Y. pestis KIM6+ and derivatives of this strain (Table S1) were inoculated from −80°C glycerol stocks onto Congo Red plates. Plates were incubated at 30°C for ~48 h. Individual colonies were inoculated onto Tryptose Blood Agar Base (TBA) slants with the appropriate antibiotic, and incubated at 30°C overnight. E. coli strains were inoculated from −80°C glycerol stocks onto TBA slants with appropriate antibiotics and incubated at 30°C overnight. For transformants, E. coli cells were grown in Luria broth (LB) or on LB agar plates.

From TBA slants, Y. pestis cells were inoculated to an OD₆₂₀ of 0.1 in chelex 100-treated PMH2 (cPMH2) at a pH of 6.0, 6.5, or 7.5 with or without 10 μM FeCl₃ and grown at 37°C at 250 rpm through 2 transfers (6-8 generations). Lysates were assayed for enzymatic activity using ONPG (4-nitrophenyl-β-D-galactopyranoside) as a substrate as previously described (Miller, 1992). The statistical significances of differences were calculated using the 2-tailed student's t-test.

Y. pestis cells were grown in cPMH2 under aerobic conditions for ~5 generations prior to use in iron transport assays under reducing conditions. Transport was initiated by adding ⁵⁵FeCl₃ to a final concentration of 0.2 μCi/ml to cultures containing 5 mM sodium ascorbate. Parallel cultures, preincubated for 10 min with 100 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP), were used to demonstrate energy-independent binding. The ⁵⁵FeCl₃ used in the transport studies was diluted into PMH2 containing 100 mM sodium ascorbate, incubated for at least 30 min at room temperature, and filtered through a 0.45-μm filter before being added to the cultures. Duplicate samples (0.5 ml) were taken at various time points after the addition of ⁵⁵FeCl₃, filtered through 0.45-μm GN-6 nitrocellulose membranes, and rinsed twice with PMH2 medium containing 20 μM FeCl₃. The membranes were added to vials containing Bio-Safe II scintillation fluid (Research Products International) and counted in a Beckman LS3801 liquid scintillation counter. Unfiltered samples were used to determine the total amount of radioactivity in each culture. The results are expressed as percent uptake/0.4 OD₆₂₀ to compensate for slight increases in cell density during the course of the assay.

For transport under non-reducing conditions Y. pestis cells were grown in cPMH1 for ~5 generations prior to use in iron transport assays. Transport was initiated by adding filtered ⁵⁵FeCl₃ to a final concentration of 0.2 μCi/ml to cultures containing cold FeCl_3. Cultures were processed and analyzed as described above.

Electrophoretic mobility shift assays (EMSAs) were used to demonstrate binding to the feo promoter region. Full length and truncated feo promoter probes were amplified by PCR using primer pairs pfeoF4 and pfeoR4, pfeoF5 and pfeoR5, or pfeoF6 and pFeoR6 (Table S2). Y. pestis KIM6+ or KIM6 fur::kan-9 (KIM6-2030) were grown with or without added iron in cPMH2 media through 2 passages to an OD₆₂₀ of 0.3–0.4, cells centrifuged and frozen at −80°C. Cell pellets were resuspended in 2 ml of B-PER II reagent, 2 μl of lysozyme (10 mg/μl), 2 μL of DNase I and 4 μL of DNase I buffer and incubated with shaking for 20 min at room temperature. Samples were centrifuged to remove debris and protein content analyzed by the Bradford assay and comparison by SDS-PAGE. Lysates were aliquoted and stored at 4°C. Binding reactions [100 mM Tris, 750 mM KCl, 10 mM dithiothreitol (DTT), 50 mM MgCl₂, 12.5% glycerol, 50 mM EDTA [pH 8], 20 fmol biotinylated DNA probe, and Y. pestis cell lysates] were incubated at room temperature for 20 min. 5X EMSA loading buffer (50 mM Tris, 250 mM KCl, 5 mM DTT, 37 μM bromophenol blue) was added and the reactions were loaded onto a 6% DNA retardation gel (Invitrogen) with prechilled 0.5X TBE buffer and run at a constant voltage of 120 V for 90 min. After electrophoresis, gels were transferred to a Biodyne B Nylon membrane (Thermo Fisher Scientific) with prechilled 0.5X TBE buffer at a constant voltatge of 120 V for 45 min. Following transfer, the membrane was crosslinked with a UV Stratalinker 2400 (Stratagene, La Jolla, CA) and incubated overnight with 20 mL blocking buffer (Thermo Fisher Scientific). The Chemiluminescent Nucleic Acid Detection Module (Thermo Fisher Scientific) was used according to the manufacturer's protocol. The membrane was visualized on CL-X Posure Film (Thermo Fisher Scientific).

Although, all other tested promoters controlling expression of iron transport systems in Y. pestis, are repressed by iron under aerobic conditions, the feoA promoter is repressed only under static (microaerobic) conditions. The other Fe²⁺ transporters (Yfe, Fet-flp, and Efe) are repressed by iron under borth aerobic and microaerobic conditionas and none of the 4 Y. pestis Fe²⁺ transporters are repressed by aerobic conditions compared to microaerobic conditions (Perry and Fetherston, 2004; Fetherston et al., 2012; Perry et al., 2012). We hypothesized that some aspect of the feoA promoter is preventing Fe-Fur regulation under aerobic conditions. To test this hypothesis we made 5′-truncations of the feo promoter region and fused them to a lacZ transcriptional reporter (Figure 1). KIM6 (Δpgm Feo⁺) strains carrying the various reporter plasmids were grown in cPMH2 in the presence and absence of iron under aerobic growth conditions at 37°C. When the intergenic region between y3913 and feoA is truncated from 396 to 335 bp, transcriptional activity increased ~2.5-fold in the presence and absence of iron (Figure 2). This activity was further increased to a 4-fold difference, both in the presence and absence of iron when the 396 bp promoter region was truncated to 271 bp. A modest but statistically significant 1.3-fold ron-mediated repression was not observed until the feoA promoter region was truncated to 208 bp. This construct still had overall increased transcriptional activity compared to the full-length 396 bp promoter region (Figure 2). A further truncation to 195 bp maintained repression in the presence of iron, while the overall transcriptional activity decreased. The 165 and 125 bp promoters, showed stronger iron-mediated repression (2.6- and 3-fold, respectively), and overall transcriptional activity that returned to the 396bp promoter levels (Figure 2). Thus, the 396 to 165 bp region somewhat prevents Fe-Fur repression aerobically, while the 396 to 335 bp region somewhat lowers overall transcriptional activity from the feoA promoter region.

In Salmonella, RstA activates transcription from the feo promoter (Jeon et al., 2008). Previous work showed that a ΔrstAB mutation does not affect transcription from the Y. pestis feoA promoter (Fetherston et al., 2012). Since the rstAB operon is activated by the PhoP/PhoQ two-component regulators, which are activated by acidic pH (Jeon et al., 2008; Choi et al., 2009), we examined whether acidic conditions affected transcription from the feoA promoter. Y. pestis cells with full-length or truncated promoters were cultured in cPMH2 at pHs of 6.0, 6.5, or 7.5. It should be noted that all of the strains grew very slowly at pH 6.

The 396 bp promoter had a 2-fold repression in the presence of iron when the cultures were grown at pH 6. A slight, but not statistically significant repression was seen at pH 6.5. Expression levels at pH 7.5 (from Figure 2) are shown for all constructs for reference (Figure 3). However, RstAB is not responsible for this effect since KIM6+ (rstAB⁺) and KIM6-2189+ (ΔrstAB) had similar levels of activity from the feoA::lacZ reporter (Fetherston et al., 2012).

When the promoter was truncated to 208 bp, there was no significant iron repression at any of the tested pH levels (Figure 3), perhaps due to the high standard deviations associated with this reporter. While the 165 and 125 bp promoter truncations, showed ~3-fold iron-mediated repression at pH 7.5, iron repression increased to 5- and 6-fold at pH 6.5, respectively. When the pH was further lowered to 6, the iron repression became even more pronounced—12- and 8-fold respectively. At all pHs, the pattern of increased expression from the 208 bp reporter, compared to the 396, 165, and 125 bp reporters was maintained (Figure 3).

The 396 bp reporter does not exhibit iron repression. However, truncation of the promoter region to 125 bp causes transcriptional repression of the reporter by iron (Figure 2). This experiment was repeated using KIM6-2030 (fur::kan mutant) carrying the 396 or 125 bp reporter. With this fur mutant the 125 bp promoter reporter no longer exhibited iron repression. This indicates that Fur is responsible for the observed repression of the 125 bp reporter (Figure 4).

To determine whether the feo promoter itself or a unique aspect of the Fur protein in Y. pestis was preventing aerobic iron repression of the feoA promoter, we used a lacZ transcriptional reporter fused to the E. coli feo promoter region. The E. coli feo::lacZ reporter showed iron repression under aerobic conditions in Y. pestis (Figure S1). This suggests that a unique aspect of the Y. pestis Fur protein is not responsible for the lack of iron repression of the Y. pestis feo promoter under aerobic conditions. Rather the sequence/structure of the Y. pestis feoA promoter region likely prevents iron repression under aerobic, but not microaerobic conditions.

We hypothesized that a repressor binding to the feo promoter region between 396 and 208 bp is the reason for increased transcriptional activity in the truncated promoters. Additionally, a second protein binding to the feoA promoter may prevent iron-repression under aerobic conditions. To examine these possibilities, an electrophoretic mobility shift assay (EMSA) was performed using whole cell lysates from Y. pestis that had been grown aerobically in the presence or absence of iron. Biotinylated probes were generated by PCR using primers that spanned the 396 bp feoA promoter, a 251 bp 3′-truncated feo promoter fragment, or a 146 bp 5′-truncated feo promoter fragment (Figure 5A). A shift was observed using the 396 bp (Figure 5B), 251 bp (Figure 5B), and 126 bp (Figure 5C) probes regardless of growth conditions.

We would not expect to see Fur binding in the EMSAs since Fur needs to be bound to a divalent cation in order to bind DNA (de Lorenzo et al., 1988). Even in the cultures where iron was added, the intracellular iron bound to Fur would likely be converted in the ferric form after cell lysis. Typically MnCl₂ is added to cell lysates or purified Fur, when studying Fur binding, since the Mn²⁺ serves as a stable divalent cation under aerobic conditions (Friedman and O'Brian, 2003). However, since there is a Fur-binding site in the feoA promoter it was necessary to ensure that Fur was not responsible for this shift. Cell lysates from the fur mutant grown with or without iron were also used, and also resulted in a shift with the 396 bp (Figure 5B) and 146 bp (Figure 5C) probes. The 251 bp probe was not tested. Therefore, this observed shift is not due to Fur binding, but some other Y. pestis protein. In order to further identify the region in which the protein(s) is binding, two additional biotinylated probes were generated by PCR, splitting the 251 bp probe into an upstream 126 bp probe and downstream 125 bp probe (Figure 5A). Regardless of growth condition, a shift was seen with the upstream 126 bp probe, but not with the 125 bp downstream probe (Figure 5C). These data suggest that there are (1) two proteins binding the feo promoter: one within the 126 bp probe (396 to 271 bp region in Figure 1) and one within the 146 bp probe (146 to 1 bp region in Figure 1) or (2) one protein binding to both regions.

Although, the pWSK vectors are considered low-copy number (Wang and Kushner, 1991) which we have used in transcriptional analysis of transcriptional regulation of numerous promoters (Fetherston et al., 1999, 2012; Gong et al., 2001; Kirillina et al., 2006; Forman et al., 2010; Perry et al., 2012), we wanted to ensure that our results were not due to titration of some negative regulatory factor by the feoA reporter plasmids. Therefore, we constructed vectors where the reporter gene would integrate into the Tn7 insertion site of Y. pestis using the Tn7-based system developed by Choi et al. (2005). We tested integrated reporters with the feoA (396 bp), feoA165, and feoA208 lacZ reporters in KIM6+ grown under static and aerobic conditions in the presence or absence of iron. The integrated reporters behaved in the same fashion as their plasmid counterparts (compare Figure S2 and Figure 2) with regard to the overall transcriptional activity and iron regulation. Thus, the feoA construct was only repressed by iron in cultures grown under static conditions while feoA165 reporter was iron-regulated under both static and aerobic conditions. The activity of the integrated feoA208::lacZ construct was much higher than the other two feo constructs and exhibited the same regulation pattern as its plasmid counterpart (compare Figure S2 and Figure 2). The integrated lacZ gene without any promoter fragment exhibited very low activity under all conditions (Figure S2).

We also used the integrated reporter system to examine regulation of an extended fragment of the feoA promoter region. Subsequent analysis and annotation of the Y. pestis feoABC operon predicts the start codon for feoA is further downstream than the originally annotated initiating ATG (at 0 to −2) in Figure 1. To determine if the additional bp separating the 2 predicted ATG start codons would affect the regulation of the feo operon, we made another feoA::lacZ reporter (pUCR6KfeoP447-lac-Gm) that included these nucleotides and integrated it into the Tn7 insertion site of KIM6+. The reporter with the extended promoter fragment exhibited the same properties as our original feoA::lacZ construct (data not shown) indicating that the additional base pairs did not play a role in the regulation of the feoABC operon.

The transcriptional and binding data caused us to re-examine the feoA promoter region. Another, putative RNA polymerase (RNAP)—binding site (labeled P1/-10/-35 in Figure 1; the putative P1 promoter spans the Fnr binding site) is located upstream of the FBS and the P2 promoter (Figure 1). To test this, we deleted 41 bp that encompass the FBS and includes the putative −35 and −10 regions for the downstream P2 promoter. This strain, designated KIM6-2208+ (attTn7:: feoAΔFBS::lacZ) had higher β-galactosidase activity (1.5–2.5X) than the integrated reporter with the original feoA::lacZ construct (KIM6-2203+; attTn7:: feoA::lacZ) under all conditions (Figure 6). Absence of the downstream P1 promoter may allow better read through from transcription initiation at the upstream P2 promoter. Interestingly, the attTn7:: feoAΔFBS::lacZ) reporter was slightly repressed (~2-fold) by iron under static but not aerobic conditions. Examination of the feoA promoter revealed additional potential FBSs with weaker matches to the Y. pestis consensus FBS (Zhou et al., 2006), located at bp 262–280 (11/19 bp) and bp 204–222 (12/19). Whether, either of these regions is responsible for the iron repression observed with the attTn7:: feoAΔFBS::lacZ reporter is unknown; however, the observed ~2-fold iron repression was alleviated in a Y. pestis fur mutant (KIM6-2030+; Figure 6).

Deletion of 20 bp that would include the putative −10 region of the upstream P1 promoter resulted in an integrated construct (attTn7:: feoAΔfeoP1::lacZ in KIM6-2207+) with lower overall β-galactosidase activity than the original attTn7:: feoA::lacZ reporter in KIM6-2203+ under both static and aerobic conditions. However, like the attTn7:: feoA165::lacZ reporter in KIM6-2204+, the attTn7:: feoAΔfeoP1::lacZ reporter was repressed by iron under both conditions. This provides additional evidence that the Y. pestis feo operon contains 2 promoter regions with the downstream P2 promoter being repressed by iron when cells are grown either statically or aerobically and suggest that the upstream P1 promoter is more active and slightly repressed by iron only under static conditions.

In examining the Y. pestis feo promoter region, we noted the presence of 3 nearly identical 11 bp direct repeat sequences, two of them located before the upstream P1 promoter (R1 and R2) and one (R3) present between the 2 promoters (Figure 1 and Table S3). To determine if these regions regulated expression of the feoABC operon, we deleted each of them in turn and analyzed the effect on the β -galactosidase activity of strains carrying the respective integrated reporters. An R3 deletion (attTn7:: feoAΔR3::lacZ in KIM6-2211+) had no significant effect on the expression of the feoA::lacZ reporter under any of the tested conditions. Similarly, deletion of R1, the most distally located repeat, had no effect on regulation in that the reporter construct (attTn7:: feoAΔR1::lacZ in KIM6-2209+) was only repressed by iron in cells grown under static conditions. Under aerobic conditions, the attTn7:: feoAΔR1::lacZ reporter was not repressed by iron but was less active (51–65%) than the attTn7:: feoA165::lacZ reporter. The attTn7:: feoAΔR2::lacZ in KIM6-2211+ reporter in KIM6-2210+ behaved essentially the same as the attTn7:: feoAΔfeoP1::lacZ reporter. Expression of the lacZ gene was lower relative to the attTn7:: feoA::lacZ reporter but was repressed by iron under both static and aerobic growth conditions (Figure S3). The R2 deletion would remove a potential −35 box for the putative P1 promoter (Figure 1) which likely accounts for the results we obtained with this construct.

To demonstrate that the Yfe and Feo systems are involved in active iron transport, transport assays were performed under reducing and non-reducing conditions. Sodium ascorbate was utilized to maintain ⁵⁵FeCl₃ in the reduced, ferrous state. An Δirp2 mutant (unable to make Ybt), transported ~40% of the ferrous iron under reducing conditions (Figure 7A) and ~35% under non-reducing conditions (Figure 7B) by 30 min of incubation at 37°C. The uptake was energy-dependent, since CCCP-poisoned cells showed very little cell-associated iron (Figures 7A,B). The Δirp2 Δyfe double mutant, transported iron at slightly reduced rates compared to the Δirp2 mutant, under both reducing (~3-fold) (Figure 7A) and non-reducing (~2-fold) (Figure 7B). The Δirp2 Δfeo double mutant had slightly reduced rates of uptake under reducing conditions compared to the Δirp2 mutant (Figure 7A). However, the Δirp2 Δyfe Δfeo triple mutant showed very little cell-associated iron, a level comparable to the CCCP-poisoned cells, under both reducing (Figure 7A), and non-reducing (Figure 7B) conditions. This indicates, that Yfe and Feo have somewhat redundant in vitro ferrous uptake functions with one system partially compensating for loss of the other. However, together they are crucial for ferrous iron transport under the short-term in vitro uptake conditions used here. In addition, these assays indicate that the Yfe and Feo systems can use ferric iron as well as ferrous.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.