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J. Biol. Chem., Vol. 277, Issue 3, 2169-2175, January 18, 2002

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In Vivo UV Laser Footprinting of the Pseudomonas putida ⁵⁴ Pu Promoter Reveals That Integration Host Factor Couples Transcriptional Activity to Growth Phase^*

Marc Valls, Malcolm Buckle^§, and Víctor de Lorenzo^¶

From the  Department of Microbial Biotechnology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Campus de Cantoblanco, 28049 Madrid, Spain and ^§ Enzymologie et Cinétique Structurale, UMR 8532 du CNRS, Institut Gustave Roussy, 94805 Villejuif, France

Received for publication, August 23, 2001, and in revised form, October 10, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

The occupation of the ⁵⁴-dependent Pu promoter of Pseudomonas putida by the integration host factor (IHF) under different growth conditions has been monitored in its native state and stoichiometry (i.e. monocopy) with UV laser footprinting technology. We present evidence that an abrupt change in intracellular IHF concentrations occurs when P. putida cells enter stationary phase. This change results in enhanced binding of the factor to the promoter and in the ensuing bending of the target DNA. Since Pu activity depends rigorously on DNA bending, promoter occupation is in turn translated into a much higher transcriptional output when cells leave exponential growth. Inspection of the residual activity of Pu in an IHF strain reveals that IHF predominantly locks the capacity of the promoter to specific growth stages and also that additional physiological signals are entered in the system through ⁵⁴-RNA polymerase. The results substantiate the notion that ⁵⁴ promoters process metabolic co-regulation signals through factor-induced changes in the architecture of the cognate DNA region. Further, they validate UV laser technology as a suitable tool to visualize nondisruptive alterations of DNA shape in vivo.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

The activity of bacterial promoters in vivo is generally subject to two levels of control. First, regulated promoters respond to particular physical-chemical signals (typically distinct nutrients) through cognate proteins that directly or indirectly up/down-regulate expression of genes for transport, metabolism, or other functions. Nevertheless, regulation of specific promoters is subject to a second level of control (generally termed co-regulation) that links transcription of individual genes or operons to the general physiological standing of the cells (1). This ensures a proper balance between all those metabolic networks operating concurrently in living cells (2). A well characterized paradigm of co-regulation is the cAMP-receptor-protein-mediated catabolite repression that certain carbon sources exert on a large number of promoters in Escherichia coli (3). However, there is growing evidence that many, if not all, bacterial promoters are influenced by global environmental stimuli in addition to their primary inducer signals (2). The mechanisms involved in such co-regulation are very diverse, involving not only signaling through chemicals and metabolites whose levels report physiological conditions (ppGpp, trehalose, fumarate, and ATP) but also phosphotransfer relays, mRNA stability, and interplay between sigma factors (for a review, see Ref. 1). Growth conditions affect also the levels and activities of nucleoid-associated proteins, which bind DNA with variable sequence specificity (4) and can thus influence expression of a large number and variety of genes.

The Pu promoter of the TOL plasmid pWW0 of Pseudomonas putida (Fig. 1) is an engaging case of integration between specific and global inputs in the outcome of transcription. Pu belongs to the class of promoters that depend on the alternative sigma factor ⁵⁴ and is activated at a distance by a toluene-responsive activator (XylR). This involves the binding of the regulator to upstream activating sequences (UASs)¹ and the looping-out of the complex drawing it into close proximity to the ⁵⁴-containing form of RNA polymerase (⁵⁴-RNAP) bound to the 12/24 region of the promoter (5). In the case of Pu, this event is assisted by the presence of an integration host factor (IHF) binding site at the intervening region between the UAS and the ⁵⁴-RNAP attachment site (Fig. 1). The IHF protein is a small (20-kDa) basic heterodimeric protein that contains a helix-turn-helix domain involved in dimerization and two antiparallel -sheets. IHF binds and sharply bends DNA (6), producing a distinct footprint on target DNA sequences (7, 8). IHF binding sites include the motif 5'-WATCARNNNNTTR-3' (where W is A or T and R is A or G) separated by 8 bp from a less conserved A/T-rich track of 4-6 bp (9, 10). These structural themes are conserved with small variations in all bacterial species. IHF binding to Pu sharply bends the target DNA sequence, an event that fixes an optimal promoter geometry and facilitates contacts between distant proteins and aids the recruitment of ⁵⁴-RNAP to 12/24 (11, 12). The lack of IHF abolishes any significant activity from Pu in vitro or in vivo (13, 14).

The performance of Pu is exquisitely dependent on the metabolic status of the cell. An excess of certain carbon sources or rapid growth in rich medium inhibits the promoter in vivo even if toluene is present in the culture or if a constitutive XylR variant is used (15, 16). At least four different physiological inputs are channeled into the promoter. First, the presence of glucose and other carbohydrates controls Pu activity through a process which involves the ptsN gene, encoding the IIA^Ntr protein of the phosphoenolpyruvate:sugar phosphotransferase system (17, 18). Second, rapid growth in a rich medium down-regulates the activity of Pu probably by keeping the performance of the ⁵⁴ protein under check, a phenomenon that has been termed exponential silencing (15, 19). Third, there seems to be a separate control connected to the heat shock response as revealed by the significant loss of activity of the promoter in cells defective in the FtsH protein (20). Finally, Pu is moderately stimulated by the alarmone (p)ppGpp that mediates the stringent response to amino acid starvation (21). None of these observations, however, explain entirely the phenomenal burst in activity of a chromosomal Pu-lacZ fusion (from undetectable to ~10,000 Miller units) when P. putida cells pass from exponential growth to stationary phase.

The absolute requirement of DNA bending for Pu activity and the growth phase variations of IHF reported for E. coli make it plausible that some co-regulation signals could be channeled through factor-mediated changes in Pu geometry. Although simple, this notion has been difficult to test properly (15, 22, 23).

In this work, we set out to clarify unequivocally the connections between the physiological standing of P. putida cells, the concentration of IHF, and the activity of the Pu promoter. The main instrument to this end has been the use of a nondisruptive UV laser footprinting technique for visualizing the binding of IHF to Pu in vivo and the ensuing bending of DNA in monocopy in the native stoichiometry of regulatory elements. While this technique has been applied in the past to follow IHF binding to target sites in multicopy plasmids in E. coli (24, 25) this is the first case that the same approach has been applied to examine the occupation of a functional promoter by the factor in vivo under physiologically significant conditions. As shown below, IHF binds to the Pu promoter only when cells cease to grow exponentially. This synchronizes with a sudden rise in intracellular concentrations of the factor and a steep increase in promoter output.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Strains and Plasmids-- P. putida MAD2 is a derivative of the reference P. putida strain KT2442 that bears a hybrid mini-Tn5 transposon that can be selected through a tellurite resistance (Tel) marker. In addition, the transposon encodes the sequence of the truncated protein XylRA deleted of amino acids 1-223 but expressed under the same translation initiation region and promoter (Pr) that drives expression of the native xylR protein. This protein variant is fully constitutive and requires no inducer for promoter activation. The chromosomal insert bears a transcriptional Pu-lacZ fusion as well (26). This design ensures that all regulatory elements controlling expression from Pu are placed in one copy per chromosome. An IHF (ihfA) variant of P. putida MAD2 was created by delivering the ihfA::Km insertion born by plasmid pJRS1 (13) into the chromosome of the target strain through a double crossover in vivo. The knockout was confirmed by PCR and also by verifying the lack of expression of IHF with an anti-IHF mouse serum (see below). The plasmid used as the template for UV laser footprinting (pEZ9) has been described before (27). This construct carries the entire region between co-ordinates 208 and +93 of the Pu sequence in respect to transcription initiation inserted as an EcoRI-BamHI fragment in pUC18.

Monitoring Promoter Performance in Vivo-- P. putida strains were pregrown overnight at 30 °C in LB medium prior to any procedure. For induction experiments, the cultures were diluted 100-fold in fresh medium and grown with vigorous shaking, and the absorbance was followed at 600 nm (A₆₀₀). Pu activity was followed in all cases by assaying the accumulation of -galactosidase in P. putida MAD2 and its ihfA variant. For a gross measurement of reporter activity, -galactosidase assays were made on cells permeabilized with chloroform and sodium dodecyl sulfate as described by Miller (44) under the conditions specified in each case. The linearity of the assay within the range of cell densities and the time of reaction with o-nitrophenyl--D-galactoside was verified in all cases. -Galactosidase activity values given throughout this paper represent the average of at least three independent experiments, each of which was conducted in duplicate samples, with deviations being less than 15%. For quantification of very low levels of -galactosidase, an alternative procedure was used based on a chemiluminescent substrate of the enzyme (Galacton-Plus^®, Tropix). In this case, cells from the culture were diluted 1:10 in lysis buffer (100 mM potassium phosphate, pH 7, 0.2% Triton X-100) and subjected to two freeze-thaw cycles. 10 µl of lysed cells were then incubated for 30 min with 80 µl of reaction buffer (100 mM sodium phosphate, pH 7.0, 1 mM MgCl₂, 1× Galacton-Plus^®). These reactions were run in triplicate in 96-well plates and recorded in a luminometer for 10 s immediately after the addition of 125 µl of a light emission accelerator (Emerald^®, Tropix) following the instructions of the commercial source.

Proteins and Protein Techniques-- Purified IHF protein and ⁵⁴ factor from P. putida KT2442 were the kind gift of F. Bartels (Gesellschaft für Biotechnologische Forschong, Braunschweig, Germany). Purification of the XylRA protein fused to a poly-His metalloaffinity tag has been described before (14). As in vivo with the xylRA allele, the XylRA protein is a constitutive variant that can fully activate transcription from Pu in the absence of any aromatic (14). The RNA polymerase core enzyme of E. coli was purchased from Epicentre.

A polyclonal serum (ascites fluid) against the IHF protein of P. putida was raised by injecting BALB/c mice at days 1 and 21 with 80 µg of the purified factor (containing both IHF-A and IHF-B subunits) suspended in phosphate-buffered saline and MPL + TDM (monophosphoryl lipid A + trehalose dicorynomycolate) adjuvant (Sigma). Production of ascites fluid in immunized mice was induced by intraperitoneal injection of 3 × 10⁵ T-180 sarcoma cells at day 35. Ascites fluid was recovered 8 days later and stored at 4 °C.

For detection and quantification of the intracellular levels of IHF, aliquots of P. putida MAD2 cells were collected at different stages of growth, centrifuged, resuspended in phosphate-buffered saline, and adjusted to an A₆₀₀/ml = 5. Cells were then lysed by sonication, the protein concentration was measured with the Bradford assay (28), and samples were standardized to 1 µg/µl with SDS loading buffer. For each specimen, 15 µg of total cell protein were subjected to SDS-PAGE and transferred to an Immobilon-P membrane (Millipore). For immunodetection of the IHF protein, the membranes were incubated with a 1:2000 dilution of the anti-IHF polyclonal serum described above in phosphate-buffered saline, 3% skimmed milk, 0.1% Tween 20. An anti-mouse IgG-peroxidase conjugate (0.03 units/ml, Roche Molecular Biochemicals) was used as a secondary antibody. The membrane was developed by a chemiluminescence reaction using the BM Chemiluminescence Blotting Substrate (POD, Roche Diagnostics GmbH), visualized, and quantified using a ChemiDoc System (Bio-Rad).

To estimate the number of IHF proteins per cell, whole protein extracts of P. putida MAD2 strains and serial 5-fold dilutions of purified IHF were subjected to Western blot with the anti-IHF serum as above. The intensity of light in the protein bands was quantified after chemiluminescence was developed. A linear relationship existed between the number of molecules of purified IHF applied per lane in the gel and the light intensity of the corresponding protein bands. This standard curve was used to judge the number of IHF molecules present in the samples with the protein extracts using as a reference a conversion factor of 10⁹ cells/ml/unit of A₆₀₀, which was determined beforehand.

In Vitro UV Laser Footprinting-- 10 nM supercoiled pEZ9 plasmid was incubated for 30 min at 30 °C in binding buffer (20 mM Tris-HCl, pH 8.0, 2 mM MgCl₂, 40 mM KCl, 0,1 mM EDTA, 100 µg/ml bovine serum albumin) with different protein combinations at the following final concentrations: XylRA, 400 nM; IHF, 100 nM; RNA polymerase core enzyme, 100 nM; ⁵⁴ factor, 280 nM. Following incubation, 20 µl of each of the mixtures were passed to 0.5-ml Eppendorf tubes and irradiated with a single 5-ns-long pulse of 266 nm UV laser beam, equivalent to an energy of >30 mJ (25, 29, 30). After irradiation, the samples were either processed immediately or stored at 20 °C.

The primer used for extension of the irradiated DNA samples (5'-CCCGCTTTGAGGATATACATGGCGAAAGC-3') was named Pu14. This oligonucleotide hybridizes with the target Pu DNA 63 bp downstream from the transcription start site. Pu14 was end-labeled in its 5'-end with [-³²P]ATP and T4 polynucleotide kinase (28). For detection of pyrimidine dimers in the DNA, 10 µl of the samples with the irradiated plasmid (see above) were added with 6 µl of an amplification mixture containing 10 pmol of each of the dNTPs, 1 pmol of end-labeled primer Pu14, and 2 units of Taq polymerase in 3× GeneAmp PCR buffer with MgCl₂ (PerkinElmer Life Sciences). Samples were then denatured for 3 min at 94 °C and subjected to 10 cycles of extension (1 min at 94 °C, 1 min at 61 °C, and 1 min at 72 °C) in a Thermal Block II thermocycler (Lab Line). A final extension was allowed for 10 min at 72 °C, after which 10 µl of formamide loading buffer was added to each specimen. Samples were then electrophoresed in 6% denaturing polyacrylamide sequencing gels (28). A sequencing reaction with the same sample DNA and identical labeled primer (Pu14) was loaded as a reference in the gels, which were subsequently dried, exposed for 3 h to a Molecular Imager System -sensitive screen, and visualized with the Quantity One software (Bio-Rad). The different lanes were scanned with the same system, and the line graphs were transferred to Microsoft Excel for quantitative analysis. Intensity normalization was performed with respect to the band appearing at position 104, which falls outside of either the IHF binding site or the UASs and is thus unaffected by protein binding.

In Vivo UV Laser Footprinting-- Cultures of P. putida MAD2 and its isogenic ihfA variant were grown at 30 °C in LB liquid medium until the desired optical densities at 600 nm (A₆₀₀) were reached. At those points, 2 ml of stationary cultures or 5 ml of exponentially growing cells were collected, and suitable volumes were irradiated with single pulses at 266 nm as described for the in vitro irradiation. Cells were chilled at once, and the genomic DNA was extracted using the miniprep protocol of Ausubel et al. (28). 10 µg of such DNA were then used for the primer extension reactions. These were run in 100-µl volumes that contained 25 pmol of each dNTP, 4 pmol of labeled primer Pu14, and 5 units of Taq polymerase in a PCR buffer with MgCl₂ (see above). Unlike the in vitro extensions, the DNA samples from live cells were submitted to 60 extension cycles (1 min at 94 °C, 1 min at 59 °C, and 1 min at 72 °C) and a final extension for 10 min at 72 °C. DNA was then precipitated by addition of 10 µl of 3 M sodium acetate, pH 7.5, and 220 µl of ethanol. DNA pellets were rinsed with 70% ethanol, dried, and resuspended in 5 µl of Tris-EDTA. After addition of 10 µl of formamide loading buffer, 6-7 µl of each reaction were run on a denaturing polyacrylamide gel and analyzed as described above. Unlike the samples from irradiation in vitro, the screens required 6 days of exposure for build-up of reliable signals.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

The Loss of IHF Suppresses Pu Activity at Every Growth Stage of P. putida-- Many promoters dependent on ⁵⁴ (Pu among them, see above) contain IHF sites (Fig. 1), which influence their performance in a number of ways. In the case of Pu, IHF helps the assembly of a productive constellation of protein-protein and protein-DNA interactions at the transcription initiation complex. Despite this, the actual effect of IHF on Pu activity during growth of P. putida has never been examined. To address this issue, we set out to generate the ihfA mutation (encoding one of the two IHF subunits in P. putida) in an assay system that could faithfully reflect the regulation of the Pu promoter. The reference strain to this end was P. putida MAD2 (26) because its chromosome is inserted with a mini-Tn5 transposon, which includes the constitutive, inducer-independent allele of xylR (xylRA), expressed through its natural promoter (Pr). This is assembled next to a transcriptional Pu-lacZ fusion, so that all regulatory elements controlling Pu activity are placed in one copy per chromosome of the native host. A strain fully isogenic to P. putida MAD2 but bearing a kanamycin (Km) insertion in ihfA was then produced as explained under "Experimental Procedures." As the factor is only active as a heterodimer, this strain lacks de facto any IHF activity. With these assay strains, all changes detected in Pu can be traced to this histone-like protein.

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Organization of the Pu promoter. A, the scheme on top is a sketch (not to scale) of the regulatory elements that intervene in the regulation of the Pu-lacZ fusion of P. putida MAD2. These include binding sites (UAS) for the cognate regulator (the enhancer-binding protein XylR), IHF (which bends sharply the target DNA), and the 12/24 motif for 54-RNA polymerase binding. IHF upholds a promoter geometry, which sets an optimal constellation of protein-protein and protein-DNA contacts. B, the sequence below is an expansion of the Pu region spanning positions 50 and 92 around the IHF binding site (56 to 86). The nucleotides of the upper strand that are either protected or hypersensitive to UV laser footprinting (groups A, B, and C) in the presence of IHF (see text) are indicated in bold and are shown lined up with the consensus binding site for the factor. The predicted center of the curvature (inferred from the known crystal structure of IHF·DNA complexes) is also depicted.

Fig. 2A shows the accumulation of -galactosidase by wild-type P. putida MAD2 and its ihfA counterpart along growth in rich medium. The induction curve in the IHF⁺ strain displayed the typical exponential silencing phenomenon (15), consisting in the absence of Pu activity during rapid exponential growth in LB followed by a rapid increase of transcription at the onset of stationary phase (Fig. 2A). However, within the detection range granted by Miller's procedure (44) for monitoring lacZ fusions, it could be concluded that the loss of IHF essentially ceased Pu activity at every growth stage. This is in contrast with the situation observed in E. coli where Pu maintains 30-50% of its activity in IHF strains (31, 32). Although P. putida contains the second nucleoid-associated protein HU (45), the results of Fig. 2A clearly indicate that the functional substitution between the two factors (IHF and HU) shown in vitro and in vivo for E. coli (33) does not operate in P. putida.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Pu output is synchronized to IHF levels. A, Pu activity along growth in IHF+ and IHF strains. Cells of P. putida MAD2 xylRA+/Pu-lacZ () and its ihfA counterpart () were collected from a stationary phase culture, diluted to A600 (OD600) ~0.05, and regrown in fresh LB medium at 30 °C. Accumulation of -galactosidase (-Gal) was then recorded along time. Note the sharp induction of Pu at the onset of stationary phase in the IHF+ strain and the loss of activity in the IHF mutant. B, growth phase-dependent accumulation of IHF in P. putida MAD2. Samples from the strain were taken at different times of growth and subjected to a Western blot assay using an anti-IHF polyclonal serum. Triangles show cell growth, and circles show the average IHF concentration calculated from two independent blots (see "Experimental Procedures"). Concentration variations in independent experiments were within the 15% range. Note that augmentation of IHF levels is manifest at late exponential/early stationary phase of growth.

Intracellular Levels of IHF Increase Sharply When Cells Enter Stationary Phase-- Since Pu is inactive in vivo in the absence of IHF, we wondered whether the inhibition during exponential growth could be related to varying concentrations of the factor in the cell. In E. coli, IHF protein and mRNA levels seem to increase as cells stop growing (22, 34, 35). In one study, the protein concentration has been shown to experience a short burst encompassing the entry into stationary phase followed by a relative decrease at late stationary phase (4). In contrast, the ihfA/ihfB mRNA levels of Neisseria gonorrhoeae decline when cells enter stationary phase (23). To ascertain this question in P. putida, we raised a mouse serum against the purified IHF protein of this species and used it to follow the intracellular concentration of IHF during growth using a Western blot assay. As shown in Fig. 2B, it is evident that IHF content was much higher in stationary than in exponential growth. Furthermore, by using an IHF standard of known concentration, we could estimate that the absolute level of the factor oscillated between <2000 molecules (monomers) per cell during rapid growth and >14,000 IHF molecules when cells were well into stationary phase. This represents almost 1 order of magnitude change in protein concentration between the two physiological conditions, the most acute shift occurring at optical densities at 600 nm of around 1.5. The intracellular IHF concentration profile in P. putida approximates that reported in E. coli by Ditto et al. (22).

Although this change in IHF concentration is synchronized with the outburst of Pu activity (Fig. 2A), this does not grant per se that the two events are related. Since the IHF site in Pu has a high affinity for IHF (27), the number of available IHF molecules, even at the lowest levels found in exponential growth, should be sufficient to always saturate this target sequence. To clarify this issue it was thus necessary to monitor the actual occupation of the IHF site at exponential and stationary stages. To this end, we resorted to match the UV laser footprints caused by IHF in vitro versus those found in the target sequence in vivo following an improved procedure explained in detail in the following paragraphs.

Visualization of Factor-mediated Structural Changes in Pu Promoter in Vitro-- The UV laser footprint approach is based on the fact that a highly intense 266 nm laser light produces intrastrand modifications within the DNA helix, the most prominent being the formation of pyrimidine dimers (especially TT and TC) (29). The efficiency of the photochemical reaction at a given base is tightly related to the local DNA topology. The presence of a bound protein has two fundamental effects. First, the protein may change the local conformation of the DNA thus affecting the probability of the excited base from undergoing a given electronic rearrangement (i.e. singlet state-mediated cyclobutane dimer formation). Second, any amino acids within Van der Waals radii distance from an excited base may form a direct covalent cross-link, the order of reactivity of bases being T > C > G A (36, 37). These rearrangements can subsequently be revealed by primer extension since they interfere with DNA polymerization (29, 30). Although operatively termed footprint, irradiation with UV laser produces what may be called more properly an imprint that reports the conformation of a DNA segment and its immediate environment and not protection caused by protein binding. In addition, the reaction is very rapid (following a laser pulse of a few nanoseconds major photoreactions occurring through the singlet state have half-lives of several picoseconds and minor excited states have half-lives in the microsecond time range) and consequently nondisruptive. UV laser footprinting was thus the method of choice to visualize IHF binding to its target AT-rich sequence in Pu (Fig. 3A).

View larger version (45K): [in this window] [in a new window]   Fig. 3.   In vitro UV laser footprint of the IHF site in Pu. A, schematic representation of the technique. A single UV laser light is applied to DNA samples with or without IHF. The observed photoreactions correspond almost exclusively to dimer formation between pyrimidines that are adjacent in the target sequence. Protein binding alters the physicochemical environment and distorts DNA, affecting such reactivity. Changes are visualized in a DNA sequencing gel following primer extension. B, photomodifications in Pu caused by factors binding the promoter sequence. Plasmid pEZ9 containing the sequence of Pu was irradiated with a short (5-ns) pulse of UV laser (266 nm) in the presence of purified XylRA, IHF, the RNA polymerase core enzyme, and the sigma factor 54. Proteins added to the samples are indicated at the top of the gel at the concentrations specified under "Experimental Procedures." A control sample to visualize the photoreactivity of naked DNA is shown also. Changes in band intensities connected to the addition of IHF are labeled A, B, and C to the right of the gel with an indication of the sequences involved and their co-ordinates in respect to the transcription initiation site. The sequence GA to the left was generated with the same oligonucleotide used for primer extension. Note that the sequence is complementary to the upper strand. C, IHF binding to Pu is not influenced by other proteins that participate in transcription. The figure shows the modifications in photoreactivity of Pu caused by combinations of IHF, XylRA, and the 54-containing holoenzyme (E54). Note that the changes caused by IHF are not affected by the presence of the additional proteins.

To attribute unequivocally the changes in photoreactivity (i.e. band intensity) of Pu to the presence of IHF, the effect of UV laser photoirradiation was first investigated in vitro. Supercoiled pEZ9 plasmid (containing the Pu promoter, see "Experimental Procedures") was exposed to a 5-ns UV laser pulse in the presence of different combinations of purified IHF, XylRA (a constitutively active regulator variant (14)), core RNA polymerase, and the ⁵⁴ factor. To reveal modifications in the upper strand, the DNA samples were then subjected to 10 rounds of primer extension, and products were separated by electrophoresis in a DNA sequencing gel. The resulting band patterns reporting the physical interaction of IHF with its core binding site and the flanking regions are shown in Fig. 3B. In this particular instance, we noted that the bands that appear in the gel correspond to termination of primer extension by the Taq polymerase in the vicinity of putative pyrimidine dimers on the template strand. The size of the labeled extension products thus coincides with termination at positions opposite to adjacent pyrimidines in the sequence. Since elongation with Taq appears to stop immediately before or opposite the first modified nucleotide in a dimer, single pyrimidine pairs can produce band doublets (e.g. see TC at positions 65/66). This is in contrast with previously reported primer extension protocols based on the Klenow fragment where termination was always at position n-1 opposite the first of the dimer pair (29, 38, 39).

Fig. 3B shows that addition of IHF to the DNA-protein mixtures caused a very distinct pattern of bands throughout the known target site at the Pu promoter. None of the other proteins tested (XylRA, core RNAP, and ⁵⁴) originated a noticeable change in the set of bands produced by the UV laser footprinting procedure even at the relatively high concentrations of the factors used in the assays. To verify whether IHF binding to Pu could be followed as an event independent of and not influenced by any of the other proteins that participate in transcription, we ran the controls shown in Fig. 3C. In this case, we compared the pattern of bands resulting from addition of IHF alone (Fig. 3B) with those of the ⁵⁴-containing holoenzyme (E⁵⁴) with or without IHF and XylRA. The results of Fig. 3C gave a clear evidence that, at least in vitro, IHF binding is an event that occurs in a fashion not influenced by any of the proteins known to bind proximal sites along the Pu promoter sequence (Fig. 1). Alterations could thus be traced unequivocally to IHF binding and bending of the corresponding site at the promoter (Fig. 1).

Informative changes in the formation of pyrimidine dimers were seen at positions 58/57 (TT), 66/65 (TC), and 86/76 (TTTCTTTTTT) in the upper strand. A simple visual inspection of the resulting extension products revealed that the addition of IHF caused a hyperreactivity of the TC dimer at 66/65 concurrent with a hyporeactivity of the TT pair at 58/57. Interestingly, the bases with altered reactivity correspond to the conserved central TC and downstream TT sequence motifs of the IHF consensus binding site (Fig. 1B). The upstream cluster of pyrimidines (86/76) was also affected by IHF binding, albeit the specific pattern was not so obvious to the bare eye. The stretch of thymines present in this region (81/77) coincided with the less conserved 5' AT-rich region found in most target sites for IHF (10). Changes in band intensities (although less informative) were observed throughout the same region as well when the lower strand was used as a template for extension in similar conditions (data not shown).

To get a more reliable reference for IHF binding, the bands shown in the gel of Fig. 3B were scanned, and the intensity of the signals was normalized and finally plotted as shown in Fig. 4. As a result of this analysis, the different samples clustered clearly in two separate profiles, depending only on IHF presence in the protein mixture prior to irradiation. The sample-to-sample variations within background noise seen in the presence of ⁵⁴-RNAP and XylR confirmed that neither of these proteins altered the photoreactivity of the Pu sequence under examination. It is clear from Fig. 4 that the principal effect of IHF in the upstream stretch of pyrimidines was to decrease dimer formation in the conserved thymidine track at positions 81/77.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Quantification of changes entered in the structure of Pu upon IHF binding in vitro as revealed by UV laser footprint. The figure shows a superimposition of the normalized scans corresponding to the bands of lanes in Fig. 3B. Intensity of each signal is represented in arbitrary units. Relevant residues are indicated below the plot. Note that IHF addition alters distinctively the photoreactivity of the groups of bands labeled A, B, and C.

The changes in the pattern of photoreactivity induced by IHF in Pu may be interpreted in structural terms. The crystal structure of IHF of E. coli bound to a synthetic 35-bp oligonucleotide shows that the DNA is bent by ~160°. The center of curvature is positioned just 5' with respect to the WATCAR motif of the consensus site (Fig. 1B). The A/T-rich sequence and the TTG feature are located one helical turn upstream and downstream of the center of curvature, respectively (6). Bearing in mind that pyrimidine dimer formation through 5-6 cyclobutane adduct formation reflects the geometry of the major groove, this would explain the increase in photoreactivity in Pu at the TC pair adjacent to the center of curvature due to changes in the wedge angle between bases following IHF binding. By the same token, the TT dimers at the conserved sites upstream and downstream of the bend center became less reactive when IHF was bound to Pu.

A close perusal of the scan shown in Fig. 4 reveals the property of the bands corresponding to positions 58/57, 66/65, and 81/77 as intrinsic descriptors of the occupation of the Pu promoter by IHF since their relative intensity increases or decreases depending on IHF binding. The altered reactivities fall within conserved sequences, suggesting that these nucleotides are structurally relevant for all IHF functions. These groups of bands will be designed hereafter as A (58/57), B (66/65), and C (81/77) as indicated in Fig. 4. That IHF caused a sharp reduction of signals A and C along with a clear augmentation of signal B gave us a useful reference to judge the occupation of the Pu promoter in vivo as explained below.

The IHF Site of the Pu Promoter Is Occupied Only in the Stationary Phase of Growth-- Once a reference for IHF binding to Pu was produced in vitro, we set out to determine whether the factor was bound to the promoter at any growth stage or whether the interaction was specific to a given physiological state. From a technical point of view, this is not straightforward since all the regulatory elements that control Pu should represent the native configuration (i.e. monocopy gene dose). Since the target DNA represents a very small fraction of the whole chromosome we had to adapt the UV laser footprint technique by introducing multiple linear extensions of the target region and suppressing background noise to thus produce the single-base resolution and signal to noise ratio compatible with the results in vitro.

To this end, we grew the Pu-lacZ strain P. putida MAD2 and its IHF counterpart in LB medium in conditions similar to those depicted in Fig. 2. Samples collected at the exponential and the stationary phase of growth were then irradiated with 5-ns UV laser pulses. The modified genomic DNA was subsequently isolated, and after 60 cycles of primer extension, the positions at which pyrimidine dimers had been formed were visualized on a sequencing gel as described above. Fig. 5 shows the results of this experiment. Fig. 5B is a normalized representation of the scans presented in 5A. In the wild-type P. putida MAD2 strain, a striking difference in band intensities was observed within the IHF binding site as a function of the growth phase. The pattern of bands observed in exponentially growing cells of P. putida MAD2 could be positively matched to that obtained in vitro in the absence of IHF suggesting that the site was not occupied during rapid growth in rich medium (e.g. compare lanes 1 and 5 in Fig. 5A). In agreement with the situation in vitro (Fig. 3, B and C), the loss of ⁵⁴ (i.e. using a strain lacking the rpoN gene, which encodes ⁵⁴) did not have a significant influence on the occupation of Pu by IHF at either the stationary or exponential growth stages (not shown). Also as expected, in the isogenic IHF strain, the pattern was also equal to that of the Pu promoter in vitro without IHF (Fig. 5A, lanes 3 and 4) and displayed no difference between the exponential and the stationary phase. It is especially apparent in the normalized representation of band intensities (Fig. 5B) that these profiles of the IHF strain totally matched that of the wild-type strain in exponential growth, demonstrating again that the IHF site is unoccupied in this physiological state. On the contrary, the kind of photoreactivity of the DNA region examined in cells of wild-type P. putida MAD2 collected from stationary phase was similar to that of Pu incubated with IHF in vitro (Fig. 5A, lanes 2 and 6). This was taken as evidence that IHF was indeed bound to the promoter during this late stage of the growth curve. Furthermore, footprints of bacterial cells at late exponential phase were intermediate between those obtained in cells at the early and the late stages of growth (not shown), indicating a partial occupation of the IHF site. In summary, these experiments established that binding of IHF to Pu is only significant when cells cease to grow exponentially, a circumstance that is coincident with an intense upshift of intracellular IHF levels.

View larger version (37K): [in this window] [in a new window]   Fig. 5.   In vivo laser footprinting of the Pu promoter. P. putida MAD2 and its ihfA derivative were grown in LB medium and subjected to UV laser irradiation at exponential (Exp.) and stationary (St.) phases of growth. Genomic DNA extracted from cells was subjected to primer extension and electrophoresed in a sequencing gel. A, comparison of footprints obtained in vivo and in vitro. B, normalized representation of in vivo footprints. Note that signals corresponding to the occupation of Pu by IHF appear only in the wild-type P. putida MAD2 strain when cells are in stationary phase. Neither exponentially growing P. putida MAD2 cells nor the ihfA strain displayed any indication of IHF binding. A quantification of the corresponding bands is shown in the normalized scan below. wt, wild type; ihf, ihfA.

Categorizing the Role of IHF in the Physiological Control of Pu-- Since IHF is strictly necessary for Pu activity, the data above could indicate that down-regulation during exponential growth simply reflects the changing levels of IHF in response to a certain physiological status. This consideration incited us to re-examine the residual activity of Pu in an IHF strain. Although Pu performance is virtually undetectable in P. putida MAD2 ihfA using the standard Miller procedure (44) (Fig. 2A), we resorted to the use of a hypersensitive substrate of -galactosidase called Galacton-Plus^® to detect changes in promoter activity along growth. Cleavage of this compound by the reporter enzyme can be coupled to a light emission scheme so that the sensitivity of the method increases by 100-1000-fold that of the standard method of Miller (44). In the absence of IHF, the residual Pu output should reflect exclusively the intrinsic ability of ⁵⁴-RNAP to activate transcription during growth. To relate the conduct of the wild-type cells with the ihfA mutants in this respect, the values of light emission for both strains were plotted as percentages of the maximum level obtained in each case (Fig. 6). Most revealing, the shape of the induction curves of Pu along growth was identical in the two strains, meaning that the promoter is still subject to physiological regulation in cells devoid of IHF. Under these criteria, the results of Fig. 6 would indicate that the polymerase is not competent to activate Pu during rapid growth but is during the stationary phase. In other words, that the effect of IHF in Pu is superimposed to the separate physiological control channeled through the performance of ⁵⁴-RNAP.

View larger version (20K): [in this window] [in a new window]   Fig. 6.   Residual Pu activity in the absence of IHF is growth phase-dependent. The experiment is basically identical to that shown in Fig. 2A except that accumulation of -galactosidase (gal) by strain P. putida MAD2 () and P. putida MAD2 ihfA () was measured with the extremely sensitive substrate Galacton-Plus®. For comparison of the induction patterns of Pu in the two strains, the vertical axis for -galactosidase activity was adjusted to a percentage of the maximal and minimal values found in each case (the absolute values differed by 100-fold). Cell growth is shown in the inset.

Conclusion-- Since IHF is required for full transcriptional activity of Pu, the data above suggest that it realizes the capacity of the promoter (i.e. its absolute output) within determined activity ranges during the growth phase. The result of this is that IHF levels lock Pu performance as a whole to the physiological situation of cells when they proliferate in a rich medium. Since IHF is in itself controlled by growth phase (Fig. 2B) as a result of a collection of metabolic signals (34), our data validate the notion that factor-mediated DNA bending is an instrument for the physiological co-regulation of prokaryotic promoters (40). Although our results apply to only one system, the notion may have a general significance for other genes dependent on ⁵⁴. It is striking that some examples of this type of promoters contain IHF sites, whereas others do not (9). Given that prokaryotic promoters (including those dependent on ⁵⁴) have a limited number of potential protein and DNA targets for transcriptional co-regulation, promoter architecture may become an asset for integration of physiological signals (40). All the data collected so far on the physiological control of Pu and on the related promoter Po of Pseudomonas sp. CF600 (41-43) suggest that ⁵⁴ systems are particularly well suited for coupling a whole range of metabolic symptoms into one or more steps of the transcription initiation process. With only two constant players (the substrate-responsive UAS-binding regulator and ⁵⁴), these promoters afford an amalgam of the effector-specific transcription with the cell physiology. The specific molecular instruments for such an amalgam could be predominantly determined by the diversity of DNA sequences at the promoter. In this case, the presence or absence of IHF sites in ⁵⁴ systems may reflect the diverse degrees of physiological co-regulation required for optimization of promoter performance under the tough selection conditions that operate in bacterial niches in the environment (2).

	ACKNOWLEDGEMENTS

We are indebted to I. Cases, L. A Fernandez, and J. Garmendia for inspiring discussions.

	FOOTNOTES

^* This work was supported by European Union Contracts QLK3-CT2000-00170 and QLK3-CT1999-00041, by Grant BIO98-0808 of the Spanish Comisión Interministerial de Ciencia y Tecnología (CICYT), and by the Strategic Research Groups Program of the Autonomous Community of Madrid. The support of the Fondation de la Recherche Medicale (FRM) for the award of a grant (to M. B.) for the establishment of a new laboratory is acknowledged.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.

^¶ To whom correspondence should be addressed. Tel.: 34-91-585-4536; Fax: 34-91-585-4506; E-mail: vdlorenzo@cnb.uam.es.

Published, JBC Papers in Press, November 1, 2001, DOI 10.1074/jbc.M108162200

	ABBREVIATIONS

The abbreviations used are: UAS, upstream activating sequence; IHF, integration host factor; RNAP, RNA polymerase.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

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