression, but in this case an overlapping divergent promoter blocks the occupation of the crp promoter by RNAP (32).

Growth Phase Regulation of fis Expression-- Recently we demonstrated that increasing levels of negative supercoiling strongly activate the fis promoter both in vivo and in vitro (22). The minimal fis promoter is sufficient for activation by negative supercoiling, but the presence of the GC-rich discriminator sequence between the -10 region and the start point of transcription is absolutely required. This latter element presents a barrier to promoter opening, especially when the superhelical densities are suboptimal (46). The linkage of fis expression to growth phase-dependent fluctuations in DNA topology remains to be elucidated. However, because the superhelical density of DNA increases after the nutritional shift-up and CRP might be involved in this increase by activating gyrA (47, 48), it is conceivable that this indirect effect would contribute to the rapid attainment of maximal fis expression levels in wild type cells (Fig. 2). In addition, CRP could directly activate the fis promoter by binding at putative site I centered at -62.5, which is close to the location typical for the CRP-activated class I promoters (49), thereby displacing the polymerase molecule from the divergent promoter.

Previous studies identified six FIS binding sites in the fis promoter region and demonstrated that autoregulation of the fis promoter is primarily responsible for the steep decrease of fis expression during exponential phase (11, 16, 19). The main FIS binding site implicated in autoregulation is the high affinity site II centered at -42 and overlapping the -35 region of the fis promoter, whereas the additional binding sites appear to either have no role in negative autoregulation or support repression to different extents (20). Consistent with previous reports we observed that in vitro the binding of FIS at site II displaces the polymerase from the fis promoter. However, because the intracellular concentration of FIS decreases rapidly with successive rounds of cellular division, an additional negative regulatory system has been implicated in keeping the fis promoter in a repressed state during stationary phase (11). Nevertheless, it is apparent that at this stage of growth cycle there is enough FIS protein in the cells to repress certain genes (6, 18). Alternatively, at low intracellular FIS concentrations the FIS effect might be reinforced by synergistic interactions with other regulators, including CRP.

The Mechanism of Synergy between CRP and FIS in Repressing fis-- After the shift-up the modulation of fis promoter activity by CRP occurs concomitantly with the accumulation of FIS protein. We assume that as soon as sufficient FIS is produced, the CRP-dependent facilitation of FIS binding to site II (and perhaps site III) will compete with and override the CRP-dependent activation of the fis promoter. Indeed, we observe that in vitro CRP greatly augments the repressing effect of low FIS concentrations on fis promoter activity (Fig. 5). Furthermore, addition of polymerase does not impair the stabilizing effect of CRP on FIS binding to site II (Fig. 6). Finally, increased binding of FIS at site II is observed even at high CRP concentrations, which abolish the CRP-dependent enhancement of RNAP binding at the fis promoter (Fig. 3).

Our finding that in late exponential phase the crp cells have a higher FIS protein content than wild type yet fail to reduce fis expression efficiently is consistent with a requirement for a co-repressor at this growth stage. The results showing lower occupancy of FIS site II in the absence of CRP both in vitro and in vivo indicate that CRP acts synergistically with FIS in repressing fis promoter activity. We propose that this synergy results primarily from the formation of a nucleoprotein complex involving CRP and affecting FIS binding at the repressing site II. In conclusion, our observations strongly support the notion that CRP and FIS cooperate in the shut-off of fis expression during the exponential growth phase (Fig. 8). We presume that in crp cells the expression of numerous genes that are under the control of both FIS and CRP will be perturbed. This will be especially prominent in cases where the regulation involves cooperative interactions. Our study provides a first example of synergistic repression by FIS and CRP, which contrasts the synergistic activation reported for the RpoS-dependent proP P2 promoter and also, for the malEp promoter (30, 50). Such cross-talk between FIS and CRP could provide selective advantage by allowing flexibility of transcriptional control and also increasing the precision of the coordination of cellular gene expression with changing growth conditions.


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Fig. 8.   Model of fis promoter regulation by FIS, CRP, and RNAP. A, the schematics of the organization of protein binding sites in the fis promoter region. The FIS sites I, II, III, and IV are indicated in roman type, the proposed CRP binding sites I and II in italic bold type. The -10 and -35 regions of the fis and divergent promoters are indicated by white boxes, and DNA is represented as a thin line. Bent arrows indicate the direction of transcription. B, changes in the composition of nucleoprotein complexes modulate the transcriptional activity of the fis promoter. Step 1, in the absence of FIS and CRP, two polymerase molecules are bound at the fis and divergent promoters. The tight binding of RNAP at the divergent promoter reduces the activity of the fis promoter under conditions of RNAP limitation. Step 2, binding of CRP displaces RNAP from the divergent promoter and activates the fis promoter. The relevance and particular role of each of the putative CRP binding sites I and II in this effect have not been evaluated at present. Step 3, after the nutritional shift-up binding of FIS at site II competes with the binding of RNAP at the fis promoter and reduces promoter activity. Step 4, simultaneous binding of CRP and FIS stabilizes FIS binding at site II and perhaps III (see Fig. 3). This complex synergistically represses the fis promoter activity during mid exponential growth phase. DNA is represented as a thin curved line. The changes in the shape of the curve are to indicate the transitions in local geometry of DNA. The fis transcript is indicated by a waved line. We note that this model does not consider the role of IHF in the activation of the fis promoter.


    ACKNOWLEDGEMENTS

We thank Annie Kolb for purified CRP protein, Hermann Heumann for purified RNA polymerase and anti-beta subunit antibodies, Regine Kahmann for generous support and critical reading of the manuscript, and Andrea Schultz for excellent technical assistance.

    FOOTNOTES

* This work was supported by a grant from the Département des Sciences de la Vie du CNRS (to W. N.) and by the Deutsche Forschungsgemeinschaft through Grant SFB 190.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Present address: Wellcome/Cancer Research Campaign Institute, Tennis Court Rd., Cambridge CB2 1QR, UK.

|| To whom correspondence should be addressed. Present address: Max-Planck-Institute for Terrestrial Microbiology, Karl-von-Frisch-Strasse, D-35043 Marburg, Germany. Tel.: 49-89-2180-6161; Fax: 49-89-2180-6160; E-mail: Georgi.Muskheli@lrz.uni-muenchen.de.

Published, JBC Papers in Press, February 23, 2001, DOI 10.1074/jbc.M100632200

2 W. Nasser and G. Muskhelishvili, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: PCR, polymerase chain reaction; bp, base pair(s); ORF, open reading frame; DMS, dimethyl sulfate.

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