Iron-sulfur cluster assembly scaffold protein IscU is required for activation of ferric uptake regulator (Fur) in Escherichiacoli

Abstract

It was generally postulated that when intracellular free iron content is elevated in bacteria, the ferric uptake regulator (Fur) binds its corepressor a mononuclear ferrous iron to regulate intracellular iron homeostasis. However, the proposed iron-bound Fur had not been identified in any bacteria. In previous studies, we have demonstrated that Escherichia coli Fur binds a [2Fe-2S] cluster in response to elevation of intracellular free iron content and that binding of the [2Fe-2S] cluster turns on Fur as an active repressor to bind a specific DNA sequence known as the Fur-box. Here we find that the iron-sulfur cluster assembly scaffold protein IscU is required for the [2Fe-2S] cluster assembly in Fur, as deletion of IscU inhibits the [2Fe-2S] cluster assembly in Fur and prevents activation of Fur as a repressor in E. coli cells in response to elevation of intracellular free iron content. Additional studies reveal that IscU promotes the [2Fe-2S] cluster assembly in apo-form Fur and restores its Fur-box binding activity in vitro. While IscU is also required for the [2Fe-2S] cluster assembly in the Haemophilus influenzae Fur in E. coli cells, deletion of IscU does not significantly affect the [2Fe-2S] cluster assembly in the E. coli ferredoxin and siderophore-reductase FhuF. Our results suggest that IscU may have a unique role for the [2Fe-2S] cluster assembly in Fur and that regulation of intracellular iron homeostasis is closely coupled with iron-sulfur cluster biogenesis in E. coli.

Keywords: ferric uptake regulator (Fur); iron homeostasis; iron–sulfur cluster biogenesis.

Figure 1

The iron–sulfur cluster assembly scaffold protein IscU is required for the [2Fe-2S] cluster assembly in Fur in E. coli cells.A, PCR confirmation of gene iscU deletion in E. coli cells. Gene iscU was deleted using the single-step inactivation procedure (52) as described previously (43). Two primers (primer 1, 5′-TGACCTTTCTCCGCTGTGGG-3′, and primer 2, 5′-CTTTACCGCGGTTAGCCAGA-3′) were used for the PCR confirmation of the gene iscU deletion. Lane M, 100 bp DNA ladder (NEB). Lane 1, from wildtype E. coli cells. Lane 2, from the ΔiscU mutant cells. B, UV-Vis absorption spectra of Fur proteins purified from wildtype E. coli cells (spectrum 1) and the ΔiscU mutant cells (spectrum 2) grown in M9 medium supplemented with Fe(NH₄)₂(SO₄)₂ (2 μM) under aerobic growth conditions. Purified Fur proteins (50 μM) were dissolved in buffer containing NaCl (500 mM) and Tris (20 mM, pH 8.0). Insert (a), the photograph of purified Fur from wildtype E. coli cells (1) and the ΔiscU mutant cells (2). Insert (b), the SDS-PAGE gel of Fur proteins purified from wildtype E. coli cells (lane 1) and the ΔiscU mutant cells (lane 2) grown in M9 medium supplemented with Fe(NH₄)₂(SO₄)₂ (2.0 μM) (lane 2). Lane M, the PAGE-MASTER protein markers (GenScript co.) with molecular weights. C, the [2Fe-2S] cluster binding of Fur in E. coli wildtype and the ΔiscU mutant cells grown in M9 medium supplemented with increasing concentrations of iron. Fur protein was purified from E. coli wildtype and the ΔiscU mutant cells grown in M9 medium supplemented with indicated concentrations of Fe(NH₄)₂(SO₄)₂. The concentration of the [2Fe-2S] cluster in Fur was determined using an extinction coefficient of 10 mM⁻¹ cm⁻¹ at 410 nm (25). The [2Fe-2S] cluster occupancies of Fur were calculated from the ratio of the [2Fe-2S] cluster to Fur and plotted as a function of iron concentrations in M9 medium. Data represent mean ± SD (standard deviation) from three independent experiments. Fur, ferric uptake regulator.

Figure 2

The Fur-box binding activity of Fur purified from E. coli wildtype and the ΔiscU mutant cells grown in M9 medium supplemented with 2 μM iron.A, the restriction site protection assays of Fur. pUC19-iuc (3.2 nM) was preincubated with increasing concentrations of purified Fur proteins, followed by digestion with HinfI (0.5 unit) at 37 ^°C for 10 min. The digested DNA products were separated by 1.5% agarose gel electrophoresis. Lanes 1 to 4, pUC19-iuc (3.2 nM) was preincubated with 2.0 μM, 1.0 μM, 0.5 μM, and 0.25 μM Fur purified from wildtype E. coli cells, respectively. Lanes 5 to 8, pUC19-iuc (3.2 nM) was preincubated with 2.0 μM, 1.0 μM, 0.5 μM, and 0.25 μM Fur purified from the ΔiscU mutant E. coli cells, respectively. Lane 9, no Fur protein was added before the HinfI digestion. Lane 10, pUC19-iuc only with no HinfI digestion. B, relative binding activity of Fur proteins purified from wildtype and the ΔiscU mutant E. coli cells. The intensities of the DNA band of 787 bp shown in (A) were quantified using ImageJ and plotted as a function of the Fur concentrations. Data represent mean ± SD (standard deviation) from three independent experiments. Fur, ferric uptake regulator.

Figure 3

Deletion of gene iscU prevents activation of Fur as a repressor in E. coli cells.A, a reporter gene fur::gfp was constructed and cloned into a plasmid pBAD as described in the Experimental Procedures. An active Fur represses the expression of the reporter gene fur::gfp and decreases the GFP fluorescence in E. coli cells. B, the repressor activity of Fur in E. coli wildtype and the ΔiscU mutant cells. The constructed plasmid containing the reporter gene fur::gfp was introduced into wildtype and the ΔiscU mutant cells. Wildtype E. coli and the ΔiscU mutant cells were grown in M9 medium supplemented with 0, 1.0 μM, 2.0 μM, and 10.0 μM Fe(NH₄)₂(SO₄)_2. After 5 h growth at 37 ^oC under aerobic conditions, the cells were subjected to the GFP fluorescence measurements. The relative GFP intensities were obtained from the ratios of the GFP fluorescence intensity to the cell density (A at 600 nm) of the culture. The E. coli cells without the reporter gene fur::gfp were used as the controls. The data represent mean ± SD (standard deviation) from three independent experiments. C, deletion of gene iscU increases the sensitivity of E. coli cells to iron in M9 medium. Wildtype E. coli and the ΔiscU mutant cells were grown in M9 medium supplemented with 0, 1.0 μM, 2.0 μM, and 10.0 μM Fe(NH₄)₂(SO₄)_2. After 5 h growth at 37 ^°C under aerobic conditions, the cell growth was measured at 600 nm. The ratios of the cell growth in M9 medium supplemented with increasing iron concentrations to the cell growth in M9 medium with no iron addition were plotted as a function of the iron concentrations in M9 medium. The A values at 600 nm (cell growth) of wildtype and the ΔiscU mutant grown in M9 medium with no iron addition were 0.74 ± 0.03 and 0.66 ± 0.04 for, respectively. The data represent mean ± SD (standard deviation) from three independent experiments. Fur, ferric uptake regulator; GFP, green fluorescent protein.

Figure 4

IscU promotes the [2Fe-2S] cluster assembly in apo-form E. coli Fur in vitro.A, UV-Vis absorption spectra of apo-form Fur (30 μM) after reconstitution with Fe(NH₄)₂(SO₄)₂ (1.0 mM), L-cysteine (1.0 mM), cysteine desulfurase (IscS) (1.0 μM), and dithiothreitol (4 mM) in the absence (spectrum 1) or presence (spectrum 3) of IscU (50 μM) at 37 ^°C for 20 min. Spectrum 2, IscU (50 μM) after reconstitution with Fe(NH₄)₂(SO₄)₂ (1.0 mM), L-cysteine (1.0 mM), cysteine desulfurase (IscS) (1.0 μM), and dithiothreitol (4 mM) at 37 ^°C for 20 min. B, the [2Fe-2S] cluster is reconstituted in apo-form E. coli Fur by IscU. Spectrum 1, apo-form Fur (30 μM) after the reconstitution without IscU. Spectrum 2, the net spectrum of apo-form Fur after the reconstitution with IscU [the reconstituted IscU (spectrum 2) was subtracted from the reconstituted apo-form Fur and IscU (spectrum 3) in (A)]. C, the restriction site protection assays of apo-form E. coli Fur after reconstitution with or without IscU. pUC19-iuc was preincubated with increasing concentrations of Fur proteins, followed by digestion with HinfI at 37 ^°C for 10 min. The digested DNA products were separated on 1.5% agarose gel electrophoresis. Lanes 1 to 3, pUC19-iuc (3.2 nM) was preincubated with 4.0 μM, 2.0 μM, and 1.0 μM apo-form Fur after reconstitution without IscU, respectively. Lanes 4 to 6, pUC19-iuc (3.2 nM) was preincubated with 4.0 μM, 2.0 μM, and 1.0 μM apo-form Fur after reconstitution with IscU, respectively. Lane 7, no Fur protein was added before the HinfI digestion. Lane 8, pUC19-iuc (3.2 nM) was preincubated with IscU (4.0 μM). Lane 9, pUC19-iuc only. The data are representative from three independent experiments. D, relative binding activity of apo-form Fur after reconstitution with or without IscU. The intensities of the DNA band of 787 bp in the agarose gel shown in (C) were quantified using ImageJ and plotted as a function of the Fur concentrations. Data represent mean ± SD (standard deviation) from three independent experiments. Fur, ferric uptake regulator.

Figure 5

IscU is also required for the [2Fe-2S] cluster assembly in the H. influenzae Fur but not for the [2Fe-2S] cluster assembly in the E. coli ferredoxin and FhuF in E. coli cells.A, UV-Vis absorption spectra of the H. influenzae Fur purified from wildtype E. coli (spectrum 1) and the ΔiscU mutant (spectrum 2) cells grown in M9 medium supplemented with 2 μM Fe(NH₄)₂(SO₄)₂ under aerobic growth conditions. Purified H. influenzae Fur (40 μM) was dissolved in buffer containing NaCl (500 mM) and Tris (20 mM, pH 8.0). B, UV-Vis absorption spectra of the E. coli ferredoxin (Fdx) purified from wildtype E. coli (spectrum 1) and the ΔiscU mutant (spectrum 2) cells grown in M9 medium supplemented with 2.0 μM Fe(NH₄)₂(SO₄)₂ under aerobic growth conditions. Purified ferredoxin (75 μM) was dissolved in buffer containing NaCl (500 mM) and Tris (20 mM, pH 8.0). C, UV-Vis absorption spectra of the E. coli FhuF purified from wildtype E. coli (spectrum 1) and the ΔiscU mutant (spectrum 2) cells grown in M9 medium supplemented with 2.0 μM Fe(NH₄)₂(SO₄)₂ under aerobic growth conditions. Purified FhuF (12 μM) was dissolved in buffer containing NaCl (500 mM) and Tris (20 mM, pH 8.0). The results are representatives of three independent experiments. Fur, ferric uptake regulator.

Figure 6

A proposed model for the interplay between iron–sulfur cluster biogenesis and regulation of intracellular iron homeostasis in E. coli cells. When intracellular free iron content is depleted, IscU cannot assemble a [2Fe-2S] cluster in Fur, and Fur is inactive. When intracellular free iron content is elevated, IscU, together with L-cysteine, cysteine desulfurase (IscS) and other iron–sulfur cluster assembly machinery, assembles a [2Fe-2S] cluster in Fur, and Fur becomes an active repressor to bind the Fur-box and regulates intracellular iron homeostasis. A structural zinc binding site is shown in apo-Fur and the [2Fe-2S] cluster-bound Fur. Fur, ferric uptake regulator.