Department of Molecular Genetics and Biotechnology, The Hebrew University, Faculty of Medicine, POB 12272, Jerusalem 91120, Israel1
Author for correspondence: Ilan Rosenshine. Tel: +972 2 6758754. Fax: +972 2 6784010. e-mail: ilanro{at}cc.huji.ac.il
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ABSTRACT |
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Keywords: EPEC, LEE, H-NS, Ler, thermoregulation
Abbreviations: EPEC, enteropathogenic Escherichia coli; GFP, green fluorescent protein; LEE, locus of enterocyte effacement; H-NS, histone-like nucleoid-structuring protein; Ler, LEE-encoded regulator; IHF, integration host factor; Tir, translocated intimin receptor
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INTRODUCTION |
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We previously showed that expression of the LEE-encoded type III secretion system is thermoregulated, being repressed at 27 °, and expressed at 37 °C (Rosenshine et al., 1996 ). Thermoregulated expression of virulence genes was reported also in enteroinvasive E. coli (Forsman et al., 1992
; Jordi et al., 1992
) and Shigella (Maurelli & Sansonetti, 1988
). In some of these cases H-NS (histone-like nucleoid-structuring protein) plays a key role in the repression at the non-permissive temperature. H-NS is a nucleoid-associated protein, frequently involved in the response of enterobacteria to environmental stimuli. In most cases it mediates negative regulation and counteracts a positive regulator (Atlung & Ingmer, 1997
). In E. coli K-12 H-NS is involved, directly or indirectly, in the regulation of about 5% of the genes (Hommais et al., 2001
).
Expression of LEE2, LEE3, LEE4, LEE5, espG and orf19 is activated by Ler (LEE-encoded regulator), a distant homologue of H-NS, encoded by the LEE1 operon (Friedberg et al., 1999 ; Mellies et al., 1999
; Sanchez-SanMartin et al., 2001
; Sperandio et al., 2000
). Therefore, the decision whether or not to activate expression of LEE1 is critical for the initiation of a regulatory cascade leading to expression of other LEE operons. LEE1 expression is dependent on activation by Integration Host Factor (IHF) (Friedberg et al., 1999
). Additional factors, including Fis, Per and quorum sensing, are involved in modulation of LEE1 gene expression (Goldberg et al., 2001
; Kanamaru et al., 2000
; Mellies et al., 1999
; Sperandio et al., 1999
). Recently it was reported that in the K-12 background, H-NS acts as a negative regulator of LEE2, LEE3 and orf19 (Bustamante et al., 2001
; Sanchez-SanMartin et al., 2001
). It was hypothesized that Ler activates gene expression by negating the H-NS-mediated repression (Bustamante et al., 2001
; Sanchez-SanMartin et al., 2001
). In conclusion, the LEE operons are subject to regulation by multiple factors; some, like IHF, H-NS and Fis are global regulators expressed by K-12 laboratory strains, and others like Ler, Per and perhaps other regulators are specific to EPEC.
The involvement of any of the above factors, including H-NS and Ler, in thermoregulation is not clear. In this communication we describe the role of H-NS in thermoregulated expression of the LEE operons. We report, for the first time, the formation of an EPEC hns mutant and an EPEC double mutant of hns and ler. We used these mutants to analyse the role of hns and ler in the thermoregulated expression of LEE genes. The genetic analysis was supported by in vitro biochemical analyses using a purified H-NS protein. We show that H-NS is involved in two levels of regulation of the LEE genes. First, it represses the expression of LEE1 (including ler) at 27 °C but not at 37 °C, leading to thermoregulated expression of all other Ler-regulated LEE operons. In addition, H-NS represses expression of the LEE2, LEE3, LEE4, LEE5 and espG operons both at 27 °C and at 37 °C. Upon shifting the culture temperature from 27 °C to 37 °C, the expressed Ler activates the expression of LEE2, LEE3, LEE4 and espG by releasing the H-NS-mediated repression. In the case of LEE5, Ler acts both by antagonizing the H-NS-mediated repression and by an additional mechanism, as yet to be defined.
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METHODS |
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Protein extraction and immunoblot analysis.
Bacterial cultures grown as indicated above (see Bacterial strains) were centrifuged and extracted by boiling for 7 min in lysis buffer (1% SDS and 50 mM Tris/HCl pH 7·5), 2 min centrifugation at 14000 g and collecting the supernatant. Protein concentrations of the samples were adjusted and samples were subjected to SDS-PAGE, transferred to nitrocellulose membranes and treated with antibodies as described (Wolff et al., 1998 ).
Construction of EPEC mutants.
To construct an EPEC hns mutant, a 1380 bp DNA fragment containing EPEC hns gene and flanking regions was amplified by PCR using EPEC DNA and primers hnsF4 and hnsR4 (Table 2). A BamHI/HindIII digest of the amplified DNA fragment was cloned into the BamHI/HindIII sites of the pBS plasmid to form pBS-hns (pDF6). A 1·2 kb DNA fragment containing the kan gene cassette was purified as a PstI fragment from pUC4K (Pharmacia), cloned into the PstI site of the hns gene in pDF6 to form pDF7 (pBS-hns::kan). A BamHIXhoI DNA fragment containing the hns::kan DNA was purified from pDF7 treated with Klenow fragment (MBI Fermentas) and cloned into the SmaI site of pCVD442 to form pDF8 (pCVD-hns::kan), in SY327
pir strain. pDF8 was then electroporated into E. coli SM10
pir. EPEC insertionally inactivated hns mutant (designated DF3) was created by allelic exchange of wild-type hns with hns::kan carried by pDF8, using the sucrose selection method (Donnenberg & Kaper, 1991
).
We adopted and modified the one-step inactivation method of chromosomal gene disruption that makes use of the Lambda Red recombinase in E. coli K-12 (Datsenko & Wanner, 2000 ), to operate in EPEC. This system, that enables recombination of linear DNA fragments in the E. coli genome (Datsenko & Wanner, 2000
), was used to construct an EPEC ler::kan hns::cat double mutant. A DNA fragment containing the cat gene was amplified from plasmid pKD3 (Datsenko & Wanner, 2000
), using primers cm-pKD3-F and cm-pKD3-R. The cat fragment was cloned into the PstI site of pDF6 to construct pTU15 (pBS-hns::cat). EPEC ler::kan (Friedberg et al., 1999
) was electroporated with plasmid pKD46 containing the Lambda Red recombinase (Datsenko & Wanner, 2000
). This strain was subsequently electroporated with a linear DNA fragment containing hns::cat, that was amplified with primers hnsF4 and hnsR4, using pTU15 DNA as a template. Cmr clones were selected at 37 °C, streaked twice and cmr amps clones that had lost pKD46 were selected. PCR analysis confirmed replacement of the wild-type hns gene by hns::cat and the formation of EPEC ler::kan hns::cat double mutant.
H-NS purification.
Native, untagged H-NS was purified to homogeneity from an H-NS-producing clone of E. coli BL21/pPD3 as described by Dersch et al. (1993) . The published procedure was scaled down to 1 l bacterial culture and the purified H-NS was concentrated to 3 mg ml-1 using Centricon YM-10 (Amicon).
DNA band shift assay with H-NS.
Three DNA fragments were used: a 186 bp DNA fragment corresponding to the sequence from position -173 to +11 relative to the LEE1 transcriptional start point (LEE1 fragment), amplified with primers 9F and 5R; a 395 bp PCR fragment corresponding to sequence from position +117 to -288 nucleotides relative to LEE2 transcriptional start point (LEE2LEE3 fragment), amplified with primers 30F and 31R; and a DNA fragment 171 bp in size containing the CIII coding region, amplified with primers 2703 and 2902. The LEE fragments and the CIII fragment were digested by XbaI and EcoRI respectively and end-labelled by filling in using Klenow fragment of DNA polymerase I (MBI Fermentas) and [
32P]dCTP (3000 Ci mmol-1; Amersham). Binding of H-NS to the end-labelled DNA (36 fmol) was performed at 25 °C for 30 min, in 10 µl binding buffer containing: 50 mM Tris/HCl (pH 7·4), 70 mM KCl, 1 mM EDTA, 10 mM ß-mercaptoethanol, 100 µg bovine serum albumin ml-1, 6% glycerol and 100 ng poly(dIdC) (Boehringer). At the end of the incubation period the samples were loaded on 4·5% polyacrylamide gel (acrylamide to bisacrylamide, 37·5:1 w/w) in 0·5x TBE buffer and electrophoresis was carried out at room temperature. The dried gels were exposed for autoradiography and the results were quantified using a phosphorimager (Fuji) as described.
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RESULTS |
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Expression of LEE genes in EPEC double mutant inactivated in both ler and hns genes
To further substantiate the role of H-NS in the regulation of LEE2, LEE3, LEE4, LEE5 and espG without any possible interference of Ler, we constructed an EPEC double mutant inactivated in both hns and ler. This EPEC mutant was constructed by using the Lambda Red recombinase system that enables recombination of linear DNA into the E. coli K-12 genome as described by Datsenko & Wanner (2000) (see Methods). We compared the expression of LEE genes in the background of the following isogenic EPEC strains: wild-type, ler::kan, hns::kan and ler::kan hns::cat. The four strains were grown at 37 °C and analysed by immunoblotting using antibodies raised against EspA, EspB (LEE4-encoded proteins), intimin and Tir (LEE5-encoded proteins). The EPEC ler hns double mutant expressed EspA and EspB at levels comparable to the hns mutant. However, this mutant expressed Tir and intimin to levels significantly lower than the hns mutant (Fig. 4A
). As expected, the EPEC hns mutant expressed these proteins at levels comparable to the wild-type EPEC, while the ler mutant did not express any of these proteins (Fig. 4A
).
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H-NS binds to the LEE1 and LEE2LEE3 promoter regions
We tested whether regulation by H-NS involves its interaction with the regulatory regions of LEE1 and LEE2LEE3. H-NS was purified as described by Dersch et al. (1993) (Fig. 5A
) and used in gel shift assays with three DNA fragments: a fragment that extends nucleotides -173 to +11 relative to LEE1 transcriptional start site (LEE1 fragment), a fragment containing the overlapping promoter region and the flanking 5' coding regions of LEE2 and LEE3, corresponding to nucleotide positions +117 to -288 relative to LEE2 transcriptional start point (LEE2LEE3 fragment), and a control fragment driven from the CIII gene of Lambda phage. The LEE1 and LEE2LEE3 fragments formed low- mobility complexes in the presence of H-NS (Fig. 5B
, D
). However, the formation of a low-mobility complex with the control CIII DNA fragment was insignificant and demanded a much higher H-NS concentration (Fig. 5F
). This indicates a preferential binding of H-NS to the LEE1 and LEE2LEE3 fragments. To assess the apparent binding affinity, we performed a quantitative analysis of the free and bound DNA in the presence of increasing (1·5 to 2-fold) H-NS concentrations (Fig. 5C
, E
). A small increase (about 1·5 to 2-fold) in H-NS concentration was sufficient to shift the majority of the LEE1 DNA, as well as the LEE2LEE3 DNA, from a free to a protein-bound form, suggesting a cooperative binding mode of H-NS. The data show an apparent dissociation constant (Kd, protein concentration that binds 50% of the DNA) of 0·95 µM and 0·80 µM with LEE1 and LEE2LEE3 fragments respectively. In both cases the curve shape confirms a cooperative binding mode of H-NS. The binding of H-NS to the LEE promoter regions is consistent with its role as a repressor of each of these LEE genes.
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DISCUSSION |
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However, it was more complicated to draw conclusions regarding the role of H-NS in the regulation of all other LEE operons, since the LEE1-encoded Ler is a positive regulator of these operons. Therefore, to examine the role of H-NS without the interference of Ler, we constructed an EPEC strain mutated in both hns and ler. The analysis of LEE gene expression in this double mutant was particularly informative. While in the absence of Ler at 37 °C the expression of LEE2, LEE3, LEE4, LEE5 and espG operons was repressed, the repression was alleviated upon further inactivation of hns. These results indicate that H-NS represses each of these operons regardless of Ler. While at 37 °C the H-NS-mediated repression of LEE1 was inefficient, H-NS efficiently repressed the other operons at 37 °C, including LEE2, LEE3, LEE4, LEE5 and espG.
We further showed that in the absence of H-NS, Ler was dispensable for full expression of LEE2, LEE3, LEE4 and espG. This suggests that Ler activates expression of these operons exclusively by preventing the H-NS-mediated repression. However, inactivation of hns was insufficient for a complete restoration of LEE5 expression, in the absence of Ler, suggesting that Ler activates expression of LEE5 both by antagonizing the H-NS mediated repression as well as by an additional mechanism, as yet to be defined.
Based on these results, our current working model, presented in Fig. 6, is as follows. At the non-permissive temperature of 27 °C, H-NS mediates repression of LEE1, LEE2, LEE3, LEE4, LEE5 and espG. The physical evidence that H-NS binds to the LEE1 and LEE2LEE3 regulatory regions supports the speculation that H-NS might act as a direct repressor of these operons. It is yet to be confirmed that this interaction occurs also in the case of the other LEE operons. According to our model, upon shifting the temperature from 27 °C to 37 °C, the expression of LEE1 is alleviated while the expression of LEE2, LEE3, LEE4, LEE5 and espG is still repressed by H-NS. The production of Ler at 37 °C subsequently activates the expression of LEE2, LEE3, LEE4, LEE5 and espG. Ler mediates the activation of LEE2, LEE3, LEE4 and espG by alleviating the H-NS-mediated repression. Activation of LEE5 by Ler involves both antagonizing the H-NS repression and an additional, as yet unknown mechanism, that is independent of H-NS.
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In conclusion, our results on the opposite role of Ler and HNS in respect to expression of the LEE2 and LEE3 operon confirm and substantiate a previous report (Bustamante et al., 2001 ). In addition we show, for the first time, the opposite role of these two regulators in the case of three additional LEE operons, namely the LEE4, LEE5 and espG. More importantly, we describe here for the first time the role of H-NS in temperature-dependent repression of the LEE1 operon including ler. This repression is the basis for the thermoregulated expression of all other LEE operons.
In enteroinvasive E. coli and Shigella, virulence genes are repressed at 30 °C and expressed at 37 °C (Maurelli & Sansonetti, 1988 ). H-NS is involved in the thermoregulation of virF (Falconi et al., 1998
), the first positive activator of a multi-step regulatory cascade leading to expression of the type III secretion system and other virulence effectors (Dorman & Porter, 1998
). It was proposed that the physical basis for the thermoregulated expression of virF resides in a temperature-dependent structural modification of the virF promoter, resulting from a temperature-sensitive curved DNA structure (Falconi et al., 1998
). H-NS also mediates repression of the virB gene downstream in the Shigella regulatory cascade (Tobe et al., 1993
). The regulatory cascade in EPEC, whereby the expression of Ler is thermoregulated by H-NS, is apparently analogous to the Shigella system. However, in EPEC the role of H-NS is more complex, given its involvement in repression of all other major LEE operons, and because of the activity of Ler as a positive regulator that prevents H-NS-mediated repression of all other LEE operons.
We hypothesize that a temperature-dependent DNA-topology modification of the LEE1 promoter region might play a role in the thermoregulation of LEE1. This hypothesis is supported by our previous data showing that binding of IHF upstream of the LEE1 promoter is essential for LEE1 expression (Friedberg et al., 1999 ). IHF that binds to DNA in specific sites bends the DNA to form nucleoprotein complexes (Rice et al., 1996
). The IHF-dependent modification of the DNA curvature might change the accessibility to H-NS in a temperature-dependent manner. It remains to be seen if IHF antagonizes H-NS and thereby alleviates the H-NS mediated repression of LEE1 at 37 °C. In DNase footprinting experiments H-NS does not show any distinct footprinting, but protects extended DNA sequences of the LEE1 promoter region, including the IHF binding site (not shown). Interplays, either synergistic or antagonistic, between histone-like proteins, including H-NS and IHF, have been implicated in several unrelated genetic systems (McLeod & Johnson, 2001
). For instance, in the case of the early promoter of bacteriophage Mu, it was shown that IHF interacts directly with the transcription initiation complex and in addition alleviates the H-NS mediated repression of the Pe promoter (van Ulsen et al., 1996
). In the case of Shigella flexneri virulence genes it was reported that IHF mediates virB expression by overcoming the repression exerted by H-NS (Porter & Dorman, 1997
).
A finely tuned regulation of the ler gene is presumably critical for the timing of LEE gene expression during infection. Fis, PerABC and quorum sensing are also involved in the regulation of LEE1 (Goldberg et al., 2001 ; Mellies et al., 1999
; Sperandio et al., 1999
). The future challenge is to define in EPEC and related organisms the role of all the various regulators and to characterize the interplay that mediates LEE gene expression in response to the host-environmental signals.
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ACKNOWLEDGEMENTS |
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Received 21 March 2002;
revised 7 May 2002;
accepted 21 May 2002.