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BIOCHEMISTRY AND MOLECULAR BIOLOGY

A σ54-dependent promoter in the regulatory region of the Escherichia coli rpoH gene

Anna Janaszak, Wiktor Majczak, Beata Nadratowska, Agnieszka Szalewska-Pałasz, Grażyna Konopa, Alina Taylor

  • Department of Molecular Biology, University of Gdańsk, Kładki 24, 80-822 Gdańsk, Poland
  • Correspondence
    Alina Taylor
    ataylor{at}biotech.ug.gda.pl

    • Microbiology 2007; 153(1):111–123 · https://doi.org/10.1099/mic.0.2006/000463-0

    View at publisher PubMed

    Abstract

    The Escherichia coli rpoH gene is transcribed from four known and differently regulated promoters: P1, P3, P4 and P5. This study demonstrates that the conserved consensus sequence of the σ54 promoter in the regulatory region of the rpoH gene, described previously, is a functional promoter, P6. The evidence for this conclusion is: (i) the specific binding of the σ54–RNAP holoenzyme to P6, (ii) the location of the transcription start site at the predicted position (C, 30 nt upstream of ATG) and (iii) the dependence of transcription on σ54 and on an ATP-dependent activator. Nitrogen starvation, heat shock, ethanol and CCCP treatment did not activate transcription from P6 under the conditions examined. Two activators of σ54 promoters, PspF and NtrC, were tested but neither of them acted specifically. Therefore, PspFΔHTH, a derivative of PspF, devoid of DNA binding capability but retaining its ATPase activity, was used for transcription in vitro, taking advantage of the relaxed specificity of ATP-dependent activators acting in solution. In experiments in vivo overexpression of PspFΔHTH from a plasmid was employed. Thus, the σ54-dependent transcription capability of the P6 promoter was demonstrated both in vivo and in vitro, although the specific conditions inducing initiation of the transcription remain to be elucidated. The results clearly indicate that the closed σ54–RNAP–promoter initiation complex was formed in vitro and in vivo and needed only an ATP-dependent activator to start transcription.

    Edited by: S. J. W. Busby

    INTRODUCTION

    The Escherichia coli heat shock stimulon consists of regulons controlled by the σ32 (Arsène et al., 2000; Zhao et al., 2005) and σ24 RNA polymerase (RNAP) subunits (Erickson & Gross, 1989; Connolly et al., 1999), the rpoH and rpoE gene products, respectively. A third regulon of the stress response has been identified from recent data. It is under the control of the σ54–RNAP subunit (the rpoN gene product). The first σ54-dependent heat shock operon, pspA–E, was described by Model's group (Brissette et al., 1991; Model et al., 1997). Later, it was found that the ibpB gene is transcribed not only from the σ32 promoter upstream of the ibpAB operon (Allen et al., 1992), but also from the internal σ54 promoter situated in the ibpA–ibpB intergenic space (Kuczyńska-Wiśnik et al., 2001). Moreover, a canonical sequence of a σ54 promoter, proximal to the rpoH coding sequence, was found by sequence analysis (Pallen, 1999). Information as to whether the sequence represents a functional promoter is currently lacking.

    Transcription of the rpoH gene can be initiated from four previously described promoters (Fig. 1⇓). Three of them, P1, P4 and P5, are recognized by σ70–RNAP (Erickson et al., 1987; Nagai et al., 1990; Ramirez-Santos et al., 2001; Solis-Guzman et al., 2001) and P3 is recognized by σ24–RNAP (Erickson & Gross, 1989; Wang & Kaguni, 1989a; Missiakas & Raina, 1998). The P2 promoter was found only in one E. coli strain, SC122 (Erickson et al., 1987), and was absent from MG1655. Transcription of the rpoH gene from these promoters is regulated by CRP–cAMP, CytR (Ramirez-Santos et al., 2001; Kallipolitis & Valentin-Hansen, 1998) and DnaA (Wang & Kaguni, 1989b; Messer & Weigel, 1997) proteins.

    Figure image not available in archive
    Fig. 1.

    Organization of the rpoH regulatory region. (a) The regulatory region of the rpoH gene [−524 to +183 bp relative to ATG (+1)]. The transcription start sites from the P1, P3, P4, P5 and P6 promoters are marked by vertical arrows. Binding sites for regulatory proteins CRP, CytR and DnaA are indicated. Horizontal arrows represent primers used in this study (listed in Table 1). The PCR products obtained with these primers are shown schematically under the figure. (b) DNA sequence of the rpoH regulatory region. The first base of the rpoH coding region is numbered +1. The IHF-binding site, the RBS sequence and the ATG codon are underlined. Vertical arrows represent transcription start sites from the P1 and P3–P6 promoters. The sequence of the P6 (σ54-dependent) promoter is boxed. The putative binding sites for PspF (UAS II) and NtrC are overlined (continuous line and dotted lines respectively). These sequences are compared to the consensus sequences of PspF (Dworkin et al., 1997) and NtrC (Weiss et al., 1992) at the bottom of the figure.

    If the potential σ54 promoter of the rpoH gene is functional, it should mean that stress responses encompass as yet unidentified processes. Functional σ54 promoters are rare in the E. coli genome: their number was estimated to be about 30, though sequences resembling canonical σ54 promoters are abundant (Reitzer & Schneider, 2001). The σ54 subunit is not essential for E. coli growth under laboratory conditions; however, it is necessary for expression of genes involved in diverse processes like nitrogen metabolism, transport of dicarboxylic acids, pilus formation, formate dehydrogenase synthesis, metabolism of aromatic compounds (xylene, toluene), and expression of the pspA–E operon and of the ibpB gene encoding a small heat shock protein.

    E. coli cells contain seven different σ subunits (Arsène et al., 2000; Wösten, 1998) that form two classes characterized by sequence homology and specificity of promoter recognition. Six of them belong to class I, represented by σ70. The σ54 subunit is a unique member of class II (Merrick, 1993; Shingler, 1996; Wösten, 1998), sharing no homology with class I. The σ54 subunit recognizes the conserved sequence YTGGCACG-N4-TTGCWNN of the promoters located at −12 and −24 bp from the transcription start site (Barrios et al., 1999). Initiation of σ54–RNAP-dependent transcription resembles eukaryotic transcription initiation by RNAP II. (Guo et al., 1999, 2000; Fu et al., 2000). A closed complex of σclass I–RNAP–promoter isomerizes spontaneously to the active, open complex. In contrast, isomerization of the σ54–RNAP–promoter closed complex requires interaction with the specific enhancer-bound, ATP-dependent activator (reviewed by Merrick, 1993; Shingler, 1996). Contact between the activator and the σ54–RNAP–promoter complex is achieved by DNA looping, facilitated either by the integration host factor (IHF) protein or by intrinsic DNA topology (Pérez-Martin et al., 1994; Carmona et al., 1997). Control, imposed on the DNA melting step, requires ATP hydrolysis and involves the promoter −12/−11 element (Guo et al., 1999, 2000) bound inside the RNAP channel (Polyakov et al., 1995; Zhang et al., 1999; Severinov, 2000; Foster et al., 2001).

    Activators of σ54-dependent promoters have a modular structure and share conserved sequence motifs with the AAA+ protein family (Shingler, 1996; Zhang et al., 2002; Cannon et al., 2003; Studholme & Dixon, 2003). The N-terminal domains of the majority of these activators act as specific sensors of inducing signals. NtrC may be regarded as a representative of this group. In response to nitrogen limitation, the ATPase activity of NtrC is positively regulated by phosphorylation of its N-terminal domain by the NtrB kinase and stimulated by DNA binding (Weiss et al., 1991, 1992).

    Members of another smaller, group of activators of σ54-dependent promoters, which lack the N-terminal regulatory domain, include PspF of E. coli (Jovanovic et al., 1996; Model et al., 1997) and HrpR of Pseudomonas syringae (Grimm et al., 1995). PspF, the activator of the pspA–E operon, occurs in cells at low concentrations and is constitutively active. Modulation of its activity, unlike that of NtrC, depends on protein regulators: PspA (negative) or PspB and PspC (positive) (Shingler, 1996; Elderkin et al., 2002; Hankamer et al., 2004). PspF responds to heat shock and ethanol treatment, but the strongest reaction is evoked by bacterial membrane impairments coincident with the dissipation of charge as by the gene IV protein of filamentous phages (e.g. f1, M13) or by carbonylcyanide m-chlorophenylhydrazone (CCCP; Model et al., 1997). The molecular mechanisms of signal reception and transduction in this system are not yet understood.

    The purpose of this work was to establish whether the σ54–RNAP holoenzyme binds the putative σ54-dependent promoter (P6) in the regulatory region of the rpoH gene, and whether it is able to initiate transcription from P6 in vitro and in vivo. We present evidence of the holoenzyme binding and initiation of transcription in vitro and in vivo, based on gratuitous activation by PspFΔHTH. The identity of the specific activator and inducing conditions remain to be elucidated.

    METHODS

    Bacterial strains, plasmids, culture conditions, primers and DNA fragments.

    The E. coli strains, plasmids and primers used in this study are listed in Table 1⇓. The bacteria were grown in Luria–Bertani (LB) medium at 30 °C (unless otherwise stated). When appropriate the following antibiotics were added: ampicillin (100 μg ml−1), tetracycline (15 μg ml−1), kanamycin (30 μg ml−1) and chloramphenicol (34 μg ml−1). Transformation of E. coli cells was performed according to Sambrook et al. (1989) by using the CaCl2 procedure.

    Table 1.

    E. coli strains, plasmids and primers used in this study

    DNA fragments W1-W2, W8-W9, W2-W5 and W3-W7 (Fig. 1a⇑) were amplified by PCR with the corresponding primers.

    Proteins.

    The His-tagged proteins listed below were purified by Ni-affinity chromatography using the BioLogic LP chromatography system (Bio-Rad). His–σ54 and His–NtrC proteins were overproduced from strains BL21(DE3)(pS54-2) and BL21(DE3)(pNTRC-3), respectively and purified as described by Rippe et al. (1997, 1998). His–PspF and His–PspFΔHTH were obtained by overproduction from SG13009(pREP,pMJ16) and K1527 (pMJ15) respectively and purified according to Jovanovic et al. (1999). The IHF protein was purified as described by Filutowicz et al. (1994) after overexpression from strain DH5α(pHNβα). σ70 was overproduced from CF1690Δlac(pVI690) and purified as described by Fujita & Ishihama (1996). E. coli core RNAP was purchased from Epicentre Technologies. Reconstituted RNAP holoenzyme was obtained by incubating the core RNAP with an appropriate σ subunit at a 1 : 4 molar ratio for 15 min at 30 °C in STA buffer (25 mM Tris/acetate pH 8.0, 8 mM magnesium acetate, 10 mM KCl, 1 mM DTT, 3.5 % PEG). NtrC was phosphorylated to NtrC-P before use by the addition of carbamyl phosphate (10 mM) to the reaction mixture.

    Electrophoretic mobility shift assays (EMSAs).

    The DNA fragments W1-W2, W2-W5 and W8-W9 were end-labelled with [γ-32P]ATP (166.5 TBq mmol−1) by using T4 polynucleotide kinase (Promega). DNA–protein binding was carried out in STA buffer supplemented with BSA (100 μg ml−1) and poly[dI–dC] (30 μg ml−1) for 20 min at 30 °C and was stopped by the addition of 2 μl loading dye (0.05 % bromophenol blue, 50 %, v/v, glycerol). The amounts of DNA and holoenzyme are given in the legends for Figs 2–5⇓⇓⇓⇓. Electrophoresis was performed in 4.5 % polyacrylamide gel in Tris/glycine buffer (25 mM Tris, 200 mM glycine) at 8 V cm−1 for 2 h. The gels were dried and submitted to autoradiography or analysed with a Molecular Imager FX (Bio-Rad).

    Figure image not available in archive
    Fig. 2.

    σ54–RNAP binding to the rpoH promoter region. (a) EMSA. The γ-32P end-labelled 277 bp DNA fragment W2-W5 (8 nM) was incubated with increasing amounts of σ54-RNAP, as indicated in the figure. The position of the DNA fragment and the shifted DNA–σ54–RNAP complex are marked with arrows. (b) Electron microscopy: photomicrographs show the position of the σ54–RNAP on the W1-W2 DNA fragment (707 bp). The conditions for holoenzyme binding to DNA were as described in Methods. σ54–RNAP (200 nM) was incubated with a W1-W2 DNA fragment (35 nM) for 15 min at 30 °C. Photomicrographs were digitized to measure the position of the holoenzyme. (c) Statistical analysis of about 100 EM images showing σ54–RNAP bound to the putative P6 promoter, within the W1-W2 DNA fragment [−524 to +183 bp relative to ATG (+1)]. The scheme below the graph represents the DNA fragment containing the putative P6 promoter.

    Figure image not available in archive
    Fig. 3.

    DNase I footprints of σ54–RNAP and σ70–RNAP in the rpoH promoter region. (a) The γ-32P end-labelled DNA fragment W3-W7 was incubated with increasing quantities of σ70–RNAP, σ54 or σ54–RNAP. Lane 1, DNA control, no protein. Lanes 2–5, σ70–RNAP: 100, 200, 500 and 700 nM, respectively. Lanes 6–9, σ54: 500, 1000, 2000 and 4000 nM, respectively. Lanes 10–12, σ54–RNAP: 100, 200 and 500 nM, respectively. Lane 13, control, core RNAP (100 nM). After a brief digestion with DNase I, the reaction products were subjected to electrophoresis alongside the products of the sequencing reactions (G, A, T, C) performed with the W7 primer. Regions protected by σ54–RNAP and σ70–RNAP are indicated with brackets. (b) The DNA sequence containing the overlapping P5 and P4 promoters and protected from DNase I digestion by σ70–RNAP is shaded. The DNA sequence encompassing the putative P6 promoter (bold letters) protected by σ54–RNAP from the DNase I digestion extends from −37 to −68 bp relative to the rpoH start codon (white box).

    Figure image not available in archive
    Fig. 4.

    IHF binding to the rpoH gene regulatory region. (a) EMSA. The γ-32P end-labelled 277 bp DNA fragment W8-W9 (7.8 nM) was incubated with increasing quantities of IHF protein (as indicated in the figure). The positions of the W8-W9 DNA and the retarded DNA–IHF complex are indicated with arrows. (b) DNase I footprint. γ-32P end-labelled W8-W9 DNA was incubated with increasing amounts of IHF protein, as indicated. After brief digestion with DNase I, the reaction products were subjected to electrophoresis alongside the products of the sequencing reactions (G, A, T, C), performed with W9 primer. The region protected by IHF is bracketed. (c) The IHF-binding site. The consensus sequence of the IHF-binding site is shown in bold letters. The region protected from DNase I digestion is shaded. The footprint extends from −337 to −383, relative to the rpoH start codon.

    Figure image not available in archive
    Fig. 5.

    PspF and NtrC-P binding to the rpoH regulatory region. (a, d) EMSA. The γ-32P end-labelled 707 bp DNA fragment W1-W2 (2 nM) was incubated with increasing amounts of PspF (a) or NtrC-P (d), as indicated. Arrows or brackets indicate the positions of the DNA fragment and the retarded DNA–protein complex. (b, e) Electron microscopy: photomicrographs show the binding of PspF (b) and NtrC-P (e) to the W1-W2 (707 bp) DNA fragment. The conditions for protein binding to DNA are described in Methods. The activator proteins (500 nM) were incubated with streptavidin end-labelled W1-W2biot DNA (35 nM) for 15 min at 30 °C. Photomicrographs were digitized to measure the position of the activator proteins on the DNA fragment. (c, f) Statistical analysis of about 100 EM images showing PspF (c) and NtrC-P (f) bound within the 707 bp W1-W2 DNA fragment containing the rpoH regulatory region. The diagrams below the graphs represent the DNA fragment with the P6 promoter and putative PspF- (c) and NtrC- (f) binding sites indicated.

    Electron microscopy (EM).

    Buffer A (50 mM Tris/acetate pH 8.0, 100 mM potassium acetate, 8 mM magnesium acetate, 1 mM DTT, 27 mM ammonium acetate, 3.5 % PEG 8000) was used for protein–DNA binding reactions (Su et al., 1990). The W1-W2 (707 bp) DNA fragment containing the putative P6 promoter (200 nM) and σ54–RNAP (35 nM) were incubated for 15 min at 30 °C in a final volume of 20 μl.

    For visualization of PspF or NtrC-P binding the W1-W2 fragment was prepared by PCR with biotinylated W2 primer (W2biot). W1-W2biot (620 ng) was incubated with streptavidin (Promega) in a total volume of 40 μl in TM buffer for 1 h at 37 °C. The excess of streptavidin was removed by filtration through Amicon Microcon-PCR (Milipore) filters. The PspF or NtrC-P proteins (500 nM) and the W1-W2biot DNA fragment (200 nM) were incubated at 30 °C for 15 min in buffer A.

    The reaction products were cross-linked by the addition of 0.2 % glutaraldehyde, and, after incubation at room temperature for 15 min, diluted 1 : 20 with TM buffer. Preparations for electron microscopy were made by adsorption to mica (Spiess & Lurz, 1988), stained with uranyl acetate, platinum–carbon coated and analysed with a Philips CM100 electron microscope. The lengths of the DNA fragments and the positions of complexes were measured by using an electronic digitizer and evaluated with a computer program as described previously (Weigel et al., 1997).

    DNase I footprinting.

    Primers were end-labelled with [γ-32P]ATP (166.5 TBq mmol−1) and T4 polynucleotide kinase (Promega). DNA fragments were PCR amplified using 32P end-labelled W7 primer and unlabelled W3 primer for σ54, σ54–RNAP and σ70–RNAP footprints. 32P end-labelled W9 primer and unlabelled W8 primer were used for IHF footprints. DNase I footprinting assays were performed as described by Leblanc & Moss (2001) in a total volume of 10 μl of STA buffer. The labelled DNA fragments at a final concentration of 10 nM were incubated, for 15 min at 30 °C, with proteins in quantities given in the figure descriptions. DNase I (5×10−4 units) was added and after incubation for 1 min at 37 °C the reaction was stopped by the addition of EDTA to a final concentration of 50 mM followed by heating for 2 min at 95 °C in formamide loading dye. The reaction products were separated on an 8 % PAGE/urea gel. In parallel, the products of DNA sequencing reactions, performed using the fmol DNA Cycle Sequencing System (Promega), were separated. Gels were subjected to autoradiography or exposed to Kodak screen and analysed using the Molecular Imager FX (Bio-Rad).

    In vitro transcription.

    Multiple-round transcription assays were performed essentially as described by Wigneshweraraj et al. (2003) with minor modifications. Briefly, the supercoiled DNA template pTE103-W1-W2 for in vitro transcription was prepared by cloning the W1-W2 (707 bp) DNA fragment digested with BamHI and EcoRI into the pTE103 transcription vector, purified by centrifugation (at 2×105 g, for 16 h) through a caesium chloride/ethidium bromide gradient and verified by sequencing (Macrogen). Reactions were conducted in STA buffer supplemented with BSA (100 μg ml−1), in a total volume of 20 μl. First, 1 μg of template DNA was incubated for 10 min at 37 °C with 100 nM σ54–RNAP and each of the activators (PspF, PspFΔHTH or NtrC-P) at concentrations indicated in the figure legends. Then ATP was added to the final concentration of 5 mM, and the reaction was continued for 5 min to allow for izomerization of the closed complex into an active, open complex. Transcription was initiated by the addition of ribonucleotide mixture (0.5 mM ATP, 0.5 mM GTP, 0.5 mM CTP, 0.06 mM UTP, 0.11-0.185 MBq [α-32P]UTP) and incubation for 10 min at 37 °C. Then heparin (100 μg ml−1) was added and incubation was continued for 5 min. The reaction was stopped by the addition of a formamide loading dye. Samples were heated (2 min at 95 °C) and subjected to electrophoresis in 4.5 % PAGE/urea gel at 250 V in TBE buffer. Gels were subjected to autoradiography or exposed to Kodak screen and analysed using the Molecular Imager FX (Bio-Rad).

    Primer extension analysis.

    Cells were grown in LB medium at 30 °C, to an OD575 of 0.4 and harvested by centrifugation at 2×103 g for 10 min. Total RNA was isolated using the RNA Prep Plus kit (A & A Biotechnology). For PspFΔHTH or σ54 overexpression from pMJ13 or pVI688, respectively, cultures at an OD575 of 0.4 were induced by the addition of IPTG (1 mM) and incubation was continued for 0.5–1 h for protein synthesis. The W7 primer complementary to nucleotides +73 to +44 of the rpoH gene was 5′-end labelled using T4 polynucleotide kinase (Promega) and [γ-32P]ATP (166.5 TBq mmol−1). RNA (50 μg) was mixed with the labelled W7 primer (1 pmol) and heated to 85 °C for 20 min. Then M-MuLV reverse transcriptase buffer was added and the W7 primer was hybridized to the RNA at 62 °C for 1 h. Then, RevertAid H Minus M-MuLV reverse transcriptase (Fermentas), ribonuclease inhibitor (1 unit μl−1) and dNTPs (2.5 mM each) were added. Primer extension reactions were carried out at 42 °C for 1 h and stopped by adding loading dye containing formamide. DNA sequencing reactions were carried out with the same primer using the fmol DNA Cycle Sequencing System (Promega). Reaction products were heated for 2 min at 95 °C and loaded on a 8 % PAGE/urea gel together with products of the DNA sequencing reactions carried out with the fmol DNA Cycle Sequencing System. Gels were subjected to autoradiography or exposed to Kodak screen and analysed using a Molecular Imager FX (Bio-Rad).

    RESULTS

    Analysis of the promoter region of the rpoH gene

    At the start of this work only the presence of canonical sequence for a σ54 promoter in the promoter region of the rpoH gene was known (Pallen, 1999), raising the question whether it might be an active promoter. Analysis of the upstream sequence of the rpoH gene by a blast search showed that the σ54 promoter was correctly situated according to the rules proposed by Reitzer & Schneider (2001) for active σ54 promoters. Moreover, an IHF–binding site homologous to the consensus sequence was found at the position −349 to −362 (Fig. 1b⇑) (Craig & Nash, 1984; Goodrich et al., 1990). The search for a possible enhancer was focused on sequences resembling those of PspF- and NtrC-binding sites, since PspF is the activator of the σ54-dependent stress-induced pspA–E operon, and NtrC activates a few σ54-dependent promoters. It seemed probable that one of these activators could participate in transcription of the rpoH gene encoding σ32, the main stress-response factor. Sequence analysis of the rpoH regulatory region indicated the presence of some imperfect NtrC- binding sites and PspF-binding sites spread over the whole region of interest (Fig. 1b⇑).

    σ54–RNAP binding to the putative P6 promoter

    σ54–RNAP binding to P6 was tested by electrophoretic mobility-shift assay (EMSA), electron microscopy (EM) and DNase I footprinting. For EMSA the 277 bp DNA fragment containing the putative P6 promoter was prepared by PCR using primers W2 and W5 (Fig. 1a⇑) and end-labelled with [γ-32P]ATP. We observed a DNA shift at a σ54–RNAP : DNA molar ratio of 5 : 1 or higher (Fig. 2a⇑), a preliminary indication of σ54 holoenzyme binding to this DNA region. EM gave strong evidence for specific binding of the holoenzyme to the putative σ54 promoter, P6 (Fig. 2b⇑). The 707 bp DNA fragment W1-W2 (encompassing W2-W5) was used. Statistical analysis of approximately 100 EM images revealed that σ54–RNAP binding was centred on the postulated promoter (Fig. 2c⇑). The precise site of σ54–RNAP binding was determined by DNase I footprinting (Fig. 3a⇑). The W3-W7 (326 bp) γ-32P end-labelled DNA fragment was prepared by PCR, as detailed in Methods. In agreement with the EM results (Fig. 2b, c⇑) σ54–RNAP bound exactly at the postulated P6 promoter, covering the sequence −37 to −68 (Fig. 3⇑). As a reference, DNase I footprinting with σ70–RNAP was performed. The protected region from −72 to −117 corresponded to the P5 and P4 promoters. The nucleotide sequence of the protected area is shown below the autoradiogram (Fig. 3b⇑). There was no binding of the σ54 protein alone, which is observed only with σ54 promoters containing a stretch of four Ts immediately upstream of the conserved GC of the promoter sequence (Buck & Cannon, 1992). P6 contains a TTGTT sequence instead. A DNA fragment with no protein added (Fig. 3a⇑, lane 1) and a DNA fragment incubated with core RNAP only (lane 13) were used as controls and showed negative results as expected.

    IHF, NtrC and PspF binding upstream of the P6 promoter

    Analysis of the regulatory region of the rpoH gene revealed the presence of a putative IHF-binding site, with remarkable homology to the consensus site (Craig & Nash, 1984; Goodrich et al., 1990). To our knowledge this has never been detected before. To determine whether this site actually binds IHF, EMSA and DNase I footprinting were performed. EMSA demonstrated IHF binding to the 277 bp DNA fragment W8-W9. Binding occurred at a molar ratio of IHF to DNA of 2 : 1 (Fig. 4a⇑). DNase I footprinting showed IHF binding at the predicted site, i.e. at −337 to −383, corresponding to the extended IHF-binding site (Goodrich et al., 1990) (Fig. 1⇑ and Fig. 4b, c⇑).

    The search for NtrC- or PspF-binding sites by sequence analysis of the 707 bp W1-W2 DNA fragment resulted in a complicated picture of several imperfect binding sites for each of these proteins (Fig. 1b⇑). Nevertheless, it seems possible that some of them act as enhancers, binding the activator protein.

    EMSA, EM and footprinting experiments were used to check the possible binding of PspF and NtrC-P (NtrC phosphorylated in vitro by carbamyl phosphate) to the 707 bp DNA fragment W1-W2. A high molar ratio of either of the two proteins (200 nM PspF monomer or 250 nM NtrC-P monomer per 2 nM DNA) was needed to produce a mobility shift (Fig. 5a, d⇑). These data suggested that both activators bind DNA nonspecifically. For EM the 707 bp DNA fragment was end-labelled with streptavidin (see Methods), which served as a reference point for measurements of the position of the nucleoprotein complex. It was determined from the photomicrographs and by statistical analysis that both proteins, PspF and NtrC-P, bound all along the DNA fragment (Fig. 5b, c, e, f⇑). Since the previously observed specific binding of the σ54–RNAP detected by EM was limited to the narrow area of the P6 promoter, it was evident that neither of the activators bound specifically. Also, the results of NtrC-P and PspF DNase I footprinting were negative (data not shown). This evidence supports the hypothesis that PspF and NtrC-P are not the specific activators for the transcription from P6.

    Transcription in vitro from the P6 promoter

    The P6 promoter was tested for in vitro transcriptional activity in multiple-round transcription. The supercoiled template was prepared by cloning the W1-W2 DNA fragment containing P1, P3, P4, P5 and the putative P6 promoters in the pTE103 transcription vector (Elliott & Geiduschek, 1984). Each reaction mixture contained template DNA (pTE103-W1-W2), σ54–RNAP and the activator protein at increasing concentration. Use was made of the relaxed specificity of activators on supercoiled templates (Dworkin et al., 1998), and PspF, NtrC-P and PspFΔHTH were employed. Each of these activators caused the initiation of transcription (Fig. 6a, b, c⇓). PspF and NtrC-P stimulated transcription at much lower protein levels (about 50 nM) than PspFΔHTH (1 μM), perhaps because PspFΔHTH not only does not bind DNA, but also has ATPase activity 30-fold lower than PspF (Jovanovic et al., 1999). We concluded that σ54–RNAP recognized the P6 promoter and formed a closed initiation complex capable of starting transcription in vitro provided that any of the ATP-dependent activators was present. Transcription in vitro (activated by PspF) was slightly inhibited by IHF (data not shown), which is consistent with the observation that IHF stimulates transcription only with a specific activator (Dworkin et al.1998).

    Figure image not available in archive
    Fig. 6.

    In vitro transcription of the rpoH gene from the P6 promoter. Transcription from supercoiled DNA template pTE103-W1-W2 with σ54–RNAP (100 nM). Reaction conditions were as described in Methods. The identified band is the ∼500 nt rpoH transcript. Activator proteins PspF (a), PspFΔHTH (b) and NtrC-P (c) were used at the concentrations indicated above each lane.

    Identification of the transcription start site in vivo

    In order to identify the 5′ end of the rpoH transcript from the P6 promoter, primer extension reactions were performed with total RNA isolated from the wild-type (wt) strains: MG1655 or ET8000, and the rpoN derivative of ET8000, ET8045. These strains were transformed with two plasmids: pMJ13, overproducing PspFΔHTH from the pspF877 gene, inducible by IPTG, and pREP, providing the lacI repressor. The specific conditions that would induce transcription from P6 were not known; however, use was made of the observation of Jovanovic et al. (1996) that overproduction of PspFΔHTH activated pspA operon expression even in the absence of inducing stimuli. P6 transcription appeared totally dependent on the overproduction of PspFΔHTH from pMJ13 induced by IPTG (Fig. 7a, b⇓). The 5′ end of the P6 transcript was found at position −30, from the rpoH start codon (+1). There was rough proportionality between PspFΔHTH concentration (dependent on the length of the induction time) and the level of the transcript (Fig. 7a⇓). This transcription was abolished by the rpoN mutation (Fig. 7b⇓, lane 9), though PspFΔHTH was overproduced to the same level in the wt and mutant strains, as confirmed by SDS–PAGE (data not shown). Complementation of the rpoN mutation by plasmid pVI688 overproducing σ54 restored transcription from P6 (Fig. 7c⇓), if PspFΔHTH was overproduced simultaneously with the σ factor. These results indicate that the transcription from P6 is σ54-dependent and that the σ54–RNAP is able to direct transcription of the rpoH gene from the P6 promoter if an activator is supplied.

    Figure image not available in archive
    Fig. 7.

    Transcriptional start site from the P6 promoter of the rpoH gene. The arrows indicate positions of the transcripts from the P3, P4, P5 and P6 promoters [locations of transcriptional start sites relative to the ATG start codon (+1) of the rpoH gene are shown in parentheses]. (a) Primer extension reactions were carried out with total RNA isolated from E. coli MG1655(pMJ13, pREP) before (lane1) and 30 (lanes 2 and 3) or 60 (lanes 4 and 5) min after induction of PspFΔHTH overproduction from pMJ13 by addition of IPTG. (b) Primer extension reactions were carried out with total RNA isolated from ET8000(pMJ13, pREP) and its rpoN derivative ET8045(pMJ13, pREP) before (lanes 6 and 8) and 30 min after (lanes 7 and 9) induction of PspFΔHTH overproduction by addition of IPTG. (c) Primer extension reactions were carried out with total RNA isolated from ET8045(pMJ13, pREP, pVI688) before (lane 10) and 30 min after (lane 11) induction of PspFΔHTH and σ54 overproduction from pMJ13 and pVI688, respectively.

    Testing factors that might induce transcription from P6

    In an attempt to identify conditions that might affect the induction of transcription, heat shock (from 30 °C to 42 or 50 °C), 10 % ethanol or CCCP treatments were applied, since these treatments induce transcription of the pspA–E operon by the PspF activator (Darwin, 2005). Also, induction of NtrC activity by nitrogen starvation was tested. None of these conditions activated the P6 promoter, as determined by primer extension. The results are consistent with neither PspF nor NtrC being a specific activator of this promoter.

    DISCUSSION

    We provide evidence that the putative σ54-dependent P6 promoter in the regulatory region of the rpoH gene of E. coli is able to direct transcription. Binding of the σ54–RNAP holoenzyme to the P6 promoter sequence was demonstrated by EMSA, electron microscopy and DNase I footprinting. The closed complex of the σ54–RNAP and P6 promoter extended from −7 to −38 bp, relative to the transcription start site (−37 to −68 bp relative to the ATG). This is consistent with the size of the DNA area protected by closed complexes observed for other σ54 promoters (Buck & Cannon, 1992; Wang et al., 1998). The closed complexes of these promoters require interaction with activators of the AAA+ protein type bound to remote enhancers. IHF is a frequent participant in the process. The presence of an IHF-binding site that corresponded to the consensus sequence (Craig & Nash, 1984; Goodrich et al., 1990) and demonstration of binding to IHF, was consistent with previous findings regarding the structure and functioning of most of the σ54 promoter regulatory regions. The dependence of in vitro transcription on the AAA+ activators NtrC-P, PspF or PspFΔHTH (Fig. 6⇑) was shown. Also an analogous situation produced in vivo by overproduction of PspFΔHTH, which does not bind DNA, from pMJ13 in E. coli cells resulted in activation of transcription from P6.

    The location of the transcriptional start site 30 nucleotides upstream of the rpoH coding sequence by primer extension experiments (Fig. 7a, b, c⇑) was in agreement with previous prediction (31 nt upstream according to Pallen, 1999). The start occurred in the statistically determined area for starts from the σ54 promoters, i.e. between 8 and 21 nt downstream of the promoter's conserved GC (Barrios et al., 1999). The rpoN mutation abolished transcription from P6 as expected (Fig. 7b⇑, lane 9).

    We interpret these data, taken together, as evidence that σ54–RNAP binds the P6 promoter and forms a closed complex able to initiate rpoH gene transcription in vitro and in vivo, provided that an ATP-dependent activator is present. However, the specific activator and conditions inducing transcription from the P6 promoter remain unknown. This makes assessment of the physiological importance of P6 difficult, but evolutionary conservation of the P6 sequence (Pallen, 1999) speaks for its usefulness in as yet unrecognized conditions.

    The P6 promoter influenced transcription starting from the upstream promoters (P1–P5) of the rpoH gene. Formation of the stable, closed complex of σ54–RNAP at the P6 promoter can be a spatial hindrance for the RNAP holoenzyme. Such a complex cannot be formed in the rpoN strain. Accordingly, an increased level of the transcription from the upstream promoters in the rpoN cells was observed (Fig. 7b⇑, lanes 8 and 9) but not further explored. The sigma competition for the core RNAP, mediated by the alarmone ppGpp (Sze & Shingler, 1999; Maeda et al., 2000; Jishage et al., 2002; Laurie et al., 2003; Nyström, 2004; Magnusson et al., 2005) could also play an important role in the complicated regulation of rpoH transcription. Since core RNAP is limiting for transcription, the amount of σ70 bound to RNAP is increased in the absence of σ54.

    In a paper by Reitzer & Schneider (2001), the authors mentioned unpublished results of experiments in which it was not possible to demonstrate the existence of a transcript from the P6 promoter in nitrogen-limited cells, which meant NtrC was not the activator of this transcription. We confirm this opinion. These results are also consistent with the negative result of our in vitro studies on PspF and NtrC-P binding to the rpoH regulatory region (Fig. 5⇑).

    There is a possibility that the unidentified enhancer for the activator may be found further upstream or downstream of the 707 bp (W1-W2) region tested. The enhancers binding ATP-dependent activators for the σ54 promoters are usually situated about 100–160 bp upstream of the coding region; they may rarely be found 700 bp apart, but a larger distance is not excluded (Gralla & Collado-Vides, 1996). Belitsky & Sonenshein (1999) described an enhancer located 1.5 kb downstream of the σ54 promoter of the rocG gene of the Bacillus subtilis. We are currently working on the construction of transcriptional fusions that should be helpful in a search for P6 activating conditions.

    Since IHF binds in the rpoH regulatory region, it might be expected that it plays a role in rpoH transcription regulation. However, under the conditions of our experiment on in vitro transcription from P6, with PspF as an activator, IHF slightly inhibited the reaction (results not shown). Considering the results of Dworkin et al. (1998), who found that IHF facilitated transcription initiation only when a specific activator was used, it has to be accepted that, as long as the specific activator is unidentified, the significance of the IHF binding cannot be evaluated. Nevertheless, the IHF binding seems to be a strong argument for its regulatory role in transcription from P6. Nyström (1995) reported that IHF overproduction caused the induction of the rpoH-dependent heat shock response and proposed that this effect could reflect an influence of IHF on the activity of one or several rpoH promoters. Our results lend support to this notion.

    One can speculate on the role of transcription from P6. Recently attention was paid to biofilm formation. This is an interesting process from the point of view of the development of unicellular organism cooperation leading to a kind of bacterial community. Beloin et al. (2004) found that biofilm formation involves, among other processes, the stress response. The pspA–E operon was strongly induced. The expression of ibpB and ibpA, genes for small heat shock proteins, increased 40- and 20-fold, respectively, compared with expression during the exponential growth phase of planktonic cells, in conditions inducing biofilm formation. The clpB and dnaK genes were also induced (Schembri et al., 2003). Elevated expression of these genes is dependent on σ32, the rpoH gene product. The molecular mechanism of this induction remains obscure.

    Acknowledgments

    We would like to thank Peter Model and Karsten Rippe for providing the strains and plasmids used in this study. We are also grateful to Victoria Shingler for helpful discussions and Grzegorz Węgrzyn for his critical reading of the manuscript. This work was supported by Grant 3 P04A 001 23 from the Polish Ministry of Scientific Research and Information Technology, State Committee for Scientific Research (to A. T.).

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