ErpA, an iron sulfur (Fe S) protein of the A-type essential for respiratory metabolism in Escherichia coli

Abstract

Understanding the biogenesis of iron-sulfur (Fe-S) proteins is relevant to many fields, including bioenergetics, gene regulation, and cancer research. Several multiprotein complexes assisting Fe-S assembly have been identified in both prokaryotes and eukaryotes. Here, we identify in Escherichia coli an A-type Fe-S protein that we named ErpA. Remarkably, erpA was found essential for growth of E. coli in the presence of oxygen or alternative electron acceptors. It was concluded that isoprenoid biosynthesis was impaired by the erpA mutation. First, the eukaryotic mevalonate-dependent pathway for biosynthesis of isopentenyl diphosphate restored the respiratory defects of an erpA mutant. Second, the erpA mutant contained a greatly reduced amount of ubiquinone and menaquinone. Third, ErpA bound Fe-S clusters and transferred them to apo-IspG, a protein catalyzing isopentenyl diphosphate biosynthesis in E. coli. Surprisingly, the erpA gene maps at a distance from any other Fe-S biogenesis-related gene. ErpA is an A-type Fe-S protein that is characterized by an essential role in cellular metabolism.

Fig. 1.

Growth analysis of E. coli LL401(ara_p::erpA) strain in aerobiosis. (A) Strain LL401 was grown on LB medium plates with added arabinose (Ara) (Upper) or glucose (Glu) (Lower). (B) Strains were grown in LB medium with added 0.2% glucose (broken lines and open symbols) or 0.2% arabinose (solid lines and filled symbols). The strains studied were LL401(ara_p::erpA)/pUC18 (squares) and LL401(ara_p::erpA)/pLUE-A(erpA⁺) (triangles). (C) Strains LL401(ara_p::erpA) harboring pUC18 (○), pLUE-A (■), pLUE-A42 (▵), pLUE-A106 (□), and pLUE-A108 (•) were grown in LB medium with 0.2% glucose added.

Fig. 2.

Growth analysis of E. coli LL401(ara_p::erpA) and LL402 (ΔerpA::cat) strains in anaerobiosis. (A) Strain LL401 was grown on LB medium plates with added arabinose (Ara) (Upper) or glucose (Glu) (Lower). (B) Strains LL402(ΔerpA::cat)/pUC18 (open bars) and LL402(ΔerpA::cat)/pLUE-A (erpA⁺) (filled bars) were grown under anaerobiosis in minimal medium supplemented with glucose or glycerol as a carbon source and with nitrate, trimethylamine N-oxide (TMAO), or fumarate as electron acceptors. The inoculum was 0.02 ml of an overnight culture grown under anaerobiosis in minimal medium with glucose. Cells were pelleted and washed before inoculation. OD₆₀₀ values were recorded after 16 h of incubation at 37°C. The experiment was performed in triplicate, and a representative experiment is shown.

Fig. 3.

Phenotypic suppression of E. coli LL402 (ΔerpA::cat) mutant by the eukaryotic MVA pathway. (A) Strains MG1655, MG1655 MVA⁺, LL402 (ΔerpA::cat), and LL405 (ΔerpA::cat, MVA⁺) were grown on an LB medium plate containing the MVA pathway inducer l-arabinose (0.2%) and the MVA substrate (1 mM) and was incubated at 37°C for 24 h under aerobiosis. (B) Strains LL402 (ΔerpA::cat) (square) and LL405 (ΔerpA::cat, MVA⁺) (triangle) were grown under aerobiosis in 0.2% LB arabinose-containing medium supplemented (open symbols) or not (filled symbols) with 1 mM MVA.

Fig. 4.

Quinone contents from E. coli LL402 (ΔerpA::cat) and wild-type (wt) strains. Strains MG1655 and LL402 (ΔerpA::cat) were grown for 15 h under anaerobiosis in LB glucose medium. Two milliliters of the culture was taken, and cells were pelleted after three washes with water, resuspended in 4 ml of LB glucose, and incubated 2 h at 37°C under aerobiosis, then cells were washed three times, pelleted, and frozen directly. Ubiquinone (UB8), menaquinone (MK8), and dimethylmenaquinone (DMK8) contents in the samples from MG1655 (black bars) and LL402 (ΔerpA::cat) (hatched bars) were determined.

Fig. 5.

Fe–S transfer from holoErpA to apo-IspG. (A) UV–visible light absorption spectra. ApoIspG (880 μM) (spectrum 1) was incubated anaerobically with 1.26 mM ⁵⁷Fe-reconstituted ErpA (two iron atoms per polypeptide chain) in 0.1 M Tris·HCl, pH 8/0.1 M NaCl/5 mM DTT for 1 h. Spectrum 2 shows reconstituted IspG after separation of proteins. (B) Mössbauer spectrum of 220 μM reconstituted IspG at 4.2 K. Solid lines above the spectra are theoretical simulations corresponding to the different doublets (see text).