Sequential induction of Fur-regulated genes in response to iron limitation in Bacillus subtilis

Expression of FrvA Imposes Iron Deprivation and Inhibits Cell Growth of B. subtilis.

Due to the high activity and redundancy of iron import systems, iron starvation can be difficult to achieve under laboratory conditions without the use of chelating agents such as dipyridyl. Here, we explored the use of the Fe²⁺ efflux transporter FrvA as a genetic tool to study iron limitation responses in B. subtilis. The resulting gradual depletion of intracellular iron levels may more closely mimic the natural situation wherein intracellular iron is depleted by growth.

To validate its use as an iron depletion system, we monitored the effects of FrvA expression on intracellular metal levels using inductively coupled plasma mass spectrometry (ICP-MS). The uninduced cells harboring an isopropyl β-d-1-thiogalactopyranoside (IPTG)-inducible ectopic copy of frvA (WT P_spac-frvA) displayed a slightly reduced level of intracellular iron compared with WT, presumably due to the leaky expression from the P_spac promoter. When FrvA was induced by IPTG, intracellular iron levels declined by twofold after 30 min of treatment (Fig. 1A). By contrast, intracellular iron levels in both WT and uninduced cells increased significantly (about twofold) after 2 h (Fig. 1A), probably due to high iron demand as the growing cells enter into late logarithmic phase (transition phase) growth, which is characterized by increased respiratory activity (29). The intracellular level of other metals such as Mn and Co was not significantly changed during the course of the experiment (SI Appendix, Fig. S1).

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Fig. 1.

Induction of L. monocytogenes FrvA imposes iron deprivation and inhibits cell growth of B. subtilis. (A) Levels of intracellular metals (Fe, Mn, and Co) were monitored for 3 h after 1 mM IPTG addition by inductively coupled plasma mass spectrometry (ICP-MS). The total concentration of ions was expressed as µg ion per gram of protein (mean ± SD; n = 3). Significant differences between WT and induced P_spac-frvA cells are determined by two-tailed t test as indicated: *P < 0.01. (B) Representative growth curves of the same strains as A in LB medium amended with 10 µM FeSO₄. For IPTG-treated cells, 1 mM IPTG was added to cell culture when OD₆₀₀ reaches ∼0.25. Experiments were performed at least three times with three biological replicates.

To evaluate the effects of FrvA expression on growth, we grew cells to early exponential phase (OD₆₀₀ ∼ 0.25) in LB medium amended with 10 µM of FeSO₄, induced expression of FrvA with 1 mM of IPTG, and monitored growth. The uninduced cells grew as well as WT in the first 3 h and displayed very minor growth inhibition afterward, presumably due to leaky expression of FrvA from P_spac (28) (Fig. 1B). In contrast, when FrvA was induced, cell growth was inhibited after ∼2 h (Fig. 1B). Thus, there is a substantial period of time (between ∼30–90 min after induction) where iron levels are significantly reduced (more than twofold reduction of total intracellular iron) with little if any apparent growth inhibition. Therefore, we chose this period of time to explore the graded response of the Fur regulon.

Fur-Regulated Genes Are Derepressed in a Stepwise Fashion.

To provide a global overview of the transcriptional changes as cells transition from iron sufficiency to iron deficiency we analyzed changes in RNA levels at 20, 30, 60, and 120 min after FrvA induction using oligonucleotide-based microarrays (SI Appendix, Fig. S2). In this analysis, most Fur-regulated operons were expressed at a low level before induction, as expected for cells grown in LB supplemented with 10 µM iron. In a direct comparison between cells carrying the P_spac-frvA construct grown with and without IPTG induction, the majority of the Fur regulon was derepressed (at least transiently) between 20 and 60 min after induction. In many cases, the maximal fold induction was comparable to that noted in a comparison of the fur mutant to WT (in strains lacking P_spac-frvA). In general, it appeared that some genes were induced at earlier time points, while induction of others was only evident at late time points, suggesting that the Fur regulon might be derepressed in a gradual manner in response to iron availability. Consistent with our prior study (28), induction of the PerR regulon in response to iron limitation was also apparent at late time points (3 h) and also in cultures with IPTG added at the time of subculture (steady state) (SI Appendix, Fig. S3A). This derepression is noted for peroxide detoxification enzymes (katA, ahpC, and ahpF) and a miniferritin that may function in iron storage (mrgA). These results were further confirmed by quantitative PCR (qPCR) (SI Appendix, Fig. S3B).

We were surprised to note that the two iron uptake systems most important for growth in LB medium (30), elemental iron uptake [EfeUOB; formerly, YwbLMN (31)] and ferric citrate import [FecCDEF; formerly, YfmCDEF (7)] were not highly induced during the course of this experiment (SI Appendix, Fig. S2). We hypothesized that these operons might be already partially derepressed in the uninduced cells due to mild iron deprivation resulting from leaky expression of FrvA. To test this idea, we used qPCR to monitor the expression of selected Fur-regulated operons in LB with 10 µM iron. We included the uptake systems for elemental iron (efeUOB), ferric citrate (fecCDEF), hydroxamate siderophores (fhuBCGD), and petrobactin (fpbNOPQ), as well as flavodoxins (ykuNOP), BB biosynthesis (dhbACEBF), BB uptake (feuABC), and iron-sparing response (fsrA, fbpAB, and fbpC) operons. Relative derepression of the first gene in each operon was analyzed by comparing gene expression in the induced cells as a percentage of derepression as observed in a fur mutant versus WT. Among all of the genes tested, three genes, fpbN, efeU, and fecC, were strongly derepressed (30–55% derepression) even at time 0 (SI Appendix, Fig. S4), presumably due to leaky expression of FrvA.

We hypothesized that a higher concentration of iron in the medium might enable a more complete repression of the Fur regulon. We therefore checked repression in LB medium amended with 25, 50, and 100 µM iron and determined that 25 µM was sufficient to fully repress Fur-regulated genes. We therefore repeated our qPCR analysis using LB medium amended with 25 µM. For this analysis we used 50% derepression as the threshold to define the order of gene induction. Three waves of derepression were revealed with 50% derepression at ∼10, 20, and 60 min (Fig. 2). As judged by monitoring of the first gene in each operon, we can resolve early (fpbN, efeU, and fecC), middle (fhuB, ykuN, dhbA, and feuA), and late (fsrA, fbpA, and fbpC) genes (Fig. 2). These results are consistent with the first set of experiments using 10 µM FeSO₄ with the exception of feuA being assigned as a middle gene (Fig. 2 and SI Appendix, Fig. S4). The late induction of the FsrA/FbpABC iron-sparing response is expected to lead to translational down-regulation of a large number of target genes, which, as shown previously, is reflected as a reduction of steady-state mRNA levels (13). Indeed, we also observed a decrease in the mRNA levels of many FsrA-regulated genes at later time points (1 and 2 h) (SI Appendix, Fig. S5).

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Fig. 2.

Fur-regulated genes are induced in a stepwise manner as a function of iron depletion. Cells were grown at 37 °C in LB medium overnight and subcultured (1:100 ratio) into fresh LB medium amended with 25 µM FeSO₄. At OD₆₀₀ ∼0.25, 1 mM IPTG was added to induce expression of FrvA. Quantitative PCR was conducted using a specific primer set for each gene (SI Appendix, Table S2). The housekeeping gene 23S rRNA was used as an internal control. Relative expression of the first gene in each operon at different times was analyzed by comparing gene expression in the IPTG-induced cells versus that in wild-type cells. The percentage of derepression was normalized based on the full derepression observed in a fur null mutant versus WT cells, which was set as 100% derepression for each gene tested.

Stepwise Induction of Fur-Regulated Genes Correlates with Operator Occupancy.

The molecular mechanism for the stepwise derepression of Fur-regulated genes may simply be that Fur operator occupancy is sequentially affected as a function of iron depletion. To test this idea, we constructed a fully functional chromosomal FLAG-tagged Fur in both WT and P_spac-frvA (SI Appendix, Fig. S6). Using chromatin immunoprecipitation (ChIP) coupled with qPCR, we found that occupancy of Fur on the operator sites tested declined rapidly upon iron depletion. Fur occupancy decreased significantly at the sites of three early genes (fpbN, efeU, and fecC) by 1 min; the differences of Fur occupancy among the three sets of genes became evident by 3 min, with only ∼10% occupancy for early genes (fpbN, efeU, and fecC), ∼35% occupancy for middle genes (fhuB, ykuN, dhbA, and feuA), and ∼55% occupancy for late genes (fsrA, fbpA, and fbpC) (Fig. 3). More importantly, the three distinct patterns revealed by ChIP (Fig. 3) correspond to the same three sets of early, middle, and late genes determined by qPCR (Fig. 2). The timescale of Fur dissociation from its operators (Fig. 3) appears to be considerably faster than the resulting accumulation of the corresponding mRNAs (Fig. 2). However, the ChIP procedure likely underestimates the time required for loss of operator binding since the times noted do not account for the time required to harvest cells by centrifugation before protein–DNA cross-linking. Moreover, the mRNA measurements reflect the cumulative effect of Fur dissociation, RNAP recruitment, and transcription, leading to an increase in mRNA levels. Despite the differences in timescale, these results support the general notion that Fur dissociates from early gene operator sites more quickly as intracellular iron levels decrease.

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Fig. 3.

Operator occupancy correlates with stepwise derepression of the Fur regulon. Fur occupancy on different promoter sites was evaluated by chromatin immunoprecipitation (ChIP) using anti-FLAG antibodies. Coimmunoprecipitated DNA was quantified by qPCR using specific primer sets to the promoter regions of the target genes as listed in SI Appendix, Table S2. DNA enrichment was calculated based on the input DNA (1% of total DNA used for each ChIP experiment). Fur occupancy at different sites was set as 100% at time-point 0. Dates are presented as the relative percentage (%) of occupancy at different time points after 1 mM IPTG induction (mean ± SD; n = 3). No significant DNA enrichment was observed for gyrA that is used as a nonspecific negative control.

Stepwise Induction of Fur-Regulated Genes Is Correlated with Protein–DNA-Binding Affinity.

As the FrvA efflux pump gradually depletes iron from the cytosol the concentration of metallated Fur protein is predicted to decrease. We therefore hypothesized that the earliest induced operons in the Fur regulon would correspond to those with operator sites that bind holo-Fur weakly and, conversely, that the late-induced genes would bind holo-Fur with higher affinity. We determined the biochemical binding affinity in vitro using electrophoretic mobility shift assay (EMSA). The equilibrium dissociation constant (K_d) for Fur binding to different target operators provides an estimate of the concentration of active protein required for half-maximal repression. In support of our hypothesis, the stepwise induction of Fur regulon correlates, at least in part, with protein–DNA-binding affinity (Table 1 and SI Appendix, Figs. S7 and S8). For example, Fur binds to the operators of early genes with relatively low affinity (K_d value of ∼3 and 5.6 nM for fecC and fpbN, respectively), to those of middle gene with higher affinity (0.8 nM for fhuB, 0.9 nM for feuA, and 2.1 nM for dhbA), and with highest affinity to those for late genes (0.5–0.6 nM for all three late genes, fsrA, fbpA, and fbpC) (Table 1 and SI Appendix, Figs. S7 and S8). However, there are some exceptions. For instance, the induction of efeU, which binds Fur with high affinity (a K_d value of 1.5 nM), occurs as early as that of fpbN (a K_d value of 5.6 nM). Fur binds to some early and middle genes with an affinity difference of three- to fourfold (Table 1 and SI Appendix, Figs. S7 and S8), suggesting that other factors may also affect the regulation of some Fur target genes. Indeed, previous studies have revealed that multiple transcription factors such as ResD, NsrR, and Fur interact with one another and coregulate transcription of ykuN and other Fur-regulated genes during anaerobic fermentation and nitrate respiration (32, 33). It is also known that the bacillibactin uptake system (FeuABC) is under regulation of both Fur and Btr (13, 14) (see below).

Stepwise Induction of Fur Regulon in Response to Iron Limitation in Strains Expressing Bacillibactin.

In most environmental isolates of B. subtilis, the mature siderophore BB is synthesized and exported by YmfD, a major facilitator superfamily transporter (34). The ferric–siderophore complex (Fe³⁺-BB) is then imported by the FeuABC-YusV system and hydrolyzed by the BesA esterase to release iron (8). Once imported, BB binds to the Btr transcription activator and stimulates transcription of the feuABC operon. BB binds to Fe³⁺ with remarkably high affinity, comparable to the chemically similar enterobactin produced by enteric bacteria (7, 12), and is therefore an effective iron-scavenging agent. Indeed, sfp⁺ strains have higher growth rate and yield in minimal medium amended with either 25 µM (SI Appendix, Fig. S9A) or 1 mM iron (SI Appendix, Fig. S9B) compared with sfp⁰ strains. Many laboratory strains (based on B. subtilis 168) are sfp⁰ and therefore only produce the BB precursors DHB(G), which bind Fe³⁺ with only modest affinity (lower than citrate) and may not function as an effective siderophore in natural settings (7, 35). Moreover, DHB(G) is ineffective in activating Btr, although Btr does have significant stimulatory activity even in the absence of siderophore (14).

To further explore the graded response of the Fur regulon in strains producing BB, we monitored the induction of Fur target genes in sfp⁺ strains harboring an IPTG-inducible copy of frvA (sfp⁺ P_spac-frvA) as cells transition from iron sufficiency (LB + 25 µM iron) to deficiency. Consistent with the studies above (Fig. 2), Fur-regulated genes were derepressed in a stepwise manner with early (ypbN, efeU, and fecC), middle (fhuB, ykuN, dhbA, and feuA), and late (fsrA, fbpA, and fbpC) genes (Fig. 4). However, a few changes were notable: induction of all early and middle genes was shifted a little earlier, while induction of the late genes (fsrA, fbpA, and fbpC) was shifted later (Fig. 4). This suggests that efficient iron acquisition through Fe³⁺-BB delays proteomic remodeling via the iron-sparing response.

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Fig. 4.

Fur-regulated genes are induced in a similar stepwise manner as a function of iron depletion in strains producing bacillibactin. Overnight cell culture was inoculated with 1:100 ratio into fresh LB medium amended with 25 µM FeSO₄. After OD₆₀₀ reaches ∼0.25, 1 mM IPTG was added to induce expression of FrvA. Total RNA was extracted and subjected to cDNA synthesis followed by qPCR. Relative expression of each gene at different times was monitored by comparing gene expression in the IPTG-induced cells versus that in sfp⁺ WT cells. The percentage of derepression was evaluated based on the full derepression observed in sfp⁺ fur mutant versus sfp⁺ WT, which was set as 100% derepression.

Inactivation of Bacillibactin Uptake Affects Induction of the Fur-Regulated Genes.

Expression of feuA is induced ∼20-fold upon iron depletion in a sfp⁰ strain (SI Appendix, Fig. S10A). Previous studies revealed that BB, but not the precursor DHBA, increases binding of Btr to the feuA promoter and stimulates transcription (13, 14). Indeed, the expression of feuA was derepressed up to 68-fold in a sfp⁺ strain (SI Appendix, Fig. S10C), indicating a more than threefold activation by BB. In the absence of Btr, feuA mRNA levels were reduced >30-fold, although Fur repression of feuA is still relieved by iron depletion (SI Appendix, Fig. S10 B and D). This effect can also be observed in disk-diffusion assays (SI Appendix, Fig. S11A). BB and DHB(G) compounds bind to Fe³⁺ and form complexes of purple color apparent around the zone of inhibition resulting from overexpression of FrvA (SI Appendix, Fig. S11A). As expected, inactivation of bacillibactin uptake by deletion of btr leads to clearly visible accumulation of DHB(G) or BB in the medium (SI Appendix, Fig. S11A). This result was confirmed by quantitation of the secreted Fe³⁺-DHB(G) or Fe³⁺-BB complex (SI Appendix, Fig. S11 B and C).

Although deletion of btr does not change the order of derepression of Fur target genes (SI Appendix, Fig. S10 E and F), most genes are induced earlier than observed in experiments done in WT cells (Figs. 2 and 4). In sfp⁰ strains, 50% derepression shifts from 10, 20, 60 min to 5, 14, or 40 min for early, middle, or late genes, respectively (SI Appendix, Fig. S10E). In sfp⁺ strains, 50% derepression shifts from 8, 15, 70 min to 3, 8, or 35 min for early, middle, or late genes, respectively (SI Appendix, Fig. S10F). These results suggest that biosynthesis and uptake of BB is critical for maintaining iron homeostasis upon iron limitation. These results also confirm that Btr is required for significant expression of the feuA operon, and in the absence of Btr the production of iron chelators (BB or its precursors) that cannot be efficiently imported back into the cell results in a more rapid onset of iron starvation (SI Appendix, Fig. S10 E and F).

Iron-Sparing Response Induced by Iron Depletion Leads to an Increase in Intracellular Labile Iron.

As shown above, when cells experience severe iron limitation the iron-sparing response is engaged to modulate cellular proteomes and prioritize the use of limited iron (Figs. 2 and Fig. 4 and SI Appendix, Figs. S4, S5, and S10). Since the translation of many abundant, low-priority iron-utilizing proteins is blocked by FsrA and its coregulators, we anticipated that intracellular labile iron pools might actually increase under these conditions, despite the fact that total intracellular iron is depleted (Fig. 1A). Indeed, in Escherichia coli expression of the small RNA RyhB, analogous to FsrA in B. subtilis, leads to an increase in the intracellular iron pool (36, 37). To test this notion, we monitored the intracellular labile pool by testing sensitivity to streptonigrin (SN), an antibiotic that responds to intracellular labile iron levels (38). Indeed, when FrvA is expressed, it depletes total intracellular iron but the intracellular labile iron level increases, as judged by the higher sensitivity to SN in both LB plates without or with supplementing exogenous iron (Fig. 5). In the absence of the FsrA-mediated iron-sparing response, this increase in intracellular labile iron no longer occurs (Fig. 5), suggesting that the iron-sparing response is chiefly, if not solely, responsible for this increase.

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Fig. 5.

The iron-sparing response induced by iron depletion leads to an increase in intracellular labile iron. Sensitivity to streptonigrin (SN) was monitored using a disk-diffusion assay in LB agar plates containing either 0 (A) or 100 μM FeSO₄ (B). Strains tested in both panels are wild-type (WT, CU1065), fsrA null mutant, P_spac-frvA, and fsrA P_spac-frvA. The data are expressed as the diameter (mean ± SD; n = 3) of the inhibition zone (mm). Significant differences determined by two-tailed t test are indicated: *P < 0.05.