(~1 [NSTITUTPAS,'EUR/ELSEVIER Paris 1998 Res. Microbiol. 1998, I49, !5-25 A role for H-NS the regulation of the virF gene of ShigeUa and enteroinvasive Escherichia coli G. Prosseda (i), EA. Fradiani (1), M. Di Lorenzo (l), M. Falconi (2), G. Micheli <3), M. Casalino t4), M. Nicoletti (5) and B. Colonna (~)(*) it; Dip.Biologia Celluhlre e dello Sviluppo, Universitgz La Sapienza, Via degli Apuli, 1, 00185 Rome, (4) Dip Biologia, 111 Universit?t, 00146 Rome, and t5) Dip.Scienze Biomediche, Universit& G.D'Annunzio, 66100 Chieti (Italy) SUMMARY We have investigated the role of H-NS, one of the major components of the bacterial nucleoid, in the exgression of the virF gene present on the large virulence plasmid of Shigeila and enteroinvasive Escherichia coil in response to different environmental conditions. VirF is an AraC-like protein which activates at least two promoters, virB and virG, both repressed by H-NS. Band shift experiments reveal that the affinity of H-NS for the virF and virB promoters is comparable, while the affinity for the virG promoter is higher. Polyacrylamide gel electrophoresis of three DNA fragments containing the virF, the virB and the virG promoters demonstrates, in agreement with computer predictions, that they have an intrinsically curved structure, confirming the preference of HNS for bent DNA. In vivo transcriptional analysis of virF mRNA shows that H-NS negatively controls the expression of virF at 30°C. The expression of a virF-lacZ translational fusion in E.cofi wild type and in an hns-defective derivative grown at 30° or 370C and at pH 6.0 or 7.0 indicates that, in the absence of H-NS, virFexpression becomes insensitive to temperature and to limited pH changes. Our results strongly suggest that H-NS controis virFexpression by binding to the virFpromoter and by repressing its expression at low temperature and at low pH. Key-words" virF gene, H-NS protein, pH, Temperature; Regulation, Shigella, EIEC, Bent DNA. INTRODUCTION H-NS, one of the major components of bacterial chromatin, affects the expression of a large n u m b e r of unrelated genes coding for Submitted June 30, 1997, accepted October 1, 1997. (*) Correspondingauthor. housekeeping functions, as well as for virulence functions (Defez and De Felice, 1981; Hulton et al., 1990; Dorman and Ni Bhriain, 1993; Atlung and Ingmer, 1997). It is an abundant, n o n - b a s i c 1 5 . 5 - k D a D N A b i n d i n g protein 16 G. PROSSEDA ET AL. (Spassky et al., 1984), able to associate with curved DNA with high affinity (Yamada et al., 1991; Owen-Hugues et al., 1992). H-NS is encoded by the hns gene (Pon et al., 1988), which has been shown to be highly conserved among enterobacterial species (La Teana et al., 1989). The H-NS protein plays a crucial role in the regulation of virulence in Shigella and in enteroinvasive E s c h e r i c h i a coil (EIEC), the causative agents of bacillary dysentery (Hale, 1991), by repressing the expression of the invasion genes (located on a large plNV plasmid) at 30°C (Maurelli and Sansonetti, 1988) or in low osmolarity medium (Porter and Dorman,1994). Activation of the plNV invasion genes occurs in a coordinate manner at 37°C and is mediated by two transcriptiona. ~'tivators: the VirF and VirB proteins (Adler et al., 1989). In a cascade model, VirF activates transcription of the gene coding for the secondary regulator VirB, which in turn activates at least three unlinked operons coding for the invasion plasmid antigens (lpa proteins) and for their type III exportation system (Mxi and Spa proteins) (Sakai et al., 1988; Tobe et al., 1991, 1993). The virG gene, which is responsible for intra- and intercellular spread of bacteria, is activated by VirF (Lett et al., 1989). Repression of virulence gene expression also occurs at permissive temperature, when the virulence plasmid is integrated into the host chromosome, due to H-NS-dependent inhibition of v i r B transcription (Zagaglia et al., 1991 ; Colonna et al., 1995). Although the exact mechanism of virF activation has not been fully elucidated, VirF has been considered as a positive activator that has the property of alleviating and/or counteracting the silencing effect of HNS on the virB and virG promoters (Sakai et al., 1988; Dagberg and Uhlin, 1992). Moreover, reduced expression of virF (and consequently of the invasion genes) at low pH has also been reported in S.sonnei (Nakayama and Watanabe, 1995). EIEC LIB = = enteroinv&sive Escherichia coli. Luria-Bertani (broth). VirF belongs to the AraC family, which consists of proteins involved in the activation of genes for carbohydrate utilization or in the activation of virulence genes in several enterobacterial species (Dorman, 1992). Like other a r a C genes, e.g. cfaD and m s , virF has an abnormally low GC content (about 30%, as opposed to about 50 % for bulk DNA of these enteric bacteria). The VirF protein is a close relative of the AraC-like CfaD and AppY proteins of E.coli, which are both negatively regulated by H-NS at 30°C (Jordi et al., 1992; Atlung et aL, 1996). In a previous work (Colonna et al., 1995) we observed that hyperexpression of v i r F fully restores invasiveness of EIEC strain HN280 at non-permissive temperature (30°C). This has led us to propose that H-NS might also control virF expression by binding to the virF promoter and that the thermoregulated expression of virB might be due to the combined repression exerted by HNS on virB and virE In this paper, we show that H-NS controls the expression of VirF by binding to the virF promoter and by repressing its expression at low temperature and at low pH. MATERIALS AND METHODS Bacterial strains, plasmids and genetic procedures The bacterial strains and plasmids used are listed in table I. Bacteria were grown in LB (Luria-Bertani) broth. Transformation and transduction with P lvir were done as described by Miller (1992). Strain HN4104 was constructed by introducing the hns 118 deletion of TP504 into MC4100 and by selecting for cotransductants with zch-506::TnlO. The presence of the hns-defective allele was monitored by observing the appearance of red colonies on MacConkey base agar containing salicine 0.2% (Bertin et al., 1994). Antibiotics and chemicals were used at the following concentrations: ampicillin 50 lxg/ml, chloramphenicol 25 Ixg/ml, kanamycin 30 lxg/ml, tetracycline 5 l.tg/ml, trimethoprim 10 ~tg/ml and Xgal 20 lxg/ml. PCR = polymerase chain reaction. H - N S R E G U L A T I O N O F virF E X P R E S S I O N Construction of the virF-lacZ fusion Plasmid pFBlac2, carrying the virF-lacZ fusion: was constructed by cloning a PCR-generated fragment containing the virF promoter into the multicloning site of the l a c Z transductional fusion vector pMCI4O3. For the amplification of the virF promoter, we used the E.coli HN280 pINV DNA as a template. The forward primer, BX71 (5'-TTGAAT'FC A A A T A C T T A G C T T G - 3 ' , coordinates - 1 4 3 to -121), was designed to have an EcoRI site at the 5' end; the reverse primer, QH7 ( 5 ' - T A G G G A T C CAAGCGAACCTTTATATC-3', coordinates +90 to +115) (Sakai et al., 1986), was designed to have a BamHl site at the 5' end. The PCR product, containing the virF transcriptional and transductionai regulato~ signals as well as the sequence coding for the 16 N-terminal amino acids of VirF, was digested with E c o R l and B a m H l and then cloned into E c o R l BamHI digested pMCI403, thus generating plasmid pFBiac2. The sequence of the PCR-generated fragment was checked by the dideoxy chain terminating method (Sambrook et aL, 1989). Beta-galactosid~ase assay Beta-galactosidase assays were performed on sodium dodccyl suiphate-chloroform-permeabilized cells as described (Miller, 1992). Cultures were grown to ODr00 0.5-0.6 in LB broth containing 0.1 M sodium phosphate buffers (pH 6.0 and pH 7). Northern analysis Bacterial strains were grown to OD6t~)0.5 in LB broth (containing ampicillin 50 lag/ml and kanamycin 30 lag/ml) at different temperatures. Aliquots of total RNA, extracted as previously described (Colonna et al., 1995), were denatured at 100°C for 5 rain in the presence of 2M formaldehyde and 50% formamide, separated on agarose gel, and then hybridized as described (Sambrook et al., 1989). The relative amounts of RNA loaded in each lane were estimated by visualization of the rRNA by ethidium bromide staining. Hybridization was performed using an 800bp BsoFI fragment of pMYSH6520 as a virF probe (Sakai et al., 1988). Densitometric analysis of autoradiograms was carried out with the aid of a "Biorad GS670" imaging densitometer. Molecular procedures Isolation of plasmids, restriction digestions, cloning, electrophoresis and purification of DNA fragments were carried out as previously described (Colonna et aL, 1995). The plasmid copy number of 17 pMYSH6504 and of pFBlac2 was determined as described previously (Falconi et al., 1993) on EcoRI linearized DNA. DNA probes were 32p-labelled by the random printing method. PCR amplification was carried out by using 50 ng of template DNA and 1 pM of each primer in a 100-~tl reaction mixture conraining 67 mM Tris-HC1 (pH 8.8), 16 mM ammonium sulphate, 1 mM MgCI v 0.01% Tween 20 and 200 p.M of each deoxynucleoside tfiphosphate. The reaction was stopped after 25 cycles, each cycle comprising 30 s of denaturation at 95°C, 3 rain of primer annealing at temperatures ranging from 44 to 48°C (depending on the primer sequence) and l min of extension with Taq polymerase at 72°C. The PCRgenerated fragments were then purified by phenol chloroform extraction and ethanol precipitation (Sambrook et al., 1989). Purification of H-NS was performed as previously described (Falconi et al., 1988). Band shift assay The fragments containing the virF, virB, and virG promoters used in this study were obtained by PCR amplification of E.coli HN280 plNV DNA. The amplification of the virB promoter was primed with the oligomers BX5 (5'-GGAGCTCTCACATCAG3') and BX6 (5'-GTCGTTGCACAAATCC-3'), corresponding to coordinates -136 to -121 and +60 to +75 of the virB sequence (Adler et aL, 1989; Tobe et al., 1993), and resulted in a 21 l-bp fragment. The virF promoter was amplified using primers BX7 (5'C A A A T A C T T A G C T T G T - 3 ' ) and BX8 (5'G C G A A C C T T T A T A T C T - 3 ' ) , corresponding to coordinates -135 to - 1 2 0 and +89 to + 104 of the virF sequence (Nakayama and Watanabe, 1995; Sakai et al., 1986) and resulting in a 239-bp fragment. The primers GU23 (5'-GAAAAGTTGCGGTCTG-3') and GT 18 (5'-AGGTAATTCTCCGGCC3'), corresponding to nucleotides - 1 0 9 to - 9 4 and +192 to +207 of the virG sequence (Lett et al., 1989), were used to amplify the virG promoter, resulting in a 316 bp-fragment. Each reaction mixture contained approximately 5 ng of the appropriate PCR-generated DNA fragment (end-labelled with 32p-dATP by a fill-in reaction with the Klenow fragment of DNA polymerase; Sambrook et al., 1989), 50 ng poly(dI-dC) (Pharmacia) as a competitor DNA and different concentrations of purified H-NS in a total volume of 15 lal of 10 mM Tris-HCl pH 8, 10 mM MgCI 2 100 mM NaCl, l0 mM KCI, 1 mM spermidine, 0.5 mM dithiothreitol and 5 % glycerol (Falconi et al., 1993). After 10 rain of incubation at room temperature, the samples were subjected to electrophoresis on 7 % polyacrylarnide gels (60/1, acryl/bis) in TAE (Tfis-HCI 40 mM pH 7.4, sodium acetate 5 mM, EDTA 1 mM). Quantitative radioactivity estimates were obtained by exposing the gels in a "BioRad GS250" molecular imager. 18 G. PROSSEDA ET AL. Bending assay The intrinsic curvature of PCR-amplified DNA fragments was studied by comparing their electrophoretic mobility to that of non-bent marker fragments (Pharmacia's 100-bp ladder) on 0.75-mm thick 5 % polyacrylamide gels (29.2•0.8, acryl/bis) run in TBE (90 mM Tris-HC1, 90 mM H3BO~, 2.5 mM NagEDTA) at 4°C and at 60°C under 5 V/cm voltage gradient. Computer predictions of intrinsic curvature were generated with a program developed by one of the authors (G,M.) using published estimates of dinucleotide wedge angles (Bolshoy et al., 1991). RESUI,TS Interaction of H-NS with the v/r promoters The current model for the regulation of virulence gene expression suggests that VirF positively regulates transcription of virB and virG, while at non-permissive temperature (30°C), HNS acts as a negative regulator toward both genes (Dagberg and Uhlin, 1992; Tobe et al., 1993). It has been reported that in Shigella (Tobe et al., 1991) as well as in EIEC (Colonna et al., 1995), the virF level at 30°C is reduced compared to 37°C. This led us to suppose that at 30°C, virF might be under the control of H-NS. In order to establish whether H-NS is able to bind to the virF promoter with the same efficiency displayed towards the virB and virG prorooters, we amplified the three promoter regions by PCR, using the plNV plasmid of E.coli HN280 (pHN280) as a template and the primers BX5/BX6 (virB promoter), BX7/BX8 (virF promoter) or GU23/GT18 (virG promoter). The PCR fragments were purified, end-labelled with 32p and subjected to an in vitro DNA binding assay with increasing amounts of purified H-NS (0400 ng of proteirg5 ng of DNA fragment). The reaction products were then analysed by gel retardation in the presence of excess synthetic poli(dldC) as a competitor. The results, shown in figure l a, indicate that H-NS associates with each of the three DNA fragments tested, albeit with different affinities. The quantitative analysis of the autoradiograms, reported in figure lb as percentage of free DNA, reveals that the affinity of H-NS for the virF and virB promoters is comparable, while the affhaity for the virG promoter is higher: 160 ng of H-NS dimer are required to bind 50% of virF or virB DNA, while only 80 ng are sufficient to bind 50% of virG (the corresponding "HNS dimer/vir fragment" molar ratios are 3.2× 109/1 for virF, 2.9× 102/1 for virB and 2.1 × 102/1 for virG). Analysis of intrinsically curved DNA in the vir promoters It has been previously shown that in vitro HNS associates with high affinity to intrinsically curved DNA (Yamada et al., 1991 ; OwenHughes et al., 1992). Computer analysis of the sequences of the PCR-generated fragments containing the virF, virB and virG promoters, based on previous estimates of dinucleotide wedge angles (Bolshoy et al., 1991), reveals a high potential for sequence-directed DNA curvature (fig. 2b). To experimentally test these predictions, we analysed the migration of the three PCR fragments in polyacrylamide gels under temperature conditions known to greatly enhance (4°C) or reduce (60°C) the effect of sequence-mediated DNA bending on electrophoretic mobility (Diekmann and Wang, 1985). As shown in figure 2a, the three fragments tested display a lower electrophoretic mobility at 4°C as compared to 60°C. In particular, at 4°C, the 239-bp virF fragment migrates as a 267-bp fragment, the 21 l-bp virB fragment migrates as a 233-bp fragment and the 316-bp virG fragment as a 415-bp fragment. At 60°C, the fragments migrate according to their "real" size. This result strongly suggests that DNA sequences around the virF and virB promoters may have an intrinsically curved structure, although the magnitude of the curvature as ~ndicated from the variation in electrophoretic raobility seems moderate. The relative increase in apparent size at 4°C with respect to the "real" size is 1!.7% for the virF fragment and 10.4% for the virB fragment. The variation is more pronounced for the virG fragment, whose apparent H - N S R E G U L A T I O N O F virF E X P R E S S I O N a 0 0 19 virB virF virG - 136 to + 7 5 - 135 to + 104 - 1 0 9 to + 2 0 7 0 tl~ 0 0 0 0 0 0 0 0 0 0 tN 0 0 0 0 0 0 0 (:3 0 0 IS} ,r- 0 0 0 0 CO H-NS (ng) b virB virF virG \ 100 v < Z r~ q) 50 ! ! ! 0 200 400 0 I I 200 : .= 400 0 I I ,o 200 : 400 H-NS (ng) Fig. 1. Interaction between H-NS and the vir promoters. Comparative electrophoretic mobility shift of virB, virF and virG promoter regions. (a) PCRgenerated DNA fragments of virB (211 bp), virF (239 bp) and virG (316 bp) were incubated in the presence of increasing amounts of H-NS and band shift assays were performed as described in "Materials and Methods". (b) After electrophoresis, the gel was exposed in a "BioRad GS250" molecular imager to quantify radioactivity associated with DNA-protein complexes and with unbound DNA. The percentage of unbound (free) DNA is plotted as a function of the amount of H-NS added. 20 a G. PROSSEDA ET AL. vi~ vi~ vi~ .... bp ..... s O * C b virF virF .......... vi~ virG 4"c vi~ -) virG Fig. 2. Intrinsic bending of the vir promoters. (a) Ethidium bromide staining of 0.75-mm thick 5 % polyacrylamide gels. The PCR-generated vir DNA fragments (see legend to figure 1) were run in TBE (Tris-HCI, H3BO 3, EDTA) buffer at 60°C and at 4°C, intercalated by marker lanes (100 bp ladder, Pharmacia). (b) Computergenerated predictions of intrinsic curvature in above vir fragments. size exhibits a 31.3 % relative increase at 4°C. This is in good agreement with the higher affinity of H-NS for this fragment observed in band shift experiments (fig. 1), and further confirms the preference of H-NS for curved DNA tracts. In vivo transcription of the v/rF gene The data on the binding of H-NS to the virF promoter prompted us to analyse the level of virF mRNA in strain MC4100 and in its h n s l l 8 deletion derivative HN4104, transformed with plasmid pMYSH6504, a pBR322 derivative containing the v i r F gene (table I). The relative pMYSH6504 copy number in strain HN4104 versus strain MC4100 was about 0.9, indicating, in agreement with previous studies (Bertin et al., 1992; Falconi et al., 1993), that mutations in hns do not significantly affect plasmid content. Equivalent amounts of total RNA extracted from strains grown at 30°C and at 37°C were denatured, separated on 1.2% agarose gels, and hybridized with a virF-specific probe (see Materials and Methods). The densitometric evaluation (fig. 3b) of the autoradiograms shown in figure 3a reveals that in the hns + strain there is a 3.7-fold reduction in the virF mRNA level at 30°C as compared to the level observed at 37°C. In the hns-defective strain, the expression of virF does not exhibit temperature dependence and attains a two-fold higher level as compared to the hns + strain grown at 37°C. These data suggest that in hns + backgrounds, the expression of virF is regulated by temperature and that this regulation is mediated Ly H NS. Involvement of H-NS in the pH-dependent expression of v/rF In order to further confirm the HNS dependence of virF expression suggested by Northern analysis, we followed the expression of virF at different temperatures. It has been recently reported that in S. sonnei, the expression of virF is reduced at pH 6 (Nakayama and Watanabe, 1995). Since H-NS has been shown to be involved in pH-dependent regulation of some genes, we analysed the expression of virF-lacZ translational fusion in E . c o l i hns + and h n s strains grown at different temperatures, as well as at different pH values. DNA of plasmid pFBlac2, a pMC1403 derivative containing the virF-lacZ fusion, was introduced by transformation into MC4100 and its hns I 18 derivative HN4104. The results reported in table II confirm that the expression of the virF-lacZ fusion is regulated by temperature (there is a 3.6-fold reduction at 30°C, in agreement with Northern analysis) and also by H-NS: indeed, the deletion of the hns gene (strain HN4104) results in constitutive expression of the reporter gene (7835 Miller units at 30°C and 7817 Miller units at 37°C). Moreover, in MC4100 pFBlac2 growth at pH 6.0 produces, both at 30°C and at 37°C, a two-fold reduction in the 13-galactosidase level, as compared to growth at pH 7.0. This indicates that, together with temperature, pH may also influence the expression of virF. T h e H-NS REGULATION OF virF EXPRESSION 21 Table L Bacterial strains and plasmids. Relevant features Strain or plasmid Source or reference Strains: HN280 MC4100 HN4104 TP504 E. coli EIEC O135; contains virulence plasmid pHN280; invasive Zagaglia et al., 1991 E. coli K12 F- araD139 zl(argF-lac) U169 rpsL150 relAl flbB5301 Casadaban, 1980 deoCl pstF25 rbsR A(hns tdk adhE opABCD) 118 zch-506::TnlO derivative of MC4100; Tc r Colonna et al., 1995 E. coli KI2 F- leuB6 serBl203 thi I zch-506::TnlO zdd-230::Tn9 Bertin et al., 1994 A(hns tdk adhE opABCD) 118; Tc r, Cm r Plasmids : virulence plasmid of strain HN280 pHN280 pMYSH6504 pMYSH6001 replicon vector (ori-ColEl) carrying the virF gene of S. flexneri 2a virulence plasmid pMYSH6000; Ap r, Km r pMYSH6520 pSCl01-derivative replicon vector carrying the virF gene of S. flexneri 2a virulence plasmid pMYSH6000; Ap r, Km r pBR322 replicon vector carrying the lac operon; Apr pMC1403 pMCI403 replicon vector containing virF-lacZ gene fusion; Ap r pFBlac2 Zagaglia et al., 1991 Sakai et aL, 1986 Sakai et aL, 1988 C~sadaban, 1980 This study Tot=tetracycline-resistant; Cmr=chloramphenicol-resistant; Apr= ampicillin-resistant ; Km TM kanamycine-resistant. introduction of an hns deletion (strain hN4104) also induces, increased temperature-independent expression of the reporter gene at pH 6.0. Interestingly, the pH 6.0 levels of 13-galactosidase at 30°C and at 37°C (6097 and 7841 Miller units, respectively) are in close agreement with those observed at pH 7.0 (7835 and 7817 Miller units). These results clearly indicate that the absence of a functional hns gene relieves, albeit not completely, the pH 6-mediated repression of virF, suggesting that H-NS might be responsible for the pH-dependent modulation of virF expression. DISCUSSION Among bacterial histone-like proteins, H-NS plays not only a purely structural role in the organization of the chromosome, but also a rather dynamic role in the regulation of gene expression in response to environmental stimuli: it is by far the most important modulator of virulence gene expression in pathogenic bacteria (reviewed by Atlung and lngmer, 1997). Shigella and EIEC cause diseases in humans by a similar and complex mechanism, which relies on the expression of genes located on the chromosome as well as on a large virulence plasmid (plNV) (Hale, 1991). The primary activator of the regulatory cascade leading to the pathogenic phenotype is the VirF protein (Sakai et al., 1988), which belongs to the AraC family of transcriptional activators (Dorman, 1992). In this paper, we show that the expression of the virF gene of Shigella and EIEC is negatively regulated by H-NS. In particular, H-NS represses the virF gene under specific conditions: low temperature or low pH. It has been proposed that H-NS might exert its efli~ct on gene expression directly, by binding to the DNA as a silencer of extended chromosomal regions (Goransson et al., 1990) or as a repressor of specific promoters (Ueguchi and Mizuno, 1993). An alternative model envisages an indirect involvement of H-NS through its effect on ~s (Barth et al., 1995) or on DNA supercoiling (Hulton et al., 1990). The gel retardation experiments we have carried out (fig. 1) show that the purified H-NS protein binds specifically to the virF promoter, strongly suggesting that H-NS acts directly as a virF repressor. It is known that H-NS thermoregulates two other plNV-located genes, virB and virG, by repressing their expression at 30°C (Maurelli and 22 G. PROSSEDA ET AL. hns + hns1 2 hns + hns3 4 a virF 30°C 37°C b strains, the expression of the virG gene is increased and temperature-independent. We show (fig. 1, right panels) that H-NS binds to the virG promoter with an affinity about two-fold higher than that for the virB promoter (fig. lb, right panel). The pronounced bent structure of the virG promoter (fig. 2a, right panel) may account for this observation. Intrinsic DNA curvature may be important in determining the architecture of nucleoprotein complexes and in facilitating looping between c i s - a c t i n g regulatory elements (Harrington, 1992). Histone-like proteins preferentially recognize bent DNA (Drlica and Rouviere-Yaniv, 1987) or induce bending (Perez-Martin et al., 1994). H-NS is known to bind DNA with a rather strong preference for curved regions (Yamada et al., 1991). The results we obtained by testing the virF, virB and virG promoter regions for the presence of intrinsically bent tracts show that in gels run at 60°C (fig. 2a, left panel), when the electrophoretic mobility is affected almost exclusively by size, the three fragments migrate according to their length. In gels run at 4°C (fig. 2a, right panel), when migration is also strongly dependent on conformation, the three fragments show a significantly lower mobility. The assay indicates that the virF and virB fragments exhibit a moderate curvature (about 11% increase in apparent size) with respect to the virG fragment (about 31% increase in apparent size). Fig. 3, In vivo expression of the virF gene. (a) Northern hybridization of total RNA extracted from MC4100 (/ms+) and HNI404 (hns118) harbouring pMYSH6504, grown at 30 ° and 37°C. Each lane was loaded with 10 Ixg of total RNA. The blot was hybridized with a 800 bp BsoH fragment of pMYSH6520 as a virF probe, (b) Densitometric tracings of the autoradiogram shown in panel a. Sansonetti, 1988; Dagberg and Uhlin, 1992; Lett et al., 1989). The virB promoter has been shown to bind H-NS specifically (Tobe et al., 1993). Our gel retardation assays (fig. l, left and centre panels) indicate that the affinity of H-NS for the virB promoter is about as high as that for the virF promoter. Genetic studies CLe~ et al., 1989; Dagberg and Uhlin, 1992) indicate that in hns-defective T a b l e II, pH and temperature-dependent expression o f virF-lacZ. Strains ~-galactosidase activityta) pH6 pH7 30°C 37°C 30°C 37°C MC4100 pMCI403 HM4104 pMC 1403 MC4100 pFBlac2(b) HN4104pFBIac2 (b) 3 2 421 6097 1 2 1594 7841 2 1 965 7835 1 2 3477 7817 (a) Units of ~-galactosidase are calculated according to Miller (Miller, 1992). The results are the average of at least four independent experiments. (b) The plasmid copy number (determined as described in "Materials and Methods") does not exhibit significant changes between the wild type (MC4 ! 00) and the hns mutant (HN4 i 04). H-NS REGULATION OF virF E gPRESSION To understand whether the binding of H-NS to the virF promoter (fig. 1) affects the expression of virE we determined the level of virF mRNA in E. coli KI2 MC4100 and in its h n s l l 8 deletion derivative HN4104 harbouring the recombinant plasmid pMYSH6504. The results of the Northem experiments carried out with RNA extracted from strains grown at 30 ° and 37°C (fig. 3) indicate that in MC4100, the virF mRNA level is temperature-regulated, in agreement with previous observations in Shigella (Tobe et al., !991) and in EIEC (Colonna et al., 1995), and that hns inactivation results in a significant increase in the virF mRNA, in particular at non-permissive ternperatures (30°C). This observation is paralleled by the 13-galactosidase activity of a virF-lacZ translational fusion (carried by plasmid pFBiac2) introduced into the wild type and into the H-NSdepleted strain (table II): hns inactivation has a significantly stronger effect at 30°C than at 37°C. These data indicate that H-NS plays a role in the thermoregulation of virE The involvement of HNS in the thermoregulation of transcription appears to be a common feature in pathogenic bacteria: other transcriptional activators of virulence operons, e.g. claD, p a p l and virB, are known to be repressed by H-NS at low temperature (Jordi et al., 1992; Gorangson et al., 1990; Tobe et al., 1993). Regulation by extracellular pH has been reported for many bacterial genes (Olson, 1993). Though not all pH-regulated loci so far described are affected by hns mutations, for some of them an involvement of H-NS has been observed (Hulton et al., 1990). Using the virF-lacZ fusion carried by plasmid pFBlac2, in the wild type strain (E. coli KI2 MC4100), we observe a reduction in virF expression at low pH, both at 30°C and at 37°C (table II). This is in good agreement with previous observations on the pH-dependent expression of virF in S. sonnei (Nakayama and Watanabe, 1995). The introduction of plasmid pFBlac2, carrying a virF-lacZ fusion, into an hnsdefective E. coli strain (HN4104) not only allows deregulated expression of virF at 30°C (in agreement with the results from Northern assays; fig. 3) but also at low pH (table II). This indicates that the pH-dependent expression of virF is also linked to H-NS control. 23 It has recently been reported (Nakayama and Watanabe, 1995) that the repression of virF is relieved at low pH in the absence of the CpxA protein, a sensor of two-component signal transduction systems. Here we report that in hns mutants, virF expression becomes insensitive to limited pH changes (table II). Taking account of these data, the expression of the virF gene appears to be under the control of at least two housekeeping proteins, H-NS and CpxA. In this context, it will be interesting to compare the expression of virF in tms/cpxA-defective strains under varying pH and temperature conditions. Although further experiments are needed to precisely locate the sites at which H-NS specifically interacts with the virF promoter region at 30°C or at pH 6.0, the present study clearly shows the key role of H-NS in modulating virulence gene expression in response to different environmental conditions that enteroinvasive microrganisms experience outside the host or in the different stages of the infectious process. Acknowledgments We thank C. Sasakawa for plasmid pMYSH6504 and pMYSIt6520; M.J. Casadaban for plasmid pMCI403 and P. Lejeune for strain TP504. We are grateful to A. Coppo and C.O. Gualerzi for suggestions and critical reading of the manuscript. This work was supported by grants from CNR (Consiglio Nazionale delle Ricerche), from MURST (Min.Universit.~ e Ricerca Scientifica e Tecnologica), from the University Rome La Sapienza (Grandi Progetti d'Ateneo 1996) and in part from the Foundation "lstituto Pasteur-Fondazione Cenci Bolognettr'. H-NS joue un r61e dans la r6gulation du g~ne virF de Shigella et de Escherichia coil ent~roinvasif Nous avons 6tudi6 le r61e jou6 par H-NS, un des composants majeurs du nucl6o'fde bact6rien, dans l'expression du g~ne virF, en r6ponse h diff~rentes conditions exp6rimentales. Le g~ne virF est localis6 sur le grand plasmide de virulence de Shigella et de Escherichia coli ent6roinvasif. La prot6ine VirF est une prot6ine du m~me type que AraC et elle active au moins deux promoteurs, virB et virG, tous deux 24 G. P R O S S E D A E T AL. rtprimts par H-NS. Des exptriences de retard sur gel de polyacrylamide nous ont permis de montrer que H-NS prtsente des affinitts comparables pour les promoteurs virF et virB et une affinit6 plus 61ev t e pour le promoteur virG. L ' a n a l y s e par 61ectrophor~se en gel de polyacrylamide de fragments d ' A D N contenant les promoteurs virF, virB et virG, dtmontre, en accord avec les prtdictions par ordinateur, que ces r t g i o n s p r t s e n t e n t une c o u r b u r e intrins~que. Cette observation confirme la p r t f t rence de H-NS pour I'ADN courbt. Des ,analyses de transcription in vivo du g~ne virF montrent que H-NS contrtle ntgativement l'expression de ce g~ne 30°C, L'expression d'une fusion traductionnelle virF-lacZ a 6t6 6tudite chez E. coli de type sauvage et chez un mutant dtfectif pour H-NS, h 30 ° ou 37°C et ~ pH 6,0 ou 7.0. Les rtsultats obtenus indiquent qu'en absence d'H-NS I'expression de virF devient insensible '~ la temptrature et "~ des changements limit,Ss de pH. Nos rtsultats sugg~rent fo~tement que H-NS contrtle l'expression de virF en se liant au promoteur de ce g~ne, et en rtprimant son expression ~ basse temptrature et bas pH. 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