The H-NS protein represses transcription of the eltAB operon, which encodes heat-labile enterotoxin in enterotoxigenic Escherichia coli, by binding to regions downstream of the promoter

Ji Yang, Marija Tauschek, Richard Strugnell and Roy M. Robins-Browne

Department of Microbiology and Immunology, University of Melbourne, Victoria 3010, Australia, and Murdoch Childrens Research Institute, Royal Children's Hospital, Flemington Road, Parkville, Victoria 3052, Australia

Correspondence
Roy M. Robins-Browne
r.browne{at}unimelb.edu.au


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Heat-labile enterotoxin, a major virulence determinant of enterotoxigenic Escherichia coli, is encoded by the eltAB operon. To elucidate the molecular mechanism by which the heat-stable nucleoid-structural (H-NS) protein controls transcription of eltAB, the authors constructed an eltAB–lacZ transcriptional fusion and performed {beta}-galactosidase analysis. The results showed that H-NS protein exerts fivefold repression on transcription from the eltAB promoter at 37 °C and 10-fold repression at 22 °C. Two silencer regions that were required for H-NS-mediated repression of eltAB expression were identified, both of which were located downstream of the start site of transcription. One silencer was located between +31 and +110, the other between +460 and +556, relative to the start site of transcription, and they worked cooperatively in repression. DNA sequences containing the silencers were predicted to be curved by in silico analysis and bound H-NS protein directly in vitro. Repression of eltAB transcription by H-NS was independent of promoter strength, and the presence of H-NS protein did not affect promoter opening in vitro, indicating that repression was achieved by inhibiting promoter clearance or blocking transcription elongation, probably via DNA looping between the two silencers.


Abbreviations: CT, cholera toxin; DRE, downstream regulatory element; EMSA, electrophoretic mobility shift assay; EPEC, enteropathogenic Escherichia coli; ETEC, enterotoxigenic E. coli; H-NS protein, heat-stable nucleoid-structural protein; LT, heat-labile enterotoxin


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Enterotoxigenic Escherichia coli (ETEC) is a major cause of watery, cholera-like diarrhoea in children in less-developed countries and in travellers to those regions (Black, 1993; Rowe et al., 1970). It is also an important animal pathogen of considerable economic significance to the pig industry, amongst others. ETEC strains express one or both of two toxic proteins, known as heat-labile (LT) and heat-stable (ST) enterotoxins (Nataro & Kaper, 1998), which perturb the transport of fluid and electrolytes across the intestinal epithelium. LT is a heterohexameric protein of 84 kDa which contains one A subunit and five identical B subunits (Spangler, 1992; Nataro & Kaper, 1998). The B pentamer binds to the host membrane receptor ganglioside, GM1, after which the A subunit is cleaved within the epithelial cells, where it activates adenylate cyclase, resulting in accumulation of cAMP and the consequent disruption of ion channels across the intestinal lumen (Spangler, 1992; Nataro & Kaper, 1998).

LT is encoded on a 60 kb virulence plasmid termed Ent (Yamamoto et al., 1984). The eltA and eltB genes encoding the A and B subunits, respectively, share extensive homology with those for cholera toxin (CT; Mekalanos et al., 1983) and appear to form a single transcriptional unit (Yamamoto et al., 1984). Transcriptional regulation of CT has been extensively studied. Expression of the ctx gene encoding CT is controlled by a regulatory cascade involving two cytoplasmic membrane pairs, ToxS/ToxR and TcpP/TcpH, and an AraC-like activator, ToxT (DiRita et al., 1991; Higgins et al., 1992; Skorupski & Taylor, 1997; Hase & Mekalanos, 1998; Krukonis et al., 2000). In contrast, little is known about the regulation of the eltAB operon. It has been reported that LT transcription is negatively regulated by the heat-stable nucleoid-structural (H-NS) protein at low temperature and that a downstream regulatory element (DRE), which covers the coding region of eltA, is involved in H-NS mediated repression (Trachman & Maas, 1998; Trachman & Yasmin, 2004). However, the detailed molecular mechanism by which H-NS controls transcription of the eltAB operon is not known.

H-NS is a small (137 aa) but abundant protein (approximately 2x104 molecules per cell) (Ussery et al., 1994; Rimsky, 2004; Dorman, 2004). It is a global regulator which controls the expression of a large number of genes whose products are involved in a wide range of cellular processes (Ussery et al., 1994; Rimsky, 2004; Dorman, 2004). In pathogenic enteric bacteria, such as enteropathogenic E. coli (EPEC) (Bustamante et al., 2001; Haack et al., 2003), ETEC (Murphree et al., 1997), enteroinvasive E. coli (Falconi et al., 2001), Shigella flexneri (Porter & Dorman, 1994) and Vibrio cholerae (Nye et al., 2000; Yu & DiRita, 2002), H-NS participates in the regulation of virulence gene expression. In these bacteria, H-NS functions mainly as a negative regulator which represses transcription in response to environmental changes, such as temperature and osmolarity (Atlung & Ingmer, 1997). Unlike most other transcriptional regulatory proteins, H-NS binds to its DNA targets in a sequence-independent fashion, but shows a strong preference for regions of intrinsic curvature (Rimsky et al., 2001). The minimum unit of H-NS protein in solution is a dimer but it exists in various oligomeric states (Badaut et al., 2002; Rimsky, 2004). Structural and functional analyses have shown that H-NS contains two domains separated by a flexible linker. The N-terminal domain (aa 1–65) is responsible for dimerization and the C-terminal domain (aa 90–137) is involved in nucleic acid binding (Ueguchi et al., 1996; Badaut et al., 2002).

In order to understand the molecular biology of H-NS-mediated transcriptional regulation of the eltAB operon of ETEC, we carried out a number of genetic and biochemical experiments to map and characterize the eltAB promoter and determine the H-NS binding sites which are required for repression. Using in-gel DNase I footprinting, we identified two regions, between +31 and +110 and +460 and +556 relative to the start site of transcription, as silencers that interact directly with the H-NS protein. In silico analysis indicated that both silencers correspond to regions that are intrinsically curved. Genetic analysis demonstrated that these silencers are critical for H-NS-mediated repression of eltAB expression, suggesting that repression occurs via DNA looping. Data from KMnO4 footprinting indicated that generation of a repressing nucleoprotein complex at the eltAB operon inhibits a step(s) following promoter open complex formation.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Strains, plasmids, oligonucleotides, reagents and media.
The prototype ETEC strain H10407 (Evans et al., 1973) was used as a source of DNA for PCR amplification of various eltAB fragments. The E. coli K-12 strains used in this study are as follows. JM101 [{Delta}(lac–proAB) supE thi F'(traD36 proA+B+ lacIqZ{Delta}M15)] (Messing, 1983) was used for cloning and propagating M13 derivatives. E. coli XL-1 Blue MRF' [{Delta}(mcrA)183 {Delta}(mcrCB–hsdSMR–mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac [F' proAB lacIqZ{Delta}M15 Tn10 (Tetr)] (Stratagene) was used for oligonucleotide-directed mutagenesis. E. coli MC4100 [{Delta}(argF–lac)U169 rpsL150 relA araD139 flb-5301 deoC1 ptsF25] (Casadaban, 1976) and E. coli PD145 (MC4100 hns : : Tn10) (Dersch et al., 1994) were used as hosts for analysis of the expression of various eltAB–lacZ fusions. Plasmids pMU2385 (Praszkier et al., 1992), pUC19 (Vieira & Messing, 1982) and M13tg130 (Kieny et al., 1983) have all been described previously. Oligonucleotides used in this study are listed in Table 1. Luria broth [1 % (w/v) tryptone, 0·5 % (w/v) yeast extract, 171 mM NaCl] was routinely used for growing E. coli strains. Trimethoprim was used at a final concentration of 40 µg ml–1 in Luria broth. Restriction enzymes and chemicals were purchased commercially. Highly purified RNA polymerase holoenzyme and native H-NS protein used in the in vitro experiments were provided by A. Ishihama (Nippon Inst. Biol. Sci., Tokyo, Japan).


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Table 1. Oligonucleotides used in this study

 
Recombinant DNA techniques and oligonucleotide-directed mutagenesis.
Standard recombinant DNA procedures were used as described by Sambrook et al. (1989). DNA was sequenced with a model 377 DNA sequencer and ABI Big Dye terminators (Perkin-Elmer). In vitro mutagenesis with synthetic oligonucleotides was performed on an M13tg130 derivative containing the eltAB1–757 fragment using commercially available kits (Amersham and US Biochemical). Mutations were confirmed by DNA sequence analysis.

{beta}-Galactosidase assay.
{beta}-Galactosidase activity was assayed as described by Miller (1974). Specific activity was expressed in units described therein. The data are the results of at least three independent assays.

Primer extension.
Primer YJ003 (Table 1) was labelled at the 5' end with [{gamma}-32P]ATP and T4 polynucleotide kinase (Promega). The labelled primer was co-precipitated with 10 µg total RNA isolated from E. coli MC4100 carrying a pUC19 derivative containing either the eltAB1–297 or eltAB1–757 fragment. Hybridization was carried out at 45 °C for 15 min in 10 µl TE buffer containing 150 mM KCl. Primer extension reactions were started by the addition of 24 µl extension solution [20 mM Tris/HCl (pH 8·4), 10 mM MgCl2, 10 mM DTT and 2 mM dNTPs and 1 U µl–1 AMVreverse transcriptase (Promega)] and were carried out at 42 °C for 60 min. Samples were then precipitated and analysed on a sequencing gel.

Construction of various eltAB–lacZ transcriptional fusions and deletion mutants.
The three eltAB–lacZ fusions, eltAB1–297lacZ, eltAB1–490lacZ, eltAB1–757lacZ, were constructed as described below. The three eltAB fragments, which carry 297, 490 and 757 bp respectively, were generated by PCR using total cellular DNA isolated from ETEC strain H10407 as template and primers described in Table 1. Each of the PCR fragments, which were flanked by a BamHI site and a HindIII site, was cloned into M13tg130 and sequenced. The three eltAB fragments were excised from the M13tg130 derivatives and cloned into the BamHI and HindIII sitesof the single-copy plasmid pMU2385, to create eltAB–lacZ transcriptional fusions.

Deletion mutant {Delta}1–95 was constructed as follows. A BamHI site between positions 95 and 100 (Fig. 1a) was created by oligonucleotide-directed mutagenesis on M13tg130-eltAB1–757 using primer YJ030 (Table 1). The eltAB fragment ({Delta}1–95) lacking the first 95 nt was cloned into pMU2385 to create the eltAB({Delta}1–95)lacZ transcriptional fusion.



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Fig. 1. Sequence of the eltAB regulatory region and determination of transcription start site. (a) The transcription start site of the eltAB promoter is marked with an angled arrow. The numbers in parentheses represent positions relative to the start site of transcription. The –35 and –10 regions of the eltAB promoter are indicated with lines above the sequence. The translation start codon (ATG) for the A subunit of heat-labile enterotoxin is underlined. The regions that are protected by H-NS protein in in-gel DNase I assays are indicated with bold lines beneath the sequence, and the hypersensitive positions revealed at high concentrations of H-NS are indicated with asterisks. The regions predicted to be curved are indicated with dashed lines above the sequence. The regions that are primed by oligonucleotides (YJ001, YJ003, YJ004 and YJ005) for PCR amplification of the various eltAB fragments are marked by arrows. (b) Determination of the start site of transcription of the eltAB operon by primer extension. E. coli MC4100 strains carrying pUC19, or a pUC19 derivative containing the eltAB1–297 or eltAB1–757 fragment, were grown in Luria broth at 37 °C to mid-exponential phase. Total cellular RNA isolated from each of the strains was hybridized with 32P-labelled primer YJ003. Lanes: control, reaction with RNA from MC4100 carrying pUC19; 1, reaction with RNA from MC4100 carrying a pUC19 derivative containing the eltAB1–297 fragment; 2, reaction with RNA from a pUC19 derivative containing the eltAB1–757 fragment. The extension product is marked with an arrow.

 
Deletion mutant {Delta}192–297 was constructed as follows. The XbaI–HindIII eltAB fragment containing the region between positions 297 and 757 (Fig. 1a) was excised from M13tg130-eltAB1–757. A BamHI–XbaI eltAB fragment containing the region between positions 1 and 192 was generated by PCR using primers YJ001 and YJ041 (Table 1). The two fragments were ligated at the XbaI site and the resulting fragment was cloned into the BamHI and HindIII sites of pMU2385 to create the eltAB({Delta}192–297)lacZ transcriptional fusion.

Electrophoretic mobility shift assay (EMSA) and DNase I footprinting.
The 32P-labelled eltAB fragments (–79 to +137, +129 to +330 and +313 to +597) used in the EMSA or in-gel DNase I footprinting experiments were generated as follows. The oligonucleotide primers YJ046, YJ020 and YJ005 (Table 1) were each labelled with 32P at the 5' end by using [{gamma}-32P]ATP and T4 polynucleotide kinase. The eltAB fragments (–79 to +137, +129 to +330 and +313 to +597) were each amplified by PCR using the 32P-labelled primers, unlabelled primers (32P-YJ046-YJ003, 32P-YJ020-YJ004 and 32P-YJ005-YJ031) (Table 1) and M13tg130-eltAB1–757. The amplified eltAB fragments were then purified on a native polyacrylamide gel. The reactions for EMSA (25 µl) were carried out in transcription buffer [50 mM Tris/HCl (pH 7·8), 50 mM NaCl, 3 mM magnesium acetate, 0·1 mM EDTA, 0·1 mM DTT and 25 µg BSA ml–1] containing approximately 3 nM end-labelled eltAB fragment and 250 or 500 nM purified H-NS protein. The samples were incubated at 22 °C for 20 min. Six microlitres glycerol (40 %) was added to each sample before electrophoresis on 5 % native polyacrylamide gels (37·5 : 1). The polyacrylamide gels and running buffer contained the following: 50 mM Tris base, 50 mM boric acid, 1 mM MgCl2 and 1 % glycerol. Following a pre-run at 4 °C for 1 h at 20 V cm–1, samples were loaded onto the gel and electrophoresis was carried out at 4 °C for approximately 12 h at 10 V cm–1.

The in-gel DNase I assay was performed essentially as described by Yang et al. (2004). Gel slices containing various protein–DNA complexes, as well as free DNA fragments from EMSA, were excised from the polyacrylamide gel. The gel slices were each incubated in 10 µl of covering buffer (10 mM Tris/HCl, pH 8·0, 2 mM DTT, 5 % glycerol, 0·5 mg BSA ml–1 and 0·4 U DNase I ml–1; Amersham Biosciences) at room temperature for 15 min. Five microlitres starting solution (50 mM MgCl2 and 50 mM NaCl) was added to each sample and the reactions continued for a further 2 min before being terminated by addition of 30 µl stop solution (100 mM EDTA and 2 % SDS). DNA was extracted from the gel slices and analysed on a sequencing gel.

KMnO4 footprinting.
A 0·5 µg sample of the linear eltAB1–757 fragment carrying the eltAB promoter and regulatory elements was incubated in 20 µl in vitro transcription buffer [50 mM Tris/HCl (pH 7·8), 50 mM NaCl, 3 mM magnesium acetate, 0·1 mM EDTA, 0·1 mM DTT and 25 µg BSA ml–1] in the presence or absence of H-NS protein at 22 °C for 10 min. RNA polymerase (100 nM) was then added to the reaction mixtures. Following a further incubation at 30 °C for 30 min, to allow open complex formation, the samples were treated with 2·5 µl KMnO4 (80 mM) for 3 min at room temperature. The reaction was quenched with 2 µl {beta}-mercaptoethanol (14·7 M). The DNA was cleaved with piperidine at 90 °C for 20 min. A primer extension experiment using {alpha}-32P-labelled oligonucleotide (YJ046) was carried out to probe for the modified bases in the template strand of the open complex. Primer extension reactions were carried out in a Thermal Cycler for 25 cycles, using PCR Mastermix (Promega) and 32P-labelled primer (YJ046). The extension products were analysed on a 6 % denaturing polyacrylamide gel next to the GA track generated by Maxam and Gilbert sequencing.


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Identification of a region involved in H-NS-mediated repression of eltAB transcription
Analysis of the DNA sequences flanking the eltAB operon of a number of clinical ETEC isolates revealed the presence of a conserved region extending from the translation start codon to a position 236 bp further upstream (Schlör et al., 2000). The sequences upstream of this conserved region contain partial insertion sequence elements and vary greatly between different ETEC strains. Therefore any cis-acting element which controls eltAB transcription should be located within or downstream of this conserved region. To determine the degree of H-NS-mediated repression of eltAB transcription, we amplified three DNA fragments by PCR using total cellular DNA isolated from the prototype ETEC strain H10407 as template, and primers based on the previously published sequence of the etlAB genes (Yamamoto et al., 1984) (Fig. 1a). All three fragments started at the same point, 217 bp upstream of the start site of translation of the A subunit (Fig. 1a), but varied at their downstream ends. The three fragments, which comprised 297, 490 and 757 bp, respectively, were cloned into pMU2385 to create eltAB–lacZ transcriptional fusions. pMU2385 is a single-copy vector which carries a promoterless lacZ structural gene with its own translational signals (Yang et al., 2004). Each of the three pMU2385 derivatives, eltAB1–297lacZ, eltAB1–490lacZ and eltAB1–757lacZ, and the control plasmid, pMU2385, were transformed into isogenic Hns+ or Hns E. coli K-12 strains, MC4100 (Hns+, LacZ) and PD145 (Hns, LacZ) (Casadaban, 1976; Dersch et al., 1994). {beta}-Galactosidase levels were assessed for each of the transformants grown in Luria broth at 37 or 22 °C.

As shown in Fig. 2, at 37 °C, in the Hns+ background (MC4100), the fusions eltAB1–297lacZ and eltAB1–490lacZ produced similar levels of {beta}-galactosidase activity of 675 and 791 units, respectively. In contrast, the level of expression from eltAB1–757lacZ was only 238 units, about one-third of that of the other two constructs. In the Hns background (PD145), the {beta}-galactosidase activities for the fusions eltAB1–297lacZ and eltAB1–490lacZ were increased 1·3- or 1·6-fold, respectively, relative to the levels in the Hns+ host. A far more pronounced increase was observed with the construct eltAB1–757lacZ, where the {beta}-galactosidase expression was approximately fivefold higher in the Hns host than in the Hns+ host.



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Fig. 2. Promoter activities of various eltAB transcriptional fusions and effects of H-NS and temperature on eltAB expression. {beta}-Galactosidase activities are the mean values from three independent assays, with variation <15 %. See Methods for details. Fold repression (Fold rep.) is the specific activity of {beta}-galactosidase of the Hns strain (E. coli PD145) divided by that of the Hns+ strain (E. coli MC4100).

 
At 22 °C, there were no major changes in expression with constructs eltAB1–297lacZ and eltAB1–490lacZ in either the Hns+ or the Hns background, but the expression of the construct eltAB1–757lacZ was only 101 units in the Hns+ strain compared to 978 units in the Hns strain, which represented a 10-fold repression of eltAB transcription by H-NS overall. The difference in the levels of expression in the Hns+ background at 22 °C (101 units) and 37 °C (238 units) indicates a twofold induction at the permissive temperature of 37 °C. The results from this analysis indicated that all three DNA fragments contain the eltAB promoter region and that the eltAB1–757 fragment carries a region responsible for temperature-mediated repression by H-NS. It was also apparent that an H-NS binding site is located in the structural gene between positions 491 and 757.

Characterization of the eltAB promoter
To determine the start site(s) of transcription for the eltAB operon, we carried out a primer extension experiment. A 32P-labelled primer was hybridized with 10 µg total RNA isolated from strain MC4100 carrying a pUC19 derivative containing either the eltAB1–297 or the eltAB1–757 fragment. Following extension using reverse transcriptase in the presence of dNTPs, the samples were analysed on a sequencing gel. Only one major extension product was seen in each sample, indicating the presence of a single promoter for the eltAB operon (Fig. 1b). Judged by the intensity of the extended product, more eltA transcripts were produced by pUC19-eltAB1–297 than by pUC19-eltAB1–757, consistent with the observation that eltAB1–757 is more repressible by H-NS than eltAB1–297. The start site of transcription was mapped to an adenine residue located 56 nt upstream of the start codon for the A-subunit of LT (Fig. 1a). Based on this start site, a putative eltAB promoter was identified (Fig. 1a). This contained a perfect –35 region (TTGACA) but had a less conserved –10 region (TAAACA), which were separated by an ideal spacing of 17 bp. In addition, a cluster of 12 AT pairs was found 5 bp upstream of the –35 sequence, which represents a putative UP element of the eltAB promoter (Gourse et al., 2000).

In order to confirm the identity of the eltAB promoter, mutations were introduced into the putative –10 region, –35 region and UP element of the eltAB1–757 fragment by site-directed mutagenesis. The promoter activities of the down mutations (–10 down and –35 down) were reduced to about 5 % of the wild-type level in both the Hns+ and Hns hosts at both 37 and 22 °C (Fig. 3). On the other hand, improving the –10 region by making it into a perfect hexamer (–10 up) resulted in a twofold increase in promoter activity at 37 °C. At 22 °C there was only an approximately 1·5-fold increase in promoter strength with this mutant in the Hns+ host. Although the genetic changes in these three mutant promoters affected eltAB expression, the levels of H-NS-mediated repression remained similar to that of the wild-type.



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Fig. 3. Effect of mutations in the eltAB promoter on transcription and repression by H-NS. The sequence of core promoter elements is shown and the various genetic changes of mutants are marked. {beta}-Galactosidase activities were determined as described in Methods.

 
To assess the role of the putative UP element in eltAB expression, the AT-rich cluster was removed from the eltAB1–757 fragment ({Delta}UP). Functional analysis showed that this mutation had no significant effect either on transcriptional expression or on repression by H-NS (Fig. 3).

Prediction of DNA curvature for the eltA gene
The DNA sequence containing the eltAB promoter region and the entire eltA structural gene was examined in silico for the presence of any intrinsic DNA curvature by using the program BEND-IT (http://www.icgeb.org/dna/bend_it.html). As shown in Fig. 4, the sequence spanning the region between positions 1 and 300 or –160 and +140, relative to the start site of transcription, which encompasses the eltAB promoter and the initially transcribed region, is predicted to contain several curved DNA motifs (with predicted curvature >5 degrees). In addition, a region in the structural gene between positions 600 and 700 on the DNA sequence, or between +440 and +540, relative to the start site of transcription, is also predicted to have intrinsic DNA curvature. This is consistent with the observation that an H-NS binding site, which is critical for temperature-mediated repression, is located at the downstream end of the 757 bp fragment (see above).



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Fig. 4. In silico analysis of intrinsic curvature of the eltA gene and regulatory region, using the BEND-IT program. The regions with >5 degrees per helical turn of DNA (dashed line) represent curved sequences.

 
Deletion of the region between positions 192 and 297 affects H-NS-mediated repression
The prediction of highly curved sections upstream of the eltA fragment, between positions 1 and 300, led us to investigate the possible involvement of this region in H-NS-mediated repression. Two deletion mutants, {Delta}1–95 and {Delta}192–297, were constructed. {Delta}1–95 lacks a region of 95 bp between positions 1 and 95 and {Delta}192–297 lacks 106 bp between positions 192 and 297 (Fig. 1a). Functional analysis of the mutants showed that removing the region upstream of the eltAB promoter ({Delta}1–95) had no significant effect on H-NS-mediated repression, whereas deleting the region downstream of the start site of transcription ({Delta}192–297) reduced H-NS-mediated repression from around fivefold at 37 °C and 10-fold at 22 °C to less than twofold at both temperatures (Fig. 5).



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Fig. 5. Effect of deletion mutations on H-NS-mediated repression of eltAB transcription. {beta}-Galactosidase activity was determined as described in Methods.

 
EMSA and in-gel DNase I footprinting analysis
To test if H-NS can bind directly to the regions that are essential for eltAB repression, we carried out an EMSA. Three 32P end-labelled fragments covering the regions between positions –79 and +137, +129 and +330, and +313 and +597, respectively (Fig. 1a), were generated by PCR. Each of the DNA fragments was incubated with varying amounts of purified H-NS at 22 °C for 30 min and the samples were analysed on native polyacrylamide gels. The H-NS protein formed discrete protein–DNA complexes with fragments –79 to +137 and +313 to +597 but not with fragment +129 to +330 (Fig. 6a). In the EMSA with fragments –79 to +137 and +313 to +597, a second retarded protein–DNA complex (II) was seen at the higher concentration of H-NS (500 nM) (Fig. 6a).



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Fig. 6. EMSA and in-gel DNase I footprinting. (a) EMSA analysis of H-NS binding to eltAB fragments –79 to +137, +129 to +330 and +313 to +597. EMSA was carried out as described in the Methods, using 32P end-labelled eltAB fragments. Lane 1, sample without H-NS; lane 2, sample with 250 nM H-NS; lane 3, sample with 500 nM H-NS. (b) Gel slices containing free DNA (F), H-NS–DNA complex I (I) and H-NS–DNA complex II (II) from the EMSA (a) were treated with DNase I, then analysed on an 8 % sequencing gel. Regions protected by H-NS are indicated by bars. Hypersensitive sites are marked with asterisks.

 
To characterize the H-NS–DNA complexes, each of the retarded bands (I and II), as well as the band containing free DNA (F) (Fig. 6a), was excised from the native polyacrylamide gels, and the gel slices were subjected to limited digestion by DNase I. The samples were then analysed on a sequencing gel. In the case of fragment –79 to +137 (Fig. 6b), two patches of sequence, between positions +31 and +46 and +91 and +110, were protected by H-NS in both complexes I and II. Several hypersensitive sites were detected in complex II, indicating distortion of DNA at higher H-NS concentrations. The in-gel DNase I footprinting with fragment +313 to +597 showed that both complexes I and II consisted of H-NS protein bound to a region between positions +460 and +556 (Fig. 6b). Complex II was formed at a higher H-NS concentration (500 nM) but it had the same protection profile as complex I which was detected at a lower H-NS concentration (Fig. 6b). This suggests that H-NS molecules may be able to pack together during oligomerization on DNA.

H-NS does not inhibit open complex formation at the eltAB promoter
To assess the effect of H-NS on promoter opening during transcription initiation of the eltAB promoter, we carried out KMnO4 footprinting. The 757 bp linear DNA fragment that contains the eltAB promoter and H-NS binding sites was incubated with purified H-NS protein (200 nM, 400 nM and 1000 nM) at 22 °C for 10 min, after which RNA polymerase (100 nM) was added to the reaction. The samples were incubated for a further 25 min at 30 °C to allow the formation of open complexes. Following treatment with KMnO4, primer extension analysis was used to measure the extent of promoter opening. In the presence of RNA polymerase, cleavage of the thymine residues at positions –7, –8 and –9 in the bottom strand was detected (Fig. 7). Addition of H-NS had no effect on promoter opening.



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Fig. 7. KMnO4 footprinting analysis of the effect of H-NS on open complex formation of the eltAB promoter. Linear template (eltAB1–757) was incubated with RNA polymerase (100 nM) in the absence or presence of H-NS (200 nM, 400 nM and 1000 nM). The positions of the DNA template strand which are reactive to KMnO4 are marked with arrows. M, size standards.

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In this study, we carried out a series of in vivo and in vitro experiments to elucidate the molecular mechanism by which H-NS protein controls transcription of the eltAB operon encoding LT, a major virulence factor of ETEC. The results from {beta}-galactosidase analysis using a single-copy plasmid carrying an eltAB1–757lacZ fusion show that H-NS exerts fivefold repression of transcription from the eltAB promoter at 37 °C, and that this repression is increased to 10-fold in response to growth at 22 °C. Deletion analysis demonstrates that efficient repression requires binding of H-NS protein to two regions that are separated by 350 bp. The results of this analysis correlated well with data obtained from in silico analysis, EMSA and in-gel DNase I footprinting of the eltAB fragment. The regions that are essential for repression in vivo were predicted to contain intrinsic curvature and can interact directly with the H-NS protein.

Both of the H-NS-binding regions were located downstream of the eltAB promoter, one between +31 and +110 and the other between +460 and +556, relative to the start site of transcription. These results are in agreement with Trachman & Maas (1998), who showed that deleting the eltA structural gene increased LT mRNA synthesis in an Hns+ strain at low temperature. The upstream site contained two patches of approximately 20 bp that were protected by H-NS, whereas the downstream H-NS-binding motif encompassed a single sequence of >90 bp. The presence of multiple H-NS-binding sites over a long distance of DNA occurs in most genes whose expression is repressed by H-NS. The observation that the presence of only one binding region did not bring about significant repression by H-NS, whereas the presence of both regions led to a maximal 10-fold repression, indicates cooperative interactions between the H-NS molecules bound at the two sites. Such interactions could induce DNA loop formation and compact the sequence of the eltA structural gene into a nucleoprotein complex, inhibiting transcription by RNA polymerase. The stronger repression observed at 22 °C was probably due to an enhanced DNA curvature, which resulted in increased affinity of H-NS for its binding sites.

In Gram-negative bacteria, H-NS is one of the few repressor proteins that can act by binding to a region located far downstream of the start site of transcription. The proU operons from E. coli and Salmonella typhimurium were the first genes identified with a DRE (Dattananda et al., 1991; Overdier & Csonka, 1992; Lucht et al., 1994). In S. typhimurium, the DRE of proU is positioned between +73 and +274, relative to the start site of transcription, and is essential for repression by H-NS. Detailed analysis of the mechanism of repression of the proU promoter has shown that binding of H-NS to DREs inhibits open complex formation by RNA polymerase (Jordi & Higgins, 2000). The bgl operon from E. coli carries two silencers to which H-NS binds preferentially (Schnetz, 1995; Dole et al., 2004). One silencer is located upstream of the promoter and the other is located 600–700 bp downstream of the start site of transcription (Dole et al., 2004). The two silencers function independently in the repression of bgl expression. While binding of H-NS to the upstream silencer represses the bgl promoter, presumably by inhibiting transcription initiation, binding of H-NS to the downstream silencer blocks chain elongation during mRNA synthesis by RNA polymerase. In the case of the eltAB operon, however, the two H-NS-binding regions appear to work cooperatively rather than additively. In addition, as demonstrated by KMnO4 footprinting, occupation of the two binding sites by H-NS molecules did not affect formation of open complexes by RNA polymerase at the eltAB promoter. Mutational studies showed that H-NS-mediated repression is independent of promoter strength, as the repression ratio was essentially the same after altering the sequence of the promoter core elements to reduce or strengthen promoter activity. Together, these observations support the idea that H-NS represses expression of the eltAB promoter either by inhibiting later stages of transcription initiation (e.g. promoter clearance) or by aborting elongation of RNA polymerase immediately after transcription initiation.

The eltAB operon is encoded by a mobile genetic element on a virulence plasmid and the structural genes for the A- and B-subunits share approximately 80 % DNA and protein sequence homology with those for CT (ctx). However, the promoter regions of the two operons have undergone considerable genetic changes during evolution with regard to regulation of transcriptional expression in their respective hosts. Although transcription of ctx is also negatively regulated by H-NS, the region responsible for H-NS-mediated repression is situated upstream of the promoter core elements, between –69 and –400 relative to the start site of transcription (Nye et al., 2000; Yu & DiRita, 2002). In the case of the eltAB operon, deleting the sequence between –65 and –160 had no effect on H-NS-mediated repression. The different binding locations of the H-NS protein may have resulted from co-evolution or incorporation with other regulatory systems specific for V. cholerae and ETEC, and could play a role in fine-tuning the levels of the enterotoxin production under different conditions.

The eltAB operon contains a moderately strong promoter, as judged by the levels of {beta}-galactosidase expression from eltAB–lacZ fusions in the Hns background. The core promoter elements include a perfect –35 region and an optimal spacer of 17 bp, which are ideal for binding of the {sigma} subunit of RNA polymerase to the promoter. In addition, the –10 region and the sequence between the –10 region and the start site of transcription are highly AT-rich, which facilitates promoter opening. All these factors contribute to the strength of the eltAB promoter and may explain why the putative AT-rich UP element is dispensable for promoter activity. Given the high intracellular levels of H-NS that vary little during bacterial growth, it is likely that the eltAB operon is normally repressed by H-NS when ETEC strains are not in their host environment. This effect would be overcome by ETEC-specific regulatory proteins which, under appropriate environmental conditions, could act as an anti-repressor to alleviate the effect of H-NS on eltAB expression. Indeed, several bacterial regulatory proteins are known to relieve H-NS repression by displacing the protein from DNA, e.g. the Fis protein at the rrnB P1 promoter in E. coli K-12 (Schneider et al., 2003), the ToxT protein at the ctx promoter in Vibrio cholerae (Yu & DiRita, 2002), the Ler protein at the LEE2, LEE3 and LEE5 promoters in EPEC (Bustamante et al., 2001; Haack et al., 2003) and the VirF protein at the virB promoter in Shigella flexneri (Beloin & Dorman, 2003). The discovery of analogous proteins in ETEC would provide further insight into the regulation of enterotoxin production by these bacteria.


   ACKNOWLEDGEMENTS
 
This work was supported by a grant from the Australian National Health and Medical Research Council. We thank J. Gowrishankar, S. Rimsky, A. Ishihama and J. Pittard for strains, plasmids and proteins used in this study.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 29 October 2004; revised 27 December 2004; accepted 5 January 2005.



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