Molecular Investigation of Iron–Sulfur Cluster Assembly Scaffolds under Stress

Fe/S biosynthesis is controlled in <i>Escherichia coli</i> by two machineries, the housekeeping ISC machinery and the SUF system that is functional under stress conditions. Despite many <i>in vivo</i> studies showing that SUF is more adapted for Fe/S assembly under stress, no molecular data supporting this concept have been provided so far. This work focuses on molecular studies of key actors in Fe/S assembly, the SufB and IscU scaffolds under oxidative stress and iron limitation. We show that the IscU Fe₂S₂ cluster is less stable than the SufB Fe₂S₂ cluster in the presence of hydrogen peroxide, oxygen, and an iron chelator

Activities of Fe-S proteins in <i>iscU</i>_<i>IM</i> and Δ<i>cyaY</i> strains.

<p>Repression of the IscR-regulated gene (<i>iscR</i>::<i>lacZ</i>) (A), Nuo (B) and Sdh (C) activities in the wt (DV901) (white bars), <i>iscU</i>_<i>IM</i> (BR755) (white bars), their <i>ΔcyaY</i> derivatives (DV925, BR756) (black bars), and Δ<i>iscU</i> (BR667) (grey bars) strains. The amount of IscR-dependent repression (fold repression) was determined by dividing the β-galactosidase activity present in the strain lacking IscR (DV915) by the β-galactosidase activity measured for each strain. Error bars represent the standard error from three independent experiments. (D) Cell extracts of indicated strains were subjected to immunoblot analysis using antibodies raised against IscU, IscR, NuoF and NuoC.</p

Analysis of IscU_IM<i>in vitro</i>.

<p>(A) Comparison of the CD spectra (expressed in mdeg) recorded in the region 190–250 nm between IscU_WT (filled line) and IscU_IM (dotted line). (B) Purified IscS, CyaY and IscU_WT (left panel) or IscU_IM (right panel) were mixed in 1:1:1 ratio (144 μM of each protein) in the presence of 4-fold excess of Fe(SO₄)₂(NH₄)₂, 10-fold excess of L-cysteine and 5 mM DTT and incubated for 40 minutes. The mixture was then loaded onto a QFF column equilibrated with 50 mM Tris pH 8. Proteins were eluted with 50 mM Tris pH 8 containing 1M NaCl. SDS-PAGE analyses have been performed on samples from the column on-put (0) and the peaks 1 and 2 for each mixture. (C) Reconstitution of [2Fe-2S] IscU_WT (filled line) and IscU_IM (dotted line) followed by UV-visible absorption spectroscopy. Apo-IscU_WT or apo-IscU_IM (144 μM) were incubated with 5 mM DTT, 1.44 μM IscS, 2 mM L-cysteine and 0.43 mM Fe(SO₄)₂(NH₄)₂ in 50 mM Tris-HCl pH 8. (D) Comparison of the kinetics of enzymatic Fe-S cluster formation on IscU_WT (black diamonds) and IscU_IM (white squares). Experiment was carried out using 25 μM IscU_WT or IscU_IM, 25 μM IscS, 100 μM Fe(SO₄)₂(NH₄)₂, 250 μM L-cysteine, 2 mM DTT. Fe-S cluster formation was followed by absorbance at 420 nm. The experiment was repeated at least three times. One representative experiment is shown.</p

The <i>iscU</i>_<i>IM</i> Δ<i>suf</i> Δ<i>cyaY</i> strain is hypersensitive to oxidative stress.

<p>The wt (DV901), <i>iscU</i>_<i>IM</i> (BR755), <i>iscU</i>_<i>IM</i><i>ΔcyaY</i> (BR756), <i>iscU</i>_<i>IM</i><i>Δsuf</i> (BR763) and <i>iscU</i>_<i>IM</i><i>Δsuf ΔcyaY</i> (BR767) strains were grown overnight at 37°C in LB medium. Cultures were diluted in sterile PBS, and 5 μL were directly spotted onto LB medium plates containing either 1 mM H₂O₂ or 250 μM paraquat. Growth was analysed after overnight incubation at 37°C. Each spot represents a 10-fold serial dilution.</p

The <i>iscU</i>_<i>IM</i> Δ<i>cyaY</i> strain is resistant to aminoglycosides.

<p>Survival of wt (DV901), <i>iscU</i>_<i>IM</i> (BR755) and their Δ<i>cyaY</i> derivatives (DV925 and BR756) without antibiotic (A) and after (B) Gentamicin (Gm) (5 μg/mL) and Kanamycin (Kan) (10 μg/mL) (C) treatment. Survival, measured by colony-forming units (CFU) per mL, was normalized relative to time zero at which the antibiotic was added (midexponential phase cells; ~5 ×10⁷ CFU/mL) and was plotted as log_<i>10</i> of % survival. Error bars represent the standard error from three independent experiments.</p

Bacterial strains and plasmids used in this study.

<p>Bacterial strains and plasmids used in this study.</p

Model for CyaY protein evolution.

<p>Schematic representation of the universal tree of life, for which complete genome sequences are available. LUCA (Last Universal Common Ancestor), LECA (Last Eukaryotic Common Ancestor), LACA (Last Archaeal Common Ancestor) and LBCA (Last Bacterial Common Ancestor). For each prokaryotic phylum (whose color code is the same as the one used in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005134#pgen.1005134.s005" target="_blank">S5 Fig</a>), the number of genomes encoding a CyaY and a IscU homolog with respect to the number of complete available genomes is given. The black arrow indicates the presence of a CyaY encoding gene in the ancestor of a given lineage. The evolutionary event at the origin of the <i>cyaY</i> gene in the Delta/Epsilon subgroup cannot be definitively inferred. One hypothesis is that the <i>cyaY</i> gene is originated in the common ancestor of the Proteobacteria which together with a probable massive loss of <i>cyaY</i> (#) in Delta/Epsilonproteobacteria subgroup explains the presence of CyaY in the species of the Delta/Epsilonproteobacteria subgroup. Dotted arrows indicate horizontal gene transfer events (HGT) (black) and the mitochondrial endosymbiosis (grey). Sequence-logo of the region 99–108 in IscU homologs is also represented using Phylo-mLogo. This region contains the LPPVK motif and amino acid residues at position 108.</p