Thermoregulation of Shigella and Escherichia coli EIEC pathogenicity. A temperature-dependent structural transition of DNA modulates accessibility of virF promoter to transcriptional repressor H-NS
1998; Springer Nature; Volume: 17; Issue: 23 Linguagem: Inglês
10.1093/emboj/17.23.7033
ISSN1460-2075
AutoresMaurizio Falconi, Bianca Colonna, Gianni Prosseda, Gioacchino Micheli, Claudio O. Gualerzi,
Tópico(s)Viral gastroenteritis research and epidemiology
ResumoArticle1 December 1998free access Thermoregulation of Shigella and Escherichia coli EIEC pathogenicity. A temperature-dependent structural transition of DNA modulates accessibility of virF promoter to transcriptional repressor H-NS Maurizio Falconi Maurizio Falconi Laboratorio di Genetica, Dipartimento di Biologia MCA, Università di Camerino, 62032 Camerino (MC), Italy Search for more papers by this author Bianca Colonna Bianca Colonna Dipartimento di Biologia Cellulare e Sviluppo, Università di Roma La Sapienza, 00185 Roma, Italy Search for more papers by this author Gianni Prosseda Gianni Prosseda Dipartimento di Biologia Cellulare e Sviluppo, Università di Roma La Sapienza, 00185 Roma, Italy Search for more papers by this author Gioacchino Micheli Gioacchino Micheli Centro Acidi Nucleici C.N.R., 00185 Roma, Italy Search for more papers by this author Claudio O. Gualerzi Corresponding Author Claudio O. Gualerzi Laboratorio di Genetica, Dipartimento di Biologia MCA, Università di Camerino, 62032 Camerino (MC), Italy Search for more papers by this author Maurizio Falconi Maurizio Falconi Laboratorio di Genetica, Dipartimento di Biologia MCA, Università di Camerino, 62032 Camerino (MC), Italy Search for more papers by this author Bianca Colonna Bianca Colonna Dipartimento di Biologia Cellulare e Sviluppo, Università di Roma La Sapienza, 00185 Roma, Italy Search for more papers by this author Gianni Prosseda Gianni Prosseda Dipartimento di Biologia Cellulare e Sviluppo, Università di Roma La Sapienza, 00185 Roma, Italy Search for more papers by this author Gioacchino Micheli Gioacchino Micheli Centro Acidi Nucleici C.N.R., 00185 Roma, Italy Search for more papers by this author Claudio O. Gualerzi Corresponding Author Claudio O. Gualerzi Laboratorio di Genetica, Dipartimento di Biologia MCA, Università di Camerino, 62032 Camerino (MC), Italy Search for more papers by this author Author Information Maurizio Falconi1, Bianca Colonna2, Gianni Prosseda2, Gioacchino Micheli3 and Claudio O. Gualerzi 1 1Laboratorio di Genetica, Dipartimento di Biologia MCA, Università di Camerino, 62032 Camerino (MC), Italy 2Dipartimento di Biologia Cellulare e Sviluppo, Università di Roma La Sapienza, 00185 Roma, Italy 3Centro Acidi Nucleici C.N.R., 00185 Roma, Italy ‡M.Falconi and B.Colonna contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:7033-7043https://doi.org/10.1093/emboj/17.23.7033 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The expression of plasmid-borne virF of Shigella encoding a transcriptional regulator of the AraC family, is required to initiate a cascade of events resulting in activation of several operons encoding invasion functions. H-NS, one of the main nucleoid-associated proteins, controls the temperature-dependent expression of the virulence genes by repressing the in vivo transcription of virF only below a critical temperature (∼32°C). This temperature-dependent transcriptional regulation has been reproduced in vitro and the targets of H-NS on the virF promoter were identified as two sites centred around −250 and −1 separated by an intrinsic DNA curvature. H-NS bound cooperatively to these two sites below 32°C, but not at 37°C. DNA supercoiling within the virF promoter region did not influence H-NS binding but was necessary for the H-NS-mediated transcriptional repression. Electrophoretic analysis between 4 and 60°C showed that the virF promoter fragment, comprising the two H-NS sites, undergoes a specific and temperature-dependent conformational transition at ∼32°C. Our results suggest that this modification of the DNA target may modulate a cooperative interaction between H-NS molecules bound at two distant sites in the virF promoter region and thus represents the physical basis for the H-NS-dependent thermoregulation of virulence gene expression. Introduction Bacteria entering the human host encounter an increase of their growth temperature to 37°C; this environmental change represents a cue that can trigger virulence gene expression in human pathogens such as Shigella species and enteroinvasive Escherichia coli (EIEC) (Maurelli and Sansonetti, 1988). These microorganisms cause disease by a similar, complex mechanism of pathogenicity which depends on the expression of chromosomal or plasmid-borne virulence genes (Sansonetti et al., 1982; Finlay and Falkow, 1997). The virulence genes carried by pINV are organized in regulons coordinately regulated by a central modulator, VirR (Maurelli and Sansonetti, 1988), corresponding to the major nucleoid protein H-NS (Lammi et al., 1984; Spassky et al., 1984; Pon et al., 1988), which represses their expression at 30°C or at low osmolarity (Tobe et al., 1991, 1993; Dagberg and Uhlin, 1992; Porter and Dorman, 1994). In addition to chromosome-encoded H-NS (VirR), two pINV-encoded activators, the proteins VirF and VirB, are involved in the transcriptional control of the invasion genes (Adler et al., 1989). In a cascade model, VirF activates transcription of the gene coding for the secondary regulator VirB which, in turn, activates several operons encoding the invasion genes. The activation of virB transcription by VirF is highly sensitive to changes in DNA topology and is antagonized at 30°C by H-NS (Tobe et al., 1993, 1995). Thus, the cellular level of VirF is crucial for the expression of the invasive phenotype of Shigella and E.coli EIEC and, while the expression of virF is not constitutive at 30°C, it is induced at 37°C (Tobe et al., 1991; Colonna et al., 1995) and may also be regulated at the post-transcriptional level through tRNA modifications (Durand et al., 1994, 1997). Furthermore, virF inactivation or hyperexpression results in a complete loss of the invasive phenotype or in the induction of invasiveness at the non-permissive temperature, respectively. More recently, it has been shown that a constitutively higher level of virF mRNA and β-galactosidase expressed from a virF–lacZ fusion can be found in cells with an hns-defective background (Prosseda et al., 1998). These data suggested that, as in the case of other virulence-regulating genes such as cfaD (Jordi et al., 1992), virB (Tobe et al., 1993) and papI (Forsman et al., 1992), virF expression might also be negatively controlled by H-NS in a temperature-dependent manner. This premise was supported further by data suggesting that this protein may interact with the promoter region of virF (Prosseda et al., 1998). H-NS is known to affect, primarily at the transcriptional level, the expression of a fairly large number of genes (reviewed in Atlung and Ingmer, 1997), and although the molecular basis of its regulatory activity probably rests on its preferential interaction with intrinsically curved DNA (Yamada et al., 1990, 1991; Tanaka et al., 1991), its ability to induce bending of non-curved DNA (Spurio et al., 1997) and its ability to induce negative supercoiling (Tupper et al., 1994), different mechanisms of repression have been proposed (Higgins et al., 1988; Goransson et al., 1990; Hulton et al., 1990; Owen-Hughes et al., 1992; Falconi et al., 1993; Ueguchi and Mizuno, 1993; Tupper et al., 1994; Barth et al., 1995). To explain how H-NS may regulate gene expression in a temperature-dependent manner is even more intriguing. In fact, while induction of gene expression by a temperature increase is not restricted to the pathogenicity genes, but also concerns the heat shock regulon, the participation of a DNA-binding protein such as H-NS in thermoregulation is a unique characteristic of the virulence genes. Furthermore, unlike the heat response, which entails a temperature increase above the normal growth temperature and which produces a transient expression of the genes belonging to the regulon, temperature-dependent induction of the vir regulon in Shigella involves a temperature shift within the normal range of growth temperatures and the stable activation of the regulon. Thus, it is clear that temperature control over virulence expression operated by H-NS entails a unique mechanism, clearly different from that responsible for the activation of the heat shock regulon. To explain the thermoregulation in Shigella pathogenicity expression, it is commonly assumed that the temperature control acts via the plasmid-encoded VirF and VirB proteins and involves an H-NS and/or environmentally induced alterations in DNA supercoiling. In fact, it has been reported that H-NS can alter DNA topology constraining negative supercoiling (Tupper et al., 1994). Furthermore, it is known that changes in DNA supercoiling can occur in response to the same environmental factors (e.g. temperature, osmolarity and phase of growth) which influence the expression of both Salmonella and Shigella virulence genes (Galan and Sansonetti, 1996). More specifically, since VirF can bind to virB in the absence of a thermal signal, it has been postulated that H-NS antagonizes this transcriptional activator only at low temperature via changes in topology (Tobe et al., 1993, 1995). A similar interpretation has been offered for thermoregulation of virulence mediated by histone-like proteins in Yersinia (Mikulskis and Cornelis, 1994). However, since the effect of hns mutations on the supercoiling of reporter plasmids is not straightforward, being sometimes negligible (Kawula and Orndorff, 1991; Yasuzawa et al., 1992) and, in other cases, of opposite sign in different types of bacteria (Dorman et al., 1990), alternative explanations have also been formulated. It has been proposed, for instance, that thermoregulation by H-NS could be mediated via the bacterial translational machinery which would produce higher levels of this protein at 30°C compared with 37°C. In turn, this increased concentration of H-NS could cause an increased competition with VirF for binding to virB promoter (Tobe et al., 1993) or a change in the H-NS quaternary structure favouring its specific (inhibitory) interaction with the upstream region of virB (Hromockyj et al., 1992). The recent data suggesting that H-NS may also directly control the expression of virF in a temperature-dependent manner opens up new perspectives for the elucidation of the mechanism responsible for thermoregulation of pathogenicity in Enterobacteriaceae. Thus, in the present study we have undertaken the task of establishing whether H-NS indeed plays a role in the temperature-dependent control of virF expression and of defining the molecular basis of this thermoregulation. By in vitro and in vivo footprinting, we have demonstrated that H-NS binds, at 30°C but not at 37°C, to two sites in the upstream region of virF, one overlapping the −35 and −10 promoter elements. Furthermore, we have demonstrated that interaction at this site is sufficient for transcriptional repression of virF by H-NS but that both sites are necessary for efficient thermoregulation. Analysis at different temperatures of the intrinsic DNA curvature of fragments containing different segments of the virF promoter showed that this parameter may cause a structural modification of DNA within the virF regulatory region, which controls the accessibility of the DNA target to H-NS. Results Temperature- and H-NS-dependent activity of the virF promoter in vivo As mentioned above, previous data had shown that the in vivo expression of virF present on a recombinant plasmid is influenced by temperature and pH in wild-type but not in hns-defective E.coli strains. Furthermore, purified H-NS was found to interact with the virF promoter, at least as judged by electrophoretic gel-shift experiments (Prosseda et al., 1998). The affinity of H-NS for the virF promoter was estimated to be similar to that displayed for virB, whose interaction with H-NS is well documented (Tobe et al., 1993) and was attributed to an intrinsic curvature predicted in this DNA. These results suggested that virF expression is under the control of H-NS which represses transcription by binding to its promoter at low pH and low temperature (Prosseda et al., 1998). To confirm this hypothesis and, more importantly, to elucidate the molecular basis of a possible H-NS-dependent thermoregulation of virF expression, we sought to characterize, both in vivo and in vitro, the nature, conditions and functional consequences of the interaction between H-NS and virF. To this end, the constructs and the DNA fragments schematically presented in Figure 1 were prepared and transformed into E.coli cells (either wild-type or carrying two different hns alleles). As seen in Table I, the level of β-galactosidase activity expressed in vivo from the virF–lacZ fusion carried by pFABlac112 (Figure 1) was found to be 4–5 times lower at 30°C compared with 37°C in the two wild-type strains (MC4100 and HN4122), while it was essentially the same at 30 and 37°C in the two hns null alleles. Figure 1.Schematic representation of the constructs used in this study. Plasmid pMYSH6504 is a pBR322 derivative containing the entire Shigella flexneri virF gene including the promoter and an extended upstream region (from −289), while pFB41 is derived from pMYSH6504 which contains a shorter virF promoter region (from −135). pFABlac112 and pFBlac2 were obtained by fusing fragments A + B (−345 to + 104) and B (−135 to +104) of virF, respectively, to the promoterless lacZ gene in pMC1403 (see Materials and methods). Download figure Download PowerPoint Table 1. Temperature-dependent expression of virF–lacZ fusions Strains/plasmids β-Galactosidase activitya Expressed at 30°C 37°C Expression ratio (37/30°C) MC4100 (wt)/pFABlac112 833 (±15%) 4180 (±10%) 5.02 HN4104 (hns118)/pFABlac112 7537 (±8%) 5781 (±9%) 0.77 HN4122 (wt)/pFABlac112 542 (±18%) 2162 (±7%) 3.99 HN4124 (hns2)/pFABlac112 2005 (±1%) 1970 (±1%) 0.98 HN4122 (wt)/pFBlac2 1051 (±9%) 1703 (±10%) 1.62 HN4124 (hns2)/pFBlac2 1929 (±6%) 1918 (±4%) 0.99 aUnits of β-galactosidase are calculated according to Miller (1992). The results represent the average of at least four independent experiments, and standard deviation values expressed as a percentage are reported in parentheses. The plasmid copy number, determined as previously described (Prosseda et al., 1998), does not exhibit a significant difference between wild-type and hns mutants strains. When pFABlac112 was replaced by pFBlac2, which carries a shorter upstream fragment of virF (Figure 1), repression of lacZ at 30°C (compared with its expression at 37°C) was <2-fold and, again, seen only in the wild-type hns background (Table I). These data confirmed and extended the previous observation that in vivo H-NS may control virF expression in a temperature-dependent manner (Prosseda et al., 1998). Binding of H-NS to the virF promoter region is temperature dependent The above result prompted us to map the binding sites of H-NS on the virF promoter by performing in vitro [DNase I and dimethyl sulfate (DMS)] and in vivo (DMS) footprinting experiments at different temperatures in the presence and absence of H-NS. DNase I footprinting carried out on supercoiled plasmid (pMYSH6504) DNA containing the whole virF gene (Figure 1) revealed the existence of two fairly extended sites (sites I and II) which were protected preferentially by H-NS when the DNA target and the purified protein were incubated at 28°C. Although the precise borders of these sites are not very sharp, it is relevant that there is a long (160 bp) non-protected fragment between site I and site II. Site I spans approximately from −46 to +46 on one filament and from −54 to +33 on the other (Figure 2A and B) and site II from −278 to −234 on one filament and −274 to −218 on the other (Figure 2C and D). Within both sites, a few DNase I-hypersensitive diester bonds (indicated by the arrows in Figure 2B and C) are visible. In contrast to the results obtained at 28°C (Figure 2A–D), very little or no protection by H-NS was observed when identical footprinting experiments were carried out at 37°C; in fact, within the limits of accuracy of this type of analysis, we cannot detect any significant difference in the DNase I digestion patterns obtained at 37°C in the absence and presence of up to 500 nM H-NS in the regions of H-NS site I (not shown) and site II (Figure 2E). These results indicate that the interaction of H-NS with these two DNA targets is strongly affected by a moderate (10°C) temperature variation. Figure 2.DNase I fooprinting of the virF promoter region by H-NS. Plasmid pMYSH6504 was incubated at 28°C (A–D) or at 37°C (E) with or without the indicated concentrations of H-NS expressed as nM dimer; the samples were processed as described in Materials and methods using BX8 (A), F322 (B), F321(C) and FO1 (D and E) as primers. Lanes G, A, T and C represent TaqI polymerase sequencing reactions using the same primers. The H-NS-protected sites are indicated with vertical broken lines and labelled I and II, the sites hypersensitive to DNase I are indicated by horizontal arrows. Download figure Download PowerPoint The interaction of H-NS with the virF promoter region was also investigated in vivo analysing the DMS modification on plasmid pMYSH6504 (Figure 1) in E.coli YK4122 (wt) and YK4124 (hns2) at 37 and 28°C. As seen in Figure 3A and B, there is a substantial difference between the in vivo footprinting patterns obtained at 37°C and at 30°C in the strain containing wild-type H-NS; two stretches of DNA with several bases, mostly G residues whose positions are indicated as C on the complementary strand, are substantially less accessible to DMS at the lower temperature. On the contrary, in the hns− background, there is no difference in the DMS reaction patterns obtained at 37 and 30°C and there is essentially no difference in the reactivity patterns of these DNA samples and the DNA sample extracted from YK4122 (wt) cells grown at 37°C (cf. the appropriate lanes of Figure 3A and B), taking into account that slightly less total DNA was loaded in the case of wild-type lanes. Since the two strains are isogenic except for the presence or absence of a functional hns gene and since the regions of reduced reactivity with DMS at 30°C closely correspond to the two H-NS-binding sites identified by DNase I footprinting in vitro, these results suggest that in vivo there is a temperature-dependent contact between these DNA segments and H-NS. Figure 3.In vivo and in vitro DMS probing of the virF promoter region. In vivo DMS footprinting of pMYSH6504 (A and B) was carried out as described in Materials and methods using E.coli YK4122 (wt) and YK4124 (hns2) cells growing at 30 or 37°C as indicated. In vitro DMS footprinting was carried out, as described in Materials and methods, at 37 and 28°C, on supercoiled plasmid DNA pMYSH6504 (C and D) and pFB41 (E), in the absence or presence of the indicated concentrations of H-NS (dimer). For the primer extension by TaqI polymerase of the DNA partially cleaved at G and A residues, the 32P-end-labelled primers used were: FO1 (A and D) and BX8 (B, C and E). Lanes A, T and C represent TaqI polymerase sequencing reactions using the same primers. Download figure Download PowerPoint To confirm this premise, DMS footprinting experiments were also performed in vitro in the presence and absence of H-NS (Figure 3C and D). As seen in the figure, most of the bands of site I (i.e. −16, +4, +15, +23, +39, +44, +52 and +55) and of site II (i.e. −249, −250, −254, −270, −274, −275, −277 and −278) which displayed a notable intensity decrease in the DMS probing in vivo are also strongly protected from chemical attack in vitro at 28°C (cf. Figure 3A and D for site II, and Figure 3B and C for site I). In vitro H-NS protection of both sites was again observed only at low (28°C) but not at high (37°C) temperature. Since DMS is known to methylate guanine residues at the N-7 position in the major groove of DNA (Borowiec and Gralla, 1986), the shielding effect of H-NS is compatible with the premise that this protein interacts with the major groove of DNA, as suggested by Tippner et al. (1994). Taken together, these experiments demonstrate that the temperature-dependent protection of sites I and II of virF detected in vivo in a wild-type hns background is due to the interaction of these sites with H-NS without the involvement of any other cellular protein. The localization of H-NS-binding sites, identified by the different footprinting techniques used in this study, is summarized in Figures 4 and 5. As seen in Figure 4, there is excellent correspondence between the sites protected by H-NS from DNase I digestion on both filaments and DMS reaction in vitro at 28°C and, in turn, between these sites and those protected in vivo by H-NS at the same temperature. Figure 4.H-NS-binding sites within the virF promoter region. The H-NS-binding sites within the nucleotide sequence (−289 to +67) of the virF promoter region are indicated by broken lines (protection from DNase I), by bold letters (protection from DMS in vivo) and by diamonds (protection from DMS in vitro). The shaded sequences indicate (from bottom right): the translation initiation triplet, the transcriptional start site and the −10 and −35 promoter consensus sequences. The continuous line indicates the predicted DNA curvature. Download figure Download PowerPoint Figure 5.Computer-generated prediction of intrinsic curvature in the virF promoter region. The fragments A (light grey) and B (dark grey) are described in the legend to Figure 1. The transcription start site is indicated by the white arrow. The H-NS-binding sites I and II are positioned according to the footprinting experiments summarized in Figure 4. Download figure Download PowerPoint The dimensions of the two sites occupied by H-NS at low temperature and the finding that H-NS has similar affinities for them (as seen in Figure 2A–D, they are protected similarly by comparable amounts of protein) suggest that both sites might be occupied simultaneously and somewhat cooperatively by several H-NS molecules. Moreover, the two sites are not entirely independent of each other, since DMS footprinting carried out on plasmid pFB41, which unlike pMYSH6504 lacks the promoter-distal H-NS site (Figure 1), revealed only a very weak protection in the region of H-NS-binding site I (cf. Figure 3C and E). These results suggest the existence of long-range protein–protein interactions among H-NS molecules occupying sites I and II of virF. These interactions could be favoured by the intrinsic DNA curvature predicted between the two H-NS-binding sites (Figure 5) which may contribute to bringing into close proximity these sites which are ∼160 bp apart (Wang and Giaever, 1988; Travers, 1989, 1995). Finally, for the interpretation of functional data (see below), it seems relevant to notice that the H-NS promoter-proximal site (site I) includes both −35 and −10 elements of the virF promoter. This H-NS-binding model is reminiscent of that proposed for the H-NS–hns interaction (Falconi et al., 1993) and consistent with the known preference of H-NS for binding curved DNA (Yamada et al., 1990, 1991; Owen-Hughes et al., 1992; Zuber et al., 1994) as well as with the essential role attributed to protein–protein interactions in the correct functioning of this DNA-binding protein (Spurio et al., 1997). In vitro transcription of virF is influenced by H-NS and temperature If H-NS alone is directly responsible for the thermoregulation of virF expression in vivo, as suggested by the results presented in Table I and by its temperature-dependent interaction with the virF promoter, it might be possible to reproduce this effect in a purified in vitro system. That this is indeed the case is shown by the results of experiments in which the virF promoter activity was examined in the presence and absence of H-NS as a function of temperature. Thus, the addition of purified H-NS was found to inhibit severely (∼80% up to 15 min incubation) transcription of virF at 30°C when supercoiled plasmid pMYSH6504 (Figure 1) was used as template (Figure 6A), while transcription at 37°C was only slightly affected by H-NS (Figure 6B). When the transcriptional activity of virF in the presence or absence of H-NS was measured as a function of temperature between 26 and 40°C, it was found that the extent of transcriptional inhibition caused by a given amount of H-NS was not constant and did not vary linearly as a function of the incubation temperature (Figure 6C). Instead, below 30°C, H-NS caused a 5-fold inhibition of virF transcription but had negligible effects on samples incubated above the critical temperature of 32°C (Figure 6C). The extent of the H-NS repression seen in this experiment at 30°C closely corresponds to that found in vivo at the same temperature (Table I). Figure 6.Effect of H-NS and temperature on the virF promoter activity in vitro. The figure presents the time course of in vitro transcription at 30°C (A and E) and 37°C (B and F) in the presence (●) or in the absence (○) of H-NS (150 nM of dimer). Transcription reactions were performed as described (Falconi et al.,1993) using supercoiled (A–C) and EcoRI-linearized (E and F) pMYSH6504 as DNA template. (C) The temperature dependence of the H-NS transcriptional repression of virF determined by monitoring transcription after 10 min of incubation with RNA polymerase at the indicated temperatures in the presence or absence of H-NS. (D) The levels of virF transcription at 30°C (□,▵) and 37°C (▪,▴) with supercoiled (S) and and EcoRI-linearized (L) pMYSH6504 as DNA template. The virF transcripts were detected by Northern analysis using the 32P-labelled 750 bp DNA fragment corresponding to the entire virF gene as probe; the signal was quantified by Molecular Imager (Bio-Rad Mod.GS-250). Download figure Download PowerPoint The relevance of DNA supercoiling for the inhibition of virF promoter function by H-NS was examined in the following experiment in which the plasmid template pMYSH6504 was linearized by EcoRI digestion prior to the transcription test. In a preliminary experiment, the activity of the virF promoter on a supercoiled and linearized (L) template was analysed at 30 and 37°C (Figure 6D). As seen from this panel, the supercoiled (S) is transcribed better than the linearized (L) template, and temperature has little and no influence on the activity of the linearized and supercoiled promoter, respectively. Unlike that seen with the supercoiled DNA template, H-NS did not inhibit transcription at either 30 or 37°C (Figure 6E and F), indicating that inhibition of virF transcription occurs only on supercoiled DNA at 30°C. The specific requirement for template supercoiling for H-NS inhibition is clearly indicated by the fact that the level of transcription of the supercoiled template at 30°C in the presence of H-NS is way below the level of transcription obtained at either 30 or 37°C on the linearized template in the presence of the same amount of H-NS (cf. Figure 6A, E and F). As seen above, the upstream region of virF contains two H-NS-binding sites designated I and II (Figure 5). Since site I overlaps the conserved elements of the virF promoter, it is reasonable to assume that this site is involved in transcriptional repression by H-NS whose interaction with DNA might occlude the access of RNA polymerase to the −35 and −10 consensus sequences. To determine whether the more upstream H-NS site II is also required for repression, a shorter construct (pFB41), which lacks ∼150 bp of the distal portion of the virF promoter region (Figure 1), was used as a transcriptional template. As seen from Figure 7, while essentially no inhibition of transcription by H-NS was seen at 37°C (Figure 7B), at 30°C this protein produced a significant decrease (85%) of virF transcription but only for very short incubation times (<4 min) while the inhibition was relieved with longer incubation, dropping to 50 and 25% after 6 and 15 min, respectively (Figure 7A). These data are in full agreement with the observation that in vivo there is only a minor difference in the transcriptional activity of virF between wild-type and hns2 strains transformed with the same pFBlac2 construct (Table I). Figure 7.Effect of H-NS on in vitro transcription from a virF promoter fragment lacking the upstream H-NS site II. Time course of in vitro transcription of supercoiled pFB41 DNA (Figure 1) at 30°C (A) and 37°C (B) in the presence (●) or absence (○) of 150 nM H-NS (dimer). The other experimental conditions are as described in the legend to Figure 6. Download figure Download PowerPoint Taken together, these transcription results and the aforementioned very weak H-NS footprints on site I in the absence of site II indicate that without the latter site, H-NS forms an unstable nucleoprotein complex which cannot withstand a prolonged competition with the RNA polymerase. Influence of temperature on DNA topology of the virF promoter Since superhelicity of pMYSH6504 was found to be essential for repression of the virF promoter activity by H-NS (Figure 6) and since the temperature may influence the supercoiling level of plasmids (Goldstein and Drlica, 1984; Lopez-Garcia and Forterre, 1997), the distribution of pMYSH6504 topoisomers extracted from wild-type (MC4100) and from two hns mutant strains (HN4104 and YK4124) grown in LB medium at 30 and 37°C was analysed. No detectable differences in the level of superhelicity were detected when the topoisomers of the plasmid isolated from wild-type or H-NS-def
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