Previous studies have established that the expression of Salmonella enterica pathogenicity island 1 (SPI1), which is essential for epithelial invasion, is mainly regulated by the HilD protein. The ferric uptake regulator, Fur, in turn modulates the expression of the S. enterica hilD gene, albeit through an unknown mechanism. Here we report that S. enterica Fur, in its metal-bound form, specifically binds to an AT-rich region (BoxA), located upstream of the hilD promoter (PhilD), at position -191 to -163 relative to the hilD transcription start site. Furthermore, in a PhilD variant with mutations in BoxA, PhilD*, Fur·Mn2+ binding is impaired. In vivo experiments using S. enterica strains carrying wild-type PhilD or the mutant variant PhilD* showed that Fur activates hilD expression, while in vitro experiments revealed that the Fur·Mn2+ protein is sufficient to increase hilD transcription. Together, these results present the first evidence that Fur·Mn2+, by binding to the upstream BoxA sequence, directly stimulates the expression of hilD in S. enterica.
Citation: Teixidó L, Carrasco B, Alonso JC, Barbé J, Campoy S (2011) Fur Activates the Expression of Salmonella enterica Pathogenicity Island 1 by Directly Interacting with the hilD Operator In Vivo and In Vitro. PLoS ONE 6(5): e19711. https://doi.org/10.1371/journal.pone.0019711
Editor: Michael Hensel, University of Osnabrueck, Germany
Received: January 20, 2011; Accepted: April 4, 2011; Published: May 6, 2011
Copyright: © 2011 Teixidó et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Ministerio de Ciencia e Innovación (MICIIN) [grants BFU2008-01078 to J.B. and BFU2009-07167 to J.C.A.]; and the Generalitat de Catalunya [2009SGR1106 to J.B.]. B.C. was the recipient of a Juan de la Cierva fellowship from the MICIIN. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Salmonella enterica is a bacterial pathogen that causes numerous diseases, ranging from gastroenteritis to systemic infections, in several hosts including humans. Moreover, it is one of the most important pathogens associated with food-borne illness worldwide , . An early step in the pathogenesis of non-typhoidal Salmonella species involves their ability to penetrate the intestinal epithelium. Invasion is mediated by the presence of a type III secretion system (T3SS), which is encoded on the tightly regulated Salmonella pathogenicity island 1 (SPI1) –. The main regulator of SPI1, HilA, directly activates the expression of the invF and prgH operons, which encode the components of the T3SS apparatus , . InvF facilitates the expression of several effector genes on SPI1 and elsewhere in the S. enterica genome –. In turn, hilA expression is under the control of other transcriptional activators, HilD and HilC, likewise encoded within the SPI1, and RtsA, located elsewhere in the chromosome –. These three transcription factors independently activate not only HilA expression but also each others' and their own, thus comprising a complex feed-forward regulatory loop . Moreover, HilC and HilD can directly activate invF independently of HilA .
T3SS expression is also modulated by several distinct environmental signals , , ensuring that the system is available by the time the bacterial pathogen reaches the distal small intestine, where invasion of the epithelial monolayer takes place . For example, it is well known that osmolarity and bile salt concentration influence T3SS production by controlling hilD expression through the EnvZ/OmpR and BarA/SirA two-component systems, respectively –. In addition, it has been shown that phosphate or Mg2+ and Ca2+ concentrations, sensed by the PhoR/PhoB and PhoP/PhoQ systems, respectively, are likewise involved in SPI1 regulation , . In both cases, the sensor system controls expression of the SPI1 repressor HilE, which is encoded by a gene located outside of SPI1. It has been suggested that the negative regulatory effects of HilE are exerted by its direct interaction with HilD, such that HilD-mediated activation of hilA is prevented . Another protein, H-NS, has also been described to control SPI1. This nucleic-acid-associated protein, binds to the promoter region of hilA, rtsA, hilD and hilC genes diminishing their expression , .
Iron concentration is also associated with T3SS expression . Fe2+ is essential for bacterial development – and S. enterica is confronted with different free-iron concentrations during its infectious process. Normally, free Fe2+ is scarce inside the host due its sequestration by several different cellular mechanisms , . However, in the lumen of the small intestine, where dietary iron is mainly absorbed , there is abundant free Fe2+. Accordingly, it has been reported that several of the Salmonella genes that are expressed when iron is scarce remain silent as long as the bacterium is confined the intestinal lumen . The Fe2+ concentration is thought to act as a signal that allows the pathogen to sense its location inside the host .
The Fur (ferric uptake regulator) protein is the main regulator of iron homeostasis in many bacteria. As a major regulator of gene expression, it not only controls genes involved in iron homeostasis but also ultimately coordinates intracellular iron levels with many other cellular processes . In transcriptional and translational gene fusions, Fur was shown to activate SPI1 expression by increasing the amount of HilD , . Recent studies described the ability of Fur to modulate hilA expression by negatively controlling the levels of the H-NS global regulator . In the presence of Fur·Fe2+, hns expression is repressed. The resulting decrease in the H-NS concentration reduces the repression that it exerts on the hilA promoter, thus allowing a rise in the expression of this gene , . Nevertheless, the previously described Fur·Fe2+-mediated activation of hilD expression remains unknown , .
In Escherichia coli, Fur exhibits Fe2+-dependent DNA-binding activity to a specific sequence, namely the Fur box, located in the promoter region of genes directly repressed by Fur . The Fur box is a 19-bp consensus sequence organized either as two inverted repeats separated by 1-bp, or as at least three contiguous hexamers, 5′-NATWAT-3′ (where N is any nucleotide and W is an A or a T), aligned in either a direct or an inverse orientation –. In the Fe2+-bound form, E. coli Fur represses genes involved in respiration, flagellar chemotaxis, the TCA cycle, glycolysis, methionine biosynthesis, phage DNA packaging, DNA synthesis, purine metabolism, and redox stress resistance –. Moreover, E. coli Fur has also an indirect positive effect on some genes by repressing the expression of the rhyB . The absence of RhyB for pairing at the ribosomal binding site of mRNAs of genes positively regulated by Fur prevents their degradation by subsequent recruitment of the RNA degradosome .
Fur has been characterized in several other bacterial species – and other Fur-regulated pathways not related with sRNA have been described . For instance, in Neisseria meningitidis, the Fur·Fe2+ complex has been shown to act directly as a transcriptional activator once it binds to the promoter region of several virulence-associated genes . Other Fur activation pathways have been reported in Pseudomonas aeruginosa, Yersinia pestis, Helicobacter pylori, and E. coli –. In S. enterica two sRNAs, RfrA and RfrB, have been identified. Both are homologous to E. coli RyhB and participate in the Fur-mediated positive control of genes such as sodB , . Nevertheless, neither RfrA nor RfrB mediates Fur control of hilD expression . Moreover, direct control by Fur of the SPI1 repressor, hilE, has been ruled out .
To understand the molecular mechanism(s) that modulate hilD expression by Fur·Fe2+, we analysed whether the Fur protein of S. enterica serovar Typhimurium directly controls hilD expression. Our results show that Fur protein, in its metal-bound form, binds to an AT-rich operator located upstream of the hilD promoter region (PhilD), and it acts directly as a transcriptional activator of hilD. These findings help to elucidate the role of iron in the regulation of SPI1 expression and provide the first evidence of a Fur-mediated direct activation mechanism in S. enterica.
Materials and Methods
Bacterial strains, plasmids and growth conditions
All bacterial strains and plasmids used in this work are listed in Table 1. Bacterial cultures were grown at 37°C in LB. When necessary, ampicillin (100 µg/ml), chloramphenicol (34 µg/ml), or kanamycin (150 µg/ml) was added to the bacterial culture. When needed 2,2-dipyrydil (DPD) was added to the medium at a concentration of 0.2 mM , .
In silico searches for Fur binding sites
The 337-bp PhilD spanning −247 to +90 (relative to the transcription start site ) was used for in silico searches with the Virtual Footprint online framework program . The searches were carried out using the pre-existing P. aeruginosa (16-mer) matrix .
S. enterica fur was PCR-amplified using suitable oligonucleotides (Table S1), cloned into the pET15b expression vector, and transformed into E. coli BL21(DE3) pLysE strain. The Fur protein was purified using the TalonTM Metal Affinity Resin Kit (Clontech), as reported , and eluted from the affinity column by thrombin cleavage in buffer A (50 mM Bis-Tris/borate pH 7.5, 5 mM MgCl2, 10% glycerol) containing 500 mM NaCl. Fur was then loaded onto a Q-Sepharose equilibrated with buffer A containing 100 mM NaCl and eluted with a 600–1000 mM NaCl gradient. S. enterica Fur, which is free of E. coli H-NS protein, is expressed as dimers. The activity of purified S. enterica Fur was confirmed by electrophoretic mobility shift assays (EMSAs), which tested the ability of the protein to bind the promoter region of a confirmed Fur-regulated gene, foxA . Figure S1 shows the Fur binding region in PfoxA and the EMSA results using purified Fur protein.
Electrophoretic mobility shift assays
Appropriate DNA probes were obtained by PCR using suitable DIG-labeled oligonucleotides (Table S1). Fur EMSAs were done as previously described, with slight modifications . In each case, 100 ng of each DIG-labeled DNA probe (20 nM) was incubated with increasing concentrations of Fur in buffer B (10 mM Bis-Tris/borate pH 7.5, 5% glycerol, 1 mM MgCl2, 40 mM KCl, 100 µg BSA/ml, 0.2 mg salmon sperm DNA/ml) with or without 100 µM MnCl2. For competitive assays, at least a 200-fold excess of either specific or non-specific non-labeled DNA was added. To assay the binding ability of Fur in its apo form, EDTA chelator was included in the binding mixture at a concentration of 1 mM. The binding mixture was incubated for 10 min at 37°C, after which the samples were separated by 5.5% polyacryamide gel electrophoresis (PAGE) in Bis-Tris buffer . The DIG-labeled DNA-protein complexes were detected by following the manufacturer's (Roche) protocol.
For the footprinting assay, the 375-bp [α-32P]-NcoI-HindII PhilD DNA (10 nM) from pUA1111 was incubated with increasing concentrations of Fur (1.5–100 nM) for 15 min at 37°C in buffer C (50 mM Bis-Tris/borate pH 7.5, 5% glycerol, 10 mM MgCl2, 1 mM MnCl2). DNase I digestion was carried out by addition of the enzyme in binding buffer containing 5 mM CaCl2 followed by incubation for 5 min at 37°C; the reactions were stopped by the addition of 25 mM EDTA. The samples were ethanol precipitated, resuspended in 6 µl of loading buffer, and fractionated on 6% denaturing (d) PAGE . As the molecular weight marker, a G + A sequence reaction  was carried out and run in parallel with the corresponding footprinting reactions.
Construction of S. enterica mutant derivatives
S. enterica UA1888 and UA1889 strains, containing a KanR cassette (inserted at position –192) and either wild-type (PhilD) or the Fur BoxA mutant variant (PhilD*), and the hilD knock-out mutant (UA1891) were constructed using the one-step PCR-based gene replacement method as described  and the appropriate oligonucleotides (Table S1). The PCR products were transformed in UA1875 carrying the pKOBEGA plasmid (Table 1). In the PhilD or PhilD* mutant derivative, transcription orientation of the kan gene was opposite that of the hilD gene, thus avoiding promoter interference. Indeed, the expression of neither hilD nor the downstream prgH genes was affected by the presence of the KanR cassette in UA1888, and the expression levels were the same as those obtained using the SV5015 wild-type strain. All constructs were transferred as described  into the SV5015 wild-type strain or the null fur mutant derivative (Δfur, UA1880) by transduction using the P22int7(HT) bacteriophage and the suitable constructed strain as donor. The absence of the prophage in the transductants was determined by streaking them onto green plates, as described previously . The obtained mutants were verified by PCR, using the appropriate primers (Table S1), and by nucleotide sequencing.
Quantitative RT-PCR assays
Quantitative real time RT-PCR (qRT-PCR) assays of hilD or foxA expression in different genetic backgrounds were carried out. To maximize the iron effect, bacteria were grown in LB medium, which contains saturated iron concentrations. When needed and to generate an iron-limiting environment DPD was added to the medium. All bacterial strains were overnight cultured and then diluted 1/100 in the appropriate media (with or without DPD) and incubated aerobically at 37°C. Once the bacterial culture reached OD550 = 0.8, the cells were harvested and then the RNA was extracted using the Quiagen RNeasy kit following the manufacturer's instructions. The qRT-PCR assays were performed as previously reported  using suitable oligonucleotides (Table S1). It should be noted that the hilD oligonucleotide pair is located upstream the KanR cassette insertion site in the hilD knock-out mutant (UA1891), thus allowing determination of the mRNA level in this genetic background. The results were normalized with respect to recA, a standard control gene not associated with the Fur regulon . A change in the recA expression pattern was not observed in any of the genetic backgrounds assayed in this study (see Figure S2, in which 16S RNA is used as the standard). The relative expression level was defined as the ratio between the expression of hilD or foxA in each mutant derivative and that observed in the UA1888 strain containing a wild-type upstream Fur promoter region (PhilD).
In vitro transcription assays
The PhilD upstream region, spanning −247 to +90 (relative to the transcription start site), with either a wild-type BoxA or BoxA* mutant variant was cloned into pGEM®-T, generating plasmids pUA1111 and pUA1112, respectively (Table 1). In vitro transcription assays (run-off transcription) were performed using 10 nM of HindII-cleaved pUA1111 (containing PhilD) or pUA1112 (PhilD*), or with pGEM®-T plasmid DNA (Pν) as an unrelated Fur control. The pUA1111 vector was also used as a supercoiled DNA RNAP template. All of the DNAs were pre-incubated with increasing concentrations of Fur protein (1.5–200 nM) for 15 min at 37°C in buffer D (50 mM Tris-HCl pH 7.5, 5% glycerol, 5 mM MgCl2. 1 mM MnCl2, 2 mM spermidine, 10 mM DTT) in a 25-µl reaction. One unit of E. coli RNAP Eσ70 holoenzyme (USB, Cleveland) and 0.5 mM of each rNTP (with [α-32P]-rUTP) were added. The reactions were incubated for 60 min at 37°C and then stopped by the addition of 15 µl of loading buffer followed by heating to 75°C for 10 min. The in vitro generated transcripts from the linear templates, which contained the cloned PhilD or PhilD*, the vector promoter (Pν), and the supercoiled pUA1111 vector, were separated in 6% dPAGE, visualized, and quantified as described .
Fur·Mn2+ binds PhilD DNA with high affinity
A search for putative Fur cognate sites in S. enterica SL1344 PhilD, spanning −247 to +90 (relative to the transcription start site ), was carried out using the Virtual Footprint online framework . Accordingly, two putative Fur boxes were predicted (Figure 1A): (i) BoxA, at position −191 to −163, corresponding to an AT-rich region (dG + dC content <15%, vs. 50% for the total genome) located ∼ 100-bp upstream of the HilD and HilC binding sites and (ii) BoxB, at position −48 to −30 (dG + dC content ∼31%), overlapping the promoter −35 element and situated ∼ 30-bp downstream from the HilD and HilC binding sites  (Figure 1A).
A. Upstream region (−247 to +90, FrgA) of the S. enterica SL1344 hilD gene. The putative Fur Boxes A (−191 to −163) and B (−48 to −30), identified through the Virtual Footprint framework program, are boxed in blue and gray, respectively, with the HilD and HilC binding region (−57 to −91 ) shown in green. The −35 and −10 promoter (PhilD) consensus regions and the +1 transcription start site  are underlined. The coding region of hilD is denoted in lower-case characters. B. EMSA of PhilD DNA (FrgA) (20 nM) in the presence of increasing concentrations (2.5, 12.5, 50, and 187 nM) of Fur protein in buffer B. The mobility of PhilD DNA in the absence of Fur protein is shown as a control (−). C. EMSA of the FrgA probe in the presence of the chelator EDTA or with no Mn2+ added to buffer B. The mobility of the PhilD DNA probe in buffer B containing Mn2+ in the absence or presence of 50 nM Fur protein is shown as a negative and positive control, respectively. D. EMSA of the FrgA probe in the presence or absence of non-labeled PhilD or pGEM®-T DNA used as a specific or non-specific competitor, respectively. The specificity of Fur binding was determined usig a 200-fold excess of the corresponding non-labeled DNA. The presence or absence of a component is indicated by + or −, respectively. FD, free PhilD DNA; PD, the protein-DNA complex.
To validate the function of these putative Fur binding sites, the S. enterica Fur protein was purified and EMSA studies were performed using the PhilD region (position −247 to +90) as probe (Figure 1B). All EMSAs were done in buffer containing Mn2+ as a common substitute of Fe2+ due to its greater stability under aerobic conditions , . As shown in Figure 1B and Figure S1, in the presence of Mn2+, Fur dimers bound with high specificity and affinity to PhilD, with an apparent binding constant (KDapp) of about 4.5±1.5 nM, defined as the protein concentration necessary to complex 50% of labeled DNA. In contrast, in the absence of Mn2+ or following the addition of EDTA, Fur was unable to form a complex with PhilD DNA (Figure 1C), suggesting its metal-dependent DNA-binding activity . In addition, the Fur-Mn2+ complex displayed a higher DNA affinity than Fur-Mg2+ (Figure S1). Together these observations suggest that Fur binds to the PhilD promoter with the same characteristics as those reported when it acts as a repressor .
To test the specificity of the reaction, competition experiments were performed. A large excess (200-fold) of non-specific DNA (pGEM®-T DNA) was unable to compete with PhilD for Fur·Mn2+ binding, whereas an excess of non-labeled PhilD DNA fully competed for binding with labeled PhilD DNA (Figure 1D). It is therefore likely that at least one Fur·Mn2+ binding site is present in PhilD, supporting the in silico predictions.
Fur·Mn2+ binds to BoxA DNA
To elucidate which of the two putative Fur boxes, or even both, binds Fur·Mn2+, serial deletions of the PhilD region were obtained, generating FrgB, FrgC, and FrgD (Figure 2A). As shown in Figure 2B, FrgD (spanning the −247 to −123 interval) but not FrgB (−170 to +90) or FrgC (−16 to +90) bound Fur·Mn2+ (Figure 2B). It is therefore likely that: (i) Fur·Mn2+ interacts with the region spanning −247 to −123 (containing the putative BoxA, −191 to −163), and (ii) Fur·Mn2+ interacts neither with the core PhilD region nor with the −91 to −57 interval, which includes the HilD and HilC binding sites , .
A. Diagram of the PhilD region. The putative Fur BoxA, located from −191 to −163, and BoxB, from −48 to −30, are framed in blue and gray, respectively. The HilC and HilD binding sites , are shown in green. The positions refer to the +1 transcription start site of the hilD gene . The hilD coding region is striped. B. EMSA experiments using FrgA (PhilD), FrgB, FrgC, and FrgD DNA probes (20 nM) from the PhilD region in the absence (−) or presence (+) of 50 nM Fur protein.
Fur·Mn2+ recognizes BoxA DNA and spreads to adjacent regions
DNAse I footprinting experiments were carried out to further determine the specific Fur·Mn2+ binding site in the PhilD region. At low Fur·Mn2+ concentrations (0.5 Fur dimers/PhilD DNA), only the −189 to −170 region, including BoxA, was protected from DNase I attack (Figure 3, lane 4). The BoxA site contains three copies of the hexameric 5′-NATWAT-3′ Fur consensus sequence separated from each other by 4-bp. Two hexamers (subsites I and III) are in the direct (→) orientation and one (subsite II) is in the inverse (←) orientation (→4-bp←4-bp→), conforming to a typical Fur box . At limiting Fur·Mn2+ concentrations, the protected sequence included subsites I and II spaced by 4-bp, but subsite III was poorly protected (see Figure 3). At sub-saturating and saturating Fur·Mn2+ concentrations (2.5∶1 to 10∶1 Fur·Mn2+:PhilD DNA ratios), an extended DNase I -protected interval (from −147 to −219) was observed (Figure 3, lanes 1–3). Sites hypersensitive to DNase I attack were not apparent, suggesting that upon Fur·Mn2+ binding no obvious major distortion of the DNA occurred. It is likely that, by cooperative interaction, Fur·Mn2+ showed limited spread onto the 5′ (∼ 30-bp) and 3′ (∼ 23-bp) regions of PhilD DNA (Figure 3). Fur·Mn2+, under the concentrations used, halted before reaching the HilD, HilC, RstA binding region (−91 to −57). Similar results were described in E. coli, when Fur·Mn2+ acts as a transcriptional repressor , , .
The 375-bp [α-32P]-NcoI-HindII PhilD (top strand) DNA (5 nM) was incubated with increasing Fur concentrations (1.5–100 nM). The positions are related to the transcription start site (+1) . The three BoxA subsites are boxed and enlarged, with arrows denoting their relative orientation. Abbreviations: −, absence of Fur; C, a G + A sequence ladder of the DNA probe was used as molecular size marker.
To test the contribution of the AT-rich upstream operator (BoxA) to Fur recognition of PhilD DNA, a mutant BoxA DNA (PhilD*) was constructed and then used in EMSA experiments (Figure 4). As expected, S. enterica Fur specifically recognized PhilD DNA with wild-type BoxA in the upstream region but failed to bind PhilD* carrying mutations in the three subsites of the Fur box region (Figure 4B).
A. Sequence of wild-type BoxA in PhilD and the mutated BoxA* variant in PhilD*. The three subsites of the Fur box and their relative orientation are also indicated. B. EMSA using 20 nM PhilD or PhilD* DNA and 50 nM Fur·Mn2+. The presence or absence of a component is indicated inside the table by + or −, respectively.
Fur activates hilD gene expression in vivo
To determine whether Fur increases PhilD utilization in vivo, the transcription level of hilD was assayed in several S. enterica strains. Wild-type PhilD and the PhilD* variant, each with an upstream KanR cassette, were integrated into their native locus, leading to strains UA1888 and UA1889, respectively (Table 1). It is worth noting that the mutations in BoxA* (PhilD*, Figure 4A) were located upstream of the HilD, HilC, RstA , and RNA polymerase (RNAP) binding sites (, Figure 1A). Also, the presence of the KanR cassette upstream of BoxA did not modify hilD expression, since the transcription level of this gene was similar to that obtained using the SV5015 wild-type strain (Figure 5).
Relative hilD mRNA were estimated by qRT-PCR in PhilD (UA1888), PhilD* (UA1889), Δfur PhilD (UA1890), or wild-type (SV5015) strains grown in high (LB) or low (DPD) iron concentration, as described in the Materials and Methods section. To demonstrate that PhilD utilization is not affected by the upstream KanR cassette, the hilD mRNA level in the wild-type SV5015 strain (wt) is also shown. The expression level of foxA, which is repressed by Fur , in all genetic backgrounds and conditions was determined as a control. For each condition, the relative gene expression levels were calculated as the ratio of each relative mRNA concentration with respect to that obtained in the isogenic wild-type strain (PhilD) and normalized to that of the S. enterica recA gene. The mean value from three independent experiments (each in triplicate) is shown.
The hilD mRNA levels were analyzed by qRT-PCR and normalized with respect to the recA gene, which is not associated with the Fur regulon . As a control, expression of the foxA gene, previously shown to be Fur repressed , was also measured under the same growth conditions. There was no evidence that the PhilD* mutation affected foxA expression (Figure 5). In the absence of Fur or when iron was scarce (DPD addition), foxA expression levels increased (Figure 5), consistent with the ability of Fur·Fe2+ to repress PfoxA utilization . Under iron-saturated conditions, hilD mRNA level was ∼10-fold higher than under iron-limiting conditions (DPD addition) (Figure 5). Similar results were observed when 1.5 mM EDTA was added to the media as a chelator (data not shown). The fact that hilD expression level in the UA1888 (PhilD, DPD addition), UA1889 (PhilD*), and UA1890 (Δfur) strains were similar suggests that inactivation of BoxA (in PhilD*) had no effect on basal expression of the gene.
Under iron-saturated conditions, the expression of hilD mRNA in the PhilD* mutant derivative was similar to that measured under iron-limiting conditions (Figure 5), suggesting that PhilD activation does not occur by a mechanism involving transcriptional de-repression. In the PhilD* mutant derivative, hilD mRNA levels were also similar to those obtained with the wild-type PhilD in a null Fur mutant strain (Figure 5), implying that a wild-type BoxA sequence in cis is necessary for the increased accumulation of hilD mRNA.
There is a caveat to these findings, however, as there was no decrease in hilD expression in the absence of both HilD and Fur proteins, determined using transcriptional fusions . This was confirmed by qRT-PCR experiments using the UA1892 (Δfur ΔhilD) strain, in which, as expected, hilD expression was increased by a factor of 1.2±0.18 with respect to the wild-type strain. Nonetheless, even though the absence of Fur or a decrease in the iron concentration resulted in a clear reduction in hilD expression (Figure 5), both HilD and Fur appear to be necessary for full in vivo expression of the gene. This complex response might be explained by the fact that only when the HilD protein reaches a significant threshold it is able to activate the expression of hilA and further induce its own expression .
Fur·Mn2+ activates hilD expression in vitro
To address whether the presence of Fur·Mn2+ is sufficient to activate PhilD utilization, in vitro transcription experiments with linearized PhilD (pUA111) or PhilD* (pUA112) DNA were performed (Figure 6). A single transcript band was obtained with HindII-linearized DNA, indicating that hilD is expressed from a single promoter (Figure 6A, lane 1). The length of the transcript was in full agreement with transcripts initiated at PhilD, as determined by primer extension , confirming that the 116-nt transcript was the genuine transcript from PhilD. Minor transcripts with small molecular masses were attributed to RNAP pausing sites since no obvious promoter sequences could be predicted in the putative upstream regions.
A. Linear HindII-cleaved pUA1111 DNA (10 nM) containing PhilD was pre-incubated with increasing concentrations of Fur (1.5–200 nM). Transcription reactions, performed in the absence or presence of increasing concentrations of Fur, were initiated by adding RNAP and 0.5 mM of each rNTP (with [α-32P]-rUTP). The reactions were incubated for 60 min at 37°C. Transcripts generated in vitro from the PhilD template DNA were separated in 6% dPAGE. The in vitro transcripts from PhilD and their lengths are shown. The sizes of the markers are indicated. B. Linear HindII-cleaved pUA1111 or pUA1112 DNA (10 nM) containing PhilD or PhilD* was pre-incubated with increasing concentrations of Fur (1.5–200 nM). Relative mRNA synthesis from PhilD (filled circles) and PhilD* (empty triangles) in the absence or presence of increasing Fur was compared and is denoted in arbitrary units (AU). Also, the pUA1111 vector containing PhilD (PhilD sc) (open squares) and the pGEM®-T plasmid vector promoter (Pv) (empty circles) were used under the same conditions as supercoiled DNA and the non-specific control, respectively.
In the presence of Mn2+ and the absence of Fur, steady-state levels of the 116-nt transcripts from PhilD and PhilD* were similar (37±3 arbitrary units, AU), suggesting that the BoxA mutations did not affect PhilD* utilization (Figure 6B). In the presence of Mn2+, PhilD utilization, or accumulation of the 116-nt transcript, increased with increasing Fur concentration, with an optimum reached when ∼ 2.5 Fur dimers/DNA molecule were added (123±5 AU) (Figure 6A). Similar levels of PhilD utilization were obtained using supercoiled DNA (PhilDsc) as template, ruling out any topological requirement for transcription activation (Figure 6B). At sub-saturating Fur·Mn2+ concentrations, PhilD utilization increased by more than 3-fold over the control without Fur·Mn2+ (Figure 6). However, the addition of Fur·Mn2+ did not significantly increase the levels of transcription of an unrelated promoter (Pv) (27±5 AU) (Figure 6B).
PhilD* utilization was not significantly increased at Fur·Mn2+ concentrations equal to or higher than those required to activate this promoter (Figure 6B). As seen in Figure 6B, Fur·Mn2+, at sub-saturating or half-saturating concentrations, interacted with BoxA, suggesting that the protein facilitates RNAP utilization of the PhilD region. It is therefore likely that Fur·Mn2+ acts as a transcriptional activator of S. enterica hilD expression. Since transcription activation was not observed when BoxA was inactivated by mutations (PhilD*), half-saturating Fur·Mn2+ concentrations are apparently necessary for transcription activation of the hilD promoter in vitro. However, in the presence of ∼ 20 Fur dimers/DNA molecule, similar activation was not observed (Figure 6B). Since ∼ 20 Fur dimers/DNA molecule similarly did not affect the expression of PhilD* or an unrelated promoter (Pv) (Figure 6), a contaminant RNase or any other non-specific effect can be ruled out as responsible for the reduced RNA synthesis at constant Mn2+ and higher Fur concentrations. It could be hypothesized that Fur·Mn2+ prevents PhilD activation based on its reported cooperative spreading. Thus, nucleoprotein assembly along the promoter region may interfere with, rather than stimulate, the interaction of RNAP with PhilD, returning hilD expression to its basal level.
We show that the S. enterica hilD gene, whose product is the most important regulator of the HilA activator and therefore of SPI1 T3SS expression, contains a Fur binding site (BoxA) in the upstream region of PhilD (−191 to −163). Fur, in its metal-bound form, bound with high affinity to BoxA in PhilD DNA but not to the BoxA mutant variant (BoxA*) in PhilD*. In vivo and in vitro experiments revealed that Fur bound to the upstream element of PhilD activated hilD expression, but did not activate transcription from PhilD*. Thus, metal-bound Fur appears to be a direct transcriptional activator of the S. enterica hilD gene. Being this the first evidence of Fur acting directly as an activator in this bacterium.
In most bacterial species, Fur·Fe2+ is a transcriptional repressor, binding to cognate sites within position −35 to +12 in the promoter region , , , –. In these cases, transcription of the target promoters is blocked by steric hindrance rather than by preventing transcription elongation , , . Fur·Mn2+ bound to BoxA in PhilD, which is situated upstream of HilD, HilC (see Figure 1A ), or even RtsA binding sites , activates RNAP utilization of PhilD. A similar upstream location of the Fur cognate site has been described for Fur-activated genes in other microorganisms , , , , , suggesting a close relationship between the location of the Fur binding site in the promoter of the controlled gene and its role as an activator.
The majority of transcriptional activators, upon binding to their cognate upstream element adjacent to the core RNAP sites, either drive the recruitment of the latter to the target promoter or alter the conformation of the promoter DNA to facilitate RNAP loading –. Here we provide the first evidence of a direct mechanism of S. enterica hilD transcription activation by showing that Fur·Mn2+ binds to a distal upstream-activating sequence. We thus propose that Fur·Fe2+ is sufficient, in vitro, to increase RNAP recruitment. It is unlikely that Fur·Fe2+ alone alters the DNA conformation, because DNase I hypersenstive sites were not observed. There are not evidences for Fur·Mn2+ and RNAP interaction and activation via a looping mechanism, but Fur·Mn2+ bound to its target site are sufficient for increased PhilD utilization. It can be envisaged, however, that the HilD regulation in vivo is complex as suggested the hilD expression results obtained in the double hilD fur mutant and the fact that RtsA, HilC and HilD recognize a sequence downstream Fur and apparently antagonize H-NS- and Hha-mediated repression, suggesting that these complex control region (−120 to −57) laid within BoxA (−191 to −163) and the core promoter region (−60 to + 10) , .
The expression of SPI1 is known to be modulated by environmental signals, which indirectly control hilD transcription . Among these signals, a relationship between extracellular iron concentrations and SPI1 expression has been suggested  and an indirect association of Fur and hilA expression through H-NS described . In addition, several reports have shown that low-oxygen concentrations, such as those present in the intestinal lumen, increase SPI1 expression , , . Free iron is scarce inside the host, but Fe2+ is abundant in the intestinal lumen, where it is efficiently absorbed by intestinal epithelial cells . Fur does not sense oxygen concentrations directly but is instead able to monitor the redox signal via the equilibrium between Fe2+ and Fe3+ . It should be noted that the Fur·Fe3+ complex is not functional and that, as Fe3+ is insoluble, it cannot be translocated inside the cell , .
This work describes the direct activation of SPI1 by Fur through its interaction with an upstream region on PhilD and thus adds new information to Fur SPI1 regulation models , ,  (Figure 7). Taken together, our data shed light on the role of Fur in SPI1 control. Specifically, Fur is able to control, either directly (in the case of HilD) or indirectly through H-NS (in hilA, hilD, hilC, and rtsA), all the main regulators of SPI1. These results strengthen the relationship between S. enterica invasiveness and both iron and oxygen concentrations inside the host. Accordingly, the expression of hilD, hilA, hilC, and rtsA, and consequently that of T3SS1, should be stimulated once S. enterica reaches the epithelial surface, where iron concentrations are high and those of oxygen low. T3SS expression has been reported as essential for epithelial invasion . Once Fe2+ levels decrease or those of O2 increase, the number of Fur·Fe2+ complexes should diminish markedly, as should T3SS, which is no longer needed and must remain silent in subsequent steps of the infection process . This sequence of events is supported by a previously published report in which, the epithelial invasiveness of Fur-defective mutants was shown, in an acid-sensitive independent manner, to be lower than that of the wild-type strain . Direct regulation by Fur of hilD expression would allow rapid signal transduction once the Fe2+ concentration increases, situating PhilD in a similar hierarchic position as sRNAs that indirectly control other genes positively regulated by Fur.
A Fur SPI1 regulation model after Ellermeier et al.  and Troxell et al. . Green arrows indicate a direct activation effect; red lines correspond to direct repression. The thick green arrow shows the herein described pathway of direct activation by Fur of hilD gene expression. H-NS control of the hilD, hilC, rtsA and hilA promoters is also shown , , .
A. SDS-PAGE showing the Fur purified protein. Lanes 1 and 2 correspond to non-induced and IPTG-induced cell crude extracts of BL21(DE3)pLys containing the S. enterica fur gene cloned in the pET15b vector. Lane 3 is the purification fraction containing the Fur native protein after thrombin digestion. B. Scheme of PfoxA indicating the location of the Fur binding site in blue. The ATG start codon is indicated in bold. C. EMSA performed using DIG labeled PfoxA probe (20 nM) and the purified Fur protein at increasing concentrations (2.5, 12.5, 50, and 187 nM). Lane (-) indicates the mobility of the DNA probe without Fur in the binding mixture.
Fur-Mn2+ binds with high affinity to PhilD DNA. The 375-bp [a-32P]-NcoI-HindII DNA (2 nM) fragment containing PhilD was incubated with increasing Fur concentrations (3–400) for 15 min at 37°C in buffer A (50 mM Bis-Tris/borate buffer pH 7.5, 5% glycerol, 10 mM MgCl2, 1 mM MnCl2) or E (50 mM Bis-Tris/borate buffer pH 7.5, 5% glycerol, 1 mM MgCl2, 0.1 mM MnCl2). The absence of a component is indicated by -; FD, protein-free PhilD DNA; IC, intermediate complexes; PD, protein-DNA complexes.
qRT-PCR assays of recA expression in the different genetic backgrounds used in this work. For each condition, the relative recA expression levels were calculated as the ratio of its mRNA concentration with respect to that obtained in the isogenic wild-type strain (PhilD) and normalized to that of the S. enterica 16S RNA. The mean value from three independent experiments (each in triplicate) is shown.
We are deeply indebted to Prof. G.M. Ghigo for the generous gift of plasmid pKOBEGA and to Maria Pilar Cortés and Joan Ruiz for their excellent technical assistance.
Conceived and designed the experiments: LT BC JCA JB SC. Performed the experiments: LT BC SC. Analyzed the data: LT BC JCA JB SC. Contributed reagents/materials/analysis tools: JCA JB. Wrote the paper: JCA JB SC.
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