SufU is an essential iron-sulfur cluster scaffold protein in Bacillus subtilis

Abstract

Bacteria use three distinct systems for iron-sulfur (Fe/S) cluster biogenesis: the ISC, SUF, and NIF machineries. The ISC and SUF systems are widely distributed, and many bacteria possess both of them. In Escherichia coli, ISC is the major and constitutive system, whereas SUF is induced under iron starvation and/or oxidative stress. Genomic analysis of the Fe/S cluster biosynthesis genes in Bacillus subtilis suggests that this bacterium's genome encodes only a SUF system consisting of a sufCDSUB gene cluster and a distant sufA gene. Mutant analysis of the putative Fe/S scaffold genes sufU and sufA revealed that sufU is essential for growth under minimal standard conditions, but not sufA. The drastic growth retardation of a conditional mutant depleted of SufU was coupled with a severe reduction of aconitase and succinate dehydrogenase activities in total-cell lysates, suggesting a crucial function of SufU in Fe/S protein biogenesis. Recombinant SufU was devoid of Fe/S clusters after aerobic purification. Upon in vitro reconstitution, SufU bound an Fe/S cluster with up to approximately 1.5 Fe and S per monomer. The assembled Fe/S cluster could be transferred from SufU to the apo form of isopropylmalate isomerase Leu1, rapidly forming catalytically active [4Fe-4S]-containing holo-enzyme. In contrast to native SufU, its D43A variant carried a Fe/S cluster after aerobic purification, indicating that the cluster is stabilized by this mutation. Further, we show that apo-SufU is an activator of the cysteine desulfurase SufS by enhancing its activity about 40-fold in vitro. SufS-dependent formation of holo-SufU suggests that SufU functions as an Fe/S cluster scaffold protein tightly cooperating with the SufS cysteine desulfurase.

FIG. 1.

Phenotypes of B. subtilis scaffold protein mutants. (A) Scheme of the B. subtilis suf gene cluster (black, energy-producing system; medium gray, putative scaffold; light gray, cysteine desulfurase). (B) Growth of WT (•) without xylose and sufU conditional mutant without xylose (□) and with 1% xylose induction (▴) in Belitsky minimal medium. (C) Growth in minimal medium of WT in the presence (•) or absence (▴) of glutamate compared with the sufA mutant in the presence (○) or absence (▵) of glutamate.

FIG. 2.

Activities of the Fe/S cluster proteins aconitase (Aco) and succinate dehydrogenase (SDH) and the non-Fe/S protein malate dehydrogenase (MDH) in cell lysates of the wild type (light gray), the ΔsufA mutant (dark gray), and the sufU conditional mutant (black) without xylose induction. Error bars indicate standard deviations from four independent experiments.

FIG. 3.

UV/visible absorption spectrum of reconstituted holo-SufU shows spectral features similar to those for known Fe/S cluster proteins. SufU was reconstituted by using FeCl₃ and Li₂S in one- to fourfold molar excesses over SufU as indicated in 50 mM Tris, 150 mM NaCl, pH 8.0. The UV/visible absorption spectra were recorded. The inset shows spectra of SufU after reconstitution without and with reduction (*) with 2 mM sodium dithionite.

FIG. 4.

The UV/visible absorption spectrum of purified SufU_D43A shows spectral features similar to those for known Fe/S cluster proteins. SufU_D43A was purified aerobically, and a UV/visible spectrum was recorded (solid line). The dashed curve represents the spectrum of SufU_D43A after reduction with 0.5 mM sodium dithionite.

FIG. 5.

holo-SufU activates the isopropylmalate isomerase Leu1. (A) Time course of Leu1 activation by the addition of holo-SufU reconstituted with fourfold (•) molar excesses of Fe and S and 13.3 μM (final concentration after reconstitution) concentrations of FeCl₃ and Li₂S in the absence of SufU (○). (B) Time course of Leu1 activation by the addition of holo-SufU reconstituted with the indicated molar excess of Fe and S. All Leu1 activation reaction mixtures contained 2.9 μM apo-Leu1 in 50 mM Tris, 150 mM NaCl, pH 8.0. Leu1 activity was measured at the indicated time points by the formation of isopropylmalate in 20 mM Tris, pH 7.4, and 50 mM NaCl by measuring the absorption at 235 nm. The reaction was started by the addition either of holo-SufU or of Fe³⁺ and S²⁻ at the same concentrations.

FIG. 6.

SufU activates the cysteine desulfurase SufS. (A) Specific activity of SufS in the absence and presence of SufU. (B) SufS-dependent Fe/S cluster reconstitution on SufU. SufU (50 μM) and SufS (0.5 μM) were preincubated with 5 mM DTT for 1 h. After preincubation, 250 μM ferric ammonium citrate was added and incubated until color stability. Then 2.5 mM cysteine was added and incubated for 30 min.