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
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ABSTRACT |
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INTRODUCTION |
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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 165) is responsible for dimerization and the C-terminal domain (aa 90137) 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.
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METHODS |
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-Galactosidase assay.
-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 [
-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 eltAB1297 or eltAB1757 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 µl1 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 eltABlacZ transcriptional fusions and deletion mutants.
The three eltABlacZ fusions, eltAB1297lacZ, eltAB1490lacZ, eltAB1757lacZ, 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 eltABlacZ transcriptional fusions.
Deletion mutant 195 was constructed as follows. A BamHI site between positions 95 and 100 (Fig. 1a
) was created by oligonucleotide-directed mutagenesis on M13tg130-eltAB1757 using primer YJ030 (Table 1
). The eltAB fragment (
195) lacking the first 95 nt was cloned into pMU2385 to create the eltAB(
195)lacZ transcriptional fusion.
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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 [
-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-eltAB1757. 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 ml1] 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 cm1, samples were loaded onto the gel and electrophoresis was carried out at 4 °C for approximately 12 h at 10 V cm1.
The in-gel DNase I assay was performed essentially as described by Yang et al. (2004). Gel slices containing various proteinDNA 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 ml1 and 0·4 U DNase I ml1; 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 eltAB1757 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 ml1] 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 -mercaptoethanol (14·7 M). The DNA was cleaved with piperidine at 90 °C for 20 min. A primer extension experiment using
-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.
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RESULTS |
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As shown in Fig. 2, at 37 °C, in the Hns+ background (MC4100), the fusions eltAB1297lacZ and eltAB1490lacZ produced similar levels of
-galactosidase activity of 675 and 791 units, respectively. In contrast, the level of expression from eltAB1757lacZ was only 238 units, about one-third of that of the other two constructs. In the Hns background (PD145), the
-galactosidase activities for the fusions eltAB1297lacZ and eltAB1490lacZ 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 eltAB1757lacZ, where the
-galactosidase expression was approximately fivefold higher in the Hns host than in the Hns+ host.
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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 eltAB1297 or the eltAB1757 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-eltAB1297 than by pUC19-eltAB1757, consistent with the observation that eltAB1757 is more repressible by H-NS than eltAB1297. 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 eltAB1757 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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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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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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DISCUSSION |
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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 600700 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 -galactosidase expression from eltABlacZ 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
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.
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ACKNOWLEDGEMENTS |
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Received 29 October 2004;
revised 27 December 2004;
accepted 5 January 2005.
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