O₂ availability impacts iron homeostasis in Escherichia coli

Significance

Our understanding of how cells regulate intracellular iron pools has been largely shaped by studying cells grown under aerobic conditions, in which the barrier to iron acquisition is dominated by O₂-dependent insolubility. However, less is known about how bacteria meet their iron demands in the O₂-limiting or anaerobic environments that are common to many ecosystems and reflective of the ancient atmosphere of early Earth. Because the transcription factor ferric-uptake regulator (Fur) plays a central role in controlling iron homeostasis in many bacterial species, we use Fur activity as a surrogate for understanding how anaerobiosis alters iron homeostasis. Our finding that levels of active Fur are increased during anaerobiosis emphasizes fundamental differences in bacterial iron requirements between aerobic and anaerobic growth conditions.

Abstract

The ferric-uptake regulator (Fur) is an Fe²⁺-responsive transcription factor that coordinates iron homeostasis in many bacteria. Recently, we reported that expression of the Escherichia coli Fur regulon is also impacted by O₂ tension. Here, we show that for most of the Fur regulon, Fur binding and transcriptional repression increase under anaerobic conditions, suggesting that Fur is controlled by O₂ availability. We found that the intracellular, labile Fe²⁺ pool was higher under anaerobic conditions compared with aerobic conditions, suggesting that higher Fe²⁺ availability drove the formation of more Fe²⁺-Fur and, accordingly, more DNA binding. O₂ regulation of Fur activity required the anaerobically induced FeoABC Fe²⁺ uptake system, linking increased Fur activity to ferrous import under iron-sufficient conditions. The increased activity of Fur under anaerobic conditions led to a decrease in expression of ferric import systems. However, the combined positive regulation of the feoABC operon by ArcA and FNR partially antagonized Fur-mediated repression of feoABC under anaerobic conditions, allowing ferrous transport to increase even though Fur is more active. This design feature promotes a switch from ferric import to the more physiological relevant ferrous iron under anaerobic conditions. Taken together, we propose that the influence of O₂ availability on the levels of active Fur adds a previously undescribed layer of regulation in maintaining cellular iron homeostasis.

Iron homeostasis requires coordination between iron uptake, storage, and cofactor synthesis to supply iron for the iron-containing proteome (1–3). In bacteria, this coordination is orchestrated by transcription factors that are predicted to provide a direct readout of the cellular iron status (4). In particular, the transcription factor ferric uptake regulator (Fur) plays a leading role in many bacteria to maintain iron homeostasis by directly sensing levels of ferrous iron (Fe²⁺) and altering gene transcription accordingly (3, 5). Recently, we reported that transcriptional repression of many genes of the Escherichia coli K12 Fur regulon is enhanced under anaerobic growth conditions (6), suggesting that O₂ provides an additional input into Fur regulation.

The regulation of Fur activity by iron has provided the paradigm for understanding how bacteria maintain appropriate iron levels in vivo. When iron is available, each subunit of a Fur dimer binds one molecule of Fe²⁺ ion (7, 8). In this Fe²⁺-bound form, Fur exhibits increased affinity for specific DNA sequences [referred to a Fur boxes (9, 10)] and represses transcription of many iron assimilation pathways. In contrast, when iron is limiting, Fur shifts to an iron-free form, thereby inactivating Fur function. The low micromolar binding affinity of Fe²⁺ for Fur (11–13) affords a window into the physiologically relevant iron concentration that Fur responds to in cells. Indeed, many studies suggest that under aerobic growth conditions, the intracellular iron pool available for Fur binding is in the low micromolar range (14, 15). However, our recent finding that Fur-mediated repression is enhanced under anaerobic conditions (6) raises the question of whether the intracellular iron pool is altered during anaerobiosis.

O₂ tension influences the oxidation state of iron, and thereby the means by which it is acquired by cells (1, 3, 16, 17). When O₂ is absent, iron is primarily in its soluble, reduced Fe²⁺ form and can be taken up directly by bacteria via dedicated pathways (e.g., the FeoABC system). In contrast, when O₂ is present, iron is oxidized to an insoluble ferric (Fe³⁺) state. The uptake of Fe³⁺ involves the synthesis and secretion of Fe³⁺-chelating molecules called siderophores (e.g., enterobactin). Transport of the Fe³⁺-bound siderophore into bacterial cells involves specific outer membrane receptors, the energy-transducing TonB–ExbB–ExbD complex, and ABC-type transporters that ultimately deliver the Fe³⁺-siderophore to the cytoplasm, where the iron is reduced and released. Given the impact of O₂ on the oxidation state of iron and the fact that Fur controls expression of both ferrous and ferric iron assimilation pathways, an O₂-dependent input into the expression of the Fur regulon would make physiological sense.

In this study, we show that such an O₂-dependent mechanism exists by probing how O₂ impacts Fur activity. We investigated the correlation between increased anaerobic Fur DNA binding across the genome and increased transcriptional repression mediated by Fur. To dissect the mechanisms by which the amount of active Fur is elevated during anaerobiosis, we measured the levels of intracellular chelatable Fe²⁺ and Fur protein in aerobic and anaerobic cells, and we assayed Fur activity in strains lacking proteins involved in iron uptake or storage. Finally, we measured the response of Fur to O₂ following a shift from anaerobic to aerobic conditions and addressed the influence of other O₂-responsive transcription factors on expression of genes in the direct Fur regulon. Together, our findings provide insight on the means by which O₂-dependent regulation of Fur occurs, which involves the up-regulation of Fe²⁺ uptake under anaerobic conditions.

Results

Fur DNA Occupancy Increases During Anaerobiosis.

A previous genome-wide study of Fur–DNA interactions using ChIP-sequencing (ChIP-seq) revealed that under anaerobic conditions, Fur bound more regions in the E. coli K12 genome than under aerobic growth conditions (6). This finding led us to hypothesize that during anaerobiosis, an increase in the amount of active Fe²⁺-Fur could explain Fur binding to DNA sites that are more varied from the consensus Fur binding motif (6) than those occupied by Fur in both the presence and absence of O₂. Here, we build upon these results to address whether the O₂-dependent changes in expression observed for the Fur regulon (6) can be attributed to changes in Fur DNA occupancy genome-wide.

By quantifying the ratio of Fur enrichment for 74 of the highest intensity iron-dependent ChIP-seq peaks (6) using the statistics-based differential binding algorithm DBChIP (18), we found that, overall, the ratio of anaerobic to aerobic Fur ChIP-seq signal increased, ranging from ∼1.5- to 10-fold, with a median of threefold (Tables S1 and S2). The increase in the ratio of anaerobic to aerobic Fur ChIP-seq signal at each site suggested an increase in Fe²⁺-Fur binding at these sites.

The anaerobic increase in genome-scale Fur DNA binding appears to be transcriptionally relevant since, for most of the Fur regulon, the ratio of anaerobic to aerobic Chip-seq signal correlated positively with the fold increase in Fur-mediated repression calculated from anaerobic and aerobic transcriptomic data, respectively (Table S1). For example, Fur binding to the promoter regions of exbB and tonB (both involved in Fe³⁺-siderophore transport) and bfd (involved in iron storage/release from bacterioferritin) increased 6.2-, 5.2-, and 3.9-fold under anaerobic conditions, respectively (Fig. 1 and Table S1). This corresponded to a 6.0-, 3.3-, and 7.0-fold respective increase in Fur-mediated repression of these genes under anaerobic conditions compared with aerobic conditions (Table S1). In instances where the ChIP-seq ratio measured for Fur occupancy in anaerobic and aerobic cells was closer to 1, smaller O₂-dependent changes in gene expression were detected (e.g., entC and fhuE, involved in Fe³⁺-siderophore transport; ryhB, encodes a regulatory small RNA) (Table S1). This latter group of promoters represents some of the more strongly repressed Fur genes (6, 19), and the lower ratio likely stems from nearly comparable Fur binding under both aerobic and anaerobic conditions. In summary, these data indicate that under anaerobic conditions, Fur binding is increased at many promoters, which can lead to comparable changes in Fur-mediated gene repression.

Fig. 1.

Occupancy of Fur binding regions increases during anaerobiosis. Fur ChIP-seq peak regions for exbB, tonB, and bfd from cells grown under anaerobic (blue) or aerobic (red) conditions in MOPS minimal glucose media containing 10 μM FeSO₄, were plotted from data reported by Beauchene et al. (6). Shown are three replicates for each growth condition. The x axis indicates the genomic position of the ChIP-seq peak, and the y axis indicates the read count after each dataset was normalized to 20 million reads. The ratio of the anaerobic ChIP signal to aerobic ChIP signal for all Fur ChIP-seq peaks is presented in Tables S1 and S2.

A Fur Binding Site Is Sufficient to Confer Regulation by O₂.

To test if the presence of a Fur DNA binding site is sufficient to confer O₂-dependent regulation as suggested by our genome-wide analysis, we engineered a synthetic Fur-dependent promoter (P_fepBS), in which the tac promoter (P_tac) contains the Fur binding site from P_fepB (which drives expression of FepB, an enterobactin binding protein). This binding site is predicted to bind two Fur dimers (20, 21) and was positioned in P_tac to maintain the same spacing (−19 to +21 bp) with respect to the promoter −35 hexamer from P_fepB. In a strain containing P_fepBS driving expression of a chromosomal lacZ reporter gene, β-galactosidase activity was significantly decreased when Fur was present (Fig. S1), indicating that Fur mediates repression of P_fepBS in vivo. Furthermore, Fur-dependent repression was increased threefold under anaerobic growth conditions compared with aerobic growth conditions (Fig. 2), demonstrating a direct effect of O₂ availability on Fur-dependent regulation of P_fepBS. Taken together, the enhancement in Fur occupancy at genomic sites, along with the increase in Fur repression under anaerobic conditions, provides evidence for a direct role of Fur in the global response to anaerobiosis.

Fig. 2.

Fur-dependent repression of a synthetic promoter containing a Fur DNA binding site is influenced by O₂ and the FeoABC ferrous uptake system. Fur-mediated repression of a chromosomal P_tac-lacZ fusion bearing the E. coli fepB Fur binding site (P_fepBS) is shown in wild-type, Δfur, ΔfeoB, ΔfecC, ΔentA, and ΔftnA Δbfr Δdps strains grown under aerobic (gray bars) or anaerobic (white bars) conditions in MOPS minimal glucose media containing 1.0 μM FeSO₄. Fold repression was calculated by dividing the average promoter activity in the ∆fur strain by the average promoter activity in each of the other strains tested (at least two replicates of each; promoter activity was determined using β-galactosidase assays, as shown in Fig. S1), and error bars represent the propagation of errors calculated according to the method of Ku (45).

Additional Transcription Factors Can Tailor O₂-Mediated Expression of Some Members of the Fur Regulon.

For a few members of the Fur regulon, we hypothesized from previous results that the impact of O₂ on their transcription might also require the O₂-regulated transcription factors FNR and ArcA (22, 23). Therefore, we measured expression of lacZ fusions to P_exbB, P_fiu, P_cirA, or P_feoA in strains lacking one or more of these transcription factors. In the absence of any mutations, expression of P_exbB, P_fiu, and P_cirA decreased under anaerobic conditions (Fig. 3 A–C), in agreement with transcriptomic data (6). For P_exbB, we found that deletion of fur increased expression to comparable levels under both aerobic and anaerobic conditions, indicating Fur-mediated repression was sufficient to explain its decreased expression under anaerobic conditions (Fig. 3A). Deletion of fnr had no effect on P_exbB expression, despite a reported FNR binding site (22). In contrast, for P_fiu and P_cirA, we found that decreased expression of these promoters under anaerobic conditions was only partially dependent on Fur (Fig. 3 B and C). For both promoters, we found that ArcA also contributes to anaerobic repression (Fig. 3 B and C), in agreement with ArcA binding these promoter regions in vivo (23). This additional role of ArcA likely explains why the increase in Fur binding to P_fiu and P_cirA anaerobically did not correlate well with the fold increase in Fur-mediated repression (Table S1).

Fig. 3.

Fur and other O₂-regulated transcription factors tailor the expression of iron-uptake systems. Strains bearing lacZ fusions to P_exbB (A), P_fiu (B), P_cirA (C), and P_feoA (D) were assayed for β-galactosidase activity under aerobic (gray) or anaerobic (white) growth conditions. Deletion of the relevant transcription factor in the reporter strain is indicated by (−). Cultures were grown in MOPS minimal glucose media containing 1.0 μM FeSO_4. Error bars represent the SE from triplicate experiments.

Similar analysis of the promoter driving transcription of feoABC, encoding a ferrous uptake system, revealed several distinctive features (Fig. 3D). First, in contrast to the ferric uptake systems, P_feoA is poorly expressed aerobically even in the absence of Fur. Second, unlike the ferric uptake systems (e.g., cirA, exbB, fiu), expression of P_feoA increased a small amount under anaerobic conditions compared with aerobic conditions, despite increased anaerobic Fe²⁺-Fur levels. Third, we found that this increased anaerobic expression of P_feoA required both of the anaerobic transcription factors FNR and ArcA, as suggested by genome-wide and other data (22–24). This positive effect of both ArcA and FNR appears to limit the negative effect of Fur under anaerobic conditions. Thus, FNR and ArcA produce an expression pattern for P_feoA under anaerobic conditions opposite to the expression pattern of the ferric transport systems. Taken together, these data support the notion that Fur plays a central role in reprogramming gene expression profiles in response to O₂ because of changes in levels of Fe²⁺-Fur available for DNA binding and by Fur acting in concert with other transcription factors.

The Labile Fe²⁺ Pool Increases During Anaerobiosis.

To determine the basis for increased levels of Fe²⁺-Fur under anaerobic conditions, we measured the levels of both Fur protein and iron. Western blot analysis revealed that Fur protein levels are comparable under aerobic (22 μM) and anaerobic (17 μM) growth conditions (Fig. S2). To test if increased intracellular Fe²⁺ availability could explain the increase in active Fe²⁺-Fur protein levels under anaerobic conditions, we used chelator-assisted electron paramagnetic resonance (EPR) spectroscopy (25) to measure the intracellular chelatable Fe²⁺ pool. This labile pool is estimated to be 1.0% of cellular iron (14) and is defined as iron not tightly bound to cellular proteins, and possibly associated with metabolites, such as glutathione, citrate, or phosphorylated sugar compounds (25–27). We found that the amount of chelatable Fe²⁺ was approximately sevenfold higher in anaerobic cells compared with aerobic cells (Fig. 4 A and B). When total cellular iron levels (which encompass both Fe²⁺ and Fe³⁺ in proteins and the labile iron pool) were measured by inductively coupled plasma mass spectrometry, there was only a slight increase in the total iron content of anaerobically grown cells (Fig. 4C). These results indicate that while total cellular iron in aerobically and anaerobically grown cells is comparable, the labile iron pool (that which is accessible for Fur binding) is higher in the absence of O₂.

Fig. 4.

Intracellular labile Fe²⁺ levels increase during anaerobiosis. EPR spectra showing the signal at g = 4.3, normalized for OD₆₀₀ from aerobic or anaerobic cells grown in MOPS minimal glucose media containing 1.0 μM FeSO₄. Aerobic (red) or anaerobic (blue) cells were treated with desferoxamine mesylate (DFO) or were left untreated (black). (A) Four to five replicates of each experiment are shown. (B) Chelatable Fe levels in aerobic (gray) or anaerobic cells (white) calculated from EPR spectra. (C) Inductively coupled plasma mass spectrometry was used to measure whole-cell Fe levels in three biological replicates of aerobic (gray) or anaerobic cells (white), which were grown in MOPS minimal glucose media containing 1.0 μM FeSO₄ and normalized for cell pellet (milligrams). (D) Viable cell counts (assayed in triplicate) for aerobic (gray) or anaerobic cells (white) grown in MOPS minimal glucose media containing 1.0 μM FeSO₄, with and without treatment of 2.5 mM H₂O₂ for 15 min. For B, C, and D, error bars represent the SE.

To provide further support for an elevated labile Fe²⁺ pool in anaerobic cells, we assayed the sensitivity of cells to hydrogen peroxide (H₂O₂). H₂O₂ is reduced by Fe²⁺, resulting in the formation of hydroxyl radicals via the Fenton reaction (28). Hydroxyl radicals are highly detrimental to the integrity of cellular components, such as DNA (29, 30), and can lead to cell death. We determined the sensitivity of aerobic and anaerobic cells to H₂O₂ using a strain lacking RecA, which is defective in DNA repair and thus more sensitive to H₂O₂ compared with a wild-type strain (31). Anaerobically grown cells showed 10-fold more sensitivity to killing by H₂O₂ than aerobically grown cells (Fig. 4D), suggesting that Fe²⁺ is more readily available in anaerobic cells to promote hydroxyl radicals and DNA damage. These results agree with previous findings that anaerobic cells are highly sensitive to H₂O₂ (31, 32).

Taken together, these data show that increased levels of Fe²⁺-Fur under anaerobic conditions are not due to elevated Fur protein levels, but rather to an increase in the labile Fe²⁺ pool. Consequently, these higher Fe²⁺ levels would allow for increased formation of Fe²⁺-Fur, and thus explain increased Fur DNA binding and transcriptional repression during anaerobiosis.

Regulation of the Labile Iron Pool by O₂.

To determine how the labile iron pool is increased under anaerobic conditions, we asked whether the source of iron (Fe²⁺ or Fe³⁺) or media composition (rich versus minimal) influences Fur-mediated repression under aerobic or anaerobic conditions. Fur-mediated repression was assayed using the synthetic P_febBS-lacZ fusion. We did not find any changes in O₂-dependent regulation of Fur activity when the media were varied from our standard 3-(N-morpholino)propanesulfonic acid (MOPS) minimal media (Fig. S3). We also considered the possibility that decreased repression by Fur under aerobic conditions could be explained if iron uptake was less efficient due to the decreased solubility of iron in the presence of O₂. However, increasing external iron levels 10-fold resulted in no change in the levels of Fur-mediated repression under aerobic conditions, suggesting that iron availability was not limiting (Fig. S3). In contrast, under anaerobic conditions, repression by Fur was increased even further, suggesting that iron transport could be contributing to Fur regulation under anaerobic conditions.

Since the FeoABC system has a major role in anaerobic Fe²⁺ uptake (17, 33, 34), we investigated whether anaerobic regulation of Fur was disrupted in a strain lacking feoB. Indeed, we found that elimination of the FeoB iron transporter caused a threefold defect in P_fepBS repression by Fur under anaerobic conditions (Fig. 2). Furthermore, O₂-dependent regulation of Fur required FeoB since Fur repressed P_fepBS to a similar extent under aerobic and anaerobic conditions in the ΔfeoB mutant (Fig. 2). The effect of feoB on Fur activity did not appear to reflect a general defect in iron-containing transcription factors since we did not find any change in activity of two Fe-S cluster-containing transcription factors (FNR and IscR) when similarly assayed under anaerobic conditions (Fig. S4). These data suggest that FeoABC contributes to the O₂-dependent regulation of Fur by increasing the anaerobic labile iron pool available for Fur binding.

We also tested whether other iron transport systems affected Fur activity (3). Enterobactin-mediated Fe³⁺ uptake was disrupted by deletion of entA. The inability to synthesize the enterobactin siderophore caused a twofold loss in the ability of Fur to repress P_fepBS under both aerobic and anaerobic conditions (Fig. 2). However, the EntA⁻ mutant retained O₂-dependent regulation of Fur, presumably because of the continued function and regulated expression of FeoABC (Fig. 2). As a control, we showed that Fur-mediated repression was not affected in a strain lacking fecC, involved in transport of ferric citrate, which was not added to our media (Fig. 2).

Finally, we tested whether iron storage proteins affected the amount of iron available for Fur binding by measuring Fur-mediated repression in iron storage-deficient strains. The only known mechanisms of iron storage in E. coli require O₂ or H₂O₂ (35), suggesting that less iron might be stored anaerobically, and potentially contributing to the increase in the labile iron pool. However, elimination of iron storage proteins by deletion of ftnA, bfr, and dps did not alter the level of Fur-mediated repression under either aerobic or anaerobic conditions (Fig. 2).

Fur Activity Responds to a Shift in O₂ Availability.

Adapting to changes in O₂ tension is key for most organisms. Given the significant differences in the bioavailability of iron in the presence and absence of O₂, we wanted to test if the sudden introduction of O₂ to anaerobic cells altered Fur activity. To do this, we assayed β-galactosidase from lacZ fusions to either our synthetic promoter (P_fepBS) or the fepA promoter (P_fepA), which drives expression of the FepA Fe³⁺-enterobactin transporter. Upon exposure to either O₂ or α,α′-dipyridyl, a known cell-permeable iron chelator, we observed an increase in P_fepBS and P_fepA expression (Fig. 5 and Fig. S5). Although the rate of increase in expression of these promoters after exposure to O₂ was slower than with α,α′-dipyridyl, de-repression of these promoters occurred on a similar time scale.

Fig. 5.

Anaerobic Fur activity decreases upon exposure to O₂. A strain containing P_fepBS-lacZ was grown anaerobically and was then either shifted to aerobic growth conditions (●) or treated with an iron chelator (200 μM α,α′-dipyridyl; ▲) at time 0. Samples were removed and assayed for β-galactosidase at various time points. Untreated cells (♦) are indicated. Cultures were grown in MOPS minimal glucose media containing 1.0 μM FeSO_4. Error bars represent the SE from three experiments. (Inset) Expanded view of O₂- and α,α′-dipyridyl–treated cells from time 0–20 min.

To determine how other genes that are directly repressed by Fur respond to O₂ exposure, we analyzed time series transcriptomic data from RNA isolated from E. coli at 0.5, 1, 2, 5, and 10 min after a shift from anaerobic to aerobic conditions [first reported by von Wulffen et al. (36)]. Similar to what we observed for P_fepBS and P_fepA, expression of these genes showed an immediate response to the addition of O₂, with almost all RNA levels increasing by 2 min (Table S3). Thus, these data indicate that expression of the Fur regulon responds to sudden changes in environmental O₂, conditions in which E. coli would experience in its natural habitat.

Discussion

Our data provide insight into how O₂ availability alters iron homeostasis in the facultative bacterium E. coli. Overall, we propose that an increase in the intracellular labile Fe²⁺ pool, mediated by the FeoABC ferrous transport system, leads to an increase in Fur-mediated repression under anaerobic conditions. The rise in anaerobic Fe²⁺ availability increases the population of Fur protein that is bound to Fe²⁺ and, accordingly, drives Fur DNA binding and transcriptional repression. The global change in expression of the Fur regulon under iron-sufficient anaerobic conditions promotes a shift in expression from Fe³⁺ to Fe²⁺ transport systems, allowing E. coli to use the most physiologically relevant form of iron.

The Labile Iron Pool Increases Under Anaerobic Conditions.

It is generally considered that the affinity of a metal sensor for its metal, and the subsequent homeostatic control the regulator exerts on its regulon, defines intracellular free metal concentrations (37–39). Thus, before this work, one would expect a priori that the intracellular free (labile) Fe²⁺ pool would be comparable in aerobically and anaerobically grown cells. Despite this expectation, we found that intracellular chelatable iron levels increased nearly sevenfold (∼26 μM in aerobic cells and ∼177 μM in anaerobic cells) under anaerobic conditions. Previous studies have shown that the FeoABC transporter, which is widely distributed in bacteria (17), contributes to anaerobic iron uptake (33, 34). Therefore, our finding that FeoABC was required for increased Fur activity under anaerobic conditions provided a link between increased iron import under anaerobic conditions and the anaerobic labile Fe²⁺ pool. The ability of FeoABC to mediate an increase in the iron pool under anaerobic conditions appears to be due, in part, to its unique mechanism of transcriptional regulation (discussed below).

Fur Fe²⁺ Occupancy Varies with O₂ Tension.

Since the affinity of Fur for Fe²⁺ is estimated at 1.2–55 μM (11–13), and the cellular Fur concentration is comparable between aerobic (∼22 μM) and anaerobic (∼17 μM) cells, our data suggest that the fraction of Fur bound by Fe²⁺ is impacted by O₂-dependent changes in Fe²⁺ levels. We propose that under aerobic conditions even when iron levels are sufficient, Fur is not completely occupied with Fe²⁺, whereas under anaerobic conditions, Fur-Fe²⁺ occupancy increases due to the increase in the labile iron pool (Fig. 6). It was also surprising that the decrease in iron available for Fur binding observed with the ΔfeoB mutant, did not impact gene regulation mediated by the transcription factors FNR and IscR that ligate Fe-S clusters. This result could imply that the affinity of iron for Fe-S cluster biogenesis machinery is much stronger than the binding affinity to Fur, creating a hierarchy of iron usage in the cell.

Fig. 6.

O₂-dependent regulation of the Fur regulon. How O₂ influences the Fe²⁺ occupancy of Fur under aerobic and anaerobic growth conditions is depicted. In the absence of O₂, increased intracellular labile Fe²⁺ levels, which are mediated by the FeoABC system, promote Fur Fe²⁺ occupancy, and thus lead to increased repression of Fur-dependent promoters. In the presence of O₂, cells shift to using Fe³⁺ uptake systems and intracellular labile Fe²⁺ levels are lower compared with those in anaerobic cells. As a result, levels of Fe²⁺-Fur are decreased, leading to partial occupancy and less repression of promoters containing less conserved Fur binding sites. However, promoters containing more conserved Fur binding sites remain repressed.

The Strength of Fur DNA Recognition Sites May also Contribute to O₂ Regulation of the Fur Regulon.

The effect of O₂ on Fur-mediated repression varied for individual members of the Fur regulon. Some genes exhibited only small differences in Fur-mediated regulation between aerobic and anaerobic conditions, whereas others showed a much stronger dependence on anaerobic conditions to observe regulation by Fur. To explain these findings, we propose that the increase in Fe²⁺-Fur levels under anaerobic conditions allows binding to Fur sites of a wider range of affinities. In contrast, under aerobic conditions, the reduced levels of Fe²⁺-Fur bias binding to stronger affinity Fur sites. In support of this model, bioinformatic analysis of sites bound by Fur only under anaerobic conditions was much further from consensus than that of sites bound by Fur both in the presence and absence of O₂ (6). Although, there is no resource to compare Fur affinities obtained under similar solution conditions for individual sites across the regulon, information theory predictions of Fur binding site strength suggest that many of the DNA sites, which bound Fur well both in the presence and absence of O₂ (e.g., ryhB, entC, cirA, yjjZ, fepA), should be high-affinity sites (21). Thus, the strength of the Fur DNA binding site likely contributes to how much of an effect O₂ has on an individual regulon member. For the Fur homolog Zur, the affinity of the transcription factor for its DNA sites also correlates to the fold repression (40). Further, it has been shown that stepwise metallation of the Zur Zn binding sites fine-tunes the degree of Zur repression of its target promoters (41, 42).

Physiological Significance of O₂ Regulation of Fur.

Our data reveal that the O₂-dependent changes in levels of active Fur play a role in tailoring expression of iron uptake systems to the growth conditions in which their substrate (Fe²⁺ or Fe³⁺) is most likely to be found. In most cases, Fur-mediated repression was sufficient to explain decreased expression of Fe³⁺ uptake systems under anaerobic conditions. However, for a few Fe³⁺ uptake systems (e.g., fiu, cirA), decreased expression under anaerobic conditions required repression by both ArcA and Fur. In contrast, anaerobic expression of the FeoABC Fe²⁺ uptake system is under positive control of ArcA and FNR. This regulatory mechanism prevents additional Fur-mediated repression of P_feoA (unlike that of the ferric uptake systems) and explains how expression increases under anaerobic conditions even when Fur is more active. By wiring feoABC expression in this manner, Fe²⁺ can enter cells through a transport system whose expression is not strictly controlled by Fur under anaerobic growth conditions. In turn, anaerobic up-regulation of feoABC facilitates the increase in the iron pool and recalibrates iron homeostasis.

It might be counterintuitive that even small changes in Fur-mediated repression between aerobic and anaerobic conditions could be physiologically significant. However, it is important to note that under iron-sufficient conditions, the low expression levels of Fur-repressed iron uptake systems are sufficient to drive iron uptake. Iron uptake systems only become further induced when external iron becomes scarce. Thus, it is logical to assume that even small changes in levels of these transport systems will affect iron uptake rates.

In summary, our data show that O₂ has a direct effect on levels of active Fur in vivo and that several factors contribute to the expression profile of the Fur regulon in response to O₂. The expression output of each gene is a summation of Fur activity, which depends on the extent of Fur Fe²⁺ occupancy, the Fur DNA binding sequence, and coordinated regulation by other transcription factors. Our data highlight the complexity of coordinating a central cellular activity like iron homeostasis to multiple environmental cues. This fine-tuning of transcriptional regulation ensures that the necessary gene products are available for cellular function. Finally, we predict that the differential effect of O₂ on iron homeostasis observed in this study is likely to be conserved in other Fur-containing facultative bacteria since it reflects an adaptation to the intrinsic sensitivity of Fe²⁺ to oxidation in aerobic environments.

Methods

Detailed materials and methods can be found in SI Methods.

Differential Fur DNA Binding Analyses.

Differential binding of Fur between aerobic and anaerobic conditions was assessed using the algorithm DBChIP (18). This analysis was performed on 74 previously reported iron-dependent Fur ChIP-seq peaks from anaerobically grown E. coli with read counts >4,000 at the peak summit (6). Read counts of each dataset were normalized to 20 million reads. The fold change between aerobic and anaerobic Fur DNA occupancy, reported here as the ChIP ratio, was calculated by dividing the average ChIP signal from three anaerobic replicates by that of three aerobic replicates. Fur was annotated as differentially bound if the adjusted P value of the fold change was <0.05.

Growth Conditions.

For the β-galactosidase assays (Figs. 2, 3, and 5 and Figs. S1 and S3–S5) and H₂O₂ sensitivity assays (Fig. 4D), strains were grown at 37 °C in MOPS minimal 0.2% glucose media (43) containing either 1.0 μM or 10 μM FeSO₄ under aerobic conditions (by shaking) or anaerobic conditions (using filled screw-capped tubes) as described previously (44). In Fig. S3, different media and iron sources are indicated in the figure legend. β-galactosidase assays were performed as described (6).

For the in vivo O₂ shift experiments (Fig. 5 and Fig. S5), strains bearing P_fepA-lacZ or P_fepBS-lacZ were grown at 37 °C in MOPS minimal 0.2% glucose media containing 1.0 µM FeSO₄ by sparging with an anaerobic gas mix of 95% N₂ and 5% CO₂. At time 0 min, which corresponded to an OD₆₀₀ of 0.1, cultures were either shifted to aerobic conditions by switching the gas mix to 70% N₂, 5% CO₂, and 25% O₂, or were treated with α,α′-dipyridyl (final concentration of 200 µM). Samples were removed at various time points for measurement of OD₆₀₀ and β-galactosidase activity as described by Beauchene et al. (6).

For EPR spectroscopy and Western blot analysis, cultures were grown at 37 °C in MOPS minimal glucose media containing 1.0 μM FeSO₄ to an OD₆₀₀ of ∼0.4 (Lambda 25 UV/Vis Spectrophotometer; PerkinElmer) by sparging with either an aerobic (70% N₂, 5% CO₂, 25% O₂) or anaerobic (95% N₂, 5% CO₂) gas mix (44).

Chelator-Assisted EPR Spectroscopy.

A MG1655 culture volume of 450 mL (OD₆₀₀ of ∼0.4) was pelleted, resuspended in 10 mL of fresh media containing either 20 mM membrane-permeable iron chelator desferoxamine mesylate (DFO; Sigma Aldrich) and 10 mM membrane-impermeable iron chelator diethylenetriaminepentaacetic acid (DTPA; Sigma Aldrich) or just 10 mM DTPA (to remove contaminating iron) as described previously (25). After a 15-min incubation at 37 °C, cells were washed twice with 5 mL of 20 mM Tris⋅HCl (pH 7.4) and resuspended in 350 μL of 20 mM Tris⋅HCl (pH 7.4) and 30% glycerol. A portion of these cells (200 μL) was transferred to EPR tubes (4-mm Thin Wall Quartz EPR sample tube with a length of 250 mm; Wilmad Lab Glass) and frozen at −80 °C. The remaining cells were diluted to measure the final OD₆₀₀ (Lambda 25 UV/Vis Spectrophotometer; PerkinElmer). All manipulations were completed in a Coy anaerobic chamber. Four to five replicates of each sample were analyzed.

H₂O₂ Sensitivity Assays.

Cells were treated with 2.5 mM H₂O₂ (equilibrated in an anaerobic chamber for anaerobic cultures) for 15 min at 37 °C (31) and then immediately diluted 1,000-fold into fresh MOPS glucose minimal media containing 1.0 μM FeSO₄. Treated and nontreated cultures were plated on TYE agar plates to determine the number of viable cells. Anaerobic conditions were maintained by use of a Coy anaerobic chamber and GasPak anaerobic jar.