Fur activates the expression of Salmonella enterica pathogenicity island 1 by directly interacting with the hilD operator in vivo and in vitro

Abstract

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 (P(hilD)), at position -191 to -163 relative to the hilD transcription start site. Furthermore, in a P(hilD) variant with mutations in BoxA, P(hilD*), Fur·Mn(2+) binding is impaired. In vivo experiments using S. enterica strains carrying wild-type P(hilD) or the mutant variant P(hilD*) showed that Fur activates hilD expression, while in vitro experiments revealed that the Fur·Mn(2+) protein is sufficient to increase hilD transcription. Together, these results present the first evidence that Fur·Mn(2+), by binding to the upstream BoxA sequence, directly stimulates the expression of hilD in S. enterica.

Figure 1. Fur·Mn²⁺ specifically binds P_hilD DNA.

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 [12]) shown in green. The −35 and −10 promoter (P_hilD) consensus regions and the +1 transcription start site are underlined. The coding region of hilD is denoted in lower-case characters. B. EMSA of P_hilD 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 P_hilD 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 P_hilD 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 P_hilD 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 P_hilD DNA; PD, the protein-DNA complex.

Figure 2. Fur·Mn²⁺ specifically binds BoxA in P_hilD.

A. Diagram of the P_hilD 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 (P_hilD), FrgB, FrgC, and FrgD DNA probes (20 nM) from the P_hilD region in the absence (−) or presence (+) of 50 nM Fur protein.

Figure 3. Footprint assay of P_hilD DNA using increasing Fur concentrations.

The 375-bp [α-³²P]-NcoI-HindII P_hilD (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.

Figure 4. Fur·Mn²⁺ does not bind to a mutated BoxA (P_{hilD *})

A. Sequence of wild-type BoxA in P_hilD and the mutated BoxA* variant in P_hilD*. The three subsites of the Fur box and their relative orientation are also indicated. B. EMSA using 20 nM P_hilD or P_hilD* DNA and 50 nM Fur·Mn²⁺. The presence or absence of a component is indicated inside the table by + or −, respectively.

Figure 5. Relative hilD mRNA levels in several bacterial strains and iron concentrations.

Relative hilD mRNA were estimated by qRT-PCR in P_hilD (UA1888), P_hilD* (UA1889), Δfur P_hilD (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 P_hilD utilization is not affected by the upstream Kan^R 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 (P_hilD) and normalized to that of the S. enterica recA gene. The mean value from three independent experiments (each in triplicate) is shown.

Figure 6. In vitro transcription of P_hilD in the presence of Fur protein.

A. Linear HindII-cleaved pUA1111 DNA (10 nM) containing P_hilD 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 P_hilD template DNA were separated in 6% dPAGE. The in vitro transcripts from P_hilD and their lengths are shown. The sizes of the markers are indicated. B. Linear HindII-cleaved pUA1111 or pUA1112 DNA (10 nM) containing P_hilD or P_hilD* was pre-incubated with increasing concentrations of Fur (1.5–200 nM). Relative mRNA synthesis from P_hilD (filled circles) and P_hilD* (empty triangles) in the absence or presence of increasing Fur was compared and is denoted in arbitrary units (AU). Also, the pUA1111 vector containing P_hilD (P_hilD sc) (open squares) and the pGEM®-T plasmid vector promoter (P_v) (empty circles) were used under the same conditions as supercoiled DNA and the non-specific control, respectively.

Figure 7. Scheme of SPI1-Fur mediated regulation.

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 , , .