Role of Porins in Iron Uptake by Mycobacterium smegmatis

Chemicals and enzymes.Hygromycin B was purchased from Calbiochem. All other chemicals were purchased from Merck, Roth, or Sigma at the highest purity available. Enzymes for DNA restriction and modification were from New England Biolabs, Invitrogen, and Boehringer. Oligonucleotides were obtained from Integrated DNA Technologies. ⁵⁵FeCl₃ was purchased from Perkin-Elmer, and [1,5-¹⁴C]citric acid was purchased from GE Healthcare.

Bacterial strains, media, and growth conditions.All bacterial strains used in this study are listed in Table 1. Unless otherwise noted all mycobacterial strains were grown at 37°C in Middlebrook 7H9 liquid medium (Difco Laboratories) supplemented with 0.2% glycerol, 0.05% Tyloxapol, or on Middlebrook 7H10 agar (Difco Laboratories) supplemented with 0.2% glycerol. Escherichia coli DH5α was used for all cloning experiments and was routinely grown in LB medium at 37°C. Hygromycin was used when required at the following concentrations: 200 μg ml⁻¹ for E. coli and 50 μg ml⁻¹ for mycobacteria. For iron-dependent growth experiments a minimal medium consisting of 500 μM MgCl₂·6H₂O, 7 μM CaCl₂·2H₂O, 1 μM NaMoO₄·2H₂O, 2 μM CoCl₂·6H₂O, 6 μM MnCl₂·4H₂O, 7 μM ZnSO₄·7H₂O, 1 μM CuSO₄·5H₂O, 15 mM (NH₄)₂SO₄, 12 mM KH₂PO₄ (pH 6.8), 1% (vol/vol) glycerol was supplemented with ammonium ferric citrate as an iron source as indicated in the text and figures. Ferric citrate was made using a Fe³⁺/citrate molar ratio of 1:200. To minimize trace iron contamination, bottles containing medium stock solutions were washed in 6 M HCl and solutions were prepared with highly purified water (Barnstead Nanopure Diamond; 18.2 MΩ-cm). All low-iron growth experiments (see Fig. 1A and 4 below) were carried out in polystyrene culture tubes (Becton Dickinson) to avoid iron leaching from glass. Low-iron growth was operationally defined as iron-dependent growth with high IdeR activity and high chrome azurol S (CAS) activity compared to cultures grown at higher iron concentrations.

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FIG. 1.

Loss of porins in M. smegmatis results in increased expression of iron-repressed genes. (A) Validation of a cytoplasmic iron reporter. M. smegmatis harboring an fxbA-gfp fusion (pML1801) was grown to mid-logarithmic phase in minimal medium containing different ferric citrate concentrations. (B) The porin mutant showed increased IdeR activity. M. smegmatis SMR5 (wt), ML16 (ΔmspA ΔmspC ΔmspD), and ML56 (ΔmspA ΔmspC ΔmspD attB _L5::p_imyc-mspA) harboring the iron reporter were grown in a high-iron minimal medium containing 150 μM ammonium ferric citrate. In both panels, fluorescence intensities of M. smegmatis cells were determined in triplicate and normalized to the optical densities of the samples. Standard deviations are shown as error bars. AU, arbitrary units.

Construction and validation of an intracellular iron reporter for mycobacteria.An iron-regulated reporter was constructed similar to that described previously (8). The M. smegmatis gene fxbA and its corresponding cis-regulatory elements were transcriptionally fused to the codon-optimized gfp _m²⁺ gene (33) by overlap extension PCR. A region encompassing the first 23 codons of fxbA down to −187 from the transcription start site of fxbA was amplified from the M. smegmatis genome by PCR using primers 1534 (5′-CACGCTCTAGATCGTTGACCAGGACCACG-3′) and 1559 (5′-TCCTCGCCCTTCGAGATATCCATGACCACGCGCACAGG-3′) (the introduced restriction sites are underlined). gfp _m²⁺ was amplified with a 5′ fxbA overhang separated by an EcoRV restriction site by using the primers 1558 (5′-GTGCGCGTGGTCATGGATATCTCGAAGGGCGAGGAGCTG-3′) and 1535 (5′-CCTGCGAAGCTTCTACTTGTACAGCTCGTCCATG-3′) and pMN437 as a template (Table 2). The products of these two reactions were amplified using primers 1534 and 1535 to create the fusion construct, which was then cloned into pMS2 via the XbaI and HindIII sites to create pML1801.

M. smegmatis SMR5 (wt), ML16 (ΔmspA ΔmspC ΔmspD), and ML56 (ΔmspA ΔmspC ΔmspD attB_L5 ::p_imyc-mspA) were grown on 7H10 plates for 3 days. A single inoculum was prepared from plates by resuspending bacteria into a minimal medium without any added iron. This resuspension was used to inoculate 4 ml minimal medium containing 0.25, 0.5, 1.0, 5, 10, or 20 μM ferric citrate in 14-ml polystyrene tubes to an optical density at 600 nm (OD₆₀₀) of 0.01. Precultures were incubated for 30 h at 37°C, washed, and resuspended in minimal medium containing identical iron concentrations. When the cultures were in the mid-logarithmic growth phase (OD₆₀₀, 0.8 to 2.0), 650-μl aliquots were harvested by centrifugation. The cells were washed twice in Tris-buffered saline with Tween buffer, and green fluorescent protein (GFP) fluorescence intensities were determined in 96-well plates by using a Biotek Synergy HT plate reader with an excitation at 485 nm and a 528-nm ± 20-nm emission filter. Fluorescence intensities were normalized to the optical density of the same samples.

Photometric determination of secreted siderophores.The CAS assay was modified based on a previously published protocol (29). A working solution containing 100 mM potassium acetate (pH 5.5), 600 μM hexadecyltrimethylammonium bromide, 150 μM chrome azurol S, and 15 μM FeCl₃·6H₂O was prepared. For activity determinations media and culture filtrate were mixed in a 1:1 ratio with the working solution. Solutions were incubated for 30 to 45 min at 37°C, and absorbance was measured at 630 nm. The difference in the absorbance at 630 nm between medium and culture filtrates was normalized to the optical density of the cultures. All samples were analyzed in triplicate. The signal from the CAS assay is highest when cultures are grown with iron concentrations less than 5 μM for more than 16 generations, which presumably exhausts intracellular iron stores and promotes the production of siderophores. We chose 0.25 μM ferric citrate for our experiment (see Fig. 4B) to promote siderophore-dependent growth and limit the amount of interference from the 200-fold excess of citrate.

Uptake assays.Uptake experiments were done as published previously (42) with the following modifications. M. smegmatis SMR5 (wt), ML16 (ΔmspA ΔmspC ΔmspD), and ML56 (ΔmspA ΔmspC ΔmspD attB_L5 ::p_imyc-mspA) were grown on 7H10 plates for 3 days. Cells were scraped into 4 ml 7H9 medium and were filtered through a 5-μm filter to remove cell clumps. These cells were used to inoculate 4 ml of 7H9 Middlebrook medium. The preculture was grown overnight and used to inoculate 100 ml of 7H9 Middlebrook medium. This culture was grown to mid-logarithmic phase (OD₆₀₀, 0.6 to 1.0) and harvested by centrifugation (3,250 × g for 10 min at 4°C). Cells were washed twice in a buffer consisting of 50 mM Tris, pH 6.9 [⁵⁵Fe(Cit)₂ ⁵⁻ and Fe(¹⁴Cit)₂ ⁵⁻ uptake], or 50 mM KH₂PO₄, pH 7.0 (⁵⁵Fe-exochelin MS [ExoMS] uptake), supplemented with 15 mM KCl, 10 mM (NH₄)₂SO₄, 1 mM MgSO₄, or 200 μM sodium citrate [⁵⁵Fe(Cit)₂ ⁵⁻ and Fe(¹⁴Cit)₂ ⁵⁻ uptake only] and resuspended in the same buffer to an OD₆₀₀ of approximately 2.5 on ice. For uptake experiments 2 ml of cell suspension was equilibrated at 37°C for 5 min and shaken at ≈400 rpm. Radiolabeled substrates were added to cells as indicated in the figure legends and the text. Samples of 200 μl were removed at 1, 2, 4, 8, and 16 min and added to 400 μl of a killing buffer consisting of 100 mM LiCl, 50 mM EDTA in 4% formaldehyde in Spin-X filter microcentrifuge tubes. Cells were immediately centrifuged and washed twice in killing buffer. The radioactivity of the cells was quantified by liquid scintillation counting (Beckman Coulter LS6500). All experiments were performed in triplicate. Radioactive counts were normalized to the dry weights of cells by determining the dry mass of 4 ml of the washed cell suspensions.

Purification of ferri-exochelin MS.ExoMS was purified from M. smegmatis as published previously (30). Briefly, 2 liters of a minimal medium supplemented with 0.25 μM ammonium ferric citrate and 50 μM sodium citrate was inoculated from an identical overnight preculture of the porin mutant M. smegmatis ML16 (34). The culture filtrate was isolated by filtration through a 0.2-μm filter after 72 h of growth and saturated with FeCl₃ until a precipitate formed. The iron-saturated culture filtrate was incubated at room temperature for 1 h to ensure formation of Fe-ExoMS. The mixture was filtered to remove insoluble iron and passed through cation exchange resin (AG50W X-8, NH₄ ⁺ form; 80 cm by 1.6 cm) by high-performance liquid chromatography at 2.5 ml min⁻¹, washed with deionized water at 2.5 ml min⁻¹, and eluted with 1 M NH₄Cl-NH₄OH (pH 9.6) at 5 ml min⁻¹. Deep-orange fractions eluted after 70 ml and had a characteristic absorbance at 422 nm. Fractions with absorbance at 422 nm exceeding the baseline were pooled (∼50 ml), evaporated to dryness, resuspended in approximately 5 ml deionized water, and desalted by gel filtration (Sephadex G-10). Desalted material was quantified by UV-visible spectroscopy with a λ_max of 422 nm and ε of 2,860 M⁻¹ cm⁻¹ as published elsewhere for exochelin MS (7). Iron was removed from this purified material by mixing with an appropriate amount of 8-hydroxyquinoline (8HQ) overnight in a 60:40 MeOH:H₂O solution (∼50 ml; [Fe-ExoMS], ∼100 μM; [8HQ], ∼500 mM). Fe-8HQ was extracted with 5 volumes of chloroform. The aqueous phase containing deferrated ExoMS was evaporated to dryness and resuspended in 2 ml deionized water and titrated with FeCl₃ to determine its concentration. This preparation yielded approximately 300 μg Fe-ExoMS per liter of culture of M. smegmatis ML16.

Preparation of radioactive ferric citrate.A ⁵⁵Fe(Cit)₂ ⁵⁻ solution was prepared that consisted of 50 mM Tris (pH 6.9), 15 mM KCl, 10 mM (NH₄)₂SO₄, 1 mM MgSO₄, 100 μM FeCl₃ (74 μCi ⁵⁵Fe), and 20 mM sodium citrate. The solution was centrifuged at 16,000 × g for 1 h to precipitate insoluble iron. This method takes advantage of the insolubility of Fe³⁺ at neutral pH and assumes that, in the presence of a 200-fold molar excess of citrate and at a pH between 6.0 and 9.0, the predominant species in solution is Fe(Cit)₂ ⁵⁻ (11, 18). To verify the formation of the ferric citrate complex, the above concentration of iron was added to a solution in the absence of citrate and to 0.5 M HCl. It was observed that 96% of iron precipitated in the absence of citrate, compared to the same amount of iron added to a 0.5 M HCl reference solution, indicating that iron is complexed by citrate under these conditions. An Fe([¹⁴C]Cit)₂ ⁵⁻ uptake solution was prepared consisting of 50 mM Tris (pH 6.8), 15 mM KCl, 10 mM (NH₄)₂SO₄, 1 mM MgSO₄, 30 μM FeCl₃, and 6 mM citrate (12.5 μCi [1,5-¹⁴C]citric acid). The pH of this solution was adjusted with NaOH to 6.8. This solution was diluted 30-fold for uptake experiments. A ⁵⁵Fe-ExoMS uptake solution was prepared that consisted of 50 mM KH₂PO₄ (pH 7.0), 15 mM KCl, 10 mM (NH₄)₂SO₄, 1 mM MgSO₄, 100 μM deferrated-ExoMS, and 100 μM FeCl₃ (269 μCi ⁵⁵Fe³⁺). All iron added to this mixture remained in solution after centrifugation.

Loss of porins leads to increased IdeR activity by Mycobacterium smegmatis.Mycobacteria are routinely cultured in Middlebrook medium containing 150 μM ferric citrate (20). Given the small size and hydrophilicity of ferric citrate and the fact that these concentrations repress siderophore biosynthesis (27), we reasoned that the mechanism of iron penetration into cells under high-iron conditions occurs by diffusion through porins. If the number of porins were diminished, the cells might sense an iron-deficient state and respond by upregulating iron acquisition genes to maintain homeostatic levels of iron. To test this hypothesis we constructed an iron-regulated reporter in which the cis-regulatory elements and the first 23 codons of the siderophore biosynthesis gene fxbA were fused to a gfp gene in a similar manner as published for an iron-responsive lacZ-based reporter (8). FxbA transcription is regulated by the global mycobacterial iron-dependent regulator IdeR, such that under high-iron conditions IdeR is bound to its operator, preventing the transcription of 92 genes involved in iron acquisition (43), and conversely, it derepresses the same genes under low-iron conditions (12). M. smegmatis transformed with the fxbA-gfp reporter construct displayed GFP fluorescence levels inversely proportional to the iron concentration in the medium, establishing that this reporter is indeed sensitive to extracellular iron concentrations (Fig. 1A). At iron concentrations greater than 5 μM, siderophore biosynthesis gene expression was minimal (Fig. 1A). This is consistent with previous observations that mycobactins are not detected at these concentrations (27), reaffirming that iron homeostasis is maintained in the absence of siderophores in mycobacteria. These results suggest (i) that an alternative route of entry for iron exists under these conditions and (ii) that the gfp-based reporter can be used as an iron sensor in M. smegmatis.

To examine whether diffusion of ferric citrate through porins is sufficient to meet the iron requirements of M. smegmatis, we measured iron reporter activity in the wt, the triple porin mutant (ΔmspA ΔmspC ΔmspD; ML16 [Table 1]), and the triple porin mutant complemented with an integrated copy of mspA expressed from a constitutive promoter (ML56 [Table 1]). The strains were grown in minimal medium containing 150 μM ammonium ferric citrate, cells were harvested in the mid-logarithmic growth phase, and iron reporter activity was analyzed (Fig. 1B). The signal of the iron reporter in the porin mutant ML16 was increased by 64% compared to wt cells. Reporter activity was restored to wt levels by expressing mspA, indicating that lack of porins results in a derepression of IdeR-regulated genes to increase iron uptake and thereby maintain iron homeostasis.

Porins are used by M. smegmatis to take up iron from ferric citrate.To rule out indirect effects of porins that might result in derepression of IdeR in the porin-deficient strain, uptake of ferric citrate was measured directly. The wt, ML16, and ML56 strains were grown overnight in minimal medium containing 150 μM ammonium ferric citrate. Cells were washed and incubated with ⁵⁵Fe-labeled ferric citrate (see Materials and Methods). The triple porin mutant ML16 accumulated approximately 40% less iron than wt M. smegmatis (Fig. 2). Expression of mspA in the porin mutant restored ferric citrate uptake to wt levels, demonstrating that the reduced uptake was caused by the loss of Msp porins and not by an indirect effect. These results showed that M. smegmatis does indeed acquire iron from ferric citrate in a porin-dependent manner under high-iron conditions. These experiments also validated the GFP-reporter construct as a reliable indicator of iron availability in M. smegmatis.

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FIG. 2.

Uptake of ferric citrate by M. smegmatis is dependent on porins. Radioactive ferric citrate [⁵⁵Fe(Cit)₂Na₅] was produced and added to wt M. smegmatis cells grown under high-iron conditions. Triplicate samples were removed at the indicated time points. The radioactivity of the cells was determined by liquid scintillation counting and normalized to the dry mass of cells. The amount of accumulated iron was calculated from the molar ratio of ⁵⁵Fe³⁺ to Fe³⁺ in the uptake stock solution. Standard deviations are shown as error bars.

We were also interested in whether the entire ferric citrate molecule was taken up into cells. It has been reported that citrate from ferric citrate is not taken up into M. smegmatis cells grown under low-iron conditions (18). It is possible that citrate was not taken up in these experiments due to the presence of de novo-synthesized siderophores, which would outcompete citrate as iron chelators. Hence, we analyzed the uptake of ¹⁴C-labeled ferric citrate in M. smegmatis under high-iron conditions, in which siderophore biosynthesis activity was minimal (Fig. 1A). However, no accumulation of ¹⁴C-labeled citrate was observed (Fig. 3).

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FIG. 3.

Citrate from ferric citrate is not taken up by M. smegmatis under high-iron conditions. 1,5-¹⁴C-labeled ferric citrate was added to M. smegmatis cells grown under high-iron conditions to a final concentration of 200 μM citrate (∼420 nCi [1,5-¹⁴C]citrate) and 1 μM FeCl₃. Triplicate samples were removed at the indicated time points. The radioactivity of the cells was determined by liquid scintillation counting and normalized to the dry mass of cells. Standard deviations are shown as error bars.

The threshold for siderophore production is reduced in the porin mutant.To examine whether the iron deficiency experienced by the porin mutant ML16 would lower the threshold for siderophore production in order to compensate for reduced iron uptake (i.e., exhibit derepression of IdeR at higher iron concentrations), we compared iron reporter activities in wt, ML16, and ML56 grown in minimal medium containing ferric citrate concentrations ranging from 20 μM to 0.25 μM. Cells were isolated from precultures containing identical iron concentrations. The iron reporter activity was then determined from cells in the mid-logarithmic growth phase. The reporter activity of all three strains increased above baseline levels at around 1 μM ferric citrate. However, basal expression of siderophore biosynthesis genes was higher in ML16 than in wt M. smegmatis and ML56 (Fig. 4A). Of note, ML16 induces full transcriptional activity from IdeR-regulated genes at higher iron concentrations than wt cells, indicating that the threshold for the production of siderophores is indeed reduced by fewer porins in the outer membrane of M. smegmatis.

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FIG. 4.

Effects of the number of porins on the derepression of IdeR. (A) Threshold analysis. M. smegmatis wt, the triple porin mutant ML16 (ΔmspACD), and the triple porin mutant ML56 constitutively expressing mspA (ΔmspACD::p_imyc-mspA) harboring the iron reporter were grown to mid-logarithmic phase in minimal medium containing the indicated ferric citrate concentrations. Fluorescence intensities of M. smegmatis cells were determined in triplicate and normalized to the optical densities of the samples. Percent maximal IdeR activity is reported as the percentage of the fluorescence intensity from cultures grown in 0.25 μM ferric citrate. (B) The porin triple mutant ML16 produces more siderophores than the wild type. Indicated strains were grown in minimal medium containing 0.25 μM ferric citrate. The culture filtrates were analyzed for siderophore activity in a CAS assay and normalized to the optical density of the samples. Error bars represent standard deviations from biological triplicates. AU, arbitrary units.

To examine whether iron reporter activity correlates with siderophore production, we used the CAS assay to quantify siderophores in culture filtrates of M. smegmatis wt, ML16, and ML56. The CAS assay is based on the removal of iron from the iron-binding chromogenic dye CAS by siderophores (29). wt, ML16, and ML56 were grown in a minimal medium containing 0.25 μM ferric citrate for 27 h. These cultures were washed and used to inoculate fresh minimal medium and grown for another 27 h. Then the cells were harvested, and culture filtrates were analyzed for CAS activity. ML16 produced nearly twice the amount of siderophores than wt cells under these conditions (Fig. 4B). Expression of MspA in ML16 diminished CAS activity. These data indicate that the lowered threshold for siderophore production in ML16 indeed results in the production of more siderophores than from wt M. smegmatis when cells are grown over an equal time period.

The uptake of Fe-ExoMS by M. smegmatis does not depend on porins.The mechanism of uptake for the predominant siderophore of M. smegmatis, Fe-ExoMS, is not known. To rule out the possibility that porins of M. smegmatis mediate the passage of Fe-ExoMS across the outer membrane and thus account for the high CAS activity of ML16, the uptake of ⁵⁵Fe-labeled ExoMS was analyzed. ⁵⁵Fe-labeled ExoMS was prepared from low-iron culture filtrates of ML16 as published previously (30) (see Materials and Methods). M. smegmatis wt and ML16 were grown under high-iron conditions (150 μM ammonium ferric citrate), and uptake of ⁵⁵Fe-labeled ExoMS was measured at a final concentration of 1 μM Fe-ExoMS (Fig. 5). Fe-ExoMS was taken up into cells under the conditions examined; however, there was no difference in the amount of accumulated iron between wt and ML16, indicating that Fe-ExoMS is shuttled across the outer membrane of M. smegmatis by a porin-independent process.

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FIG. 5.

Porins do not affect uptake of Fe-ExoMS by M. smegmatis. Purified Fe-ExoMS was deferrated and labeled with ⁵⁵FeCl₃. ⁵⁵Fe-labeled ExoMS was added to M. smegmatis cells grown under high-iron conditions at a final concentration of 1 μM (∼50 mCi/mg Fe³⁺). Triplicate samples were removed at the indicated time points. The radioactivity of the cells was determined by liquid scintillation counting and normalized to the dry mass of cells. The amount of accumulated iron was calculated from the ⁵⁵Fe³⁺-to-Fe³⁺ molar ratio in the uptake stock solution. Standard deviations are shown as error bars.