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Turning Escherichia coli into a Frataxin-Dependent Organism.

Roche B, Agrebi R, Huguenot A, Ollagnier de Choudens S, Barras F, Py B - PLoS Genet. (2015)

Bottom Line: We found that in the double mutant IscUIM ΔcyaY, the ISC pathway was completely abolished, becoming equivalent to the ΔiscU deletion strain and recapitulating the drastic phenotype caused by FXN deletion in eukaryotes.Biochemical analyses of the "eukaryotic-like" IscUIM scaffold revealed that it exhibited a reduced capacity to form Fe-S clusters.Subsequent acquisition of SUF, the second Fe-S cluster biogenesis system, in bacteria, was accompanied by diminished contribution of CyaY in prokaryotic Fe-S cluster biogenesis, and increased tolerance to change in the amino acid present at the 108th position of the scaffold.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Chimie Bactérienne, UMR 7283, Aix-Marseille Université-CNRS, Institut de Microbiologie de la Méditerranée, Marseille, France.

ABSTRACT
Fe-S bound proteins are ubiquitous and contribute to most basic cellular processes. A defect in the ISC components catalyzing Fe-S cluster biogenesis leads to drastic phenotypes in both eukaryotes and prokaryotes. In this context, the Frataxin protein (FXN) stands out as an exception. In eukaryotes, a defect in FXN results in severe defects in Fe-S cluster biogenesis, and in humans, this is associated with Friedreich's ataxia, a neurodegenerative disease. In contrast, prokaryotes deficient in the FXN homolog CyaY are fully viable, despite the clear involvement of CyaY in ISC-catalyzed Fe-S cluster formation. The molecular basis of the differing importance in the contribution of FXN remains enigmatic. Here, we have demonstrated that a single mutation in the scaffold protein IscU rendered E. coli viability strictly dependent upon a functional CyaY. Remarkably, this mutation changed an Ile residue, conserved in prokaryotes at position 108, into a Met residue, conserved in eukaryotes. We found that in the double mutant IscUIM ΔcyaY, the ISC pathway was completely abolished, becoming equivalent to the ΔiscU deletion strain and recapitulating the drastic phenotype caused by FXN deletion in eukaryotes. Biochemical analyses of the "eukaryotic-like" IscUIM scaffold revealed that it exhibited a reduced capacity to form Fe-S clusters. Finally, bioinformatic studies of prokaryotic IscU proteins allowed us to trace back the source of FXN-dependency as it occurs in present-day eukaryotes. We propose an evolutionary scenario in which the current mitochondrial Isu proteins originated from the IscUIM version present in the ancestor of the Rickettsiae. Subsequent acquisition of SUF, the second Fe-S cluster biogenesis system, in bacteria, was accompanied by diminished contribution of CyaY in prokaryotic Fe-S cluster biogenesis, and increased tolerance to change in the amino acid present at the 108th position of the scaffold.

No MeSH data available.


Related in: MedlinePlus

The iscUIM Δsuf ΔcyaY strain is hypersensitive to oxidative stress.The wt (DV901), iscUIM (BR755), iscUIMΔcyaY (BR756), iscUIMΔsuf (BR763) and iscUIMΔsuf ΔcyaY (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.
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pgen.1005134.g003: The iscUIM Δsuf ΔcyaY strain is hypersensitive to oxidative stress.The wt (DV901), iscUIM (BR755), iscUIMΔcyaY (BR756), iscUIMΔsuf (BR763) and iscUIMΔsuf ΔcyaY (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.

Mentions: Because frataxin deficiency, in yeast, led to hypersensitivity to oxidants, we also tested the importance of CyaY in the “eukaryotized” background. Fig 3 shows that introduction of the cyaY deletion in the iscUIM Δsuf strain led to hypersensitivity to hydrogen peroxide and to paraquat, a superoxide generator.


Turning Escherichia coli into a Frataxin-Dependent Organism.

Roche B, Agrebi R, Huguenot A, Ollagnier de Choudens S, Barras F, Py B - PLoS Genet. (2015)

The iscUIM Δsuf ΔcyaY strain is hypersensitive to oxidative stress.The wt (DV901), iscUIM (BR755), iscUIMΔcyaY (BR756), iscUIMΔsuf (BR763) and iscUIMΔsuf ΔcyaY (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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4440780&req=5

pgen.1005134.g003: The iscUIM Δsuf ΔcyaY strain is hypersensitive to oxidative stress.The wt (DV901), iscUIM (BR755), iscUIMΔcyaY (BR756), iscUIMΔsuf (BR763) and iscUIMΔsuf ΔcyaY (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.
Mentions: Because frataxin deficiency, in yeast, led to hypersensitivity to oxidants, we also tested the importance of CyaY in the “eukaryotized” background. Fig 3 shows that introduction of the cyaY deletion in the iscUIM Δsuf strain led to hypersensitivity to hydrogen peroxide and to paraquat, a superoxide generator.

Bottom Line: We found that in the double mutant IscUIM ΔcyaY, the ISC pathway was completely abolished, becoming equivalent to the ΔiscU deletion strain and recapitulating the drastic phenotype caused by FXN deletion in eukaryotes.Biochemical analyses of the "eukaryotic-like" IscUIM scaffold revealed that it exhibited a reduced capacity to form Fe-S clusters.Subsequent acquisition of SUF, the second Fe-S cluster biogenesis system, in bacteria, was accompanied by diminished contribution of CyaY in prokaryotic Fe-S cluster biogenesis, and increased tolerance to change in the amino acid present at the 108th position of the scaffold.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Chimie Bactérienne, UMR 7283, Aix-Marseille Université-CNRS, Institut de Microbiologie de la Méditerranée, Marseille, France.

ABSTRACT
Fe-S bound proteins are ubiquitous and contribute to most basic cellular processes. A defect in the ISC components catalyzing Fe-S cluster biogenesis leads to drastic phenotypes in both eukaryotes and prokaryotes. In this context, the Frataxin protein (FXN) stands out as an exception. In eukaryotes, a defect in FXN results in severe defects in Fe-S cluster biogenesis, and in humans, this is associated with Friedreich's ataxia, a neurodegenerative disease. In contrast, prokaryotes deficient in the FXN homolog CyaY are fully viable, despite the clear involvement of CyaY in ISC-catalyzed Fe-S cluster formation. The molecular basis of the differing importance in the contribution of FXN remains enigmatic. Here, we have demonstrated that a single mutation in the scaffold protein IscU rendered E. coli viability strictly dependent upon a functional CyaY. Remarkably, this mutation changed an Ile residue, conserved in prokaryotes at position 108, into a Met residue, conserved in eukaryotes. We found that in the double mutant IscUIM ΔcyaY, the ISC pathway was completely abolished, becoming equivalent to the ΔiscU deletion strain and recapitulating the drastic phenotype caused by FXN deletion in eukaryotes. Biochemical analyses of the "eukaryotic-like" IscUIM scaffold revealed that it exhibited a reduced capacity to form Fe-S clusters. Finally, bioinformatic studies of prokaryotic IscU proteins allowed us to trace back the source of FXN-dependency as it occurs in present-day eukaryotes. We propose an evolutionary scenario in which the current mitochondrial Isu proteins originated from the IscUIM version present in the ancestor of the Rickettsiae. Subsequent acquisition of SUF, the second Fe-S cluster biogenesis system, in bacteria, was accompanied by diminished contribution of CyaY in prokaryotic Fe-S cluster biogenesis, and increased tolerance to change in the amino acid present at the 108th position of the scaffold.

No MeSH data available.


Related in: MedlinePlus